4 5784891476752204819

Biomedical Science Practice
fundamentals OF
biomedical science
Biomedical Science
Practice
Experimental and professional skills
Edited by
Hedley Glencross
Institute of Biomedical Science
Nessar Ahmed
Manchester Metropolitan University
Qiuyu Wang
Manchester Metropolitan University
1
1
Great Clarendon Street, Oxford ox2 6dp
Oxford University Press is a department of the University of Oxford.
It furthers the University’s objective of excellence in research, scholarship,
and education by publishing worldwide in
Oxford New York
Auckland Cape Town Dar es Salaam Hong Kong Karachi
Kuala Lumpur Madrid Melbourne Mexico City Nairobi
New Delhi Shanghai Taipei Toronto
With offices in
Argentina Austria Brazil Chile Czech Republic France Greece
Guatemala Hungary Italy Japan Poland Portugal Singapore
South Korea Switzerland Thailand Turkey Ukraine Vietnam
Oxford is a registered trade mark of Oxford University Press
in the UK and in certain other countries
Published in the United States
by Oxford University Press Inc., New York
© Oxford University Press 2011
The moral rights of the authors have been asserted
Database right Oxford University Press (maker)
All rights reserved. No part of this publication may be reproduced,
stored in a retrieval system, or transmitted, in any form or by any means,
without the prior permission in writing of Oxford University Press,
or as expressly permitted by law, or under terms agreed with the appropriate
reprographics rights organization. Enquiries concerning reproduction
outside the scope of the above should be sent to the Rights Department,
Oxford University Press, at the address above
You must not circulate this book in any other binding or cover
and you must impose the same condition on any acquirer
British Library Cataloguing in Publication Data
Data available
Library of Congress Cataloging in Publication Data
Data available
Typeset by MPS Limited, a Macmillan Company
Printed in Italy on acid-free paper by
L.E.G.O. S.p.A.
ISBN 978-0-19-953329-9
1 3 5 7 9 10 8 6 4 2
Contents
An introduction to the Fundamentals of 3 Communications in laboratory
Biomedical Science series x medicine 42
Acknowledgements xiv
Contributors xv Georgina Lavender
Abbreviations xvi
Introduction 42
3.1 Communication and the clinical laboratory 43
1 Biomedical science and
3.2 Communication and confidentiality 48
biomedical scientists 1
3.3 Methods of communication 49
Hedley Glencross
3.4 Data protection 55
Introduction 1 3.5 Record keeping 61
1.1 What is biomedical science? 2 Summary 63
1.2 Biomedical science degree programmes 3 Further reading 63
1.3 What is a biomedical scientist? 4 Questions 64
1.4 Professional skills 6
4 Health and safety 66
1.5 Initial employment as a biomedical
Alison Taylor
scientist 10
1.6 What do biomedical scientists do? 12 Introduction 66
Summary 18 4.1 Hazards 67
Further reading 19 4.2 Routes of entry into the body by chemical
and biological agents 70
Questions 19
4.3 Statutory framework for health and safety 70
4.4 Risk assessment 76
2 Fitness to practise 20
Hedley Glencross 4.5 Control of substances hazardous to health 80
4.6 Fire regulations 83
Introduction 20
4.7 Personal protective equipment 85
2.1 Healthcare regulation 20
4.8 Reporting of Injuries, Diseases and Dangerous
2.2 Health Professions Council 21 Occurrences Regulations 87
2.3 HPC Standards of Conduct, Performance 4.9 Transport regulations 88
and Ethics 24 4.10 Personal health and safety 89
2.4 Renewal of HPC registration 30 4.11 Universal or standard precautions 92
2.5 Institute of Biomedical Science 30 Summary 92
2.6 IBMS Code of Conduct and Good Further reading 93
Professional Practice 31 Questions 93
2.7 IBMS and education 34
Summary 39 5 Statistics and handling data 95
Andrew Blann
Further reading 39
Questions 40 Introduction 95
vi CONTENTS
5.1 Types of information and how to 8.2 Components of a microscope 187

describe them 96
8.3 Magnification 192
5.2 Analysis and interpretation of data 105
8.4 Dark field microscopy 192
5.3 Statistical packages 120
8.5 Phase contrast microscopy 193
Summary 122
8.6 Polarization microscopy 195
Further reading 123
8.7 Fluorescence microscopy 195
Questions 123
8.8 Confocal microscopy 198
6 Preparing and measuring 8.9 Inverted microscopes 199

reagents 125 8.10 Electron microscopy 200
Ian Graham Summary 209
Introduction 125 Further reading 209

Questions 209
6.1 Balances and weighing 126
6.2 Volume measurements and delivery 131
6.3 Pipettors 135
9 Electrochemistry 211
Peter Robinson
6.4 Preparing reagents: concentrations
and dilutions 141 Introduction 211
Summary 148 9.1 Basic concepts and definitions 212
Further reading 149 9.2 Principles of electrochemical
Questions 149 techniques 213
9.3 Potentiometric techniques 215
7 Samples and sample collection 151 9.4 Voltammetric techniques 225
Joyce Overfield
9.5 Biosensors 228
Introduction 151 Summary 233
7.1 Blood 152 Further reading 233
7.2 Urine 161 Questions 234
7.3 Fluids other than blood and urine 163

7.4 Cytopathology and histopathology samples 167 10 Radioactivity and radiation 236
John Gaffney and Mark Slevin
7.5 Samples for microbiology testing 169
Summary 176 Introduction 236
Further reading 176 10.1 Atomic structure 236
Questions 177 10.2 Types of radioactive decay 238
10.3 Rate of radioactive decay 241
8 Microscopy 179 10.4 Units associated with radioactivity 242
Tony Sims
10.5 Interaction of radiation with matter 243
Introduction 179
10.6 Detection and measurement
8.1 Microscopy and image formation 181 of radioactivity 244
CONTENTS vii
10.7 Safety precautions when using 13 Chromatography 311

radioisotopes 247 Qiuyu Wang, Nessar Ahmed, and Chris Smith
10.8 Clinical applications of radioactivity 249
Introduction 311
Summary 254
13.1 Partition or distribution coefficient 312
Further reading 254
13.2 Planar chromatography 314
Questions 255
13.3 Column chromatography 319
11 Spectroscopy 257 13.4 High performance liquid
Qiuyu Wang, Helen Montgomery, chromatography 329
Nessar Ahmed, and Chris Smith 13.5 Gas–liquid chromatography 334
Introduction 257 Summary 337
11.1 Radiation and the electromagnetic Further reading 338
spectrum 257 Questions 338
11.2 Interactions between radiation and matter 259
14 Electrophoresis 341
11.3 Radiation, particles, and quanta 260
Qiuyu Wang, Nessar Ahmed, and Chris Smith
11.4 Absorbance 261
Introduction 341
11.5 Absorption spectra 266
14.1 Principles of electrophoresis 341
11.6 Light scattering methods 272
14.2 Factors affecting electrophoretic
11.7 Fluorescence and fluorimetry 273
separations 343
11.8 Nuclear magnetic resonance
14.3 Detection of separated components 345
spectroscopy 274
14.4 Paper or cellulose electrophoresis 348
11.9 Mass spectrometry 279
Summary 287 14.5 Cellulose acetate electrophoresis 349
Further reading 288 14.6 Starch electrophoresis 351

Questions 288 14.7 Polyacrylamide gel electrophoresis 352
14.8 Agarose gel electrophoresis 362
12 Centrifugation 290
14.9 Capillary electrophoresis 367
14.10 Isoelectric focusing 370
Introduction 290
14.11 Two-dimensional electrophoresis 372
12.1 Basics of centrifugation theory 291
Summary 374
12.2 Types of centrifuges 295 Further reading 374
12.3 Tubes and rotors 298 Questions 375
12.4 Separation methods using centrifuges 299
12.5 Safety and centrifuges 303
15 Immunological techniques 377
Christine Yates
12.6 Examples of clinical centrifugation 305
Summary 308 Introduction 377
Further reading 308 15.1 Outline of the immune system 378

Questions 309 15.2 Synthesis of antibodies in vivo 380
viii CONTENTS
15.3 Production of polyclonal and monoclonal 17.4 Tracked automation systems and
antibodies in vitro 382 the core automated laboratory 470
15.4 Antigen–antibody interactions 385 17.5 Automation in wider laboratory settings 473
15.5 Immunological techniques used in clinical Summary 475

laboratories 389 Further reading 476
Summary 409 Questions 476
Further reading 410
Questions 411 18 Point of care testing 478
Jan Still and Lynda Petley
16 Molecular biology techniques 412 Introduction 478

18.1 Standards and guidelines 479
Introduction 412 18.2 Advantages and limitations
of point of care testing 480
16.1 Structure and properties
of macromolecules 413 18.3 POCT committee and POCT policy 484
16.2 Molecular biology-based techniques 422 18.4 Procurement 488
16.3 Isolation of nucleic acids 422 18.5 Evaluating a POCT device 489
16.4 Hydrolysis of nucleic acids 18.6 Use of POCT devices 492

and restriction endonucleases 428
18.7 Connectivity 494
16.5 Gel electrophoresis 434 18.8 Training for POCT 495
16.6 DNA sequencing 435 18.9 Quality and POCT 496
16.7 Blotting techniques 439 18.10 Audit 498
16.8 Enzyme-linked immunosorbent assays 18.11 Problems, incidents, and litigation 499
and fluorescence in situ hybridization 446
Summary 502
16.9 DNA cloning 449 Further reading 502
16.10 DNA microarrays 457 Questions 503
Summary 458
Further reading 459 19 Quality assurance
Questions 460 and management 505
Elaine Moore
17 Laboratory automation 461 Introduction 505

Tim James
19.1 Quality control 506
Introduction 461 19.2 Quality management 508
17.1 Benefits of automation 462 19.3 Documentation in the clinical laboratory 512
17.2 Collection of suitable samples 19.4 Standards and regulatory requirements
and delivery to the laboratory 465 in the medical laboratory 513
17.3 Specimen reception considerations 19.5 Tools and techniques for continual
in the core automated laboratory 467 improvement 517
CONTENTS ix
Summary 524 20.6 Opportunities for self-guided

Further reading 524 personal development 541
Questions 525 20.7 Reflective learning 541

20.8 Study skills 542
20 Personal development 526 20.9 Evidence supporting personal
Georgina Lavender development 547
Introduction 526 20.10 Performance appraisal and

personal development plans 549
20.1 Continuing professional development 528
Summary 550
20.2 Opportunities for personal development
Further reading 551
in the workplace 529
Questions 551
20.3 Training and development 532
20.4 Professional bodies and personal Glossary 553
development 534 Index 563
20.5 Commercial concerns and personal

development 540
An introduction to the
Fundamentals of Biomedical
Science series
Biomedical Scientists form the foundation of modern healthcare, from cancer screening to
diagnosing HIV, from blood transfusion for surgery to food poisoning and infection control.
Without Biomedical Scientists, the diagnosis of disease, the evaluation of the effectiveness of
treatment, and research into the causes and cures of disease would not be possible.
However, the path to becoming a Biomedical Scientist is a challenging one: trainees must not
only assimilate knowledge from a range of disciplines, but must understand—and demonstrate
—how to apply this knowledge in a practical, hands-on environment.
The Fundamentals of Biomedical Science series is written to reflect the challenges of biomedi-
cal science education and training today. It blends essential basic science with insights into
laboratory practice to show how an understanding of the biology of disease is coupled to the
analytical approaches that lead to diagnosis.
The series provides coverage of the full range of disciplines to which a Biomedical Scientist
may be exposed – from microbiology to cytopathology to transfusion science. Alongside vol-
umes exploring specific biomedical themes and related laboratory diagnosis, an overarching
Biomedical Science Practice volume provides a grounding in the general professional and ex-
perimental skills with which every Biomedical Scientist should be equipped.
Produced in collaboration with the Institute of Biomedical Science, the series
• Understands the complex roles of Biomedical Scientists in the modern practice of medi-
cine.
• Understands the development needs of employers and the Profession.
• Places the theoretical aspects of biomedical science in their practical context.
Learning from this series

The Fundamentals of Biomedical Science series draws on a range of learning features to help
readers master both biomedical science theory, and biomedical science practice.
Case studies illustrate how the biomedical science theory and practice
CASE STUDY 6.1 Choosing an appropriate Cross ref
method for volume measurement and Electropho presented throughout the series relates to situations and experiences
are descri
delivery ‘Electroph
that are likely to be encountered routinely in the biomedical science
The purpose of this case study is to help you to think about the importance of choosing laboratory. Answers to questions posed in some Case Studies are avail-
an appropriate volume measurement method for a particular situation. Details about
alternatives will be given later in this section. able in the book’s Online Resource Centre.
Consider the following scenarios, A and B.
AN INTRODUCTION TO THE SERIES xi
BOX 9.1 Ohm’s Law

Additional information to augment the main text appears in boxes
Ohm’s Law can be rearranged to describe the current, voltage, or resistance. The easiest
way to remember these relationships is to use a VIR triangle as shown in Figure 9.2. To
use this, simply use your finger to hide the value that you want to calculate. The remain-
ing two values then show you how to do the calculation.
Method boxes walk through the key protocols that the reader is likely
METHOD How to set up and use a pipettor
to encounter in the laboratory on a regular basis.
■ Choose a pipettor appropriate to the task in hand. For P1000, 085 represents 850 lL. The me
a given model, it is essential to use the pipettor only digits on the display varies with the mo
within the volume range indicated. Use beyond this must be checked otherwise an incorre
range can damage the pipettor mechanism. delivered.
■ Organize the work area so that all materials and items ■ To measure and deliver a volume of sa
of equipment to be used are conveniently placed. The is first depressed as far as the first st
work surface needs to be at a comfortable height. If nec- be felt) with the pipettor held away
Further features are used to help consolidate and extend essary, this can be achieved by using a height adjustable Next the tip should be placed below
students’ understanding of the subject
The concentration of a solution is determined by the quantity of solute and the volume of
Key points reinforce the key concepts that the reader should master solvent as described in Box 6.6.
from having read the material presented, while Summary points act
Key Points
as an end-of-chapter checklists for readers to verify that they have re- Solutions consist of a solute dissolved in a solvent; the concentration of the solution is
membered correctly the principal themes and ideas presented within determined by the relative proportions of solute and solvent.
each chapter. The different ways in which concentration may be expressed will now be considered. The
commonest form is molarity and this along with the concept of the mole will be considered
in some detail followed by other common forms of concentration molality % (per cent)
Key terms provide on-the-page explanations of terms with which the al lines represent a phase boundary (electrode/electrolyte interface)
A cation (pronounced cat-ion)
lines the salt bridge. Note that according to this notation the negative
reader may not be familiar; in addition, each title in the series features ced to the left, the positive electrode (cathode) is placed to the right,
is an ion that has fewer electrons
in its electron shells than
erefore flow from left-to-right in the connecting wire. protons in its nucleus. It thus
a glossary, in which the key terms featured in that title are collated. carries a net positive charge and
will move towards the negative
electrode during electrolysis.
of electrochemical techniques
An anion (pronounced an-ion)
is an ion that has more electrons
an be categorized as galvanic or electrolytic. A galvanic cell is
than protons. Thus it has a net
ns occur spontaneously at the electrodes when they are connected negative charge and will move
ctor. In an electrolytic cell, however, the electrochemical reactions towards the positive electrode
Self-check questions throughout each chapter and extended ques- A potentiometric electrode is said to have Nernstian characteristics if it obeys Nernst’s law. If
the changes in potential relative to concentration are less than the theoretical values, it may
tions at the end of each chapter provide the reader with a ready means indicate an electrode malfunction or interference from other ions.
of checking that they have understood the material they have just en- SELF-CHECK 9.3
Given that the universal gas constant is 8.314 J mol−1 per K, T is the temperature in Kelvin
countered. Answers to these questions are provided in the book’s On- (=273 + T°C) and that the Faraday constant is 96,485 coulomb mol−1, calculate the change in
potential in mV for a ten-fold change in the concentration of H+ when the temperature of the
line Resource Centre; visit www.oxfordtextbooks.co.uk/orc/glencross system is 35°C.
Cross references help the reader to see biomedical science as a uni- The system is allowed to achieve equilibrium, at which time the pH is m
compared with a calibration curve of pH against known PCO2 values.
fied discipline, making connections between topics presented within Severinghaus’s modifications doubled the sensitivity of the original devic
Cross reference ity, and improved the response time. In 1958, Severinghaus described a
each volume, and across all volumes in the series. Measuring HCO3−, PCO2, and both his PCO2 electrode and a Clark O2 electrode, which you can read
pH in clinical samples are to form a combined blood gas analyser. Commercial manufacture of th
described in Chapter 6 of
electrode began in 1959 and such electrodes are still found in PCO2 ana
Clinical Biochemistry.
ratories today.
The Severinghaus electrode is a gas-sensing electrode and as it is based

the electrical output is a logarithmic function of PCO2. At 37°C an output
for a 10-fold change in PCO2. The response is also temperature depend
xii ONLINE LEARNING MATERIALS
Online learning materials

The Fundamentals of Biomedical Science series doesn’t end with the printed books. Each title in
the series is supported by an Online Resource Centre, which features additional materials for
students, trainees, and lecturers.
www.oxfordtextbooks.co.uk/orc/fbs
Guides to key experimental skills and

methods
Multimedia walk-throughs of key experimental skills—including
both animations and video—to help you master the essential
skills that are the foundation of Biomedical Science practice.
Biomedical science in practice

Interviews with practising Biomedical Scientists working in a range of disciplines, to
give you valuable insights into the reality of work in a Biomedical Science laboratory.
ONLINE LEARNING MATERIALS xiii
Digital Microscope
A library of microscopic images for you to investigate using this
powerful online microscope, to help you gain a deeper appre-
ciation of cell and tissue morphology.
The Digital Microscope is used under licence from the Open

University.
‘Check your understanding’ learning modules

A mix of interactive tasks and questions, which address a variety
of topics explored throughout the series. Complete these mod-
ules to help you check that you have fully mastered all the key
concepts and key ideas that are central to becoming a proficient
Biomedical Scientist.
We extend our grateful thanks to colleagues in the School of

Health Science at London Metropolitan University for their in-
valuable help in developing these online learning materials.
Answers to self-check, case study,

and end of chapter questions
Answers to questions posed in the book are provided to aid self-
assessment.
Lecturer support materials

hair shaft
sweat pore
The Online Resource Centre for each title in the series also fea- dermal papilla
sensory nerve ending
for touch
tures figures from the book in electronic format, for registered stratum corneum
pigment layer EPIDERMIS
stratum germinativum
adopters to download for use in lecture presentations, and other stratum spinosum
stratum basale DERMIS
arrector pili muscle
educational resources. sebaceous gland
hair follicle
To register as an adopter visit www.oxfordtextbooks.co.uk/orc/ papilla of hair

SUBCUTIS
(hypodermis)
glencross and follow the on-screen instructions. nerve fiber

blood and vein
lymph vessels artery
sweat gland
FIGURE 7.1
pacinian corpuscle Diagram of the skin.
Any comments?
We welcome comments and feedback about any aspect of this series.
Just visit www.oxfortextbooks.co.uk/orc/feedback/ and share your views.
Acknowledgements
I would like to thank the following individuals and organizations for their help, without
whom it would have been difficult to complete this project.
Katy Linton and Sidonie Kamtchouang for the case studies reproduced in Chapter 1. The
Health Professions Council for their advice on regulatory matters and their degree approval
process, for permission to use the HPC logo and to reproduce their documents. I would like
to thank Chris Smith for his comments and support during the various stages of planning and
writing, and would especially like to thank my fellow editors Qiuyu Wang and Nessar Ahmed
whose help and advice have proved invaluable to an editorial novice. Particular thanks must
also go to my assistant Sandra Shevlin without whom it would have been far more difficult to
complete this project.
Finally, I would like to thank my wife Jill, for her support and encouragement.
Hedley Glencross
London, April 2010
I would like to thank Chris Smith for his friendship over the years. His support and guidance
whilst working on this and other books, has been invaluable. He has been an excellent
mentor and I have learnt a lot from him for which I shall be eternally grateful. I would also
like to take this opportunity to acknowledge the friendship, companionship, and help of
the late Professor Ed Wood. Finally, I would like to acknowledge my family for their support,
patience, and encouragement.
Dr Nessar Ahmed
Manchester, April 2010
I would like to thank my co-editors, Nessar and Hedley for their invaluable help and advice
and for giving me such an enjoyable co-editing experience. I would like to express my
appreciation to Jonathan Crowe of OUP for his organizational skills and constant support and
help. My thanks are also due to Mr Mick Hoult for the beautiful illustrations he has provided.
Lastly, but not least, is my gratitude to my husband Chris Smith for his morale boosting and
academic support.
Qiuyu Wang
Manchester, April 2010
Contributors
Nessar Ahmed Tony Sims

School of Healthcare Science, Manchester Metropolitan Formerly of the Histopathology Department, University
University Hospitals of Leicester NHS Trust
Andrew Blann Mark Slevin

Haemostasis, Thrombosis and Vascular Biology Unit, School of Healthcare Science, Manchester Metropolitan
University Department of Medicine, City Hospital, University
Birmingham
Chris Smith
John Gaffney Formerly of School of Biology, Chemistry and Health
School of Healthcare Science, Manchester Metropolitan Science, Manchester Metropolitan University
University
Jan Still
Hedley Glencross Department of Chemical Pathology, Watford General
Institute of Biomedical Science, London Hospital
Ian Graham Alison Taylor

Formerly of School of Biology, Chemistry and Health Histology Department, Basingstoke and North
Science, Manchester Metropolitan University Hampshire Hospital
Qiuyu Wang
Tim James
School of Healthcare Science, Manchester Metropolitan
Department of Clinical Biochemistry, Oxford Radcliffe
University
Hospitals NHS Trust
Christine Yates
Georgina Lavender
Immunology Department, Hull and East Yorkshire
Laboratory Training Officer and freelance tutor
Hospitals NHS Trust
Helen Montgomery
Kratos Analytical, Manchester Online materials developed by
Elaine Moore
Pathology Department, St Peter’s Hospital, Surrey Sheelagh Heugh, Principal Lecturer in
Biomedical Science, Faculty of Human Sciences,
Joyce Overfield London Metropolitan University
School of Healthcare Science, Manchester Metropolitan
Dr Ken Hudson, Lecturer in Biomedical Science,
University
Faculty of Human Sciences,
Lynda Petley London Metropolitan University
Partnership Pathology Services, Frimley Park Hospital
William Armour, Lecturer in Biomedical Science,
Foundation Trust
Faculty of Human Sciences,
Peter Robinson London Metropolitan University
Institute of Postgraduate Dental Education, University of
Central Lancashire, Preston
Abbreviations
% v/v volume–volume percentage ccc supercoiled covalently closed circular
% w/v mass–volume or weight–volume CCD cooled charge-coupled device
% w/w mass percentage CD cluster of differentiation
μ electrophoretic mobility cDNA complementary DNA
A&E Accident and Emergency department CDC Centers for Disease Control
a absorptivity CE capillary electrophoresis
A absorbance CEP Centre for Evidence-based Purchasing
ABTS azino-bis-ethylbenzthiazoline sulfonic acid CF cystic fibrosis
ACDP Advisory Committee on Dangerous Pathogens CH100 total haemolytic complement test
ACOP Approved Codes of Practice CHART continuous hyperfractionated radiotherapy
ADR accord européen relative au transport CHIP Chemicals (Hazard, Information and Packaging
international des marchandises dangereuses for Supply) Regulations
parroute CI chemical ionization
AGE agarose gel electrophoresis CIC Connectivity Industry Consortium
AHG anti-human globulin CID collisional dissociation cell
AIDS acquired immune deficiency syndrome CJD Creutzfeldt–Jakob disease
ALP alkaline phosphatase CNS central nervous system
AMP adenosine 5’-monophosphate CNST Clinical Negligent Scheme for Trusts
R
Amp ampicillin resistance gene COSHH Control of Substances Hazardous to Health
ANCA anti-neutrophil cytoplasmic antibodies CPA Clinical Pathology Accreditation
ANOVA analysis of variance CPD continuing professional development
AP alkaline phosphatase CPSM Council for Professions Supplementary to
ATP adenosine 5’-triphosphate Medicine
BAC bacterial artificial chromosome CSF cerebrospinal fluid
BIVDA British In-Vitro Diagnostics Agency CSU catheter sample of urine
BJBS British Journal of Biomedical Science CT computed tomography
bp base pairs CTP cytidine 5’-monophosphate
BRI Bristol Royal Infirmary CV coefficient of variation
BSA bovine serum albumin d distance
BSE bovine spongiform encephalopathy dAMP deoxyadenosine 5’-monophosphate
BSHI British Society of Histocompatibility and dATP deoxythymidine 5’-monophosphate

Immunogenetics dCTP deoxycytidine 5’-monophosphate
BSI British Standards Institute ddNTP dideoxynucleoside triphosphate
BSQR Blood Safety and Quality Regulations DDT dichloro-diphenyl-trichloroethane
c concentration DGA DiGeorge anomaly
c velocity of light dGMP deoxyguanosine 5’-monophosphate
ABBREVIATIONS xvii
DGSA Dangerous Goods Safety Advisor G centrifugal field or acceleration

DIG digoxygenin GALT gut associated lymphoid tissue
DNA deoxyribonucleic acid GHS globally harmonized system
DPA Data Protection Act GLC gas–liquid chromatography
dps disintegrations per second GLP good laboratory practice
dRib deoxyribose GMP guanosine 5’-monophosphate
DSE display screen equipment H&E haematoxylin and eosin
DSEAR Dangerous Substances and Explosive Atmosphere h Planck’s constant

Regulations H height of a theoretical plate in a
DSS 3-trimethylsilyl 1-propane suphonate chromatography column; magnetic
component of electromagnetic
dTDP deoxythymidine 5’-diphosphate
radiation
dTMP deoxythymidine 5’-monophosphate
H0 applied magnetic field
E electric field or potential gradient; energy of
HASAWA The Health and Safety at Work Act
electromagnetic radiation
Hb haemoglobin
E electronic component of electromagnetic
S
radiation Hb haemoglobin S
A
ECD electron capture detector HB adult haemoglobin
EDTA ethylenediamintetraacetic acid HETP height equivalent to a theoretical plate
EI electron ionization HIS Hospital Information System
EIA enzyme immunoassay HIV human immunodeficiency virus
EIm electron impact HPC Health Professions Council
ELISA enzyme-linked immunosorbent assay HPCE high performance capillary

electrophoresis
ELISPOT enzyme-linked immunospot
HPLC high performance liquid chromatography
EQA external quality assessment
HRP horseradish peroxidase
EQF European Qualification Framework
HSA human serum albumin
ESI electrospray ionization
HSC Health and Safety Commission
ESR erythrocyte sedimentation rate
HSD Higher Specialist Diploma
EU European Union
HSE Health and Safety Executive
eV electron volt (1.6 × 10−19 J)
HSG Health and Safety Guidance publications
F Faraday constant
HTA Human Tissue Act (also used for Human
ƒ frictional coefficient Tissue Authority)
FAB fast atom bombardment I intensity of transmitted light
FACS fluorescence activated cell sorter I0 intensity of incident light
FBC full blood count IBMS Institute of Biomedical Science
FID flame ionization detector ICR ion cyclotron resonance
FISH fluorescent in situ hybridization IEF isoelectric focusing
FOI Freedom of Information Ig immunoglobulin
FTP fitness to practise IIF indirect immunofluorescence
FVU first voided urine IMS industrial methylated spirits
g earth’s gravitational field INR international normalized ratio
xviii ABBREVIATIONS
IOSH Institute of Occupational Safety and Health MM multiple myeloma

IPTG isopropylthio-β-galactoside mol mole – the SI unit denoting the amount of a
substance
IQC internal quality control
MOSFET metal oxide semiconductor field effect
IQR inter-quartile range
transistors
IR infrared
Mr relative molecular mass
IRMA immunoradiometric assay
MRI nuclear magnetic resonance imaging
IRT immunoreactive trypsin
mRNA messenger RNA
IS internal standard
MRS magnetic resonance spectroscopy
ISE ion selective electrode
MRSA methicillin resistant Staphylococcus aureus
ISFET ion selective field effect transistor
MS mass spectrometry
ISO International Standards Organization
MSDS Material Safety Data Sheet
IT information technology
MSU midstream urine
IUPAC International Union of Pure and Applied
N Avogadro’s number
Chemistry
N the number of theoretical plates in a
IVD in vitro diagnostic device
chromatography column
JBL journal-based learning
NAS sodium (Na), aluminium (Al), silicon (Si) glass
Kbp kilobase pairs used in ion selective electrodes
Kd distribution coefficient NEQAS National External Quality Assessment Service
Kow octanol–water partition coefficient NHS National Health Service
KSF Knowledge and Skills Framework NHSLA NHS Litigation Authority
L or l path length NMR nuclear magnetic resonance
L length NPSA National Patient Safety Agency
LAS lithium (Li), aluminium (Al), silicon (Si) glass nt number of nucleotides
used in ion selective electrodes oc open circular or nicked conformation
LDH lactate dehydrogenase OH occupational health
LET linear energy transfer OJEU Official Journal of the European Union
LEV local exhaust ventilation p probability
LIMS laboratory information management system PAC phage artificial chromosomes
LIS laboratory information system PAGE polyacrylamide gel electrophoresis
LOLER Lifting Operations and Lifting Equipment PAH phenylalanine hydroxylase
Regulations
PAS periodic acid-Schiff
m mass
PASA NHS Purchasing and Supply Agency
mAB monoclonal antibody
PAT portable appliance testing
MALDI matrix-assisted laser desorption ionization
PBC primary biliary cirrhosis
MALDI-MS matrix-assisted laser desorption ionization-
mass spectrometry PCR polymerase chain reaction
MALT mucosa associated lymphoid tissue PCV packed cell volume
MBL mannose-binding lectin PDP personal development plan
MHO manual handling operations PET positron emission tomography

PFGE pulsed field gel electrophoresis
MHRA Medicines and Healthcare products Regulatory
Agency Phage bacteriophage
ABBREVIATIONS xix
pI isoelectric points RIDDOR The Reporting of Injuries, Diseases, and

Dangerous Occurrences Regulations
PKU phenylketonuria
RNA ribonucleic acid
PMF peptide mass fingerprinting
rRNA ribosomal RNA
POCT point of care testing
RSI repetitive strain injury
PPE personal protective equipment
RT-PCR reverse transcriptase-PCR
PQQ pre-qualification questionnaire
s sedimentation coefficient
PUWER Provision and Use of Work Equipment
Regulations S Nernst factor
PVA polyvinyl alcohol SABRE serious adverse blood reactions and events
PVC polyvinyl chloride SABS Safety Alert Broadcast System
PVDF polyvinylidene difluoride SCOT support coated open tubular
PX partial pressure (concentration) of the gas X SD standard deviation
Q quadrupole SDS sodium dodecyl sulphate
QA quality assurance SDS-PAGE sodium dodecyl sulphate polyacrylamide gel
QAA Quality Assurance Agency for Higher electrophoresis
Education SE standard error
QC quality control SETS Standards of Education and Training
QCDA Qualifications and Curriculum SHOT serious hazard of transfusion
Development Agency
SI Le Système International d’Unités
QIT quadupole ion-trap
SLE systemic lupus erythematosus
QMS quality management system
SOP standard operating procedure
QP quality policy
SRID single radial immunodiffusion
qPCR quantitative real time polymerase chain
t retention time
reaction (PCR). Also called real-time (PCR)
T transmitted light
R electrical resistance
to dead time
R universal gas constant
t1/2 half-life
Rad radiation absorbed dose
TAG triacylglycerol
RBE relative biological effectiveness
TAT turnaround time
RCA root cause analysis
Tb turbidity
RCF relative centrifugal field
TB tubercle bacilli/tuberculosis
RE restriction endonuclease
TCA trichloroacetic acid
Real-time PCR real-time polymerase chain reaction. Also
called quantitative real time or qPCR TEM transmission electron microscopy
Rem roentgen equivalent man TEMED N,N,N’,N’-tetramethylethylenediamine
rev min−1 revolutions per minute TLC thin layer chromatography
RF radio frequency TMB tetramethylbenzidine
Rƒ relative to front TMS tetramethylsilane (Si(CH3)4)
RFLP restriction fragment length polymorphisms TOF time of flight
Rh Rhesus blood group TREM transport emergence card
RIA radioimmunoassay Tris 2-amino-2-hydroxymethylpropane-1,3-diol
Rib ribose tRNA transfer RNA
xx ABBREVIATIONS
TAT turnaround time α selectivity

UKAS United Kingdom Accreditation Service β –
positron
UMP uridine 5’-monophosphate ε molar extinction coefficient
UPS uninterruptible power supply λ wavelength of electromagnetic radiation
UV ultraviolet ρ density of the solvent
v frequency of electromagnetic radiation
v (sedimentation) velocity Units
v’ wave number Ω Ohm
v partial specific volume A Ampere – the SI unit of electrical current
V0 void volume
Å angstrom (10−10 m)
VDU visual display unit
Bq becquerel
Ve elution volume
C coulomb
Vis visible
Ci curie
VLCFA very long chain fatty acids
Da dalton
VOSA Vehicle and Operators Service Agency
Gy gray
Vt total volume
Hz hertz
W width at the base of a peak
nm nanometre
WCOT wall coated open tubular
ppm parts per million
WEL workplace exposure limit
s second
Wh width at half the height of a peak
Sv Sievert
WRULD work-related upper limb disorders
S Svedberg unit
X-gal artificial substrate, 5-bromo-4-chloro-3-indolyl-β-
galactoside, of β-galactosidase T temperature in kelvin
z charge V volt
1
Biomedical science
and biomedical
scientists
Hedley Glencross
Learning objectives
After studying this chapter, you should be able to:
■ Define biomedical science as a scientific discipline

■ Outline the academic and scientific contents of biomedical science degree programmes
■ Discuss the training required to become a biomedical scientist
■ Discuss how the education and training of a biomedical scientist are interrelated
Introduction
Biomedical science is often described as the application of the basic sciences, but especially
the biological sciences, to the study of medicine – in particular, the causes, consequences,
diagnosis, and treatment of human diseases. Biomedical scientists are registered practition-
ers of the subject, who normally work in departments of clinical pathology. Thus, they are
vital healthcare, scientifically qualified professionals who, for example, perform diagnostic
tests on clinical samples of blood and urine. Given their role(s), biomedical scientists are typi-
cally active in more laboratory-based work and tend to have a relatively limited contact with
patients compared with other healthcare professionals.
Approximately 70% of medical decisions or interventions – from diagnoses to monitoring med-

ical treatments to screening populations at risk of a particular clinical condition – are based on
the activities of biomedical scientists or require the knowledge and skills of biomedical science.
This may be as simple as a preoperative blood test or something more complex, such as using
an image-guided needle biopsy to remove tissue samples to help diagnose breast or prostate
cancers. Unfortunately, while everyone knows their doctor and, indeed, doctors and nurses
2 1 BIOMEDICAL SCIENCE AND BIOMEDICAL SCIENTISTS
have a high degree of recognition in society, in many ways the same is not true of biomedical
scientists. Perhaps considering the following scenario may reinforce the point. A 52-year-old,
overweight male smoker presents to his local Accident & Emergency department with chest
pains, shortage of breath, and complaining of a generalized weakness and tiredness. It is appar-
ent that he may be experiencing any one of a number of problems. These include, for example,
a panic attack, a chest infection, an allergic reaction, or he may be at the early stage of a heart
attack. How can an appropriate clinical decision be reached? Biomedical scientists earn their
living by helping in these decision-making processes!
Routine tests, such as measuring the patient’s height, weight, temperature, and blood pres-
sure, as well as taking X-rays and performing detailed laboratory investigations on samples
from a patient may all help provide answers. The laboratory investigations may include blood
tests to look for increased activities of enzymes or substances that act as markers for a heart
attack, microbiology tests to identify infection-causing microorganisms, while immunological
tests may help eliminate the possibility of an allergic reaction. Hence, clinical laboratories are
often busy places (Figure 1.1).
The education and training needed to become a biomedical scientist are detailed and lengthy.
However, if this is your career choice, then the knowledge you learn and the skills you acquire
have the potential to impact greatly upon many individuals and their lives. They will also give
you an interesting and worthwhile career.
1.1 What is biomedical science?

Biomedical science is more than a broad collection of scientific topics.
Cross reference Instead, it refers to a defined area of study related to the clinical investigation of disease within
The HPC will be discussed a degree programme. Degree programmes of these types are designed to allow the student to
further in Chapter 2 ‘Fitness
meet the educational standards and, in some cases, the professional skills required of a regis-
to practise’.
tered biomedical scientist by the Health Professions Council (HPC). Only those individuals
FIGURE 1.1
A typical, modern clinical laboratory.
1.2 BIOMEDICAL SCIENCE DEGREE PROGR AMMES 3
who have met the appropriate standards of the HPC will be legally allowed to call themselves
a biomedical scientist in the UK. The HPC is a statutory regulatory body for a variety of thera-
peutic and scientific health professions.
The definition of biomedical science that this book and the accompanying volumes in the
series will be using is that of the Quality Assurance Agency for Higher Education (QAA) and
is described in Section 1.2 below.
1.2Biomedical science degree

programmes
Biomedical science degrees are concerned with the study of a variety of subjects including:
• Biology
• Chemistry
• Pathobiology of human disease
• Analytical methods
Although biology and chemistry are key elements of the academic programme, pathobiology is
a fundamental component of a biomedical science degree. Pathobiology involves studying anat-
omy, physiology, biochemistry, genetics, immunology, microbiology, cell biology, and molecular
biology. It is also concerned with knowledge of disease processes, which are investigated in the
clinical laboratory specialities of histology, cytology, biochemistry, immunology, haematology,
transfusion science, and microbiology. Any student successfully completing such a course of
study will have a graduate level proficiency in the science of the causes, consequences, diagnosis,
treatment, and management of human diseases.
Biomedical science requires multidisciplinary approaches to the study of human diseases and,
as such, you must know why and how the development of diseases affects the normal struc-
tures and functions of the human body. You will also need to know the current methods used
for investigating, diagnosing, and monitoring diseases, as well as research methodologies,
which will include an appreciation of how new methods to combat diseases are developed,
evaluated, and integrated into clinical practice. Biomedical science also requires knowledge of
several scientific and clinical subject areas, which have been summarized by the QAA. You can
see a list of these subjects in Table 1.1.
TABLE 1.1 Quality Assurance Agency for Higher Education

(QAA) subject areas in biomedical science
Scientific subject areas Clinical subject areas

Human anatomy and physiology Histology
Cell biology Clinical biochemistry
Biochemistry Cytology
Genetics Clinical immunology
Molecular biology Haematology
Immunology Transfusion science
Microbiology Medical microbiology

Practical and transferrable skills

Biomedical science is very much a hands-on science and acquiring some of the discipline or
subject-specific skills associated with its practice is an essential part of a biomedical science
degree programme. These skills include:
• Learning how to handle patient samples in a safe and responsible manner when preparing
these samples for clinical investigation
• Using aseptic procedures
• Using laboratory instruments safely and appropriately to ensure accuracy, precision, and
reproducibility
• Interpreting patient-related data in an appropriate manner
Biomedical science, as described by the QAA, is an honours degree programme of study and so
this will incorporate a project or dissertation, which are the definitive components of hon-
ours degree programmes. They are individually undertaken by a student although, of course,
they are under the guidance of and studied with the support of professional staff. An hon-
ours degree will help in the development of transferrable skills, such as literature searching,
appraisal of literature, experimental design, scientific communication, research methodology
related to ethics, governance, audit, and statistical analysis.
As part of the assessment of your dissertation or project you may be required to present this
work to a group of peers (fellow students).
You have now seen the extensive and complex nature of biomedical science degrees in terms
of their scientific and professional contents, but these degree programmes are not only just
about science. They will enable you to develop a wide range of generic skills related to team-
working, communication, negotiation, numeracy, data analysis, and information technology.
Developing these generic skills is as essential as the gaining of underlying scientific knowledge
upon which biomedical science is based. Although the knowledge and skills acquired from
biomedical science degrees are essential for work in National Health Service (NHS) laborato-
ries of the UK, they also provide a sound academic and professional base and the potential to
work in any of the organizations and areas you can see in Table 1.2.
SELF-CHECK 1.1
What generic skills may be acquired during the study for a biomedical science degree?
1.3 What is a biomedical scientist?

This section title may seem a slightly odd question to ask as you may naturally infer that gradu-
ates with a biomedical science degree course are all biomedical scientists. However, as we
have already explained in Section 1.1, the title biomedical scientist is legally protected and
only those persons who are registered as such with the HPC are permitted to use it within the
UK. Thus, the title describes a person who has met the appropriate minimum or threshold
standards of proficiency required by the HPC (and have demonstrated that their character and
health are satisfactory, too). These standards are met by the successful completion of an HPC
approved degree programme.
One of the more important HPC proficiency standards for biomedical scientists to consider is
standard 3.a1, which states: ‘a registrant biomedical scientist must know the key concepts of
1.3 WHAT IS A BIOMEDICAL SCIENTIST? 5
TABLE 1.2 Summary of organizations and

work areas for biomedical science graduates
Organizations
The National Blood Service
The Health Protection Agency
Independent pathology laboratories
Veterinary laboratories
Agricultural laboratories
The World Health Organization
Universities
Areas of practice
Clinical genetics
Forensic science
Research
Pharmaceutical diagnostics
Medical devices
Clinical trials
Commerce
Education
Food and food safety
Biotechnology
the biological, physical, social, psychological and clinical sciences which are relevant to their
Cross reference
profession-specific practice’. To meet this standard you will need to demonstrate an under-
The HPC, their Standards of
standing of the knowledge and practice of all of the major areas of biomedical science. The Proficiency, and the degree
underlying scientific knowledge was outlined in Section 1.2 and the associated professional approval process will be
skills will be discussed in Section 1.4. discussed further in Chapter 2
‘Fitness to practise’.
Amongst the HPC approved programmes are a number of honours degrees that are also
accredited by the Institute of Biomedical Science (IBMS), which is the professional body
that biomedical scientists may join; its offices are shown in Figure 1.2.
The IBMS offers professional qualifications and sets standards of professional behaviour for
its members. As part of its work, the IBMS has also developed a training programme using
a portfolio of professional evidence (the registration portfolio) that leads to the award of a Cross reference
Certificate of Competence, which you can see in Figure 1.3. The certificate may be used The Institute of Biomedical
as suitable evidence when applying to the HPC for registration. The concept of a portfolio Science, their professional
should not be new to most readers. However, a portfolio worthy of the award of a Certificate guidelines, and the degree
of Competence needs to focus on the evidence necessary to an external verifier that you have approval process will be
discussed further in Chapter 2
acquired the knowledge and skills to meet the threshold standards of proficiency necessary
to join the HPC.
FIGURE 1.2
Institute of Biomedical Science, 12 Coldbath Square, London.
1.4 Professional skills

The professional training of a biomedical scientist can only be delivered and the relevant
skills acquired by physically working in a clinical laboratory to gain the necessary hands-on
experience.
Key Points
Throughout this chapter and the rest of the accompanying volumes in the series, clinical
laboratory will be used to describe a collection of departments. Department refers to an
individual discipline-specific area of work.
1.4 PROFESSIONAL SKILLS 7
FIGURE 1.3
Institute of Biomedical Science Certificate of Competence. Copyright © Institute
of Biomedical Science.
Professional skills may be acquired in a variety of ways. One way may be through the com-
pletion of a full-time HPC approved degree. However, these skills may also be gained during
employment, while simultaneously completing an IBMS accredited part-time degree pro-
gramme and the portfolio of professional evidence described in Section 1.3. Professional skills
may also be acquired after graduation, during employment in a relevant department by com-
pleting the portfolio of professional evidence, whilst also undertaking some discipline-specific
training. This is the route followed if you have gained an unaccredited degree whose content
needs to be supplemented with further study. Case study 1.1 illustrates how this was achieved
by a now successful biomedical scientist.
CASE STUDY 1.1 Supplementary study

and professional skills
I graduated with a first class honours degree in anatomy. there learning how a diagnostic histology department oper-
Biology had always been my strength throughout school, ates, whilst also refreshing my practical skills.
and I enjoyed every minute of my degree, especially the
Shortly after completing my undergraduate modules, I was
pathology elements. My final year project involved a lot of
offered a full-time, paid, 14-week placement in the histol-
practical histology. Consequently, I decided to investigate
ogy department, which allowed me to make a proper start
histology as a potential career option and I was told that to
on my IBMS Registration Portfolio. I made good progress
do histology for a living, I would have to either undertake
with it, and during that time I applied for and was offered
a medical degree, or ‘do’ a PhD and become a researcher.
a permanent trainee biomedical scientist position in the
I chose the latter option, as I have always preferred to be
same laboratory. For the pieces of work that made up my
‘behind the scenes’, finding out how and why things work,
portfolio, I tried to pull in as many aspects of biomedical
rather than dealing with the public directly. I embarked on
science as I could, to show that I was aware of how histol-
a PhD studying the morphology and formation of the min-
ogy related to the other disciplines in pathology. The case
eral phase of bone, and how it alters in osteoporosis and
osteoarthritis. studies were particularly useful in demonstrating this, and
by researching how other disciplines applied to particular
Part-way through my PhD, I found information about bio- areas of pathology, I was able to deepen my understanding
medical science and biomedical scientists. I had never pre- of histopathology at the same time.
viously given much thought to who tested patient samples
in hospitals, assuming those people to be doctors. I real- Following success in my PhD, soon afterwards I completed
ized that this was what I had been looking for in a career – my registration portfolio and verification visit. The labora-
investigating anatomy, histology, and pathology ‘behind the tory tour went well, although I was understandably nervous.
scenes’. Deciding that the PhD would still be very useful to me, I passed, but my assessor did say that I should have made
and given that I was enjoying it and wanted to complete it, my portfolio less impersonal – it was all neatly typed, but
I continued with my research, while finding out what I would the evidence should have been annotated with my com-
need to do to become a biomedical scientist. I contacted ments or reflections.
the IBMS, who assessed my BSc, and I was told that since my
Currently, I am working in another department and I am
degree was not accredited by the IBMS, I would have to take
about to be assessed for my Specialist Diploma in Cellular
a course of supplementary education.
Pathology. I contacted the IBMS regarding my MSc and
Once I had completed my PhD laboratory work, and had was told that because my PhD was relevant to my field,
begun the daunting task of writing up my thesis while study- an MSc was not needed in my case. This means I am able
ing as an undergraduate again! I undertook six 10-credit to move directly on to the Higher Specialist Diploma, and
undergraduate modules in clinical microbiology, haematol- I plan to spend the next 3 years gaining experience and
ogy and immunohaematology, transfusion science, immu- gathering evidence for this, before sitting the examina-
nology, clinical biochemistry, and cytopathology. While tions. Eventually, I hope to train in histological dissection,
studying and writing up, I contacted a local laboratory to as to me this is the most exciting and appropriate use of
ask if they would be willing to have me visit for work experi- the varied skills I have developed over the years of my
ence. The laboratory agreed, and I spent one day each week training.
Finally, these skills can be gained through full-time study and completion of an accredited
biomedical science degree programme that includes both academic study and professional
training through a series of laboratory placements. Case Study 1.2 shows a typical reflec-
tive essay written at the end of a laboratory placement. A Certificate of Competence is then
awarded upon graduation.
1.4 PROFESSIONAL SKILLS 9
CASE STUDY 1.2 Example of a placement

reflective essay
My placement took place at a District General Hospital in chemiluminescence), the basic principles behind the test
Scotland, in a multidisciplinary laboratory consisting of the procedures were the same as those taught in the immunol-
histopathology, biochemistry, haematology & blood trans- ogy module. I made similar observations when I moved on
fusion, and microbiology departments. The placement was to other departments.
scheduled for the second semester of the third year of the
course and the outcome was to gather a training portfolio It was a relief not to have to perform manual cell counting
over a 12-week period. Being trained in a multidisciplinary in haematology or in microbiology as at university; mean-
laboratory was particularly advantageous for me in that while, it was good to see that the university was in line with
I was able to experience work in different fields, so this gave new advances in biomedical science, such as molecular
me an insight on what were my disciplines of preference. methods, which are increasingly used in clinical labora-
tories. It was also interesting to see how most of the tech-
It all started with an induction week, which involved tutori- niques learned at university could be used interchangeably
als on important laboratory requirements such as labora- across disciplines, namely molecular techniques, micro-
tory accreditation, the roles of regulatory and professional scopic techniques, and immunological methods. My only
organizations (HPC and IBMS), quality management and reservation was about the content of a particular university
quality assurance, health and safety, and data protection, practical class where no practical work was actually done
and this list is not exhaustive. My view of that first week was (not even the haematoxylin and eosin staining routinely
a little bit of apprehension because not only was the ‘9 to done in histopathology laboratories) except for micro-
5’ routine difficult to cope with, most of the concepts were scopic identification on ready-made slides.
new, very prescriptive, and appeared to have nothing to do
with what was being taught at university. However, it didn’t The importance of maintenance, calibration of equipment,
take long for me to realize that the life of patients is what is and quality control (QC) was much more emphasized than
at the end of the line, explaining why the biomedical pro- at university. Other important notions, such as prioritiza-
fession is so tightly regulated. tion, effective communication, time management, and cost
effectiveness are only some of the other topics with which
Two weeks discipline-specific training were then received students are not really familiarized; it was a privilege to be
in each of the departments and it was interesting to see how introduced to these at an early stage and this gave me a feel
the knowledge and skills gained at university were applied of what is expected in the working place.
to work in the laboratory, this in terms of test principles,
test procedure and techniques, interpretation, and statis- To sum it up, I would say that going on to a placement was an
tical analysis of results. My first 2 weeks of training were excellent opportunity for me to appreciate the practical rele-
in the biochemistry department where I was introduced vance of every single module taught in the biomedical science
to the modular analysers. Despite the fact that the system course, from the first year of study. It is evident that without
was highly automated, standard operating procedures the basic knowledge and skills gained from clinical modules
(SOPs) indicated that different test procedures and detec- it would be very difficult to understand work in the labora-
tion principles were exactly the same as those performed tory. Working in a NHS laboratory is not just about applying
manually in practical classes at university. For instance, a SOP to patient samples to obtain some results. I’ve learned
most analytes (for example, glucose, lipids) were measured that a biomedical scientist should be able to know why a
using colour reactions and spectrophotometer readings; test is being requested, when and how the sample should be
hormones, enzymes, and tumour markers were measured properly collected, what sort of additives (if indicated) are
using immuno-assay systems, and depending on their needed and what will be their impact on the tests, what test
molecular size, either sandwich methods (measurement methods are more appropriate for a particular specimen, and
of Troponin T) or competitive assays (as for free thyroxin) what precautions should be taken during processing. He/she
were used. Although these methods were also auto- must also be able to analyse the results obtained and make
mated and the detection principles varied (fluorescence, informed comments about their significance.
The rewards of the placement were, in fact, greater than independent and more confident approach to it compared
I initially thought, as it wasn’t until I started working on my with the execution of my previous laboratory assignments.
honours project that I realized how much I had learned in
In addition, the placement turned out to be an integral work
just a few weeks. I became conscious as soon as I started
the project that my planning, organizational, and practi- experience on my CV, which helped earn me a job in a NHS
cal skills were much more enhanced. I also had a totally laboratory so I’m happy today to say that it was worth doing.
SELF-CHECK 1.2
How are professional skills acquired?
1.5Initial employment as a
biomedical scientist
Following registration with the HPC, it is likely that newly qualified biomedical scientists will be
employed in a discipline-specific department, such as histology, biochemistry, cytology, hae-
matology, immunology, microbiology, blood transfusion, or virology. However, it is becom-
ing apparent that the divisions between these traditional disciplines are becoming blurred
and new departments are beginning to emerge through advances in science and technology
or through changes in clinical practice. For example, some laboratories combine the routine
diagnostic investigations performed in haematology or biochemistry into a department often
called blood science or blood sciences. Further, the use of point of care testing (POCT) by
bench-top or hand-held devices in settings outside of the laboratory, including a patient’s own
home, is increasing (Box 1.1). Often POCT devices allow simple microbiological, biochemical,
or haematological tests to be performed in combination.
Cross reference It is only after registration and employment that you will acquire the full range of more detailed
Point of care testing is explored discipline-specific skills. This may involve completing an IBMS Specialist Portfolio, which is
in Chapter 18. explained in Chapter 2 Fitness to practise.
Training
Properly structured and delivered training programmes are essential to the development of a
biomedical scientist. Whether the training is part of a work placement or as a new employee
BOX 1.1 Point of care testing
Point of care testing involves using hand-held or bench-top analysers to test patient
samples in settings other than a clinical laboratory. These relatively simple analysers
often use capillary blood sampling and may be encountered in hospital areas such as
intensive care or within a primary care setting, for example, an anticoagulation clinic.
Increasingly, POCT devices and tests are being offered directly to patients for part of self-
testing or as part of the self-management of chronic diseases such as diabetes.
1.5 INITIAL EMPLOYMENT AS A BIOMEDICAL SCIENTIST 11
in a laboratory, all new biomedical scientists will be assigned a training lead who will facilitate
their training. All of the training will be guided by the contents of an individual training pro-
gramme and the HPC requirements for minimum standards of proficiency that are illustrated
in Figure 1.4.
Training programmes are, by their nature, individual to the person concerned because of
a variety of issues, such as prior knowledge and expertise, and the individual’s own learn-
ing style. However, any one programme is likely to contain the basic underlying knowledge
needed, together with an indication of the level of skill, a definition of competency and the
assessment processes. As training is a continuous process, any training programme will need
to be negotiated and agreed by a trainee and the training lead. Training is also an interactive
FIGURE 1.4
Front cover of the Health Professions Council Standards of Proficiency for
Biomedical Scientists. Copyright © Health Professions Council.
process that requires equal commitment from the trainee and the employer and must take
place without affecting the service provided by the laboratory. So its delivery must be in a
manner that minimizes its effects on the normal working of a department, but maximizes the
benefits to the trainee.
During training it may be necessary to work alongside support staff or undertake some of the
basic work of the department, such as loading machines, making solutions, and filing. Hence,
professional training may have to fit around the needs of an individual department. Such tasks
should not affect the delivery of training. In fact, these types of tasks are likely to benefit a
trainee, as they will increase his or her understanding of all the functions of a laboratory and
the associated benefits of collaborative work. All individuals are members of a team, and need
to help colleagues and support all the different areas and activities of a department. This will
also provide time for more experienced colleagues to deliver professional training to others.
After successful completion of such a training programme, the trainee should have become a
competent, confident, and independent practitioner known as a registered biomedical scientist.
1.6 What do biomedical scientists do?

Until now we have considered the knowledge and skills that are necessary to be a biomedical
scientist, but what will you do as a professional biomedical scientist in your daily working life?
Much of the work undertaken by a laboratory is determined by a combination of the popula-
tion served by the hospital, the associated range of illnesses that this population may develop,
and the clinical specialities that are practised within the hospital. For example, in a teaching
hospital, you would probably undertake more specialized work or research-related activities,
along with the routine work associated with investigating, diagnosing, and treating disease. In
a general hospital, the work may be less specialized, but there may also be opportunities for
involvement in other activities. Wherever you work, it is likely that you will be busy, and it will
be expected that you and the other biomedical scientists perform your duties and fulfil your
roles in a helpful and professional manner, remembering that all samples originated from a
patient and, in the end, all test results affect him or her.
Laboratory tests
It is estimated that over 70% of clinical or medical decisions or interventions are based
wholly or partly on the results of laboratory tests. The significance of these tests cannot be
underestimated.
Laboratory tests can be divided into three general categories, diagnostic, screening, and moni-
toring, which you can see outlined in Table 1.3.
Diagnostic tests are used to investigate a patient who has signs or symptoms of illness. For
example, someone who experiences pain on urinating may have an infection of the renal tract.
Samples of urine from the patient must be investigated for evidence of disease-causing micro-
organisms. Screening tests are used to identify an ‘at risk’ individual from a defined population,
who does not demonstrate symptoms, but may have an undiagnosed illness or condition.
Thus, for example, in the UK all females of reproductive age (and slightly beyond) are invited
to have a sample taken from their cervix to investigate for any potential cellular changes that
may indicate the development of cancer. Monitoring tests are used to check the status of an
individual before a medical procedure, following diagnosis and treatment, or to monitor his
or her progress during a long-term condition. For example, someone who goes into hospital
1.6 WHAT DO BIOMEDICAL SCIENTISTS DO? 13
TABLE 1.3 Types and role of laboratory tests
Type of test Role of test

Diagnostic Investigating an individual showing signs or symptoms of an illness
Screening Identification of ‘at risk’ individuals within a defined population who

do not show any symptoms, but may have an underlying undiagnosed
clinical condition
Monitoring Checking the health status of an individual before undertaking a

medical procedure
Assessing an individual following diagnosis and treatment
Monitoring the progress of long-term or chronic conditions
for an operation will be subjected to blood and urine tests designed to give information about
their overall health and physiological functions. Increasingly, microbiological investigations
form part of this test profile to ascertain if an individual has one of the so-called ‘super bugs’,
such as methicillin-resistant Staphylococcus aureus (MRSA). It may be that the blood and urine
tests show the patient’s liver, kidneys, and heart are healthy, and are not a bar to the planned
operation. However, the microbiology tests may show he or she is suffering an infection that
needs to be treated before an operation can take place. This type of situation shows why it
is essential for biomedical scientists to understand the work of all the main departments and
why training programmes (discussed above) emphasize these aspects of biomedical science.
We will now, briefly, consider the work carried out in each of the main discipline-specific
departments.
Histopathology
Histology involves the microscopic study of tissues to identify disease that may be present.
Tissues may be removed from living individuals following an operation or from deceased
individuals at post-mortem. The tissue samples range from small biopsies to whole organs. Cross reference
Slides are prepared from these samples, which are stained and examined using a microscope. You can read more about the
Diseases such as inflammation, infections, benign and malignant tumours can all be diagnosed roles of biomedical scientists in
histology in the companion text,
using histology, as you can see in Figure 1.5. Biomedical scientists are involved at all stages of
Histopathology.
the preparation processes and work closely with medical histopathologists in this respect.
Clinical biochemistry
Clinical biochemistry involves the study of biochemicals and biochemical mechanisms, and
their imbalances within the body. Clinical biochemistry tests are usually performed on blood
or urine samples, but other fluids may also be tested, for example, cerebrospinal fluid or fluids
that abnormally collect around the heart, lungs, or abdominal organs. Many of the tests are
performed automatically on machines (Figure 1.6) that use robotics to manage many samples
simultaneously. Malfunctions of organs or diseases, such as diabetes, heart attacks, and kidney
failure, may be diagnosed using biochemical tests. Other aspects of biochemistry are related to Cross reference
screening for drug use and abuse, which may involve investigating living patients or deceased You can read more about the
work of clinical biochemists in
individuals. Again, biomedical scientists are involved at all stages of the preparation process,
and work closely with clinical scientists or chemical pathologists in this respect.
FIGURE 1.5
Thin section of breast stained
with haematoxylin and eosin
showing abnormal cells
infiltrating connective tissue.
FIGURE 1.6
Samples waiting to be loaded
onto a chemistry analyser.
Cytopathology
Cytology (like histology) involves microscopic studies, but in this case cells are studied, rather
than tissues, to identify disease that may be present. The cells are obtained through natural
shedding, aspiration, or mechanical abrasion, and often involve the analysis of body fluids.
Slides are prepared from these samples and examined using a microscope. Diseases such
as inflammation, infections, benign and malignant tumours are diagnosed using cytology.
Probably the best-known use of cytology is the examination of samples obtained from the Cross reference
uterine cervix as part of the UK cervical screening programme as can be seen in Figure 1.7. You can learn more
Once again, biomedical scientists are involved at all stages of the preparation and interpreta- about clinical cytology in
tion processes, and work very closely with medical cytopathologists and clinical medical staff Cytopathology.
in this respect.
Immunology
Immunology involves the study of the immune functions of the body, as a system or its
responses to a stimulus. Immunological specimens include blood (Figure 1.8) and other fluid
or tissue samples that are subjected to a variety of manual or automated clinical tests. Diseases
of the immune system include immunodeficiency where it fails to respond to appropriate
stimuli, autoimmune disease when the immune system ‘attacks’ the tissues it is meant to pro- Cross reference
tect, or hypersensitivity when it responds inappropriately to harmless stimuli. Immunology is Read more about clinical
also involved in tissue and organ transplantation through the matching of donor and recipient aspects of immunology in
the companion volume,
tissues, or the determination of rejection. Biomedical scientists, clinical scientists, and medical
Immunology.
immunologists are all concerned with such studies.
FIGURE 1.7
Normal and abnormal cells in a cervical smear stained by Papnicolaou’s method.
FIGURE 1.8
White blood cells showing green fluorescence after being labelled with fluorescein.
Haematology
Haematology is the study of the morphology and physiology of blood. Haematological tests
Cross reference
are performed, as you might expect, on blood samples; such tests are increasingly carried out
You can read more about
haematological investigations in by automated instruments. Diseases such as anaemia or leukaemia can be diagnosed follow-
Haematology. ing analyses of the numbers of erythrocytes or the haemoglobin content, or the numbers
of leukocytes present in the sample haematology investigations. Other aspects of haematol-
ogy are related to abnormalities of blood coagulation or the monitoring of individuals on
long-term medication using anticoagulants. Biomedical scientists and medical haematologists
collaborate during these studies. If you examine Figure 1.9 you can see the presence of normal
blood cells on a microscope slide of a stained blood film.
Transfusion science
Transfusion science involves the provision of blood and blood products to patients who have
anaemia, blood clotting problems, and those who are suffering from acute blood loss or have
been involved in an accident. Donated blood is the primary source of blood for these purposes.
Acquiring donations of blood for transfusion requires its appropriate collection, storage, prep-
Cross reference aration, and the issue of suitable blood and blood products. Other work in transfusion science
The companion volume, is related to the collection, storage, and preparation of donated cells and tissues. Work in
Transfusion and Transplantation blood transfusion is highly regulated and is subject to EU Directives that are now part of UK
Science will give you more law. Biomedical scientists tend to work very closely with clinical staff due to the nature of the
detailed information on
supply aspects of these products. Figure 1.10 shows blood that has been cross-matched in
this topic.
order to establish its ABO and Rhesus blood group.
FIGURE 1.9
Normal blood cells in a blood film stained by May–Grünwald and Giemsa’s method.
Microbiology and virology

Microbiology and virology are involved in the detection, isolation, and identification of micro-
organisms, including bacteria, fungi, parasites, and viruses that cause disease. Samples may be
taken from any part of a body, which may be of a living or deceased individual. Traditionally,
microorganisms were grown on nutrient agar plates as shown in Figure 1.11, in broths or cell
cultures, but increasingly the use of molecular detection methods (see Chapter 15) is becom-
ing more common. Tests that used to take weeks to confirm the identity of a microorganism
FIGURE 1.10
Cross-matched blood showing blood group types.
FIGURE 1.11
Chromogenic (colour-identifying) agar plate showing the presence of colonies of
methicillin-resistant Staphylococcus aureus (MRSA).
Cross reference
You can read more about the can now be done in days. Often when examining a bacterium, its sensitivity to antibiotics is
roles of biomedical scientists in also determined, to ensure the optimal treatment for the patient is chosen and to minimize
medical microbiology in Medical
the misuse of these drugs. Medical microbiologists and virologists work closely with biomedi-
Microbiology.
cal scientists in microbiology and virology investigations.
SELF-CHECK 1.3
Using histopathology as the discipline, briefly explain the uses of diagnostic tests.
SUMMARY
■ Biomedical science is a term used to describe the study of the causes, consequences,
diagnosis, and treatment of human diseases
■ Biomedical science is also an academic subject and can be studied as part of an honours
degree programme, the content of which has been defined by the QAA
■ To become a registered biomedical scientist, an individual must meet defined minimum

standards of proficiency by successfully completing an approved degree programme that
includes a period of professional training
1.6 WHAT DO BIOMEDICAL SCIENTISTS
QUESTIONS
DO? 19
■ The study of biomedical science will include acquiring knowledge and professional skills
in all of the major discipline-specific areas
■ Once registered, biomedical scientists normally pursue their career by professional prac-
tice and the extended study of one of the major discipline-specific areas
FURTHER READING
● Halushka PV, Krug EL. Is the training of biomedical scientists at a crossroads?
Academic Medicine, 2009; 84: 459–463.
● Burnett D, Poyser KH, Sherwood RA. A Practical Guide to Accreditation in Laboratory

Medicine. ACB Venture Publications, London, 2002.
Useful Website
■ Clinical Pathology Accreditation. www.cpa-uk.co.uk
QUESTIONS
1. Which of the following statements is/are TRUE?
(a) Screening tests are used to investigate signs and symptoms

(b) Populations are subjected to screening tests
(c) Screening tests can identify illnesses
(d) Screening tests are used to identify cancers only
(e) Screening tests are 100% accurate
2. Discuss why it is necessary for prospective HPC registrants to study all of the major areas
of biomedical science.
3. If the outcomes are similar, why do training programmes differ between organizations
and individuals?
4. Which of the following are role(s) of a test to monitor health?
(a) Checking the general health status of an individual prior to performing a medical
procedure.
(b) Identification of at-risk individuals who have no symptoms
(c) Investigating an individual who is showing symptoms
(d) Assessing the progress of long-term clinical conditions
(e) Follow-up assessments of treated individuals
Answers to self-check and end of chapter questions are available in the Online Resource
Centre accompanying this book.
Go to www.oxfordtextbooks.co.uk/orc/glencross
2
Fitness to practise
Hedley Glencross
Learning objectives
■ Define the functions of a regulatory authority

■ Discuss the roles of a professional body
■ Explain how a regulatory body determines and maintains the fitness to practise of its
members
■ Demonstrate how a professional body may help you meet the requirements imposed by
regulatory authorities
■ Explain how a professional body may help you develop your career
Introduction
Fitness to practise is the ability of an appropriately trained and registered individual to perform
his or her professional tasks in a safe manner, and to make appropriate judgements with a
degree of autonomy. In Chapter 1 you were introduced to the Health Professions Council
Cross reference (HPC) and the Institute of Biomedical Science (IBMS). In this chapter we describe these organi-
Chapter 1 ‘Biomedical science zations (Figure 2.1a,b), discuss how their respective, but complementary approaches to fitness
and biomedical scientists’. to practise differ, and explore their contributions to the training and career development of
biomedical scientists.
2.1 Healthcare regulation

Biomedical scientists have been regulated by legal statutes since 1960 under the Professions
Supplementary to Medicine Act, when biomedical scientists, or rather medical laboratory tech-
nicians as they were then known, were registered and directed by the Council for Professions
Supplementary to Medicine (CPSM). The CPSM regulated nine professions and maintained
a list of individuals who had met their threshold standards. However, the CPSM did not set
other professional standards, so that once a biomedical scientist was admitted to the then
CPSM state register there was no requirement to demonstrate any continuing competence or
2.2 HEALTH PROFESSIONS COUNCIL 21
(a) (b)
FIGURE 2.1
Logos of (a) Health Professions Council (HPC) and (b) Institute
of Biomedical Science (IBMS). Both logos reproduced with
permission from the respective organizations.
further education to remain on the register. As a consequence, it was difficult for the CPSM
to remove someone from the register, other than for a criminal offence. However, following
a number of then well-publicized incidents, such as the murder of four children by the nurse
Beverley Allitt in 1991 and the murders of many elderly patients by the general practitioner
Cross reference
Harold Shipman between 1995 and 1998, the issue of regulation of all healthcare profession-
Chapter 4 ‘Health and safety’
als (including medical staff) was brought into focus during the 1990s. Consequently, in 2001 a
discusses some of the legal
Parliamentary Order for the Health Professions, which included provision for the formation of framework relevant to
the HPC, was approved. The HPC was given (and maintains) a UK-wide remit for the registra- healthcare professionals.
tion of competent healthcare professionals.
2.2 Health Professions Council

The HPC is principally concerned with patients and their safety. Indeed, it was established as a
regulator of healthcare standards with a duty to protect the public. Thus, the work of the HPC
is designed to safeguard the public from being harmed by the actions or inactions of health-
care professionals. Parliament has granted legal powers for the HPC to carry out its statutory
roles by establishing and maintaining a register of competent individuals who meet its stand-
ards of training, professional skills, behaviour, and health. Competence is defined as the ability
of a registered individual to repeatedly perform any action related to his or her profession to a
defined standard, and in a safe and effective manner. Any individual who wishes to be admit-
ted to the HPC register must demonstrate minimum or threshold standards of proficiency and
provide evidence that they are of excellent character and in good health. Appropriate evi-
dence that these professional standards have been satisfied would be by successfully complet-
ing an approved degree course, as we discussed in Chapter 1. Students who have not studied Cross reference
Chapter 1 ‘Biomedical science
in the UK may apply to the HPC for registration by its international route, as they will need to
and biomedical scientists’.
have their qualifications, experience, and competence individually assessed.
The HPC currently registers 15 professions, including therapeutic professionals, such as physio-
therapists and radiographers, as well as scientific professionals, for example clinical scientists and
biomedical scientists (Table 2.1). The HPC fulfils its principal role of regulating the activities of
these professionals by restricting the use of protected titles, such as physiotherapist or biomedi-
cal scientist to those individuals it deems competent. The HPC also has the legal rights to inves-
tigate and remove from its register any individuals who it finds to be incompetent. Individuals
may be removed from the register because of unprofessional conduct, inappropriate personal
actions, or because of criminal activities.
22 2 FITNESS TO PR ACTISE
TABLE 2.1 Professionals regulated by the HPC
Therapeutic professionals Scientific professionals

Arts therapists Biomedical scientists
Chiropodists/podiatrists Clinical scientists
Dieticians
Hearing aid dispensers
Occupational therapists
Operating department practitioners
Orthoptists
Paramedics
Physiotherapists
Practitioner psychologists
Prosthetists and orthotists
Radiographers
Speech and language therapists
The HPC sets both generic and profession-specific standards in the following areas:
• Proficiency
• Education and training
• Conduct, performance, and ethics
• Continuing professional development
Standards of proficiency are the knowledge and skills that a registrant must demonstrate
before he or she can be admitted to the register. To remain registered, all registrants must
maintain their knowledge and skills as appropriate to their developing role during the progress
of their careers. The standards of education and training (SETS) are those that providers of
education and training must meet to ensure all students who complete an HPC approved
educational programme meet their standards of proficiency. Successful students are then
able to apply to HPC for registration. Standards of conduct, performance, and ethics apply
to registrants. These are the codes of behaviour, attitude, and aptitude to which registrants
must adhere if they are to remain a registered professional. It is the continuing enforcement
of these standards that allows the public, patients, and the registrants’ colleagues to be con-
fident of their suitability to practise and remain on the register. Standards of continuing pro-
fessional development (CPD) also apply to registrants. Individual registrants are required to
demonstrate that they are keeping up to date in their developing role. They must also show,
through a range of CPD activities, how these activities benefit themselves professionally, the
wider health service, and patients alike. The Institute of Biomedical Science also contributes
to CPD as outlined in Box 2.1.
Cross reference The SETS and CPD will be discussed in more detail in Chapter 20. The standards of profi-
Chapter 20 ‘Personal
ciency and the standards of conduct, performance, and ethics are explored in more detail in
development’.
Section 2.3.
2.2 HEALTH PROFESSIONS COUNCIL 23
BOX 2.1 Continuing professional

development
Upon joining the IBMS, new members are automatically enrolled on the IBMS
Continuing Professional Development Scheme (although there is an option to opt out).
The variety of activities associated with the scheme and their independent, annual veri-
fication with its systematic and structured approach means it has been recognized by
the HPC as appropriate for their purposes. Thus, registrants can meet many of the HPC
requirements for CPD without needing to provide evidence of each individual activity
undertaken.
HPC approval of biomedical science degrees

The HPC has developed standards of education and training that education providers must
satisfy if their biomedical science degree courses are to achieve HPC approval. These generic
standards ensure that any individual who successfully completes an approved programme
will also reach a minimum standard of proficiency. As a consequence, he or she will then be
eligible to apply to be admitted to the HPC register.
Key Points
The major advantage of successfully graduating from an HPC approved degree is that
you will be able to apply immediately for admittance to the HPC Register.
The key areas considered when approval for a biomedical science degree is sought are:
• Level of entrance qualifications
• Programme admissions
• Programme management and resources
• Curriculum
• Practice placements
• Assessments
The HPC approval process is designed to be robust, but not too onerous for the education
providers. The approval of a new degree programme by the HPC will require that they make
an initial visit to the education provider. The HPC delegation may visit as part of a wider assess-
ment group or in isolation. Once approval has been granted by the HPC, it is ‘open ended’ and
subject to satisfactory monitoring. For example, there is an annual review of the degree pro-
gramme to ensure its structure and content have not been subject to major revisions and that
it continues to meet the HPC Standards of Education and Training. If there have been major
changes in an approved programme, the HPC will consider their effects on the standards.
Thus, an annual review may be the trigger for further visits by the HPC for the re-approval of
significantly altered biomedical science degree programmes.
Although degree approval is normally done on behalf of individuals who wish to gain HPC
registration, a number of post-registration degree programmes are also approved by the HPC.
These include, for example, prescribing or local anaesthetic programmes, and do not apply to
biomedical scientists.
2.3HPC Standards of Conduct,

Performance and Ethics
The HPC has developed 14 ‘duties’ that define their Standards of Conduct, Performance and
Ethics (Figure 2.2) and to which all registrants from the 15 regulated professions must conform.
These duties are listed in Table 2.2. The duties describe in general terms what is expected of
registrants as an autonomous and accountable professional. A rather simpler summary is that
the standards describe the levels of honesty, integrity, and probity required of a registered
individual at all times in his or her personal and professional life. These standards have neces-
sarily been written in a rather stilted style as they are used (in conjunction with the standards
of proficiency) to decide on a course of action if any concerns are raised about the practice of
a specific individual.
Of the duties listed in Table 2.2, only 9 and 14 are outside the current normal practice of bio-
medical scientists, given that they are unlikely to treat patients or advertise their services.
These standards may be met in a number of ways. For example, a registrant may seek profes-
sional advice and support from professional bodies as part of their roles in representing and
promoting the interests of their members. This may be as simple as joining and maintaining
membership of a professional body or it may be by seeking expert advice from that professional
body. As will be discussed later (Section 2.6), the IBMS also defines good practice for its members
and other individuals by providing guidance to help clarify professional or scientific matters.
FIGURE 2.2
Front cover of HPC Standards of
Conduct, Performance and Ethics.
Copyright © Health Professions
Council.
2.3 HPC STANDARDS OF CONDUCT, PERFORMANCE AND ETHICS 25
TABLE 2.2 Health Professions Council standards of conduct, performance and ethics
Duty You must:
1 Act in the best interests of service users
2 Respect the confidentiality of service users
3 Keep high standards of personal conduct
4 Provide any important information about your conduct and competence
5 Keep your professional knowledge and skills up to date
6 Act within the limits of your knowledge, skills, and experience and, if necessary, refer
the matter to another practitioner
7 Communicate properly and effectively with service users and other practitioners
8 Effectively supervise tasks that you have asked other people to carry out
9 Get informed consent to give treatment (except in an emergency)
10 Keep accurate records
11 Deal fairly and safely with the risks of infection
12 Limit your work or stop practising if your performance or judgement is affected by

your health
13 Behave with honesty and integrity, and make sure your behaviour does not damage
the public’s confidence in you or your profession
14 Make sure any advertising you do is accurate
To meet duties 3 and 12 (Table 2.2), individuals are required at registration to provide a signed
reference from an appropriate person. Duty 3 is often satisfied by a reference from an HPC-
registered individual as they are familiar with the level of professional conduct required.
However, with regard to duty 12, it is most likely to be provided by a GP or other medical
practitioner who is able to comment on the applicant’s current state of health.
Once an individual has joined the register, he or she is only required to sign (every 2 years at
renewal of registration) a declaration that their ability to practise has not been affected by any
changes in his or her health status. This declaration covers all aspects of health and abilities,
in particular the decision-making skills and awareness required by registered individuals. It is
expected that the HPC will be informed immediately if the health status of a registrant changes
in a way that affects their role(s) as a registered individual. From 2007 this declaration has also
included a statement regarding CPD, as discussed in Section 2.4.
HPC Standards of Proficiency

The HPC has also developed standards of proficiency for each of the 15 professions (Figure 1.4).
Some of the standards are generic in that they apply to all of the regulated professions, while oth-
ers are related to individual professions. However, both groups of standards cover three areas:
1. Expectations of a health professional

2. Skills required for the application of practice
3. Knowledge and understanding of these skills
Each of these areas is subdivided into a number of individual statements that describe
the details regarding each standard. The top-level generic standards are illustrated in
Table 2.3.
TABLE 2.3 Health Professions Council Standards of Proficiency
1. Expectations of a health 2. Skills required for the 3. Knowledge, understanding,

professional application of practice and skills
Registrant biomedical Registrant biomedical Registrant biomedical scientists
scientists must: scientists must: must:
1a.1 2a.1 3a.1

Be able to practise within the Be able to gather Know and understand the
legal and ethical boundaries appropriate information key concepts of the bodies of
of their profession knowledge that are relevant to
their profession-specific practice
1a.2 2a.2 3a.2
Be able to practise in a Be able to select and use Know how professional
non-discriminatory appropriate assessment principles are expressed and
manner techniques translated into action through a
number of different approaches
to practice, and how to select or
modify approaches to meet the
needs of an individual, groups,
or communities
1a.3 2a.3 3a.3
Understand the importance Be able to undertake or Understand the need to
of and be able to maintain arrange investigations as establish and maintain a safe
confidentiality appropriate practice environment
1a.4 2a.4
Understand the importance Be able to analyse and
of and be able to obtain critically evaluate the
informed consent information collected
1a.5
Be able to exercise a
professional duty of care
1a.6
Be able to practise as an
autonomous professional,
exercising their own
professional judgement
1a.7
Recognize the need for
effective self-management of
workload and resources and
be able to practise accordingly
1a.8
Understand the obligation to
maintain fitness to practise
1. Expectations of a health 2. Skills required for the 3. Knowledge, understanding,

professional application of practice and skills
1b.1 2b.1
Be able to work, where Be able to use research,
appropriate, in partnership reasoning, and problem-
with other professionals, solving skills to determine
support staff, service users, appropriate actions
and their relatives and
carers
1b.2 2b.2
Be able to contribute Be able to draw on
effectively to work appropriate knowledge
undertaken as part of a and skills in order to
multidisciplinary team make professional
judgements
1b.3 2b.3
Be able to demonstrate Be able to formulate
effective and appropriate specific and appropriate
skills in communicating management plans
information, advice, including the setting of
instruction, and professional timescales
opinion to colleagues,
service users, their relatives,
and carers
1b.4 2b.4
Understand the need for Be able to conduct
effective communication appropriate diagnostic or
throughout the care of the monitoring procedures,
service user treatment, therapy, or
other actions safely and
skilfully
2b.5
Be able to maintain
records appropriately
2c.1
Be able to critically
evaluate the impact of or
response to the registrant’s
actions
2c.2
Be able to monitor and
review the ongoing
effectiveness of a planned
activity and modify it
accordingly
2c.3
Be able to audit, reflect on,
and review practice
In summary, these standards describe the minimum level of knowledge and skills required by a
registered biomedical scientist to be able to practise. Indeed, it is essential that these standards
are met as they are the threshold standards needed for an individual to be admitted to the
HPC register and describe the scope of practice for a newly registered biomedical scientist.
Scope of practice defines the range of activities in which a registered individual has been
trained, assessed, and deemed competent to perform. The initial scope of practice defines
the threshold standards for admittance to the HPC register, which are met on successfully
Cross reference completing an HPC approved degree or obtaining an IBMS Certificate of Competence (Figure
We described the Certificate 1.3). Although the statements (standards) are too numerous to fully explore within this book,
of Competence in Chapter 1 the education and training programmes undertaken by a prospective biomedical scientist will
‘Biomedical science and provide the knowledge, skills, and evidence to ensure that these minimum standards of pro-
biomedical scientists’.
ficiency are met.
SELF-CHECK 2.1
Why is it considered necessary that the HPC apply standards of proficiency for prospective
registrants?
Scopes of practice are not static and change during a registrant’s career. Thus, as a career
progresses the associated scope of practice also evolves as new skills are acquired or new
roles undertaken. However, any new scope of practice also provides a different context for
the HPC Standards of Proficiency and their Standards of Conduct, Performance and Ethics.
For instance, if a registrant no longer practises in the laboratory, but works as, for example, a
researcher, lecturer, or manager, and can show that he or she continues to meet the standards
of the HPC, they can remain on the register. It is only when these standards cannot be met
that a registrant may wish to voluntarily remove themselves from the register or be removed
by the HPC itself.
If a registrant has voluntarily come off the register or been removed from it, the HPC does
allow a suitable individual to be readmitted and has developed processes, which include
remedial training, supervised practice, or a combination of both, for this purpose.
SELF-CHECK 2.2
How may registrants who no longer practise in a laboratory, continue to meet the HPC standards?
HPC sanctions on practice

The HPC has statutory powers to apply sanctions or restrictions that limit a registered individ-
ual’s ability to practise as a biomedical scientist (or as 1 of the other 15 appropriately registered
professionals). Information regarding the competency of a registered individual will result in
an investigation by the HPC. If this raises sufficient concern, it will be followed by a Fitness
to Practice (FTP) hearing on either competence or health grounds. You can see an outline of
the FTP hearing in Figure 2.3. Depending on the outcome, the hearing can lead to sanctions
against the individual concerned, which include restrictions on the right to practise, supervi-
sion of practice, suspension from the register or, ultimately, removal from the register. Fitness
to practise hearings are detailed and rigorous, but apply the lesser civil, not criminal, court
burden of proof, as these hearings relate only to professional practice and public safety.
Summaries of three FTP hearings are given in Case studies 2.1, 2.2, and 2.3 that indicate
the detail in which the HPC investigates all allegations made against registrants from all of its
15 registered professions.
Allegation
Investigating committee
panel meeting
Preliminary meeting Preliminary meeting
Further investigation Further investigation
Conduct and competence Health committee

committee panel hearing panel hearing
FIGURE 2.3
Schematic illustrating the HPC Fitness to Practise process.
CASE STUDY 2.1 Example of FTP hearing relating to

an occupational therapist
An occupational therapist was struck off the register follow- concerned basic competencies. The allegations relating to
ing allegations that he or she failed to maintain adequate note keeping, which included the falsification of records,
records, provided inappropriate treatment to patients, wrote demonstrated a marked lack of honesty and integrity.
up case notes retrospectively, falsely wrote up case notes, and Furthermore, the panel concluded that the registrant had
incorrectly closed cases that required further assessment. not shown insight into his or her failings, or the conse-
quences arising from them.
The panel determined that the misconduct found to be
proven was wide ranging, covered a period of time, and
CASE STUDY 2.2 Example of FTP hearing relating to

an operating department practitioner
An operating department practitioner was struck off the The panel considered that a caution order would not
register for self-administration of the drug propofol having reflect the severity of the matter and that a ‘conditions of
accessed the employer’s drug store without authorization. practice’ order would not be appropriate, given that the
The registrant had also received a police caution for this registrant was not present at the hearing. It was not known
offence. if the registrant was working, with the result that condi-
tions could not be considered. The panel gave careful con-
The panel took into account the fact that for the police cau-
sideration to imposing a suspension order, but concluded
tion to have been given, a full admission of the allegation had
that there had been a serious breach of trust on the regis-
to have been made. Accordingly, the panel were satisfied that
trant’s part, which had the effect of putting patients and
the theft of the drugs had occurred. The panel were also satis-
colleagues at risk.
fied that the registrant had self-administered the drug.
CASE STUDY 2.3 Example of FTP hearing relating to a

biomedical scientist
A biomedical scientist was struck off the register follow- over a significant period, but at no stage had the regis-
ing allegations that the registrant had failed to maintain trant shown any insight into their inappropriate nature, or
adequate records and not ensured staff were adequately remorse for the consequences of these actions on patients
trained, had behaved in an intimidating and aggressive and colleagues. The panel concluded that these behaviours
manner towards staff, and discussed confidential personal were misconduct and showed a clear breach of several of
details of staff with others. These behaviours had continued the Standards of Conduct, Performance and Ethics.
All registered professionals who are found guilty of a criminal offence will have the details of
the offence, trial, and any sentence passed to the HPC for investigation, hearing, and possible
sanctions as described above.
Full transcripts of all FTP hearings, including those for biomedical scientists, are made public
and may be found on the HPC website.
SELF-CHECK 2.3
List five reasons for which a biomedical scientist may be suspended or removed from the HPC
register.
2.4 Renewal of HPC registration

To remain on the HPC register, an individual registrant is required to sign a personal declara-
tion every 2 years confirming that he or she continues to meet the HPC Standards of Conduct,
Performance and Ethics and their Standards of Proficiency. Since November 2007 this declara-
tion by biomedical scientists also included a statement that they also continue to meet the
HPC standards of health and CPD. To check the validity of this latter statement, 2.5% of the
total registrants from each of the regulated professions are required to provide evidence of
their CPD activities during the preceding 2 years. Failure to provide sufficient evidence within a
given time period results in action by the HPC against those individuals, such as suspension or
even removal from their register, although further time to submit evidence may also be given.
All new registrants are automatically exempt from this requirement for the first 2 years of their
registration period. The first audits for biomedical scientists occurred in 2009.
The HPC register currently contains over 200,000 professionals. Whilst it is mandatory for
individuals providing services to the NHS to be registered (or in training to be registered or
appropriately supervised by a registered individual if unregistered), membership of a profes-
sional body is a matter of personal choice. The professional body for biomedical scientists is
the Institute of Biomedical Science.
2.5 Institute of Biomedical Science

The Institute of Biomedical Science (IBMS) is the professional body for biomedical scientists
in the United Kingdom and, as such, it:
2.6 IBMS CODE OF CONDUCT AND GOOD PROFESSIONAL PR ACTICE 31
• Consists of a number of individuals who have voluntarily joined the Institute to maintain
the knowledge and skills that defines biomedical science
• Sets standards of professional conduct appropriate to individuals who practise biomedical

science
• Administers the award of qualifications appropriate to this knowledge and skills
Within the UK, the IBMS has a highly developed network of regions and branches that provide
a forum for the discussion of scientific and/or professional matters. Commonly, there is also a
CPD officer and a network of local hospital representatives, who may also be approached for
information or help. Some regions and branches also support discipline-specific discussion
groups that are able to provide another arena where scientific or professional matters may
be discussed. Upon joining the IBMS, an individual is assigned to a specific branch, which is
normally determined by the work address of the individual. It is possible to change branch if
he or she wishes to join a branch closer to their home address.
The vast majority of IBMS members reside and practise in the UK; however, it also has members
in many countries and supports branches in Cyprus, Gibraltar, and Hong Kong.
The IBMS promotes and develops biomedical science and those who practise biomedical sci-
ence, and is therefore principally concerned with professional matters and issues related to
its members; you should not, however, infer that it is not concerned about patients or with
patient safety. The activities of the IBMS are supportive and allow appropriate members to
meet the HPC standards described in Section 2.2.
Classes of IBMS membership

The IBMS has four classes of membership: Associate, Licentiate, Member, and Fellow. You can
see a summary of the classes of membership and how to progress through them in Table 2.4.
As an undergraduate student, an individual is admitted to the class of Associate upon joining
the IBMS. Following graduation and the award of a Certificate of Competence (Section 2.2
and Chapter 1) he or she will be able to upgrade to Licentiate membership. Once admitted
as a Licentiate, further upgrade of membership class to Member and ultimately to Fellow
is encouraged. Equivalent routes for entry and membership class upgrade for all corporate
classes of IBMS membership exist for those individuals whose education and training are dif-
ferent to what might be described as ‘standard’.
Whatever their membership class, members of the IBMS are expected to apply the same prin-
ciples of good professional conduct and practice in biomedical science.
SELF-CHECK 2.4
How may the structure of the IBMS membership help biomedical scientists develop their careers?
2.6IBMS Code of Conduct and Good

Professional Practice
The IBMS Code of Conduct is embedded within the IBMS publication, Code of Good Professional
Practice for Biomedical Scientists. Like the HPC Standards of Conduct, Performance and Ethics
(Section 2.3), this publication states in general terms what is expected of a member of the
TABLE 2.4 Classes of the Institute of Biomedical Science membership effective from 1 January 2009
Membership class Comments and progression

Licentiate Licentiate is the initial class of corporate membership for someone who has been awarded the IBMS
Certificate of Competence.
Licentiates are entitled to use the letters LIBMS after their name.
Licentiates of the IBMS have the opportunity to work towards an IBMS Specialist Diploma.
This qualification will allow individuals to apply for the next class of IBMS membership: Member
Member Member is the next class of corporate membership for someone who is already a licentiate and
holds an IBMS Specialist Diploma.
Members are entitled to use the letters MIBMS after their name.
Members of the IBMS have the opportunity to work towards an IBMS Higher Specialist Diploma.
This qualification will allow individuals to apply for the highest class of IBMS membership:
Fellow.
Fellow Fellow is the highest corporate class of IBMS membership for someone who is already a Member
and holds an IBMS Higher Specialist Diploma.
Fellows are entitled to use the letters FIBMS after their name.
Associate Associate is the non-corporate class of IBMS membership for someone who is not eligible for
corporate membership.
Categories of Associate include: students on accredited biomedical science degree courses and
graduate trainees employed in an IBMS approved training laboratory.
IBMS in both their personal and professional lives. The IBMS Code of Conduct is reproduced
in Table 2.5.
Compare the seven major points of the code with the 12 of the 14 statements in the Standards
of Conduct, Performance and Ethics of the HPC (Table 2.2) pertinent to biomedical scientists;
you can see that both are essentially similar. However, the ways in which they are written are
TABLE 2.5 Institute of Biomedical Science Code of Conduct
All members of the Institute of Biomedical Science will at all times:

1. Exercise their professional judgement, skill, and care to the best of their ability.
2. Fulfil their professional role with integrity, refraining from its misuse to the detriment of patients, employers, or
professional colleagues.
3. Seek to safeguard patients and others, particularly in relation to health and safety.
4. Treat with discretion all confidential and other information requiring protection, and avoid disclosing to any unauthorised
person the result of any investigation or other information of a personal or confidential nature gained in the practise of
their profession.
5. Act in good faith towards those with whom they stand in professional relationship and conduct themselves so as to uphold
the reputation of their profession.
6. Strive to maintain, improve, and update their professional knowledge and skill.
7. Promote the study and development of biomedical science, and the education and training of practitioners of biomedical
science.
2.6 IBMS CODE OF CONDUCT AND GOOD PROFESSIONAL PR ACTICE 33
rather different. For example, duty 6 of the HPC requires biomedical scientists to act within the
limits of their knowledge and skills, and to refer problems to another (senior) practitioner as
necessary. This duty is incorporated into statements 1, 2, 5, and 6 of the IBMS code.
SELF-CHECK 2.5
Which statements in the IBMS Code of Conduct would allow you to meet the HPC standard 1
that requires a biomedical scientist to act in the best interests of service users?
The IBMS Code of Good Professional Practice for Biomedical Scientists is only one of a number
of its publications (Box 2.2).
BOX 2.2 IBMS publications and website
The IBMS produces a variety of publications and leaflets, 1 The Biomedical Scientist
however, its two principal journals, which have been pub- 2 British Journal of Biomedical Science
lished for over 50 years and are sent to its members are:
The Biomedical Scientist is published monthly and contains
news, articles of scientific interest, CPD activities, IBMS
activities, and a classified advertising section. The British
Journal of Biomedical Science (BJBS) is published quarterly
and is the largest worldwide circulation peer-reviewed
journal dedicated to biomedical science (Figure 2.4). It
includes wide-ranging papers on basic science, scientific,
and clinical practice of biomedical science.
The website of the IBMS is of increasing interest to mem-

bers and visitors alike. You can see the welcome page in
Figure 2.5
FIGURE 2.4
Front cover of an issue of the British Journal of FIGURE 2.5
Biomedical Science. Welcome page of the IBMS website (www.ibms.org).
Good professional practice

The IBMS has defined what it regards as good professional practice in the context of bio-
medical science and the behaviour of biomedical scientists. Its code of conduct describes
the attributes and the professional behaviour expected of IBMS members. This code comple-
ments that of the HPC Standards of Conduct, Performance and Ethics, which we described in
Section 2.3. The IBMS code is relatively comprehensive as you can see from the areas listed in
Table 2.6, and is explained in more detail in the IBMS publication, Code of Good Professional
Practice for Biomedical Scientists (Figure 2.6). However, in many ways the IBMS code does not
describe the full range of good professional practice. This would be difficult anyway, since
Cross reference observations as to what is good or bad practice are subjective, and it can often be difficult to
Health and safety, quality give an objective viewpoint. In this volume, we will discuss in detail health and safety, quality
assurance and management, assurance, and professional development in the relevant chapters. However, the education
and personal development are and training programme required of a biomedical scientist in the UK will ensure an individual
discussed in Chapters 4, 19, and
works within the context of the IBMS Code of Good Professional Practice and meet the various
20, respectively.
standards of the HPC.
2.7 IBMS and education

The IBMS has been involved in formal educational activities since its founding in 1912; indeed,
the IBMS motto is ‘Learn that you may improve’. Its major educational activities in higher educa-
tion institutions are the accreditation of appropriate degree programmes and its involvement
in the provision of professional qualifications.
Degree accreditation
The programme of biomedical science degrees is defined for registration purposes by the
Quality Assurance Agency for Higher Education (QAA), while their standards are subject to
approval by the HPC (Section 2.2). However, the IBMS, in its role as a professional body also
TABLE 2.6 Areas to which the IBMS

code of practice applies
Functions of a biomedical scientist
Professional relationships
Management and leadership
Staffing and skill mix
Staffing and workload
Records and archives
Training
Professional development
Quality assurance
Performance
2.7 IBMS AND EDUCATION 35
FIGURE 2.6
Front cover of IBMS Code of
Good Professional Practice.
Copyright © Institute of
Biomedical Science.
accredits the contents of degrees in biomedical science. Accreditation of a professional

degree is a process of peer review set against a number of defined learning outcomes. The
IBMS accredits both undergraduate and postgraduate degree programmes for membership
purposes. Accreditation of any particular degree in biomedical science by the IBMS is not
designed to replace or replicate the HPC approval process or, indeed, the internal validation
exercises undertaken by the universities concerned. The IBMS considers the content of the units
or modules constituting the curriculum of the degree and uses the definitions and subject areas
already discussed in Chapter 1, as the basis for their undergraduate accreditation process.
Accreditation by the IBMS requires the formation of a local liaison group consisting of academic
staff of the university, NHS employees (largely hospital laboratory staff), and any independent R E D I T
part-time teachers who may be involved. This group is required to meet on a regular basis to C
C
oversee matters relevant to the course. Written evidence regarding the formation of the group
A
and its meetings must be provided for scrutiny by the IBMS. It is generally expected that senior
and experienced laboratory staff from local hospitals will contribute to the delivery of both
undergraduate and postgraduate courses through part-time lecturing as ‘experts’ in clinical
subjects, particularly where expertise in those areas cannot be provided by university staff.
P
E
R
O
Successfully completing an IBMS degree at an accredited university (Figure 2.7) offers a G R A M M
number of advantages to the graduate. Thus, it eases the entry of a graduate into the IBMS
and contributes towards registration with the HPC.
FIGURE 2.7
The accreditation of a specific degree in biomedical science provided by a particular univer- Logo for use on, for example,
sity may only apply to the academic component. This allows successful students to work in a stationary by an IBMS
variety of occupations or areas, which we listed in Chapter 1. It also allows the IBMS to tailor accredited university.
its accreditation of postgraduate education and degrees to the needs of biomedical scientists
during their professional careers. A number of universities have developed co-terminus or
applied biomedical science degrees. These degrees consist of an accredited programme of
academic study together with laboratory-based teaching and training during a series of work
placements. The advantage of completing this type of degree is that professional training is inte-
grated into the degree programme. A Certificate of Competence (Figure 1.3) is awarded upon
graduation allowing an individual to immediately apply for inclusion on the HPC register.
Graduates who have completed a degree course that is not accredited by the IBMS may
be required to undertake supplementary or extra undergraduate studies to make their
non-accredited degree equivalent to an accredited honours degree in biomedical science.
Normally, the major areas of extra study involve the clinical disciplines, which are often not
fully covered in non-accredited degrees. If a degree programme of study does not include
any laboratory-based professional training, the Certificate of Competence must be obtained
by a different route. For example, it can be obtained by working in a laboratory following
graduation. Alternatively, it can be gained while working in a laboratory and studying for an
FIGURE 2.8 appropriate degree part-time. Such study pathways require the registration portfolio to be
Logo for use on, for example, verified by IBMS appointed examiners, who must visit the place of work. The verifiers must
stationery by an IBMS also assess the role of the laboratory in the training and have the remit to award training
approved training laboratory. approval to appropriate laboratories (Figure 2.8).
Professional qualifications awarded by the IBMS

The IBMS has developed the following professional qualifications:
• Specialist Diploma
• Certificate of Expert Practice
• Higher Specialist Diploma
• Diploma of Expert Practice
• Advanced Specialist Diploma
These qualifications are linked to classes of Institute membership (Section 2.5) and also to a
career in the National Health Service (NHS). The IBMS qualification structure is outlined in
Figure 2.9.
The Specialist Diploma (Figure 2.10) is likely to be the first IBMS qualification encountered
following registration at the beginning of a career in biomedical science. Qualification for a
diploma involves the professional study of a single discipline. Diplomas are awarded in the
principal, discipline-specific areas of biomedical science:
• Clinical biochemistry
• Cytology
• Histology
• Haematology with hospital transfusion practice
• Histocompatibility and immunogenetics
• Immunology
• Microbiology
• Transfusion science
• Virology
HCS Career Additional Membership

framework optional class
stage qualifications
9 Professional Doctorate
8 Advanced Specialist
Diploma
Diploma of
Expert Practice Fellow
7 Higher Specialist
Diploma
Certificate of
6 MSc Expert Practice Member
Specialist Diploma
5 Licentiate
Certificate of
Competence
BSc (Hons) Biomedical FIGURE 2.9

Science Hierarchy of IBMS membership classes related
to academic and professional qualifications.
Some of these programmes have been produced in partnership with other organizations, such
as the British Society of Histocompatibility and Immunogenetics (BSHI). The content of all
specialist diplomas have a common element related to professional roles and responsibili-
ties, health and safety, receipt and processing of samples, laboratory computers, laboratory
equipment, and quality assurance. However, they also include discipline-specific elements
that naturally differ from subject to subject.
The IBMS recommends that 12–18 months of study be undertaken to collect the evidence
required for the award of a specialist diploma. Its award requires an assessment by an IBMS
appointed assessor. The assessment process consists of three parts:
1. Candidate presentation
2. Assessment of the portfolio
3. Candidate-led tour of the relevant hospital department
Each process will assess knowledge, practice, and skills, but is also designed to test an indi-
vidual’s confidence in demonstrating these points to the assessor.
The presentation by the candidates to the assessor will be expected to last 20 minutes and
demonstrate that a transition from a newly qualified and generically trained individual to
a specialist biomedical scientist has occurred. The candidate will also need to explain how
their training relates to the daily practice within a single discipline of biomedical science.
FIGURE 2.10
IBMS Specialist Diploma.
The assessor will examine the portfolio of evidence to ensure all sections have been completed.
The candidate will then take the assessor on a tour of their department during which they may
be asked questions about their activities or the functions of the department. Finally, there will
be feedback to the candidate, their training co-ordinator, and department manager.
If successful, the candidate will be notified of this on the day.
Certificates of Expert Practice are intended to recognize scientific practice in specific areas that
would only be performed by a few individuals who have chosen to specialize, and for whom
alternative qualifications do not exist. They are at a level commensurate with a specialist
diploma, but in areas that do not warrant the development of a full portfolio. In this way, they
complement the broader discipline-specific post-registration awards already undertaken.
Examples of such qualifications include the Certificate of Expert Practice in Electron Microscopy,
Certificate of Expert Practice in Quality Management and Certificate of Expert Practice in
Training.
The Higher Specialist Diploma (HSD) is also a discipline-specific qualification, although an

HSD in Management and Leadership has also been developed. The HSD is designed to test
and demonstrate a deeper professional knowledge of a discipline than that at Diploma, and
may not be entered without first holding an appropriate postgraduate degree either an IBMS
accredited MSc or an IBMS approved masters or doctorate-level qualification.
The HSD is at the centre of the IBMS qualification process in that although the HSD is the nor-
mal requirement for the upgrade from Member to Fellow, it represents only an intermediate
FURTHER READING 39
step in terms of professional practice. The HSD assessment runs annually over two days and
includes case studies or scenarios as a major component of the examination. In this way, the
ability to draw strands of information or practice together and demonstrate a deeper under-
standing is assessed. Ironically, although related to practice, this qualification has no practical
component in its assessment!
Diplomas of Expert Practice and Advanced Specialist Diplomas of the IBMS recognize knowl-
edge and skills at the highest level of scientific or clinical practice within areas of biomedical
science or in the application of biomedical science. They are designed for those who wish to
practise at the level of a consultant biomedical scientist.
Examples of Diplomas of Expert Practice qualifications include the Diploma of Expert Practice
in Clinical Transfusion, Advanced Specialist Diploma in Cervical Cytology, and Advanced
Specialist Diploma in Ophthalmic Pathology. The cervical cytology advanced diploma was
introduced in 2001 and holders have seen their role expand in the intervening period from
a starting point of reporting of abnormal cervical cytology results to a number of other areas
including case presentations at multidisciplinary team meetings, audit of invasive cancers,
and commissioning.
SUMMARY
■ The principal duty of the Health Professions Council is to protect the public.
■ Biomedical scientists are regulated by the Health Professions Council, which has the legal
powers to admit individuals to its register.
■ The Health Professions Council posseses the legal powers to restrict practice and remove
from its register those registrants who it considers not competent to practise.
■ Biomedical scientists may join their professional body, the Institute of Biomedical
Science.
■ The Institute of Biomedical Science acts for its members by helping them meet the require-
ments of the Health Professions Council but also provides other support and benefits to
members.
■ The functions and activities of the Health Professions Council and Institute of Biomedical
Science are complementary, and they work closely together.
FURTHER READING
● Wood J. The roles, duties and responsibilities of technologists in the clinical labora-
tory. Clinica Chimica Acta 2002; 319: 127–132.
● Burnett D, Poyser KH, Sherwood RA. A Practical Guide to Accreditation in Laboratory

Medicine. ACB Venture Publications, London, 2002.
● Institute of Biomedical Science. Good Professional Practice for Biomedical Scientists,

2nd edn. Institute of Biomedical Science, London, 2005.
Useful Websites
■ Health Professions Council. http://www.hpc-uk.org/index.asp
■ HPC international route. http://www.hpc-uk.org/apply/international/
■ HPC degree approval. http://www.hpc-uk.org/education/programmes/
■ HPC readmittance process. http://www.hpc-uk.org/apply/readmission/
■ HPC Fitness to Practise. http://www.hpc-uk.org/complaints/making/
■ Institute of Biomedical Science. http://www.ibms.org/
■ IBMS Code of Good Professional Practice. http://www.ibms.org/go/professional:good-
professional-practice
■ IBMS degree accreditation process. http://www.ibms.org/go/education-development:
ibms-courses:accreditation
■ IBMS portfolio verification process. http://www.ibms.org/go/registration:reg-
qualification-assess
■ IBMS laboratory training approval. http://www.ibms.org/go/registration:lab-standards
■ IBMS regions and branches. http://www.ibms.org/go/ibms-networks
■ IBMS CPD Scheme. http://www.ibms.org/go/education-development:cpd
■ Department of Health England. http://www.dh.gov.uk/en/index.htm
■ Health of Wales Information Service. http://www.wales.nhs.uk/
■ Scotland’s Health on the Web. http://www.show.scot.nhs.uk/
■ Department of Health, Social Services and Public Safety Northern Ireland. http://www.
dhsspsni.gov.uk/
QUESTIONS
1. Which of the following statements are applicable to biomedical scientists?
(a) Must be able to practise in a non-discriminatory manner

(b) Are not required to use problem-solving skills
(c) Be able to critically evaluate collected information
(d) Be able to review their individual knowledge and skills
(e) Be able to maintain appropriate records
(f) Have to always practise under direct supervision
(g) Always work in isolation
2. Which of the following statements from the HPC are applicable to biomedical
scientists?
(a) You must keep your professional knowledge and skills up to date
(b) You must get informed consent to give treatment (except in an emergency)
(c) You must keep accurate records
(d) You must limit your work or stop practising if your performance or judgement is
affected by your health
QUESTIONS
(e) You must make sure any advertising you do is accurate

(f) You must provide any important information about your conduct and competence
3. Why is it considered necessary for an individual to be registered?
4. Why should it be appropriate for registrants to join a professional body, if this is not
mandatory?
5. Discuss the value of continuing professional development.
3
Communications in
laboratory medicine
Georgina Lavender
Learning objectives
■ Explain the need for clear policy for communication

■ Explain why there is a need for a generic telephone policy
■ Discuss the attributes required of a biomedical scientist when communicating results
■ Discuss the regulations that apply to the storage and transmission of electronic data with
reference to the current guidelines from the Institute of Biomedical Science
■ Explain the roles of regulatory bodies that have interests in clinical laboratory record keeping
Introduction
Communication skills, including written, verbal, and electronic communications, are an essen-
tial part of being a biomedical scientist. Biomedical scientists are required to communicate
clearly and precisely on all aspects of their work. They must communicate within their local
workplace with their colleagues, within the organization that employs them, and they must
be able to communicate with professionals and any of the public with whom they may have
indirect contact.
Communication from the clinical laboratory includes the results of laboratory tests, specimen
requirements, changes to methodology, and their effects on reference ranges, changes to ser-
vice provision, significant changes in laboratory personnel, the introduction of new policies and
procedures, or any other information that may affect the relationships between the laboratory
and its service users.
One of the principal roles of biomedical scientists is to provide information to appropriate

healthcare professionals that may affect the diagnosis and treatment of patients. They pro-
vide information regarding prescription drugs, what drugs are suitable and in what amounts
3.1 COMMUNICATION AND THE CLINICAL L ABOR ATORY 43
for a variety of diseases. They also contribute to screening programmes for large num-
bers of ‘well’ individuals to detect disease in the early stages before symptoms are noticed.
The whole process of receiving specimens, performing analytical work, and reporting results
from start to finish relies on adequate communications between all the groups involved. This
communication may not immediately be obvious to less experienced members of a labora-
tory team; hence, the purpose of this chapter is to highlight areas of both verbal and non-
verbal communication that may impact on the relationships between laboratory staff, and
other professionals and users. As biomedical scientists, you must be aware of your limitations
and the consequences of your actions.
This chapter highlights the importance of adequate communication skills within the clinical
laboratory and how communications by laboratory personnel can affect the perceptions of
users of the service they provide.
3.1Communication and the clinical

laboratory
Laboratory medicine has a reputation for being one of the more under-rated services within
a hospital environment. However, studies and audits have shown how essential the clinical
laboratory is in diagnosis and in the treatment expected in the demanding healthcare organi-
zations of today. It has been estimated that the clinical laboratory will have an input into 70%
of patients receiving healthcare. Laboratory medicine plays a vital role in the overall function
of the healthcare system, but its role may be overlooked, particularly by the general public.
This has come about because, traditionally, clinical laboratory personnel appear to have little
contact with the other hospital staff and with patients. Little has been known about the work
carried out behind the laboratory doors, with patients largely accepting that the ‘path lab’ is
a somewhat unpleasant entity where doctors dig around with bits of dead people. However,
the reality could not be further from these preconceived ideas. The World Wide Web has raised
public awareness of healthcare to new levels, and evidence-based medicine and audit have
increased the importance of clinical laboratory investigations with the other healthcare pro-
fessional groups within the patient care pathways. Laboratory medicine is now recognized by
a wider audience within the healthcare community as an essential discipline and given due
appreciation. Staff in clinical laboratories have a much higher profile in the provision of patient
care. The corollary is that clinical laboratory staff must be aware of the impression that they give
to patients, health professionals, and laboratory users (those who either request tests or receive
results as part of their duties) during the course of their work. Just because the laboratory staff
are not necessarily seen in person does not detract from the importance of being aware of the
often latent signals that are picked up by other groups with which they have dealings.
Laboratory staff in public areas of hospitals

Clinical laboratory personnel rarely wear a uniform, and are often not immediately recogniz-
able as hospital staff when in public areas, such as shops, dining rooms, and hospital corridors.
As healthcare professionals, individuals have a responsibility to dress and act in a manner that
will not cause offence to others, breach their hospital trust policy, or bring the profession into
question by any aspect of their behaviour.
Some organizations have a dress code to which all staff are expected to adhere. Dress codes
are often viewed as draconian by laboratory staff who (a) are rarely seen outside the laboratory
44 3 COMMUNICATIONS IN L ABOR ATORY MEDICINE
and (b) are usually largely covered with a white laboratory coat. However, there are attempts
by employers to bring standardization and professionalism to their workforces. Clothes are
perceived, rightly or wrongly, as a form of non-verbal communication. Staff outside the labo-
ratory, patients, and their relatives and visitors are quickly judgemental of the standard of
dress worn throughout the hospital. Biomedical scientists must remember that the general
public may not share their personal views regarding what is or is not an acceptable standard
of dress at work, and that a dress code has been written by an organization with its total staff in
mind. When considering standards of dress within a hospital, aspects of infection control and
Cross reference health and safety have to be considered, and the reason behind certain aspects of dress codes
Chapter 4 ‘Health and safety’. may not immediately be apparent. While biomedical scientists may not usually be present in
clinics and on wards, they are still members of the wider hospital community and cannot be
given preferential treatment. Where biomedical scientists are free to dress as they please, they
should remember to be clean, tidy, and not wear clothing that may cause offence to others,
or that may constitute a breach of the Health Professions Council (HPC) standards of conduct,
Cross reference performance, and ethics. These were introduced in Chapters 1 and 2. Likewise, if they are
Chapter 1 ‘Biomedical science provided with a full uniform, as opposed to a laboratory coat and other personal protective
and biomedical scientists’ and equipment (PPE), then it would be seen that the dress code of the department is to wear that
Chapter 2 ‘Fitness to practise’. uniform as and when appropriate.
The HPC has clear standards of behaviour that are expected from its registrants at all times in
their working life, and also in their private and personal life. Standards of behaviour are also
viewed as non-verbal means of communication, and any unacceptable private behaviour may
spill over and be extended to the type of behaviour that should be shown to a member of the
public. The biomedical scientist involved in a street brawl may possibly be deemed as having a
short temper and be capable of assaulting a patient under certain circumstances. Such behav-
iour will be investigated by the HPC where a criminal offence has been proven to have taken
place and could jeopardize the registrant’s fitness to practise.
Communications within the clinical laboratory

Clinical laboratories are increasingly subject to extensive legislation. This has led to descrip-
tive procedures and documents being made available to all personnel within the laboratory.
The advantage of having such documentation is that it ensures all staff follow procedures to
high professional and scientific standards. The advantage of strict written instructions or stan-
Cross reference dard operating procedures (SOPs) is that all staff who are required to carry out a particular
You can read more about SOPs task or procedure do so in the same way. Not only are such SOPs necessary for Clinical
in Chapter 19 ‘Quality assurance Pathology Accreditation (CPA) purposes, there is also the guarantee that if a member of staff
and management’. is run over by a bus in their lunch hour, another member of staff will know exactly what to do
to complete the day’s work, even though there has been no direct communication between
the two individuals!
Communications within a clinical laboratory are varied. They include the transfer of instructions
and information between staff who work together to provide the end products of the laboratory.
These products are the final results of the various measurements and tests performed on clini-
cal specimens. Teamwork is the way in which a group of individuals must interact together to
achieve a common goal. Thus, maintaining a team structure and working together in an appro-
priate fashion is crucial. Most working days within a clinical laboratory follow a given pattern in
that the work is generated and must be carried out to specific deadlines. It is essential that every
member of the team is aware of their responsibilities and the responsibilities of others around
them. Each person working in a laboratory, regardless of his or her grade, has their own, some-
times unique place in that team. Team members have a responsibility to meet deadlines.
Communication within the team is necessary so that the team leader is able to maintain appro-
priate control and the rest of the team recognize his or her leadership. It is essential that any
information that may affect the processing of specimens is communicated throughout the team,
both upwards to the most senior member with the responsibility of the service, and downwards
to the support staff. Figures 3.1 (a and b) illustrate the ends of the spectrum when information
needs to be communicated effectively between members of staff in the same department.
For example, the major failure of a large piece of equipment in the clinical chemistry labora-
tory may only seem important to those involved. Such events, while unlikely, require a number
of immediate remedial actions in addition to repairs to the equipment; all staff must know
what is required of them. Any major delays in the processing of samples must be reported
to staff in specimen reception, so that they are aware of the situation and can inform other
departments or service users as necessary that clinical samples may not be processed with
the usual speed and efficiency. Staff involved in the issuing of results and dealing with tele-
phone calls asking for results must also be informed so that they are able to explain to service
users why the results they require may not be ready. There is also the need for clear written
(a) “May I start the meeting by thanking you all for your exceptional effort over the last
two weeks in dealing with an unprecedented number of clinical samples.”
(b)
YOU’RE
FIRED
FIGURE 3.1
(a) Appropriate and (b) inappropriate ways of informing staff of departmental or
procedural changes.
communication in the laboratory to invoke contingency plans that may involve staff who do
not directly work in the laboratory, such as those involved in point of care testing (POCT)
Cross reference using equipment in remote locations or using another laboratory on a different hospital site,
Chapter 18 ‘Point of care testing’. or even in another hospital or organization.
Other examples where effective communication is essential include the following:
• Occasions when staff are temporarily absent and their duties must be covered by others
• The reporting of failures of quality control procedures
• Clinical samples that must be processed immediately because test results are required
urgently
• Stock control where there is a danger of a particular reagent running out before a new
delivery is expected
All these factors may critically affect the routine functioning of the laboratory and will almost
certainly require attention from more than one member of staff.
It is also essential to communicate any updated or changed information within the labora-
tory team, no matter how trivial it may appear. Thus, the introduction of a new method may
require a change in the stated reference range of a particular analyte, and this, of course, has
implications for the final result appearing to be within normal limits. A change in the batch of
a particular reagent on a large analyser may require its recalibration and a re-evaluation of the
performance of the method. Even something as apparently simple as a broken door may result
in a fire exit becoming unusable and require the selection of an alternative route.
SELF-CHECK 3.1
Why is it necessary to write down any changes in the consignment lot numbers of reagents
used in laboratory procedures?
Procedures are often changed by senior members of staff who are not actually involved in the
performance of the daily tasks. In practice, often new or inexperienced members of staff are
the persons to point out a flaw in a change because they are the ones who can most easily
see possible consequences of any changes. However, it is also possible that junior members
of staff may feel that particular tasks have no real benefit, and are irrelevant and so stop doing
them. This may have adverse effects further along the laboratory chain. It is essential that any
changes to SOPs are discussed fully by the whole team and only implemented when there is
a written instruction to do so.
Communication within the laboratory also includes completing critical paperwork, such as
Cross reference quality control charts, reagent books, and troubleshooting sheets in the case of an instru-
Chapter 19 ‘Quality assurance ment failure. If a staff member carries out maintenance or runs successful quality control, and
and management’. does not record it in the correct manner in accordance with recognized laboratory procedure,
other members of staff will not know that the tasks have already been carried out.
Communications between clinical laboratory staff

and service users
Service users may be described as any person or group of people who interact with the labo-
ratory. This, of course, includes patients and their relatives. It also includes doctors or other
healthcare professionals (both within and outside of a hospital trust), and visitors to clinical
laboratories. Table 3.1 gives you an indication of possible service users; it is not exhaustive and
should only be used as a guide.
Patients and their relatives form a group that are generally described as direct service users
given the manner in which results and information generated by the laboratory affect them.
They will be given information about their own health, or that of a relative or friend that may
have a profound impact on their lives. They are rarely medically qualified and communication
with this group must be appropriate to their level of understanding, but without being in any
way patronizing. The communication must also comply with local policy, professional codes
of conduct, and current legislation.
Hospital staff groups are varied and information given to them must also be appropriate to
their qualifications. It also depends on their need-to-know as part of their own professional
duties; not all hospital employees need to have access to confidential information about
patients. Different departments will have their own rules regarding who needs to know certain
pieces of information, and if there is any doubt regarding the level of information that can be
passed between staff groups, it is better to be cautious and give too little in the first instance.
More detailed information can always be given at a later date, but it is far more difficult to
retract something inappropriate.
Community medicine groups are healthcare professionals who do not necessarily have direct
connections to the clinical laboratory, but are external users of the laboratory services. They
need to communicate with clinical laboratories regarding patient samples and clinical test
results as part of their daily activities. Levels of information and communications between
laboratories with these groups depend on local policies.
Visitors to the clinical laboratory are wide ranging, from healthcare professionals to patients.
The common link in this group is that they are entering an environment with which they are
unlikely to be familiar. Their levels of knowledge are equally widespread and may be very spe-
cialized. Each visitor must be treated appropriately to the needs of the visit.
TABLE 3.1 List of possible service users
Hospital staff Community medicine groups Visitors to the laboratory

Doctors General practitioners Company representatives
Nurses Community nurses Research scientists and
Radiographers Midwives doctors
Physiotherapists Surgeries Instrument engineers
Occupational therapists Administrative staff Formal inspectors
Cardiographers Patients
Pharmacists
Dieticians
Midwives
Phlebotomists
Porters
Domestic staff
Maintenance staff
Ward support staff
Secretarial staff
It is often difficult for biomedical scientists to build relationships with user groups that are
not based on preconceived ideas. Biomedical scientists are often disadvantaged in a hospital
structure because there are largely unseen, and so is the environment in which they work.
Often they will be judged by an unseen, audience responding to telephone conversations
and reports. However, as biomedical scientists, we must also realize that the majority of the
work done during the course of a normal day passes in and out of the laboratory without inci-
dent, and is processed in a timely manner in accordance with the expectations of the service
users. There is no need for a service user to have any direct contact with laboratory staff. It is
only when there are deviations from the normal process, such as when a patient’s condition
becomes critical and a result is needed urgently, a sample is lost and a result not available as
expected, or there is a major failure of a piece of laboratory equipment and test results are
therefore not available in the usual timeframe. Such occurrences may prompt a user to contact
the laboratory staff. It can be that service users who need to communicate with the laboratory
are under more pressure or stress than usual, and may be abrupt or rude. However, biomedi-
cal scientists should remain professional and act appropriately at all times.
Key Points
It is in everyone’s interest that good working relationships are fostered with all service
users.
Communication and
3.2
confidentiality
In direct face-to-face contact or over the telephone, laboratory staff must quickly establish
exactly who they are communicating with. It is fundamental for a biomedical scientist to
know to whom they are speaking before releasing information or patient details that could be
considered confidential or sensitive. Confidential information must only be given to certain
groups of users. For example, it is usually inappropriate for a biomedical scientist to discuss
the results of clinical tests or give such results to a patient, a patient’s relative, or members of
the public unless there are specific circumstances within written guidelines. Such behaviour,
Cross reference even inadvertent, would be in breach of the standards laid down by the HPC, and could
You can read more about the lead to both disciplinary action by the employer and investigation of the action by the HPC,
HPC in Chapter 2 ‘Fitness to
and that could then result in the biomedical scientist being removed from the professional
practise’.
register.
Key Points
In some cases patients may need to know the result of their test, including glucose
Cross reference measurements in diabetes or when monitoring anticoagulation.
You can read more about point
of care testing in Chapter 18,
and Haematology in the series
These considerations may also apply in some circumstances to commercially sensitive infor-
companion book.
mation. For example, discussions about pricing structures or costing regarding laboratory
3.3 METHODS OF COMMUNICATION 49
equipment with competitor companies as this may be sensitive information if a major pur-
chase is about to go out to tender.
3.3 Methods of communication

The major form of communication between laboratory staff and most of the user groups listed
in Table 3.1 is verbal, but this is not exclusive as written material is still exchanged. Verbal
communication may be one-to-one, an example being telephoning a doctor with a set of test
results concerning a particular patient. It may also be used to inform a large group of a change
in policy or procedure. The introduction of a new method may be presented to a large group
of hospital staff in the form of a case study to demonstrate why the new method is preferable
over the old. Written communications are used to transfer both clinical test results and mes-
sages, and may be in paper or electronic forms. Whether meant for a single recipient or for a
wide audience, the message must be clear and unambiguous, and presented at a level suitable
for the intended recipient.
The most important interface for the transfer of results through a hospital is the nurses’ station
(Figure 3.2) on a ward or clinic.
Telephone communications
Telephone communications are often the means by which a laboratory is judged by its users.
While few people have face-to-face contact with laboratory staff, a relatively large number use
the telephone as the primary means of contacting laboratory staff. Their reasons for telephoning
may include:
FIGURE 3.2
A typical nursing station on a children’s ward. This normally extremely busy area is the
major interface between patients and the clinical laboratory. Photograph courtesy of
Central Manchester University Hospitals NHS Foundation Trust, UK.
• Incoming requests
• Incoming questions that can be answered in general terms
• Incoming requests that need to be answered by a specific person
• Incoming calls for a named person
• Outgoing results
• Outgoing information of a general nature
• Specific information to a named individual in response to an outstanding request
Individual laboratories will each have their own telephone policies, although all such
policies should conform to certain basic principles. All workers must be familiar with the
telephone policy and associated administration of their own department and strictly adhere
to it. Figures 3.3 (a and b) illustrate the correct and incorrect manner in which to deal appro-
priately with a telephone enquiry.
Generally the following basic rules apply: biomedical scientists and other laboratory staff must
remember that the same rules apply to the telephone as to face-to-face and paper transfers
(a) “Certainly, I will e-mail

those test results to you
immediately”
(b)
“Your call is important to
us and I’ll deal with your
emergency tomorrow”
FIGURE 3.3
(a) Appropriate and (b) inappropriate ways of
answering telephone enquiries.
of confidential and sensitive information. It is essential that the recipient of any telephone
conversation be identified at the start of a call, either incoming or outgoing, so that only
appropriate discussions take place. Information of a clinical nature should never be given to
patients, their relatives, or members of the public. Nor should messages that relay clinical and
confidential details be left on an answer phone.
General incoming requests for specific patients

All incoming requests, such as those relating to urgent work, additional clinical tests, patient
demographic changes, and similar instructions must be recorded, together with the time and
date of the request, the name of the requestor, and the name of the person recording the Cross reference
Chapter 19 ‘Quality assurance
message. This is necessary so that all requests can be subsequently tracked and are traceable
and management’.
for audit purposes. You can read about audit in Chapter 19.
An example of the use of retaining a paper or electronic record may be when the clinical testing
of a patient’s sample becomes urgent due to the deterioration in his or her condition. If a sample
from a patient is already in the laboratory, this will save time. However, should the requesting
clinician telephone the laboratory and the individual fails to record the details of this call, it is
possible that sample urgency will not be known. This may cause an inappropriate delay in the
patient’s treatment. An adverse incident complaint may then be made by the doctor against the
laboratory. If the doctor is able to provide the name of the person to whom he or she spoke, then
this may be sufficient evidence that the member of staff is not following laboratory protocol. Cross reference
See Chapter 1 ‘Biochemical
Many laboratory information management systems (LIMSs) have a facility that automati- investigations and quality
cally records the time, date, and person entering information, as well as auditing any changes control’ in the accompanying
to requests. This useful facility allows a LIMS manager to see who has done what to a particular textbook, Clinical Biochemistry
request, and will also bring changes to the attention of other staff without the need for confor more information on
versation or passing paper notes. However, information held on LIMS is only as accurate as laboratory management
the person who inputs the data. systems.
Incoming questions that can be answered in general terms

General enquiries, such as What colour tube is used for a particular test? do not usually require
documenting, but it is important for the receiver of the call to realize that there is always a
laboratory user at the other end of the telephone. When talking to users, the biomedical sci-
entist must remain professional at all times, no matter how irrelevant the enquiry may seem.
However, it is also important that the identity of the user be established and any information
given by telephone is appropriate to the level of understanding of the user.
Incoming requests that require answering by a specific or

named person
Incoming calls that request specific information are sometimes beyond the professional
expertise of the person taking the call. These requests should promptly be passed to a more
relevant staff member, with as much detail as possible, so that the requestor does not have
to repeat themselves. It can be irritating to a service user to have to keep repeating the same
information when handed from person to person on the telephone, and reflects poorly on the
communication strategies within the department.
If someone receives a call asking for a named member of staff it is advisable to find out if
the named individual is available to receive the call. If so, the requestor should be informed,
particularly if the call needs to be transferred to another extension. Again, it is frustrating
for a service user to be transferred to an ever-ringing extension with no prospect of an
answer. The caller then has no other option, but to put down the telephone and start the
process again when there may have been an opportunity to ask for someone else at an
early stage.
Outgoing results
The telephone policy of all organizations may cover giving results or this may be guided by a
separate policy. At a minimum, when telephoning a result you must identify the person taking
the call. This individual should write down the result, the time, and date of the call, and a record
of the person giving the result. This information should then be read back to the caller in the
laboratory to ensure the correct information has been communicated and understood. This
type of information may form a paper record, but is more likely to be held electronically on a
LIMS. This forms part of an audit trail and, while at first glance may seem somewhat lengthy, it
is necessary in case something goes wrong. It is essential to ensure that any contradictions are
corrected. If there are any doubts about the abilities of the receiver to correctly transcribe and
communicate the results, then the biomedical scientist should request that someone more
suitable be asked to take over the call. This is essential for the safety of the patient; biomedi-
cal scientists must be able to demonstrate independent and confident practice, and not feel
intimidated in challenging the competence of the receiver of the results. A cautionary example
is outlined in Case study 3.1.
CASE STUDY 3.1 Transposing results
A post-operative patient had a short stay on the intensive care worker, and a check of the results book on intensive care
unit following major heart surgery before being deemed well revealed that the ward clerk had also followed the correct
enough to be transferred to a surgical ward. Prior to transfer, procedures, and the results matched. The nurse transfer-
the ward clerk on intensive care telephoned the laboratory ring the patient had taken the results from the record book
to ask for the most recent test results on the urea and elec- on intensive care and written them onto a scrap of paper,
trolyte concentrations of the patient. Results were given by which she had taken with the patient to the surgical ward.
the support worker in accordance with the laboratory pro- She then verbally gave the results to the nurse receiving the
tocol and recorded by the ward clerk in accordance with the patient, who also wrote the results on a scrap of paper. Later
intensive care protocol. The electrolytes and creatinine were this nurse transferred the results into the patient’s notes.
normal and the urea was 7 mmol dm−3 (with a reference Both nurses agreed that they had written results on paper,
range of 3–8). Although this value is near the top of the refer- but no record of these handwritten notes remained. The
ence range it is not an unexpected finding after major sur- patient notes revealed that there had been a transposition
gery. In the afternoon, a telephoned request came into the of the values of urea and K+ results when compared with
clinical biochemistry laboratory urgently requiring repeat the electronic record held in the LIMS. The consequence
determinations of urea and electrolytes on the patient, fol- was that when the surgical junior doctor came to review
lowing treatment of the hyperkalaemia (high concentration the patient, he noticed an apparent hyperkalaemia, which
of K+ in the plasma) together with measurements of blood he immediately treated, reducing the concentration of K+
insulin and glucose concentrations. The laboratory noticed in the patient’s blood.
that there had been no previous hyperkalaemia and quickly
This course of events ended with the patient receiving an
assayed the sample, to find the K+ to be 1.2 mmol dm−3 (ref-
infusion of K+ and suffering no ill effects. However, this
erence range 3.8–5.2). So, what had happened and where
could easily have resulted in the tragic death of the patient
had communications broken down?
and highlights the importance of following an SOP when
A check of the record held in the LIMS revealed that the communicating results verbally, either by telephone or
correct procedures had been carried out by the support face to face.
Outgoing information of a general nature

There are occasions where the laboratory has to use the telephone as a means of quickly dis-
seminating specific or general information to its users. This use will be covered by a telephone
policy but, as always, the persons receiving the information and whether it has been under-
stood must be established. Any questions relating to the information should be answered pro-
fessionally and courteously, no matter how trivial they may seem. Sometimes it is necessary to
tell users that there is a major breakdown in, for example, equipment or information technol-
ogy, so that the normal service is likely to be disrupted. Again, the recipient of this information
may well react to it in a discourteous or unprofessional manner; however, laboratory staff must
maintain professional standards of conduct, even when severely provoked to do otherwise.
Returning specific calls

On occasions, laboratory staff will be asked to return a call to a particular individual. It is
essential that all such calls are returned, even if the matter seems unimportant or the reason
for the call cannot be dealt with. Not responding as promised is the lack of communication
that reflects badly on the laboratory service. There is little that is more frustrating to a service
user than waiting for an expected piece of information that never arrives. Unfortunately, users
are far more likely to remember the one negative experience, rather than the 10 positive times
the laboratory was helpful.
SELF-CHECK 3.2
What is the simplest check one can make to ensure data given out over the telephone has
been correctly received?
Paper reports
In our increasingly paperless society, the number of hard-copy laboratory reports is in decline.
It is felt by many (and there have been many studies to show this) that the majority of paper
reports never reach their final destination, usually the patient’s written medical record, and
is largely a waste of resources and the time needed to produce them in quantity. However,
because the majority of reports are available electronically does not mean that the presenta-
tion of laboratory data as a written document, as a means of visual communication can be
disregarded or dismissed as unimportant. The written report is often the professional standard
that the outside world uses to judge the status of the laboratory. Poor reports imply poor
mechanisms to produce the data. The production of pathology reports of appropriate quality
may not be considered as a means of communication in the first instance but, for many users,
the request form and the final report are the only direct interface they have with the clinical
laboratory service. The report must be clear and unambiguous, and written with the correct
use of English grammar and language. This is particularly important for added comments.
Many laboratories now only offer coded comments or only allow certain groups of staff to add
comments. While an individual may understand the comment they are writing, it is essential
to ask how that comment may be viewed by the reader. Is the comment of relevance and does
it add value to the report? Is it written in good English? Will the service user understand the
comment? Has the correct medical terminology been used?
When information is presented as a paper report, all laboratory data should be accompanied
by appropriate units, reference ranges, useful clinical information, details of the issuing labora-
tory, as well as the obvious information, such as complete patient demographics and specimen
collection details. When such information is not complete, time and effort will be wasted by
the recipient of the report in trying to find the missing data, as indicated in Box 3.1.
BOX 3.1 Details are required
As examples, a patient with sepsis is likely to have a variety of microbiological investiga-

tions. The results of a swab with no details of the site may render the clinical information
available useless. Likewise, serial blood samples are often taken (a poorly controlled
diabetic may have several blood glucose samples taken over a 24-hour period) and the
date on a report with no indication of the time the blood sample was taken may make
the serial results of little value.
Electronic data
As the generation of paper communication is decreasing, so the transfer of electronic data is
increasing. The transfer of such data is the primary method of communication in many labo-
ratories. This allows requests to enter the laboratory using ward–based requesting programs
linked to pathology networks. Results can also be returned automatically to the patient elec-
tronic record and downloaded directly to general practitioner surgeries and outreach clinics.
The use of websites and email to ‘cascade’ information is reducing the need for writing letters,
telephone conversations, and the need to print information on paper.
Electronic data must be viewed as being the same as written communication. The rules applied
to paper reports about, for example, including units, reference ranges, useful clinical informa-
tion, details of the issuing laboratory, complete patient demographics and specimen collec-
tion details, also apply to electronic reports. Again, only standard, recognized abbreviations
should be used, and any possibly ambiguous language such as ‘text speak’ must be avoided.
The NHS and the Department of Health have standardized acronyms for medical and other
frequently applied terminology. Previous references to use of LIMS in respect to coded com-
ments and standardization apply, as in the same way a paper report may be the only user–
laboratory interface, for some groups of users, the electronic copy of the report may be the
only interface. The use of coded comments through a LIMS in the laboratory makes for much
easier standardization.
Email is a popular method of communication. It is fast, easy to use, and an excellent mecha-
nism for cascading information. However, there is no excuse for not writing emails in correct
English, with complete spelling and no shorthand. Emails should be proofread before sending,
as the standard of the English used may be the only basis a service user has to make a judge-
ment on the overall educational standards of a member of the laboratory staff. While elec-
tronic data is a boon in today’s busy society, it also has serious consequences when things go
awry and, like all methods of communication, they are not 100% reliable. All systems can fail
and data be lost or fall into the wrong hands. Thus, steps must always be taken to ensure that
procedures are in place to support the protection and recovery of electronic data.
All electronic data must be held on a back-up system. Anyone who has lost personal work
due to the failure of a personal computer has experienced the frustration and extra work
that could so easily have been prevented by saving the data on a secondary data storage sys-
tem! Whereas CD-ROMs and memory sticks may be useful ways for students to protect their
personal educational material, this method of storage is unsuitable for patient information.
3.4 DATA PROTECTION 55
Indeed, government data was lost in two high-profile cases involving the Ministry of Defence,
who admitted losing personal details of service employees, and the Child Benefit offices, which
lost bank details of some claimants. Such cases involving the loss of personal information
demonstrate the need for the secure storage of sensitive data pertaining to the general public.
Imagine the scale of such a system failure if the data, such as that stored on a LIMS, or on a
hospital-wide patient information system, was lost due to insufficient back-up mechanisms
or stolen due to poor security. All large systems within the NHS have a back-up mechanism
that implements some type of housekeeping and back-up of all accessed files at regular time
intervals. This frequently goes unnoticed by the majority of staff, but the usual practice is that
a named person has overall responsibility for the security and protection of laboratory data
and sets protocols that must be followed to prevent loss of data or even to prevent data falling
into the wrong hands. The NHS technology systems have carefully controlled access, with all
data being password protected and tracked with audit trails. It is essential that all staff follow all
local and Trust-wide policies to ensure the protection and security of electronic data.
The storage and transfer of electronic data is covered by the Data Protection and the Freedom
of Information (FOI) Acts, and the consequences of both pieces of legislation require their
examination in detail.
3.4 Data protection

The Data Protection Act (DPA) 1998 governs the use of personal information by businesses
and other organizations. It came into law in March 2000, and regulates how a business or
organization may store and use personal details about individuals, including their names and
addresses, bank details, medical records, and opinions they may have expressed. A specific
problem arising from the Act is that it only applies to living individuals. Following their death,
any data stored about them need no longer be protected in the same way as when they were
living. This is seen as a loophole in the existing legislation that is particularly pertinent to
health records.
The DPA 1998 applies eight principles for the purpose of protection of personal information.
You can see these listed in Table 3.2.
TABLE 3.2 Summary of principles listed in the Data Protection Act (DPA)
Processed fairly and lawfully
Processed for one or more specified and lawful purpose, and not further processed in any
way that is incompatible with the original purpose
Adequate, relevant, and not excessive
Accurate and, where necessary, kept up to date
Kept for no longer than is necessary for the purpose for which it is being used
Processed in line with the rights of individuals
Kept secure with the appropriate technical and organizational measures taken to protect the
information
Not transferred outside the European Economic Area (The European Union member states,
plus Norway, Iceland, and Liechtenstein), unless there is adequate protection for the personal
information being transferred
These principles ensure that personal information may only be held and used for the purpose
for which it was originally intended and cannot be used for anything else subsequently. It also
gives individuals the right to view the data that is held about themselves. Thus, for example,
the DPA gave individuals the following legal rights:
• The right to a copy of the data
• The right to prevent processing likely to cause damage or distress
• The right to prevent processing for the purpose of direct marketing
• The right to have inaccurate data rectified, blocked, erased, or destroyed
• Rights in relation to automated decision making
In addition, the Act classifies some personal information as sensitive, about which there are
even stricter rules. Sensitive information includes:
• Racial or ethnic origin
• Political opinions
• Religious or similar beliefs
• Trade union membership
• Physical or mental health or conditions
• Sexual orientation
• Offences or alleged offences committed
• Proceedings related to these offences or alleged offences
An organization is only allowed to use sensitive information where they are able to conform with
a narrow set of conditions to ensure that such information is only used where the organization
has an essential need to use it, or that the individual has given explicit consent for its use.
It is immediately clear that records held in a clinical laboratory, either in paper or electronic
forms, and also any paper reports issued by the laboratory, are considered sensitive personal
information for the purpose of the DPA and must be treated accordingly. Considering the scale
of sensitive information held within a hospital, and indeed the NHS, it is a significant task, and
to that end, in 1997, a report was commissioned by the Chief Medical Officer of England, and
a committee established under the chair of Dame Fiona Caldicott. A report followed, referred
to as the Caldicott Report, highlighting six key principles and making 16 specific recommenda-
tions to be applied to all data held by the NHS.
The six key principles as set out in the Caldicott Report are:
1. Justify the purpose

2. Do not use patient-identifiable information unless it is absolutely necessary
3. Use the minimum necessary patient-identifiable information
4. Access to patient-identifiable information should be on a strict need to know basis
5. Everyone with access to patient-identifiable information must be aware of their
responsibilities
6. Understand and comply with the law
These principles mean that every single use or transfer of patient-identifiable information that
moves within or outside of an organization, such as the NHS, should be necessary and its
purpose defined. All transfers of such information must be regularly scrutinized and reviewed
by an appropriate person to establish that the continued transfer really is necessary. All NHS
trusts have a named Caldicott Guardian, who is usually a senior member of the medical staff
with the overall responsibility for ensuring compliance within the trust.
Patient-identifiable information that is not strictly necessary should be excluded unless it would
be dangerous to remove that data. Patients should only be identified where it is essential to the
successful movement of that data and, even then, the minimum amount of identification should
be used. Current legislation often dictates the amount and type of data needed. Once data has
been collected and stored, that data must only be accessed by those people to whom the data is
absolutely necessary and those with access must be sure of their own responsibilities to protect
the data and how they themselves fit into the legal requirements of the Caldicott principles.
So, having described the legislation, we will now consider how the information held in pathol-
ogy departments is held in relation to the Caldicott principles. First, justify the purpose. Clinical
requests must be made on the basis of providing information for diagnosis and treatment and
not for research purposes unless consent has been specifically sought. It is clearly paramount
that the correct reports be identified with the correct patient, so in terms of using little or no
identifying information, this must always be balanced against patient safety. It is generally
accepted that patients be identified by full name, date of birth, and a unique number (hospital
allocated or NHS number, Box 3.2) as minimum information for the purpose of pathology
records. Problems do arise when using data transfer mechanisms, such as fax machines or
electronic links, where there is a danger that personalized data may not end up in the hands
of the person for whom it was intended. There are circumstances where it is necessary to
code or encrypt information so that the true identity can only be recognized by the intended
recipient. An exception to this general rule, where sets of results need not have a true identity,
are data arising from clinical trials or research programmes; in these cases, anonymous reports
are acceptable. Access to information on a need to know basis has already been discussed in
this chapter in relation to use of the telephone as a method of communication (Section 3.3).
All healthcare professionals and laboratory support workers have policies and protocols that
outline their specific responsibilities with regard to patient information.
BOX 3.2 Use of the NHS number
The NHS number is now the identifier of choice across different NHS organizations. It
is a 10 digit number that is unique to every individual who is registered with the NHS in
England and Wales. The NHS is now required to use this number on all formal patient
documentation. The NHS number is issued to all babies born in England and Wales,
and will be given to anyone not born in those countries that is eligible to register with a
general practitioner and use NHS facilities.
SELF-CHECK 3.3
Why is the NHS number now the recognized means of identifying an individual?
Freedom of Information Act

The Freedom of Information Act 2000 (FOI) came into force in January 2005. The Act gives
a general right of access to all types of recorded information held by public authorities and
places obligations on them to disclose information (subject to a range of exemptions); indeed,
the information applied for may be retrospective in nature. The FOI gives applicants two
related rights:
1. The right to be told whether the information exists

2. The right to receive the information within 20 working days, where possible in the manner
requested
Thus, a public sector organization has a statutory duty to implement the Act and regulates how
an organization, such as the NHS, must respond to a request for information that it may have
stored. The Act applies to everyone within the organization of a Public Authority (inclusive of
all aspects of the NHS) and has a huge impact on the public sector. The purpose of the FOI Act
is to contribute to breaking down barriers within the NHS and help organizations learn from
each other, as well as improving on their own performance. There are specific exceptions to
the data that can be released that need to be discussed.
It does appear at first glance that the DPA and the FOI Act are at odds with one another, but
really they are complementary in nature. Under the DPA, individuals have a right to access
information about themselves, but third parties do not have rights to personal information.
Under the FOI Act, individuals are not able to access to any personal information, either about
themselves or others, but are able to access most other types of information. The main strate-
gic objectives of the combined Acts are listed, and are not specific to the NHS or the hospital
environment.
• To improve people’s knowledge and understanding of their rights and responsibilities
• Seek to encourage an increase in openness in the public sector
• Monitor the Code of Practice on Access to Government Information
• Develop a data protection policy, which properly balances personal information privacy
with the need for public and private organizations to process personal information.
Both the DPA and FOI Act are the responsibility of the Lord Chancellor’s Department at
Government level, namely the Information Commissioner.
Informed consent
The 2000 NHS Plan pledged that proper consent would be sought from all NHS patients and
research subjects. The plan promised a review of consent procedures and the establishment of
good practice in seeking consent throughout the NHS. The recommendations are that before
a doctor or other healthcare professional treats a patient, they need to obtain their consent.
The consent may be verbal, but it is better in writing, and outlines the key points and deci-
sions of the procedure. A patient has the right to withdraw his or her consent at any time. The
healthcare professional seeking consent has an obligation to ensure that the patient knows
enough to be able to make an informed decision, and has had the opportunity to ask ques-
tions and consider alternative treatments and procedures. Patients must be told in advance if
their treatment is likely to involve the removal of part of their body, and also if invasive sam-
pling, such as taking blood samples is likely to be part of their care plan. Sample taking during
operations may routinely be used for teaching, research, or public health monitoring and, if
this is the case, the patient must be made aware of the possible use of such samples and their
consent obtained. If prior consent has been provided in writing, particularly in the case of the
retention and storage of histological samples, there is no longer ambiguity on the outcome
of a given series of events. Likewise, regarding consent to taking blood samples. It is readily
assumed that if a patient has a request form and has allowed the phlebotomist to take a blood
sample, they have given consent for those tests marked on the form, but to no others. It is no
longer considered appropriate to use excess samples for research purposes or for additional
tests to be added without discussion with the requesting clinician and in some circumstances,
with the patient.
Human Tissue Acts

The Human Tissue Act (HTA) 2004 (England, Wales, and Northern Ireland) or HTA and the
Human Tissue (Scotland) Act 2006 have replaced a variety of earlier legislation, including The
HTA 1961, the Anatomy Act 1984, and the Human Organ Transplant Act 1989. All consent
for the use of pathological samples for prolonged storage and/or research activities must be
obtained in writing, and the paperwork be sufficiently detailed to establish that the patient
is fully aware of the nature of the consent sought. The consent must be given voluntarily, be
accompanied by sufficient information, be made by a legally competent individual, and be
given with no form of coercion. Research must have the dignity, rights, safety, and well-being
of the participant as the primary considerations. All studies must have appropriate written,
informed consent, with clear arrangements about how that consent should be obtained. It is
for the protection of the researcher that the consent record shows that the process has been
two way, with the participant having the opportunity to ask questions and receive answers
and, even better, if a friend or relative of the participant was also present at the interview and
allowed to participate in the questioning. It needs to be understood that informed consent is
a process and not simply a signature on a form, and that the informed consent form indicates
that there has been adequate communication between the healthcare professional and the
participant or patient. It is increasingly common that, rather than signing consent to a list of
inclusive events, patients are asked to sign a general disclaimer; thus, specific consent is not
required at a later date.
The HTA was passed by Parliament in the wake of public response to the activities at the Bristol
Royal Infirmary and Alder Hey Hospital, which are discussed further in Chapter 19, where
specific consent to remove, store, and carry out research on tissues retrieved at post-mortem Cross reference
from children having undergone unsuccessful major surgery had not been requested. It was Chapter 19 ‘Quality assurance
clear at that time, that many of the public had little grasp of post-mortem procedures, nor the and management’.
fairly common practices of storing tissues for research and teaching purposes. The HTA has
made the processes of post-mortem and retrieval of tissue much more transparent. Although
Scotland has its own Act, the content of both Acts are expressions of the same principles, such
that the HTA Act:
• Regulates removal, storage, and use of human tissues, which are defined as material that
has come from a human body and consists of, or includes, human cells. Now the HT Act is in
force, it is unlawful to carry out these licensable activities without an appropriate licence
• Created a new offence of deoxyribose nucleic acid (DNA) ‘theft’, which is having human
tissue with the intention of analysing its DNA without the consent of the person from
whom the tissue came, from 1 September 2006
• Made it lawful to take minimum steps to preserve the organs of a deceased person for
use in transplantation, while steps are taken to determine the wishes of the deceased or,
in the absence of their known wishes, obtain consent from someone in an appropriate
relationship
• Give specified museums in England discretionary power to move human remains out of
their collections, if the remains are reasonably believed to be those of a person who died
less than 1000 years ago
These acts also have a list of enforceable offences, with penalties ranging from fines to impris-
onment or exceptionally both. These offences include:
• Removing, storing, or using human tissue for Scheduled Purposes without appropriate
consent
• Storing or using human tissue donated for a scheduled purpose for something else
• Trafficking human tissue for transplantation purposes
• Carrying out licensable activities without holding a licence from the HTA (with lower penal-
ties for related lesser offences such as failing to produce records or obstructing the HTA in
carrying out its legal responsibilities)
• Storing or using human tissue, including hair, nail, and gametes with the intention of its
DNA being analysed without the consent of the person from whom the tissue came or of
those close to them if they have died
The scheduled purposes mentioned in the above list is a general term used to cover the
description given to the patient or relative of the procedures and purposes for which that
tissue was collected. Scheduled purposes differ between tissue samples and also between
patients; they include whether the tissue is to be used only for diagnostic purposes or also
for research, whether the tissue can be used by a single institution or be shared, and if it can
be stored after use. If is it to be stored, then the scheduled purpose will include details of the
storage including its site and duration.
Clearly, the HTA and the need for informed consent go hand-in-hand. The written com-
munication between healthcare professional and patient/participant acts as a legal docu-
ment to provide the necessary proof that informed consent has been properly sought and
agreed with regard to blood, fluid, and tissue samples requiring investigation, processing, and
storage within laboratory medicine. This is particularly important in histopathology when
acquired tissue from surgery or post-mortem (Figure 3.4) may be further used for teaching or
FIGURE 3.4
Interior of a modern mortuary. The removal of any tissues must comply with the Human
Tissue Act. Photograph courtesy of the London Borough of Haringey, UK.
3.5 RECORD KEEPING 61
research purposes. Tissue must only be used and stored in the manner for which prior consent
has been obtained.
3.5 Record keeping

While record keeping has been mentioned briefly as a method of communication within the
laboratory (Section 3.1) and is also an HPC standard, it deserves consideration in its own right
within the remit of the Data Protection and the FOI Acts. It has already been mentioned that
communication within the laboratory also includes completion of documents such as quality
control charts, reagent log books, and troubleshooting sheets in the case of an instrument
failure. The primary purpose of this type of documentation is so all laboratory staff can assess
situations at a glance, whether certain basic procedures have been followed, whether a par-
ticular instrument is functioning correctly, or whether the availability of certain stock is below
a certain level without having to find and question a specific member of staff. Record keeping
in the laboratory is not only limited to practical procedures carried out on benches, but also
covers complete patient and staff records.
Key Points
When completing patient records, clearly it is essential that a report correctly identifies
the patient but it is also necessary that an historical record be kept of all the laboratory
investigations pertaining to a patient.
When accessing a new set of results on a LIMS for authorization, there is frequently a so-called
delta check performed, where the new result is compared with the previous existing record.
If there is a significant change, the result may not be automatically passed for authorization
and release directly by the LIMS, but may be held in a queue and require manual validation or
authorization by a biomedical scientist who can investigate further (see Case study 3.2).
Another area of laboratory medicine where it is essential that full historical records be main-
tained is within the discipline of blood transfusion. If a patient develops an antibody to a
particular red cell antigen, there is a possibility that the titre of that antibody may change with
time. Examples of these are the antibodies of the Kidd system, such as anti-Jka. Not all anti-
bodies to erythrocyte antigens remain detectable in the serum at a consistent titre and some
may decrease with time. A patient may have presented several years previously with a positive
CASE STUDY 3.2 Identifying samples

is essential
A patient with a blood haemoglobin of 140 g dm−3 (reference range 140–180) 24 hours
later has a haemoglobin concentration of only 75 g dm−3, and therefore requires a blood
transfusion. If the patient has undergone major surgery, such as requiring a prolonged
period of time on a cardiac bypass machine, or has just had a massive gastrointestinal
bleed, the discrepancy between the two results is both explained and appropriate. If
there has been no such clinical event to explain the change, then it is unlikely that the
samples have originated from the same patient and there has been an error in correctly
identifying the sample.
antibody screen and such an antibody identified. When returning in the present day for major
surgery and possible transfusion, the antibody screen may be negative, as titres are so low as
Cross reference
to be undetected by routine screening methods. If that patient required a blood transfusion, it
would still be necessary that Jka negative blood be used since the introduction of the antigen
immunology in Chapter 15
‘Immunological techniques’. would promote a massive response in terms of antibody production, and the patient would be
likely to suffer an incompatible transfusion reaction that could possibly be fatal.
Staff records
Staff records must also be kept by departmental managers. Records must exist for human
resource purposes, such as sickness and absence rates, the availability of staff on different
parts of shift systems, names of staff on professional registers, and details of mandatory train-
ing, competence assessment, and continuous personal development, that may be needed to
fulfil professional responsibilities.
External bodies
Record keeping in the clinical laboratory also has to satisfy the many external regulatory
bodies that have a bearing on current laboratory practices. There are many legal requirements
to be met, as well as guidelines for good practice. Such bodies include Clinical Pathology
Accreditation (UK) Ltd (CPA), Medicines and Healthcare products Regulatory Agency (MHRA),
the Health and Safety Executive (HSE), and the NHS Cervical Screening Programme. We will
examine briefly the role of each in this respect.
Clinical Pathology Accreditation (UK) Ltd

The CPA is a peer reviewed organization that provides an external review of clinical pathology
services and external quality assessment schemes. Currently, CPA accreditation is desirable,
rather than mandatory, but this situation may change in the future. CPA assessors visit labora-
tories and examine services to confirm that defined standards of practice are being met in all
areas of laboratory medicine. Thus, all laboratory records must comply with the most up-to-
date and relevant of the CPA Standards to meet the requirements of the inspectors.
Medicines and Healthcare products Regulatory Agency

The MHRA is an agency of the Department of Health and thus a government organization, that
was established to protect the health and safety of the public in relation to the dispensation
of medicines and use of medical devices. The activities of the MHRA are particularly relevant
in the field of blood transfusion, as blood products are classed as pharmaceuticals and admin-
istered to a patient following suitable prescription by a qualified person. The MHRA assesses
against several European Union directives (the majority of which are now part of UK law) that
have set standards of practice in blood transfusion, and which apply to blood establishments
and hospital blood banks. Inspectors for the MHRA require evidence that these directives are
being met for quality systems of working, the use of equipment and consumables, and that
there are suitable traceability and record-keeping requirements for blood banks and facilities
that may receive blood products for transfusion.
Health and Safety Executive

The HSE protects people against risks to health or safety arising out of work activities. Its
role is discussed more fully in the following chapter. The HSE is a government body, which
regulates all employers to ensure that all aspects of health and safety law are met in full.
FURTHER READING 63
The HSE inspectors have a right to visit workplaces, and inspect practices and records to ensure
Cross reference
that all relevant UK and European Union legislation is being adhered to in full. The HSE has
Chapter 4 ‘Health and safety’.
the power, in extreme circumstances, to close down and prosecute any employer found to be
in breach of these regulations, particularly if previous warnings have been issued. All records
concerning health and safety, which constitute UK and European Union law, must be up to
date and available for health and safety inspectors to access at any time.
SUMMARY
■ Effective communication both within a clinical laboratory, and between its staff and a
variety of external people and organizations is necessary. This is because biomedical sci-
entists work as part of a laboratory team, must follow local policy and procedures and
comply with legislation, and are involved in a process that provides essential information
contributing to patient care
■ Biomedical scientists are healthcare professionals, and must never act in a manner that
causes offence to others or to bring the biomedical science profession into disrepute by
inappropriate communications of any means
■ Biomedical scientists and other laboratory staff are required to work in structured teams,
following set procedures and protocols, and must co-operate and communicate appro-
priately with others within the laboratory environment
■ Biomedical scientists must communicate in a professional manner and at an appropriate

level when dealing with the public, patients, and the wider healthcare groups of profes-
sionals and support workers
■ Biomedical scientists may communicate by non-verbal or verbal means, and verbal com-
munication may be written or spoken, and indirect communication may be the only way
that a biomedical scientist is judged by the wider healthcare community
■ Biomedical scientists must conform to current legislation, such as the Data Protection,
Freedom of Information, and the Human Tissue Acts, and be aware of the consequences
of their actions if inappropriate records are kept and audit trails are not maintained
■ Biomedical scientists must ensure that all work carried out within laboratory medicine has
appropriate consent and that confidentiality is always maintained
FURTHER READING
● Divan A. Communication Skills for the Biosciences. Oxford University Press, Oxford,
2009.
Useful guide on how to effectively communicate scientific knowledge.
● Pitt SJ, Cunningham JM. An Introduction to Biomedical Science in Professional and

Clinical Practice. Wiley-Blackwell, Chichester, 2009.
Chapter 3. ‘Communication for biomedical scientists’ is worth browsing through.
64 3 COMMUNICATIONS IN L ABORATORY MEDICINE
● Standards of Conduct, Performance and Ethics. HPC, London, 2008.
● Standards of Proficiency – Biomedical Scientists. HPC, London, 2007.
Useful Websites
■ www.ibms.org
■ www.hpc-uk.org
■ www.cpa-uk.co.uk
■ www.mhra.gov.uk
■ www.hse.gov.uk
■ www.hta.gov.uk
■ www.businesslinks.gov.uk
■ www.nhsprofessionals.nhs.uk
QUESTIONS
1. Guidelines issued by the Institute of Biomedical Science concerning the communication
of results by telephone:
(a) Are a legal requirement

(b) Can be adapted for individual situations
(c) Are an example of best practice
(d) Must be adhered to by all members of the Institute of Biomedical Science
(e) Are only of relevance to NHS employees
2. Under the Data Protection Act, sensitive information is defined as:
(a) Information that relates to the age of an individual

(b) Data that cannot be sold on to another party
(c) Information that can be released to a third party under the Freedom of Information
Act
(d) Information that can only be held with explicit consent
(e) Information that must be removed from a database after 5 years
3. The key principles of the Caldicott report do not include:
(a) The appointment of a Caldicott Guardian

(b) Information held outside of the NHS
(c) Instructions stating which personnel should have access to medical records held by
the NHS
(d) Instructions informing NHS staff of their legal obligations under the Data Protection
Act
(e) Instructions that all staff must comply with the law
4. Indicate which of the following statements are TRUE:
(a) The NHS number is given to all babies born in the UK

(b) Telephone calls to the laboratory requesting tests to be added to a previous sample
must be documented
QUESTIONS
3.5 RECORD KEEPING 65
(c) Acronyms for laboratory tests may be used freely, providing the laboratory responds
with a published list that explains what they mean
(d) The Data Protection Act applies only to those members of NHS staff who are
named on a professional register
(e) Electronic data on a screen must be viewed as a written method of communication
5. The text refers to point of care blood glucose concentrations at a diabetic clinic and
international normalized ratio (INR) results at a coagulation clinic as examples where it
may be appropriate to give results to patients. List other occasions where results may be
given directly to the patient.
6. With reference to stored electronic data, what are the potential consequences for an
individual if medical data are lost?
4
Health and safety
Alison Taylor
Learning objectives
■ Identify hazards that represent a risk to yourself or to the safety of others

■ List your statutory duties under the Health and Safety at Work Act
■ Discuss the regulations that drive health and safety practices in the workplace
■ Conduct a basic risk assessment and be able to outline more specialized assessments
■ Use personal protective equipment safely and effectively, and be aware of their limitations
and maintenance requirements
Introduction
Occupational health and safety are any system or procedure established to ensure the health,
safety, and welfare of every person coming into a work place. Health is preventing illness
occurring to the body or mind, and safety is the prevention of physical injury. The term wel-
fare describes facilities, such as sanitation, heating, lighting, and changing rooms, which are
not direct causes of ill health or injury, although deficiencies in these areas could and almost
certainly would, contribute to poor health and safety.
The law defines standards of health and safety (Health and Safety at Work Act, Section 4.3)
and, once identified any hazards or deficiencies must be corrected. In health and safety
practice, a key way to identify deficiencies is by workplace inspection, which is a special-
ized audit, and by performing an assessment of the risk (Section 4.4). Risks are the combined
likelihood of a hazardous event occurring, and the severity of injury or ill health that it could
cause. Any deficiencies or hazards identified by either route must have corrective actions or
control measures implemented to ensure they do not cause ill health or injury. The effective-
ness of these controls needs to be assessed when the inspections and/or risk assessments are
repeated or reviewed. The regulatory body for health and safely legislation is the Health and
Safety Executive (HSE), which carries out these types of inspections and publishes guidance
to help employers and employees comply with the law (Figure 4.1a). The HSE also produce
reference materials and institute advertising campaigns to raise awareness of health and safety
risks. Within the UK, the Institute of Occupational Safety and Health (IOSH) is the recognized
4.1 HA ZARDS 67
(a) (b)
FIGURE 4.1
Logos of (a) Health and Safety Executive
(HSE) and (b) Institute of Occupational
Safety and Health (IOSH). Logos reproduced
with kind permission from the respective
organizations.
body for health and safety professionals, providing training, guidance, and information for
professionals in this field (Figure 4.1b).
This chapter will examine the legal requirements and regulations governing occupational
health and safety, including the methods used to assess and control risks. In combination, these
measures attempt to ensure the health and safety of everybody in a workplace. Naturally, how-
ever, we will focus on the risks and controls particular to biomedical science laboratories.
4.1 Hazards
A hazard is any substance, activity, or process that may cause harm. On a daily basis, you may
encounter many potential hazards (for example, crossing a busy road). However, by using sub-
conscious risk assessments and applying actions to minimize the dangers of the hazard, you
will (we hope!) avoid injury and ill heath. Unfortunately, in working environments, hazards,
especially those found in clinical laboratories, may not be easily identifiable. Thus, it is dif-
ficult to take actions to minimize their effects. Training, labelling of substances, using standard
operating procedures, and acting on all the relevant information, such as risk assessments, will
assist in identifying hazards related to each task undertaken.
In this section we will identify common laboratory hazards and discuss the process of con-
ducting formal risk assessments to assess these hazards, the risks they pose, and the controls
required to reduce or eliminate them.
Hazards may be classified into six main types:
1. Biological
2. Physical, mechanical, or equipment
3. Chemical
4. Electrical
5. Fire
6. Environmental
Biological hazards
Biological hazards take five main forms:
• Bacteria
• Viruses
68 4 HEALTH AND SAFET Y
• Fungi
• Moulds
• Prions.
Cross reference Bacteria are single-celled prokaryotic microorganisms that can live independently or depend-
You can read more about the ent on host organisms. Examples of bacteria include the causative organisms of tuberculosis
roles of biomedical scientists in and legionella. Viruses are nucleoprotein complexes, which are about 200 times smaller than
Chapter 1 ‘Biomedical science bacterial cells. All viruses are obligate parasites in that they can only grow or replicate within a
and biomedical scientists’.
host cell. Examples of viruses include agents that cause influenza and acquired immune defi-
ciency syndrome (AIDS). Fungi are single-celled or multicellular eukaryotic organisms, which
have cell walls rich in chitin. They reproduce by producing spores. Fungi cause skin conditions
such as athlete’s foot, some forms of eczema, or they may cause allergic reactions. Moulds are
microscopic fungi, which can have similar effects on health to the above, but can also lead to
respiratory sensitization because their small size allows them to evade filtration by the respira-
tory system. Thus, they can cause or contribute to conditions such as asthma. Prions are pro-
tein molecules that adopt toxic conformations and cause infectious diseases such as scrapie,
bovine spongiform encephalopathy (BSE), and Creutzfeldt–Jakob disease (CJD).
The Advisory Committee on Dangerous Pathogens (ACDP) classifies all biological hazards
into one of four categories or groups (1 to 4). These are published by the Health and Safety
Commission (HSC). The group assigned to an agent defines the level of containment required
to control the risks associated with it, which are defined in Control of Substances Hazardous
to Health (COSHH, Section 4.5) regulations.
Group 1 hazards are biological agents that are unlikely to cause human disease. In contrast,
hazard group 2 are those agents that can cause human diseases. However, effective treat-
ments and/or prophylaxes are available to combat these agents and so this group is unlikely
to spread in the community. Biological agents of hazard group 3 can cause severe human
disease and present a high risk to health. There is a risk of the disease spreading in the com-
munity but there are usually effective prophylaxis or treatments available. Hazard group 4
agents can cause severe human disease and present a serious risk. There is also a likelihood of
the disease spreading in the community because usually effective prophylaxis or treatments
are not available.
Physical, mechanical, and equipment hazards

Physical hazards or those associated with mechanical devices or other equipment include:
entanglement, entrapment, being struck by or striking an object (impact injuries), abrasion,
cutting, and shearing. These hazards most often cause injury but in some cases lead to ill
health. Slips/trips/falls also come into this category and are common causes of occupational
injuries. Health problems associated with extensive computer or display screen equipment
(DSE) work or repetitive muscular actions are included in this group.
Chemical hazards
Chemical hazards can be present in a number of physical forms; these include solids, dusts,
gases, vapours, mists, fumes, and liquids. The severity of ill health effects they cause can vary
considerably.
Chemicals agents can enter the body in a variety of ways, dependent on their physical form.
For example, an irritant chemical in solid pellet form is unlikely to enter the body but on
contact with the skin, can lead to relatively minor health effects. Hence, its effects are easily
4.1 HA ZARDS 69
controlled by wearing safety gloves (Section 4.7). However, in powder form the same chemi-
cal could enter the body by inhalation and have significant effects, such as shortness of breath
and asthma. The control of this greater hazard may possibly require the use of a ventilation
hood. Dusts are categorized separately as they have significant specific risks dependent on the
size of the particle. A flammable substance in the form of a fine dust will mix easily with air and
can become explosive. Fine dusts, otherwise known as respirable dusts, present a significant
hazard because of the ease with which they are inhaled.
Liquids exist in different forms depending on the ambient temperature and their boiling Cross reference
points. Liquids are less predictable and more difficult to handle than solids. Splashes and spills Safety issues concerning
increase the chance of contact with the skin or entry into the body by ingestion or through radiation are discussed in
the eyes. Gases and vapours are produced by heating liquids or when working in higher tem- Chapter 10 ‘Radioactivity and
radiation’.
peratures. They can enter the body through the skin, gastrointestinal tract, and eyes, or by
inhalation. Spraying processes can form mists, even at low temperatures, and this increases
the chances of the liquid causing ill health. Fumes are mists or vapours containing very small
metallic particles. In a laboratory, a fume could be formed when the bulb in a mercury micro-
scope ‘blows’.
The different types of chemical hazards and their classification are discussed under the Control
of Substances Hazardous to Health (COSHH) in Section 4.5. Some chemicals are also radiation
hazards and so are classified differently; their use is governed by the Radiation Protection Act.
Electrical hazards
Any electrical equipment, either fixed position or portable, has the potential to cause an elec-
tric shock. This can be particularly so in a clinical laboratory when electricity and water are
often used in close proximity. They can pose electrical hazards, with consequences ranging
from minor to serious injury or even death. Portable appliance testing (PAT) must be carried
out regularly to check the safety of the instrument, but there must also be a more frequent
inspection of plugs, cables, and casing. Any potential hazards arising from the use of electrical
equipment in close proximity to chemicals need assessing and controlling.
Fire hazards
Fires can only start if all parts of the fire triangle, oxygen, fuel, and a heat source, are present
(Section 4.6). Therefore, flammable substances, including liquids and gases, or substances or
equipment giving off heat can all fall into this group of hazards.
Environmental hazards
Environmental hazards include loud noises, vibrations, light, excessive humidity, and/or tem-
peratures, and a lack of ventilation. Sometimes these are the most difficult hazards to identify,
as they are often experienced daily and so become accepted or tolerated. These hazards often
cause welfare-related issues. In extreme situations, or when particularly vulnerable people are
affected, they can result in ill health.
SELF-CHECK 4.1
List at least two examples of each of the major types of hazard discussed above.
4.2Routes of entry into the body by

chemical and biological agents
Chemical and biological hazards can only cause harm if they enter the body. Controlling these
hazards therefore requires knowledge of their possible routes of entry.
Airborne biological and chemical agents can gain entry into the lungs following inhalation
through the mouth or nose. Here, they can be absorbed and then transported around the
body in the bloodstream. Thus, they can potentially affect every organ in the body. The skin
prevents the absorption of many agents into the bloodstream. However, some chemicals can
enter the body this way through pores on the surface of the skin. More commonly, absorption
occurs through wounds, burns, or breaks in the skin in, for example, conditions like eczema.
A number of biological and chemical agents can enter the body through the gastrointestinal
tract following their ingestion, and potentially gain access to the bloodstream. This is not a
common route of entry into the body, but may occur if good hand hygiene and basic labo-
ratory health and safely practices are not followed. Finally, some agents can enter the body
by injection due to accidents with hypodermic needles and compressed air lines, which can
puncture the skin giving direct access into the bloodstream.
SELF-CHECK 4.2
To what types of hazards would a person collecting a venous blood sample be potentially
exposed? What are the possible routes of entry of the hazard(s) into the body?
There are many regulations governing health and safety; however, in this chapter we will focus
on the principal ones most likely to affect you while working in a biomedical laboratory within
the UK. Health and safety across Europe is governed by the European Union (EU) through
directives, which are incorporated into the laws of the member states.
Statutory framework for health

4.3
and safety
Prior to 1974, health and safety legislation in the UK was only targeted at specific industries,
leaving many workers unprotected. Other people, such as contractors and visitors entering
a work place were simply not protected in law. The introduction of new regulations was too
slow to reflect changing practices leading to a system that was reactive, rather than proactive.
However, the legislative framework changed with the Health and Safety at Work Act.
Health and Safety at Work Act 1974

The Health and Safety at Work Act (HASAWA) resulted from a review of occupational health
and safety in 1970 by Lord Robens. The HASAWA is an enabling act, which means the Secretary
of State can pass new regulations without the lengthy process of passing them into law by Acts
of Parliament. Thus, the process of developing health and safety legislation became more
proactive.
The HASAWA established the HSC whose duties are to draw up new regulations and to enforce
them through the actions of the HSE. In April 2008, the two bodies merged under the name, HSE.
4.3 STATUTORY FR AMEWORK FOR HEALTH AND SAFET Y 71
The HSE is responsible for issuing Approved Codes of Practice (ACOP) for most regula-
tions. The role of ACOPs is to explain the regulations and the detailed requirements necessary
to comply with them. The HSE produces two types of health and safety guidance (HSG)
documents, legal and best practice. The legal guidance series of booklets covers the technical
aspects of the regulations and usually includes the regulations and ACOP. Best practice guid-
ance is published in the HSG series for specific areas; an example being the Decontamination
of Equipment Prior to Inspection, Service and Repair HSG(93)26. Both ACPOs and HSGs are
good sources of information and are readily available on the HSC website, which is listed at
the end of the chapter.
The HSE has teams of inspectors to cover all types of workplaces, including hospitals and
laboratories, assisted by local authority inspectors covering premises such as hotels, shops,
and restaurants. They can inspect at any time and have similar powers to the police in being
able to collect evidence, and take and retain samples and photographs. One of two levels
of enforcement notification is issued if deficiencies or dangerous practices are identified. An
improvement notice follows contraventions of a regulation, together with a set date for its
resolution. Alternatively, a prohibition notice halts the activity until a satisfactory resolution
of the problem is established.
Should the HSE prosecute a health and safety offence successfully, the penalties imposed
range from fines of up to £5000 for employees and £20,000 for employers, if the case is tried
in a magistrates’ court. Failure to comply with an enforcement notice can result in imprison-
ment for up to 6 months. In the crown courts, fines are unlimited with imprisonment for up to
2 years for the same offence.
SELF-CHECK 4.3
List the role(s) of the HSC.
Duties of employers
The HASAWA places a duty on employers ‘to ensure, so far as reasonably practicable,
the health, safety and welfare of all employees’. It also details what that may entail; such
as providing a safe place in which to work and ensuring all procedures associated with
work are safe to perform. In addition, the act sets out the need for set policies with asso-
ciated processes for organizing arrangements such as risk assessments and consultation
with employees. The act protects visitors to a workplace, outside contractors, and people
living near it or passers-by, by placing a duty of care on the employers to protect all those
affected by its activities. Duties are also placed on the employer to ensure all materials,
including equipment and substances, are without risk to health. The Act also affects the
practice of suppliers by placing on them a duty to design, manufacture, import, and sup-
ply equipment or substances that are, again, as far as is reasonably practicable, without
risk to health.
Duties of employees
The HASAWA places two main duties on employees:
1. To take reasonable care for the health and safety of yourself and others affected by your
acts or omissions
2. As an employee, you must co-operate with your employer to enable them to fulfil their
legal obligations
A further section, often known as the ‘horseplay section’, impresses a duty on everyone not
to interfere or misuse safety equipment. The act also states that employers must provide staff
with all personal protective equipment (PPE, Section 4.7) required to fulfil their work duties
free of charge.
The ‘six pack’

The UK is part of the EU and European law, passed by the European Commission, is therefore
an integral part of our legal system. The resulting uniform approach to health and safety in the
workplace across the EU is designed in part to remove barriers to trade between its member
states. The first effect of the EU on UK health and safety law occurred in 1992, when six sets of
regulations were introduced:
1. Management of Health and Safety at Work Regulations

2. Workplace (Health, Safety and Welfare) Regulations
3. Health and Safety (Display Screen Equipment) Regulations
4. Personal Protective Equipment at Work Regulations
5. Manual Handling Operations
6. Provision and Use of Work Equipment Regulations
These regulations are contained in a set of documents familiarly called the ‘six pack’, which you
can see in Figure 4.2.
FIGURE 4.2
Documents contained in the ‘six pack’; the six sets of regulations that originated in the
European Union and which became part of United Kingdom health and safety legislation in
1992. © Crown copyright.
Management of Health and Safety at Work Regulations

The Management of Health and Safety at Work Regulations (known colloquially as the ‘man-
agement regs’) were updated in 1999. They provide the framework for the risk assessment
process that we will discuss in the following section. None of these regulations contradicts
those of the HASAWA but, in general, expands on them and redefines some of the duties
of both employer and employee. Thus, the employer must undertake suitable and sufficient
risk assessment, and establish health and safety arrangements that include planning, control,
monitoring, and review of all work-associated procedures. To achieve effective health and
safety management, the employer should use competent specialists to advise them on how to
comply with their legal duties.
Employers must ensure that all health and safety information, particularly findings of risk
assessments are communicated to all employees. They must also provide specific health and
safety training. Employers must put in place emergency procedures and ensure all workers are
aware of them. The employer must also co-operate on health and safety matters with other
employers sharing a workplace.
With regard to employees, Management of Health and Safety at Work Regulations require
that they follow their training and instructions whenever they use equipment or handle sub-
stances. They must also report anything that may be dangerous, and any problems with health
and safety equipment or arrangements to their manager or health and safety officer.
SELF-CHECK 4.4
Give an appropriate example of something that has to be reported to a manager under the
duties imposed on employees by the Management of Health and Safety at Work Regulations.
Workplace (Health, Safety and Welfare) Regulations

The Workplace (Health, Safety and Welfare) Regulations, known as the ‘welfare regs’, give the
minimum requirements for a workplace to ensure the welfare of employees. These are gen-
eralized regulations, and cover elements of the working environment such as its ventilation,
temperature, lighting, floors, furniture, wastes, and accommodation space (room dimensions).
The associated ACOPs give information that is more detailed. The regulations also cover the
use of corridors and staircases within premises that give access and egress to and from places
of work.
The safety aspects covered by the welfare regs include maintenance of equipment, windows,
doors, and traffic routes. Maintenance also includes general cleaning and the emphasis should
be on planned maintenance, which is preventative, rather than repair after breakdown. The
welfare section covers the provision of sanitary and washing facilities, drinking water, facilities
where workers can rest and eat meals, and secure space to store items such as outdoor coats.
Health and Safety (Display Screen Equipment) Regulations

The Health and Safety (Display Screen Equipment) Regulations, usually known as the ‘Display
Screen Equipment (DSE) Regs’ are designed to establish systems that prevent ill health occur-
ring when using a computer or any instrument that incorporates a display screen or visual
display unit (VDU). The regulations define a user as someone who works continuously with
DSE in excess of 1 hour each working day. They entitle a user to an eye test to be paid for by the
employer. If glasses are required specifically for DSE work, only the employer must pay for them.
The regulations prescribe suitable and sufficient risk assessment of the workstation, its sur-
rounding area, the software being used, and the user.
The regulations also set minimum specifications for the workstation, giving details on the
type of chair, dimensions of the desk or workspace, and how adjustable the DSE must be.
The DSE regulations have been written to help prevent the occurrence of ill health, such as
visual and musculoskeletal problems and stress. Therefore, taking regular short breaks, away
from the DSE when possible, is a requirement. Regular short breaks, to answer a phone or to
talk to someone in a different work area are good examples of this, but if these opportunities
do not occur, formal breaks must be taken. There is also a duty on the employer to provide
information on these aspects of the work, and to train staff to be aware of the risks and how
to avoid them.
The study of how well a person is suited to their work in combination with their workstation
is known as ergonomics. Ergonomic studies assess the physical and mental capabilities of the
individual, including their perceptions, interactions, movements that occur while doing the
job, and the surrounding work area. This includes elements of the task such as the positioning
of equipment, how easily the individual can reach it without excessive stretching that over
time might cause discomfort or injury, and avoiding stress to the individual by allowing them,
rather than the process, to control the speed of the work. Workstations must be adjustable, as
individuals have different physical and mental capabilities. The study of variations in physical
characteristics, such as height, arm, and leg length, is known as anthropometry and is a key
component to ergonomic assessments. Poor ergonomics can lead to work-related upper limb
disorders (WRULDs) such as repetitive strain injury (RSI), tenosynovitis (inflammation of the
tendons), carpal tunnel syndrome (pain in the wrist and hand), and frozen shoulder.
SELF-CHECK 4.5
Name the elements that should be considered in a DSE risk assessment?
Personal Protective Equipment at Work Regulations

The Personal Protective Equipment at Work Regulations place a duty on the employer to pro-
vide and maintain PPE when its requirement is identified by risk assessment. Personal protec-
tive equipment, by definition, only protects an individual and so potentially leaves others in
the workplace at risk. Thus, it should only be used as a last resort, although it can be combined
with other control measures such as working in a fume cupboard or other local exhaust ven-
tilation (LEV) system. As with the other six-pack regulations, there is a duty on the employer
to provide information, instruction, and training to anyone who is required to use PPE. These
must include the reasons why PPE should be used, including the risks it is designed to control,
and how the equipment should be used and maintained. The regulations place a duty on
employees to use PPE as they have been instructed and trained to do so, to maintain and store
it after use, and to report any obvious defects.
Any item of PPE that is provided to the employee must be to control the risk associated with
the process, and be appropriate for the conditions of use and the characteristics of the wearer.
For example, face masks provided to prevent inhalation of a dust may not adequately protect
someone with facial hair, which prevents them fitting correctly. The regulations place a duty
for these considerations to be formally assessed for risk and, when multiple PPE is required,
that all of the items must be compatible and not introduce additional hazards. The risk assess-
ment must be reviewed if any part of the process changes to ensure that PPE is still required
and that what is provided is adequate for the purpose.
All PPE must be regularly maintained; the level of maintenance should be proportional to the
potential risk and type of PPE. It may also be necessary to keep a written record of all mainte-
nance. For example, a respirator requires regular inspections and planned preventative main-
tenance that must be recorded, whereas safety gloves may only require a visual inspection by
the user. The use of disposable PPE can simplify this process, but all users must be aware of the
necessity to discard and replace disposable PPE items. All PPE must be stored appropriately.
SELF-CHECK 4.6
What factors should be considered when choosing PPE for a task?
Manual Handling Operations

Manual handling operations (MHO) cover those actions required to move any load by human
effort. These include pushing, pulling, lifting, putting down, and carrying loads. The regula-
tions place a duty on the employer to put in place processes that avoid the need for MHO.
When this is not possible, the MHO must have been subject to a suitable and sufficient risk
assessment. Risk assessments for MHOs must cover five areas:
1. Environment
2. Task
3. Individual
4. Load
5. Equipment used
These can be remembered using the acronym ETILE. To make an assessment of the possible
load, manufacturers are required to indicate its total weight and the heavier side of uneven
loads. Once a risk assessment has taken place, there is a duty on the employer to reduce the
risk of injury by introducing appropriate controls such as task layout, work rotation, PPE, team-
work, and training. As with all risk assessments (Section 4.4), they must be reviewed if any of
the processes change.
These MHO regulations also place duties on employees to follow the safe systems of work and
use any equipment provided in a proper manner. In practice, in a laboratory setting, MHOs
are not usually a significant part of the work, but MHO should be considered when ordering
stock to ensure materials arrive in units that can be moved, stored, and used without risk of
causing injury.
SELF-CHECK 4.7
What does the acronym, ETILE stand for?
Provision and Use of Work Equipment Regulations

The Provision and Use of Work Equipment Regulations (PUWER) are designed to ensure that
work equipment is safe to use no matter which country it originated in or how old it is. The
Lifting Operations and Lifting Equipment Regulations (LOLER) 1998 apply to equipment used
such as hoists and trolleys, for lifting or lowering loads, but the duties placed upon employers
and employees are similar to those in PUWER.
The PUWER regulations place a duty on the employer to ensure all work equipment is
suitable for its purpose and, when deciding what equipment to purchase, the health and
safety of its users is taken into consideration. Furthermore, all work equipment must be
maintained in an efficient state and working order, and kept in good repair with planned
preventative maintenance as required. All maintenance activities and inspections must be
recorded. The regulations are prescriptive about numerous features including systems such
as emergency stop controls, guarding of dangerous parts, protection against high tempera-
tures, and procedures to isolate equipment from power supplies. As with all the six-pack
regulations, the employer has a duty to provide information, instruction, and training to
anyone who uses the equipment so that they are fully aware of its operation, risks, and
emergency procedures.
4.4 Risk assessment

There is a legal requirement to carry out specific risk assessments for certain types of work
such as DSE and MHOs (Section 4.3). The general duty for an employer to conduct risk assess-
ments is imposed by the Management of Health and Safety at Work Regulations. Risk assess-
ments and other arrangements must be fully described in writing (‘documented’) when there
are more than four employees at a workplace.
To clarify the processes involved in risk assessment we should first consider some of the defini-
tions used:
• Hazards are any substance, equipment, or process with the potential to cause harm
• Risk is the likelihood that the substance, equipment, or process will cause harm
Using the terms in these specific ways makes it possible to clearly define an activity, for exam-
ple, a process could present a high hazard, but a low risk because the hazard is well controlled.
The level of risk is governed by the severity of the harm that could be caused, often referred to
as impact, and the likelihood that harm will occur.
The phrase used to describe the level of risk assessment required in many of the regulations is
suitable and sufficient. All risk assessments must identify the major risks and ignore trivial ones,
identify controls and list them in order of importance. Assessment should remain valid for a
reasonable period of time. In practice, this means that the level of detail in a risk assessment
must reflect the degree of risk, so that high risk activities have more detailed assessments
that focus on the most dangerous hazard. Risk assessments must be regularly reviewed. The
timescale for review should reflect the level of risk, with high risk activity assessments being
reviewed more frequently than those of low risk. However, if any part of the process changes,
which may include a change in the person performing the activity, the current risk assessment
must be reviewed immediately. This cycle is shown in Figure 4.3.
Identify hazards
Assess risks Apply controls
FIGURE 4.3
Review effect
Outline of the risk assessment cycle.
4.4 RISK ASSESSMENT 77
Types of risk assessment

Risk assessment can either be qualitative, using terms such as high, medium, and low, or quan-
titative giving a numerical value. Quantitative risk assessments (Figure 4.4) are usually done for
high risk activities, but also in large organizations when a guidance tool of numerical values to
be used for different levels of risk is provided to give consistency.
Increasingly, risk assessments are carried out at an organizational level. These assessments look
at the same hazards, but also consider the risks to the service or business, and service users or
patients. For example, if a fire hazard in a laboratory that stored flammable liquids was poorly
controlled, a fire might occur. If a fire does start, it is unlikely that anyone would be seriously
hurt provided that the employees followed the correct evacuation procedures. However, a fire
that is not rapidly extinguished may cause considerable damage to the laboratory, which may
mean it could not operate for some time. The cost of this in terms of repair, effect upon patient
care, and the need for a contingency plan would form a part of a business risk assessment.
Risk assessment process

The HSE publishes guidance that outlines the five steps on how to carry out a risk assessment:
1. Identify the hazards

2. Decide who might be harmed and how
3. Evaluate the risks and decide on precautions
4. Record your findings and implement them
5. Review your assessment and update if necessary
Some systems regard step 3 as two separate steps, because once evaluation of the risks has
occurred, they should be arranged in order of severity (‘prioritized’) before precautions are
considered.
Generally, at least two people should perform a risk assessment: one who is familiar with the
activity and the workplace, and another who is less familiar with both, as it is easy to overlook
potential, but well controlled problems if one is used to the activity and environment. A per-
son less familiar with the workplace will see it with ‘fresh eyes’. They may be able to identify
hazards and suggest alternative controls that those working in the area had not considered.
The use of two contrasting assessors therefore increases the likelihood of identifying hazards,
5 5 10 15 20 25
4 4 8 12 16 20
Likelihood
3 3 6 9 12 15
2 2 4 6 8 10
1 1 2 3 4 5
FIGURE 4.4
1 2 3 4 5 Example of a 5 × 5 risk matrix used to perform
Consequence quantitative risk assessments.
which must be recorded together with details of the persons carrying out the assessment, and
details of the activity and work area being assessed.
When deciding who might be at risk, it is necessary to consider vulnerable persons, such as
young workers or expectant mothers, in addition to anyone else that comes into the work
place including visitors and contractors. The HSE provides a sample form in their written guid-
ance, although many organizations have their own format.
Hazards should be evaluated in terms of their potential danger, and then arranged in order
of decreasing danger. In most systems, controls already in place are also considered when
evaluating risks, but these processes must be checked as part of the assessment. Any actions
and controls must have completion dates or priorities set to them. The degree of risk associ-
ated with the process (high, medium, or low), and the action completion dates will indicate
the timescale for reviewing the assessment. A specific individual must take responsibility for
any required action and the individual identified in writing.
Involving all staff in risk assessment is essential and, indeed, a legal requirement. It also identi-
fies and introduces controls that will be workable and have the support of the team needing
to implement them.
Hierarchy of risk control

The following options must be considered when deciding what are appropriate controls for an
identified hazard. If possible, the risk should be removed, but if this is impossible, something
less hazardous should be used as a substitute, for example, by replacing a dangerous chemical
with one that is non-hazardous or less harmful. When this is not possible, access to the hazard
needs to be prevented, such as by placing hazardous liquids in drip bottles, rather than in
open baths. Alternatively, exposure should be reduced, for example, by enclosing the process
within a fume cabinet or minimizing the time spent doing the activity. As always, defining and
following safe systems of work, using PPE, providing adequate information and training, all
help to eliminate risks. In reality, a combination of all these approaches is usually required to
control the hazards associated with any one particular activity, and provision of training and
information is always essential.
Legal interpretation
The HASAWA impresses a duty on the employer to ‘ensure, so far as reasonably practicable,
the health, safety and welfare of all employees’. This is the most common level of duty in health
and safety law, and was first described by Judge Asquith in 1949 in the case of Edwards versus
the National Coal Board. That case decided that ‘reasonably practicable’ was different to ‘physi-
cally possible’. This means that the level of risk has to balance against the sacrifice involved in
implementing its control. If the sacrifice grossly outweighs the risk, the employer would have
discharged his duty if he did not implement the control, as it was not reasonably practica-
ble. Put in another way, if the risk is relatively small compared with the cost of the measures
required to control it no action is necessary. The level of risk must consider both likelihood and
severity and the cost of implementing the control should include time, trouble, and expense.
During any risk assessment process, this balance needs consideration for each of the types of
controls set out in the hierarchy of control. At some point, the cost of the control will be at an
acceptable balance to the risk; this gives the minimum level of control required. You can see
in Figure 4.5 a diagrammatic representation of how time and cost must be considered along
with the degree of risk when deciding what controls to put in place.
4.4 RISK ASSESSMENT 79
Risk Cost FIGURE 4.5

(Likelihood (Time, expense, Schematic indicating how costs should be considered against the degree
and severity) and trouble)
of risk when deciding which controls should be established.
Specialist risk assessments

Some of the regulations we have discussed, have a requirement for specialized risk assess-
ments that consider specific risks and control hierarchy. In addition to these, the Management
of Health and Safety at Work Regulations recognize that some types of people may be more
susceptible to risk. Specific risk assessments are necessary for such people because they have
different capabilities or may be more susceptible to hazards, therefore increasing the risk of
the activity. These categories of people include the young, expectant, or new mothers, and
lone workers who do not have the support of a team, and so are subject to additional hazards
and higher levels of risk; hence, their need for different systems of work and emergency
procedures.
Reporting of incidents
An incident is an unplanned event that causes an adverse outcome, such as injury, ill health,
and/or damage to property, equipment, or the environment. Near misses are events that
could have had an adverse outcome. Incident reporting and an appropriate investigation of
any incident are essential parts of a health and safety management system. This can turn into
a reactive approach, but the system can be proactive, contributing to improving health and
safety, when used to report near misses and hazards. Reporting and investigating near misses
are essential as generally, 10 near misses occur for every minor injury and the number of
minor injuries relates to the occurrences of serious injuries. The Birds Accident Triangle of
1969 shows how many minor incidents occur for every major event (Figure 4.6). Thus, near
misses are ‘free lessons’ that help prevent more serious incidents.
There is a legal requirement, under the Social Security (Claims and Payments) Regulations for
employers to keep a written record of any accidents that occur in the workplace. Stationary
offices produce an accident book, but the records can be in any form as long as sufficient
detail about the accident and persons affected is recorded. The employer is also required to
investigate the cause of the accident and enter this into the record.
All organizations have their own systems for reporting incidents and near misses. In gen-
eral, information about the event, the people involved, and contact details are collated and
recorded. In some cases, damaged equipment is quarantined because it may be necessary to
examine it as part of the investigation. Often immediate actions are necessary to make the area
and/or activity safe, and these must be noted. If a medical practitioner is consulted, he or she
must record details of any injuries.
1 Major injury
10 Minor injury
30 Damage only
FIGURE 4.6 600 No injury or damage

Bird’s accident triangle (1969) showing how many minor
occurrences underpin every major accident. See also Figure 19.7.
The severity of an incident is often quantified by scoring its impact against the likelihood of
re-occurrence in the same way that risk levels are assessed. Near misses are assessed in the
same way, by scoring the likely impact against the likelihood of occurrence. If the resulting
score is relatively high, a more formal investigation, including specialist methods, such as root
cause analysis, can determine the cause and suitable controls. Root cause analysis is a struc-
tured approach to investigating an incident, with the aim of identifying and examining its base
cause, as implementing corrective actions at this level will produce the most effective control.
If a specimen has been mislabelled, the person responsible may have simply made a mistake;
however, checking training and competency records may identify deficiency in the training or
induction process. Corrective actions aimed at improving training and induction will affect all
staff in the area and potentially prevent them making a similar error. Hopefully, this will lead to
much better control and lower the level of that type of risk.
4.5Control of substances hazardous

to health
The Control of Substances Hazardous to Health (COSHH) Regulations, first introduced in 1988,
were the most significant piece of legislation introduced in the UK after the HASAWA; since
that time, they have been subject to many amendments. Control of Substances Hazardous to
Health Regulations set out the requirement for a management system to assess health risks,
put in place suitable control procedures, and to monitor these controls. The COSHH regula-
tions pre-dated the Management Regs, and were the first regulations to require employers to
perform risk assessments. Thus, it brought about significant changes to health and safety prac-
tices. These regulations also established the need to monitor the health of employees. Two
potential types of ill health can occur – acute and chronic. Acute conditions include asthma or
fainting. They are usually of short duration with a rapid onset of symptoms following a single
or short-term exposure to the causative agent. Although acute ill health can be severe, it is
usually reversible. Chronic ill health includes cancers caused by exposure to industrial and
environmental carcinogens. Chronic ill health normally develops over a long period following
repeated exposures to a harmful agent. The onset of symptoms is often so gradual that the
effects on health can be undetected and the condition remains undiagnosed for years. Unlike
acute conditions, the effects of chronic diseases tend to be irreversible.
4.5 CONTROL OF SUBSTANCES HA ZARDOUS TO HEALTH 81
The COSHH regulations cover almost all hazardous substances, however, some agents are
subject to separate, and more specific regulations. These agents include lead, asbestos, and
sources of radiation.
Classification of hazards
A chemical substance is considered hazardous if it is classified as dangerous to health under
the Chemicals (Hazard, Information and Packaging for Supply) (CHIP) Regulations 1994. The
CHIP regulations place a duty of care on all suppliers when they package and transport haz-
ardous substances. All packages must be clearly labelled with hazard information contained in
the form of a Material Safety Data Sheet (MSDS). Both hazard warning labels and MSDSs are
essential sources of the information required to perform a COSHH assessment. The packaging
and labelling of biological substances is legislated by a number of transport regulations, and
is discussed in Section 4.9.
There are four main types of chemical hazards: irritant, harmful, corrosive, and toxic, which
are represented by the symbols you can see in Figure 4.7, or by recognized abbreviations.
Irritants, (Xi), are non-corrosive substances that cause inflammation of the skin or respiratory
(a)
TOXIC HARMFUL IRRITANT CORROSIVE Flammable
(b)
GHS - Hazard Pictograms and correlated exemplary Hazard Classes
Physical Hazards
Explosives Flammable Liquids Oxidizing Liquids Compressed Gases Corrosive to Metals
Health Hazards Env. Hazards
Acute Skin Corrosion Skin Irritation CMR1, STOT2 Hazardous to the

Toxicity Aspiration Hazard Aquatic Environment
1
carcinogenic, germ cell mutagenic, toxic to reproduction
2
specific target organ toxicity
FIGURE 4.7
(a) Traditional hazard symbols commonly used on chemical packages. Since 2006 these
have been gradually replace worldwide by (b) the globally harmonized system (GHS)
of classification and labelling of chemicals. The European Union adopted GHS at the
end of 2008.
tract after repeated exposure; acetone and formaldehyde are two examples of irritant chemi-
cals. They may also cause individuals to become sensitized or allergic to that substance, or may
increase the severity of other allergies from which an individual may suffer. Many everyday
substances are harmful (Xn), for example, cleaning products and paints. These substances
may cause minor health effects if swallowed or inhaled, or if they penetrate the skin. Fortunately,
the use of PPE (Section 4.7) is usually sufficient to be able to work safely with them. Corrosive
(C) substances are usually strong acids or alkalis, which can attack or burn living tissues. Many
cleaning agents contain corrosive substances. Toxic (T) substances, otherwise know as poi-
sons, reduce or prevent the function of major organs such as the heart, liver, and kidneys.
Mercury and carbon monoxide are examples of toxic substances.
Even pure chemicals may have more than one hazard classification, for example, some clean-
ing agents are both harmful and corrosive. Some laboratory reagents contain a number of
substances of these types. Under the COSHH regulations, the controls identified and put in
place must be adequate to control the most serious hazard to health. Risks associated with
Flammable (F) substances are not covered by COSHH, but if the substance has other classifi-
cations, such as being harmful or an irritant, these risks must be considered under COSHH.
SELF-CHECK 4.8
What is the correct abbreviation for each of the classified hazards shown in Figure 4.7(a)?
Complying with COSHH

Seven basic steps must be satisfied, if an organization is to comply with the COSHH regulations:
1. Risk assessment
2. Identify precautions
3. Prevent or adequately control exposure
4. Ensure control measures are used and maintained
5. Monitor exposure of employees to hazardous substances
6. Carry out appropriate health surveillance where necessary
7. Ensure employees are informed, trained, and supervised
A COSHH risk assessment must be detailed and focus on the hazards to health from the sub-
stance or mixture of substances, and the way they are used and the environment it is used in.
Thus, for example, different hazards are presented when a substance is sprayed compared
with those present when it is painted onto a surface; spraying is more difficult to control, may
affect a larger area, and can be inhaled far more easily, and therefore can potentially affect
more people.
To identify precautions that prevent or control exposure, COSHH regulations prescribe a more
specific hierarchy of control than the management regulations described in Section 4.3 that
give priority to mechanical or engineering controls when a substance cannot be removed or
substituted. The COSHH regulations also specifically state that PPE should be used as a control
when no other measures are available, although, of course, PPE can be used in combination
with other control methods. To ensure control measures are maintained, COSHH regulations
include a specific requirement to ensure that engineering controls, such as safety cabinets and
LEV, undergo thorough examinations at least every 14 months with written records made of
them, which must be kept for at least 5 years.
4.6 FIRE REGUL ATIONS 83
Employers must monitor the exposure of employees to hazardous substances that have
exposure limits assigned to them and this must be recorded. Hazardous substances have
workplace exposure limits (WELs) set for them, which are available in EH40, a publication of
the HSC. The limits are set at a level at which ill health from the substance would be unlikely
to occur in the majority of workers. The limits may be short-term (15 minutes) or long-term
(8 hours) depending on whether the agent usually causes acute or chronic ill health effects.
General records must be kept for at least 5 years, but a record of an individual’s (personal)
exposure must be available for 40 years. Substances that require the monitoring of exposure
usually require the employer to carry out health surveillance. In large organizations, an occu-
pational health department provides and reviews health screening questionnaires for staff
exposed to such hazards. The department may also conduct tests, such as skin checks and lung
function tests, to monitor the health of staff and detect any possible ill health effects.
Control of Substances Hazardous to Health Regulations also place a duty on employers to

inform employees of any risks and the relevant control measures in use. They must also train
staff in how to use and maintain all control measures, and perform activities safely. There is
also a duty on the employer to supervise employees, and ensure the control measures are
used correctly and are well maintained.
Dangerous Substances and Explosive

Atmosphere Regulations
The Dangerous Substances and Explosive Atmosphere Regulations (DSEAR) are designed to
protect employees from the risks of substances likely to cause fire, create dangerous atmos-
pheres, and/or explosions. The use, handling, and storage of flammable substances, such as
industrial methylated spirits (IMS), often used in clinical laboratories, are controlled by these
regulations. Flammable substances also fall under these regulations, although like any other
hazards to health, they are also covered by COSHH regulations. Like all regulations, DSEAR
prescribe systems to be established that allow appropriate risk assessments to be performed
and which implement controls that must be monitored and reviewed.
4.6 Fire regulations

In 2005, a major change to fire legislation in the UK was made with the introduction of the
Regulatory Reform (Fire Safety) Order. The need for fire certification of premises by the fire bri-
gade was removed and replaced by a requirement by employers to carry out a fire risk assess-
ment. Employers should appoint a person responsible for fire safety and this person should:
• Carry out a fire risk assessment to identify the risks and hazards
• Consider who may be especially at risk
• Eliminate or reduce the risk from fire as far as is reasonably practical
• Provide general fire precautions to deal with any residual risk
• Take additional measures to ensure fire safety where flammable or explosive materials are
used or stored
• Plan procedures to deal with any emergency and document these plans
• Review the findings of risk assessments as necessary
The regulations also contain minimum standards for the provision of fire fighting equipment,
and fire-related safety signs to show the location of this equipment, alarm call points, and
fire exits. There is also a requirement that all employees receive annual training on fire proce-
dures, which should include training in the use of the fire fighting equipment available in the
workplace.
at
The control of fire risks requires knowledge of how a fire starts. The initiation of a fire has three
Fu
He
requirements: oxygen or air, fuel, and a source of heat, which are often represented as a fire
el
triangle, which you can see in Figure 4.8. The absence or removal of any one of the three will
prevent a fire starting.
Oxygen
SELF-CHECK 4.9
FIGURE 4.8
Fire triangle showing the three List two sources of heat and two types of fuel that are routinely found in clinical laboratories.
elements required to start a
fire.
Fire fighting equipment, such as fire blankets and fire extinguishers, dampen fires by removing
one or more of the three elements required for a fire to burn.
Fire extinguishers are predominately red, but have colour-coded labels that easily identify the
different types of extinguisher. The commonest types found in laboratories are, for example,
red and black labelled extinguishers, which are water- and carbon dioxide-based, respec-
tively. Water extinguishers function by removing heat, while carbon dioxide extinguishers
reduce oxygen levels. Each type of extinguisher must be used correctly: water extinguishers
must not be used on electrical fires as this carries the risk of an electric shock. Figure 4.9
shows some of the different types of extinguishers and the types of fires they are able to
extinguish.
Fire Extinguisher Chart

Extinguisher Type of fire
Solids
Colour Type (wood, paper, Flammable Flammable Electrical Cooking
cloth, etc.) Liquids Gases Equipment Oils & Fats
Water
Yes No No No No
Foam
Yes Yes No No Yes
Dry
Powder
FIGURE 4.9 Yes Yes Yes Yes No

Fire extinguisher selection
chart showing the type of Carbon
extinguishers and their colour Dioxide
(CO2)
codes, together with their
No Yes No Yes Yes
recommended uses.
4.7 PERSONAL PROTECTIVE EQUIPMENT 85
4.7 Personal protective equipment

We looked at the regulations designed to ensure the provision and use of PPE to control risks
that cannot be controlled by other means in Section 4.3. We will now look at some of the com-
mon types of PPE that you might use in a biomedical laboratory. Figure 4.10 shows a selection
of the types of PPE that are used in biomedical laboratories.
Body protection
Laboratory coats are worn to protect everyday clothing and skin from exposure to hazardous
substances. Depending on the nature of the hazards, laboratory coats may need to be supple-
mented by aprons, or the use of a disposable coat or overalls. For example, disposable coats
are used in Category 3 containment laboratories. The fastenings, cuffs, and sleeves of labora-
tory coats should be inspected before first use and the fit must enable the person to fully close
the coat. Overly long coats or sleeves are additional hazards, as parts of the coat could get
trapped, or caught on equipment or furniture, causing an accident.
It may be necessary to have transport coats for staff, for use when moving between risk areas
of the laboratory to relatively clean ones, such as corridors. A clean coat must always be worn
when leaving a laboratory to go to a clean site or to visit clinical areas for laboratory-related
purposes. This ensures that the clean area is not contaminated with substances from the labo-
ratory transported on a used laboratory coat.
Hand and foot protection

Many different types of safety gloves are routinely used in clinical laboratories. As with all
equipment, the type of glove selected must effectively control the risk; the circumstances
FIGURE 4.10
Selection of personal protective equipment (PPE) that might
be used in a biomedical laboratory. Courtesy of Sperian
Protection, UK.
of use must also be considered. Liquid resistant gloves can control the risks associated with
handling substances where contact between them and the skin must be avoided. However,
many types of glove perish upon contact with commonly handled laboratory reagents, such
as xylene; therefore, careful consideration must be given to the material of the glove. Vinyl,
latex, nitrile, and rubber gloves are all available. An additional consideration when selecting
safety gloves is that of latex skin allergies or sensitivities. Most workplaces try to remove all
latex material from use; however, some non-latex gloves do not, in some people’s opinion,
give the same dexterity and sensitivity of touch so they may still be used in a limited number
of areas. A detailed risk assessment justifying their use should be carried out and anyone at
risk clearly identified.
Safety gloves made of a suitable material may also be used to protect against sharp or rough
surfaces in MHOs and when handling substances at, or working in, extremes of heat.
Gloves must also be of the correct size to ensure safe handling of the substance: loose gloves
can lead to reduced grip and tight gloves to skin irritations. You can see an example of a glove
selection chart, showing the type of gloves used when handling some common chemical and
biological hazards in Table 4.1.
Foot protection is sometimes required to control the risk of hazardous substances falling onto
feet. In most circumstances, rigid, fully enclosed shoes offer sufficient protection. In others,
reinforced footwear might be necessary to protect against falling sharp objects or chemicals.
TABLE 4.1 Example of a glove selection chart showing the type of gloves to be used with some common types of chemical
and biological hazards
Glove material Uses Recommended for: Not recommended for: Comments

Latex (natural rubber) Incidental contact Weak acids and Oils, greases, and organic Good for biological and
bases, alcohols, solvents water-based materials.
aqueous solutions Poor for organic solvents.
Little chemical protection.
Can puncture easily. Can
cause or trigger latex
allergies
Nitrile (synthetic rubber) Incidental contact Oils, greases, acids, Aromatic solvents, many Good for solvents, oils,
caustic solutions, ketones, esters, and greases, some acids and
aliphatic solvents chlorinated solvents bases. Clear indication
of tears and breaks.
Good alternative for those
with latex allergies
Polyvinyl alcohol (PVA) Specific uses Wide range of Acids, alcohols, bases, Good for aromatic and
aliphatic, aromatic water chlorinated solvents.
ketones (except Poor for water-based
acetone), esters, solutions
ethers and
chlorinated solvents
Polyvinyl chloride (PVC) Specific uses Strong acids and Aliphatic, aromatic, Good for acids, bases,
bases, salts and other chlorinated solvents, oils, fats, peroxides, and
aqueous solutions, nitrocompounds amines. Good resistance
alcohols, glycol to abrasions.
ethers Poor for most organic
solvents
This table is for general reference only; for specific recommendations contact the glove manufacturer or MSDS.
4.8 REPORTING OF INJURIES, DISEASES AND DANGEROUS OCCURRENCES REGUL ATIONS 87
Specialist footwear is sometimes used to control other risks, for example, using non-slip soles
in hazardous floor conditions, or electrically insulated shoes to protect against electric shock,
and shoes incorporating anti-fatigue properties for people that spend long periods of time
standing.
Eye, head, and ear protection

Numerous types of spectacles, goggles, and face shields are available to protect the eyes
and/or face from contact with hazardous substances. Here, again, the material used to
construct them must be sufficient to control the risks involved. The comfort of the person
required to wear the PPE must be considered, as otherwise they are unlikely to wear them
consistently.
Head and ear protectors are not commonly used in laboratories. Head protection involves
wearing hard hats or bump caps to protect against objects falling from above or when working
in confined spaces. Ear plugs or defenders can be used to protect against noise that at some
levels and frequencies damages hearing.
Respiratory protection
Filtration masks, respirators, and breathing apparatus can be used to control the hazards of
airborne agents when engineering solutions, such as ventilation systems and safety cabinets
cannot be used. Often these are used in non-routine procedures like conducting maintenance
of the normal engineering controls or after a spillage. All masks, respirators, and breathing
apparatus must be tested to ensure they fit the individual who will use them. Factors such as
beards can compromise the fit and, therefore, the level of protection given will be reduced.
Fume hoods do not suffer this disadvantage; however, they must be fitted with the correct air
filters and checked as part of a detailed maintenance schedule.
Reporting of Injuries, Diseases and

4.8
Dangerous Occurrences Regulations
The Reporting of Injuries, Diseases and Dangerous Occurrences Regulations (RIDDOR) set out
the processes of reporting injuries, ill health, or other dangerous events to the HSE or, in some
cases, the local authority. The aim of the regulations is to provide data on those significant
events that can be analysed to reveal any national patterns and so lead to new health and
safety advice and/or regulations. When serious events are reported, the HSE reviews the infor-
mation it has received about the incident and may decide to inspect the workplace. The data
is also used to collect information about occupational illnesses, which may lead to a better
understanding of how they develop and how to control the hazards that cause them.
There are several levels of reporting that depend on the severity of the situation:
• Major injuries and dangerous occurrences
• Three-day-plus accidents
• Reportable diseases
Major injuries and dangerous occurrences include deaths, major injuries, or those requir-
ing hospital treatment, fires, electrical shocks, and the release of biological agents.
Such events must be notified immediately. If an injury sustained at work prevents a person
from doing all of their normal work for more than 3 days, it must be reported to the HSE on
a specific form within 10 days of the incident becoming reportable. Should an employee be
diagnosed with a reportable disease by a medical practitioner, the employer must report
this to the HSE on the specified form. Examples of such diseases include occupational
dermatitis, occupational asthma, mesothelioma, tuberculosis, and some musculoskeletal
disorders.
The Health and Safety (First Aid) Regulations 1981 require an employer to provide first aid
equipment, facilities, and personnel. The specific nature of the provision required is related to
the findings of both general and COSHH risk assessments.
SELF-CHECK 4.10
How should a dangerous occurrence be reported to the HSE?
4.9 Transport regulations

Transporting dangerous goods is regulated by several pieces of legislation. These include, for
example, the CHIP regulations we discussed in Section 4.5, the Carriage of Dangerous Goods
and Use of Transportable Pressure Equipment Regulations (2004), and a European agreement
know as accord européen relative au transport international des marchandises dangereuses par-
route (ADR) of 2009. The Department for Transport publishes guidance on these regulations,
and, with the HSC and Vehicle and Operators Service Agency (VOSA), is also responsible for
enforcing them.
Dangerous goods include pathology samples, gas cylinders, chemicals, explosives, flammable
liquids, and radioactive substances. Each specific class has defined controls for their packaging
and labelling, and what must be used when they are transported.
As with all other modern regulations, risk assessment, training, and information about safe
systems of work, risks, and controls is an essential part of the duties imposed. Every employer
that transports dangerous goods as part of their business must have a Dangerous Goods
Safety Advisor (DGSA), who must have attended a training course and passed the associated
examination. The role of the DGSA is to ensure all staff involved in the processes are aware
of the requirements needed to meet the regulations. Drivers who transport dangerous goods
by road must carry instructions in writing, known as a Transport Emergence (TREM) card,
detailing the substance, its category name, UN number and class, details of what to do and
who to contact in an emergency, and first aid information in case of spillage of the substance
being carried.
Pathology samples
Blood, urine, faeces, swabs, tissue samples, and clinical waste are infectious substances under
the regulations, and assigned to class 6.2, which is further subdivided into A and B groups. The
majority of samples fall into category B and may contain hazard group 1, 2, or 3 pathogens.
They must be labelled: UN3373 – Biological substance, category B. Category A samples are
defined as cultures of hazard group 3 pathogens or samples suspected of containing hazard
group 4 pathogens. Packages with category A samples must be labelled: UN2814 – infectious
substance affecting humans. Only specified carriers may transport category A samples.
4.10 PERSONAL HEALTH AND SAFET Y 89
Packaging and labelling of biological materials

Pathological specimens may be transported by road, rail, or air if the packaging and labelling
instructions given in Packaging Instructions P650 (found in part 4 of ADR) are followed. These
instructions may be summarized as follows. The primary layer (the sample container or tube)
must be watertight and leak proof. There must be an absorbent material, sufficient to soak up the
volume of sample, between the primary and secondary layers. The secondary layer (sample bag)
must be watertight. A rigid outer packaging that has passed a 1.2-m drop test and has minimum
dimensions (on one face) of 100 × 100 mm is required. Cushioning material must be inserted
between the secondary layer and the outer packaging. Either the primary or the secondary pack-
aging must be able to withstand a pressure differential of 95 kPa or more. Usually, the request form
is attached to the outside of the bag to protect it from damage by any leakage. The package must
be labelled with the UN3373 diamond and biohazard label as shown in Figure 4.11 below.
Samples that do not have an associated risk of infection, such as processed tissue blocks, can
be transported as ‘exempt human specimen’, but must be packed according to the instructions
contained in P650.
4.10 Personal health and safety

Every workplace will have its own specific health and safety instructions that you must follow
at all times. The initial training you receive when you first take a job in a laboratory must always
cover the hazards, risks, and controls of each process you undertake. However, you will later
receive supplementary specific and mandatory training related to the regulations we have
already discussed. If you believe that you have received inadequate information, you should
consult a senior member of staff. However, some rules and practices are common to all labo-
ratories, and include the following:
• A waterproof dressing must cover all cuts, abrasions, or other types of wounds to the skin
especially of the hands before you start work. If you puncture your skin in an accident
at work, gently encourage it to bleed under cold running water. Report the incident to a
senior member of staff and seek medical advice as necessary
• You must never take personal items, such as pens, pencils, cosmetics, toiletries, and hair-
brushes, into the laboratory. Cosmetics are not to be applied in the laboratory; indeed,
hands must never be in contact with the mouth, face, eyes, and nose. Always wear a cor-
rectly fastened coat when in the laboratory. It must be cleaned regularly and immediately
changed if it becomes contaminated
FIGURE 4.11
Labelling required for transporting category B
BIOLOGICAL SUBSTANCE, CATEGORY B specimens.
• Never take food or drink (including sweets and medication) into a laboratory nor eat or
drink there. Always follow the correct laboratory procedures. Never deviate from them
unless you have received alternative instructions from a senior member of staff for a spe-
cific reason. Should circumstances arise that prevent you from following the procedure,
always inform a senior member of staff before continuing. Wear gloves when necessary;
if this is a requirement, it will be specified in the standard operating procedure. Replace
the gloves if they become contaminated or perforated. Avoid all practices that can
cause splashing or the release of airborne substances unless they are performed in an
appropriate containment cabinet. Report all accidents, breakages, spillages, or damaged
equipment to a senior member of staff immediately
• Always wear a clean coat if visiting a clinical area. Clean coats must be stored in a separate,
clearly marked area, separate from that for other laboratory coats. Remove your coat and
wash your hands before leaving the laboratory
• If you become ill and consult a doctor, always inform him or her where you work, and ask
them to talk to your manager if they require further details
• Lastly, never take any unnecessary risks in a laboratory
Occupational health
Most employers will have their own occupational health (OH) service, or access to one. The
role of OH is to assess whether individuals are fit to perform the role in question before an offer
of employment is given, and to give advice on health issues that relate to the specific area of
an employee’s work. Occupational health may also provide services such as vaccinations and
health monitoring. Staff can also discuss concerns related to their health and welfare with
them, in confidence.
Risk awareness
Some people are natural risk takers; others are naturally more cautious. It is essential that in
the workplace you take a responsible attitude to risk and avoid it at all times. You should also
remember that the severity of a hazard is rarely the same as the level of risk; laboratory proce-
dures contain a wide range of controls that, if followed, greatly reduce the level of risk.
Infection control
Hand hygiene is an essential control to minimize the risk of infection. It is essential that you take
great care every time you wash your hands. The seven-step technique, which is usually displayed
above sinks designated for hand washing (Figure 4.12), should be followed to ensure every area
of the hands, fingers, and wrists is cleaned. Great care should also be taken to dry hands thor-
oughly as leaving them damp can cause dermatitis. False or long nails and jewellery can harbour
dirt, or infectious and hazardous material: never wear them in areas where they pose a risk.
Clothing and personal appearance

Many workplaces have a clothing and personal appearance policy. Such policies are partly
intended to encourage health and safety elements, such as wearing enclosed footwear, hav-
ing hair tied up, and not wearing large earrings or necklaces, but they are partly intended to
ensure staff present a professional appearance. Standards of dress are subjective; hence, these
4.10 PERSONAL HEALTH AND SAFET Y 91
Step 1 Squeeze a small

amount of sanitizer
gel or soap over left
palm. Dip all the
fingers of the right
hand into the left
palm and vice versa
Step 2
Join hands palm to

palm
Step 3
Rub right palm over

left hand as shown
and then left palm
over right hand
Step 4
Interlace fingers with

hands joined palm to
palm
Step 5
With fingers still
interlaced, rub the
backs of fingers
against opposing
palms
Step 6
Clasp thumbs in
opposing palms and
wash with rotational
movements
Step 7 Wash wrists with

rotational movements
of opposing hand
FIGURE 4.12
Rinse and dry Seven-step technique of hand
thoroughly washing. Once clean, the hands
must be thoroughly dried.
policies can set clear guidelines for staff to follow. Dress standards are closely related to infec-
tion control policy as long-sleeved tops or shirts and ties can transport infectious material
around the workplace. Thus, a balance between presenting a professional appearance, per-
haps by wearing a tie, and infection control, must be established. A professional appearance
by staff is especially important in service industries such as healthcare, and is an essential

part in developing trust and positive working relationships with professionals in other depart-
ments, as well as with patients.
4.11 Universal or standard precautions

The Centers for Disease Control (CDC) in the USA first recommended universal or standard
precautions 1985, in response to the risk of transmitting human immunodeficiency virus (HIV).
It is not always possible to tell if a clinical sample poses an infection risk; hence, universal pre-
cautions is a system of work and controls that assume all patient-related contact carries a risk
of infection.
The key points of universal precaution systems are:
• Good hand hygiene
• The wearing of PPE at all times
• The correct use and disposal of sharps, such as needles and blades
• Disinfection of body fluid spills
• Careful disposal of waste
Using universal precautions in the wider healthcare setting has been the driver for decontami-
nating hands after every patient interaction, irrespective of the level of infection risk from the
examination or procedure. This has lead to the increased use of hand gel cleaning agents.
In a clinical laboratory, implementing universal precautions means treating all specimens as

if they are extremely hazardous, and wearing gloves and possibly eye protection at all times.
Wearing of gloves would even be necessary when using equipment such as telephones and per-
sonal computers. This approach would certainly simplify the use of precautions and, therefore,
make it easier to follow and monitor the use of controls. However, the long-term use of PPE can
lead to ill health issues, for example, wearing gloves for extended periods can lead to dermatitis.
SUMMARY
■ The Health and Safety at Work Act and the Management of Health and Safety at Work
Regulations form the legal framework that ensures the safety of everyone in the work-
place. They place numerous duties upon employers, but also some on employees.
■ All employees have a duty to ensure their actions or omissions do not cause a risk to
themselves or to the safety of others working with them. They also have a duty to use and
to care for equipment as instructed, and to report defects in equipment or hazards to the
health and safety officer.
■ Risk assessment is the primary tool prescribed in legislation to ensure high standards
of health and safety. Risk assessments identify hazards and those who are potentially in
danger; they evaluate the risks associated with those hazards and suggest how the risks
can be reduced to acceptable levels. All risk assessments need reviewing on a regular basis
to ensure they are still adequate. Other regulations that require a more detailed form of
risk assessment include COSHH, DES, Manual Handling and Fire Safety.
QUESTIONS 93
■ The ethical duty of everyone to ensure the safety of everybody affected by the workplace
is upheld in law. Ensuring a high standard of health and safety in the workplace is also
desirable because the overt costs associated with incidents and injuries are considerable;
hidden costs, such as insurance, time lost in investigations, and negative publicity, are also
substantial.
FURTHER READING
● HSE Books Safe Working and the Prevention of Infection in Clinical Laboratories and
Similar Facilities. HSE Books, Bootle 2003.
Gives detailed information on health and safety specific to clinical laboratories.
● HSE Books 5 Steps to Risk Assessment. HSE Books, Bootle, 1998. Available at: http://
www.hse.gov.uk/risk/fivesteps.htm.
This is an essential guide to performing risk assessments.
● Hughes P, Ferrett E. Introduction to Health and Safety at Work, 4th edn. Butterworth-
Heinemann, Oxford, 2009.
This is an excellent book, covering all areas of health and safety, including the legal aspects
and case law.
Useful Websites
■ www.hse.gov.uk
■ www.hsebooks.co.uk
■ http://www.coshh-essentials.org.uk/
These websites are excellent sources of information about general health and safety issues and
COSHH information. Many of the publications are available to download.
QUESTIONS
1. The abbreviation, Xn is used in chemical hazard classification to represent which of the
following?
(a) Flammable
(b) Irritant
(c) Harmful
(d) Corrosive
(e) Toxic
2. Which of the following statements are TRUE?
(a) The penalties for failing to comply with HSE enforcement notices include up to 6
months’ imprisonment
(b) The act introduced to ensure workplaces were safe was The Safety Act
(c) Under DSE rules you are entitled to free glasses if you require them to do
your job
(d) RSI stands for recent strain injury
(e) You must wear gloves when opening specimen containers to get a better grip on
the lid
3. Arrange the following two lists into their most appropriate pairings.
ACD Display screen equipment

ACOP Health and Safety at Work Act
DSE Personal protective equipment
HSE Dangerous Substances and Explosive Atmosphere Regulations
WRULD Reporting of Injuries, Diseases and Dangerous Occurrences Regulations
DSEAR Lifting Operations and Lifting Equipment Regulations
HASAWA Health and Safety Executive
HSC Provision and Use of Work Equipment Regulations
IOSH Repetitive strain injury
LOLER Advisory Committee on Dangerous Pathogens
MHO Work-related upper limb disorder
WELs Approved codes of practice
PUWER Manual handling operations
RSI Workplace exposure limits
RIDDOR Institute of Occupational Safety and Health
PPE Health and Safety Commission
4. What are the five steps required to perform a general workplace risk assessment?
5. (a) What are the possible routes of entry to the body for a chemical mist? (b) Suggest
possible control measures to prevent its entry.
6. What are your duties as an employee under the Health and Safety at Work Act?
7. List your duties as an employee under the Management of Health and Safety
Regulations?
8. (a) What are the three elements of the fire triangle? (b) Which of these is eliminated when
using a carbon dioxide fire extinguisher?
9. (a) Give an example of a mechanical control, as described by the COSHH regulations.
(b) What are the requirements under COSHH to maintain such equipment and associ-
ated records?
10. What are the differences between a laboratory using universal precautions compared
with one applying control measures based on risk assessment?
5
Statistics and
handling data
Andrew Blann
Learning objectives
■ Explain the concepts of quantitative and qualitative data

■ List the different modes of distribution: the normal distribution and the non-normal
distribution
■ Describe simple statistical tests to analyse data
■ Be familiar with correlation
Introduction
Biomedical science is dominated by information, almost all of which refers to the health status
of an individual or groups of individuals. Many biomedical scientists are directly involved in
the generation of numerical or descriptive data derived directly from human body tissues.
For haematologists, the data generated may be the number of red and white blood cells in a
sample of venous blood, while for biochemists the data may be the concentration of glucose
in that blood. Microbiologists may collect data that tells which particular microbes are present
in a sample of sputum from a patient with a lung problem, and histologists may study lung
tissues from that patient, and the data they handle may determine the presence or absence of
a cancer. Data may not only be at a single timepoint; in some cases we are interested in how
an index (for example, cholesterol concentration) changes as someone undergoes a change in
their diet and lifestyle.
The data handled by a biomedical scientist is typically generated in one of two ways: either
directly by the biomedical scientist (for example, using microscopy, as in many microbiology,
96 5 STATISTICS AND HANDLING DATA
histology, and cytology analyses) or indirectly, through the use of some kind of automated
analyser (which are used for most haematology and biochemistry tests). Immunologists and
haematologists often use both analysers and microscopy. Whatever the means through which
the data are collected, the value of data, and how it can be interpreted must be understood.
The limitations of the techniques and what steps can be taken to ensure these techniques are
delivering the correct data must also be addressed. Failure to grasp these concepts may lead
to errors and possibly the loss of life.
This chapter gives a broad overview of the issues surrounding data handling and statistical
analyses as it applies to biomedical science. It will explore the different ways in which data
can be described and analysed, and consider issues that relate to best practice when handling
data.
5.1Types of information and how to

describe them
Let us first consider why data is needed. The understanding and treatment of many human
diseases and conditions relies heavily on the generation and interpretation of numerical
data. For example, a person with diabetes needs to monitor and control their blood glucose
concentration: they need to collect data, such as measuring blood glucose, and interpret this
data. Is the value too high, too low, or within acceptable limits? In another setting, such as
the study of large numbers of people, it is important to be able to understand those factors
(such as high blood pressure and smoking) that predict the major health issues of a stroke or
heart attack.
Often, a biomedical scientist handles more than just a single value (as in the case of a single
blood glucose measurement as above), but needs to compare sets of data (that is, multiple
measurements from different groups). The analysis of data in this way starts to draw upon
reasonably sophisticated numerical and statistical tools. Quite often, these tools seem quite
intimidating at first glance.
The nature of information

As data comes in many different forms, it is helpful to be able to characterize it in some way so
that it provides an idea of how it is best to present and also perform an analysis. In biomedical
science, information generally comes in one of two types:
• Quantitative data is that where the information is assigned a directly measurable, numeri-
cal value. Examples of this are height, weight, age, red blood cell count, temperature,
serum potassium concentration, or perhaps the proportion or percentage of patients with
a particular problem (such as an infection) or a particular ABO blood group. In almost all
cases, the value of the data is defined not by an individual, but by an objective observation
or scale, itself often derived from a machine.
• Qualitative data, on the other hand, is formed from information in words (often descrip-
tive terms such as good, reasonable, occasional, frequent, moderate, severe). These items
of information are generally obtained directly from people being investigated, not merely
patients with a particular health problem, and use of a questionnaire is common. However,
at least one observer has made the distinction of qualitative research methods. A major
5.1 T YPES OF INFORMATION AND HOW TO DESCRIBE THEM 97
aspect of word-data of this type is that the value or relative importance of the word itself is
defined by the individual and, therefore, has a high degree of subjectivity.
Despite the possible value of these two types of data, some types of information lie some-
where between the two. It is also possible for some types of qualitative data to be converted
into numerical forms for rigorous analysis, that is, for it to be changed from a qualitative to a
quantitative form. A weakness with qualitative data is in the subjective definition of the infor-
mation. Consider pain. What one person may consider as being moderate, to another person
could be severe. This gives a degree of uncertainty. However, another study may compare the
number of people who use the word ‘moderate’ to describe their pain, with those that use the
word ‘severe’, or even ‘excruciating’. In the latter case, the assessment of pain is being evaluated
in a qualitative way (i.e. the ‘quality’ of the pain).
Quantitative data
Broadly speaking, we can classify just about all quantitative data into one of two types: that
which is categorical and that which is continuous.
Categorical data fits into one of any number of discrete boxes or categories; there are no
‘in-betweens’. An example of this is the number of men and the number of women in a par-
ticular group. In the case of sex there are generally no in-betweens. Here, it is important that
data lies only in one of a discrete number of categories, and sex is a good example of this.
Another example is the consequence of sex: children. In real life you can’t have a fraction or
proportion of a child, only whole numbers.
This concept can be extended to more than two discrete groups. Just about everyone belongs
to one of the four ABO blood groups of A, B, AB, and O; there are few (if any) exceptions that,
if they do occur, will be so rare as to be ignored in the final analysis (unless you are an expert in
blood transfusion!). Our colleagues in microbiology can say with a good degree of confidence
that an organism (such as methicillin resistant Staphylococcus aureus – MRSA) is present or
absent – there should be no in-between. Immunologists can say ‘yes’ or ‘no’ to the presence of
antibodies to organisms such as a bacterium or a virus, or if the patient has high levels of cer-
tain antibodies to a particular infective agent. Histologists will tell us that a tissue either is or is
not invaded by a cancer, whilst cytologists report the presence or absence of certain abnormal
cells, such as in cervical cancer.
Continuous data includes factors such as height, weight, blood pressure, and just about all
haematology and biochemistry results (such as the number of red blood cells and concentra-
tions of triacylglycerols and Na+ in the serum). The data, consisting of individual numbers,
can be described by almost any figure in a given range (such as age, which can be anywhere
between 0 and 120 years or so).
The presentation and analysis of continuous data is more complex than if the data is categorical.
When the data is continuous we need to consider three aspects: the central point or tendency,
the variance, and the distribution. These three aspects will now be explained in turn.
The central point

The most important statistical aspect of continuous data is the consideration of the central
point of that particular data set. Some statisticians prefer to use the word ‘tendency’ (which
may seem rather inexact) instead of ‘point’, which is not unreasonable as there are actually two
such values within a single data set: the average value and the middle value. These two differ-
ent central points are the mean and the median, respectively. However, some consider there
to be a third point – the mode – but this is very rarely used.
• The mean point of a set of data is obtained by simply adding up all the data, and then divid-
ing by the number of the individual data points. As such, the mean may also be seen as the
‘average’ number. How the mean point is obtained is described in Box 5.1.
BOX 5.1 The mean value
Consider a group of 11 data points with individual values of 25, 30, 21, 27, 35, 28, 22, 25,
29, 27, 24. These data (which may be the ages of a football team) sum to 293. Thus, the
mean value is 293 divided by 11, which (to three significant figures) is 26.6.
By placing the eleven data points in order from lower to highest, i.e.
21, 22, 24, 25, 25, 27, 27, 28, 29, 30, 35
The mean value (26.6) is indeed, very close to the middle value of the whole set (the 6th,
with a value of 27). The importance of ‘closeness’ of these two figures will be discussed
further in this section.
• One way of arranging a set of numbers is to list them in rank order, i.e. from the lowest
to the highest. The data point that is in the middle of the entire series is the median, and
this value is arrived at by a completely different set of rules than is the mean value. This is
described in Box 5.2.
BOX 5.2 The median value
Consider the data set of 21, 60, 15, 32, 19, 25, 113, 24, 29, 35, and 22. If these are arranged,
or ranked, in order from the smallest (on the left) to the largest (on the right), a series is
obtained:
15, 19, 21, 22, 24, 25, 29, 32, 35, 60, 113.
In this series of 11 data points, the one in the middle is the 6th one – i.e. with 5 data points
larger (greater than 25) and 5 data points smaller (less then 25). This central point is the
value 25 – also known as the median (highlighted in bold). Note that it is not the mean.
In this data set the mean (average) would be the sum of all the individual points (394)
divided by the number of individual data points (11) to give 35.8 – a very different figure
to 25. This difference is important and will be discussed later in this section.
If the data set is of an even number of data points, when there is no exact middle point,
this is taken as the average of the two middle points. For example, in the series . . .
10, 11, 12, 14, 18, 26, 39, 60
… there is no one single middle point, so we must create one, which in this series is the
midpoint between 14 and 18, i.e. 16.
So each set of data has both a mean and a median. Sometimes they are the same number (or
are close together), but in other data sets they may be very different.
SELF-CHECK 5.1
We can classify just about all quantitative data into one or two classes. What are these
groupings?
SELF-CHECK 5.2
What are the mean and the median of the data set: 156, 217, 199, 306, 224, 256, 178, 267,
317? A calculator will probably be useful in working out the mean, although it can be done
by hand.
Variance
A second concept in getting to grips with a set of data is its variance, a term derived from its
root word ‘variety’. This word helps give us information such as the highest and lowest points in
a particular group of data, and the extent to which the data clusters tightly around the central
point of the mean or the median. If it is tightly clustered, the data is said to have low variance: if
more diverse (spread out), it has high variance. We use one of two particular measures of vari-
ance depending on the nature of the central point (the mean or the median). When the central
point of a data set is the mean, we use standard deviation (SD) to describe the variance as
illustrated in Box 5.3. However, if the data is such that the central point is the median, then the
variance is described in terms of the inter-quartile range (IQR) as described in Box 5.4.
The SD provides an idea of the degree to which the data is clustered close about the mean
value, or is more spread out, i.e. the variance. For example, suppose a data set has a mean of
100 and a SD of 20. The SD is telling us that most of the data points (in fact, about two-thirds
of them) lie within 1 SD either side of the mean, that is between 80 and 120. However, we
can go further, and often find that nearly all the data points (actually, about 95% of them) are
between 60 (the mean minus two SDs) and 140 (the mean plus two SDs). However, if another
data set has a similar mean of 100, but a much smaller SD, such as 8, then 95% of the data
points should range between 84 (100 minus 16) and 116 (100 plus 16). So a small SD tells us
that the set of data is tightly clustered near to the mean, and when the SD is large, this means
that the data is more spread out.
When describing the first set of data we would generally say that the mean and SD are 100
(20), although some use the notation 100 ± 20. Similarly, the second set of data can be writ-
ten as 100 (8), or perhaps 100 ± 8. The SD is quite hard to obtain on a simple calculator, but is
easier to obtain using a programmable calculator and, of course, on a computer loaded with
a statistical software package.
Figure 5.1 illustrates variance with two different patterns. It shows two sets of data, pattern A
and pattern B, which both have the same central point value of about 95. However, the two
patterns have very difference variances. One of them (pattern A, lower) covers a much greater
range (from about 65–120) compared with the other (pattern B, upper), which runs from 90
to about 98. So in both cases the mean is 95, but the SD of pattern A is 9, whilst the SD from
pattern B is only 2.
The second method of describing variance, the inter-quartile range is derived from the data
points that are one-quarter and three-quarters of the way into the complete data set when it
is ranked from lowest to highest. So starting from the lowest point, and working our way up,
BOX 5.3 Variance, standard deviation,

and standard error
The expressions variance, standard deviation, and standard error are worthy of further
explanation. Take, for example, the data set we have already seen in Box 5.1.
21, 22, 24, 25, 25, 27, 27, 28, 29, 30, 35
The mean value is 26.6, but some numbers (such as 35) are greater than this and some
(such as 21) smaller. The difference between these two numbers and the mean is 8.4 and
5.6, respectively. The variance of a data set is simply the degree to which individual data
points are far from the mean, and this can be quantified. This difference (i.e. 8.4 or 5.6)
is officially called the ‘deviation’.
So how is the variance of this data set calculated? The first step in defining the variance is
to take the square of each individual deviation (i.e. 8.4 becomes 70.54 and 5.6 becomes
31.36). Next we add up all the squares of the individual deviations, giving something
called ‘sum of the squares’, which in this case adds up to 154.56. The number of data
points in the set is called ‘n’, so in this case n = 11. To obtain the variance we divide the
sum of the square by n – 1, hence 154.56/10 = 15.46 (rounded up).
The standard deviation is the square root of the variance (15.46) and = 3.93.
If we divide the SD by the square root of the number of data points (n) then we get
another index, the standard error (SE) which is therefore: 3.93/3.31 and = 1.19.
So we can summarize our data set as:
Mean = 26.6
n = 11
Variance = 15.46
SD = 3.93
SE = 1.19
Thus, the variance of a data set is simply a stepping stone to the standard deviation, and
similarly the standard deviation is simply a stepping stone to the standard error. Note
that the standard deviation is a fixed property of a data set, whereas the standard error is
dependent on the number of items of data (i.e. on ‘n’), and so is not fixed but can fluctuate
widely depending on the sample size. What this means in practice is that if we summarize
our data set as mean with standard deviation, we get a different result than if we summa-
rize it as mean with standard error, i.e. 26.6 (3.93) versus 26.6 (1.19). This is why, in order
to minimize confusion, data sets are always described in terms of mean and SD.
The SE is a badly misused index, especially when put together with the mean in analys-
ing differences between groups. For example, if there were twice the number of data
points in the sample, we would obtain the same mean (26.6) and almost exactly the
same SD (3.8). However, the SE will be very different, coming down from 1.19 with 11
sets of data to 0.82 with 22 sets of data. Nevertheless, the SE does have a place as it gives
us information about the accuracy of a particular technique or method.
All this can be checked by inputting the dataset into the statistical package of your
choice.
BOX 5.4 The inter-quartile range
Consider the data set of 15 points:
65, 43, 79, 250, 82, 49, 148, 55, 77, 40, 516, 121, 59, 47, 156
In order to obtain the median and IQR, we must unravel, or sort out, the data and place
it in a rank order, from lowest on the left to highest on the right, as follows:
40, 43, 47, 49, 55, 59, 65, 77, 79, 82, 101, 148, 156, 250, 516
The middle or median value is the 8th one (i.e. has 7 points above and 7 points below) is
77 and is underlined. Similarly, the middle value of the lower group of 7 (from 40 to 65)
is 49 (highlighted in bold), and the middle value of the higher group (from 79 to 516) is
148 (also highlighted in bold). So the IQR is 49 to 148.
after we have reached 25% of the data points, we have the 25th percentile. Continuing to work
up the data set, the half-way point (after 50% of the data points) is the 50th percentile. This is
the median, as the middle of the data set has been reached. Continuing up the rank, after 75%
of the data points have been passed, the 75th percentile is achieved. Finally, the highest value
is the 100th percentile, as 100% of the data points will have been assessed. So unlike the SD,
the IQR is relatively easy to obtain from the raw data, as is illustrated in Box 5.4.
Pattern A
Frequency
60 80 100 120 140
Pattern B
Frequency
60 80 100 120 140
FIGURE 5.1
Illustration of the differences in variance between two different data sets that have a normal
distribution and an approximately equal mean value (i.e. the ‘peak’ of each distribution).
Despite the equivalence of a mean value, variances are different – the upper figure is more
rounded or spread out, whereas the lower figure is more of a sharp peak. It is likely that the
standard deviation of the upper distribution is greater than that of the lower distribution.
The IQR, like the SD, also gives us an idea of the spread of the data. We would write a summary
of the data in Box 5.4 of the median and IQR as 77 (49–148):
25th percentile
77 (49–148)
Median (50th percentile) 75th percentile
Note that the median value here (77) is certainly not in the middle of the IQR of 49–148.
Clearly, 77 is much closer to 49 than it is to 148, and this fact is an important feature of this
type of data.
Distribution
We bring the concepts of the central point and variance together to describe the distribution
of a set of data. This is effectively what the data set ‘looks’ like when represented graphically.
The vast majority of data in biomedical science is generally of a normal distribution, and
includes many laboratory and physiological indices, such as haemoglobin and albumin con-
centrations in serum, heights, and body mass indices. In some cases, it may be described as
‘bell-shaped’. An important component of data with a normal distribution is that the mean is
taken to be the central point of the data set. When data is normally distributed, the mean and
median are close together (such as 26.6 and 27, respectively, in the data set in Box 5.1), and
the SD is relatively smaller than the mean [e.g. 100 (20)]. Figure 5.2 provides an illustration of
a data set that is normally distributed.
Data that is non-normally distributed is less common, perhaps the best example from bio-
chemistry being concentrations of serum triacylglycerols in a healthy population, and from
physiology the duration of a pregnancy. This type of data may also be described as ‘skewed’.
30
Frequency
20
10
0
60 70 80 90 100 110 120
Example 1
FIGURE 5.2
Representation of a normal distribution. The data is presented as histograms, with the
tallest (i.e. of greatest frequency, with a value of about 100 units) roughly in the middle of
the entire data set. The histograms of lesser frequencies are smaller as the data sets move
further from the middle towards the two polar extremes (where, on the left, the data is as
low as perhaps 65 units, and on the right, where the data has a point at 120 units).
150
Frequency
100
50
0
0 500 1000
Example 2
FIGURE 5.3
Representation of a non-normal distribution. The data is presented as histograms, with
the tallest (i.e. of greatest frequency, with a value of about 100 units) far to the right of the
entire data set. Indeed, there is only one histogram to its left. Conversely, there are nine
histograms to the right, and the frequencies fall roughly as the value of the data set rises,
with a maximum to 500 units.
A characteristic of data with a non-normal distribution is that the mean and the median are
always far apart (e.g. the mean of 35.8 and the median of 25 in Box 5.2), so that the average of
the data set is not the middle point. Furthermore, when data is non-normally distributed, the
SD is often quite large compared with the mean. For example in Box 5.3, the mean is 118, but
the SD is 120. This helps confirm that the data set has a non-normal distribution. So in these
cases, we use the median to define the central point and the IQR to define the variance. These
points are illustrated in Figure 5.3.
Why the fuss about these two different modes of distribution? First, we need to know about
distribution so we can accurately find out if there is a statistically significant difference,
between different sets of data, such as the blood pressures of one group of people compared
with those of a different group. You might think that the blood pressures of one group at, for
example, 156 (25) mmHg is significantly different from that of another group, which may be
132 (21) mmHg. Indeed, this difference of about 18% seems large and so may be significantly
different. However, as scientists we must apply scientific methods and ensure that any differ-
ence does not merely ‘seem’ significant, but actually is statistically significant.
We can establish whether or not a statistically significant difference exists by using a suitable
statistical test. These generally involve complex mathematics that are best left to a computer.
Nevertheless, the actual test that we would choose to use depends on the way in which the
data are distributed: we would use a different test if both the data sets we seek to compare are
normally distributed than we would if one or both sets of data are distributed non-normally.
These tests will be introduced to you in Section 5.2.
A second occasion when we need to know about the distribution is in order to be able to inter-
pret a particular result from a certain individual. For example, is a serum triacylglycerols result of
2.1 mmol dm−3 normal or abnormal? This is important as a high level may indicate a particular
syndrome, the presence of a particular disease, or an increased risk of a heart attack and, if
so, we should be concerned. In this case, we need to compare the value from that individual
with the results of the same test from a large number of people we know to be healthy. Data
from this large group make up what is called the reference range. It is necessary to know the
distribution of triacylglycerol data in this healthy population to be able to make a judgement
of whether or not a result from an individual is within or is outside this reference range.
Let us suppose that 100 healthy people provide blood for a triacylglycerol test, and that the
results that come back have a non-normal distribution with 95% of the results lying between
0.5 and 2.0 mmol dm−3. So under these conditions the result for the individual in question of
2.1 mmol dm−3 only just exceeds the top of this reference range, and so may possibly require
additional investigation.
However, if the reference range has a normal distribution, it will provide different values that
we would consider to be healthy, such as between 0.8 and 1.5 mmol dm−3. If so, then the
result from the individual patient of 2.1 mmol dm−3 is of greater significance because it is much
higher than the top of the reference range and so may have significant clinical repercussions.
How can I tell if my data is normally or

non-normally distributed?
The nature of the distribution is important because you will need this to be able to apply the
correct statistical test to your data (this will be explained in the section that follows). If you chose
the wrong test, you might well get an incorrect answer. Broadly speaking, there is a small number
of simple rules to determine whether or not data is distributed normally or non-normally.
• If the SD is a lot smaller than the mean, then the data is more likely to have a normal dis-
tribution. We have already seen data like this, i.e. mean of 100 with a SD of 20. However, if
the SD is large compared with the mean, such as mean/SD of 100/90, then the data is more
likely to be distributed non-normally
• If the mean and median have similar values the data is likely to be normally distributed, e.g.
mean 26.6 and median 27 (a difference of only 1.5%), as in Box 5.1. If the mean and median
are far apart, such as 35.8 and 25 (a difference of 43%) as in Box 5.2, then the data is likely
to be non-normally distributed
• A problem with these two methods is that they work very well only for data where the
nature of the distribution (e.g. mean 34, SD 2.5) is reasonably obvious and/or you have a
great deal of experience. For this reason, use of professional statistical software packages
capable of determining the type of distribution is strongly advised
Once the nature of the distribution of sets of data has been determined, we can look to see if there
are differences between them applying the correct statistical test. This will be explained shortly.
Qualitative data
Here, information is gathered and analysed primarily as words (single or in phrases), possibly in
narrative form or with descriptive quotations. The proponents of qualitative research quite rea-
sonably argue that not all information can or should be reduced to a number. For example, how
can you reasonably quantify (place a numerical value on) someone’s feelings, attitudes, or opin-
ions, such as a belief, fear, and love? Qualitative data is often obtained from people using surveys,
interviews, or observations, generally from a validated or structured questionnaire. An alternative
would be to observe, and record verbal or written comments from a defined focus group.
A further distinction between the qualitative and quantitative approaches is not so much with
the collected information itself, but the purpose to which that data is directed. The tradition
from which quantitative research has evolved is of prediction and causal analysis, both of
which are central in asking research questions. For example, we can suppose that a given
group of patients will experience a change in a defined index (blood pressure, blood choles-
terol) following treatment with a particular drug. In contrast, qualitative research generally
5.2 ANALYSIS AND INTERPRETATION OF DATA 105
does not generate numerical data that can easily answer a precise research question. Instead,
the approach often aims to develop a theoretical explanation for the subject of study, the
strength of which is therefore not in predicting an outcome, but in an increased understanding
of the topic being investigated. Thus, the qualitative researcher may attempt to gain insights
into an individual’s feelings and thoughts, often within their own natural setting.
The questionnaire is often at the centre of many qualitative research projects. Considerable
effort must be directed to ensure, as far as is possible, that questions are appropriate, unbi-
ased, and cannot be misunderstood. For example, whilst ‘permit’ may be seen as the opposite
of ‘forbid’, the latter word is viewed by many as pejorative, whilst ‘not permit’ elicits more
responses. Questions themselves may be open or closed. In open questions, the respondent
is allowed to use their own words and phrases, which can result in dozens of different answers
that have no clear theme from only a handful of subjects.
Alternatively, the questioner may offer a dozen or more alternative words, and let the subject
choose those he or she is happiest to use. In closed questions, the respondent is offered only
a small number of answers, such as yes, no, and don’t know, or may be asked to provide a
response from a scale such as agree strongly, agree mildly, neither agree nor disagree, disagree
mildly, and disagree strongly. A weakness of this latter scale of five is the temptation to sit on
the fence and give the middle response, and be neutral, a position easily changed by simply
removing the middle expression and reducing the number of options to four, thus forcing a
choice. An additional problem with questionnaires is that respondents sometimes give a dif-
ferent response if re-tested, giving rise to fears of poor reproducibility, and so questionable
reliability.
5.2Analysis and interpretation

of data
Once we have data, what do we do with it? As biomedical scientists we use scientific methods
to understand the disease process with a view to providing a diagnosis and then monitoring
treatment. For many of us, this will be in comparing the pathology result from an individual
whose health status is unclear with that from a group of individuals we know to be healthy. We
have already seen how we use a comparator group of healthy people to provide a reference
range for serum concentration of triacylglycerols in order to determine whether or not the
result from a single patient gives us cause for concern.
In other studies (possibly in research), we may compare data from a group of individuals (such
as with a particular complaint, symptom, or frank disease, often referred to as the cases) with
that from another group generally considered to be free of the problem (the controls – hence,
a case-control study). In both types of analysis, the control group provides the normal or refer-
ence range.
One reason to be able to correctly collect and analyse data is to produce good research. A key
step in research is to be able to fashion what it is you want to find out in terms of a question,
and then turn it into a statement, generally called a hypothesis. Indeed, much of the research
process is called hypothesis testing. People with an interest in heart disease may ask ‘Does the
blood cholesterol level in one group differ from that in another group?’ This can be turned into
a hypothesis statement such as ‘the blood cholesterol level of one group is higher than that in a
different group’. Other researchers may form their hypothesis as ‘the blood pressure of a group
of patients will be reduced if they regularly take a particular drug’. A third group may hypo-
thesize that workers in one industry have more cancer than workers in a different industry.
The first two hypotheses stated above rely on data (i.e. blood cholesterol concentration and
blood pressure, respectively) that is continuously variable, and this type of data will be exam-
ined first. Later in this section, we will consider the type of data that will test the third hypoth-
esis, which is categorical (i.e. people fall into only one of a small number of discrete groups,
such as those with or without a well-defined disease or condition like cancer or diabetes).
In another, perhaps more formal research setting, suppose there is interest in determining the
levels of cholesterol not as above in an individual, but in a group of individuals such as workers
at a particular factory or people following a particular diet. One may therefore test the hypoth-
esis that cholesterols are significantly greater in the blood of workers at the factory compared
with levels in the blood of people working in an office. Once the hypothesis has been formed,
the method for testing the hypothesis needs to proceed along a number of formal steps.
• First, it is essential to find out how many people need to be recruited to make sure the
findings are reliable. An appropriate degree of power is required in the study. If the study is
underpowered and not enough people have been recruited, then the difference in levels of
cholesterol between the two groups, although seeming large, may not be significantly large.
This can lead to a false negative (i.e. we have failed to find a particular difference to be sta-
tistically significant when in fact it is indeed significant). A statistician should be consulted
at an early stage to determine the number of subjects to be recruited. In this example, we
should recruit 35 people in each group
• Before embarking on a study the approval of the local Ethics Committee must be secured.
This body ensures that patient safety is considered and that good research practice is fol-
lowed. In the United Kingdom, every hospital should have a Research Department from
whom advice (and permission) should be obtained. It may also be necessary to obtain the
funding to perform the blood test and cover the costs of analysis
• The third step is to recruit the people themselves, take the blood samples, and then analyse
them in the laboratory
• The statistician will then be consulted once more to analyse the data. The first step will
be to enter the data into a statistical package (such as Minitab or SPSS) on a computer
which can then define the nature of the distribution of the data (normal or non-normal)
for each group. Although it is well established that cholesterol data is distributed normally,
it is nonetheless good statistical practice to check this is so
Probability
It is well established that normal, healthy adult men are generally taller than normal, healthy
adult women. However, this may not be the case in all populations. Women who have grown
up with high levels of growth hormone are likely to be taller than men who have grown up
with low levels of growth hormone. Despite this, at the practical level, we can predict that the
average height of even a small group of men is likely to be taller than the average height of
a similar sized group of women. However, statisticians use the word probability (p) to give a
more scientific and secure basis to this likelihood. In biomedical science, we are keen to estab-
lish whether or not a difference in two sets of data is genuinely due to, for example, a patho-
logical process, or is due simply to chance.
Statisticians have developed a consensus that says that we are prepared to accept a difference
as real if the probability of it being a real effect (that is, not due to chance) is greater than 95%.
We express 95% in the decimal form as 0.95. If we’re accepting that there is a 95% probability
that the difference is real, then we are also accepting that there is a 5% probability that the
difference could be coincidental, that is, due to chance. We express 5% in decimal form as
0.05. Hence, our requirement for p to be less than 0.05 (i.e. p < 0.05). It follows that if p = 0.06
(i.e. we have a likelihood of 94% that the difference is genuine), we do not consider this to be
of sufficient reliability, and so describe it as statistically not significant.
Key Points
p < 0.05 – the level of probability at which we accept that the difference is real and true,
and not merely due chance, co-incidence, or some other factor.
It follows that if the chance of an effect being spurious is only 1 in 10, i.e. p = 0.1, then this
difference is not statistically significant because 0.1 is greater than 0.05. What would be even
more significant would be that a difference is so large that the probability of such a difference
being spurious or co-incidental in only 1 in 200 (i.e. 0.5%), which means that the chances of
the effect being real are 199 chances in 200 (i.e. 99.5%). Thus, a probability of 1 in 200 gives
p = 0.005, a result that is considerably less (indeed, 10 times less) than 0.05. Overall, the smaller
the p value, the greater is the likelihood that the difference is real and is not due to chance.
SELF-CHECK 5.3
Why is it necessary to have enough power in a research study?
Analysis of data from one individual

Suppose that one of your close relatives has had a blood test for lipids and tells you that he or
she has a serum total cholesterol result of 5.9 mmol dm−3. Are you concerned? This depends
on the age, sex, and some other health issues of your relative. However, an initial question
would be to ask ‘Is this result within the reference range?’
As we have discussed, the reference range is composed of results from hundreds or even thou-
sands of people who are supposed to be healthy, such as blood donors. Using the example
of cholesterol, we would expect data from a large pool of healthy people (say, 400) to have a
normal distribution, with a mean of 4.5 mmol dm−3 and a SD of 0.5. As already described, an
essential component of the relationship between the mean and SD of a normally distributed
index (such as cholesterol) is that the results from 95% of those people (i.e. about 380 of them)
will lie between 3.5 and 5.5 mmol dm−3; this range being derived from the mean value plus two
SDs and the mean value minus two SDs.
The value of 95% of the data points (as opposed to, for example, 70 or 80%) is chosen because
it is most likely to provide representative and reliable information. This is why we often see
mean and SD data being presented, as in this example, 4.5 ± 0.5 mmol dm−3. So if 95% of the
results of this population are within two SDs either side of the mean, what about the remain-
ing 5%? There are likely to be as many at the bottom end of the scale as at the top end of the
scale, and in our example it means that 10 people will have a cholesterol concentration below
3.5 mmol dm−3, and another 10 will have a value greater than 5.5 mmol dm−3. These people are
not immediately to be considered as being in ill health. This is illustrated in Figure 5.4.
So given the above criteria, a serum cholesterol of 5.9 mmol dm−3 is certainly above the top of
the reference range of 3.5–5.5 mmol dm−3. However, is this person in ill health? This we can-
not say as more details are needed before we give the individual a potential diagnosis, which
Mean
Total cholesterol
5.9 mmol dm–3
3.5 4.5 5.5

4SDs
Total cholesterol
FIGURE 5.4
A representation of a normal distribution of concentrations of total cholesterol in a healthy
population. The mean value is 4.5 mmol dm-3. If the standard deviation of this data set is
0.5 mmol dm-3, then 95% of the individual data points should lie within 4 SDs of the
mean, that is, between 3.5 and 4.5 mmol dm-3. It follows that a total cholesterol result of
5.9 mmol dm-3 is outside this 95% region. It is necessary to stress that we do not take this
result of 5.9 mmol dm-3 to be abnormal. However, in the face of other information, such as
a family history of premature cardiovascular disease, then it may prompt action.
in this case would be hypercholesterolaemia. Certainly, we feel that the individual should be
Cross reference
made aware of this high cholesterol result, and generally instructed on the risk factors for
You can read about
hypercholesterolaemia in atherosclerosis and their contribution to heart attack and stroke.
Chapter 9 ‘Abnormalities of lipid Whilst experienced biomedical scientists will become familiar with the reference range over
metabolism’ in the companion
a period of years, so that they will be able to spot an unusual result promptly, at the practical
book, Clinical Biochemistry.
level, many blood analysers in pathology laboratories (principally in haematology and bio-
chemistry) can be programmed to create an alarm if they consider a result to be so unusual
(and possibly life-threatening) as to warrant immediate attention. This extends to other results
that are outside the normal range. The results sheets that go to the ward or clinic will also have
abnormal results ‘flagged’ – often simply marked with an asterisk, or the letters H (for high)
or L (for low) to draw attention to them.
Although the cholesterol result mentioned above (5.9 mmol dm−3) may be markedly raised
compared with the reference range, it is generally inappropriate to say it is ‘significantly’ raised
in the statistical sense of the word. However, it is possible to define the exact probability that
a single laboratory result is outside the reference range, but this analysis is complex and is
beyond the scope of this chapter. We reserve the use of expressions such as significance and
probability for situations where there is a different mode of analysis, such as that of data from
groups of individuals or sets of data, as we shall now examine.
Analysis of data from two groups of individuals

In looking at data from two groups of individuals, different statistical tests should be used,
depending on the nature of the data and what hypothesis is being tested:
• In comparing continuously variable data from two different groups (such as certain blood
lipids), the Student’s t-test or the Mann–Whitney U-test would be used, depending on
the distribution of the data. i.e. normal or non-normal, respectively
• If a relationship between two sets of continuously variable data within a single group of
individuals is sought (such as between height and weight), then a test of correlation should
be used
• If the data is categorical (such as the proportions of people with cancer in one group com-
pared with another group) the chi-squared test is used
• Paired tests are used to determine any difference between two sets of data from the same
individuals taken at different timepoints, or that are linked in some other manner
We will now look at each of these types of analysis in turn.
Continuously variable data from two different

groups of subjects
Suppose a researcher wants to see if there is a difference in the levels of certain blood lipids
(such as total cholesterol and triacylglycerols) between different groups of people, such as
30 factory workers and 30 office workers. When comparing two groups of data that are con-
tinuously variable, we use the Student’s t-test or the Mann–Whitney U-test to determine if
such a difference is present. Which test to use depends on the distribution of the two sets
of data. If both sets of data are of normal distribution, then the Student’s t-test should be
used. Alternatively, if one or both sets of data are non-normally distributed, we use the Mann–
Whitney U-test. Thus, a key step is to determine the distribution of the data.
It is accepted that total cholesterol is normally distributed, and so we would present the data
as mean and SD. Let us say that the results from the factory workers is that the total cholesterol
is 5.9 (0.7) mmol dm−3, and that the results for the office workers is 5.1 (0.6) mmol dm−3. By
applying the Student’s t-test, a p value of 0.025 is obtained. This is less than our cut-off point
of p < 0.05, so it can be said with confidence (i.e. p = 0.975, or 97.5%) that the difference is
statistically real and is not spurious.
By contrast, serum triacylglycerols have a non-normal distribution, and as such would be pre-
sented with a median and IQR. The results for the factory workers is 1.7 (1.2–2.8) mmol dm−3,
whilst in the office workers it is 1.1 (0.9–1.6) mmol dm−3. This data would be analysed by the
Mann–Whitney U-test, and gives a probability value of p = 0.002. This is a highly significant
difference, as it can be said that the probability that the difference is real is 99.8%, whilst the
probability that the difference is spurious is only 0.2%.
Therefore, there is a difference in total cholesterol levels of p = 0.025, and a difference in

triacylglycerol concentrations of p = 0.002. The questions that therefore follow may include,
‘why are these differences present?’ and, perhaps later, ‘if these differences have clinical con-
sequences, do we need to act on either of them?’
Two sets of continuously variable data

from a single group of subjects
Not only can we compare a set of data (such as serum cholesterol) from two different populations
(such as in factory workers and office workers), we can also look at two different sets of data (such
as serum cholesterol versus blood glucose concentrations) within a single population (perhaps
just the office workers). Another two sets of illustrative indices are the age of an athlete and their
athletic performance (such as distance they can run in a set time). Age is not the sole determinant
of the performance of an athlete, and a fit older athlete with more experience and determination
may well out-perform someone younger and less fit. Correlation is explained in Box 5.5.
BOX 5.5 Correlation
Consider these data from 12 people: their height, weight, age, and distance that they can
run in a certain fixed time period such as 30 minutes.
Subject Height (m) Weight (kg) Age (years) Distance run (m)
1 1.56 70 43 6100
2 1.23 65 56 5200
3 1.70 72 29 6900
4 1.81 84 59 4800
5 1.46 72 34 7100
6 1.50 70 56 4800
7 1.59 66 45 4500
8 1.66 79 72 3700
9 1.70 74 56 5100
10 1.48 69 45 4800
11 1.85 88 67 4200
12 1.66 75 55 5250
By plotting the height and weight for each person in a graph we can obtain a plot such as . . .
2.5
Height (metres)
1.5
0.5
50 60 70 80 90 100
Weight (kilograms)
As it happens, the tallest person (1.85 m) is also the heaviest (88 kg), whilst the shortest
(1.23 m) is also the lightest (65 kg). However, in between these polar extremes, the rela-
tionship is not clear-cut, although an overall trend is present. The correlation coefficient
(r) gives an index of how well this data adheres to a straight line and so is a linear rela-
tionship. Because the two indices have a normal distribution, we use Pearson’s method.
In doing so, we obtain r = 0.85, which indicates a strongly positive relationship. It also
happens that this is statistically significant, as p = 0.001.
Similarly, if we plot each person’s weight against their age we can also get a graph. A
quick look at the plot below also gives the impression that as someone’s age increases,
so does their weight. Indeed, the correlation coefficient for this data is r = 0.56, which
would normally be a ‘respectable’ value. However, the probability that this data is a true
reflection of the relationship between age and weight just fails to reach statistical signifi-
cance as p = 0.06 (i.e. the likelihood of a difference being genuine is 94%).
100
90
Weight (kilograms)
80
70
60
50
20 30 40 50 60 70 80 90
Age (years)
A close look at this data can provide clues as to why this seemingly good data is in fact
not significant. Compared with the first height/weight plot, the data in this weight/age
plot is more spread out (i.e. it has greater variance) and is not as good a straight line. A
second reason why we do not have significance is because of the small numbers of data
points and therefore the low level of statistical power. What this means in practice is
that had we recruited an appropriate number of subjects we would have undoubtedly
obtained significance with p < 0.05.
These first two plots indicate a positive or direct relationship between the indices, i.e. as
one index increases, the other index also increases. However, we say that the relation-
ship is inverse if when one index increases, the other falls. This can be illustrated by the
plot of age versus the distance that the subject can run in 30 minutes.
80
70
60
Age (years)
50
40
30
20
3000 4000 5000 6000 7000 8000 9000
Distance run (metres)
Analysis of this data points to an excellent correlation coefficient where r = 0.87, slightly
better than the relationship between height and weight. Indeed, the probability that this
data truly does reflect a real association is highly significant, with p < 0.001. However, as the
relationship is inverse, then we have to place a minus sign before the correlation coefficient,
i.e. r = −0.87. If all the data points were to be found on a straight line, then the correlation coef-
ficient would be −1. In practice, however, this is almost never found in biomedical science.
An essential aspect of correlation is to be able to define the extent to which the two indices
correlate, and for this one uses a correlation coefficient. This is represented by the Greek
letter rho (r) to define this value. A strong association between two sets of indices would be
represented by a correlation coefficient where r is close to 1 (say, 0.92), whereas one would
consider the relationship to be weak if a small r value of perhaps 0.15 was obtained.
We must be cautious about interpreting correlations. Just because two indices correlate
strongly, does not mean that one causes the other. A crucial comment is always to recall that:
correlation does not imply causation
A suitable example of this is the relationship between height and weight. It is quite well estab-
lished that, in general, tall people are heavier than short people, but it is just as well known
that two people of the same height can have very different weights (and vice versa). It is also
evident to most people that, in general, height correlates with weight. However, is this because
the taller someone is, the heavier they become, or is it that the heavier one is, the taller they
become? The former seems more likely. A better example from biomedical science is from
human epidemiology and clinical studies where it is known that in a large population systolic
blood pressure generally correlates very strongly with diastolic blood pressure. However, the
increased systolic value does not cause the diastolic value to rise; and both systolic and diasto-
lic blood pressures rise in parallel and do so independently of one another.
An amusing example of a relationship between correlation and causation occurred dur-

ing the 1950s, when the rise in the import of bananas into the United Kingdom correlated
strongly with an increase in the divorce rate. It could be argued that the increased consump-
tion of bananas caused marital problems and, hence, a rise in the divorce rate or, equally,
that newly single people eat more bananas once they are divorced. Both hypotheses are
interesting.
As with comparing two groups of data from different populations, where we use the Student’s
t-test if the data is normally distributed, and the Mann–Whitney U-test if it is non-normally
distributed, we have a choice of different tests of correlation. If the two sets of data have
a normal distribution (such as height and weight), we use Pearson’s correlation method.
However, if one or both sets of data have a non-normal distribution (such as total cholesterol
and triacylglycerols) we have to use Spearman’s correlation method.
Analysis of continuous data obtained

at two time points
So far, we have been looking at data that is generally obtained at a single time point, but it is
also necessary to be able to assess whether or not there is a change in a particular index in a
group of people at two time points. This type of data is called ‘paired’ because there are always
pairs of figures (i.e. before and after), never just a single point. In clinical medicine, as part of
the development of new therapeutics, we need to know how the new drug genuinely influ-
ences the biological system it is designed to act upon. For example, to be convinced that a new
drug designed to lower the blood pressure actually does so, one needs data on the reduction
of systolic blood pressure and/or diastolic blood pressure from the same people before they
are placed on the drug, and then again later once they have been taking the drug for perhaps
weeks or months. This is shown in Box 5.6.
As before, the exact statistical test used depends on the nature of the distribution of the data:
if the difference between the two sets of data is normally distributed, then a paired t-test
BOX 5.6 Data at two time points
Suppose we wish to determine the effect of a new drug on the functioning of an organ
like the kidney. One of the most useful indices to test renal function is the concentra-
tion of creatinine in the blood. We must first identify a group of patients with kidney
problems who will therefore be likely to have abnormal levels of serum creatinine. We
need to obtain a blood sample before the drug is issued, and then place the patients
on the new drug for perhaps several months, and then take a second sample of blood.
Typical results may be:
Concentration of creatinine (mmol dm -3)
Patient number Before the drug After the drug Difference

1 150 126 –24
2 225 196 –29 Cross reference

Renal function is described in
3 176 168 –8
Chapter 3 ‘Kidney disease’ in the
4 166 172 +6 textbook Clinical Biochemistry.
5 145 140 –5
6 226 196 –30
7 189 168 –21
8 168 173 +5
9 173 146 –27
10 149 144 –5
11 171 155 –16
12 156 149 –7
Mean (SD) 174 (27) 161 (21) –13 (13)
Median (IQR) 169 (151–186) 161 (144–172) –12 (–5–26)
Note that in 10 of the 12 patients, the level of creatinine has decreased. However, in two
patients (numbers 4 and 8) levels have increased. Nevertheless, overall, there has been
a decrease in creatinine that, assuming no other changes in the lives of the patients,
implies that the drug may be active in alleviating the renal disease in these patients.
However, to be fully confident, we need to apply the correct statistical test to the data.
The choice is a paired t-test, or the Wilcoxon test. Although the mean difference (–13)
is the same as the SD (13), it is virtually the same as the median difference (–12), so we
can be fairly confident that the data is normally distributed and so the test to be used
would be a paired t-test. This test will give p = 0.004, which is considerably smaller than
the required cut-off point for statistical significance of p < 0.05. In fact, this p-value tells
use we can be 99.6% confident that the effect is real, and only 0.4% confident that the
difference is due to chance.
NB. Strictly speaking, in this type of study, we should also measure creatinine in a similar
group of patients over the same time period who have not been taking this particular
drug. We call these subjects the control group – this is a vital consideration when design-
ing and carrying out experiments.
is an appropriate test. However, if the difference between the two sets of data is distributed
non-normally, then Wilcoxon’s test should be used.
Paired analyses need not be linked in time – they may be linked in other ways, such as the
circumference of the left arm compared with that of the right arm in the same person, or the
difference in a component of the blood measured in serum compared with plasma.
Analysis of two sets of categorical data

An example of categorical data is the number of people who fall into only one of a small
number of well-defined categories, such as being male or female, whether or not you smoke
cigarettes, your ABO blood group, or one’s marital status. In pathology, alternatives may be
the presence of a certain cancer, the presence of a condition such as diabetes, or a history of
heart disease.
Let us use diabetes and heart disease as an example, and form an hypothesis such as ‘people
with diabetes have more heart disease than people without diabetes’. In order to test this
hypothesis we need to examine the presence of heart disease in two groups of people: those
with diabetes and those free of diabetes. Those free of diabetes we call the control group. First,
as with the example of levels of cholesterol in factory workers versus office workers, we need
to find out how many people to recruit to have sufficient statistical power for our result to be
reliable; this may be 100 people in each group. As with the definition of a normal or a non-
normal distribution, there are statistical programs available that can tell you the exact number
of people you need to recruit.
If we find that 35 of the 100 people with diabetes have heart disease, and that 20 people
free of diabetes have heart disease, is this seemingly large difference in proportion (i.e. 35%
versus 20%) statistically significant? The most appropriate test to use in this instance is the
chi-squared test, which effectively compares the proportions of those with and without heart
disease in the diabetes group and control group free of this risk factor. We summarize this data
in Table 5.1.
TABLE 5.1 Number of people in each group
Diabetics Non-diabetics Total

With heart disease 35 65 100
Free of heart disease 20 80 100
Total 55 145 200

The chi-squared test gives us a probability that the difference is real at p = 0.018 (that is, the
probability that the results are down to chance is just 1.8%, making it 98.2% likely that the
results are real, and are due to some genuine difference). The result of p = 0.018 is less than
the cut-off point of 0.05 so our results support the hypothesis that diabetes is associated with
excess heart disease – an established finding in human pathology.
Data from three or more groups of individuals

Analysis becomes increasingly complicated once we move away from two to three or more
groups. The biggest difference is that the simple t-test and the Mann–Whitney U-test are
appropriate only in the analysis of two groups. When we have three on more groups to analyse,
we have to use a different test. Analysis of variance (ANOVA) is the correct test to use when
all the groups have a normal distribution. However, if the distribution of even one of these
multiple groups has a non-normal distribution, we have to use the Kruskall–Wallis test.
Figure 5.5 illustrates these points. The top part shows three sets of data: ‘A’, ‘B’, and ‘C’. A cursory
‘eyeball’ view of the spread of this data gives the impression that it is normally distributed, as it
indeed is, with most of the individual data points clustered around the central section of each
of the data sets. So we have to use the ANOVA test to look for differences in this data, and in
doing so we get p = 0.001. What this means in practice is that the probability of this difference
being due to chance is only 0.1%. So around the other way, this means it is highly likely (to
99.9%) that there is a real difference between some of the three individual data sets. What can’t
be done is a series of different t-tests between each pair in turn, i.e. ‘A’ versus ‘B’, then ‘A’ versus
‘C’, and then ‘B’ versus ‘C’. This may well lead to error.
SELF-CHECK 5.4
The mean, median, and SD for the data sets ‘A’, ‘B’, and ‘C’ in Figure 5.5 are as follows:
‘A’: 100, 100, 15
‘B’: 79, 82, 17
‘C’: 146, 150, 17
Although we should use a formal test to confirm the distribution of the data, it is possible to
get an impression of distribution by looking at the values of the mean, median, and SD, as we
did in Section 5.1. Now confirm with yourself that the three sets of data all seem to have a
normal distribution.
However, ANOVA does not tell us exactly where the difference is to be found. Is it between ‘A’
and ‘B’, or between ‘A’ and ‘C’, or is it between ‘B’ and ‘C’? To do this we have to use a second
test. There are several to choose from, but Tukey’s post-hoc test is one of the most popular
(another is Dunnett’s test).
Using this test, we find that there is a significant difference between ‘A’ and ‘B’, between ‘B’ and
‘C’, and between ‘A’ and ‘C’, each with a probability value of p < 0.05.
The lower part of Figure 5.5 has three different data sets: ‘X’, ‘Y’, and ‘Z’. We can again do an
‘eyeball’ examination of the spread of this data, but it is distributed in a different manner to
groups ‘A’, ‘B’, and ‘C’. In each of ‘X’, ‘Y’, and ‘Z’ most of the data points are clustered not in the
central part of the entire set (as they are in ‘A’, ‘B’, and ‘C’), but instead are skewed over to the
far left of each group, suggesting that each set has a non-normal distribution, which indeed
is correct.
Dotplot for A–C
A
50 100 150
B
50 100 150
C
50 100 150
FIGURE 5.5
The top figure (Set A–C) shows three data sets on a common
scale. As each data set has a normal distribution, the combined
group must be analysed by one way analysis of variance. If Dotplot for X–Z
significance is found (i.e. p < 0.05), then an additional test (such
as Tukey’s test) must be performed to find out where such a
difference lies. In the present set, it is likely that each individual
set is statistically different from the two other sets. The lower
figure (Set X–Z) shows three data sets on a different scale. Unlike X
50 100 150 200 250 300
A–C, the patterns in X–Z are non-normal, as the bulk of the
data points are skewed to the left. Because these data sets have
a non-normal distribution, then the Kruskal–Wallis test must
be used to determine if there is a difference between sets X, Y,
Y
and Z. However, if there is indeed a significant difference (i.e. 50 100 150 200 250 300
p < 0.05), we cannot use Tukey’s test to determine where the
difference is because the latter can only be used when the data
has a normal distribution. One way round this is to convert the
distribution of the non-normal data set to a normal distribution, Z
and then perform Tukey’s test. 50 100 150 200 250 300
SELF-CHECK 5.5
The mean, median, and SD for ‘X’, ‘Y’, and ‘Z’ are as follows:
‘X’: 150, 115, 121

‘Y’: 106, 85, 71
‘Z’: 257, 190, 176
As in the previous self-check, it is possible to get an impression of distribution by simply look-

ing at the values of the mean, median, and SD. Again, look at the data – what factors lead you
to suspect that these data have a non-normal distribution?
Just as it is inappropriate to use a series of individual t-tests when three groups of data are
normally distributed (as in ‘A’, ‘B’, and ‘C’), one cannot simply do a series of individual Mann–
Whitney U-tests between ‘X’ and ‘Y’, then between ‘X’ and ‘Z’, then between ‘Y’ and ‘Z’. Instead,
the correct test to use is the Kruskall–Wallis test, which in this case again gives a high probability
(i.e. p = 0.001) that there is a genuine difference between some of the groups. However, like
the ANOVA test, the Kruskall–Wallis test does not tell us exactly which pairs of data are signifi-
cantly different from each other, and to achieve this we have to use an additional test, such as
Tukey’s test, to which you have already been introduced.
The problem is that Tukey’s test is designed to analyse data that has a normal distribution, and
our data sets (‘X’, ‘Y’, and ‘Z’) all have a non-normal distribution. One way of getting around
this is to convert data that is non-normally distributed into data that is normally distributed.
This can be achieved by transforming the data into a logarithmic form that frequently (but not
always) converts it to a normal distribution. This is called logarithmic transformation.
So once the data sets ‘X’, ‘Y’, and ‘Z’ data has been log transformed, and is effectively normal, an
ANOVA followed by the Tukey test can be performed. In this case we find a statistically significant
difference between ‘X’ and ‘Z’, and between ‘Y’ and ‘Z’ (both with a probability of p < 0.05), but
the difference between ‘X’ and ‘Y’ was not significant.
Once more, analyses of these sorts are virtually impossible to do without a good statistical
software package running on a computer.
Correlation of multiple groups

The ability to correlate three or more continuously variable sets of data at the same time is
extremely complex and is very rarely required in biomedical science. Consequently, it is far
beyond the scope of this chapter.
Analysis of multiple groups of categorical data

In analysing categorical data, we generally use the chi-squared test. Almost exactly the same
analyses are performed when we look at three or more groups as for two groups. An example
of this could be the number of children born to 293 women who live in different areas (143
live in Area One and 150 live in Area Two). This data is summarized in Table 5.2.
If we simply look at the percentages of the women in each of the three groups (no children,
one child, or two or more children), we find 24, 46, and 30%, respectively, in each group for
the women in Area One. Similarly, the percentages for women in Area Two are 17, 62, and
21%, respectively. A quick view of this data seems to suggest a difference – for example, 46%
would generally be thought of as being considerably less than 62%. However, statisticians do
not make claims simply by suggestion – a formal statistical test must be used to confirm our
suspicion.
If we apply the chi-squared test to this data we get p = 0.025. This tells us that there is a high
probability (i.e. 97.5%) that there is a meaningful difference, and only a 2.5% chance that the
TABLE 5.2 Number of children born to women in different areas
No children One child Two or more Total number

children of women
Women in Area One 35 66 42 143
Women in Area Two 26 93 31 150
Total 61 159 73 293

TABLE 5.3 Comparing women with no children or with one child
No children One child

Women in Area One 35 66
Women in Area Two 26 93
TABLE 5.4 Comparing women with one child or with two or more children
One child Two or more children

TABLE 5.5 Comparing women with no children or with two or more children
No children Two or more children

differences are spurious. So it appears that women in Area One do indeed have a different
distribution of the number of their children than the women in Area Two. However, as for the
ANOVA and Kruskall–Wallis tests, we cannot tell exactly where this difference is. In Area One,
is the group of 66 women (i.e. 46% of the whole group) with one child really different from the
93 women (62% of the whole group) with one child in Area Two? In order to do this we have
to break up the data set and then perform individual chi-squared tests on each pair of data
points as shown in Tables 5.3, 5.4, and 5.5.
A chi-squared analysis of the data in Table 5.3 gives a probability of p = 0.034. This means that
the difference in proportions between the women in Area One compared with the women in
Area Two is 96.6% is likely to be due to the ‘Area’ and not due to some other unknown factor
(which would have a likelihood of 3.4%). A chi-squared analysis of the data (Table 5.4) gives a
probability of p = 0.023 which means that the difference in proportions between the women
in Area One compared with the women in Area Two is 97.7% likely to be due to the ‘Area’ and
not due to some other unknown factor (which would have a likelihood of 2.3%). A chi-squared
analysis of the data in Table 5.5 gives a probability of p = 0.985. This indicates that the differ-
ence in proportions is too small to be statistically meaningful. Indeed, that the likelihood of the
difference being due to the difference in area is only 1.5%.
So in summary we can say that the difference in the proportions of the number of children
born to 293 women living in two different areas is statistically significant to the level of
p = 0.025 (i.e. we are 97.5% confident that the difference is genuine and only 2.5% as being
due to chance). Looking at this group in more detail, we can say that this difference is due
to a difference in the proportion of women with no children versus women with one child
(p = 0.023), and also in the proportions of women with one child or with two or more
children (p = 0.034). However, the difference in the proportion of women with no children
compared with the proportion of women with two or more children was not statistically
significant (p = 0.985).
Analysis of three or more sets of linked data

You have already seen how to analyse paired samples of plasma taken at two different time
points or linked together in some other way. Similar analyses are possible for the assessment
of data obtained at three or more time points or linked by three or more factors.
Consider the development of a new drug designed to reduce the numbers of platelets in the
blood. It is necessary to know how long the drug takes to have an impact. Is it days, weeks, or
months? Such an experiment would have to obtain blood samples on different occasions, then
determine the platelet counts, and then submit the data to two-way analysis of variance,
although this may also be known as repeated measures analysis of variance, or Friedman’s
ANOVA. It is described as two-way ANOVA because we need to be able to factor in the pos-
sible effects of differences (a) between patients, and also (b) between different stages of the
experiment. This is described in Box 5.7.
BOX 5.7 Two-way analysis of variance
Consider these data from 10 people: the platelet counts experiment in subjects 5 and 9 both seem to be consider-
(units: 109/cm3) have been measured before, and on three ably less (256 and 265, respectively) than the averages from
weekly occasions after the introduction of a new drug. subjects 4 and 10 (369 and 390, respectively). Indeed, the
probability that the difference between subjects is statisti-
Although the mean platelet count falls from 324 to 312 in
cally significant is p = 0.001.
just a week, is this a significant drop? Note that levels then
marginally rise to a mean of 315 after 2 weeks, then fall to Thus it may be that any difference in the overall result may in
a mean of 292 after 3 weeks. Overall, there is a significant fact be due to changes in only some of the subjects, which may
change in the mean values with a likelihood of p = 0.002. not be truly representative of the entire data set. The com-
However, note also the variation in results from different plicated and powerful mathematics of the two-way ANOVA
subjects. The average platelet counts over the whole of the adjusts for any possible differences in particular groups.
Subject Before drug use After 1 week After 2 weeks After 3 weeks
of the drug of the drug of the drug
1 299 278 284 256
2 314 325 311 289
3 287 281 294 271
4 379 376 366 356
5 269 252 261 241
6 312 311 335 326
7 325 329 356 313
8 378 311 278 251
9 267 275 261 258
10 412 387 404 356
Mean 324 312 315 292
SD 50 44 49 43
Data need not be linked by time in order to be analysed by this method. For example, we
would use a two-way ANOVA to analyse levels of a component of the blood from the same
person that has been measured in serum, in plasma anticoagulated with sodium citrate, and
also in plasma anticoagulated with EDTA.
Finally, Figures 5.6 and 5.7 provide summaries and flow paths for some of the different meth-
ods of analysis we have been looking at, based on the nature of the data, and on how to
present the data. Figure 5.6 refers to two sets of data, Figure 5.7 is three sets of data.
5.3 Statistical packages

Efficient handling and analysis of data is impossible without a good statistical package. As
effective giant calculators, these packages can be purchased direct from the manufacturer,
although in practice many large organizations, such as hospitals and universities will have site
licences, allowing a bona-fide individual almost immediate access. Indeed, higher and fur-
ther Education Institutions are almost certain to have a Department of Mathematics and/or
Statistics from where packages, and advice, can be sourced.
Simple analysis of two groups of

quantitative data
Is your data
categorical or
continuous?
Categorical:
e.g. male or female, Continuous:
with or without a e.g. blood pressure
particular disease
Use the chi-squared Determine the nature of the

test for proportions distribution: is it normally
or non-normally
distributed?
If normally distributed, If non-normally

present your data with distributed, present your
standard deviation data as median with
inter-quartile range
FIGURE 5.6
Flow chart for analysing two
sets of data. For categorical
data, use the path on the left. Analyse the two groups Analyse the two
For continuous data, use the using the Student’s groups using the
t-test Mann—Whitney test
path on the right.
5.3 STATISTICAL PACK AGES 121
Simple analysis of three or more

groups of quantitative data
Is your data
categorical or
continuous?
Categorical: Continuous:
e.g. ABO blood group e.g. height, blood pressure
Use the chi-squared Decide on the nature of the

test for proportions distribution: is it normally
or non-normally
distributed?
If p > 0.05, use individual
chi-squared tests
between groups
If normally distributed, If non-normally
present your data as distributed, present your
mean with standard data as median with
deviation inter-quartile range
Analyse the groups Analyse the groups using

using analysis of the Kruskall—Wallis U
variance to see if there test to see if there is an
is an overall difference overall difference
Only if p < 0.05, move on to determine if there are any

differences between the groups
FIGURE 5.7
Flow chart for analysing three
or more sets of data. For
Seek differences between the Convert the data to a categorical data, use the path
groups with post-hoc analysis normal distribution
on the left. For continuous
such as Tukey’s test e.g. by taking the log
data, use the path on the right.
Although claiming to be user-friendly, these packages are often complicated and are certainly
not for the untutored. It is essential that those fresh to the subject complete a formal training
programme, generally offered by their host organization (hospital, university, pharmaceutical
company), although these can also be offered by commercial training organizations.
However, even the most sophisticated statistical packages are unable to counter the biggest
problem in data management that is, the reliability of the data that is being entered. Even the
most powerful computers and statistical packages are unable to correct data that is either
incorrectly derived at source or is incorrectly entered by an operator – hence, the concept of
‘garbage in – garbage out’. This is why the concepts of quality control and quality assurance
are so important.
The best known statistical packages are (in no particular order), Minitab, SPSS, SAS, and Stata.
All of these offer the most simple tests (such as Student’s t-test and the chi-squared test) to
the most complex analyses, but some are certainly more user-friendly to the occasional user,
whilst others are designed for the full-time statistician. These packages often come with soft-
ware to generate plots and figures (such as the figures in this chapter). Advice can generally be
obtained from experienced users, themselves often in formal statistical units. Table 5.6 sum-
marizes key aspects of the uses of statistical software.
TABLE 5.6 Examples of the need for statistical software
• Determination of the nature of the distribution of continuously variable data (i.e. normal
or non-normal)
• Determination of the number of subjects to be recruited in order to adequately test your

hypothesis (i.e. a power calculation)
• Analysis of continuously variable data (e.g. Student’s t-test and the Mann–Whitney U-test)
• Analysis of categorical data (e.g. the chi-squared test)
• Generation of a correlation coefficient and graph showing the relationship between two
indices
SUMMARY
■ Information can be as letters and words (i.e. qualitative) or as numbers (i.e. quantitative).
Quantitative data can be categorical or continuously variable
■ The central point of a continuously variable data set is the mean (the average) or the
median (the middle)
■ Variance is used to describe the extent to which the individual points of a data set are
tightly bunched together around the central point, or are widely spread out. The standard
deviation is used to quantify the variance when the central point is the mean. The inter-
quartile range is used to quantify the variance when the central point is the median
■ When data is normally distributed, the central point is the mean. When data is non-
normally distributed, the central point is the median
■ Statistically significant difference is present when a difference between two data sets is
proven by a mathematical analysis to be significant
■ The reference range is a group of numbers used to provide an idea of expected or desir-
able results
■ Correlation describes the extent to which there is a relationship between two sets of
continuously variable data
■ The Student’s t-test is the method for determining a difference between two sets of
data that are both normally distributed. The Mann–Whitney U-test is the method for
QUESTIONS 123
determining a difference between two sets of data where at least one is non-normally
distributed
■ Statistical power is the extent to which your data set can be relied on to give valid results
■ Probability (abbreviated to p) is the extent to which your result is truly a consequence

of a real effect (such as the disease process), or is different merely because of chance or
co-incidence. Generally, when p is less than 0.05 (i.e. p < 0.05) a difference is statistically
significant
■ The chi-squared test is used to look for a difference in the proportions of a particular index
(such as ABO blood groups) between groups of individuals
■ Data at two time points can be analysed by a paired t-test or Wilcoxon’s test
■ When looking for a relationship between two sets of data from the same individuals, we
use Pearson’s or Spearman’s correlation methods
■ When analysing three or more sets of data, we use ANOVA or the Kruskal–Wallis test.
The chi-squared test can be used to search for difference in the proportions of data from
three or more groups. Two-way ANOVA is used to analyse three or more sets of data that are
linked, perhaps by time
■ An appropriate statistical software package is essential to allow accurate statistical analy-

ses. These packages can provide all the common statistical tests, including t-test, Mann–
Whitney, and chi-squared tests. The package will also provide a correlation coefficient and
tell you how much power you have.
FURTHER READING
● Altman DG. Practical Statistics for Medical Research. Chapman & Hall, London, 1991.
● Daly F, Hand DJ, Jones MC, Lunn AD, McConway KJ. Elements of Statistics. The Open
University/Addison Wesley, Maidenhead, 1995.
● Holmes D, Moody P, Dine D. Research Methods for the Biosciences. Oxford University
Press, Oxford, 2006.
● Petrie A, Sabin C. Medical Statistics at a Glance, 2nd edn. Blackwell, Oxford, 2004.
● Swinscrow TDV. Statistics at Square One, 9th edn (revised by Campbell MY). BMJ
Books, London, 1996.
QUESTIONS
1. Calculate (to the nearest whole number) the mean and median of the data set: 25, 37, 29,
30, 43, 32 and 22.
2. A project assessed the relationship between indices A and B in 30 people, and found a
correlation coefficient (r) of 0.36. Another project found the correlation between C and D
in the same population to be r = 0.45. Which relationship is likely to be more significant?
3. Which test is used to search for a difference between three data sets, all of which have a
normal distribution?
4. True or False: A data set with a mean of 23.4 and a standard deviation of 21.2 is likely to
have a normal distribution.
5. Consider the two sets of data:
Set A: 124, 99, 346, 150, 115, 140, 77

Set B: 120, 179, 81, 96, 143, 118, 285
Which of the following statements is TRUE?
(a) Both sets of data are normally distributed

(b) The median value of Set A is 124
(c) A difference between the two sets can be determined by the chi-squared test
(d) The two data sets can be correlated and will give a positive r value
(e) The inter-quartile range of Set B is 96–179
6
Preparing and
measuring reagents
Ian Graham
Learning objectives
■ Recognize the key features of top pan and analytical balances, and be able to use them to
weigh materials
■ Demonstrate the need for correct installation, calibration, and maintenance of balances
■ Distinguish between ways of measuring and delivering volumes of liquids, and demonstrate
an appropriate selection and application of volume measurement
■ Recognize the key features of pipettors and demonstrate their correct use in measuring and
delivering volumes
■ Know why there is a need for calibration and maintenance of pipettors
■ Distinguish between different ways of expressing concentrations and show how dilution
affects concentration
■ Demonstrate the ability to calculate concentrations, know how to prepare solutions of a
particular concentration and make dilutions
■ Show an awareness of correct practical procedures and health and safety requirements
Introduction
As a biomedical scientist, you will undertake much practical work where quantities of materi-
als and solutions must be measured and delivered. You may have to dilute a blood sample
with a solution such as saline, mix volumes of different solutions in a colorimetric assay or, per-
haps, dilute a microbial suspension. Solutions may be bought in ready prepared or they may
be prepared by yourself or others within the laboratory. In large laboratories, many routine
tests will be automated and autoanalysers will make volume measurements and mix together
different solutions. Although you may not have to prepare many solutions or undertake sig-
nificant amounts of volume measurement and delivery, it is essential you understand how to
126 6 PREPARING AND MEASURING REAGENTS
BOX 6.1 The SI system and units

of volume
Europe and much of the rest of the world uses a set of units of measurement called
SI units (SI stands for Le Système International d’Unités). Within the SI system, volume
measurements are based upon the m3, which is equivalent to 103 L. However, the USA
and some other countries have not fully adopted SI units, and volumetric glassware
and pipettes are usually calibrated in L, mL and lL. For these reasons, volumes will be
quoted in both sets of units as appropriate. Table 6.1 shows the relationship between SI
(based on the m3) and non-SI (based on the L) volume measurements.
TABLE 6.1 A comparison of SI (m3) and non-SI (L) volume

measurements
SI based volume m3 equivalent dm3/L based equivalent

3
m 103 dm3/L
dm3 10−3 m3 1 dm3/ L
cm3 10−6 m3 1 cm3/mL
mm3 10−9 m3 1 μL
undertake these activities as some assays may be performed manually. You need to be able to
use accurately balances and pipettes, or other volume measurement devices and know how
Cross reference to make solutions, perform dilutions, and understand the effects of dilution upon concentra-
You can read more about tion (see Box 6.1 for the SI system and units of volume). These are essential and fundamental
automation in Chapter 17
skills for all biomedical scientists and the quality of your work depends upon their correct
‘Laboratory automation’.
application.
6.1 Balances and weighing

This section will consider:
• Key features of balances and their use
• Care and maintenance
• Safe working practices
Types of balance
Several types of balance are available. The simplest are beam balances, but more sophisticated
top pan and analytical balances are normally used in clinical laboratories. You should be able to
Cross reference recognize Figure 6.1 as balances. Top pan and analytical balances are mainly used to measure a
Centrifugation techniques particular amount of a solid for preparing a solution, but they can also be used to weigh liquids.
are described in Chapter 12
Simple beam balances have limited applications in laboratories. For example, they can be used
‘Centrifugation’.
in balancing a pair of centrifuge tubes that contain a suspension prior to centrifugation.
6.1 BAL ANCES AND WEIGHING 127
(a) (b)
FIGURE 6.1
Photographs showing
(a) a top pan and (b) an
analytical balance.
The type of balance used determines whether mass or weight is being measured. An item
is ‘weighed’ on a balance, but there is an important difference between mass and weight.
Weight is a force (measured in, for example, Newtons, grams, or pounds) reflecting the effect
of gravitational force at a particular location upon a mass (measured in, for example, grams or
kilograms). Mass does not vary and is a measure of the amount of matter present. Although
the two are related, the weight of a given mass will change if the gravitational force varies.
A beam balance measures mass while spring balances and top pan balances measure a force,
the weight of the item. Why this difference?
A beam balance consists of a beam supported at the midpoint by a near friction-free fulcrum.
The sample is placed in a weighing or measuring pan at one end of the beam and one or
more standard masses are placed in a scale pan at the other end to achieve equilibrium. The
sum of the standard masses in the scale pan when balance is achieved gives the mass of the
sample because gravity applies equally to each side of the fulcrum point. Thus, beam balances
measure mass not weight. Variants of this arrangement have been introduced over the years.
An asymmetric fulcrum arrangement reduces the requirement for large reference masses. For
larger-scale applications, a floating platform may be provided by using a cantilever system.
By comparison, spring and top pan balances are force-measuring devices, and have different
forms of construction and operation that give weight measurements. Although they measure
weight, the requirement for top pan and analytical balances to be calibrated means they can
be used to measure mass. In practice, the terms weight and weighing have such common
usage, they tend to be applied whether mass or weight is actually being measured.
SELF-CHECK 6.1
If you were a scientist on a manned lunar mission with a top pan balance as part of your
equipment, would the mass and weight of an object be the same on earth and the moon or
different?
Top pan and analytical balances differ in appearance to a beam balance, they include elec-
tronics, have digital displays, and are easier and more convenient to use. High levels of meas-
uring precision are achieved by combining the application of mass changes, and a spring force
in achieving the final balance.
Top pan and analytical balances

Top pan and analytical balances work in a different way from the simple mechanical beam
balance. They are compact, robust, and measure to high levels of accuracy and precision.
The routine top pan balance may weigh to within 0.1 g or 0.01 g and cover a weighing range
or capacity, up to 200 g, 1000 g, or more. They have a broad range of weighing applications,
are simple to use, relatively cheap and robust if not misused. Analytical balances are a variant
of the general top pan balance, measuring to within 1 mg (10−3 g) or less, and fully enclosed
within a draught and dust shield that incorporates several sliding doors. Inevitably, these are
more expensive and used for more demanding purposes where only small quantities are to be
measured. Catalogues for electronic balances refer to readability, which is simply the number
of decimal places to which that particular balance can be used. A balance with a readability of
0.1 g would be used in general applications that differ from the more demanding applications
of one with a readability of 0.0001 g.
SELF-CHECK 6.2
Which one of the following sets of features would you associate with an analytical balance?
(a) Cheap to buy, high levels of readability, includes a draught and dust shield, generally used
for weighing smaller quantities
(b) Relatively expensive to buy, low levels of readability, includes a draught and dust shield,
generally used for weighing larger quantities
(c) Relatively expensive to buy, high levels of readability, includes a draught and dust shield,
generally used for weighing smaller quantities
(d) Relatively expensive to buy, high levels of readability, lacks a draught and dust shield, used
for weighing larger quantities.
Whatever the type, modern electronic balances are designed for a long service life with minimal
maintenance if not abused. However, they do require regular recalibration to ensure accurate
and consistent measurements.
Uses of laboratory balances

Normally, a balance would be used to weigh dry or partially hydrated solid materials to be
used in the production of a solution. Section 6.4 of this chapter considers matters related
to the production of solutions and the effects of dilutions. The amount of material needed
depends upon both the concentration and the total volume of the solution to be prepared.
Although it is relatively easy to calculate the required amount to be weighed, in a clinical labo-
ratory a standard protocol is usually followed, with amounts already determined. Balances can
also be used to measure volumes as shown in Box 6.2.
Correct installation of a balance

Top pan and analytical balances are portable. It is essential that they are correctly located and
installed, since several factors can affect their use, including:
• Draughts, which are caused by open windows and doors, rising warm air from radiators,
other hot air sources, and air conditioning ducts
• Sources of vibration or sudden movements
• Magnetic fields from some types of adjacent equipment
• A non-level, unstable support surface

6.1 BAL ANCES AND WEIGHING 129
BOX 6.2 Measuring liquid volumes using

a balance
Balances are usually associated with the weighing of solid materials. There are occasions
where it is more convenient to weigh a liquid as opposed to measuring and delivering
a particular volume of that liquid. To do this a weighing container suitable for the liquid
will be required and the density of the liquid at the ambient temperature (density is
temperature dependent) needs to be known.
density = mass/volume
Therefore
mass = density × volume
The required mass can be calculated using the density of the liquid. For example, if a
volume of 25.0 cm3 is required where the liquid density is 1.06 g cm−3 at laboratory
temperature, the calculation is:
1.06 g cm−3 (density) × 25.0 cm3 (volume) = 26.5 g (mass required)
SELF-CHECK 6.3
Glycerol is a viscous liquid sugar alcohol with a density of 1.261 g cm−3. How could you use a
top pan balance to measure a 15 cm3 volume of this liquid?
These are relatively easy to identify and rectify. For example, to level correctly, balances have a
level indicator and several adjustable feet.
Choice of balance
The choice of an appropriate balance to use is dictated by two key factors, the weight to
be measured (capacity of the balance) and the degree of accuracy of the measurement
(readability of the balance). For example, if 15.624 g of glucose were required to prepare a
solution for use in an assay, a 1000-g capacity balance with a readability of 0.1 g would be
inappropriate. However, a 65-g capacity balance with a readability of 0.1 mg (10−4 g) would
be a sensible choice.
Balance calibration
Some anticipation of use is required as balances may take some time to warm up and
self-check. In a general purpose laboratory, it is likely that balances will be kept switched on.
Once on, the balance needs to be set for the weighing units and mode of use that is required.
These are generally gram units and tare operation, respectively. The process of taring sets the
balance readout to 0 g when an empty container is placed on the pan. When material is added
to the empty container the readout is therefore an indication of the total weight of material in
the container. This makes the weighing simple. Prior to use, any spilt material left by a previ-
ous user must be carefully removed to avoid contamination of future samples and damage to
the balance.
Key Points
Balances are often operated in tare mode so that the displayed weight only measures
the material added to the weighing container, excluding the weight of the container
itself.
There must be an established schedule of balance calibration in the laboratory, and it is

useful to understand how it is achieved. When supplied new, a balance will have been cali-
brated at the factory, but regular rechecking is required to ensure that the calibration and,
hence, accuracy of the instrument is not lost. This is particularly necessary when a balance
has been moved due to the influences of location, temperature, and possibly risk of physical
knocks. The commonest balances are top pan types, which measure weight. Their calibration
METHOD How to use a balance
■ Before commencing the weighing process, you must be before adjusting the amount of added material in order
aware of any issues that may affect how you proceed. to achieve the desired weight. Many balances have a
Relevant health and safety information needs to be read communications port that allows the attachment of a
and potential problems with the material understood. printer or computer, and the recording of data if this
This will determine how the material can be safely is required.
handled, how solid and solution containers should be ■ The weight of the container into which the material is
labelled and how the material should be disposed if placed needs to be as low as possible compared with
spilt. Where handling of the material may release a dust that of material to be added so as to maximize the weight
you will need to proceed with caution. change. For instance, the addition of 0.50 g of material
■ Samples should reach ambient temperature before to a 0.50 g weighing boat would result in a 0.50–1.00 g
being weighed to avoid condensation onto the mate- weight change. The addition of the same amount to
rial and to reduce air currents around the weighing pan. a 100.00 g glass container would result in a change to
Material that is hygroscopic (absorbs or adsorbs water 100.50 g, a proportionally smaller weight change.
from the surroundings) or that loses water rapidly
■ Electronic balances are precision instruments and the
should be placed in a sealed container before weigh-
self-checking procedures, occasionally result in error
ing. The same precaution will be required where vola-
codes on the digital display. Should an error code
tile liquids are weighed.
appear, its meaning can be found in the manufacturer’s
■ Some form of container for the material to be weighed operation manual.
will be required, a plastic or foil weighing boat for small
■ A balance must be cleaned of spilt material after each
amounts or possibly some form of glassware for larger
period of use for which a brush can be left near the
amounts. A clean weighing container and spatula are
balance for this purpose.
essential to avoid contamination.
■ Balances require minimal cleaning and a wipe with
■ The commonest mode of using a balance is with tare
a soft cloth moistened with a dilute mild detergent
selected, but the procedure will vary with the model of
solution is usually sufficient.
balance. First, the empty weighing container should be
placed centrally on the pan, followed by a pause until ■ There may be circumstances where a balance is located
there is an indication that the balance has stabilized away from the open laboratory because of some poten-
(the response time varies with model). The balance is tially harmful feature of the material handled. The rea-
then tared so that the readout is zero. Material is gradu- sons for this must be clearly identified near the temporary
ally added to the weighing container using a spatula, location, so that other laboratory users are not inadvert-
remembering to pause until the balance is stabilized ently exposed to an unnecessary risk.
6.2 VOLUME MEASUREMENTS AND DELIVERY 131
involves checking against known standard calibration weights, which may be either built in, or
externally supplied and placed on the weighing pan. Two common approaches to calibration
are the simpler span calibration and more sophisticated linearity calibration methods.
Span calibration uses two weight values, zero and a near to or at capacity value to check
the balance. The linearity calibration approach uses an additional value near the middle of
the weighing range. This latter approach reduces the likely extent of deviation between the Cross reference
true and displayed weight. The procedure used will depend upon the balance make and Health and safety issues are
described in Chapter 4 ‘Health
model. Calibration checks on a regular basis are clearly essential. The Method Box ‘How to
and safety’.
use a balance’ summarizes these points.
SELF-CHECK 6.4
Why is it necessary that top pan and analytical balances are correctly located and calibrated?
Volume measurements
6.2
and delivery
• The selection of appropriate procedures for measuring volumes
• Advantages and disadvantages of different approaches
When preparing solutions by dissolving a solute in a solvent, diluting a more concentrated

solution, or delivering specific sample volumes, an appropriate means of volume measure-
ment and delivery must be chosen. Consider the two scenarios given in Case Study 6.1 and
think about the differing requirements. This will make you aware of the need for an appropri-
ate level of accuracy in different situations.
CASE STUDY 6.1 Choosing an appropriate Cross reference
method for volume measurement Electrophoretic methods

are described in Chapter 14
and delivery ‘Electrophoresis’.
The purpose of this case study is to help you to think about the importance of choosing
an appropriate volume measurement method for a particular situation. Details about
alternatives will be given later in this section.
Consider the following scenarios, A and B.
Scenario A
You need to prepare 200 cm3 of electrophoresis buffer for gel electrophoresis to sepa-
rate serum proteins. The laboratory stores a stock solution of the buffer that is 20 times
more concentrated than required.
What approach might you use to produce sufficient electrophoresis buffer of the
required dilution?
Scenario B
You are setting up an assay to measure the concentration of glucose in an unknown
sample. A standard (calibration) curve is required to estimate the unknown concentra-
tion. To produce an appropriate range of known glucose concentrations for this stand-
ard curve, a series of different volumes (in the range 0–1 cm3) of a stock glucose solution
will be required. The volume of each glucose sample will then be made up to a total
volume of 1 cm3 with buffer prior to the addition of a colour generating reagent. This
will react with the glucose to generate a colour that will be measured colorimetrically
and used in the production of the standard curve.
How might you approach the measurement and delivery of these volumes?
Are the specific volume measuring requirements for B similar to or different from A? You
do not need to consider dilutions in detail at this point, only how you would proceed in
terms of measuring and delivering the required volumes. The effects of dilutions and the
approach to working out how to dilute will be considered in the last section of this chapter.
Alternatives for measuring and delivering

volumes of liquid
Figure 6.2 shows a selection of equipment that is generally available for measuring and delivering
volumes of liquids. Such equipment can be grouped in various ways, for example, convenience
of use or volume range that can be delivered. The most useful grouping is probably according to
level of accuracy of the delivered volume. Accuracy relates to how close the measured volume
FIGURE 6.2
A selection of equipment to
measure volumes of liquids.
6.2 VOLUME MEASUREMENTS AND DELIVERY 133
is to the actual volume, whereas the precision of volume measurements refers to the closeness
of repeat deliveries of (supposedly) the same measured volume. At the low end of the accuracy
scale lie Pasteur pipettes (droppers) together with beakers and conical flasks that contain a vol-
ume mark. Measuring cylinders with a graduated scale provide an intermediate level of accuracy.
Higher levels of accuracy are provided by various types of pipettes, burettes, volumetric flasks,
and certain syringes. You may be familiar with many if not all of these, but may not have thought
much about their relative strengths and weaknesses. Certain precautions need to be taken when
Cross reference
measuring and handling certain liquids (Box 6.3). As mentioned in the Introduction, volumes
Chapter 5 ‘Statistics and
can also be delivered by weighing a liquid. This approach can deliver volumes to a high level of
handling data’.
accuracy, but lacks some convenience compared with the use of a mechanical pipettor.
The range of volume measurement procedures commonly available will now be considered.
Most emphasis will be upon mechanical pipettors (such as Gilson pipettes) as they are the most
sophisticated and accurate volume measurement devices available in the routine laboratory.
Pasteur pipettes, beakers, and conical flasks

These provide a relatively simple means of measuring and delivering specific volumes, but at
a low level of accuracy. The original Pasteur pipette was made of glass with a rubber teat to
create a partial vacuum to draw up liquid. It is possible to make an approximate calibration of a
fixed volume by drawing that volume of sample into the Pasteur pipette, marking the level on
the glass. Disposable one-piece plastic Pasteur pipettes are available with several approximate Cross reference
volume markings on the body of the pipette. Pasteur pipettes are cheap and easy to use where You can read about ELISA in
approximate volumes are required, such as dispensing a few drops of a cell suspension or for Chapter 15 ‘Immunological
completely filling wells on an enzyme-linked immunosorbent assay (ELISA) plate when a wash- techniques’.
ing step is required. Where their limitations are acceptable, Pasteur pipettes are useful: they
BOX 6.3 Precautions to be taken when

measuring and handling certain liquids
In addition to the practical considerations related to accuracy and precision, conven-

ience, flexibility, reliability, and the particular nature of a liquid may all influence how it
is measured and handled. For instance:
■ Some solutions are viscous and care must be taken to ensure complete measure-
ment and delivery of the desired volume. This will include allowing sufficient time
for the liquid to be drawn into and to drain from the measuring device
■ Sampling from a cell suspension, such as blood or microorganisms requires that the
suspension is first gently swirled to ensure an even distribution of cells
■ Protein solutions can denature at air–water interfaces if excessively shaken, stirred,
or poured, and it is necessary to keep frothing to a minimum
■ Some solutions such as strong acids are highly toxic or corrosive and care has to be
taken to avoid spillage and contamination. It may be appropriate to use disposable
‘plasticware’ where suitable
■ Some organic solvents may evaporate and so exposure to air needs to be minimized
by keeping them in appropriate sealed containers. Care has to be taken if using plas-
ticware as some organic solvents can cause deterioration of the plastic.
(a) are simple to use, quick, and the problems of cross-contamination are easily avoided with the
use of disposable plastic Pasteur pipettes.
Beakers and conical flasks are available in a range of volumes with a horizontal line indicating
the fill level for the quoted volume at the stated temperature. They are useful for measuring
approximate volumes, but must be kept horizontal when checking the volume measurement,
and are best used as holding receptacles in solution preparation and mixing.
Burettes, measuring cylinders, and volumetric flasks

Burettes and volumetric flasks are calibrated for volume measurement with high levels of
accuracy at or close to room temperature. The temperature at which the calibration has been
made is stated on the glassware. Measuring cylinders provide a lower level of accuracy (but
higher than that for beakers and conical flasks). Whereas a volumetric flask is used for meas-
uring a fixed volume (although a range of flask sizes are available), measuring cylinders and
burettes have calibration subdivisions over a range of volume and, again, can be obtained in
(b) various sizes. For making a solution, the use of a volumetric flask containing solute would be
the preferred approach, solvent being added up to the defined volume measurement line on
the flask. This is both simple and convenient.
Burettes are glass cylinders with a range of volume calibrations along their length and a tap
that can be opened and closed near one end. Burettes are useful for measuring and delivering
small volumes (aliquots), such as in titrations where, for instance, an alkaline solution is added
in small incremental volumes to a solution of acid that is stirred in a conical flask beneath the
burette. Care must be taken to clean a burette both before use, by washing with the solution
to be used, and also after use, usually by washing with water. The latter is necessary when using
solutions such as sodium hydroxide, which can cause the burette tap to seize in its mounting.
The use of measuring cylinders, volumetric flasks, burettes, and pipettes requires care in
observing the volume measurement mark at the base of the liquid meniscus. A meniscus is the
curved surface of liquid when viewed in a narrow cylindrical space, such as the neck of a
volumetric flask or the body of a pipette. The meniscus forms due to capillary action; aqueous
(c) solutions and most other liquids produce a concave meniscus in which the centre is lower
than the edges. For consistent measurement, the centre point at the base of the meniscus
should be observed at eye level against the appropriate volume calibration mark, as you can
see in Figure 6.3. Observing the level of the meniscus is a potential source of measuring error
if a consistent and appropriate approach is not adopted. When completing the filling of a
volumetric flask, or, indeed, a measuring cylinder, with solvent the final small volume needed
to take the solution to the correct meniscus position on the calibration mark have to be made
by small, dropwise additions using a Pasteur pipette. All solutions need to be appropriately
labelled, stating what they are, their concentration with any associated hazards.
SELF-CHECK 6.5
What aspects of the uses of a burette and volumetric flask could lead to inconsistencies of
measurement?
FIGURE 6.3
Although the items mentioned above have traditionally been made of glass, plastic alterna-
(a) Correct alignment of the
tives are becoming more common. Plasticware is safer to use as it is less likely to be damaged
meniscus with the indicated
than glass and can also be disposable. Glassware needs to be checked before use to identify
calibration line. (b) and
any cracked or broken items. In solution measurements and transfers between containers,
(c) show the volume
care must be taken to avoid spillages and cross-contamination. Spillages must be dealt with
incorrectly indicated.
according to local health and safety requirements.
6.3 PIPET TORS 135
Pipettes
Together with certain types of syringes, pipettes measure small volumes in a reproducible and
accurate manner. They are commonly used to deliver volumes in the 0–20 cm3 range, while
pipettors can deliver much smaller volumes, as low as 1 μL. Pipettors are complex, precision
measuring devices whose construction and operation need to be fully understood to operate
Cross reference
correctly and reliably. They will be considered in Section 6.3.
Pipettes were originally made from glass, although they have increasingly been replaced by
plastic alternatives. Two types are available. Bulb pipettes deliver a fixed volume and have a
large bulbous region part way along their length with a single, fixed volume mark near the top.
Although they only measure a single fixed volume, different sizes are available. There is only
one calibration mark and so volume measurement mistakes are unlikely. Bulb pipettes are
simple to use and potentially more accurate than a graduated pipette. Graduated pipettes are
straight-walled and calibrated across a particular volume range such as 0–10 cm3 in 0.1 cm3
increments. Care must be taken to check the way the scale is presented, for example, 10–0 cm3
or 0–10 cm3, and whether the pipette is of the ‘blowout’ type or not. Blowout pipettes require
that the last drop of liquid is expelled from the tip to deliver the intended volume. Non-
blowout pipettes rely upon gravity to deliver the volume and allow for a small volume of liquid
to remain in the tip of the pipette. This difference is not an issue if the volume to be measured
and delivered is less than the total volume of the pipette. For example, a 5 cm3 pipette could be
used to measure and deliver 4.0 cm3 with the pipette filled to the top mark (5.0 cm3) and liquid
delivered until the level had dropped to the 1.0 cm3 mark. In this situation, it would not matter
whether you used a blowout or non-blowout pipette. With both types of pipette, care has to
be taken to be consistent in reading the meniscus against the graduated scale.
SELF-CHECK 6.6
What do the terms accuracy and precision mean in the context of measuring volumes with a
pipette?
From a health and safety perspective, pipettes must always be operated with a pipette filler,
which creates a partial vacuum to draw the liquid into the pipette. This avoids any chance of
liquid entering the mouth as occasionally used to happen before pipette fillers became avail-
able. It is good practice to draw the liquid to just above the required mark, slowly releasing the
vacuum to allow the meniscus to fall to the mark. The tip of the pipette is placed above the
receiving receptacle and the pipette filler operated to release the liquid as required. As with
all laboratory procedures, a correct and consistent approach to the operation of a pipette will
deliver accurate and consistent results.
6.3 Pipettors
• The design features and use of mechanical pipettors

• Optimal performance and calibration of pipettors
Pipettors are also known as autopipettes and sometimes micropipettes. They are usually adjust-
able volume mechanical pipettes and have become the commonest type of pipette in many
laboratories. Despite their cost, they are the volume measuring tool of choice because they
are highly accurate, precise, and reliable. They are sophisticated mechanical instruments and
so have to be operated and handled correctly to avoid damage. Electronic versions of pipet-
tors are also available.
In the early 1970s Warren Gilson developed an adjustable volume, high precision pipette with
Cross reference a volume readout display. Although many manufacturers produce pipettors, they are still often
Molecular biological techniques called ‘Gilsons’ for this reason. Pipettors are of particular value in biomedical science because they
are outlined in Chapter 16 can deliver small volumes, as small as 1 μL or less, which are required in applications such as
‘Molecular biology techniques’.
in molecular biology. They use a disposable plastic tip that can be autoclaved and this adds to
their versatility. Why might this be an advantage? There are a number of situations where the
use of a ‘clean’ tip may be essential, such as for work with nucleic acids where a tip contaminated
(a) with nucleases could degrade nucleic acids in the sample. Plastic tips are cheap and so avoid-
ance of cross-contamination is easy to achieve by using different removable tips. Pipettors can be
obtained in both single and multi-channel forms, different models covering various volume ranges
and to different levels of accuracy. Multi-channel pipettors have a special manifold to which can be
attached multiple tips (such as 8- or 12-channel types). This permits simultaneous deliveries of a
particular volume of solution such as to the wells of an ELISA plate (a multi-well plate) in immunol-
ogy. You can see examples of single channel and multi-channel pipettors in Figure 6.4.
Features and types of pipettors

The features of a pipettor are as follows:
• A disposable plastic tip placed by the operator on the tip holder (different pipettor models
and capacities require different types of tips)
• A ceramic or metal piston within an airtight channel that can displace the air and create a
vacuum, allowing liquid to be drawn into the disposable tip
• A thumbwheel that can be rotated to adjust the position of the piston and change the
volume setting, which is indicated on a digital display on the side of the pipettor body
(b)
BOX 6.4 Air-displacement and

positive-displacement pipettors
Air-displacement pipettors have general purpose uses for dispensing aqueous solutions
and are the type you are most likely to use. You can see the general features of their
construction in Figure 6.5. These include a piston that moves within the chamber of
the pipettor. The piston moves down to release air, and the volume of air remaining in
the chamber is inversely proportional to the volume of liquid drawn from the sample
when the piston is allowed to rise. The smaller the cushion of air left within the pipet-
tor body, the larger the sample volume. The cushion of air between the liquid being
sampled and the piston prevents the piston being contaminated with liquid. In contrast,
positive-displacement pipettors work more like a syringe, as you can see in Figure 6.6,
which contrasts the workings of the two types. Thus, they lack an air cushion between
liquid and piston, which are in direct contact with one another. Hence, the pistons must
be disposable because of contamination with liquid. Positive-displacement pipettors
have the advantages of being more accurate than air-displacement types because they
FIGURE 6.4 are less subject to variations in temperature and because they do not form aerosols,
(a) A single and (b) multi- which can be produced by the air-displacement types. They are mainly used to measure
channel pipettors. and dispense viscous, volatile, corrosive, or radioactive liquids.
6.3 PIPET TORS 137
Plunger
Tip release button
Spring
Coupling
Seal
Piston
Shaft
Pipette tip FIGURE 6.5

Schematic showing the construction
of an air-displacement pipettor.
Piston
Aerosol
FIGURE 6.6
Comparison of the actions of (a) air-displacement and (b) positive-displacement
pipettors. See text for details.
TABLE 6.2 The Gilson Pipetman®

P range of pipettors
Model Volume range/μL

P2 0.2–2
P10 1–10
P20 2–20
P100 20–100
P200 50–200
P1000 200–1000
P5000 1000–5000
P10 mL 1000–10000
Information taken from the Gilson online

brochure at http://www.gilson.com
• A moveable sleeve above the tip that allows the tip to be removed without direct contact
with the operator, hence avoiding physical contact with the tip following use and possible
cross-contamination with new tips
There are two different types of pipettor, air-displacement and positive-displacement (see
Box 6.4). Pipettors are available in various volume ranges. For many applications, a range such as
the Gilson Pipetman® P is suitable. Table 6.2 shows the volume ranges for this family of pipettors.
SELF-CHECK 6.7
Which of the following features would you NOT associate with a pipettor?
(a) Disposable tip
(b) Permanent or disposable piston
(c) Moveable sleeve for ejecting the tip
(d) Thumbwheel for adjusting the position of the tip
Using a pipettor
If the steps for use of a pipettor given in the the Method box ‘How to set up and use a pipet-
tor’ are followed in a systematic manner, accurate and reproducible measurements will be
achieved. It is worth practicing these steps a number of times to achieve consistency.
SELF-CHECK 6.8
In what ways does a pipettor differ from a pipette?
6.3 PIPET TORS 139
METHOD How to set up and use a pipettor
■ Choose a pipettor appropriate to the task in hand. For P1000, 085 represents 850 lL. The meaning of the three
a given model, it is essential to use the pipettor only digits on the display varies with the model so instructions
within the volume range indicated. Use beyond this must be checked otherwise an incorrect volume will be
range can damage the pipettor mechanism. delivered.
■ Organize the work area so that all materials and items ■ To measure and deliver a volume of sample, the plunger
of equipment to be used are conveniently placed. The is first depressed as far as the first stop (resistance will
work surface needs to be at a comfortable height. If nec- be felt) with the pipettor held away from the liquid.
essary, this can be achieved by using a height adjustable Next the tip should be placed below the liquid surface
seat. Ideally, while holding the pipettor you should be with the pipettor body held in a near vertical position.
able to comfortably rest your elbow of the same arm on The plunger should be released slowly to allow the
the work surface. This ensures the arm can be rested for liquid to be drawn into the tip. Care must be taken to
some of the time in lengthy procedures whilst holding ensure that the tip remains within the liquid and that
the pipettor in a near vertical position. When not in use, air bubbles are not drawn into the tip. After a few sec-
the pipettor must be placed vertically in an appropriate onds, the pipettor should be withdrawn, keeping it near
rack. to vertical.
■ Fit an appropriate tip onto the pipettor, applying a little ■ To dispense the liquid, the tip is allowed to touch the
force to get a good, leakproof fit. This is most readily side of the receiving container by slightly tilting the
achieved with the tips stored vertically in a tip rack. pipettor away from the vertical, avoiding any other
A dry tip should be wetted with solution before use. liquid that may have been added already. The plunger
This reduces the surface tension of the inside surface of is slowly depressed through the first stop and as far as
the tip and will help to minimize evaporation when the the second stop (the final resistance). After a few sec-
sample is drawn. onds, keeping the plunger fully depressed, the pipettor
■ Set the volume to be measured by rotating the adjuster. is removed. Only when the pipettor has been removed
A Gilson Pipetman® P20 can deliver volumes between should the plunger be released.
2 and 20 lL. The digital display on the P20 shows three ■ The used tip can be removed by pressing the ejector
digits. The third, which is in red, represents tenths of a button which moves a collar onto the top of the tip, loos-
microlitre. Therefore, 020 represents 2.0 lL, 130 is 13.0 lL ening and releasing it. The used tip must be ejected into
and 200 is 20.0 lL. For a P200, 095 is 95 lL and for a an appropriately labelled and suitable waste container.
Obtaining optimal performance from a pipettor

Pipettors are designed to deliver high levels of performance, but inconsistencies in results
may occur if certain precautions are not taken. Thus, the mode of operation must be fol-
lowed in a consistent manner. The metal components of low volume pipettors can expand
if heat is transferred from the operator to the plastic handle part of the pipettor. Appropriate
pipettor design can partly reduce such heating but the practice of using several pipettors
on a rotational basis during prolonged use can help to overcome the potential effect of
temperature changes upon the volume determining mechanism. Extended use, particularly
if the pipettor is not held in the most comfortable position, may lead to repetitive strain
injury. This type of discomfort can lead to the operator holding and using the pipettor in a
way that is not consistent with high levels of performance. These problems can largely be
overcome by using an electronic pipettor, which removes the need for the operator to exert
a significant force to fill and empty the pipettor, and eject the tip. As with all experimental
devices, there is a limit to the period of continuous use before operator fatigue begins to
compromise performance. The work regime needs to address this issue where concentrated
periods of pipettor use occur.
Inexperienced users of pipettors tend to make one or more of the following mistakes which
can be overcome with practice:
• The tip may leak if it is not fitted securely to the pipettor; the fitting of the tip must be
airtight
• The pipettor can be damaged if used outside the designated volume range
• Incorrect volumes may be drawn if the plunger is not depressed to the first stop or if the
pipettor is raised during filling so that the tip is above the liquid level
• An incorrect volume may be delivered if the plunger is not fully depressed to the second
stop or if the pipettor tip is still in contact with the delivered sample while the plunger is
released
• Liquid may enter the internal mechanism and damage it, if the pipettor is held near to or
horizontal when liquid is in the tip. To avoid this, pipettors should be held as close as pos-
sible to the vertical when in use
It is possible to check for consistency of approach by delivering a measured volume into a

small, tared container on a balance. For water, it can be assumed that 1 μL weighs 1 mg. Even
if the pipettor is miscalibrated (accuracy is compromised), successive deliveries of the same
volume should still give the same measured weight (precision is maintained).
Calibrating a pipettor
In addition to routine inspections of pipettors, calibration checks are also required. An under-
standing of why pipettors can deteriorate can help minimize deterioration in performance,
although this can be compromised in several ways. For example, misuse by the operator
including incorrect volume adjustment, inappropriate handling that results in physical knocks,
and dropping of the pipettor can all lead to internal damage. Clearly, the more skilled the
operator, the less likely that such damage will occur. It helps if pipettors are stored in a vertical
rack when not in use. The use of certain liquids results in volatile, corrosive vapours entering
the internal mechanism and can damage the piston, spring, or seals that will affect the airtight
seal of the pipettor and the volume that is delivered.
Regular calibration checks are necessary where high levels of accuracy and precision are
required. Local laboratory procedures and practices will indicate the required frequency of
pipettor servicing and calibration.
How is pipettor calibration checked? This is quite straightforward: the easiest way is to deliver
a measured volume of water into a tared receptacle on an analytical balance. Ambient con-
ditions, such as temperature and pressure together with the density of water are taken into
account in converting the weight of the sample into the corresponding volume. This volume
can then be compared with the volume originally selected on the pipettor. Calibration checks,
as well as routine physical inspections will inform the need for maintenance, which will either
be undertaken locally in the laboratory or by the manufacturer.
SELF-CHECK 6.9
Look at Figure 6.7, which shows the digital display of a pipettor. What volume would the
FIGURE 6.7 display represent if the pipettor was from the Gilson Pipetman P range and was (a) a P20,
Digital volume display of a (b) a P100, and (c) a P200?
pipettor.
6.4 PREPARING REAGENTS: CONCENTR ATIONS AND DILUTIONS 141
Preparing reagents:
6.4
concentrations and dilutions
• A solution preparation checklist
• The mole (mol) and molar concentrations

• Alternative ways of expressing concentration
• Different types of dilution and their impact upon concentration
A solution, for instance of glucose in water, consists of a quantity of solute (such as glucose)
dissolved in a volume of solvent (water in this case). The use of balances and volume meas-
urements are the two essential techniques for the preparation of a solution and its dilution.
If a set method is to be followed, where all quantities are documented, calculations need not
be performed. However, do you know how to weigh an amount of glucose equivalent to
0.1 mol, can you prepare a 0.2 mol dm−3 sodium acetate solution, or are you able to calculate
the final concentration of a 0.0005 mol dm−3 protein solution when 1 cm3 is diluted to 25 cm3?
If not, then this final part of the chapter will be of help. The precautions to be observed before
preparing solutions are explained in Box 6.5.
BOX 6.5 A precautions checklist

when preparing solutions
Several matters need to be considered and understood before proceeding to prepare

and dilute solutions:
■ The health and safety assessments for all materials being used, any precautions that Cross reference
must be observed, procedures for appropriate labelling of solutions in storage contain- Chapter 4 ‘Health and safety’.
ers, dealing with spillages, and safely disposing of waste must be fully adhered with
■ Appropriate protective equipment must be used, including a fully fastened labora-
tory coat, together with safety glasses and gloves, as determined by the nature of the
materials to be handled and procedures to be undertaken
■ Solutions must never be pipetted by mouth
■ The location of all relevant safety equipment in the laboratory, such as an eye wash,
safety shower, first aid kit, fire extinguisher, and fire blanket must be known
■ Additional precautions must be observed in some situations, such as the addition of
a strong acid to water (and not water to the strong acid) because of the heat gener-
ated in the process. This is minimized by keeping the concentration of acid low ini-
tially and by adding it in small quantities to a much larger volume of water
■ Where a generic health and safety assessment for a particular procedure is docu-
mented and followed, a check must be made to ensure that the details of what is
being undertaken do not significantly differ from it. For instance, a risk assessment
for preparing and using a 1 × 10−5 mol dm−3 solution of a slightly hazardous material
would need to be modified where a more concentrated, 0.1 mol dm-3 solution is
being prepared and used, as concentration is a key part of the risk assessment of a
potentially harmful solution
Solutions and concentrations

The concentration of a solution is determined by the quantity of solute and the volume of
solvent as described in Box 6.6.
Key Points
Solutions consist of a solute dissolved in a solvent; the concentration of the solution is
determined by the relative proportions of solute and solvent.
The different ways in which concentration may be expressed will now be considered. The
commonest form is molarity and this along with the concept of the mole will be considered
in some detail followed by other common forms of concentration, molality, % (per cent)
concentration, and parts per million (ppm). Finally, we will discuss what the effects of dilution
upon concentration.
Moles, molar concentrations, and molarity

The mole is one of the base units of the SI system of units (Section 6.1). It is a measure of
the amount of a substance. The mole is that amount of substance that contains as many
elementary entities, such as photons, atoms, or molecules, as there are atoms in 0.012 kg
(12 g) of carbon (12C). This value is called Avogadro’s number and is equal to 6.022 × 1023,
and is a constant for a mole of any substance. Although a molecule of water (Mr, relative
molecular mass, 18) is small compared with a molecule of haemoglobin (Mr 64,500) (see
below for an explanation of Mr), a mole of each would contain this same number of indi-
vidual molecules. Of course, the mass of a mole of these two different molecules would be
vastly different.
This numerical nature of the mole is of little help when measuring a particular amount of a
substance as counting individual molecules is not a practical proposition! Instead, it is much
more convenient to use molar mass, which is the mass of all the elementary entities in one
mole of a substance and has units of g mol−1. For glucose the value is 180.16 g mol−1. A numeri-
cally equivalent alternative to molar mass is Mr, which makes reference to the mass of 12C. For
an atom the corresponding term is relative atomic mass (Ar):
Mr = (mass of one molecule of substance)/(1/12 mass of one molecule of 12C)
BOX 6.6 Concentrations of

molecules and ions
The numerical nature of the mole means that a solution contains a particularly large
number of individual solute molecules. Aqueous sodium chloride solutions contain
sodium and chloride ions (1 NaCl → 1 Na+ + 1 Cl−). A 0.04 mol dm−3 aqueous solution of
sodium chloride would have a concentration of 0.04 mol dm−3 for each of the individual
Na+ and Cl− ions, but 0.08 mol dm−3 for the combined concentrations of both.
BOX 6.7 Molecular size and the dalton
The unit called the dalton (Da) is sometimes used to express a mass equal to one-twelfth
of that for 12C. It is often adopted in biochemistry and molecular biology texts when refer-
ring to the sizes of macromolecules. A protein with Mr of 45,000 could be described as
being 45,000 Da or 45 kDa in size.
SELF-CHECK 6.10
Distinguish between a mole, molar mass, and relative molecular mass.
As Mr is a ratio of masses, it has no units, but may be expressed in daltons as described in Box
6.7. Mr is equivalent to the now outdated term molecular weight, although the latter term is
still found on many reagent bottle labels, many textbooks, and is still used by many biomedi-
cal scientists. To obtain one mole of glucose, you need to weigh 180.16 g of it; that is, obtain a
mass equivalent to the value of its Mr.
SELF-CHECK 6.11
The amino acid glycine has the formula C2H5NO2. How much glycine would you need to weigh
to obtain 0.8 moles?
Method Box ‘Preparation of solutions’ gives practical hints on how to prepare, in this example,
a solution of glucose.
How are concentrations expressed

using the mole?
A one molar solution contains one mole of solute in a total solution volume of one dm3 or litre.
This forms the basis of the molarity form of concentration. Technically, this is not in accord-
ance with the SI system of units as the SI unit of volume is the m3. However, as the m3 is 1000
times larger than a dm3 (which is equivalent to a litre ), the use of molarity is numerically more
convenient.
The number of moles in a particular volume of a solution can be calculated by applying the
following equation:
Number of moles (mol) = molarity (mol dm−3) × volume (dm3)
Key Points
Molarity is the most common representation of concentration and is the number of
moles of solute in 1 L of solution.
SELF-CHECK 6.12
What is the molar concentration of a 6 mg cm−3 solution of lactose (Mr = 360.31)? How many
moles would be in 50 cm3 of this solution?
METHOD Preparation of solutions
How could you prepare 1 dm3 of a 1 mol dm−3 aqueous ■ 1 dm3 of a 1 mol dm−3 solution will contain 180.16 g of
solution of glucose? glucose
You would: ■ 1 dm3 of a 0.2 mol dm−3 solution will contain 180.16 ×
0.2 g glucose, which = 36.032 g
■ Find out the Mr value for glucose from the reagent
■ If a volume of 100 cm3 is needed, the amount of glucose
bottle label or from data sheets (Mr = 180.16)
to be weighed is:
■ Weigh this amount using an appropriate balance then
place the glucose in a 1 dm3 volumetric flask 100 cm3 × (36.032 g/1000 cm3) = 3.603 g
■ Add distilled water to the mark on the volumetric flask Although 3.603 g is equivalent to 0.02 mol glucose
■ Mix well (mol = mass/Mr), the concentration is 0.2 mol dm−3 because
the 0.02 mol glucose is dissolved in a 100 cm3 (0.1 dm3)
However, you do not always prepare one dm3 volumes of volume. Molarity expresses concentration as though the
solutions, most likely much less than this. How could you pre- volume is 1 dm3 (1 L).
pare 100 cm3 of a 0.2 mol dm−3 solution of glucose? It is easi-
est to split the calculation into several simple, logical steps: Molarity (mol dm−3) = (mass/Mr) dm−3
Other common representations of concentration

Molarity is the commonest way of expressing concentrations and is therefore the one you are
most likely to be familiar with. However, there are several other ways in which concentration
may be expressed, three of which we will now consider.
Molality is a form of concentration where the mass of the solvent is used, rather than the
volume of solution, as used in molarity. For example, a 0.1 molal solution would contain
0.1 mol solute/kg solvent. For a dilute aqueous solution where the mass of the solute is relatively
low compared with the volume of solvent and where water has a density of 1 g cm−3, molal
and molar concentrations will be similar in value. With greater amounts of solute, the val-
ues will differ as the addition of solute will affect the final volume of solution compared to
the volume of solvent used. However, using molality can be convenient because the only
measurements are those of mass, unlike molarity, which involves both mass and volume
measurements.
SELF-CHECK 6.13
Would you expect any differences between the molal and molar concentrations of 1 dm3
volumes of aqueous solution containing (a) 0.1 mg of solute and (b) 50 g of solute?
Per cent concentrations are expressed as parts of solute per 100 parts solution. For example, %
w/v (mass–volume or weight–volume percentage) means mass parts of solute per 100 volume
parts of the resulting solution. Thus, a 10% w/v solution of glucose contains 10 g glucose per
100 cm3 of the glucose solution. Mass percentage (% w/w) expresses the concentration as sol-
ute mass parts per 100 total mass parts. It is also known as weight percentage or weight–weight
percentage. This could be used in the preparation of an aqueous solution of an alcohol. For
instance, 25 g alcohol plus 75 g water would make a 25 % w/w solution. Finally, % v/v (volume–
volume percentage) expresses the concentration as volume parts of solute per 100 volume
parts of solution.
SELF-CHECK 6.14
Convert a 0.1 mol dm−3 concentration of glucose into a mass–volume percentage (%w/v).
Parts per million (ppm), like per cent concentrations, is a non-SI expression of concentration.
Similar forms of expression include parts per thousand. You will have come across ppm if you
have carried out flame photometry or atomic absorption spectroscopy work as these tech-
niques detect relatively low concentrations of analytes. A concentration of 1 ppm means 1 g
solute per 106 g of solution or the aqueous solution volume equivalent, per 106 cm3. This is the
same as 1 μg per g or 1 μg per cm3.
Per cent concentrations and parts per million are useful ways of expressing concentrations
where the Mr of the analyte in question is unknown.
Dilutions
This final part of the chapter will consider how to dilute a concentrated solution (often called
a stock solution) to make other less concentrated solutions of known value. Where assays and
other procedures that require a range of solutions are undertaken on a regular basis, it is
convenient to store pre-prepared concentrated stock solutions for subsequent dilution as and
when required. This is, of course, only possible where the solution is stable and unaffected by
long-term storage, and the dilutions are performed in an accurate and precise manner.
Preparing diluted samples from a stock solution

We will consider the preparation of diluted samples from a stock solution using the practical
example given in Case study 6.2.
Using the approach in Case study 6.2 will allow you to handle any situation that involves
dilutions. The equation c1v1 = c2v2 also allows you to prepare a particular dilution, where the
required final concentration is known because v1, the volume of stock solution needed for the
preparation of the final volume of solution, v2, can be calculated.
Key Points
The equation c1v1 = c2v2 facilitates the calculation of the effect of a dilution and how to
produce a specific dilution from a stock solution.
It is helpful to remember that for any dilution the following points apply:
• The final concentration will be less than that of the stock solution because the number of
moles of solute taken in the measured volume of stock solution is now dispersed in a larger
volume
• The extent of the dilution will determine the concentration change, so the greater the dilu-
tion the lower the concentration of the final solution
• The effect of a dilution can be calculated using the expression c1v1 = c2v2 (Case Study 6.2)
• The accuracy of a dilution will be influenced by the choice of methods for measuring and
delivering the required volumes
• Information about a dilution needs to reference both the initial volume to be used and the
final volume. For example, a 20-fold dilution would involve adding nineteen volumes of
CASE STUDY 6.2 Quantitatively

diluting a stock solution
A common form of assay is one where a range of known concentrations of a solution is
prepared and a standard curve (calibration curve) produced. The curve can then be used
Cross reference to estimate the concentration of an unknown sample. A property of the solute (such as
You can read more about the absorbance due to a coloured compound) that varies with concentration is measured.
Beer–Lambert law in Chapter 11
The Beer-Lambert law provides the basis for a linear relationship between absorbance
‘Spectroscopy’.
and concentration.
Usually solutions are colourless and some form of colorimetric assay will be needed
to form a coloured derivative of the solute, whose absorbance can then be measured
using a colorimeter or spectrophotometer. For instance, there are several reducing
sugar-based colorimetric assays for the detection of glucose. The Somogyi–Nelson
method utilizes an alkaline copper solution that reacts with glucose resulting in the
conversion of oxidized Cu(II)2+ to reduced Cu(I)+. The latter reacts with arsenomolyb-
date to form a blue coloured complex. The absorbance of the blue solution can be
measured.
A simple assay to assess the concentration of haemoglobin in a sample will be used to

illustrate the effects of dilution of a stock solution. Look at Table 6.3 which has a blank
row and requires your input of the concentration values. The table shows a series of
dilutions from a stock solution of haemoglobin which is, of course, coloured. You should
be able to see that the dilutions have been achieved by aliqoting a range of volumes of
the haemoglobin solution between 0 and 3.00 cm3 and adding an appropriate volume
of saline to each to give a total volume of 3.00 cm3 in each case. The stock haemoglobin
solution has a concentration of 2.00 mg cm−3. You need to work through the following
information so as to be able to complete the Table.
How might you proceed to fill in the concentration values? Tubes 1, 5, and 7 should
pose little difficulty. Tube 1 lacks haemoglobin and so the concentration of its con-
tents will be zero. At the other end of the range, tube 7 contains undiluted haemo-
globin and the concentration will therefore be the same as the stock solution (2.00
mg cm−3). Tube 5 has a mixture of equal volumes of haemoglobin and saline, so the
concentration in this tube has halved. How can the other concentrations be deter-
mined? The extent of dilution will need to be calculated by comparing the initial
TABLE 6.3 Preparation of a range of haemoglobin (Hb) concentrations for

the construction of a standard curve. Stock [Hb] = 2.00 mg cm−3. The final row in
the table is for you to complete
Tubes 1 2 3 4 5 6 7
vol Hb/cm3 0.00 0.25 0.50 1.00 1.50 2.25 3.00
vol saline/cm3 3.00 2.75 2.50 2.00 1.50 0.75 0.00
[Hb]/mg cm−3
volume of haemoglobin with the final volume of haemoglobin plus saline. This is
readily achieved by using the equation:
c1v1 = c2v2
where c1 = concentration of stock solution; v1 = volume of stock solution used; c2 = con-

centration of the diluted solution; and v2 = total volume of solution after dilution.
It is essential that both c1 and c2 have the same units of concentration. Similarly, v1 and v2
must have the same units of volume.
Applying this equation to tube 6:

c1 = 2.00 mg cm−3
v1 = 2.25 cm3
c2 = unknown concentration
v2 = 3.00 cm3 (2.25 + 0.75)
N.B. v2 will be the same value for all tubes.
By rearranging the above equation, the unknown concentration c2 can be obtained:
c2 = (c1 × v1)/v2
= (2.00 mg cm−3 × 2.25 cm3)/3.00 cm3
= 1.50 mg cm−3 TABLE 6.4 Cumulative
effects of a two-fold serial
By repeating this approach you can work out the remaining concentrations and com-
dilution
plete the last row of Table 6.3.
Dilution step Dilution
1 2×
diluent to 1 volume of the stock solution to give 20 volumes of diluted solution (this could
be called a 1 in 20 dilution) 2 4×
3 8×
SELF-CHECK 6.15
4 16 ×
What is the concentration of the coenzyme NADH in a dehydrogenase enzyme assay in which
0.20 cm3 of stock 150 mmol dm−3 NADH solution is mixed with several other solutions to give 5 32 ×
a final volume of 3.00 cm3? 6 64 ×
7 128 ×
Specific types of dilution 8 256 ×
There are certain situations where a particular pattern of dilution (a dilution series) is required.
9 512 ×
These may be a linear or a non-linear series. Where a standard curve is being constructed, equal
or near to equal increments in the dilution series will ensure that there is an even spread of 10 1024 ×
data points. The range of concentrations in your completed version of Table 6.3 is of this type.
Sometimes adjustments to the pattern of dilutions are necessary so as to spread evenly the recip-
rocals of substrate concentrations, as in the Lineweaver–Burk enzyme kinetics plot. There are,
however, circumstances in which a wider range of dilutions using non-linear incremental changes
are required. This may, for example, reflect an uncertainty of knowledge about the density of Cross reference
See Chapter 15 ‘Immunological
cells in a cell suspension or concerning the level of biological activity in a sample, such as may be
techniques’.
encountered with the ELISA. In these situations, some form of serial dilution is undertaken.
A two-fold or log2 dilution is where successive dilutions halve the concentration. This is achieved
by measuring a volume of sample and mixing it with an equal volume of diluent and repeating
this ‘1 + 1’ approach for each successive dilution. Table 6.4 illustrates the effect of this type of
dilution. You can see that after only a few cycles of dilution, the concentration falls rapidly.
A ten-fold or log10 dilution occurs when each successive dilution reduces the concentration ten
fold. This is accomplished by measuring one volume of solution and adding nine volumes of
diluent to it: a ‘1 + 9’ approach for each successive dilution. After several cycles of serial dilutions,
the low concentration can become difficult to comprehend easily in a table of data. Ten-fold
dilutions make graphical representation of concentration data on a linear scale rather meaning-
less. Table 6.5 shows how logarithmic values can be used to represent such data. The log values
could be used on a linear graph scale as an alternative to using logarithmic graph paper.
SELF-CHECK 6.16
How many cycles of dilution will achieve a minimum of a 1,000-fold dilution using (a) a two-
fold and (b) a ten-fold dilution series?
TABLE 6.5 The effect of ten-fold dilution steps upon antigen

(Ag) concentration in an ELISA. The stock [Ag] is 1 mg cm−3. A log10
value change of 1 represents a 10× change in the concentration.
Dilution step [Ag]/mg cm−3 log10 [Ag]

1 0.1 −1
2 0.01 −2
3 0.001 −3
4 0.0001 −4
5 0.00001 −5
SUMMARY
■ The use of balances for weighing and pipettors and other volume measurement methods
for volume delivery are key techniques in the production of solutions and their dilution
■ Top pan and analytical balances measure weight; with calibration they can be used to
measure a required mass
■ For correct operation, balances must be appropriately sited and calibrated
■ The choice of method for measuring and delivering a volume of liquid depends upon the
level of required accuracy and precision
■ Burettes, pipettes, and volumetric flasks provide high levels of accuracy if used
correctly
■ Pipettors are the volume measurement tool of choice for highly accurate and precise
work. They use disposable tips for convenience, require calibration and, being precision
instruments, must be used with care
■ Pipettors must be used in a correct and consistent manner to achieve accurate and precise
performance; to avoid operator fatigue and discomfort in extended use, a comfortable
posture must be adopted
6.4 PREPARING REAGENTS: CONCENTR ATIONS AND QUESTIONS
DILUTIONS 149
■ Solutions are prepared at particular concentrations of solute in solvent, most frequently

expressed as molarity, but also in other forms such as molality
■ The production of solutions of a particular molarity requires an understanding of the mole

and how the relative proportions of moles of solute and volume of solvent affect the con-
centration of the solution
■ Dilutions increase the proportion of solvent compared to solute and, therefore, reduce
concentration; the application of the equation c1v1 = c2v2 simplifies the calculation of
dilutions and concentrations
■ Dilution series can be linear, such as in standard curves or non-linear such as successive
two-fold and ten-fold dilutions
FURTHER READING
● Graham I (1983) Difficulties encountered by biology students in understanding and
applying the mole concept. Journal of Biological Education, 1983; 17: 339–342.
This article addresses some of the common areas of confusion and misunderstanding in
applying the mole concept and considering concentrations and dilutions.
● Reed R, Holmes D, Weyers J, Jones A. Practical Skills in Biomolecular Sciences 3rd edn.
Pearson Education Limited, Harlow, 2007.
Chapters 22–24 of this comprehensive basic laboratory skills textbook complement much
of this chapter.
Useful Websites
■ http://www.fisher.co.uk/phpfiles/techzone/question.php?questionid=181 (accessed on
25 January 2008). This web page provides useful tips on choosing the right pipettor and
tip, recommendations for correct use, and a link to a fuller article on liquid handling.
■ http://www.gilson.com/Products/product.asp?pID=67 (accessed on 25 January 2008).
This web page introduces the Gilson Pipetman® P range of pipettors, one of the most useful
ranges of pipettor, and also links to illustrated PDF documents on the use of pipettors.
QUESTIONS
1. Indicate which of the following statements are TRUE or FALSE. Justify your answer.
(a) It does not matter what size of pipette or pipettor is used to measure a particular
volume of solution
(b) When purchasing a balance for a laboratory, it is necessary to obtain the most
sophisticated model that funds permit
(c) Dilutions for the production of a standard curve should be prepared using equip-
ment with high levels of accuracy and precision
150 PREPARING
6 AND
PREPARING MEASURING
AND REAGENTS
MEASURING REAGENTS
2. An acid has a Mr of 192 and is used in the preparation of various aqueous solutions. What
are the concentrations in mol dm−3 of the following solutions?
(a) 100 cm3 contains 2 g of acid

(b) 200 cm3 contains 4 g
(c) 5 cm3 contains 0.05 g
(d) 2.5 dm3 contains 45 g
(e) 10 g of the acid is diluted in 1 dm3, which is then diluted 25-fold.
3. (a) Which of the following represents a 10 parts per million concentration of an

aqueous solution?
I 10 g per dm3
II 1 mg per 100 g
III 100 μg per kg
IV 1 μg per mL
(b) Assuming that (a) (I) is a solution of glucose, calculate (I) the molar and (II) mass–
volume percentage concentrations.
4. There has been an electrical fault in your section of the laboratory and you have to
temporarily relocate the analytical balance that you are using. List the key issues that you
would need to address before being able to operate the balance in the new location.
5. Construct a table with the column headings cost, simplicity of construction, convenience
of use, and level of accuracy. Under each heading, use a simple scoring system ranging
from low (−−) to high (++) for a Pasteur pipette, beaker, measuring cylinder, pipette, and
pipettor. This exercise will help you to summarize the key features of different items of
volume measuring equipment. Looking at your table, do you think that ‘you get what
you pay for’?
6. What are the advantages and disadvantages of using molarity and mass percentage
(% w/w) forms of concentration?
7. Thinking about how it is achieved, what is the main precaution that must be taken when
preparing a two-fold dilution series?
7
Samples and sample
collection
Joyce Overfield
Learning objectives
■ Identify major factors that contribute to ‘sample quality’

■ Outline guidelines and procedures for the collection of samples
■ Describe the procedures for processing a range of blood samples
■ Discuss sampling of histological specimens
■ Describe the key steps in obtaining a sample for microbiology testing
■ Describe how samples should be handled during the pre-analytical stages
Introduction
Blood and other tissues, and body fluids can be tested in clinical laboratories to aid in
preventing, diagnosing, and treating diseases and disorders. A variety of analytical techniques
are used, which are being continually developed and improved in accuracy and ease of use by
biomedical scientists. Samples for clinical testing include whole blood, plasma, serum, urine,
and faeces for chemical and cellular analyses; tissues, and curettings, which are scrapings
of tissues, for histological examination to identify pathological changes; swabs may be taken
from various parts of the body for microbiological culture. Examples of body fluids that may
be tested for infection include blood, urine, cerebrospinal, bronchial, pleural, synovial, and
bone marrow.
Samples obtained from patients must be placed in suitable containers, transported to the
clinical laboratory, and stored correctly, if clinical test results from them are to be accurate.
Most clinical laboratories, including those within the National Health Service in the UK, have
standard operating procedures (SOPs), which specify the manner in which samples must be
treated. These procedures must always be adhered to.
152 7 SAMPLES AND SAMPLE COLLECTION
This chapter will describe the range of samples and the procedures for their collection for
analyses. It is not possible here to include sample preparation for every clinical test available
and if you have particular interests, the further reading at the end of the chapter is wider
ranging.
SELF-CHECK 7.1
Describe the key factors that contribute to pre-analytical quality assurance of clinical
samples.
7.1 Blood
Blood is the most commonly tested type of clinical sample because of its accessibility and
the wealth of information that can be obtained by analysing its many different constituents.
A clinician or general medical practitioner can request that a range of different blood tests be
performed in the clinical diagnostic laboratories by simply ticking or writing on a request form,
an example of which you can see in Figure 7.1.
Only a limited number of tests are performed on whole blood; most are done using plasma or
serum. Blood tests are performed for a number of clinical reasons, some of which you can see
listed in Table 7.1. These include, for example, to assist in the diagnosis of diseases or condi-
tions, or in screening tests, such as testing a cohort of individuals for the presence of human
immunodeficiency virus. Appropriate testing can also eliminate the possibility of a particular
condition even if the patient’s symptoms have suggested its possibility. Testing of samples of
blood from patients is also used to monitor the activity and severity of some clinical condi-
tions. A blood test may help to see if the patient is responding to treatment. For example, a
case of iron deficiency anaemia treated with oral iron supplements should begin to show an
increase in the concentration of haemoglobin in the blood. Samples of blood are also required
to test the effectiveness of specific organs, such as liver and kidney function, and of the effects
of medical or surgical treatment on these organs.
Naturally, a blood sample from a patient is also needed to establish his or her blood group in
cases where a blood transfusion is required.
FIGURE 7.1
Example of a request form
for laboratory tests. See also
Table 17.3.
7.1 BLOOD 153
TABLE 7.1 Examples of tests performed on blood samples
1
A full blood count (UK) or complete blood count (US). This repertoire of tests checks
for anaemia, and other conditions affecting erythrocytes, leucocytes, and platelets
2
Blood chemistry profiles
2
Kidney function (urea, Na+, K+, creatinine)
2
Liver function tests
2
Hormone (thyroxine, thyroid stimulating hormone) concentrations
2
Blood glucose (sugar) concentrations
3
Blood clotting tests Cross reference
1
4
See the Haematology book
Tests for inflammation of this series; 2 see the Clinical
2 Biochemistry book of this
Blood lipids and cholesterol concentrations
series; 3 see the Transfusion
2
Monitoring the doses of specific medications and Transplantation Science
book of this series; 4 see the
5
Immunology or serology tests such as testing for antibodies to specific viruses and bacteria Histopathology book of this
series; 5 see the Immunology
3
Blood grouping and screening for blood group antibodies book of this series.
Blood sampling procedures

The healthcare worker responsible for drawing blood samples may be a specialist phleboto-
mist, a clinician, or other healthcare worker trained in blood sampling techniques.
Samples of blood may be peripheral or venous depending on whether the sample is obtained
by pricking a finger or by venepuncture. Finger prick blood is used mostly for point of care
tests and is only acceptable providing a free flow of blood can be obtained. However, for
laboratory analyses, it is more common to obtain blood by venepuncture. Blood can be drawn
from a vein in one of several ways depending on the age of the patient and clinical tests
required. Usually, the antecubital vein, which is located in the crook of the forearm, is used as
a source (Figure 7.2). Occasionally, when veins are difficult to locate or have been used many
times, blood samples may be taken from the veins on the back of the hand.
In the UK, USA, and Australia most blood samples are collected using commercial vacuum
tube systems called vacutainers. Under certain circumstances, a syringe may be used, often
with a butterfly needle, which is a short needle with an attached plastic cannula; the name is
derived from the characteristically shaped plastic flanges that aid handling as you can see in
Figure 7.3(b).
Antecubital vein
FIGURE 7.2
Schematic illustration of a
venepuncture, indicating the
position of the patient’s arm
and antecubital vein.
(a) (b)
FIGURE 7.3
(a) Selection of conventional syringe needles and (b) a butterfly needle. Note the
characteristically shaped plastic handling vanes that give the needle its name.
However, in most parts of the developing world, needles and syringes continue to be the com-
monest method of obtaining venous blood.
Key Points
All blood is a potential source of infection and poses a risk to the phlebotomist; in par-
Cross reference
ticular, blood may carry human immunodeficiency or hepatitis viruses. Thus suitable
precautions must be taken at all times.
Irrespective of whether blood is collected by vacuum venepuncture or using a syringe and

needle, a number of general procedural rules are the same. First, any dietary requirements and
other appropriate preparatory instructions should be noted before performing investigations.
Some blood samples must be taken without a tourniquet (Figure 7.2) so it may be necessary
to check with the laboratory for the appropriate procedure. Blood samples must not be con-
taminated by patient infusions such as a saline drip or blood transfusion.
The correct type of sample tube (see later) as specified by the pathology laboratory must
always be used. If a blood sample is added to the wrong type of tube by mistake, it should
never be decanted into a second (correct) tube. Blood collection tubes must not be shaken as
this can cause haemolysis, which is the breakdown of the membrane surrounding the eryth-
rocytes and leakage of their contents. If this does occur, it is likely to invalidate subsequent
test results; hence, tubes should be mixed gently by inversion. Finally, it is essential to avoid
over- or under-filling tubes that contain an anticoagulant.
Venous blood sampling using vacutainers

Vacuum tubes were first marketed by the U.S. Company, Becton Dickinson Limited under the
trade name Vacutainer tubes. You can see a range of vacutainer tubes in Figure 7.4. Many com-
panies now sell vacuum tubes as the patent for this device is in the public domain.
7.1 BLOOD 155
FIGURE 7.4
Range of blood collection
tubes with colour-coded lids.
Each type of tube is used for
different clinical tests.
See also Tables 7.2 and 17.2
and Figure 12.14.
A vacutainer consists of a plastic hub, a hypodermic needle, and a vacuum tube. It is based on
the use of evacuated tubes so that venous blood automatically aspirates through the needle
inserted into the tube. Vacutainer systems incorporate a plastic cap known as a multiple sample
sleeve, which fits over the posterior end of the needle. This mechanism allows multiple vacuum
tubes to be attached and removed in turn from a single needle, allowing numerous blood sam-
ples to be obtained from a single venepuncture. This multiple sample sleeve also prevents blood
from leaking onto both the phlebotomist and the patient. When using the vacuum tube system,
the needle pierces the top of the sample tube and will potentially come into contact with the
additives in the tube (see below). As it is hollow, the needle can carry some additive into the
next tube and contaminate it. The additive most likely to cause trouble is ethylenediamine-
tetraaceticacid (EDTA). This will affect coagulation time assays and may interfere with some
clinical biochemistry tests by chelating and removing divalent metal ions, for example, Ca2+, and
can affect the determination of serum K+ by reducing erythrocyte integrity. Thus, blood samples
for plain tubes should be drawn first and those to be added to EDTA-containing tubes last.
Standard operating procedures for venepuncture samples must state that the healthcare
worker accurately identifies a patient by verbal confirmation, explain the reason(s) for the
procedure, and obtain the patient’s consent. The patient’s arm is examined to identify the
most easily accessible vein for the venepuncture. It is advisable not to take blood from an arm
containing a drip or transfusion as this will contaminate the sample. A tourniquet is placed in
position on the upper arm without applying pressure (to allow for patient comfort), and the
arm positioned comfortably on a support. Alcohol gel or hand washing must be performed
between each patient and latex-free safety gloves may be worn if necessary. The tourniquet is
tightened to facilitate the distension of the vein. The vein may also be gently palpated.
The vacutainer needle is now attached to the holder and its protective shield removed. The
needle is inserted into the distended vein. The vacutainer tubes are gently, but firmly pushed
in the appropriate order into the needle holder while keeping the holder steady with the other
hand. Once filled, all tubes are gently inverted as they are removed from the holder, ensuring
that the blood and anticoagulant are mixed to prevent clotting.
When all tubes have been filled, the tourniquet is released and removed. The final tube should
be removed from the vacutainer holder before removing the needle from the vein as this
practice reduces the risk of a haematoma.
The needle is withdrawn and pressure applied through a piece of gauze to prevent haemor-
rhage from the puncture wound. The patient should be asked to maintain pressure on the
needle site for approximately 5 minutes to aid wound sealing.
Venous blood sampling using a needle and syringe

A sterile, plastic syringe fitted with a suitable needle may be used to extract venous blood from
a patient. Needles for venepuncture are available in a variety of sizes, which are described by
their gauge (g) number. The most commonly used needles are:
• 21g (green top)
• 22g (black top)
• 21g (green label) butterfly
• 23g (blue label) butterfly
• 25g (orange label) butterfly
The last is only used in paediatric blood sampling or other extreme cases where the vein is par-
ticularly small. A 19g (brown top) needle has a larger bore and should be used in cases where
a large volume of blood is required, for example up to 100 cm3.
A needle and syringe may be more suitable for obtaining samples from elderly patients, or those
with difficult or inaccessible veins. Given that syringes are manually operated and the amount
of suction applied can easily be controlled, their use is particularly suitable with patients that
have small veins, which collapse under the suction of an evacuated tube. In children or other
circumstances where the quantity of blood gained may be limited, it is useful to know how
much blood has been obtained before distributing it amongst the various tubes. Needles and
syringe are also used for collecting blood for microbiological examination (Section 7.5). In all
cases, the tube contents must be mixed by inverting gently a number of times immediately
upon adding the blood to prevent clotting occurring in the tube.
Once used, the syringe and needle should be disposed of as a whole unit, that is, the needle
must not be disconnected, into a bin dedicated to sharp items. Nor must the protective sheath
be threaded back onto the needle before discarding it. Needle-stick injuries pose a risk of
infection to all healthcare workers because of the exposure to the patient’s blood. It is there-
Cross reference fore essential to follow good practice. Patients may be identified as ‘high risk’, that is already
You can read more about the diagnosed with infections, for example with HIV or hepatitis, or they may be carriers and una-
features on health and safety in
ware of their status. Box 7.1 describes the precautions necessary when obtaining blood from
a patient known to be of high risk.
Key Points
Irrespective of whether blood is collected by vacuum venepuncture or using a syringe and
needle, it must be added to the type of tube appropriate to the clinical tests required.
7.1 BLOOD 157
BOX 7.1 Procedure for the collection

of high risk blood samples
■ Samples must be collected by the clinician requesting the test or by an experienced

and fully trained phlebotomist
■ Plastic disposable apron and disposable gloves must be worn. It is recommended
that suitable eye protection such as goggles or safety spectacles be worn, but a face
mask is unnecessary
■ A waterproof disposable sheet must be placed under the patient’s arm
■ The patient’s skin must be cleaned and the needle inserted in the usual way using a
vacutainer or syringe
■ Any spillages must be cleaned with freshly made 3% hypochlorite solution
■ Used swabs must be discarded into a disposal bin specifically designated for sharp
items
■ Gloves and apron must be discarded into a plastic sack for incineration (colour-
coded yellow in the UK)
■ Hands must be washed with soap and water from elbow taps and dried with dispos-
able paper towels
■ ‘Danger of infection’ labels need to be attached to request forms and samples, which
must be placed in plastic sample bags and sealed
Blood containers
Once collected, the sample of blood can be added to one of several different containers
depending on the tests required. Some tests must be performed using serum and thus require
the prior clotting of the blood. In contrast, tests on plasma require the addition of antico-
agulants to the blood sample to prevent its clotting. If you are unclear, Box 7.2 describes the
differences between plasma and serum. If the concentration of glucose in the blood is being
measured, then the blood is added to a container containing a preservative. Sample bottles
with specifically coloured lids (see, for example, Table 7.2) indicating the additive present and
the test profiles for which they can be used have been developed to help phlebotomists in
adding blood samples to the appropriate container.
Anticoagulants and other additives

The tubes in which venous or capillary blood is transported to the clinical laboratory may con-
tain a variety of additives or none at all. It is essential to know which anticoagulant or additive
is required by the laboratory for particular tests. Table 7.2 describes anticoagulants, additives,
and their use.
For most haematology assays, particularly full blood counts (FBC), blood group determinations,
antibody screening, and transfusion compatibility tests, whole blood needs to be mixed with
the anticoagulant EDTA, which acts by chelating and removing Ca2+ from solution. This prevents
clotting, as Ca2+ is essential to the clotting cascade that forms fibrin. However, if blood clotting
tests are required, then trisodium citrate is traditionally used as the anticoagulant of choice.
BOX 7.2 What is the difference

between serum and plasma?
Plasma is formed from blood samples that have been prevented from forming a clot by
adding anticoagulants; the erythrocytes, leucocytes, and platelets are then allowed to
settle or are separated by centrifugation, leaving the plasma as a separate upper layer in
the tube. Portions of plasma can then be removed for analytical tests. Serum, however,
is obtained from whole blood that has been allowed to form a clot. The clot is formed
by the conversion of soluble fibrinogen to a fibrin gel. The gel consists of a mesh of
protein fibres that traps erythrocytes, leucocytes, and platelets. If the clot is allowed to
settle or the tube is centrifuged, the serum forms a layer above it. Samples of serum can
then be easily removed with a pipette or in a clinical analyser for testing or can be stored
in a refrigerator or freezer. Whole blood samples cannot be stored in this way as the
erythrocytes would haemolyse. In addition, many of the analytes estimated in clinical
laboratories are present in the serum and using whole blood is not satisfactory for their
analyses. Thus, plasma contains fibrinogen, which is absent in serum.
TABLE 7.2 Commonly used coloured containers and anticoagulants. See also Tables 17.2 and 17.3.
Additive Optimum concentration Mode of action Lid colour code1 Examples of clinical
tests
2
Ethylenediamine 1.2 mg anhydrous salt Chelates and removes Lilac Full blood count
tetraacetic acid per cm3 of blood Ca2+ Sickle cell tests
Glandular fever tests
2
Ethylenediamine As above As above Pink Blood group and
tetraacetic acid compatibility tests
Antibody screening
2
Trisodium citrate 1 volume 0.109 mol dm−3 As above Blue Coagulation (clotting)
trisodium citrate per 9 tests, e.g. prothrombin
volumes of blood time, activated partial
thromboplastin time
2
Trisodium citrate 1 volume 0.109 mol dm−3 As above Black Erythrocyte
trisodium citrate per 4 sedimentation rate
volumes of blood (ESR)
2
Lithium heparin, 15 (± 2.5) international Neutralizes thrombin by Green Blood glucose, Ca2+
2
Heparin units per cm3 blood. inhibiting several clotting
factors
3
Fluoride oxalate Fl– prevents pre-analytical Grey Blood glucose
breakdown of glucose
(glycolysis). Oxalate binds
Ca2+
3
Gel clot activator Gel substance Accelerates clotting of Red/brown/yellow Tests requiring serum
blood
1
Paediatric sample bottles have different coloured tops; the anticoagulant is stated on the label.
2
Anticoagulant.
3
Not an anticoagulant.
7.1 BLOOD 159
It is essential to add appropriate volumes of blood samples to the tubes as under or overfill-
ing means the proportion of blood to anticoagulant will be incorrect, which will decrease
the accuracy of test results. This is especially the case when coagulation tests are to be
performed. Some assays require whole blood and in those cases, tubes containing lithium
heparin are used.
The majority of clinical biochemistry tests are performed on serum and so a plain tube or a
tube containing a clotting accelerator (known as a gel tube, see Figure 12.14) are used. These
are usually a plastic or glass tube with kaolin-coated plastic granulate or a polymerized acryla-
mide resin or gel. The clotting accelerator can interfere with some clinical assays and so a plain
tube is recommended for certain tests, in which the blood must be allowed to clot and the
clot to retract naturally, leaving the serum freely available. If serum is removed before the clot
has retracted, it may continue to clot and form a fibrin gel. The advantage of adding a clotting
accelerator is that the rapid formation of the clot means tests can be performed immediately
after centrifugation.
Labelling of tubes
It is essential that all tubes are legibly and accurately labelled with information that allows sub-
sequent matching of the patient and sample by the laboratory. This information may include
the patient’s family and given names, date of birth, NHS/CHI number, hospital number, gen-
der, source, date, and time of sample collection. Quality assurance in laboratory professional
practice begins with sample collection. Thus, it is essential that healthcare professionals have
an understanding of the sampling procedures and the correct labelling of these samples. Each
discipline in biomedical science will have particular sample labelling requirements which are
fully explained in the companion volumes in this book series, but the bare minimum of infor-
mation is outlined in Table 7.3.
Once appropriately labelled, the sample tubes are placed in a plastic bag which is then sealed.
The patient should be checked for bleeding from the venepuncture and pressure continued
until the bleeding stops if necessary. The needle site may be covered using a fresh piece of
folded gauze and micropore tape. Finally, hands must be washed with soap and water after
removing gloves, or washed immediately if they have been contaminated with blood.
TABLE 7.3 Labelling of sample request form
Labelling of specimen containers Labelling of request form

Full name of patient Name of patient – forename and surname
Date of birth and hospital number Date of birth and hospital number
Clinical details – including any information likely
to aid the clinician or other healthcare workers
Name of consultant Name of consultant
Ward or clinic Ward or clinic
SELF-CHECK 7.2
What are the minimum identification details to be included on the label of a sample?
Factors in blood sampling that may affect

accuracy of results
Cross reference To perform tests on serum or plasma obtained from a sample of blood, the blood must first be
You can read about the centrifuged. Centrifugation must be performed at the appropriate speed and length of time to
principles of centrifugation in isolate the serum or plasma from the blood. It is essential that cellular components do not leak
Chapter 12 ‘Centrifugation’. their contents into the serum or plasma because this contamination will affect the concentra-
tions of some analytes.
Blood must be centrifuged within a suitable time of sampling, normally 4 hours post-
venepuncture. It is essential to prevent haemolysis as this will affect many biochemical and
haematological tests. High concentrations of blood lipids also affect the accuracy of many
clinical tests. This is known as lipaemia, and is due to a high fat diet or a lipid abnormality.
Figure 7.5 shows the levels of haemolysis and lipaemia in centrifuged blood samples.
Serum must be removed from erythrocytes if the sample is to be stored overnight, to ensure
accurate test results, particularly for concentrations of K+ and glucose. Samples for coagulation
tests need to be centrifuged and tested as soon as possible as coagulation factors are relatively
labile. Alternatively, the plasma may be removed and frozen prior to analysis. When freezing
samples, the permitted length of storage may depend on the temperature or the substance
being analysed. The normal recommended temperature is −20°C.
Haemolysis
(a)
0 25 50 100 200 400 800 mg 100 cm-3

0 0.25 0.50 1 2 4 8 g dm-3
Slightly haemolysed Haemolysed Grossly haemolysed

(K+, PO42–, LDH may be
affected)
Lipaemia
(b)
60 100 150 200 250 400 800 mg 100 cm-3

0.68 1.13 1.69 2.26 2.82 4.52 9.03 mmol dm-3
FIGURE 7.5
Levels of (a) haemolysis and (b) lipaemia in centrifuged blood samples. The black line gives
an indication of the opacities of the supernatants. LDH, lactate dehydrogenase.
7.2 URINE 161
Arterial blood sampling for blood gas analysis

Arterial blood samples are required when investigating conditions such as diabetes and lung
diseases. These can affect the functioning of kidneys or lungs, and result in an imbalance in
blood pH, carbon dioxide, and oxygen contents, and/or electrolyte concentrations. Arterial
blood transports oxygen to the tissues and venous blood takes waste to the lungs and kid-
neys, therefore, the gas concentrations and pH differ between these two types of blood ves-
sels. To estimate blood gases, blood is sampled from the radial artery, although in the case
of newborn infants may be taken from the umbilical cord. The radial artery is located on the
inside of the wrist, just beneath the thumb as shown in Figure 7.6, where a pulse is found. In
difficult cases an artery in the elbow (brachial) or the groin (femoral) may be used. An arte-
rial blood sample must be obtained by a clinician who is trained in this specific procedure.
Blood is drawn into a syringe, which contains the anticoagulant heparin. A small amount of
heparin is expelled from the syringe before taking the sample; adequate heparin will remain
in the 0.2 cm3 dead space of the syringe barrel and needle. A minimum of 3 cm3 of blood is
required to avoid excessive dilution by the heparin. Samples containing air bubbles must be
discarded because this will cause gas equilibration between the air and the blood, thus low-
ering the pressure of arterial CO2 and increasing the pressure of arterial O2. After sampling,
pressure must continue to be applied to the artery for at least 5 minutes to ensure bleeding
has stopped.
The sample is retained within the syringe, which is mixed gently by inversion, and sealed so
that exposure to air cannot occur. The syringe must be transported to the laboratory for test-
ing as soon as possible and should be kept at 4 oC if any delay is expected, up to a maximum
of 1 hour.
7.2 Urine
The diagnosis of many disorders is aided by collecting urine and analysing its metabolites
and chemicals. Urine may be collected as single or random samples, or as the total vol-
ume of urine voided over a 24-hour period (Table 7.4). Random or early morning samples
of urine may be tested for the presence of a variety of substances using a urine dipstick
(Figure 7.7). The glucose tolerance test used in diagnosing diabetes mellitus requires three
samples of urine, which are collected at specified intervals and analysed for their glucose
concentrations.
Radial artery
FIGURE 7.6
Schematic showing the
sampling of arterial blood
from the radial artery.
TABLE 7.4 Types of urine samples and their corresponding analytes or tests
Type of urine collection Analyte/test in urine

Random urine collection Bence-Jones protein
Reducing substances
Indicans
Osmolarity
Porphyrins (protect from light)
Pregnancy testing
24-hour collection without preservative Creatinine

Electrolytes
Protein
Urate
Citrate
24-hour collection with preservative Ca2+

Oxalate
Phosphate
5-Hydroxyindole acetic acid
Catecholamines
Collection of 24-hour urine samples

Twenty-four-hour urine samples give the most accurate estimates of the chosen analyte.
However, it is essential to obtain a collection of the total urine output over an accurately timed
24 hours. Thus, careful attention to detail is essential. The patient is supplied with a special plas-
tic container, with capacity of up to 2 dm3. If urine output in the 24 hours exceeds this, a second
container is provided. The container should not be emptied or rinsed as it is chemically cleaned,
and in some cases may contain a preservative such as hydrochloric or acetic acid. Care must be
taken to avoid spillages. It is usual to start collecting urine on rising in the morning. The patient
should initially empty their bladder and discard the urine, noting the exact time. In the next 24
hours, all urine voided should be included in the collection and added to the container. On
the following day, at the same time as the collection started on the previous day, the bladder is
emptied and all of this urine is collected into the container. Urine collection is then complete.
The container must be clearly labelled with the patient’s name and the dates and times of
collection. Additional instructions may be required for patients whose urine is to be tested for
catecholamine levels (see Box 7.3).
BOX 7.3 Foods and drugs that affect urinary

catecholamine concentrations
Many foods and drugs can disturb the biochemistry of ■ Foods: bananas, fruit, coffee, chocolate, vanilla, and
blood and produce metabolites that are detectable in flavourings.
samples of urine. The concentrations of catecholamines ■ Drugs: aspirin, monoamine oxidase inhibitors, phenothi-
in urine are particularly affected. Thus, the following foods azines, imipramine, labetalol, guanethidine, reserpine,
and drugs should not be taken for 48 hours before and for levedopa, tetracycline, or alpha methyldopa.
24 hours during, the collection of a urine sample:
7.3 FLUIDS OTHER THAN BLOOD AND URINE 163
FIGURE 7.7
Renal dipstick used for clinical
testing of urine.
Like blood, urine may also be collected for microbiological analyses (Section 7.5).
7.3 Fluids other than blood and urine

Apart from blood and urine, samples of a number of other body fluids are collected for clinical
testing. These include:
• Cerebrospinal fluid
• Bronchial fluid
• Pleural fluid
• Bone marrow
A minimum of 1 cm3 of these samples are collected by invasive procedures, which have some
degree of risk to the patient and must therefore be performed carefully by correctly trained
healthcare workers. Often, second samples tend to be unobtainable. Once collected, the
samples are added to sterile universal bottles. Some of these are mainly tested for the pres-
ence of microorganisms and so we will discuss them in Section 7.5; others may be subjected
to other clinical biochemical or immunological analyses.
Cerebrospinal fluid
Samples of cerebrospinal fluid (CSF) are required to investigate infections of the central
nervous system (Section 7.5) or cases of suspected subarachnoid haemorrhage. The fluid is
removed using a needle from the spinal canal in the lower back, in a procedure called a
lumbar puncture (Figure 7.8). Three samples of CSF are needed from patients with suspected
subarachnoid haemorrhages.
The samples should be investigated by the microbiology laboratory to determine the numbers
of cells and types of microorganisms present. In addition, the clinical biochemistry laboratory
may, for example, determine the concentration of protein in the sample.
Bronchial fluid
A fibre optic bronchoscope is used to obtain fluid from the bronchioles of the lungs, as you
can see in Figure 7.9. The tip of the bronchoscope is introduced into the main stem bronchus.
The bronchus is usually rinsed with a saline solution. Suction is then applied to the main stem
bronchus and other segments of the bronchioles to remove fluid. The bronchial fluid may be
L3 L4 L5
Spinal cord
L3
Epidural space
FIGURE 7.8 L4
Simplified illustration of the
sites and technique used to
perform a lumbar puncture.
Note how the needle is L5
inserted between the
vertebrae allowing CSF to
be collected.
7.3 FLUIDS OTHER THAN BLOOD AND URINE 165
Bronchoscope
Trachea
Bronchus
Light
FIGURE 7.9
Schematic illustration of a bronchoscopy, a procedure
that allows the respiratory system to be directly viewed.
Samples of bronchial fluid can be removed as described
in the main text.
filtered through sterile gauze before being added to a sterile container for transport to the lab-
oratory. The sample may be centrifuged to obtain cells for cytological investigations (Section
7.4), or cultured to detect and identify any microorganisms which it may contain.
Pleural fluid
Fluid that collects between the lungs and the rib cage is known as a pleural effusion. The fluid
may be sampled as illustrated in Figure 7.10, to investigate for disease. The patient is prepared
by disinfecting the skin on the patient’s back, which is then numbed using a local anaes-
thetic. A needle attached to an empty syringe is inserted into the fluid pocket, which is located
approximately 3 cm below the surface. The syringe is filled or attached to soft plastic tubing to
Fluid in pleural
cavity
FIGURE 7.10
Schematic illustration of the removal of a sample of
pleural fluid.
remove the pleural fluid into a bag or jar. The total sample, or a portion of it, is labelled with
the patient’s details and transported to the laboratory for clinical investigation.
Synovial fluid
Synovial fluid cushions and lubricates the mobile joints of the body where it is found in small
quantities in the spaces contained within the synovial membranes. Analysis of synovial fluid
may identify diseases that affect the structure and function of the joints, including osteoarthri-
tis, rheumatoid arthritis, and gout. Synovial fluid sampling is performed by a clinician using a
sterile syringe and needle in a procedure called arthrocentesis, which you can see outlined in
Cross reference Figure 7.11. A local anaesthetic is used to numb the area surrounding the joint and a needle
Gout is described in Chapter 4 is then gently inserted into the space between the bones, and a sample of fluid sucked out
‘Hyperuricaemia and gout’ in into a syringe. The fluid is placed in a sterile container and transported to the laboratory for
Clinical Biochemistry. analysis. Microscopical (Section 7.4), microbiological culturing (Section 7.5), and biochemical
tests, such as those for uric acid and glucose may be performed on the sample.
Bone marrow
Samples of bone marrow are used diagnostically to observe changes to cellular morphology,
and for clinical and immunological investigations. Samples are obtained by aspiration or by
trephine biopsy. Bone marrow may be sampled from a variety of sites but the commonest
are the posterior iliac crest and sternum. As you can see in Figure 7.12, aspiration involves
inserting a hollow needle, to which a syringe is attached, into the marrow. The marrow is then
sucked into the syringe and spread directly onto glass microscopical slides for analysis or into
a sample pot to be used for cytogenetic investigations, culturing the cells present, biochemical
analyses, and a variety of other specific investigations.
A trephine biopsy is performed using a trephine needle, known as a Jamshidi bone trephine
needle. The sample comprises a core of solid bone, which includes the marrow. This is placed
into a solution of formalin for fixation and examined by the histology laboratory.
Bone
Cartilage
Synovial fluid
Patella
FIGURE 7.11
Bone
Schematic illustration of
the removal of a sample of
synovial fluid, in this case
from the knee joint, using a
needle and syringe.
7.4 CY TOPATHOLOGY AND HISTOPATHOLOGY SAMPLES 167
Iliac crest
Hollow aspiration
needle FIGURE 7.12
Schematic illustration of the
sites and technique used
to obtain a sample of bone
marrow from an iliac bone
using a robust intra-osseous
Skin Bone Bone marrow needle and syringe.
7.4Cytopathology and
histopathology samples
Cytology is the study of cells and histology that of tissues. Cytopathology and histopathology
are the branches of pathology that study and diagnose diseases at the level of the cell and tis-
sue, respectively. The study of cytological and histological abnormalities requires samples of
tissues and cells.
In general, the majority of histopathology samples are transported to the laboratory in plastic
pots of varying sizes or glass ‘universal’ bottles containing a fixative (Table 7.5). In the UK, 4%
formaldehyde (as formalin or formal saline) is used to fix the majority of samples. The ideal
amount of fixative is 10 times the volume of the sample. Failure to use adequate amounts will
potentially result in a loss of tissue and cellular morphology, and could adversely affect subse-
quent staining and molecular techniques that may be required for diagnosis.
Cells
Cytological samples are normally provided unfixed, as cell suspensions in transport media
or as smears of cells on glass slides. The latter are fixed using alcoholic spray fixatives.
The commonest cytological samples are taken from the cervix by swabbing (Section 7.5)
and are used in smear testing to detect cervical cancer at an early, treatable stage (see
Figure 8.2).
TABLE 7.5 Fixative solutions for histology samples
Fixative Constituents Use
Formal saline 100 dm3 40% formaldehyde General preparation of

9 g NaCl paraffin embedded tissue
900 dm3 tap water sections from e.g. liver, kidney
Alcoholic formalin 100 dm3 40% formaldehyde Specific cases, as advised by

900 dm3 95% ethanol the clinical laboratory
Bouin’s fluid 75 dm3 saturated aqueous picric acid Biopsies of testicular, prostate,
25 dm3 40% formaldehyde and other tissues
5 dm3 glacial acetic acid
Glutaraldehyde 5 dm3 glutaraldehyde Ultrastructural studies

95 dm3 0.1 mol dm−3 cacodylate
buffer, pH 7.4
Fine needle aspirates

A clinical sample can be taken from superficial sites such as breast, lymph nodes, thyroid,
and salivary glands, and subcutaneous lesions in general by aspiration using a fine needle.
Aspirations can usually be performed in an outpatient department immediately by a patholo-
gist or on in-patients in their ward. Aspirations of deep-seated sites may be undertaken using
needles of the correct length and using some form of image guidance, such as ultrasound or
X-rays. If a pathologist is not available or the clinician wishes to perform the aspiration then a
member of the cytopathology laboratory may assist in the spreading, fixation, and transport of
the sample. Aspirations are generally considered to be cytopathological samples, unless larger
aggregations of cells, akin to tissues, are removed. A diagnosis can normally be reached the
same day, allowing for more rapid determination of a treatment programme for the patient.
Tissues
Post-mortem and post-operative samples may be major parts of bodies, portions of organs,
or small pieces of tissue taken as biopsies. Sections cut from pieces of tissue are processed and
stained on glass slides in a variety of ways to allow them to be visualized using a microscope.
Samples of tissue obtained during surgery may be rapidly frozen using liquid nitrogen, which
prevents ice crystals forming and disrupting the morphology of the tissues. Thus, intra-operative
Cross reference samples should be sent immediately to the laboratory, without fixation. Sections of the frozen
Chapter 8 ‘Microscopy’. sample can then be cut and stained for microscopic observation. Sections prepared in this way
can aid a rapid diagnosis and guide the surgeon during an operation. Samples may also be
obtained from patients by curettage, which are essentially scrapings of tissues.
Cross reference Biopsies such as skin or renal tissue samples, which require testing by immunofluorescence,
You can read more about must be sent to the clinical laboratory as soon as possible in normal or physiological saline
immunofluorescence in Chapter solution (9 g dm–3). These are then frozen and sections of tissue prepared. Removing biopsies
15 ‘Immunological techniques’.
of muscle tissues may require specific techniques that can be obtained by direct discussion
with the laboratory. Testicular and prostatic biopsies are recommended to be placed in Bouin’s
fixative (see Table 7.5). Lymph nodes and other samples on which microbiological or bio-
chemical examination is required must be divided and a portion placed in a sterile container.
Lymph nodes removed from patients with suspected lymphoma should be fresh and intact
and sent immediately to the histopathology laboratory.
7.5 SAMPLES FOR MICROBIOLOGY TESTING 169
Tissues may also be sampled to investigate specific enzyme activities, such as that of ATPase;
activities of this change in different types of muscle fibres in some muscle disorders. In such
circumstances, the sections are fixed following the investigative technique to ensure that
enzyme activity is not compromised. Muscle biopsies may have specific requirements, which
can be obtained after direct discussion with the laboratory.
Transport of cytological and histological samples

Samples of cells and tissues for cytological and histological investigations are placed in pots
with tightly closing lids to prevent leakage. The pots are enclosed in plastic bags for transport
to the laboratory. Samples must be accompanied with an appropriate request form in a man-
ner that keeps the form and sample together, but avoids contamination of the form. As with
all samples, it is essential to complete the request form, as incorrect or incomplete forms may
result in a delay in results. Any fresh samples must be sent to the laboratory as quickly as pos-
sible to avoid deterioration of these samples.
When the sample is received in the laboratory, it must be matched to its request form, ensur-
ing all details are legible and correct. Due to the diagnostic nature of many histological or
cytological samples, it is important that full clinical details are provided on any previous medi-
cal history, biopsies, or treatments. Failure to provide this information may result in the sample
being processed in a sub-optimal way.
Key Points
Samples for histology or cytology are often unique and it is not possible to take a repeat
sample if something goes wrong
SELF-CHECK 7.3
List three ways in which a sample of tissue can be obtained for histological examination.
Storing cytological and histological samples

Cross reference
Cytological and histological samples are often stored for longer periods than samples for other More material about the human
disciplines, being kept (as a minimum) until a definite diagnosis has been established. The tissue acts and blood regulations
is given in Chapter 19 ‘Quality
resulting prepared specimens are also stored for long periods of time, which presents its own
assurance and management’.
problems.
Storage of samples and specimens is now governed by the requirements of the various human
tissue acts and is the subject of professional guidance from the Institute of Biomedical Science
and the Royal College of Pathologists.
7.5 Samples for microbiology testing

Microbiology laboratories of hospitals investigate samples for viruses, bacteria, and fungal
infections. Virology laboratories offer routine and specialist tests for the presence of viruses,
including the detection of those causing hepatitis, acquired immunodeficiency syndrome
(human immunodeficiency virus, HIV), German measles (rubella) and, despite being caused
by a bacterium, often syphilis. The bacteriology section of the microbiology laboratory deals
with the isolation, identification, and antibiotic sensitivity testing of clinically significant bac-
terial isolates obtained from a range of patient samples. These include blood, urine, sputum,
and other body fluids, faeces, swabs from for example wounds, eyes, nose, throat, vagina, and
groin. In addition, scrapings and nail clippings are also cultured for microbiological growths.
Fungal infections are investigated by the mycology section of the microbiological laboratory.
In all cases, the healthcare worker must ensure that the sample is correctly labelled with the
patient’s identifying details, and that the appropriate details, including medical and treatment
history, are provided on the request form.
Blood
Samples of blood for microbiological culture in aerobic and anaerobic conditions are obtained
using a needle and syringe (Section 7.1) to keep the environment sterile. Bottles used for the
blood cultures do not fit in the standard vacuum tube holders, although it is possible to obtain
blood culture bottles with long narrow necks and which do fit, or to obtain adaptors that allow
the culture bottles to be used as a vacuum tube. When blood cultures are collected using a
winged blood collection set, air in its tubing means the phlebotomist must collect the blood
sample for aerobic culture first. A blood sample for anaerobic blood culture can then be col-
lected once the air has been flushed out of the tube.
It is essential that the collecting procedure is performed in as sterile a manner as possible

to prevent skin commensals contaminating the samples. To reduce this danger, an abrasive
swab is used to prepare the site by removing upper layers of dead skin cells along with their
contaminating bacteria. Povidone-iodine has traditionally been used to clean the skin, prior to
sampling, but in the UK a 2% chlorhexidine in 70% ethanol or isopropyl alcohol solution is pre-
ferred and time must be allowed for it to dry. The tops of all blood containers should also be
disinfected using a similar solution. The bottles are then incubated in specialized incubators
for 24 hours. This step allows the very small numbers of bacteria (potentially only one or two
organisms) to multiply sufficiently for identification and antibiotic resistance testing. Modern
blood culture bottles have an indicator in their base that changes colour in the presence of
bacterial growth and can be read automatically by machine. The bar-coded stickers found
on these bottles must not be removed as they are used for identification by the automated
systems.
Urine
Urinary tract infections are common in certain groups, such as the elderly, immunocompro-
mised patients, or pregnant women. The aim is to collect urine from the patient with minimal
contamination so that any pathogenic infection can be detected by culturing and identify-
ing bacteria present in the sample. Patients need to be given clear instructions as to how to
collect the urine sample. Obviously, if they require assistance this must be given. Samples
of urine should be collected midstream (MSU) or from a urinary catheter (see below). Early
morning urine samples contain the highest numbers of bacteria, as the urine has been ‘incu-
bating’ in the bladder overnight. The genitals should be thoroughly washed with soap and
water, and the first part of the urine stream discarded into the toilet or bedpan. The urine
sample is then collected into a sterile utensil such as a foil bowl or sample pot, by intercept-
ing a continuous stream of urine. Sterile plastic screw-capped bottles are used for all samples.
Sample pots for MSU contain boric acid as this keeps bacterial numbers constant for at least
24 hours.
If the urine sample is to be used to investigate the possible presence of mycobacteria that
cause tuberculosis (TB) then complete (that is not midstream) early morning urine sam-
ples need to be collected over 3 days. If testing is to be for Chlamydia trachomatis, at least
15–20 cm3 of the first voided urine (FVU) of the day is required.
In all cases, a request form should accompany the urine sample, indicating the type of sam-
ple and any patient history of antibiotic treatment. If there is to be a delay in transporting
the sample to the laboratory, the sample should be stored refrigerated, but never in a food
refrigerator!
Collecting urine with a catheter

Sometimes, for example, following an operation or when the patient cannot urinate for them-
selves, the urine is drained directly from the bladder through a catheter (catheter sample urine,
CSU) into a drainage bag. Urine samples should not be collected directly from the drainage
bag, but taken from the sampling port by clamping the drainage tube immediately below the
sampling port and waiting for 10 minutes until enough urine has accumulated. Hands must be
washed prior to cleansing the sampling port with a medi-swab. A needle and syringe is then
used to collect approximately 10 cm3 of urine from the sample port and transfer it to a sterile
pot. The sample pot should be labelled with the patient’s details and sent to the microbiologi-
cal laboratory for appropriate testing.
Collection of sputum
The aim when collecting sputum is to obtain samples of deep respiratory secretions that are
not contaminated with upper respiratory tract bacteria. Sample collection begins by explain-
ing the procedure to the patient, encouraging them to breathe deeply, and on exhalation
to cough directly into a sterile screw-capped container. Microbiological culturing of sputum
samples should use freshly collected specimens. If sputum is being sampled to investigate for
the possible presence of TB-causing mycobacteria, at least three separately collected samples
should be tested over three consecutive days.
All samples of sputum should be labelled and sent to the microbiological laboratory in a sam-
ple bag separate from those for other types of samples, for example blood.
Other fluids
Other fluids collected for microbial analysis include CSF, bronchial, pleura, and ascitic. These
fluids are obtained by invasive techniques and are from normally sterile sites, as we described
in Section 7.3. Samples must be added to sterile containers. Care must be taken by healthcare
workers to avoid contaminating the sample with normal microbial flora. If the patient has
started antimicrobial therapy prior to sampling, this must be noted. In many cases, the results
of culturing these fluids to detect microorganisms present are particularly useful clinically.
Therefore, samples should be tested immediately by the laboratory.
The skin must be thoroughly disinfected prior to taking samples of CSF by lumbar puncture
(Figure 7.8). In addition to patient identifier on the sample bottles, the bottles need to be
labelled in the order the samples were collected Three samples are collected; the first two are
used for biochemical analyses. The third should be sent to the microbiological laboratory for
examination by Gram staining, appropriate culturing of the bacteria present, which depends
on the findings of the Gram stain, and counting any cells that may be present in the sample.
Ascites is the accumulation of fluid in the abdominal cavity in a number of pathological condi-
tions, such as cirrhosis of the liver, heart failure, certain cancers, and infections, for example,
pancreatitis, chronic hepatitis, and TB. Fluid may be removed by draining (Figure 7.13) or dur-
ing surgery, and subjected to relevant clinical testing.
Sweat testing involves the collection of a sample of body sweat to determine its concentrations
of Na+ and Cl−. This has been the most widely used diagnostic test for cystic fibrosis. Sweat collec-
tion requires specific training as a ‘good’ sample depends on care and skill in avoiding evapora-
tion and contamination, both of which lead to falsely increased concentrations of Na+ and Cl−.
Collecting a sample for a sweat test uses a small, painless electric current to help draw sweat to
the surface of the skin, from where it can be collected for analysis. The sweat may be drawn from
the thigh or the forearm, depending on the age and size of the patient. The area is prepared by
washing and drying, after which two metal electrodes are attached and fastened with straps. Two
gauze pads, one soaked in saline or hydrogen-carbonate and the other in pilocarpine, a drug that
stimulates sweating, are placed under the electrodes. The electric current is applied to the skin for
five to 10 minutes to carry the pilocarpine into the skin where it then stimulates the sweat glands.
Once the sweat is being produced the electrodes are removed, and the skin is washed with dis-
tilled water and redried. A dry piece of filter paper is taped to the area where the pilocarpine
was applied. The filter paper, known as a sweat patch, is covered with wax or a sheet of plastic to
prevent evaporation. After 20–40 minutes, the plastic is removed and the filter paper is placed in
a container, which is sealed and labelled with the patient’s details for transport to the laboratory.
Collection of faeces
The microbiological examination of faeces is complex. A full clinical history must be taken from
the patient, including foods eaten because of the possibility of food poisoning, any travel to
foreign countries, any medication being taken, as well as the more usual basic information.
Abdominal cavity
Lining of
abdomen
Ascites
Drain
FIGURE 7.13
Schematic illustration of the collection of ascites
fluid by drainage.
A sample of faeces is collected using a spatula. A piece the size of a large pea is adequate. The
sample, usually called stool, is transferred to a plastic screw-cap faeces container. If the faecal
sample is liquid, the pot is filled to about one-third. Hand-washing and the wearing of safety
gloves are essential. The presence of blood in faecal sample, known as occult blood, can be
detected using specialized cards as indicate in Figure 7.14. The faecal material is smeared on
the relevant portion of the card, which is then sent to the laboratory for analysis. When test-
ing for occult blood, patients must not eat any of the following for 3 days prior to the test: red
meat, black pudding, liver, kidneys, fish with dark meat (salmon, tuna, sardines, mackerel),
cauliflower, horseradish, tomatoes, radishes, melon, bananas, soya beans, alcohol, iron tablets,
aspirin, and ascorbic acid.
Collection of samples using sterile swabs

Simple sterile swabs consist of a plastic stick ending with a pad made from cotton, Dacron,
or alginate, which is usually contained in a plastic sleeve containing a transport material
to preserve the sample or swabbing (Figure 7.15). Swabbings usually consist of body tis-
sues and fluids for the detection of microbiological pathogens. They are transported from
the patient to the microbiological laboratory in media contained in the corresponding
sleeve.
(a) (b)
FIGURE 7.14
(a) Photograph of an open and closed occult blood card. (b) Schematic illustration of a
faecal sample being added to an occult blood card. Note three cards are provided for
collecting samples on different occasions.
Transport medium Sleeve
FIGURE 7.15
Photograph of a sterile swab
and its container, which
Swab
contains transport medium.
The commonest areas sampled by swabbing are:
• High vagina
• Nose and ears
• Throat
• Conjunctiva (eye)
• General wounds
Obtaining a high vaginal swab requires a speculum being placed into the vagina to dilate
its walls. The sterile plastic envelope containing the swab is discarded. The swab is removed
from its sleeve and inserted as high as possible into the vagina and gently rotated. Any visible
discharge is particularly sampled. The swab is returned to its plastic sleeve, which contains the
transport medium and sent to the laboratory for suitable analyses. Plain swabs are suitable for
the detection of all vaginal pathogens except Chlamydia or Herpes, which require the use of
specialized types of swabs.
Nasal swabs are obtained by moistening the swab in sterile saline, and then rubbing and rotat-
ing it against the anterior nasal hairs. The swab is then placed into its sleeve, where it is mois-
tened in its transport medium. Ear swabs are collected by placing a sterile swab into the outer
ear cavity, and rotating it to collect any pus or discharge. The swab is then placed into the
sterile sleeve, labelled with the patient’s details, including whether the sample is from the left
or right ear, and transported to the microbiological laboratory.
Swabbing the throat involves depressing the tongue with a wooden spatula. The swab is
then moved to the back of the throat and the area around both tonsils rubbed using gen-
tle rotary movements. The swab is then placed in its plastic sleeve containing transport
medium.
Microbial infections of the eyes can lead to a discharge of pus or other fluid. Conjunctival (eye)
swabbing involves thoroughly soaking the swab in the exudate by rubbing it across the lower
eyelid from the inner to outer corners. It is necessary that the patient remains still during the
procedure. The swab is then added to its sleeve with transport medium.
Wound swabs should be obtained before wounds have been cleaned, patients bathed, or
antibiotic treatment commenced or changed. Swabbing should only be applied directly to
an infected site to avoid contaminating undamaged skin or mucous membranes. The swab
should be rotated in areas showing pus and then placed in transport media. Both the swab
and its request form must be appropriately labelled; particularly relevant information regard-
ing antibiotic therapy and the site of the wound.
It is essential to detect the presence of methicillin-resistant Staphyloccocus aureus (MRSA) in

or near wounds. When MRSA has previously been shown to be present in a patient, this must
be stated in the clinical details and MRSA tests asked for on the request form. This allows the
laboratory to carry out additional tests to check for MRSA at the same time as routine cultur-
ing is being performed. Patients may need screening for the presence of MRSA prior to their
admission to hospital. In such cases, swabbings from the throat, nose, axillae, and groin are
required.
SELF-CHECK 7.4
List the key steps in obtaining a wound swab for microbial culture.
Samples for investigating fungal pathogens

Samples for mycology testing should be collected into special collection envelopes supplied
by the laboratory (Figure 7.16). As with other samples, they are sent with a request form includ-
ing details of the patient’s clinical symptoms and history.
Material from skin lesions is collected by scraping material from their outer edges, usually with
Cross reference
the edge of a glass microscope slide or scalpel blade. The edge of the lesion is the part most
likely to contain viable fungal material. Scrapings from the scalp are also obtained in this way, medical microbiology in the
but should include hair stubs. Hairs may be plucked from the scalp, but cut hairs are unsuitable companion book of that name.
for testing as fungal infections are usually below the surface of the scalp.
Nail clippings for mycological investigation should be taken from the discoloured or brittle parts
of the nail, and cut back as far as possible from the free edge as the growth of some fungi are
restricted to the lower parts. Scrapings can also be taken from under the nails to supplement clip-
pings; however, even if fungi are present in the nail clippings, they often fail to grow on culturing.
High risk, hazardous microbiology samples

If it is thought that there is a likelihood of group III pathogens (Box 7.4), such as tubercle bacilli
and hepatitis B and C viruses, being present in clinical samples, then the samples must be
Cross reference
placed in a plastic sample bag, which is then enclosed in a second sample bag attached to the
request form. The request form and sample must be marked with a Danger of Infection label.
FIGURE 7.16
Photograph of an envelope
for collecting samples for
mycological testing.
BOX 7.4 Categories of pathogenic

microorganisms
The Advisory Committee for Dangerous Pathogens has divided pathogenic organisms
into four groups, according to their level of danger. Group I are the least dangerous, IV
the most pathogenic. Mycobacterium tuberculosis, one of the two pathogenic organisms
that cause tuberculosis is a group III pathogen.
SUMMARY
■ The quality assurance in obtaining samples for testing by clinical laboratories includes cor-
rectly identifying patients, obtaining samples using correct procedures, meticulous label-
ling of samples and requests forms, adhering to health and safety procedures, and being
aware of standard operating procedures for sample processing
■ Procedures for obtaining and processing blood samples require selection of the appro-
priate container. An anticoagulant or additive is necessary for some laboratory analyses.
Venepuncture procedures may employ a needle and syringe or needle and vacutainer.
All blood samples should be free from haemolysis. Plasma or serum samples are obtained
by centrifuging blood samples
■ Samples for histological examination may be obtained as tissue biopsies. Sections cut
from pieces of tissue are processed and stained on glass slides, and visualized using a
microscope. Samples of tissue obtained during surgery may be rapidly frozen using liquid
nitrogen. In some situations, samples may be obtained by aspiration using a fine needle
■ Key steps in obtaining a range of samples for microbiology testing including the relevance
of sterility and appropriate use of transport media in which to transport samples to the
laboratory
■ Urine samples for biochemical analysis may be collected randomly or over a 24-hour
period. Containers for the latter may require the inclusion of a preservative
FURTHER READING
● Ageneye F. Pre-analytical quality assurance: a biomedical science perspective.
The Biomedical Scientist, 2007; 51: 86–87.
A short paper in a professional journal from the Institute of Biomedical Science.
● Bowen RA, Hortin GL, Csako G, Otañez OH, Remaley AT. Impact of blood collection
devices on clinical chemistry assays. Clinical Biochemistry, 2010; 43: 1–2, 4–25.
QUESTIONS
● Ford M. (editor) Medical Microbiology. Oxford University Press, Oxford, 2010.

A companion book in this series.
● Haverstick DM, Brill LB, Scott MG, Bruns DE. Preanalytical variables in measurement
of free (ionized) calcium in lithium heparin-containing blood collection tubes.
Clinica Chimica Acta 2009; 403: 1–2, 102–104.
Two articles that provide details of the types of blood collection tubes and discuss their
effects on blood chemistry analytes.
● Jacobs DS, DeMott WR, Oxley DK. Laboratory Test Handbook, 2nd edn. Lexi-Comp,
Inc, Ohio, 2002.
An excellent pocket-sized reference book (1348 pp.) containing sample details for all tests
imaginable, clinical reference ranges and much more.
● Pitt SJ, Cunningham JM. An Introduction to Biomedical Science in Professional and

Clinical Practice. Wiley-Blackwell, Chichester, 2009.
This textbook covers many aspects of laboratory practice, including an introduction to the
organization of sample analyses by clinical pathology laboratories.
● Polack B. Evaluation of blood collection tubes. Thrombosis Research, 2007;119:

133–134.
A journal article focusing on types of tubes for blood samples required for coagulation and
thrombosis investigations.
Useful Websites
■ Human Tissue Authority. The Human Tissue Act. Available at: http://www.hta.gov.uk/
legislationpoliciesandcodesofpractice/legislation/humantissueact.cfm
■ Epsom & St Helier NHS Services. Available at http://www.epsom-sthelier.nhs.uk/
our-services/a-to-z-of-services/diagnostics-and-pharmacy/pathology/chemical-
pathology/ (accessed 26 December 2009). A detailed National Health Service (UK)
website that describes specimen reception for pathology samples.
■ Lab Tests Online-UK. Available at: http://www.labtestsonline.org.uk/. A peer reviewed
public resource on clinical laboratory testing with a patient-centred approach. This web-
site was established with the aid of a grant from the Health Foundation, and is maintained
by the Royal College of Pathologists and the Department of Health, UK.
■ The retention and storage of pathological records and specimens. Available at: http://
www.rcpath.org/resources/pdf/g031retentionstorageaugust09.pdf
QUESTIONS
1. Trisodium citrate is used as an anticoagulant for which of the following?
(a) Catecholamine tests

(b) Coagulation tests
(c) Investigating suspected cholera cases
(d) Investigating suspected Chlamydia cases
(e) Clinical chemistry testing
2. Which of the following statements are TRUE?
(a) The commonest type of clinical sample analysed is blood

(b) A sample of serum does not contain fibrinogen
(c) Using an insufficient amount of fixative in preparing histopathology samples results
in a loss of cell morphology
(d) A bone marrow biopsy is called a trephine biopsy
(e) Patients providing faecal samples for occult blood tests must avoid eating bananas
3. List the common additives used in blood collection tubes, their modes of action, and the
clinical tests with which they are compatible.
4. Explain why clinical samples must be appropriately centrifuged and stored prior to clini-
cal analysis.
5. What instructions would you give to a patient whose urine is to be tested for its concen-
trations of catecholamines?
8
Microscopy
Tony Sims
Learning objectives
■ Define magnification, contrast, and resolution

■ Explain the principles of image formation
■ Identify the various components of a microscope and describe their functions
■ Describe the major types of microscopes used in clinical laboratories
■ Discuss the applications of the various forms of light and electron microscopy used in
clinical laboratories
■ Set up a microscope with optimum illumination
Introduction
A microscope is an instrument that forms an enlarged image of an object that would normally
be too small to be seen with the naked eye as shown in Figure 8.1 (a) and (b). Microscopy is
the use of a microscope to examine and analyse such objects. Microscopes that use a single
lens are called simple microscopes; those with more than one are compound microscopes.
Microscopes are perhaps the most widely used instruments in biomedical science. They have
contributed greatly to our knowledge and understanding of pathological processes, and are
used in all branches of biomedical science. There are several different types of microscope,
each of which has its own specific applications. Light microscopes are used to look at cells
and tissues. They are used in many areas of biomedical science and form the work horses of
histological examinations or in screening programmes, such as that for cervical cancer (Figure
8.2). The electron microscope, with its vastly increased magnification and resolution, is used
to visualize virus particles, explore the structures of bacteria, and observe more fully the sub-
cellular components seen in both normal and diseased cells and tissues.
In this chapter we will describe how the various types of microscope are constructed and
work, and how they can be applied to diagnostic biomedical practice.
180 8 MICROSCOPY
(a) (b)
FIGURE 8.1
Microscopical examinations of (a) blood smear and (b) the bacterium Staphylococcus
aureous. Courtesy of M Hoult, School of Healthcare Science, Manchester Metropolitan
University, UK.
FIGURE 8.2
Light micrograph of a cervical smear showing pink stained normal squamous cells
from the superficial layer of the cervix. The blue stained cells are from the cervical wall
immediately below that are squamous cells; their comparatively large nuclei indicate that
they are abnormal and the patient is at risk of developing cancer of the cervix. Courtesy of
Manchester Cytology Centre, Manchester Royal Infirmary, UK.
8.1 MICROSCOPY AND IMAGE FORMATION 181
8.1Microscopy and image

formation
Three main features limit the use of any microscope:
1. Magnification
2. Contrast
3. Resolution
The magnification of a specimen given by any microscope is simply the apparent size of the
image formed divided by its real size. We will discuss how a microscope produces a magni-
fied image in Section 8.3. The contrast of an image is the difference between the darkest and
lightest coloured areas, which in microscopy reflects differences in the densities of different
parts of the structure viewed. In general, a biological specimen will show poor contrast and
will therefore be difficult to see unless the contrast between different parts of it are increased
by differential staining. Specimens for light microscopy are generally stained with coloured
dyes, which are only absorbed by some areas of the specimen so boosting the contrast, as you
can see in Figure 8.3. You can read on outline of how clinical specimens are prepared for light
microscopy in Box 8.1.
The contrast of specimens for electron microscopy is increased by staining specimens with the
salts of heavy metals, such as lead, silver, or gold. Even for specimens with excellent contrast,
the magnification any microscope can give is limited by the resolution, which we will discuss
below.
Follicular cell
FIGURE 8.3
Histological structure of the thyroid gland.
Note how staining of the different portions has
increased the contrast of the image. Courtesy of
Follicle containing Dr A.L. Bell, University of New England, College
thyroglobulin of Osteopathic Medicine, USA.
182 8 MICROSCOPY
BOX 8.1 Preparing samples for

light microscopy
In general, tissue samples are transported to the laboratory in containers holding a fixa-
tive (Table 8.1) to preserve structure. In the UK, 4% formaldehyde (known as formalin) is
the most commonly used fixative. Ideally, about a 10-fold volume of fixative to sample is
required. Failure to use adequate amounts will potentially result in a loss of tissue and cel-
lular morphology, and could adversely affect subsequent staining and molecular techniques
that may be required for diagnosis. Large tissue samples may be sliced open to facilitate
optimal fixation.
Once fixed, samples of tissue for histopathological examination must be processed to
Cross reference
allow thin (2–3 lm thick) sections to be produced for microscopical study. For routine
You can read more about health
and safety in Chapter 4. processing into paraffin wax, tissues are exposed to a number of reagents, for exam-
ple, transferring the samples through a series of solutions with increasing concentra-
tions of alcohol, to remove fully their water content. The alcohol is then replaced by an
intermediate (or link) reagent that is not miscible with water, but allows molten wax to
penetrate the sample block. In the UK, USA, and Australia, this is routinely undertaken
on automated, fully enclosed tissue processors. In developing countries, this can still be
undertaken manually; however, the nature of the solutions used to process the tissue
does pose a health and safety risk to the laboratory worker.
Sections are cut from the wax-embedded tissue using a microtome; these can also be
automated to produce sections of consistent and reproducible thickness.
Situations may arise that require the production of sections for rapid diagnosis or inves-
tigation of materials. For example, samples of tissue obtained during surgery may be
rapidly frozen using liquid nitrogen (−196°C), which prevents ice crystals forming that
would disrupt the architecture of the tissue. Sections of the sample are cut and stained
TABLE 8.1 Fixative solutions for histology samples
Fixative Constituents Use
Buffered formalin 100 dm3 40% formaldehyde General preparation of

9g NaCl paraffin-embedded tissue
sections from e.g. liver,
kidney
900 dm3 tap water
Alcoholic formalin 100 dm3 40% formaldehyde Specific cases, as advised

by the clinical laboratory
900 dm3 95% ethanol
Bouin’s fluid 75 dm3 saturated aqueous Biopsies of testicular,
picric acid prostate, and other tissues
25 dm3 40% formaldehyde
5 dm3 glacial acetic acid
Glutaraldehyde 100% glutaraldehyde Ultrastructural studies

for microscopic observation to provide a rapid diagnosis and guide to the surgeon
during the operation.
In some instances, for example, renal biopsies, it may be necessary to produce sec-
tions of less than 2 lm thickness. Often, this requires processing the tissue sample into
resin, rather than wax. Sections are cut on an automated microtome using a knife blade
strengthened, for example, by coating its cutting edge with tungsten carbide or dia-
mond. Using the more solid medium of resin allows thinner sections to be cut, but it
does suffer the disadvantage of absorbing many of the stains used to heighten contrast.
Consequently, interpreting these sections can be more difficult. Resin is also used to
embed small pieces of tissues that require examination using an electron microscope
(see Box 8.3).
Once the tissue has been processed and sectioned, it must be stained to provide contrast
between the different tissue and cellular components. There is a multitude of different
methods that may be utilized to highlight different tissue components, but the routine
stain used is haematoxylin and eosin (H&E). Such stains highlight the general architec-
ture of tissues and determine if a pathological process is occurring, or if any further
techniques are required to reach a definitive diagnosis.
Samples received in histopathology laboratories are varied: it is not unusual to receive

samples of bone or other mineralized materials. These cannot be processed straight
to paraffin wax and sectioned using a microtome because the mineralized areas are
too tough for normal microtome knives to cut. However, the mineral content of the
fixed tissue can be reduced by exposing it to decalcifiying agents. For samples with
relatively small levels of mineralization, gentle chelating agents such as ethylaminedi-
aminetetraacetic acid (EDTA) may be used. For heavily mineralized tissues, such as bone,
harsher agents such as formic or hydrochloric acid must be applied. Over-exposure to
the decalcification agents can be detrimental to tissue architecture and have a nega-
tive impact on the staining properties of the tissue. In particular, antigenic sites may be
destroyed, leading to false negative results in subsequent investigations. Thus, samples
should be examined regularly using for example X-rays, to determine the optimal extent
of decalcification. The tissue should then be thoroughly rinsed and returned to fixative
solution. In samples where the degree of mineralization is crucial to diagnosis, as for
example when investigating osteoporosis, decalcifying agents cannot be used and tis-
sues should be processed into resin.
In cytopathology, the cells of the sample must be made available on a slide for viewing.
This can be achieved in different ways. The sample may be smeared onto the slide, for
example, from a swab or a spatula. A specialized centrifuge called a cytospin may be
used, to force the cells out of suspension (be it urine, transport medium) and onto the Cross reference
slide. If a sample has very few cells in relation to its volume, it is first subjected to con- Chapter 7 ‘Samples and sample
ventional centrifugation to concentrate the cells. The pellet of cells is then resuspended collection’ describes how swabs
in a relatively small volume, which is subjected to the cytocentrifuge. can be used to obtain clinical
samples.
In the UK, many cervical samples (Figure 8.2) are received as part of the national cervical
screening programme. These samples are delivered to the cytology laboratory in trans-
port fluid or fixative and the cells are separated by using membrane filtration or density
gradient systems that deposit the cells onto glass slides. As with histopathological sec-
tions, once the cells are fixed on the slide, they must be stained to increase contrast for
visualization by light microscopy.
184 8 MICROSCOPY
Key Points
All microscopes used in biomedical science are likely to be compound in nature. That
is they consist of two main elements, called objective and eyepiece lenses. By using
lenses in this way, vastly improved magnifications can be achieved over the use of a
single lens.
Resolution
Resolution is a measure of the clarity or acuteness of an image. It is the smallest distance that
can be distinguished between two points and objects smaller than the resolution cannot be
observed. The resolution of the unaided eye is approximately 200 μm.
SELF-CHECK 8.1
Define what is meant by the term resolution.
Resolution is a limiting feature of all microscopes, as simply magnifying an image will not always
provide more information. As magnification increases, resolution or the resolving power of a
microscope must also improve. Consider, for example, a digital image that is enlarged to the
point of pixellation: clearly, the clarity of the image has been decreased despite the increase in
magnification and is known as empty magnification.
In mathematical terms, resolution can be represented by the formula:
R = 0.61λ / NA
Where R is resolution, λ is the wavelength of the radiation or light and NA is the numerical
aperture of the lens.
Numerical aperture is a quantitative representation of a lens’ light (or radiation) gathering

capacity and is represented by the formula:
NA = n sin α
Where n is the refractive index of the medium between the glass coverslip (or the prepared
specimen) and the front of the objective lens, and α is the angle between the outermost ray of
light (or radiation) that enters the front of the objective lens and the optical axis of the objec-
tive lens, as shown in Figure 8.4. If the medium between the coverslip and objective is air then
the maximum theoretical numerical aperture is 1.0. However, it is not possible to achieve this,
as the lens would need to lie directly on top of the coverslip. If, however, we substitute oil or
water for air then the theoretical numerical aperture is increased to 1.3–1.5. In practice, the
theoretical values cannot be attained and in air the best possible value of numerical aperture
is approximately 0.95 and in oil approximately 1.4. Thus, the resolution of a light microscope
is approximately half (0.61/1.4) of the wavelength of visible light.
SELF-CHECK 8.2
Calculate the approximate resolution obtained with a microscope fitted with an oil immersion
objective lens and using light of wavelength 600 nm.
Objective lens
RI air α Coverslip
1.00
Slide
FIGURE 8.4
Schematic illustrating the angle
Optical axis of the light path when calculating
of lens the numerical aperture of a lens.
Light and waveforms

Light emitted from a single source can be regarded as a series of waves travelling in straight
lines in all directions. The length or periodicity of these waves is termed the ‘wavelength’ and
gives the light its colour. White light consists of a spectrum of light of different wavelengths
(and different colours) ranging from short ultraviolet through to long infrared light.
When light passes through air it travels in a straight line. If, however, it strikes a different medium
such as glass, the light path is both retarded and refracted, or deviated at the air/glass interface. Cross reference
Light of different wavelengths is refracted to variable amounts. Refraction is best seen when Chapter 11 ‘Spectroscopy’,
light passes from air into a glass prism and out again into air and is shown in Figure 11.7(c). gives a fuller description of
electromagnetic radiation.
Monochromatic light consists of light of a single wavelength whose amplitude determines
the brightness of light. The higher the amplitude of the wave, the brighter the light will be.
It is necessary to understand these two properties of the waveform of light to appreciate the
effects they have on any image seen in a microscope (Figure 8.5).
Image formation and lens defects

When parallel light rays pass through a lens, they are refracted by the lens and brought to
a common focus by it forming an image. This focus is called the focal point. The distance
between the centre of the lens and the focal point is called the focal length. However, lenses
can suffer from a number of defects or aberrations, which may prevent light being brought to
Amplitude
Wavelength
FIGURE 8.5
Schematic illustrating the definitions of amplitude and wavelength.
186 8 MICROSCOPY
this common focus. This is because at its edge, a lens will also act as a prism due to its shape
and the greater curvature of the lens which will exacerbate any aberrations.
These defects include:
• Chromatic aberration
• Spherical aberration
• Astigmatism
Chromatic aberration
Chromatic aberration is the inability of a lens to bring light of different wavelengths to a
common focal point resulting in a blurred image with multiple coloured fringes. It is caused by
different wavelengths being refracted to different degrees, as seen in a simple prism, as lenses
also act as prisms. Thus, light rays of shorter wavelengths are brought to different focal points
than ones of longer wavelengths, as you can see in Figure 8.6.
Key Points
Chromatic aberration is the inability of a lens to bring electromagnetic rays of different
wavelengths to a common focal point.
Spherical aberration
Spherical aberration is the inability of a lens to bring light passing through the periphery of
the lens to the same point as light passing through the central part of the lens (Figure 8.7). This
is caused by the lens refracting the light to different degrees, which depends upon the curva-
ture of the lens and the angle at which light enters the lens.
Key Points
Spherical aberration is the inability of a lens to bring electromagnetic rays of different
wavelengths to a common focus.
Both spherical and chromatic aberration result in images that are significantly reduced in qual-
ity. These aberrations can both be corrected by using different glass additives and lenses in
combination that have differing amounts of curvature and different shapes.
Focal point
White light
Lens axis
Blue Red
FIGURE 8.6
chromatic abberation in a
glass lens.
8.2 COMPONENTS OF A MICROSCOPE 187
Focal point
Lens axis
FIGURE 8.7
spherical abberation in a
glass lens.
SELF-CHECK 8.3
How can chromatic aberration be corrected?
Astigmatism
Astigmatism results when a lens is unable to bring light passing through one part of a lens to
the same focal point as light passing through another part. This results in a distorted blurred
image and is best illustrated when viewing a lattice. Astigmatism is perhaps the least important
lens aberration since correction of either chromatic or spherical aberration usually results in
the elimination of astigmatism.
Key Points
Astigmatism is the inability of a lens to bring electromagnetic rays passing through
different parts of the lens to a common focal point.
8.2 Components of a microscope

The basic construction of a compound light microscope has changed little since the mid-
nineteenth century. All consist of a system of lenses and their associated components, which
are used to form a magnified image of the specimen. However, since the 1800s, significant
improvements have been made in the quality of lenses, resulting in greatly improved images
(Figure 8.8). Much emphasis has also been placed on the ergonomic design of microscopes to
provide the user with the most comfortable working position.
Light source
All microscopes require a reliable light source. In early designs, this source came from the sun
and a mirror was used to reflect its rays into the microscope condenser. As optics improved,
this form of illumination became inadequate, since it was not possible to adjust the bright-
ness. Tungsten filament lamps fitted with a variable rheostat that allows the brightness to be
adjusted have proven to be a reliable mechanism. However, tungsten filament bulbs gener-
ate considerable heat and may need cooling to be used safely. Consequently, most modern
188 8 MICROSCOPY
(a) (b)
Eye
Eyepiece
Objective lens
Specimen
Specimen stage
Condenser
Light
Mirror source
FIGURE 8.8
(a) Modern compound light microscope. (b) Schematic illustrating the lightpath of a
compound microscope.
microscopes now use low voltage halogen light sources. These provide a more intense light
and a brighter image without the heat associated with tungsten filament light sources.
Fluorescent microscopes require a light source capable of emitting a high level of ultraviolet light,
which neither tungsten nor halogen lamps are capable of producing. For this purpose the best
light source is a high-pressure mercury vapour lamp. This lamp, however, requires a specialist
lamp holder and power supply and generates a significant amount of heat that needs to be dissi-
pated. Care must also be exercised regarding health and safety issues (Health and Safety Box ‘The
use and disposal of high-pressure mercury vapour lamps’).
HEALTH & SAFETY

The use and disposal of high-pressure mercury vapour lamps
The mercury vapour lamp contains mercury gas at high pressure within a glass housing. The
intensity of light emission from these lamps diminishes with use and, in addition, the glass
becomes increasingly unstable and prone to explode. To ensure that the lamp operates optimally,
manufacturers incorporate an elapsed timer to record how long the lamp has been in use and
recommend its replacement and correct disposal after a known length of use. Disposal must be
controlled to ensure mercury does not enter the environment.
It is also essential that after the lamp has been switched off, it is not re-used until it has cooled
Cross reference down. Failure to observe this cooling period may result in the lamp exploding. Manufacturers
Health and safety issues are
now incorporate switching timers into the lamp power supply that prevent the lamp being re-
discussed in Chapter 4. used until an adequate cooling period has elapsed.
Illumination of specimens
Effective microscopy relies upon the correct illumination of specimens. This is done by using the
condenser or sub-stage condenser as it is sometimes called, to focus the light produced by the
lamp into a cone onto the specimen giving it a maximum and even illumination. In its simplest
form, the sub-stage condenser consists of one or two lenses with an adjustment that allows
vertical focusing of the light to compensate for the varying thickness of microscope slides.
For best illumination, it is necessary to be able to accurately align the axis of the condenser
with the objective lens, and to be able to adjust the size and brightness of the cone of light
hitting the specimen. The condenser is fitted with adjustable screws to move the condenser
in the x–y axes to allow the light cone to be centred, and with an iris diaphragm to adjust the
diameter of light cone.
It is possible to set the condenser in one of two ways to optimally illuminate the specimen:
• Köhler illumination
• Critical illumination
Each method has its advantages and applications within biomedical science.
Köhler illumination requires the condenser to be set in such a way that light reaching the
specimen evenly illuminates a wide area. This form of illumination is particularly useful when
recording photographic images to ensure even illumination across the whole field. Critical
illumination is the focusing of the light source at a point directly onto the specimen using the
sub-stage condenser. It is an effective way of ensuring that the maximum level of illumination
reaches the specimen. This method is particularly necessary when using high magnification
objective lenses that have short working distances, but when combined with low magnifica-
tion objective lenses result in uneven illumination.
You can read an outline of how to set up a microscope for optimal illumination in the Method
Box ‘Setting up of microscope for optical illumination’.
METHOD Setting up of microscope

for optimal illumination
■ Whatever type of microscope is being used, the basic principles of setting it up for
critical or Köhler illumination are essentially the same. First, adjust the condenser to
as high a position as possible and then focus on a sample using a low power objec-
tive lens (normally 10ë or 4ë). Secondly, close the field diaphragm in the condenser
and sharply focus the diaphragm onto the image of the sample using the condenser
focus controls.
■ The field diaphragm can be considered to represent the illumination axis and should
be adjusted using the x and y condenser adjustment screws. The condenser lens
system focuses as much light onto the sample and the lamp is focused onto the plane
of the field diaphragm of the condenser. This is termed Köhler illumination. Now
open the field diaphragm until it is just outside the field of view.
■ If the microscope does not have a condenser fitted with a field diaphragm, it is nec-
essary to place a sharply pointed object, for example a pin, onto the glass of the
coverslip and focus the illumination onto this point by raising or lowering the con-
denser. This is termed critical illumination.
190 8 MICROSCOPY
Specimen stage
The specimen stage is the rigid flat surface immediately above the condenser on which the
specimen sits. Most stages incorporate sample clamps attached to mechanical manipula-
tors that allow accurate movement of the specimen. Vernier scales are incorporated into
the mechanism, which means that when a point of interest in the specimen is observed, its
Vernier values can be recorded and, therefore, the position is known and returned to later
with ease.
Stand and tube

The stand of the microscope is the sturdy, rigid, and vibration-free base to which all the other
components are attached. It incorporates the coarse and fine focus adjustment mechanisms
of the microscope that allows for fine vertical movement of either the stage or the objective
lens depending on the manufacturer.
Above the stage the stand has a nosepiece for holding between three and five objective lenses
of different magnifications. The nosepiece rotates between set points allowing the appropriate
objective lens to be moved into the light path. Above the nosepiece is the observation tube
that houses the eyepiece lens. There are several types of observation tube: the simplest consists
of a single eyepiece lens, but more usually consists of a binocular or trinocular attachment. A
trinocular tube allows a camera to be attached for simultaneous viewing and recording of the
image. The distance from the front of the objective lens and the eyepiece is termed the tube
length. The length of the tube has a significant impact on the final magnification as described
in Section 8.3.
Recent developments in lens construction have resulted in the production of ‘infinity cor-
rected’ lenses. Here, the focused image leaving the lens is not brought to a focal point, but
rather remains parallel at the same focal plane to infinity with no change in magnification.
This development has allowed manufactures to vary the tube length without altering the mag-
nification allowing them to develop and design more ergonomic stands and multi-headed
microscopes.
Key Points
Microscopes are able to have multiple viewing heads (up to 20) that allow all observers
to see exactly the same image. This facility is particularly useful for teaching or demon-
stration purposes.
Objective lens
The objective lens collects the light from the specimen and forms a real image of the speci-
men. It is perhaps the most important component of the microscope in that it contributes
most to the ultimate quality of the image. There are numerous types of objective lenses; each
have specific uses and qualities. The selection of an objective lens is determined by the func-
tion to which the microscope is to be put.
There are three main types of objective lenses:
1. Acromatic
2. Semiapochromatic or fluorite
3. Apochromatic
All lenses have other features that contribute to their effective use and objective lenses are
no exception. Two features of note are depth of field and working distance. Depth of field is
the amount of a specimen in focus at any one time. Generally lower power lenses have a
greater depth of field and higher power lenses have a smaller depth of field so require con-
stant refocusing. Working distance is the distance between the coverslip and the bottom of
the objective lens and again lower power lenses have a longer working distance than higher
power ones.
Achromatic
Achromatic lenses are the simplest types of corrected lens available. They correct for chro-
matic aberration in two wavelengths and for spherical aberration for only one wavelength.
Achromatic lenses have a relatively low numerical aperture, which allows a good working
distance and depth of field. When using this type of lens, the quality of the image is best in
the lens axis. Blurring occurs at the periphery of the image, which makes achromatic lenses
unsuitable for colour photomicrography.
Semiapochromatic
Semiapochromatic or fluorite lenses incorporate the mineral, fluorite into the glass of the
lens. This allows lenses to be constructed with improved numerical apertures and increased
image resolution. These lenses still only compensate for chromatic aberration in two colours
and spherical aberration is still present, however, they provide superior performance over
simple achromatic lenses.
Apochromatic
Apochromatic lenses are the highest quality lenses available. They correct for three colours,
and do not exhibit spherical aberration or astigmatism. These lenses provide the highest
numerical aperture and, consequently, the highest resolution. The working distances of these
lenses are usually short but this can be used to advantage by the addition of high refractive
index immersion oil or water between the specimen and the objective lens. As we described in
Section 8.1, this has the effect of increasing numerical aperture and thus increasing the poten-
tial resolving power of the lens. Using apochromatic lenses means it is possible to achieve
numerical apertures in excess of 1.4 with full colour correction.
Eyepiece
The eyepiece is the last lens system through which the image passes. It magnifies the image
formed by the objective lens and tube stand and allows a virtual focused image to fall on the
eye of the observer. Some eyepieces are capable of accommodating a range-measuring grati-
cule. Graticules are glass discs onto which are etched micrometer scales, grids, or points, and
which can be used for quantitative and accurate measurements of images. Modern micro-
scopes are fitted with wide field, flat field, and possibly high focal point eyepieces especially
192 8 MICROSCOPY
designed for spectacle users. In choosing the eyepiece, it is necessary to match its quality to
that of the objective lens. It is inappropriate to use the best quality apochromatic objective
lens with the simplest of eyepiece lenses.
8.3 Magnification
The magnification produced by a lens can be defined by using the distance from the object to
the lens and that from the lens to the image, using the formula:
Magnification = Real image distance/Specimen focal distance
The real image distance or optical tube length has been standardized in microscopes to 160 mm.
The focal distance is the length from the optical centre of the lens to its focal point.
You can see from the formula above that the smaller the focal distance, the larger the mag-
nification obtained. The surface curvature of the lens can be increased to shorten the focal
distance, but maximum curvature is limited to that of a spherical lens. By adding multiple
lenses it is possible to significantly increase magnification: in contemporary microscopes both
objective and eyepieces lenses are constructed from numerous lenses of differing curvatures
and diameters.
The total magnification obtained using a compound microscope when the microscope has a
standard optical tube length of 160 mm can be obtained by simply multiplying the magnifica-
tion values of the objective and eyepiece lenses.
For non-standard optical tube lengths the formula in Box 8.2 must be used.
8.4 Dark field microscopy

Conventional bright field illumination will in itself provide only minimal contrast (Section 8.1)
and unstained biological samples are virtually invisible since they have insufficient variations
in optical densities. To overcome this problem, it is conventional to introduce contrast into
specimens by staining them with coloured dyes. However, when it is necessary to examine
samples in the living state, this ‘normal’ preparation may result in death and disruption of the
sample so alternative methods of illuminating the specimen are needed to prevent this.
BOX 8.2 Magnification of a compound

microscope
The final magnification produced by a compound microscope can be expressed as

objective magnification × tube lens factor × eyepiece magnification:
Tube lens factor = Optical tube length/Objective focal length
Example: Magnification using a 10× objective lens with a tube lens factor of 1.25 and an
8× eyepiece
Magnification = 10 × 1.25 × 8 = 100 ×

8.5 PHASE CONTR AST MICROSCOPY 193
Objective
lens
Specimen
Mirrored
surfaces
FIGURE 8.9
Schematic outline of lightpath in dark field illumination.
The simplest method of introducing contrast into unstained samples is to use oblique light.
In doing so, the sample is illuminated by light from an angle that has the effect of increas-
ing refraction while creating a decrease in direct illumination. Unstained samples thus appear
bright against a dark background. The dark field condenser was developed to take advantage of
this effect. This consists of a parabolic mirror placed in the substage condenser that only allows
light to reach the specimen stage at an acute angle as you can see illustrated in Figure 8.9. When
a specimen is not present on the microscope stage, light does not pass through to the objective
lens. When a sample is placed on the stage, the small variations in refraction created by the
specimen are amplified to make the sample appear bright against the dark background.
Dark field condensers are occasionally used in conjunction with the transmission fluorescence
microscope and this application will be described more fully in Section 8.7.
8.5 Phase contrast microscopy

As you learned in Section 8.1, light is a waveform. When two rays of light originate from the
same source they are said to be coherent, because they are in phase with each other and
their amplitudes coincide. As a consequence, they are able to combine to form a ray with an
amplitude twice the height of the original rays resulting in a brighter one. If, however, the two
rays become out of phase then they are able to interfere with one another. If the rays were out
of phase with one another by half of the wavelength, then this would result in the extinction
of the ray. This phenomenon of interference is utilized in the phase contrast microscope. You
can see an outline of its operation in Figure 8.10.
The sample is illuminated with a cone of light produced by an annular stop, which consists of
a ring positioned in the bright field substage condenser. The cone of light passes through the
sample, which is positioned at its focal point. Light leaving the sample enters the objective lens
of the microscope.
Objective lenses in phase contrast microscopes have a phase plate, which consists of a disc of
glass with a ring etched into it that corresponds exactly to the shape and size of the annulus.
The depth of the etching is critical: it must be of such a depth that it retards the light rays pass-
ing through the full thickness of the phase plate by ¼ wavelength when compared with the light
passing through the etched ring. This retardation, while resulting in interference, does not in itself
produce the desired increase in contrast. The sample is responsible for producing this effect.
194 8 MICROSCOPY
Retarded light ray Direct unretarded

light ray
Phase plate
Objective lens
Subject
Condenser
FIGURE 8.10
Schematic illustration of the lightpath Annulus
in a phase contrast microscope.
Tissues will also retard light by approximately ¼ of a wavelength; hence, the combined retar-
dation from the phase plate and the specimen produces the total interference.
When light passes through a sample, some of it is scattered and passes into the objective lens
in the normal way and passes to the eye of the observer through the non-etched part of the
phase plate. When the unaltered rays are focused with the retarded rays they combine to form
the real image of the specimen. Subtle changes in refractive index within the sample are seen
as varying degrees of brightness in the image against a dark background.
When setting a microscope for phase contrast it is essential to ensure that the annulus and
the phase plate are exactly aligned. It is usual to use an auxiliary microscope to view the back
focal plane of the phase contrast microscope, which allows the operator to view the phase
plate and use adjusting screws fitted in the condenser to align it with the annulus with great
accuracy. In practice each objective lens and phase plate has a matching annulus fitted into a
rotating disc set into the base of the substage condenser. Each annulus can be adjusted and
set individually to match its objective. If properly adjusted, a good phase contrast microscope
should be able to distinguish differences in refractive index of less than 5% easily, producing
Cross reference
images of high contrast without the use of staining.
urinary infections in Chapter 3 Phase contrast microscopy is used extensively in biomedical science when it is necessary to
‘Kidney disease’, in the examine unstained tissue samples, such as examining cell deposits from urine samples from
companion text, Clinical
patients suffering from urinary tract infections. Phase contrast microscopy is used to examine
Biochemistry and in Medical
and identify the cellular component of such samples.
Microbiology.
SELF-CHECK 8.4
In a phase contrast microscope, by how much does the phase plate retard the rays of light?
8.7 FLUORESCENCE MICROSCOPY 195
8.6 Polarization microscopy

As described in Section 8.1, light emitted from a single source can be regarded as electro-
magnetic waves whose amplitude represents brightness and the wavelength its colour. This
waveform is emitted and vibrates in all directions. Polarized light, however, only vibrates in
a single direction. Certain crystals are capable of producing plane polarized light by splitting
incident rays entering the crystal into two components, which are refracted by the crystal in
two different planes. This phenomenon is termed birefringence.
If we place two similar polarizing crystals so that their planes of polarization are parallel to
each other, then any light entering the first crystal would be capable of passing through the
second. However, rotating the second crystal through an angle of 90° would result in the
extinction of light and none would exit from the second crystal. This phenomenon is exploited
in the polarization microscope, which is used to examine and measure substances that are
capable of polarizing light.
The polarizing microscope has a polarizer filter situated in the substage condenser. This illu-
minates the specimen with plane polarized light. A second polarizing filter or analyser is Cross reference
situated below the eyepiece and is capable of being rotated through 180°. When using the You can read about the
polarizing microscope, the two filters are orientated so that their planes of polarization are at deposition of urate in
a right angle resulting in light extinction. However, any specimen that contains a birefringent gout patients in Chapter 4
substance will rotate the illuminating plane polarized light so that it is able to pass through the ‘Hyperuricaemia and gout’, in
the companion volume, Clinical
analyser and appear bright against a dark background. This form of microscopy is particularly
Biochemistry.
useful in the examination of fluids that may contain crystals, such as urine and joint fluids.
8.7 Fluorescence microscopy

The use of shorter wavelength light in the ultraviolet range is outside the visible spectrum so
its use to improve resolution is limited. However, certain substances when illuminated with
high energy light of a short wavelength (that is ultraviolet) will re-emit light of a lower energy
of longer wavelength that is within the visible spectrum. This is called fluorescence. The use
of fluorescent microscopes allows us to visualize small quantities of biological components
within tissue samples since they appear bright against a dark background.
Some substances are auto-fluorescent or exhibit primary fluorescence, (that is they fluoresce Cross reference
spontaneously under UV light). Other substances do not fluoresce but can have fluorescent Fluorescence is also discussed in
dyes called fluorochromes attached to them, which will produce a secondary fluorescence Chapter 11 ‘Spectroscopy’.
when stimulated with ultraviolet light. Examples of substances that exhibit auto-fluorescence
are the vitamin As and some porphyrins. A number of dyes which exhibit fluorescence can be
used to demonstrate the presence of specific cellular components in specimens.
The light source in all fluorescent microscopes must be capable of producing a high propor-
tion of light rays in the ultraviolet range. Halogen lamps produce significant amounts, but
almost all of it is in the ultraviolet blue range and so is of limited value. The commonest light
sources in use are high pressure mercury vapour or Xenon arc lamps. Both are highly efficient,
but requiring specific power supplies and appropriate lamp housings.
There are two types of fluorescence microscope, which differ in their manner of illuminating
the specimen. In transmission fluorescence microscopes, the light source is situated beneath
the specimen as in a conventional light microscope. In incident light fluorescence microscopes,
however, the specimen is illuminated from above and the viewer sees visible light transmitted
from the specimen against a black background.
196 8 MICROSCOPY
Transmitted light fluorescence

To gain the highest performance from the fluorescence microscope it is necessary to use
filters that are able to transmit the specific wavelength of the excited light, while removing
unnecessary and unwanted wavelengths that may reduce specificity. A transmission fluores-
cence microscope has two types of filters (Figure 8.11): an exciter filter placed in the light
path between the lamp and the condenser and a barrier filter situated in the body tube
before the eyepiece. The filters must be specifically manufactured to correspond to each type
of fluorochrome in use.
The exciter filter allows the maximum amount of ultraviolet light of an appropriate wavelength
to excite the specimen while filtering out the levels of visible and inappropriate ultraviolet
light from the illumination. This ensures that the maximum level of fluorescence is emit-
ted from the sample. The light entering the objective lens is a combination of unscattered
ultraviolet light and the longer wavelength visible light produced by the fluorochrome(s). It
would be dangerous for the observer if this light were to fall directly onto the eye and may
cause blindness. This is prevented by the barrier filter that removes all of the unused ultra-
violet light and allows only the fluorescent visible light to be transmitted to the eye, thus
protecting the observer. Unfortunately, to produce a filter capable of both blocking ultra-
violet light and allowing transmission of specific wavelength visible light, means the overall
Eye
Eyepiece
Barrier filter prevents

ultraviolet light reaching
eye but transmits visible
light from specimen
Objective lens
Specimen
Condenser
Exciter filter transmits

ultraviolet light to
specimen
FIGURE 8.11 Light

Outline of a transmission light Mirror source
fluorescence microscope.
8.7 FLUORESCENCE MICROSCOPY 197
level of transmission is lowered and significantly reduces the intensity and brightness of any
fluorescence produced.
An alternative to the use of a barrier filter is to use a dark ground condenser. This ensures
that the only light reaching the eye of the observer is that emitted by the specimen since
unscattered, indirect ultraviolet light does not reach the eye. This has the added advantage of
forming a dark contrasting background that gives an apparent increase in the brightness of the
fluorescent light.
SELF-CHECK 8.5
Which light has the shorter wavelength: ultraviolet or blue? (You might find it helpful to con-
sult Figure 11.2.)
Incident light fluorescence

Since the fluorescent light from a fluorochrome is emitted in all directions it is possible to illu-
minate the specimen in a fluoresence microscope from above. This is called epi-illumination.
Some of the excitation light from the fluorochrome is directed back into the objective for
observation by the user.
In operation, the exciting light passes from the lamp and through the excitation filter to a
dichroic mirror or prism, as you can see in Figure 8.12, which filters out all the unwanted light,
only allowing ultraviolet light of a specific wavelength to pass through. The resultant ultraviolet
light passes to the specimen where the fluorochrome is excited to generate visible light. The
visible light passes into the objective and then to the observer through the same dichroic prism,
which is able to transmit visible light in the opposite direction to the exciter light, and to the eye
of the observer. The image therefore appears brightly coloured against a dark background.
Incident light fluorescence microscopy has significant advantages over transmitted light fluo-
rescence. The reduced intensity of light resulting from the use of barrier filters is eliminated, a
substage condenser is not required and the level of light reaching the specimen is consider-
ably increased giving a much brighter image. In addition, since the fluorescence microscope
uses light that is produced from sources above the specimen it is possible to simultaneously
use other forms of transmission microscopy. This allows both morphological and fluorescent
images of the specimen to be seen simultaneously. This is of particular value when additional
staining of specimens may mask the fluorescence produced. In this case a phase contrast or
dark ground condenser can be used to introduce additional contrast.
Uses of fluorescence microscopy

Cross reference
Both transmitted and incident light fluorescence microscopy are used extensively in the study You can read more about
of immunological disorders. The technique of immunofluorescence is used to identify spe- immunofluorescence in
cific immune responses in disease. These methods rely on specific interactions and binding Chapter 15 ‘Immunological
between antibodies labelled with fluorescent dyes (fluorochromes) and antigens present on techniques’ and the
accompanying volume,
cell surfaces. Once bound, the visualization of the fluorescence label confirms the presence of
Immunology.
the antigen and also identifies its specific location within the tissue (Figure 8.13).
SELF-CHECK 8.6
How does an incident light fluorescence microscope differ from a transmitted light fluores-
cence one?
198 8 MICROSCOPY
Eye
Eyepiece
Visible light
emitted from
specimen
Dichroic mirror Light

reflects UV light source
to specimen
Incident light from

UV source
Objective lens
Specimen
FIGURE 8.12
Outline of an incident light fluorescence microscope.
8.8 Confocal microscopy

In all forms of microscope, the final image is affected by the thickness of the specimen being
examined because of the relatively small depth of focus. This is a particular problem in fluo-
rescence microscopy where the fluorescent light emitted from the specimen is derived from
the full thickness of the specimen resulting in blurring of the image caused by those portions
of the specimen that are above and below the focal plane. This blurring is minimized in the
confocal microscope.
Confocal microscopes use a narrow beam of light from a laser, thus, the specimen is illuminated
with coherent light of high intensity. The beam of laser light is scanned through the speci-
men using a system of galvanometer mirrors that oscillate at adjustable speeds to divert the
laser beam to produce a scan. Reflected light produced is re-focused by a confocal aperture or
pinhole which only allows light from a specific focal plane to fall onto a photomultiplier and
produce a digital signal. A computer then processes this signal to produce an image that has
a great depth of field giving the specimen a three-dimensional appearance. If the specimen is
8.9 INVERTED MICROSCOPES 199
FIGURE 8.13
Incident light fluorescence micrograph of a section of breast tumour stained using
fluorescent in situ hybridization (FISH) to demonstrate amplification of the HER2 gene on
chromosome 17. This method is used as a prognostic marker for treatment with the drug
herceptin. See also Figure 16.26 and its associated text.
successively scanned through a range of focal planes, the images at each level can be electroni-
cally stored and used by the computer to produce a full three-dimensional image of it.
To-date, confocal microscopy has found limited application in diagnostic biomedical science.
It is, however, used extensively in scientific research (Figure 8.14) since biological material can
be examined in three dimensions without the need for extensive sample preparation.
SELF-CHECK 8.7
How is the quality of an image improved using confocal microscopy?
8.9 Inverted microscopes

When examining living material, for example certain types of cells grown in tissue culture, it
is necessary to use an inverted microscope. This allows the bottom surface of a culture flask,
where the cells grow and adhere, to be examined with minimal disturbance to the specimen.
The inverted microscope is constructed so that the specimen stage has a large flat surface
capable of accommodating tissue culture flasks or Petri dishes (Figure 8.15). The objective lens
and condenser are designed to work at a much longer working distances than those of con-
ventional microscopes to allow for the thickness of the culture vessel. Inverted microscopes
200 8 MICROSCOPY
FIGURE 8.14
Confocal image of a group of stem cells. The green
areas are parts of the plasma membranes and the
purple portions are nuclei. Courtesy of
Dr Q. Wang, School of Healthcare Science,
Manchester Metropolitan University, UK.
FIGURE 8.15
Myoblasts growing in culture viewed using an
inverted microscope. Courtesy of Dr Q. Wang, School
of Healthcare Science, Manchester Metropolitan
University, UK.
also have the facility to utilize phase contrast or dark field illumination and fluorescence giving
the instrument maximum flexibility.
8.10 Electron microscopy

In Section 8.1 we noted that the resolution of a microscope depends largely upon the
wavelength of the illuminating light; using visible light limits resolution to about 200 nm.
Using radiation of short wavelengths is a way of increasing the resolution of a microscope.
8.10 ELECTRON MICROSCOPY 201
This feature is exploited in the electron microscope. Like light, moving electrons are part of the
Cross reference
electromagnetic spectrum; the faster they move the shorter their wavelength.
The electromagnetic spectrum
Modern electron microscopes have resolutions that are less than 0.2 nm when used with is described in more detail in
Chapter 11 ‘Spectroscopy’.
biological specimens, which is 1000× the resolution of a light microscope.
Components of the electron microscope

Electron microscopes were first developed in the 1920s. Their components are analogous
to those of the conventional light microscope, as you can see if you compare Figure 8.16
with 8.8.
Electron microscopes have a source of electrons, commonly called an electron gun, a con-
denser system that collects the electrons and focuses them on the specimen, a system of
(a) (b)
Filament
Wehnalt shield
Column Anode
Condenser
lens
Specimen airlock
Specimen stage
Objective lens
Projector lens
Viewing
window
Viewing
screen
To vacuum
pumps
Camera air lock Camera
FIGURE 8.16
(a) Modern transmission electron microscope ( JEOL JEM-ARM200F). Courtesy of JEOL Ltd.
(b) Cross-section diagram of a transmission electron microscope. See text for details.
202 8 MICROSCOPY
objective lenses to magnify the image formed and a projector lens system to focus the image
onto a phosphorescent screen, thus making it visible. Glass cannot focus beams of electrons,
but they can be focused using electromagnetic lenses.
The electron beam in an electron microscope is energetic and if they were allowed to travel
far in air, collisions with its molecules would soon dissipate the beam. Thus, to produce a suf-
ficiently long electron path within the microscope a high vacuum is required. Electromagnetic
lenses generate a large amount of heat so a suitable cooling system is also required.
The electron gun

The electron gun of an electron microscope consists of a tungsten filament bent into a V-shape,
which is heated by a low voltage direct current. This causes a cloud of electrons to be released
from the tip of the V. The number of electrons emitted from the filament can be varied by
altering the size of the current passing through the filament.
A beam of moving electrons is formed by a Wehnelt cap and a positively charged (anode)
plate, as shown in Figure 8.17. The Wehnelt cap is made of metal and encases the filament. It
is maintained at a potential difference of about 20 V negative to the filament resulting in an
attraction to the electrons. A hole in the cap directs the electrons towards the anode plate,
which is maintained at earth potential. The potential difference between the filament and the
anode determines the acceleration voltage and in most electron microscopes can be varied
between 20,000 and 100,000 V.
For maximum efficiency and the production of a suitable electron beam, it is essential that the
filament is heated to the correct temperature, often referred to as the saturation point, that
the Wehnelt cap is accurately aligned, and that the distance between the filament and cap is
critically adjustable.
Electromagnetic lenses
Electromagnetic lenses consist of a coil of wire encased in soft iron. They have a pole-piece
opening at the point through which the electron beam passes (Figure 8.18). When a direct
Heater current
Tungsten filament
Cathode shield
Electron cloud
Anode plate
(earth)
FIGURE 8.17
Schematic illustration of an electron gun. See main text
for details.
Annulus Upper pole piece
Iron lens
covering
Lens coils
FIGURE 8.18
Schematic showing a cross-section of an electromagnetic lens.
Lower pole piece
See main text for details.
current is applied through the wire it induces a strong magnetic field through the axis of the
coil. Since electrons are electromagnetic radiation, an electron beam passing through the
pole-piece opening is affected by the magnetic field in the metal coil, which allows the beam
to be focused. Varying the strength of the current passing through the coil we can vary the
degree by which the electrons are focused allowing the beam to be convergent or divergent.
A number of such lenses are built into the column of the electron microscope.
As with all lenses, electromagnetic ones suffer from several aberrations. As you learned ear-
lier in Section 8.1, optical lenses suffer from chromatic and spherical aberrations, as well as
astigmatism. Electromagnetic lenses suffer similar problems; however, the ways in which these
defects are overcome differ.
To recap: chromatic aberration is the inability of a lens to bring electromagnetic rays of differ-
ent wavelengths to a common focal point. Chromatic aberration in electromagnetic lenses is
overcome by using a beam of monochromatic electrons. Manufacturers go to great lengths
to ensure the high tension voltage between the filament and the anode plate within the
electron gun is stable, which ensures a constant accelerating voltage and thus a monochro-
matic beam of electrons. A further potential source of chromatic aberration in the electron
microscope is the energetic interactions between the electron beam and the specimen under
examination. This reduces the energy of the electrons and so changes their wavelength to
produce a potential polychromatic beam. This form of chromatic aberration can be mini-
mized by using higher acceleration voltages and preparing extremely thin sections of speci-
mens for examination.
Spherical aberration is caused by the inability of a lens to bring electromagnetic waves to a

common focal point. it is not possible to correct this type of aberration in electromagnetic
lenses because the magnetic field they produce varies across their diameter, and the physical
dimensions of the lenses prevents the production of compound lens. However, spherical aber-
ration can be overcome by using only very small apertures in the centres of the lenses, which
only allow electrons at the central axis of the lens to pass through and illuminate the speci-
men. Electrons at the periphery of the lenses are prevented from doing this. Given electrons
travelling at the central axis of the lens exhibit the least spherical aberration, this approach
minimizes the defect.
Astigmatism, the inability of a lens to bring electromagnetic rays from a single source to a
common focus, is caused by the asymmetry of the electromagnetic field within the lens. It
is usually caused by defects in pole piece manufacture or, more commonly, contamination
on the pole piece. This results in electrons passing through different parts of the lens being
204 8 MICROSCOPY
focused to different degrees. Naturally, contamination on the pole pieces must be minimized
to reduce astigmatism effects. This can be achieved by ensuring that the microscope column
is kept scrupulously clean and by using an anticontamination trap or cold finger comprising a
small metal plate cooled by liquid nitrogen situated close to the specimen. Thus, any contami-
nating vapour liberated from the specimen during irradiation condenses on this cold plate,
rather than the lens pole pieces.
It is not possible to remove the asymmetries within lenses that lead to astigmatism, but it is
possible to compensate for them by surrounding the lens with a number of small secondary
electromagnets. Individual adjustments of these so-called astigmatism coils make it possible
to adjust the degree of astigmatism within the lens and to compensate for the defect.
Vacuum systems
Electrons do not travel far in air since they collide and react with the gas molecules. The dis-
tance that an electron can travel without collision is called the mean free path; in modern
electron microscopes this needs to be in excess of 2 m. To achieve this distance, it is necessary
to create a very high vacuum in the region of 1.33 × 10−2 to 1.33 × 10−3 Pa (10−4 to 10−5 mm of
mercury) within the column of the microscope. This high vacuum also has the effect of reduc-
ing oxidation of the tungsten filament and thus extending its life. In addition, it also helps to
maintain a clean environment within the column and reduce contamination of the lens pole
pieces and specimens.
Several types of vacuum pumps are employed, for example, oil rotary pump and oil diffusion
pumps, which are typically used in combination.
Oil rotary pumps have a simple mechanism consisting of a series of vanes that rotate in an
oil bath. This mechanism traps gas within a chamber forcing gas molecules out of the col-
umn. They are usually employed in the initial stages of evacuating the microscope column. Oil
rotary pumps are reliable and have the advantage that they can start to operate at atmospheric
pressure. They do, however, have the disadvantage that they can only produce a vacuum of
approximately 1.33 Pa (10–2 mm of mercury) so they must be used with other types of vacuum
pump.
Oil diffusion pumps operate by heating inert oil to its boiling point. The resultant oil vapour
is trapped within a series of cones with downward-pointing pipes that form a jet of oil vapour
directed towards a cooled surface where the oil condenses and traps gas from the surround-
ing atmosphere. They are efficient pumps; capable of forming a vacuum of approximately
1.33 × 10−3 Pa (10−5 mm mercury) and are silent in operation. However, they do not operate
from atmospheric pressure and require an initial vacuum of 1.33–0.133 Pa (10−2 to10−3 mm
mercury) to operate effectively. The assistance of a rotary pump, which is mounted in series,
is therefore useful to obtain this degree of vacuum before the diffusion pump begins to oper-
ate. It is essential that diffusion pumps are not exposed to vacuums of less than 1.33 Pa (10−2
mm mercury) when the oil is at operating temperature, since this may cause back streaming of
the oil vapour passing the cooling plates allowing it to enter the microscope column causing
considerable contamination. Manufacturers prevent this from happening by placing a pres-
sure sensitive baffle plate at the top of the pump, which automatically closes if the vacuum
fails or is insufficient.
SELF-CHECK 8.8
Why is it not possible to operate an oil diffusion pump from atmospheric pressure?
Cooling system
Electromagnetic lenses generate considerable heat as a by-product of their action. This
heat can produce instabilities in the action of the lenses and can also lead to the ther-
mal expansion of metal components around the lenses that can adversely affect the
formation of a well-focused image. To prevent these undesired effects, lenses are water
cooled. In addition, oil diffusion pumps also require cooling for them to operate. This is
simply achieved by passing water pipes around and through the electromagnetic lenses
and the pump, through which chilled water is circulated maintaining the temperature at
18–20 °C.
Viewing screen and recording images

Electrons are invisible to the human eye due to their very short wavelengths. Thus, to see the
image of a specimen produced in an electron microscope it is necessary to allow the focused
electron beam to hit a phosphorescent screen placed in the beam path. The electrons react
with the phosphor, which emits light. The intensity of this light is proportional to the number
of electrons striking the phosphor and thus a visible image of the specimen is formed.
The image formed on the phosphorescent screen is transient being only present when
illuminating the specimen. The traditional method of making permanent reproducible
records of the image was to form a photographic record. Fortunately, photographic emul-
sions are extremely sensitive to electrons and are capable of producing high resolution
copies of an image. Microscope cameras have to be mounted within the column and the
vacuum system, since it is necessary for the electron to fall directly onto the photographic
emulsion. They can be situated beneath the phosphorescent viewing screen, which is moved
out of the path of the beam to allow exposure of the film or placed above the screen imme-
diately beneath the projector lens and moved into the beam path when a photograph is
required.
Photographic film contains a large quantity of moisture, which if exposed to the high vacuum
environment of the electron microscope column would result in considerable contamination
and deterioration of the vacuum. It is necessary to dry the film prior to placing it into the
column and most manufacturers incorporate a desiccation chamber within the microscope
that can be maintained under vacuum by the rotary oil pump. Most manufacturers also incor-
porate an air lock into the camera, which facilitates film change without the need to bring the
whole column back to atmospheric pressure.
With the rapid development of digital imaging systems, manufacturers of electron micro-
scopes have been quick to realize the advantages of these types of imaging systems. Digital
images are readily accessible, easy to store electronically, and can be transferred to multiple
viewers simultaneously by email. They are also of high quality and are the medium of choice
for scientific publications.
Digital cameras can be incorporated into the electron microscope in the same way as con-
ventional film cameras: either situated beneath the phosphorescent screen or placed in the
column beneath the projector lens. The image electrons are allowed to fall directly onto a small
phosphorescent screen placed in the beam and the resultant image is directed to the camera.
To take advantage of increased magnifications and resolution possibilities of the electron

microscope requires specimens to be processed in a particular way as described in Box 8.3.
206 8 MICROSCOPY
BOX 8.3 Preparing samples for

electron microscopy
Sample preparation for transmission electron microscopy (TEM) is designed to take

Cross reference
advantage of the increased magnification and resolution that TEM offers over light
microscopy (Box 8.1). Unfortunately, the majority of the reagents and solutions used in
electron microscopy and preparing samples for EM present some form of risk to the user. Hence, sample prepa-
sample preparation in the ration must only be undertaken when wearing appropriate personal protective equip-
accompanying volume – ment and within a fume cupboard.
Histopathology – in this series.
Electron beams are destructive to tissues, so the preparation of samples must result in
specimens that can withstand being bombarded with electrons in a vacuum. This is nor-
mally achieved by using extremely potent fixatives, such as glutaraldehyde and embed-
ding the sample in hard epoxy resin, which is capable of being thinly sectioned.
Preparation of slices of tissue or suspensions of cells for electron microscopy begins by

fixing them in a strong cross-linking reagent, such as glutaraldehyde or osmium tetroxide,
to minimize the effects of autolysis and microbial degradation. This type of fixation also
produces samples that are able to withstand the subsequent processing steps. The sam-
ples are then dehydrated using a graded series of concentrations of ethanol, ending with
100% strength, before being impregnated in liquid epoxy resin. The samples are then
baked to set the resin. Once embedded, sections approximately 1 lm thick are meto-
chromatically stained with, for example, toluidine blue and examined using light micro-
scopy, to initially identify any areas of particular interest. Once such areas are exposed,
ultra-thin sections of only 50–90 nm are produced using an ultramicrotome. The sections
are mounted on support grids, which typically are about 3 mm in diameter and made
of copper. The holes in the grid allow the beam of electrons to pass though the sections,
while the lattice supports the section. Prior to viewing, the sections are stained using a
variety of heavy metal salts including those of lead, uranium, or less often gold, silver,
or platinum. Once stained, the grids may then be placed in the electron microscope for
visualization of the areas of interest, such as those shown in Figures 8.19–8.22.
Application of transmission electron

microscopy in pathology
Cross reference
You can learn about the There are several pathological processes which can only be examined using electron micros-
glomerular basement copy because the cellular changes are so small that they are beyond the resolution of light
membrane and epithelial cell microscopes (Figure 8.19). This is especially true in the case of some renal diseases. There are
foot processes of the kidney changes to the glomerular basement membrane and to the epithelial cell foot processes which
in Chapter 3 ‘Kidney disease’
can only be seen using an electron microscope, which you can see in Figure 8.20 (a) and (b).
of the companion textbook,
Clinical Biochemistry and The electron microscope is also used extensively in tumour diagnosis. Particularly where there
Histopathology. is poor cell differentiation, and it is difficult to see specific features and markers at the resolu-
tion of light microscopy. An example of this occurs in the differential diagnosis of malignant
melanoma where the quantity of melanin pigment within the tumour cells is so small that
Cross reference conventional staining and immunocytochemical demonstrations appear negative. With the
Immunocytochemistry is electron microscope it is possible to see very small numbers of melanosomes and even the
described in Chapter 15 non-pigmented pre-melanosomes within the cytoplasm of melanoma cells. The development
‘Immunological techniques’.
of specific tumour markers and the use of immunocytochemistry have, however, led to less
reliance on electron microscopy in tumour diagnosis.
FIGURE 8.19
Electron micrograph of amyloid fibres within the wall
of a small blood vessel from a patient with chronic
inflammatory disease. Note the characteristic fibrillary
nature of this abnormal protein. Electron microscopy
is the definitive method of choice for the positive
identification of amyloid.
(a) (b)
FIGURE 8.20
(a) Electron micrograph of renal glomerular basement membrane from a patient suffering
from membranous glomerular nephritis. (b) Note how its appearance differs in structure
from that seen in a healthy person. Courtesy of Dr J. C. Jeanette, Department of Pathology
and Laboratory Medicine, University of North Carolina, USA.
Viral particles are far too small to be seen with a light microscope, but are clearly visible using
electron microscopy (Figure 8.21). Early identification and diagnosis of viral infections is some-
times essential if the patient is to be diagnosed quickly and receive prompt, effective treat-
ment. This is particularly the case in patients infected with human immunodeficiency virus
(HIV) and suffering from acquired immunodeficiency syndrome (AIDS) where their immune
208 8 MICROSCOPY
FIGURE 8.21
Electron micrograph of influenza viral particles. Courtesy of H. Cotterill, Manchester Royal
Infirmary, UK.
response is severely compromised. Electron microscopic examination of suitable samples can

be used to identify viral particles by their characteristic morphology and offers a simple and
quick method of sample analysis. You can see an electron micrograph of HIV particles escap-
ing from an infected cell in Figure 8.22.
FIGURE 8.22
Electron micrograph of human immunodeficiency viral particles being released from the
surface of a cell. Courtesy of H. Cotterill, Manchester Royal Infirmary, UK.
8.10 ELECTRON MICROSCOPY
QUESTIONS 209
SUMMARY
■ Microscopes are an essential part of laboratory testing and diagnosis
■ Microscopes are used in many different disciplines
■ Various types of microscopy exist and are used for different purposes
■ Microscopes must be set up and adjusted correctly to ensure optimum visualization of
specimens
■ When significant increases in magnification or resolution are required, electron micros-
copy must be used instead of light microscopy
FURTHER READING
● Bradbury HSM. Introduction to Light Microscopy, Royal Microscopical Handbook.
BIOS Scientific Publishers, London, 1998
● Rogers K. Complete Book of the Microscope. Usborne Publishers Ltd. London, UK,
2006
● Pawley JB. Handbook of Biological Confocal Microscopy. Springer, New York, 2006.
● Bozzola J, Russell LD. Electron Microscopy, 2nd edn. Jones and Bartlett, Boston, 1998.
● Ghadially F. Diagnostic Ultrastructural Pathology A Self Evaluation and Self Teaching

Manual Hodder Arnold Publishers, London, 1998.
● Cheville N. Ultrastructural Pathology, 2nd edn. Wiley Blackwell, Oxford, 2009
QUESTIONS
1. If the diameter of a virus particle appears to be 5 mm when magnified 100,000×, its ‘real’
diameter is:
(a) 1.0 μm
(b) 0.005 mm
(c) 50 nm
(d) 500 μm
(e) 5 μm
2. Indicate which of the following statements are TRUE.
(a) Specimens for light microscopes are thicker than those for electron microscopy
(b) Electron microscopes have a greater resolving power than light microscopes
(c) Moving electrons have shorter wavelengths and therefore give poorer resolution
than light rays
210 MICROSCOPY
8 MICROSCOPY
(d) Electromagnetic lenses can focus beams of electrons as well as light rays
(e) Chromatic aberration, spherical aberration, and astigmatism can affect both glass
and electromagnetic lenses
3. (a) Describe what is meant by chromatic aberration. (b) How is this defect in a micro-
scope lens corrected?
4. Describe how you would set up a light microscope illumination system to give Köhler
illumination.
5. What two main factors affect the resolution of a microscope?
6. Why is it necessary for the column of an electron microscope to be maintained under
very high vacuum?
7. Describe with the aid of a diagram, how the incident light fluorescence microscope
works and can be used in the biomedical science laboratory. You may find it helpful to
read relevant parts of Chapters 15 ‘Immunological techniques’ and 16 ‘Molecular biology
techniques’.
8. What is the total magnification of a microscope using a 40× objective lens with a focal
length of 2 mm and an eyepiece with magnification of 10×?
9
Electrochemistry
Peter Robinson
Learning objectives
■ Define key terms related to electrochemical analyses

■ Describe the reactions that occur in a typical electrochemical half cell
■ List common substances that may be measured in a clinical laboratory using electrochemical
analytical devices
■ Describe the Nernst equation and outline its use in potentiometric analysis
■ Describe the underlying principles of amperometric electrochemical devices
■ Outline the principles and clinical applications of ion selective field effect transistors
■ Define and describe the underlying principles of operation of biosensors
■ Discuss the development of biosensor technology
Introduction
It is likely that a pH electrode was the first piece of analytical equipment that you used when you
first entered a science laboratory. Like so much modern technology, you could use such a device
without ever really understanding how it works. However, working within a clinical laboratory you
may well have to use several, possibly dozens, of different electrode devices to measure a wide
range of analytes (the term generally used for the substances being measured) in clinical samples.
Ion selective electrodes, oxygen electrodes, carbon dioxide electrodes, and various biosensors are
now found in most clinical settings. It is useful therefore to have sufficient background knowledge
to be able to understand the potential and limitations of such devices. The recognition that such
electrodes fit into a small number of ‘families’ should enable you to develop experience of using
one electrode and help you understand how other ‘close relatives’ might also function. Learning a
few fundamental principles can therefore save hours of having to ‘rote learn’ the specifics of each
device in isolation. In addition, some knowledge of the principles on which these devices are
based is particularly useful when, as can occur, an electrode appears to be malfunctioning.
In many ways electrochemical analysis is ideally suited for use in the clinical laboratory. Indeed,
many techniques are also the basis of the point of care testing devices described in Chapter 18.
212 9 ELECTROCHEMISTRY
Once set up and calibrated, electrochemical devices are simple to use, such that after even the
most basic of training you will be able to measure numerous analytes in clinical samples with
high accuracy and reliability. Because electrochemical techniques do not rely on any colour
Cross reference formation they are highly suited to measurement of samples with a high background colour, in
You can read about automated particular blood. Finally, unlike most other analytical techniques, electrochemical techniques
clinical laboratory instruments use few reagents. Once an electrode and meter have been purchased the actual running cost
in Chapter 17 ‘Laboratory
per sample is generally low, and this may be of great importance to the financial competitive-
automation’.
ness of a clinical laboratory.
The aims of this chapter are to outline the likely uses of these electrode devices within a clini-
cal laboratory and to describe their fundamental principles of operation. General advice on
the care and maintenance of electrodes is also included. However, you are always advised to
consult the manufacturer for specific information regarding such matters, as electrodes from
different manufacturers may have some quite specific requirements.
9.1 Basic concepts and definitions

Electrochemistry is the study of the chemical changes produced by electrical currents and also
the production of electrical currents by chemical reactions. For more than 200 years electro-
chemical techniques have developed through advances in chemistry, physics, electronics and,
more recently, biology. The sheer number of electrochemical techniques can be bewildering,
and while there may be fundamental differences between some groups of techniques, it is
(a)
often the case that numerous techniques differ from each other in only minor detail. Sadly,
even the naming of electrochemical analytical techniques has been applied with little or no
semblance of a systematic approach. As a consequence, the nomenclature surrounding the
current current subject can be quite confusing and, at times, plainly inconsistent!
It is therefore necessary to go back to the basics of electrochemistry before jumping into a

detailed description of the devices and techniques themselves.
+ –
What is an electrical current?
(b) An electrical current is simply a movement (flow) of electric charge. However, one fundamen-
tal inconsistency that might well lead to confusion relates to something as simple as the actual
direction of the flow itself. This inconsistency dates back to the mid 18th century when Benjamin
Franklin was studying static electricity. He found that when a wool cloth was rubbed over a paraf-
e– e– fin wax block the two materials subsequently attracted each other. Franklin considered that the
wool cloth was in some way removing an ‘invisible fluid’ from the wax block, hence the type of
charge that was associated with rubbed wax became known as ‘negative’ (because it had a defi-
ciency of the ‘fluid’), whilst that associated with the wool became known as ‘positive’ (because it
+ – had an excess of the ‘fluid’). This terminology was widely adopted such that when electric batter-
ies were later developed, it was natural to assign the direction of the flow of current to be from the
positive to the negative pole. Over a century later, however, J.J. Thomson showed that cathode
FIGURE 9.1 rays were negatively charged particles, which he called ‘corpuscles’, and claimed that atoms were
(a) Conventional and built from these corpuscles. The electron had been discovered and it was suddenly realized that it
(b) electron flow notation for was electrons that carried the current in metal wires. Thus, electrons must be moving in the oppo-
an electric current showing site direction to that of the ‘conventional’ current. Sadly, it was much too late to change Franklin’s
the apparent inconsistency in naming convention and today it must be accepted that current can be considered to move in
direction of current flow. either of these two ways. Hence, both of the conventions shown in Figure 9.1 are acceptable.
9.2 PRINCIPLES OF ELECTROCHEMICAL TECHNIQUES 213
BOX 9.1 Ohm’s Law
Ohm’s Law can be rearranged to describe the current, voltage, or resistance. The easiest
way to remember these relationships is to use a VIR triangle as shown in Figure 9.2. To
use this, simply use your finger to hide the value that you want to calculate. The remain-
ing two values then show you how to do the calculation.
V
I R
FIGURE 9.2
VIR triangle used to help calculate Ohm’s Law.
Ohm’s Law
A flow of an electrical current only occurs when there is a potential difference between two
points within a circuit. It is also influenced by resistive forces present within the system. These
relationships are usually described by Ohm’s Law (Box 9.1), which states that within an electri-
cal circuit the current passing between any two points of a conductor is directly proportional
to the potential difference, that is voltage drop or voltage, between the two points but is
inversely proportional to the resistance between them. In mathematical terms, Ohm’s Law
may be written as:
I = V/R
where I is the current in amperes (often abbreviated to amps, A), V is the potential difference
in volts (V), and R is the resistance measured in Ohms (Ω).
SELF-CHECK 9.1
Calculate the voltage required to force a current of 0.2 A through a wire with a resistance
of 1200 Ω.
9.2Principles of electrochemical
techniques
Whenever you study electrochemical reactions you will come across the concept of an elec-
trochemical cell that is made up of two electrochemical half-cells. Each half-cell contains an
electrode, which at its simplest is just a metal wire, that is surrounded by an electrolyte. The
two half-cells are joined by a shared electrolyte or by a relatively concentrated solution of
different ions, often saturated or 4 mol dm−3 KCl or KNO3, called a salt bridge.
Consider a relatively simple half-cell consisting of a copper wire dipping into a solution of
CuSO4. Within this system, oxidation may occur so that the copper electrode may lose copper
ions to the solution, leaving behind electrons delocalized on the metal surface:
Cu(electrode) → Cu2+(aqueous) + 2 e−(electrode)
This leaves the electrode negatively charged with respect to the surrounding solution.
Alternatively, the conditions may favour a reduction, where copper ions in solution combine
with electrons at the metal surface to deposit copper atoms:
Cu2+(aqueous) + 2 e−(electrode) → Cu(electrode)
This produces a deficit of electrons in the copper electrode making it positively charged with
respect to the surrounding solution. However, in both cases, a spontaneous potential differ-
ence called the electrode potential is established across the electrode/electrolyte interface.
Hence, when two half-cells are combined, a flow of electrons, that is an electrical current,
may occur. Consider, for example the Daniell cell shown in Figure 9.3, which consists of zinc
and copper wires immersed in solutions of ZnSO4 and CuSO4 respectively. If the Zn2+ and Cu2+
concentrations are approximately equal, then reactions will result in the oxidation of the zinc
metal in one half-cell to leave the zinc electrode negatively charged, and the reduction of the
copper ions in the other half-cell, which results in the copper electrode becoming positively
charged. If the two electrodes are then connected by a wire, electrons will flow from the zinc
anode to the copper cathode. The Daniell cell can therefore produce an electrical current and
is thus a simple battery.
The salt bridge connecting the two solutions of the half-cells in Figure 9.3 completes the cir-
cuit and allows migration of ions between the two compartments, but prevents gross mixing
of the two solutions. The salt bridge might be as simple as a strip of filter paper soaked with a
relatively inert electrolyte such as KCl, or a U-shaped glass tube filled with the same electrolyte
held within an agar gel.
Schematic representation of electrochemical cells

In 1953 the International Union of Pure and Applied Chemistry (IUPAC) developed a standard
notation to represent electrochemical cells that is still accepted today. The IUPAC notation for
the Daniell cell shown in Figure 9.3 may be written as follows:
– Zn | Zn2+ | | Cu2+ | Cu+
Anode (–) Cathode (+)

Salt bridge
FIGURE 9.3
Daniell cell. See text for details. Note, this is a Galvanic cell and,
therefore, the anode and cathode are negatively and positively Zn2+ SO42– Cu2+ SO42–
charged respectively, which is the opposite of the electrodes in simple
electrophoresis (Chapter 14).
9.3 POTENTIOMETRIC TECHNIQUES 215
where the single vertical lines represent a phase boundary (electrode/electrolyte interface)
A cation (pronounced cat-ion)
and the double vertical lines the salt bridge. Note that according to this notation the negative is an ion that has fewer electrons
electrode (anode) is placed to the left, the positive electrode (cathode) is placed to the right, in its electron shells than
and electrons would therefore flow from left-to-right in the connecting wire. protons in its nucleus. It thus
carries a net positive charge and
will move towards the negative
electrode during electrolysis.
Classification of electrochemical techniques
An anion (pronounced an-ion)
is an ion that has more electrons
Electrochemical cells can be categorized as galvanic or electrolytic. A galvanic cell is
than protons. Thus it has a net
one where the reactions occur spontaneously at the electrodes when they are connected negative charge and will move
externally by a conductor. In an electrolytic cell, however, the electrochemical reactions towards the positive electrode
are not spontaneous and only occur if an external voltage is applied across the two elec- during electrolysis. You might
trodes. A modern rechargeable battery is, in essence, an electrolytic cell when it is being be able to remember that it
is the anion that is negatively
recharged.
charged by the mnemonic
‘a-n(egative)-ion’
SELF-CHECK 9.2
State whether the Daniell cell shown in Figure 9.3 is a galvanic or electrolytic cell.
Electrochemical analytical techniques may be conveniently grouped according to whether their

electrochemistry is galvanic (spontaneous) or electrolytic (non-spontaneous). Potentiometry
involves the use of galvanic cells, and the measurement of the spontaneous potential of an
electrode with minimal current flow. The commonest applications of potentiometry are the
pH electrode and ion selective electrodes described in Section 9.3. In contrast, voltammetry
(including amperometry) uses electrolytic cells and the measurement of the current passing
through an electrode when an external potential is applied. By far their commonest applica-
tion is the Clark oxygen electrode. This device is described in Section 9.4 and relies on the
reduction of dioxygen at a platinum cathode to give a current that is proportional to the oxy-
gen concentration in the solution. Many biosensors also incorporate amperometric detectors,
as described in Section 9.5.
9.3 Potentiometric techniques

In potentiometric analysis, an electrode, often called the working or sensing electrode, is
used that generates a spontaneous potential at its surface with the analyte, that is, the solution
being measured. However, only potential differences can be measured, hence, this cell needs
to be connected to a second cell that produces a standard potential so that the difference
between the two can be evaluated. This second, so called reference electrode, is a necessary
component of all pH and ion selective electrodes.
The behaviour of potentiometric electrodes is generally described by the Nernst equa-

tion, where the total potential, E (in volts) developed between the working and reference
electrode is:
RT
E = Econstant + 2. 303 ___
nF
log10 A
or simplified as:
E = Econstant + S/n log10 C
where Econstant is a constant potential that depends mainly on the reference electrode; 2.303 RT/F
(=S) is the Nernst factor or slope (where R is the universal gas constant, T is the temperature in
BOX 9.2 Activity
Activity is a useful chemical concept. Consider an individual ion in solution. The ability
of this ion to participate in any chemical reaction will be reduced if it is surrounded by
other ions, which ‘shield’ it. This is more likely to occur when there are more such ions
in solution. Activity therefore is a true measure of the ion’s ability to affect chemical
equilibria and reaction rates; it is often said to be the effective concentration of the ion
in solution. In most biological situations, where the concentrations of ions are rather
low, the values of the concentration and activity are equal. At higher concentrations,
however, the activity may become significantly less than the concentration.
Kelvin and F is the Faraday constant); n is the number of charges on the ion; A is the activity of
the ion; and C is the concentration of the ion.
Potentiometric electrodes actually measure the activity of an ion in solution (Box 9.2). In
most biomedical situations the concentrations of ions are low and activity is synonymous with
concentration.
The Nernst equation shows that the responses of potentiometric electrodes will depend both
on the temperature and the number of charges carried by the ion. At 25°C the Nernst factor
(2.303 RT/F) becomes 0.059 and thus the equation becomes:
E = Econstant + 0.059/n log10 C
This means that there will be a 59 mV change in potential for a 10-fold change in the concen-
tration of monovalent ion, such as H+, Na+, K+, and Cl−.
A potentiometric electrode is said to have Nernstian characteristics if it obeys Nernst’s law. If

the changes in potential relative to concentration are less than the theoretical values, it may
indicate an electrode malfunction or interference from other ions.
SELF-CHECK 9.3
Given that the universal gas constant is 8.314 J mol−1 per K, T is the temperature in Kelvin
(=273 + T°C) and that the Faraday constant is 96,485 coulomb mol−1, calculate the change in
potential in mV for a ten-fold change in the concentration of H+ when the temperature of the
system is 35°C.
pH electrode
Virtually all textbooks, including this one, describe the pH electrode separately from ion selec-
tive electrodes (ISEs) even though it is simply one specific type of ISE that responds to H+.
However, it is much commoner in laboratories than other ISEs and more likely to be used by
students and thus tends to get a more detailed coverage by authors.
In 1906, Max Cremer found that a thin glass membrane separating the two electrodes of a gal-
vanic cell could produce a potential that was responsive to changes in the concentration of H+.
Three years later, what we would recognize as a glass electrode was constructed, and studied
by Fritz Haber and Zygmunt Klemensiewicz. However, this device did not become popular
until the 1930s, when reliable amplifiers became available to measure the output from the
electrodes. Glass electrodes are still the most convenient and accurate way of determining pH
and the pH electrode/pH meter is a basic item in any science laboratory.
What most people refer to as a glass electrode, as you can see in Figure 9.4 (a), is technically a
device, not an electrode. It consists of a thin soft glass membrane, which gives the electrode its
name, and an electrically insulating tubular body made of hard glass or epoxy. This structure
encloses an internal electrolyte solution (usually 0.1 mol dm−3 HCl) and a silver/silver chloride
internal reference electrode. The silver/silver chloride internal reference electrode is con-
nected to the pH meter. A potential difference must, however, be measured between two
points. Hence a second external reference electrode is required, which also needs to be
immersed in the same test solution. Frequently, this second electrode is made up of a second
silver/silver chloride wire surrounded by a solution of 4 mol dm−3 KCl saturated with AgCl. The
external reference electrode can be a separate probe as shown in Figure 9.4 (b), although it is
commoner to find this external reference electrode built into the glass electrode in a concen-
tric double barrel arrangement, commonly referred to as a combination electrode as you
can see in Figure 9.4 (c).
Whichever reference electrode is being used, it must also form electrical continuity with the
test solution. In many electrodes, the KCl of the reference electrode is an aqueous solution that
flows out through a small hole in the electrode body to form a liquid junction. To minimize the
loss of KCl, the junction is packed with ceramic, fritted, or fibrous material, although some flow
(a) Wire connections (b) Wire connections (c) Wire connections

to pH meter to pH meter to pH meter
AgCl covered
Reference electrode
silver wire
internal solution
Filling hole
Ag/AgCl
reference
electrode
Internal
Junction
electrolyte
solution
AgCl covered
silver wire
Glass bubble Glass electrode

internal solution
FIGURE 9.4
(a) A pH ‘electrode’, (b) a Ag/AgCl external reference electrode, and (c) a combination pH
electrode. See text for explanations.
of KCl, however small, always occurs and therefore the pH electrode will slightly contaminate
the test solution. For most analytical applications this contamination is not significant. The KCl
solution lost from the electrode must be replaced. This can be done by refilling through a small
hole, high up on the electrode body. Refilling electrodes adds to their maintenance costs and
makes them less suited to situations where portability is a factor. To overcome such problems,
the internal KCl solution may be gelled, which helps slow the loss. However, as the composi-
tion of the internal solution cannot then be restored by refilling, such gel electrodes generally
have a shorter operational lifetime, although they are easier to use and maintain.
In situations where contamination of the test solution by the KCl of the reference electrode is
an issue, or where the test solution contains materials that might diffuse into the external refer-
ence electrode and affect the electrochemistry, a specially designed reference electrode called
the double junction reference electrode may be used. This device may also be selected
simply to prolong the operational lifetime of the electrode. In a double junction electrode, an
additional chamber between the reference electrode and the external solution is introduced.
This slows the loss of KCl to the test solution and presents a further diffusional barrier to
prevent the inward diffusion of any contaminating materials. Double junction electrodes are,
however, more difficult to construct and, hence, are more expensive.
Modern pH electrodes are robust and reliable devices. However, you might be interested to
know why the glass membrane found in these devices generates a potential when separating
two solutions. Not all glasses possess this property and it has taken considerable research to
optimize the composition of the glass to allow interference-free detection of H+. Soda-silica
glasses, such as Corning 015 glass, consisting of 22% Na2O, 6% CaO, and 72% SiO2, are effective
for measuring pH. Hydrogen ions themselves do not pass through the glass membrane of the
electrode. Rather, within such glass, the molecular network of SiO2 contains both Na+ and Ca2+
coordinated with oxygen that are able to exchange with H+ from the test solution and thereby
generate a potential across the glass membrane. The glass membrane therefore acts rather like
a battery whose voltage depends on the concentration of H+ ions in the test solution in which
it is immersed. The size of the generated potential (E) is given by the equation:
E = 2.303 (RT/F) log10 [H+]t/[H+]0
where [H+]i and [H+]o are the molar concentration of H+ inside and outside the glass mem-
brane, respectively. In practice, the internal H+ concentration is fixed at 10−1 mol dm−3 since the
electrode contains 0.1 mol dm−3 HCl. Hence, the electrode is responsive only to the concen-
tration of H+ in the test solution in which it is immersed.
Within the pH electrode, this spontaneously generated potential must be measured against
that produced by a reference electrode. In practice, however, other potentials are also gener-
ated within the system. The so-called asymmetry potential is poorly understood, but is present
across a glass membrane even when the H+ concentration is the same on both sides. The liquid
junction of the reference electrode may also give rise to a potential because the K+ and Cl− it
contains may diffuse out through the junction at different rates. However, these additional
potentials can be taken into account, such that at 25°C the overall potential of a glass pH elec-
trode measured against a reference electrode will be 59 mV for a 10-fold change in the activity
of H+. Rather conveniently, since pH itself is –log10 [H+], it follows that the 59 mV change in
potential actually represents each division of the pH scale.
Electrode selectivity and interference

Glass pH electrodes are particularly useful in the clinical laboratory because they demonstrate lit-
tle interference from the other components that may be found in a wide range of clinical samples.
One exception to this is the interference by monovalent cations, such as Na+ and K+. These ions
behave in similar ways to H+ at the glass surface and generate comparable potentials. Under
acidic conditions, where the concentration of H+ in solution is high, the interference is likely
to be negligible. At alkaline pH, where there are much lower concentrations of H+ in solution,
interference can be more obvious and the measured pH is lower than the true pH of the test
solution. This effect is often called the alkaline error or sometimes the sodium error, although
not only Na+ interferes in this way.
The careful selection of the glass composition is crucial to minimize such interferences, as it
Cross reference
is the composition of the glass that is solely responsible for the selectivity. Most commercial
Measuring Na+ and K+ in
pH electrodes have selectivities that are sufficient under all, but hyperalkaline conditions and clinical samples is described
below pH 12 any interference effects should be negligible even in modestly priced devices. in Chapters 1 and 2 of Clinical
Detailed information about selectivity is available from electrode manufacturers. It is possible Biochemistry.
to take advantage of this interference: by selecting a glass that has high response to Na+ it is
possible to construct a Na+ selective electrode, as described later.
Electrode storage and cleaning

The thin glass membrane of a pH electrode is fragile. Care must be taken not to break or
scratch it, or to cause a build-up of static charge by rubbing it. Many pH electrodes have
a plastic casing surrounding the glass electrode to protect them from damage. Even so, the
temptation to use the pH electrode as a stirrer must be resisted: this is a common cause of
breakage in many laboratories!
It is essential that the outer layer of glass in the pH electrode remains hydrated, so the elec-
trode is normally immersed in a solution at all times. Most manufacturers now supply specific
storage solutions for their electrodes. If these are not available, then the electrode should
never be stored in distilled or deionized water, as this will cause ions to leach out of the glass
bulb and render the electrode useless. It is acceptable to immerse combination electrodes in a
buffer solution, those of pH 4 to 7 are generally recommended, for storage between frequent
measurements. For longer-term storage it is generally recommended to keep the electrode
immersed in 4 mol dm−3 KCl.
The electrode should be washed thoroughly with distilled water after each use. As the elec-
trode is susceptible to contamination or dirt, it is recommended that it is cleaned perhaps
every 1–2 months depending on the extent and condition of use. General cleaning can involve
the use of a mild detergent. Any scale deposit on the electrode surface can be removed with
a concentrated acid, for example 6 mol dm−3 HCl. Biological materials can be removed using a
pepsin solution, and oily deposits with acetone or methylated spirits. However, the manufac-
turer’s instructions should always be consulted before using any of these methods.
If an electrode has been allowed to dry out and has lost activity, it is worth contacting the
manufacturer first, but a rejuvenating procedure might include soaking it in concentrated
hydrochloric or nitric acid, or even boiling the electrode. Such procedures are not guaranteed
to be successful and should only be used as a last resort.
pH meter
The glass pH electrodes developed in the early part of the 20th century were not used on a
large scale because of various technical difficulties. The main problem was caused by the large
internal resistance of the glass electrodes themselves (typically 50–500 MΩ). This necessitated
the use of sensitive and expensive galvanometers, which at the time were only available in
a small number of well equipped research laboratories. However, in the mid-1930s Arnold
Beckman, an assistant professor of chemistry at the California Institute of Technology, was try-
ing to develop a technique (and technology) to make quick and accurate measurements of the
acidity of lemon juice. Beckman’s innovative approach was to use a high-gain amplifier made
using two vacuum tube valves. The amplified current could then be read with much more reli-
able and cheaper voltmeters. In 1934, Beckman filed a patent, not actually for the entire pH
meter, but rather for the amplifier component alone. Some time later he put the amplifier, a
measuring electrode, and a data meter together in a compact walnut case, which he called an
acidimeter (Figure 9.5 (a)). Today we recognize this device as a pH electrode and meter, mod-
ern versions of which you can see in Figure 9.5 (b).
When using a pH electrode and meter, calibration usually involves the use of two buffered
solutions of widely differing pH. It is increasingly common to purchase such buffer solutions
ready-made from the electrode suppliers. These are often colour-coded to make the calibra-
tion process easier: the commonest buffers used are pH 4.0 (red), pH 7.0 (yellow), and pH
10.0 (blue). Calibrating most meters initially involves the pH 7 buffer. With the electrode in the
buffer solution and after sufficient time to equilibrate, the ‘calibrate’ dial on the meter should
be adjusted to give an output of pH 7.0. The electrode is then removed, rinsed with distilled
water, and blotted dry before being placed in a pH 4 buffer (if the test solution is expected to
be acidic) or a pH 10 buffer (if the test solution is expected to be basic). This second point is
adjusted using the ‘slope’ control on the pH meter. Once the pH electrode has been calibrated
it can simply be rinsed with distilled water and blotted dry before being immersed in the test
(a)
(b)
FIGURE 9.5
(a) The first commercial pH meter and probe,
called the acidometer, developed by Arnold
Beckman in the 1930s for the measurement of
the acidity of lemon juice. Photograph supplied
courtesy of Beckman Coulter. (b) Contemporary
modern pH electrode and meter.
solution for a rapid and accurate estimation of pH (Box 9.3). Between measurements, the elec-
trode should be rinsed with distilled water and blotted dry of excess liquid.
Should commercial buffer standards not be available you can make standards yourself from
recipes available in for example the Merck Index or the US Pharmacopeia. Typical standard
buffers might include:
• 0.05 mol dm−3 potassium hydrogen phthalate (pH 4.01)
• 0.025 mol dm−3 disodium hydrogen phosphate + 0.025 mol dm−3 potassium dihydrogen
phosphate (pH 6.86)
• 0.01 mol dm−3 sodium borate (pH 9.18)
Glass pH electrodes are affected by temperature (see the Nernst equation, Section 9.3). Each
division of the pH scale will, for example, represent a potential of 54 mV at 0°C, 59 mV at 25°C,
and 62 mV at 37°C. Temperature will also affect the thermal characteristics of the electrode
and the test solution such that, in practice, the pH electrode will have an isopotential point
where its potential is 0 and where temperature will not have any effect on the potential. This is
often designed to be near pH 7. The further the pH is from this isopotential point, the greater
will be the effect of temperature and the more necessary it will be to apply temperature com-
pensation. Most pH meters have a temperature compensation control that needs to be set to
the temperature of the calibrating buffers and test solution. More advanced instruments have
a temperature probe that is dipped into the calibrating and test solutions alongside the mea-
suring electrode. This ensures automatic correction of any variability resulting from solutions
having differing temperatures.
BOX 9.3 Expressing mean pH values
Consider the case of measuring the pH of a urine specimen with a pH meter. Measurement
of three samples of urine gives results of 5.5, 6.0, and 6.5. You then calculate the mean
pH of the urine to be pH 6.0. But is this value appropriate?
This is a remarkably common situation, yet the simple analysis as described above is
significantly flawed. pH is a logarithmic scale; hence, in terms of H+ concentration the
three pH values are not equally spaced. Converting the individual pH values into H+
concentrations gives:
pH 5.5 = 3.16 × 10–6 mol dm−3
pH 6.0 = 1.00 × 10–6 mol dm−3
pH 6.5 = 0.32 × 10–6 mol dm−3
The arithmetic mean of these concentrations yields a H+ concentration of 1.49 ë 10−6

mol dm−3, which equates to a pH of 5.8. This difference (5.8 versus 6.0) might be small
enough to ignore, but when pH readings are more widely spread the effects are signifi-
cant. Try Self-check question 9.4 to see this for yourself.
You should note that many papers are published that report a simple arithmetic mean
of pH value. This may have been used either pragmatically (the pH values are very close
together and hence errors are small) or through the author’s own ignorance. Beware!
SELF-CHECK 9.4
Given three solutions of pH 5, 7, and 9, respectively, you might expect the mean pH to be pH
7. However, calculate the mean as described in Box 9.3. What is the mean value?
Severinghaus CO2 electrode

The need to measure the carbon dioxide concentration in clinical samples, especially blood,
developed out of necessity during the 1950s’ polio epidemics in Europe and the USA. In the
USA alone, nearly 60,000 cases of polio were reported in 1952 and over 20,000 children were
left with mild to disabling paralysis. Such paralysis often produced respiratory difficulties,
which were treated with the use of the negative pressure ventilator, the ‘iron lung’. During
such treatment it was necessary to measure blood gases accurately and quickly to assess the
effectiveness of the therapy.
In 1954, Richard Stow, working at the Ohio State University in Columbus, developed an
electrode capable of measuring the partial pressure (concentration) of carbon dioxide or
PCO2 in solution. The device was essentially a modified glass pH electrode. Stow realized
that carbon dioxide freely penetrated rubber and that it also acidified water. Hence, he con-
structed a glass pH electrode and surrounded this with a thin film of distilled water held in
place by a thin rubber membrane (actually, in this instance a section of rubber glove). When
this device was dipped into a sample of blood, the carbon dioxide from the blood diffused
through the membrane and acidified the water surrounding the pH electrode, resulting in
a measurable drop in pH that was proportional to the PCO2. Stow published and presented
his findings, but his device was rather insensitive and unstable, and was never produced
commercially. It was, however soon modified by John Severinghaus who added a solution
of NaHCO3 and NaCl between the membrane and the electrode. Severinghaus also pre-
ferred a Teflon (polytetrafluoroethylene) membrane, rather than Stow’s rubber membrane.
In this device, carbon dioxide from the blood sample diffuses across the membrane into the
NaHCO3 and NaCl solution, and disturbs the carbonic acid/hydrogen carbonate equilibrium
to produce H+:
CO2 + H2O H2CO3 H+ + HCO3−
The system is allowed to achieve equilibrium, at which time the pH is measured. This is then
compared with a calibration curve of pH against known PCO2 values.
Severinghaus’s modifications doubled the sensitivity of the original device, enhanced its stabil-
Cross reference ity, and improved the response time. In 1958, Severinghaus described a device incorporating
Measuring HCO3−, PCO2, and both his PCO2 electrode and a Clark O2 electrode, which you can read about in Section 9.4,
pH in clinical samples are to form a combined blood gas analyser. Commercial manufacture of the Severinghaus PCO2
described in Chapter 6 of
electrode began in 1959 and such electrodes are still found in PCO2 analysers in clinical labo-
ratories today.
The Severinghaus electrode is a gas-sensing electrode and as it is based on the pH electrode,

the electrical output is a logarithmic function of PCO2. At 37°C an output of 62 mV is expected
for a 10-fold change in PCO2. The response is also temperature dependent since temperature
affects the solubility of dissolved gases, the equilibrium constants within the HCO3− layer, and
the Nernstian characteristics of the pH electrode itself. It is therefore usual to maintain the
temperature of such devices at a constant 37°C. For the most accurate analyses, it may also be
necessary to correct for the temperature of the patient who provided the samples, especially
if the patient is known to be either febrile or hypothermic.
Ion selective electrodes

The glass pH electrode is just one of a wide range of ISEs available to the biomedical scientist.
Note at the outset that these devices are called ion selective electrodes, not ion specific elec-
trodes. All of these devices do suffer from some interference, that is, responsiveness to other
ions, although in many clinical applications this interference may well be minimal. Within the
clinical laboratory you may well need to use Na+, K+, and Cl− ISEs to measure the electrolyte
balances of samples. Lithium ion ISEs may be used to measure concentrations of Li+ in samples
of serum in cases where this is being used to treat psychiatric illness.
In general ISEs can be divided into three main groups:
• Modified glass electrodes
• Polymer membrane electrodes
• Solid-state electrodes
Modified glass electrodes incorporate a membrane of specifically modified glass, as their name
implies. Polymer membrane electrodes have an ion exchange material in an inert matrix, such
as PVC, polyethylene, or silicone rubber, while solid-state electrodes incorporate a membrane
consisting of a single crystal of a sparsely soluble salt or a heterogeneous membrane in which
the salt is incorporated in an inert binding agent.
The interfering effect of Na+ on pH measurements was discussed earlier. We noted how the
chemical nature of the glass used within a pH electrode is designed to minimize this effect.
However, to measure Na+ in clinical samples, a glass electrode with a high Na+ responsiveness
and with an inner reference solution of a fixed Na+ activity are required. In early Na+ ISEs, NAS
11–18 glass, comprising 11% Na2O, 18% Al2O3, and 71% SiO2 was found to be effective, although
this has now generally been superseded by a range of lithium-based glasses (Li2O), sometimes
referred to as LAS glasses. Unfortunately, both NAS and LAS glasses are more responsive to
Ag+ and H+ than they are to Na+. Fortunately, Ag+ is rarely encountered in clinical samples and
such interference can generally be ignored. In some clinical applications, H+ interference may
also be of little concern. Consider, for example, the analysis of Na+ in blood where in healthy
individuals the plasma Na+ concentration is maintained within the range 0.135–0.145 mol
dm−3. The pH of such samples would be expected to be close to pH 7.4, which equates to a
H+ concentration of 4 × 10−8 mol dm−3, at which concentration little observable interference
would be expected. However, in other applications, the interfering effects of H+ may well be
significant. Standardizing the H+ concentration of all samples and standards to ensure that the
electrode is responding to Na+, rather than to H+ interference must be considered.
While it is possible to produce a glass electrode that is responsive to K+ using, for example, NAS
27-4 glass, a more common approach is to use electrodes constructed by incorporating spe-
cific ion exchange materials, such as valinomycin, into a polymer membrane. Molecules of the
cyclic antibiotic valinomycin have a hexagonal ring structure with an internal space of almost
exactly the same diameter as K+. They can therefore form complexes with the K+ and preferen-
tially conduct them across a membrane. Similarly, Li+ ISEs can be constructed from Li+ selective
carriers, such as crown ethers. These are capable of transporting Li+ across the membrane and
releasing the ion, in preference to the many other cations that might be present in a sample.
Various sold-state membranes are also available. Chloride ISEs are constructed using a crystal-
line membrane that incorporates AgCl in direct contact with the test solution. This membrane
slowly dissolves and Cl− from the electrode diffuses into the test solution to leave behind a surplus
of Ag+. This generates an electrical potential that is proportional to the Cl− in the test solution.
Such electrodes are quite robust and generally have faster response times than glass-based
electrodes. However, since such devices rely on the slow dissolution of the membrane itself,
they have a finite operational lifetime.
With all ISEs, we have emphasized that the electrode responds to the activity of the ion in
solution. If the instrument is calibrated with a standard of known concentration then, provided
the ionic strengths of the solutions are similar, the concentration of the test solution will be
recorded. To ensure that the ionic strengths are similar, an ionic strength adjustor can be
added to the sample and standards. Ionic strength adjustors contain high concentrations of
ions and sometimes pH adjustors, and decomplexing agents or agents to remove chemical
species that interfere with the measurement of the ion of interest. If some of the ion is not
free in solution, but exists in a complex or as an insoluble precipitate, then ion selective elec-
trodes will give a lower value than methods such as atomic absorption spectrophotometry
that detect all of the ions present. The ISE results may, however, be more applicable because it
is often the free ions that are responsible for any clinical or biological effects.
The response of all ion selective electrodes is logarithmic and is described by the Nernst equa-
tion. Ten-fold changes in ion activity give equal increments on the meter scale. Ion selective
electrodes are not intrinsically sensitive and generally show a linear response range 10−1 to
10−5 mol dm−3. In practice, the minimum detection limit of most commercial devices is 10−6
mol dm−3 at best. As with pH electrodes, the potential produced from an ISE is temperature
dependent, except at the isopotential point which varies with the type of electrode. It is there-
fore necessary to have some form of temperature control and/or compensation. All ISEs also
require a reference electrode to generate a steady potential against which the varying poten-
tial of the working electrode is compared. If either K+ or Cl− is measured, then a double junc-
tion reference electrode is needed to prevent contamination of the sample by the internal
solution of the reference electrode.
Many ions can be measured directly or indirectly by ISEs. One form of indirect measurement
is the use of the electrode as the endpoint indicator of a titration. The electrode can be sensi-
tive to the ionic species being determined or to the titrant ion. Such titrations may be 10 times
more accurate than direct measurements, since they only require the accurate measurements
of a change in potential, rather than the absolute value of the potential itself.
Ion selective field effect transistors

A range of ion selective electrodes may be constructed by modifying field effect transistors
to respond to specific ions. Such ion selective field effect transistors (ISFETs) are available
as probe type electrodes and also as microelectrodes that are suitable for implantation and
advanced experimental studies. It is likely that these devices will have an increasing clinical
value due to their robustness and because they can be stored dry, unlike conventional pH
electrodes and ISEs.
The electrical transistors found in nearly all electrical devices are metal oxide semiconduc-
tor field effect transistors (MOSFETs). Within such semiconductor devices, as you can see
in Figure 9.6 (a), a channel of semiconductor material enables a current to flow from one
electrode, called the source, to a second electrode, the drain. The rate of flow of electrons,
that is the conductivity of the semiconductor, is changed by altering the electrical diameter of
the channel. This is the function of the gate electrode, a metallic electrode that is insulated by
a layer of silicon dioxide from the channel itself, such that it can only influence the source-
to-drain current electrostatically. Within such a device, a small change in potential difference
between the gate and the body of the semiconductor is able to change the conductivity of
the channel and produce a relatively large change in the current from the source to the drain.
9.4 VOLTAMMETRIC TECHNIQUES 225
(a) Reference
Vgs Gate oxide

Source Drain
Channel
Bulk
Vdr
MOSFET
(b)
Reference
Insulating Insulating
Vgs resin resin
Gate oxide
Source Channel Drain
Bulk
FIGURE 9.6
Schematics showing the structures of
(a) metal oxide semiconductor field
Vdr
effect transistor (MOSFET) and (b) an ion
ISFET selective field effect transistor (ISFET).
In this way, the transistor acts as an amplifier. This is somewhat like a small tap controlling the
flow of water through a pipe: a small turn of the tap can dramatically increase or decrease the
flow of water.
As you can see in Figure 9.6 (b), the source and drain within an ISFET are constructed in an
identical way to the MOSFET. However, the metallic gate of the MOSFET is replaced by an
insulating coating capable of interacting with the analyte in the test solution to generate a
potential. In a pH selective ISFET, this may simply be a layer of silicon dioxide. The source and
drain leads, as well as semiconductor itself, are encapsulated so only the gate area is open for
contact with the test liquid. The device also incorporates a reference electrode, so that when
the device is immersed in a test liquid, an electrical circuit is set up between the reference
electrode and gate surface enabling the H+ concentration in solution to influence the channel
and, hence, the current passing from source to drain.
Ion selective field transistors may be further modified by changing the chemical nature of the
gate insulator or by adding an ion sensing membrane layer over the gate insulator. Using such
techniques, multi-function ISFETs capable of measuring pH, Na+, K+, and Ca2+ are formed. It is
likely that such devices will become increasingly common for the analysis of clinical samples.
ion selective field effect transistors also make eminently suitable transducers for incorpora-
tion into biosensors, as described in Section 9.5.
9.4 Voltammetric techniques

In all voltammetric techniques, an external voltage greater than that of the spontaneous
potential of the electrochemical cell is imposed on the system to force the electrochemical
reaction to occur. In simple direct current voltammetry, if an electrode is placed into a solu-
tion of an electro-reducible molecule and the potential of the electrode is made progressively
more negative a point is soon reached when reduction of the soluble species will begin at the
electrode surface and a small current will flow. A graph of current against voltage is called a
voltammogram, which you can see in Figure 9.7, The point at which the current begins to flow
is E1. As the electrode potential becomes ever more negative, the current increases dramati-
cally as the molecular species is reduced still further. Eventually, a potential is reached where
the molecular species is being reduced at the electrode surface as quickly as it can diffuse
towards that surface. At this point, E2 in Figure 9.7, a maximum or limiting current is produced
and further increases in potential will not increase the current beyond this value.
The voltammagram shown in Figure 9.7 illustrates two notable properties:
1. The midpoint of the wave, called the half-wave potential (E½), is characteristic for each
molecular species reduced
2. The value of the limiting current is directly proportional to the concentration of this
molecular species in solution
These two features enable voltammetric techniques to be used both to identify, as well as
quantify electroactive materials in solution.
More advanced forms of voltammetry, such as pulse voltammetry, differential pulse voltam-
metry, and alternating current voltammetry all use more complex voltage changes. Stripping
voltammetry allows a preconcentration step to be introduced into the analysis. These
advanced techniques are more sensitive than direct current voltammetry, although because of
this complexity they rarely find their way into routine laboratory analyses.
Voltammetry is closely related to polarography and, indeed, some authors use these terms
interchangeably. However, the term polarography should only be used when a dropping mer-
cury electrode is employed as the working electrode. This continuously renews the working
electrode surface and ensures that the analysis is not affected by residual material from pre-
vious samples contaminating the electrode. Amperometry is a much simpler technique. It
involves the reduction or oxidation of an electroactive molecular species at a constant applied
potential, usually sufficient to generate the limiting current such that the current flowing is
proportional to the concentration of the analyte in the test solution.
Current
Id
Id /2
E1 E2
E1/2
Potential difference
FIGURE 9.7
Typical direct current voltammogram.
9.4 VOLTAMMETRIC TECHNIQUES 227
Clark O2 electrode Braided wire soldered

to anode
In 1949 Leland Clark working at the Fels Research Institute, Yellow Springs, Ohio, devised and Cathode
developed an efficient bubble oxygenator for use in the oxygenation of blood during cardiac
surgery. This invention necessitated the development of a device to measure the partial pres-
sure (concentration) of oxygen (PO2) in the blood of patients undergoing such surgery. Clark
duly obliged and in 1956 described a compact oxygen probe that could measure oxygen con- Anode
centrations in aqueous solutions. The Clark electrode is still widely used today. You can see an
example of the device in Figure 9.8. It is an amperometric device that consists of a platinum
cathode and a silver anode, both of which are immersed in a solution of saturated KCl. The two
electrodes are separated from the test solution by an oxygen permeable Teflon membrane.
When a potential difference of −0.6 V is applied across the electrodes such that the platinum Ag/AgCl reference
cathode is made negative with respect to the silver anode, electrons are generated at the anode
anode, which reduce oxygen at the cathode. Platinum cathode
At the silver anode (positive), silver chloride is formed with the liberation of electrons:
4 Ag + 4 Cl− → 4 AgCl + 4 e−
In contrast, electrons are used at the platinum cathode (negative) to form water, consuming
Electrolyte
oxygen in the process:
(saturated KCl)
O2 + 4 H+ + 4 e− → 2 H2O
Since oxygen is reduced at the cathode, the concentration of oxygen in the solution of KCl
falls, which therefore acts as a sink, so that further oxygen diffuses into the solution from the
sample through the oxygen permeable Teflon membrane. Since the rate of diffusion of oxygen
through the membrane is the limiting step in the reduction process, the current produced by Teflon membrane
the electrode is directly proportional to the oxygen concentration in the sample. Clark elec-
trodes generally require the sample be stirred or mixed such that gradients of PO2 are not set ‘O’ ring
up within the sample itself, leading to erroneous (lower) PO2 values being measured. Platinum tip
Clark electrodes need to be maintained at a constant temperature since the solubility of oxy- FIGURE 9.8
gen in solution is temperature dependent. Similarly, atmospheric pressure also affects solubil- Clark oxygen electrode.
ity. Published solubility tables generally quote dissolved oxygen in pure water, but salting out
effects resulting from dissolved solutes may mean that the oxygen saturation values in clinical
samples are below those quoted in such tables.
There are many variants of the Clark electrode. It is common to find such electrodes in com-
bined blood gas analysers alongside both Severinghaus and pH electrodes to measure blood
PO2, PCO2, and pH. Modified oxygen electrodes small enough to be inserted into blood ves-
sels have been developed, but frequently this is avoided because of the dangers of infection
or of forming a blood clot. Often it is considered preferable to remove small samples of blood
from a warmed earlobe or a finger-tip and measure the oxygen content of the blood using a
small probe-type electrode.
A type of Clark oxygen electrode commonly found in many teaching laboratories is the Rank
oxygen electrode (produced by Rank Bros Ltd, UK). This is a versatile instrument and useful for
performing enzyme assays in which oxygen depletion or generation can be used as measures
of the rate of reaction. Glucose oxidase, amino acid oxidase, and catalase are enzymes whose
properties can be studied in this way.
Following its development, it was recognized that the Clark electrode could also be used to
measure hydrogen peroxide in solution simply by reversing the potential of the electrodes.
If a potential difference of +0.7 V is applied across the electrodes then the platinum anode is
BOX 9.4 What is the charge of an anode?
You can see that in Section 9.2 the anode of a galvanic cell, such as a Daniell cell (Figure
9.3), is negatively charged, whereas in Section 9.4 the anode of a voltammetric cell is
positively charged. This is not a mistake, simply the result of rather confusing inconsist-
ency. In both cases, however, the anode is the electrode at which oxidation events occur
and where electrons therefore become liberated. Similarly, reduction events always take
place at the cathode.
In this respect, some electrochemical cells differ from the electrophoresis apparatus dis-
cussed in Chapter 14 ‘Electrophoresis’, where the anode is always positively charged and
the cathode always negative.
made positive with respect to the silver cathode and electrons are generated at the anode and
used at the cathode as follows (Box 9.4). Thus, at the platinum anode (now positive), hydrogen
peroxide is consumed in an electron releasing reaction:
H2O2 → O2 + 2 H+ + 2 e−
and at the now negative silver cathode, electrons are used to reduce silver ions:
2 AgCl + 2 e− → 2 Ag + 2 Cl−
The flow of current is therefore proportional to the concentration of hydrogen peroxide in

solution. Measuring the concentration of hydrogen peroxide is particularly useful in designing
many biosensors (Section 9.5). Indeed, approximately 65% of all biosensors incorporate some
form of peroxide sensitive transducer.
9.5 Biosensors
Biosensors may be defined as self-contained integrated devices that incorporate a biological
recognition element combined with an electrochemical transducer capable of converting its
biological or biochemical signal or response into a quantifiable electrical signal. In the major-
ity of biosensors, the biological component is an immobilized enzyme that is attached to a
membrane or embedded within a structure that holds it in intimate contact with the trans-
ducer. This generally allows the enzyme to be re-used and so reduces operating costs.
Much of the technological development of biosensors has been stimulated by the need
to determine the concentrations of glucose in clinical samples. In 2000 the World Health
Organization estimated the number of people suffering from diabetes worldwide to be 171
million, and predicted this figure would double by 2030. Thus, many companies have made
significant investments in research and development programmes that have led to the pro-
duction of a wide variety of biosensor devices. Within clinical laboratories, a range of high
throughput laboratory analysers are available to assist in the routine analysis of blood glucose
and the diagnosis of diabetes. Small hand-held devices are also readily available which enable
Cross reference diabetic patients to regularly monitor their own blood glucose concentrations (Figure 9.9)
Remember, you can learn more and so make any necessary changes in diet or influence their decision to administer insulin as
about point of care testing in
appropriate. Indeed, the availability of such portable instruments has been a major influence
Chapter 18.
in the trend towards point-of-care testing within the healthcare sector.
9.5 BIOSENSORS 229
Biosensor design and operation

In 1962 Leland Clark (of oxygen electrode fame, as described in Section 9.4) first used the term
‘enzyme electrode’ to describe a device in which a traditional electrode could be modified to
respond to other materials by the inclusion of a nearby layer of enzyme. Clark’s ideas became
commercial reality in 1975 with the successful launch of the Yellow Springs Instruments model
23A glucose analyser. This device incorporated glucose oxidase, which catalyses the reaction:
Glucose + O2 → gluconic acid + H2O2
The device also contained an amperometric peroxide-sensitive electrode (which we described

in Section 9.4) to measure the amount of H2O2 produced by the reaction. Within the device,
the rate of formation of H2O2 is a measure of the rate of reaction between glucose and oxygen,
which, in turn, depends on the concentration of glucose in solution. Thus, the device can be
used to estimate the concentration of glucose in samples.
In enzyme-catalysed reactions, the relationship between substrate concentration, in this case

glucose, and reaction rate is not a simple linear one but is described by the Michaelis–Menten
equation. This is true for the glucose oxidase found within a biosensor. The rate of reaction is FIGURE 9.9
only directly proportional to the concentration of glucose when the glucose concentration is FreeStyle Lite blood glucose
low relative to that of the enzyme. However, at higher concentrations of glucose, the concentra- meter from Abbott Diabetes
tion of enzyme is the limiting factor, and the change in rate becomes hyperbolic. You can see Care. It is about the size of a
the Michaelis–Menten curve in Figure 9.10. We can engineer a more linear relationship over credit card and weighs only
a greater range of glucose concentrations by ensuring that the enzyme is enclosed behind or 40g. The meter features a
within a membrane through which the glucose must diffuse before it reacts with the enzyme. The large and easy to read LED
effect of the membrane is thus to keep the glucose concentration low compared with that of the display with a backlight for
enzyme. In this way, the system is diffusionally, rather than kinetically, limited and the response night use. The photograph
of the biosensor is then more linearly related to the concentration of glucose in solution. shows a test strip inserted into
the meter, which is discarded
after use. Photograph
A B
supplied courtesy of Abbott
Diabetes Care.
[S]
FIGURE 9.10
Representative Michaelis–Menten curve. Note where the concentration of substrate [S]
is low relative to that of the enzyme (A), the rate of reaction (v) is proportional to the
concentration. However, the concentration of enzyme becomes the limiting factor at higher
[S], as you can see at B.
The sensing probe of a device of this type comprises a peroxide-sensitive amperometric

electrode covered by a membrane comprising three layers. The first layer is made of porous
polycarbonate and limits the diffusion of glucose into the second layer, which contains immo-
bilized glucose oxidase, thereby preventing the reaction from becoming kinetically limited.
The innermost, third layer is of cellulose acetate that allows only small molecules, such as
hydrogen peroxide to reach the electrode. This layer eliminates many larger electroactive
compounds that could interfere with the amperometric measurement.
The YSI model 23A has now been replaced by the more advanced YSI model 2300 for clinical
uses and the YSI model 2700 for food analysis. As you can see in Figure 9.11, such instruments
possess a carousel that enables batches of samples to be run, with each sample requiring
only 100 seconds for analysis. These instruments can measure the glucose content of whole
blood, plasma, or serum, and require only 25 μl of sample per analysis. The membrane-bound
glucose oxidase typically needs replacing only every two or three weeks, thereby reducing the
cost of analysis. These systems also offer advanced data handling and data storage facilities.
As shown in Table 9.1, these instruments can be modified to analyse a wide variety of sub-
stances of biological interest simply through the incorporation of other oxidase enzymes in
the membrane. Where a single oxidase enzyme is not available for a particular analyte, analy-
sis can be accomplished by co-immobilizing multiple enzymes that act sequentially.
Small hand-held instruments have been developed to satisfy the demands of individuals
who wish to measure their own blood glucose concentration. In many of these instruments,
the catalyst and transducer are more intimately linked on the sensor surface. The Medisense
Exactech Glucose Meter, for example, was launched in 1986 and soon became the world’s
FIGURE 9.11
Model 2300 STAT Plus glucose and lactate analyser. The figure shows the 24-position
turntable, which allows batches of samples to be analysed. The buffer reservoir and waste
container are both conveniently contained within the analyser. Photograph supplied
courtesy of Yellow Springs Instruments Life Sciences.
9.5 BIOSENSORS 231
TABLE 9.1 Compositions of enzyme membranes available for analysers with H2O2
measuring probes as transducers
Analyte Enzyme Reaction catalysed

Alcohol Alcohol oxidase Ethanol + O2 → Acetaldehyde + H2O2
Glucose Glucose oxidase β-D-Glucose + O2 → gluconic acid + H2O2
Lactic Acid Lactate oxidase L-lactate + O2 → pyruvate + H2O2
Lactose Galactose oxidase Lactose + O2 → galactose dialdehyde derivative + H2O2
Starch Amyloglucosidase Starch + H2O → β-D-glucose

Glucose oxidase β-D-glucose + O2 → gluconic acid + H2O2
Sucrose Invertase Sucrose + H2O → α-D-glucose + fructose

Mutarotase α-D-glucose → β-D-glucose
Glucose oxidase β-D-glucose + O2 → H2O2 + gluconic acid + H2O2
best selling biosensor. The initial device was the size and shape of a pen, and used disposable
electrode strips. This was followed by a credit card style meter in 1989. These devices, again,
relied on glucose oxidase as the biological component, but did not measure reaction rate from
the production and detection of H2O2. Rather, they relied on the direct measurement of the
rate of electron flow from the glucose to the electrode surface. The reactions that occur within
this device may be summarized as follows:
Glucose + GOx-FAD → gluconic acid + GOx-FADH2
GOx-FADH2 + Mediator(oxidized) → GOx-FAD + Mediator(reduced)

and at the electrode surface:
Mediator(reduced) → Mediator(oxidized) + e−
where GOx-FAD represents the FAD redox centre of glucose oxidase in its oxidized form,
and GOx-FADH2 the reduced form. Within the Medisense device, the mediator is ferrocene,
although other manufacturers have produced similar devices using alternative mediator tech-
nologies. In essence, electrons are stripped from the glucose and then pass from the enzyme
to the mediator, which then donates them to the working electrode surface resulting in the
generation of an electrical current directly proportional to the rate of oxidation of glucose and,
hence, proportional to the glucose concentration in the sample. As well as a working and ref-
erence electrode, later test strips also incorporated a third electrode, known as a background
compensation electrode. This is not coated with glucose oxidase and thus measured the non-
specific current generated by interfering materials present in the sample. The current gener-
ated by this electrode is subtracted from that of the working electrode to produce a response
due to glucose alone. Unlike instruments of the Yellow Springs type discussed earlier, these
devices do not have any requirement for oxygen. This is advantageous, as the concentration
of oxygen in blood can vary, and in some cases may become limiting, such that the glucose
measurement is affected.
Medisense, whose only product was their blood-glucose meter, was bought by Abbott Diagnostics
in 1996 and Abbott-branded devices continued to use and develop this technology for some
time. In 1999, however, Therasense marketed a glucose meter that represented the next genera-
tion of sensing technology, and integrated the enzyme even more closely with the electrode.
Originally developed by Adam Heller at the University of Texas in the 1990s, wired enzyme
electrodes did not rely on a soluble mediator, such as the ferrocene used in the Medisense meter.
Rather, the enzyme is immobilized in an osmium-based polyvinyl imidazole hydrogel in which

the electroactive osmium groups are firmly held in place within the polymer, and cannot par-
ticipate in molecular diffusion. Thus, the electrons are not passed from enzyme to electrode
through a soluble mediator that moves by diffusion, but by a series of fixed electroactive osmium
centres that shuttle the electrons onwards, often called electron hopping.
The FreeStyle Lite meter produced by Abbott Diabetes Care and shown in Figure 9.9 incorpo-
rates wired enzyme technology. Devices of this type are highly amenable to miniaturization:
the Abbott device shown in Figure 9.9 is only the size of a credit card. Its size is largely limited
by the dimensions required for a display that can comfortably be read by a diabetic patient,
who might well have some visual impairment due to their condition. The electrochemistry is
incorporated into a test strip only a few centimetres in length that is inserted into the meter as
you can see in Figure 9.9. A sample of blood is obtained using a sterile lancet or similar prick-
ing device and placed on the test strip. Only 0.3 μL blood is needed for analysis, equating to a
drop the size of a pin head. Once loaded with sample, the device is able to measure the glu-
cose concentration in approximately 5 seconds and can store the results of up to 400 analyses
for comparative purposes. After a single use the test strip is discarded.
You might wonder why such glucose test strips are used once only when the enzyme is immo-
bilized and could possibly be re-used. Re-using the enzyme would require that the user or the
device itself be effectively washed between samples to ensure that results are not affected by
materials remaining from previous samples. Given that these devices are developed to be used
with little training by the general public and in a discrete fashion, it is clear that avoiding such
washing procedures by incorporating a single use test strip has great appeal. Further, the test
strip is the only part of the device that is exposed to the blood sample and this can be discarded
immediately after use. Thus, it is easier to maintain hygiene and prevent possible contamination
from blood borne diseases such as acquired immune deficiency syndrome (AIDS) and hepatitis.
Even so, when using these meters, the test strips together with the lancets used to obtain the
sample should be disposed of in accordance with relevant Infection Control and Health and
Cross reference Safety guidelines. These may vary slightly from region to region, but in general lancets and simi-
Health and safety are discussed
lar pricking devices need to be disposed of in a properly designed sharps containers (available
in Chapter 4.
from hospitals and pharmacists), while the test strips should be disposed of as clinical waste.
Single measurement devices, as described above, therefore play a key role in both the diagnosis
and subsequent management of diabetes. Similar devices are available for the analysis of a
Cross reference number of other clinically important analytes, for example glucose and lactate. Concentrations
Measuring renal function of lactate in blood may be measured using biosensors to detect the high levels of lactate that
is described in Chapter 3 of
typically accompany severe sepsis or septic shock, whilst creatinine and urea biosensors are
used to monitor renal function.
Continuous measuring devices are also becoming available and may well revolutionize the
control of certain disease conditions. With respect to diabetes, devices such as the FreeStyle
Navigator system from Abbott use the same wired enzyme technology as described earlier, but
now incorporate this technology into a tiny filament about the diameter of a thin hypodermic
needle. This is inserted approximately 5 mm under the skin to measure the glucose level in the
interstitial fluid that flows between the cells. The device is designed to remain in place for up
to five days, during which time it can measure glucose concentration every minute. A wireless
transmitter sends the glucose readings to a separate receiver anywhere within a 10-foot range,
and this can then issue an early-warning alarm to alert the user to a falling or rising glucose
level in time for appropriate action to be taken to avoid a hypoglycaemic or hyperglycaemic
episode.
All electrochemical techniques, ranging from the earliest pH electrodes to the most advanced
biosensors, share common appeal to the biomedical scientist. Electrochemical devices are
FURTHER READING 233
often small and their portability is an advantage to those who wish to make analyses at the
point of care. Such devices are also quite robust and cheap, relative to other analytical tech-
nologies, and require limited use of ‘wet reagents’ in their operation. The simplicity of the
devices often means that they can be used with little training, and their selectivity and sen-
sitivity are sufficient for many, although certainly not all, clinical purposes. For these reasons,
electrochemical devices are frequently considered to be one of the real workhorses of the
clinical laboratory.
SUMMARY
■ All electrochemical techniques involve the use of two electrodes and the measurement of
the potential difference between them (potentiometry) or the current that flows between
them (voltammetry)
■ pH, PCO2, and ion selective electrodes are all potentiometric devices
■ Potentiometry involves the generation of a spontaneous potential that is measured against

that produced by a reference electrode as a potential difference
■ The behaviour of these devices is described by the Nernst equation
■ The minimum detection limit of most ion selective electrodes is 10−6 mol dm−3 at best
■ Ion selective field effect transistors can be used to construct miniaturized solid-state
potentiometric devices
■ In voltammetric techniques, electrochemical reactions are not spontaneous, but forced to

occur by an imposed external voltage
■ These advanced techniques can be used to both identify and quantify analytes
■ The oxygen electrode has widespread applications in medicine and clinical analysis
■ The hydrogen peroxide electrode is used in about 65% of biosensors
■ Biosensors incorporate a biological component for specific recognition, together with a

transducer capable of generating an electrical output
■ Biosensors are widely used in the analysis of glucose concentrations for the diagnosis and
management of diabetes but also widespread applications both in the clinical laboratory
and in point-of-care testing
FURTHER READING
● Anonymous Classification and nomenclature of electroanalytical techniques (rules
approved 1975). Pure and Applied Chemistry, 1976; 45: 81–97.
A well-accepted scheme of classifying electrochemical techniques, though currently being
updated by IUPAC.
234 ELECTROCHEMISTRY
9 ELECTROCHEMISTRY
● Pretsch E. The new wave of ion-selective electrodes, Trends in Analytical Chemistry,

2007; 26: 46–61.
An interesting review revealing the significantly enhanced sensitivity and selectivity of
modern ISEs.
● Luong JHT, Male KB, Glennon JD. Biosensor technology: technology push versus
market pull. Biotechnology Advances, 2008; 26: 492–500.
A review that describes the commercial aspects relevant to the production of and demand
for biosensors. The review also describes the current commercial activities of most of the
major companies producing such devices.
● Newman JD, Turner APF. Home blood glucose biosensors: a commercial perspective.
Biosensors and Bioelectronics, 2005; 20: 2435–2453.
A thorough review of the commercial development of glucose biosensors, though missing
some of the most recent developments in continuous monitoring.
● Hönes J, Muller P, Surridge N. The technology behind glucose meters: test strips.
Diabetes Technology & Therapeutics, 2008; 10: S10–S26.
A useful article describing the technology behind a wide range of commercially available
devices.
● Brett CMA, Brett AMO. Electroanalysis. Oxford Chemistry Primers Series, Number 64.
Oxford University Press, Oxford, 1998.
Concise and well written introductory text providing a good ‘next step’ in your reading
around the subject. Particularly useful in its coverage of more advanced voltammetric
techniques.
● Cooper J, Cass AEG. (eds) Biosensors, Practical Approach Series. Oxford University
A second edition of the classic1990 text that explains how to implement diverse tech-
niques, such as protein engineering, optical and electrochemical instrumentation, and
numerical modelling in the context of producing biosensors.
Useful Website
■ http://www.freepatentsonline.com. A free and searchable database of patents. Very
useful for investigating recent developments in sensors and similar devices.
Useful Journals
■ The Analyst
■ Biosensors and Bioelectronics
QUESTIONS
1. If an electrode is described as an ISE, what does the acronym ISE mean?
(a) Ion specific electrode

(b) Ion selective electrode
(c) Ion sensitive electrode
QUESTIONS
9.5 BIOSENSORS 235
(d) Ion sensing electrode

(e) Ion standard electrode
2. Which of the following occur within an electrochemical analytical device?
(a) The anode always carries a negative charge

(b) The anode always carries a positive charge
(c) The anode is the electrode at which the oxidation reactions occur
(d) The anode is the electrode at which the reduction reactions occur
(e) None of the above
3. Which of the following statements are TRUE or FALSE?
(a) All biosensors contain an enzyme able to recognize the analyte and a transducer
that converts the response into an electrical signal.
(b) In simple direct current voltammetry, the limiting current is the highest one that
will flow when an analyte is being reduced at the electrode surface.
4. Given that the total potential, E (in volts) developed between the working and reference
electrodes of an ISE is given by the Nernst equation:
E = Econstant + 2.303 RT/nF log10 C
(a) Calculate the potential developed by a pH electrode and a Na+ ISE at 37°C. (b) Is the
accuracy of analysis of these two devices likely to be the same?
5. While your main interest in electrochemistry might be for the analyses of clinical sam-
ples, such devices do have many other uses. List some other analytical situations in
which these devices might be used, and consider why electrochemical techniques might
be favoured in these particular situations.
6. Section 9.3 gives general information on pH electrodes. Having read this section, locate
the manufacturers’ instructions for the pH electrodes in your laboratory. (a) What type
of electrodes are you using, for example, combination electrodes, gel electrodes, double
junction reference electrodes? (b) Do they all have the same mode of operation, that is,
calibration, cleaning, and storage procedures?
7. Section 9.5 describes the YSI glucose biosensor in which glucose is measured using the
enzyme glucose oxidase and a peroxide sensitive electrode as the transducer. Suggest
two other types of electrode transducers that could in theory be used to measure the
rate of this reaction and form the basis of a glucose biosensor. Why is a device measuring
peroxide preferable to either of these alternative instruments?
10
Radioactivity and
radiation
John Gaffney and Mark Slevin
Learning objectives
■ Describe radioactivity in terms of atomic structure

■ Discriminate between the main types of radiation
■ Demonstrate how the rate of decay of radionuclides can be calculated
■ Describe the methods available for the measurement of radioactivity
■ Explain the safety issues involved when handling and disposing of radionuclides
■ Identify uses of radioisotopes in biomedical science
Introduction
Radioactivity is the spontaneous emission of particles and/or rays or waves of energy from
the unstable nuclei of some atomic species. Such atoms are called radioisotopes, and as they
decay they form more stable atoms. The rays or waves, and the particles are called radiation.
There are three forms of radiation: alpha- (α-) and beta- (β-) particles, and gamma (γ-) radiation.
It is possible to detect and measure all three types of radiation.
Radiation is potentially harmful and so precautions must to be observed when using and han-
dling radioisotopes. However, it is also of use in biological and medical research and in the
diagnosis and treatment of some types of clinical conditions.
10.1 Atomic structure

The atomic nucleus contains most of the mass of the atom in the form of protons (p), and
neutrons (n). The mass of a proton is 1.67 × 10−27 kg. This is approximately the same as that
of the neutron; however, they differ in that protons are positively charged while neutrons are
10.1 ATOMIC STRUCTURE 237
electrically neutral. The nucleus or nuclide can be thought of as being surrounded by a cloud
of negatively charged electrons. The number of electrons is the same as that of the protons
so an atom is electrically neutral. Electrons are responsible for the chemical properties of the Cross reference
element. They occur only at specific energy levels called orbitals, which increase in energy You can read more about
with the distance of the orbital from the nucleus. Electrons can move between different orbit- the interactions of electrons
als if they absorb or emit energy. The lowest energy state and therefore the most stable for an and radiation in Chapter 11
‘Spectroscopy’.
electron in an atom is called the ground state. If an electron gains energy by interacting with
radiation, the electron may be excited or energized sufficiently to move to a higher energy
orbital; from there it can fall back to its original orbital with the emission of light or heat.
Nuclides can be represented in the abbreviated form, AZ X, where X represents the symbol of
the chemical element, A is the mass number, which is numerically equal to the sum of the
numbers of n and p, and Z is the atomic number, that is the number of p in the nucleus. For
example, 126C describes a carbon nucleus that contains 6p and 6n and therefore has an atomic
mass of 12 units. Although this form of carbon is generally written as 12C, it is pronounced
C-12. The atomic number is a characteristic value for each element.
SELF-CHECK 10.1
Draw a table listing the atomic mass, and the number of protons, electrons, and neutrons in
the following elements: 1H, 73 Li, 65 106
30 Zn, 46 Pd.
Isotopes
In any given nuclide, the number of protons is the same as the number of electrons. However,
although all the atoms of an element have the same atomic number, the number of neutrons,
and therefore the mass number, can vary forming isotopes. For example, carbon has isotopes
with mass numbers 10C, 11C, 12C, 13C, 14C, and 15C. These contain between four and nine neutrons,
but all have six protons. Hydrogen occurs as one of three isotopes: hydrogen (1H), deuterium (2H),
and tritium (3H); all contain a single proton, but 0, 1, or 2 neutrons, respectively. Approximately
one-third of the known elements exist as different isotopes. Many nuclides are unstable and with
time will decay to a more stable form with the emission of energy, that is the nuclides are radio-
active. However, isotopic species have variable stabilities, but not all are radioactive.
SELF-CHECK 10.2
Naturally occurring chlorine (Cl) is a mixture of 75% 35Cl and 25% 37Cl. What would be the
observed atomic weight of chlorine?
Nuclear stability
Two forces operate in the nucleus: the short-range, attractive strong nuclear force between
neutrons and between protons, and Coulomb (electrical) repulsive forces between the posi-
tively charged protons. The protons and neutrons are held in a stable state in the nucleus by
the binding energy of the strong nuclear force. Thus, the stability of the nucleus is a balance
between these attractive and repulsive forces and depends on the numbers of protons and
neutrons. The most stable nuclei, those of low atomic number, have equal numbers of these
particles. A straight line relationship occurs between the numbers of protons and that of neu-
trons up to atomic number 26 (that of iron (Fe)); which is the most stable element. You can
see this in Figure 10.1.
238 10 R ADIOACTIVIT Y AND R ADIATION
100
Neutron number
N=Z
50
0 50 100
Atomic number
FIGURE 10.1
A graph of the atomic number (Z) plotted against the numbers of neutrons (N) for the
elements of the periodic table. The hypothetical curve where N equals Z is a straight line
(red), but naturally occurring elements deviate from this (blue) because many have more
neutrons than protons.
An unstable nucleus with an excess of protons or neutrons, emits radiation until a stable ratio
occurs. However, elements of atomic number greater than 30 require an excess of neutrons
for stability. Indeed, all nuclei that contain 84 (that of polonium) or more protons are unstable
and decay. Although many isotopes are found naturally, others, especially ones used in medi-
cal procedures, are produced artificially by bombarding lighter nuclei with radiation in nuclear
reactors or in cyclotrons.
10.2 Types of radioactive decay

Radiation is energy travelling Radioactive decay involves the disintegration of atomic nuclei. The radiation released by
in the form of electromagnetic unstable nuclides until they reach a stable state can be particulate (small particles) or photons
waves or as a stream of particles. of electromagnetic radiation called:
• Alpha (α)-particles
• Beta (β)-particles and
• Gamma (γ) rays
Although lighter elements may decay and emit radiation in a single step, heavier ones, with
atomic numbers greater than 70, undergo a series of changes before reaching a stable state
and release α-particles in the process.
α Radiation
An α particle is a helium nucleus consisting of two protons and two neutrons (42He2+). Thus, the
release of an α-particle (α-decay) decreases the atomic number and the mass of the nucleus
10.2 T YPES OF R ADIOACTIVE DECAY 239
TABLE 10.1 Summary of the types of radiation emitted by unstable nuclei
Name Symbol Nature Range of Range of penetration Protective

energies/MeV* in air/m shielding
Alpha particle 4
2 α Helium ion 4–8 0.025–0.080 Not needed
0 −
Electron +1 B Energetic and fast 0.01–3 0.15–6 Plastic
moving electrons
0 +
Positron −1 B Energetic positively 0.01–3 0.15–6 Plastic
charged electrons
Gamma ray γ Electromagnetic 0.03–3 1.3–13 Lead

radiation
*1 MeV = 1.6 × 10−13 J.
by 2 and 4 units, respectively. For example, 226radium decays to 222radon by α-emission; how-
ever, the latter is also radioactive and can decay into further products:
226
88 Ra → 222
86Rn + 2He → 84Po + 2He → 82Pb → → 82Pb
4 + 216 4 + 212 208
SELF-CHECK 10.3
Uranium (U) decays to thorium (Th) by α-emission. Insert the appropriate numerical values to
complete the following equation.
238
? U → 90? Th + 42He
Decay by α-particle emission does not have many applications in biology. α-Particles have limited
penetration in air (Table 10.1) but they are toxic if the radionuclide is ingested due to their high
energy. For example, the presence of 226Ra at internal sites in the body may cause bone cancer.
β Radiation
Nuclei that are unstable due to an excessive number of neutrons may lose energy when a
neutron changes to a proton with the emission of a β-particle.
1
0 n → 11 p + −10 β−
The atomic number increases by 1 but the atomic mass does not change. A positron is emitted
if a nucleus has more protons than neutrons. Thus, when a proton changes to a neutron there
will be a decrease in atomic number of 1.
1
1 p → 10 n + +10 β+
A negatively charged β-particle (β−) is an electron and a positively charged β-particle (β+) is a
positron. These particles have the same mass, but opposite charges.
In these reactions, the sums of the subscripts and charges are balanced. For instance, the car-
bon isotope, 14C decays by β− emission to 13N:
14
6 C → 147 N + −10 β−
while 33Cl can decay to 34S by positron emission:
33
17 Cl → 33
16 S + +1 β
0 +
SELF-CHECK 10.4
The general equation for β-decay of a neutron-rich isotope is:
A
Z X → A?Y + −10 e
Insert the appropriate symbol to replace the question mark(?).
γ Rays
γ Rays are electromagnetic energy released in association with the rearrangements that occur
in a nucleus after it has emitted an α- or β-particle. γ-Rays, and X-rays, are emitted if electrons
that overlap with a nucleus combine with a proton to form a neutron. When this happens,
energy in the form of γ-and X-rays (see Box 10.1) can be emitted. Again, this will decrease the
atomic number of the nuclide by 1, but leave the atomic mass unchanged. You can see an
example of this in the way an isotope of iodine, 125I, decays:
125
53 I → 125
52 Te + X-rays + γ
The newly formed element, tellurium (Te) is deficient in an electron. However, a rearrange-
ment of its outer orbital electrons is able to fill the vacancy and occurs with the accompanying
emission of a photon of γ- or X-rays.
BOX 10.1 Discovery of radiation

and radioactivity
Wilhelm Röntgen discovered radiation in 1895 while he experimented with the dis-
charge of electricity in a vacuum. He used a glass tube, which contained two metal plates
connected to a battery. When he evacuated the tube and switched on the electric flow,
the negative charged plate or cathode glowed. Röntgen later showed that the glow was
due to the emission of energy produced when electrons in the plate jumped to a higher
orbital and fell back to their original level. Some of the electrons can be dislodged from
the plate and interact with atoms in the glass wall. In interacting, the electrons slow down
and emit radiation which Röntgen called X-rays because their nature was unknown.
Henri Becquerel discovered radioactivity in 1895 shortly after that of X-rays. He sprin-
kled a uranium salt onto a photographic plate, which he kept in the dark. When the plate
was developed, he noticed dark areas on the plate that corresponded to the position of
the uranium. Initially he thought this was due to X-rays interacting with silver atoms on
the photographic plate. Later, however, it was found that the ‘Becquerel rays’ produced
from uranium salts were different to X-rays as they ionized gases, which could be used to
measure the activity of the source. In 1897, Pierre and Marie Curie repeated Becquerel’s
experiments and showed the intensity of the energy released was proportional to the
amount of uranium. They called this energy radioactivity. In attempting to isolate a pure
emitter of radioactivity from uranium and thorium ores, they discovered a new element,
polonium that had a high activity.
Ernest Rutherford and Martha Thompson working in Manchester and Cambridge inves-
tigated the phenomena of radiation in more detail and recognized two types of radiation
emitted from uranium. These were called alpha (`) and beta (a) radiation respectively.
10.3 R ATE OF R ADIOACTIVE DECAY 241
γ-Radiation consists of high energy, short wavelength electromagnetic photons, which are
emitted during α and β decay. It has great penetration, for example, it will pass through 15 cm
of steel, but its cytotoxicity is about the same as that of X-rays. X-rays are penetrating electro-
magnetic radiation emitted when the inner orbital electrons of an atom are excited and release
energy in the same energy range as γ-rays (0.010−10 meV), but it is of non-nuclear origin,
Table 10.1 summarizes some of the major features of these types of α, β, and γ radiations.
10.3 Rate of radioactive decay

The rate of radioactive decay is a measure of how fast the conversion of an unstable radio-
isotope decaying to a stable form with the emission of energy occurs. Each radionuclide has a
different but characteristic rate of decay. The rate is unusual in that it is not affected by chemi-
cal or physical parameters such as concentration, temperature, or pressure. If a graph of the
number of atoms (N) present at set time intervals (t) is drawn, it gives an exponential curve as
you can see in Figure 10.2.
A simple relationship can be derived from this curve, that is –dN/dt is proportional to N or:
–dN/dt = λN
The term, –dN/dt is the rate of decay for the nuclide, and is commonly called the activity,
while λ is the decay constant, which differs for each radionuclide. The relationship between
these terms given above can be converted to a second equation:
ln N1/No = −λt
The symbol ‘ln’ represents the natural logarithm, No and N1 are the number of radioactive
atoms present initially and after time t, respectively. Natural logarithms can be converted to
logarithms to the base 10 by multiplying by 2.303. It is usual to calculate and quote the rate of
decay for a radioisotope as its half-life (t1/2), which is the time taken for half of the atoms in a
sample to decay. Manipulating the decay equation gives the following relationship:
t1/2 = 0.693/λ
100
80
Remaining activity / %
60
50%
40
FIGURE 10.2
20 A graph of half-life of an isotope against the
amount of radioactivity shows that radioactive
decay follows an exponential curve. At each
0
1 2 3 4 half-life the amount of radioactivity has
Half-life decreased by half.
You can see in Figure 10.2, that after one half-life, only 50% of the original atoms remain, after
two half-lives, 25% remain and so on.
The half-lives of common elements used in biology and medical science vary from hours to
Cross reference
years as you can see in the examples given in Table 10.2. Unfortunately, the half-lives of the
You can read about mass
spectrometry in the following biologically important elements 15O and 13N are 2.03 and 10.0 minutes, respectively. These are
chapter ‘Spectroscopy’. so short, that the isotopes are of little practical value. Instead, non-radioactive isotopes, for
example 15N, that can be detected and quantified by mass spectrometry are used.
SELF-CHECK 10.5
The half-life of 32P is 14.2 days. Use the appropriate equation to calculate the corresponding
value of λ.
10.4Units associated with

radioactivity
A number of units are associated with radioactivity. These include the:
• Curie
• Becquerel
• electron volt
A Curie (Ci) is the amount of a radionuclide that gives 3.7 × 1010 disintegrations per second
(dps). This pre SI unit has been superseded by an SI unit called the Becquerel (Bq), which is
defined as 1 disintegration per second (dps) although disintegrations per minute are also used.
The Bq is such a small unit that only multiples of it are commonly used.
TABLE 10.2 Half-lives of radionuclides commonly used in biology
Isotope Half-life Type of decay Examples of uses

3
H 12.26 years β −
Suitable for radiolabelling organic molecules with high specific activity for
metabolic and enzyme studies
14
C 5760 years β− Suitable for labelling organic molecules at different positions in the molecule
for investigating metabolism
22
Na 2.58 years Transport studies
32
P 14.3 days β− Labelling proteins and nucleotides (see Chapter 16)
35
S 87.2 days β− Labelling proteins (see Chapter 16)
51
Cr 27.7 days Electron capture Haematological investigations (see the companion textbook, Haematology)
59
Fe 60 days β− Haematological investigations (see the companion textbook, Haematology)
99m
Tc 6.0 hours Isomeric transition Diagnosis of disease (see the companion textbook, Biology of Disease)
125
I 60 days Electron capture Labelling proteins (see Chapter 16)
131
I 8.04 days β− Thyroid function (see the companion textbook, Clinical Biochemistry)
10.5 INTER ACTION OF R ADIATION WITH MAT TER 243
SELF-CHECK 10.6
Convert 1 μCi into the equivalent number of kBq.
The specific activity of a radioisotope is the amount of radioactivity per unit mass, which can
be expressed in cps or dpm per μmol of the substance. However, other combinations of units
are possible:
Specific activity = radioactivity (Bq, cpm, dpm etc)/amount of substance (mol, g, etc)
In experiments using radioisotopes, the radiolabelled compound is often diluted with its unlabelled
form, called a carrier, before use. This changes the specific activity of the compound. However,
there are a number of advantages to using compounds with a high specific activity, including:
• Only a relatively small amount of labelled compound need be used
• Interference in metabolic pathways is minimized
• Normal physiological conditions can be maintained
• The products of metabolic reactions can be isolated at high specific activity
SELF-CHECK 10.7
A sample of [14C]-glucose (Mr of glucose is 180) was diluted with unlabelled glucose to give a
concentration of 2 μg cm−3 and activity of 1 μCi. Calculate the specific activity of the prepara-
tion in μCi/mol.
The electron volt (eV) is the energy gained by an electron in accelerating through a potential
difference of one volt. Electron volts are used when describing the energy released during
radioactive decay. It is, however, an extremely small amount of energy, only 1.6 × 10−19 J, and
for practical purposes keV (103 eV) or MeV (106 eV) are used.
We have summarized these units in Table 10.3.
10.5Interaction of radiation
with matter
Radiation interacts with matter in ways that are dependent on the energy of the radiation.
Kinetic energy of motion is contained in α- and β-particles, while γ rays contain energy in the
form of electromagnetic radiation. All α-particles and γ-radiation from a particular nuclide are
TABLE 10.3 Terms and units used in describing radionuclides
Unit Abbreviation or symbol Definition of unit

Counts per minute (or second) cpm or cps Measured rate of decay
Disintegrations per minute dpm or dps Real rate of decay

(or second)
Curie (μCi) Ci 3.7 × 1010 dps (Ci × 10−6)
Becquerel Bq 1 dps
Electron volt eV Energy equal to 1.6 × 10−19 J

of the same energy, and are in the range of 2–9 MeV and keV to MeV, respectively. The energy
of β-particles from a given nuclide is more variable, with a range up to a maximum value,
Cross reference Emax. Their mean energy is 1/3 Emax. β-Particles vary in their energies because the amount of
You can read more about energy available is shared between the β-particle and a second particle, either a neutrino or
electromagnetic radiation in an antineutrino. If decay is by β+ emission, a neutrino is also released. Decay by β- emission is
Chapter 11 ‘Spectroscopy’. associated with the release of an antineutrino. In both cases, the energy is distributed between
the β-particle and the neutrino or antineutrino in a random manner. Hence, the energy pos-
sessed by a β-particle differs at each disintegration.
Excitation and ionization

When radiation interacts with matter, it can lead to excitation and ionization of electrons. If
the electron gains sufficient energy, it will be lost from the atom or molecule: a process called
ionization. Ionization results in the formation of an ion pair, consisting of a positively charged
ion and a free electron. Particulate radiation, α- and β-particles, cause excitation and ioniza-
tion because they directly interact with electrons. Depending on the energy of the particles,
they may interact with many atoms before losing energy. For example, the particles lose about
35 eV of energy for every ionization, therefore an α-particle of 3.5 MeV will produce about
105 ion pairs. The effect of γ-radiation depends on its energy. At low energy, it may be unable
to remove an electron and so expends its energy as heat. At higher energies, electrons will be
excited or removed.
SELF-CHECK 10.8
Suggest what may happen to the free electron generated by ionization.
10.6Detection and measurement

of radioactivity
Detecting and measuring the amounts of radioactivity relies on its abilities to excite electrons
and ionize atoms and molecules as described in Section 10.5. Sources of α-particles are rarely
used in biomedical science, and methods to detect β-particles and γ rays are emphasized.
Three methods are available to do this, based on the ionization of a gas (gaseous detection), or
of a liquid (liquid scintillation counting), or the interaction of the radiation with a photographic
emulsion (autoradiography).
Gaseous detectors
Gaseous detectors consist of a gas-filled chamber that contains two electrodes across which
a voltage is applied. When ionizing radiation interacts with the gas it generates ion pairs.
The electrons move to the positive electrode and the positive ions to the negative plate
generating a current. The magnitude of the current is proportional to the numbers of ion
pairs, which, in turn, is proportional to intensity of the source of radiation. A commonly used
gaseous detector is the Geiger counter, whose typical appearance can be seen in Figure 10.3.
The Geiger counter can detect about 99% of the β-particles from a source, but cannot detect
γ-radiation.
10.6 DETECTION AND MEASUREMENT OF R ADIOACTIVIT Y 245
Meter
Cathode surface
Glass window
Anode Wire mesh

FIGURE 10.3
Glass bead A Geiger—Muller detector.
Scintillation counters
In a scintillation counter, the radiation interacts with chemicals called fluors, which give off fluo-
rescent energy when they interact with radiation. If the energy of an electron in its ground state
is increased to an excited, higher energy level, then the subsequent decay back to the ground
state is associated with the emission of light. This phenomenon is called fluorescence. Hence,
the interaction of the radiation with the fluor can be detected as flashes of fluorescent light.
Liquid scintillation counting is used to detect isotopes that decay by β emission and can be
used to detect the isotopes commonly used in biomedical science, such as those of carbon,
phosphorous, and hydrogen. The sample is dissolved in a liquid containing the fluors called
the scintillant. The flashes of light produced by interactions of the β-particles with the fluor
are detected by photomultiplier tubes as you can see in outline in Figure 10.4. The photomul-
tiplier converts the flashes of light into an electronic signal, whose magnitude is proportional
to the number of β-particles detected. Scintillation counters can separate the flashes of light
produced according to the energy spectrum of the original radionuclide. This allows the simul-
taneous counting of two different isotopes in a single sample. For example, many biological
compounds can be labelled using both 14C and 3H for use in biomedical research.
Solid scintillation counters are also available, but are only used for penetrating radiation, for exam-
ple, isotopes of iodine and involve a crystal of fluorescent material held close to the sample.
A drawback to liquid scintillation counting is quenching, where the flash of light produced by
the fluor is absorbed by a contaminant before it can be detected. This leads to undercount-
ing of the amount of radionuclide in the sample. This problem is encountered in biomedical
science research since organic solvents, such as alcohols or chloroform, which are efficient
quenchers, are often used to extract biological compounds from tissues. Liquid scintillation
counters have quench correction facilities that use standards of known activity, thus their
counting allows the quenching of the sample to be estimated and corrected for.
Autoradiography
Radioactively labelled compounds separated by, for example thin layer chromatography
or gel electrophoresis, can be detected by exposing the paper or gel to a photographic film.
(a) β β-particle
Solvent molecule
Excited fluor
Emitted photon
(b) Teflon tube
Reflecting surfaces
Scintillant containing fluors

and radioactive sample
Photon
Photosensitive surface
Photomultiplier
FIGURE 10.4
(a) The use of fluors to detect radioactive decay
by stimulating the production of flashes of light
(photons). (b) The detection of radiation in a
scintillation counter. Processing device
The radiation released by the radionuclide interacts with silver grains in the film in a similar
manner to that of light during photography, reducing silver halide crystals to metallic silver.
Thus, an image showing the distribution of radioactivity in the paper or gel is produced. Once
visible on the photographic plate, the corresponding materials in the paper or gel must then
be identified. A disadvantage of autoradiography is the sample must contain enough energy
to be able to interact with the film; if the sample is of low specific activity it may require weeks
or months to produce a detectable image.
Autoradiography can also be performed on whole animal sections to investigate the distribu-
tion of a radiolabelled drug in the organism. Following exposure, the film will show an image
10.7 SAFET Y PRECAUTIONS WHEN USING R ADIOISOTOPES 247
of the section where the radioactivity is concentrated in tissues and organs. Generally, only
weak β-emitting isotopes, such as 3H, 14C, 35S, are used in localizing materials in tissues and Cross reference
cells using autoradiography. Their low energy and therefore limited penetration means they You can read more about
produce a sharp image on the photographic films. autoradiography in Box 13.1.
Limitations in detecting radiation

Whatever the type of detector, it will not be 100% efficient because not all disintegrations
of a radioactive nuclide are detectable. In addition, some of the emitted radiation may be
absorbed by contaminants, such as organic chemicals like chloroform or acids, and so are
missed by the counter. Thus, only a proportion of the disintegrations can be detected and
these will be recorded as counts per second (cps) or counts per minute (cpm); often the effi-
ciency of liquid scintillation counters is only 50 to 70%. However, if the efficiency of the count-
ing process can be determined, it is possible to convert the recorded cpm to disintegrations
per minute (dpm). For example, if a counter detects 50% of the known disintegrations in a
standard, then the counts obtained for the samples will have to be doubled.
SELF-CHECK 10.9
A scintillation counter has a counting efficiency of 82% for 14C and gave a reading of 45 000
cpm for a sample. Calculate the true value in dpm.
10.7Safety precautions when

using radioisotopes
Radiation absorbed by tissues causes ionization and the formation of free radicals leading to
cell damage, mutations in DNA and may possibly lead to cancer. However, there is no simple
relationship between the amount of radiation emitted by a source and the degree of cell dam-
age. The energy of the radiation, the amount of it absorbed by the body and the susceptibility
of some tissues to radiation damages all need to be taken into account.
Absorbed dose
The absorbed dose is the energy deposited in tissues when they interact with radiation. The unit
of absorbed dose is the gray (Gy), which is the absorption of 1.0 J of energy per kilogram of tis-
sue. The pre-SI unit of absorbed dose was the rad (from radiation absorbed dose), which is the
dose of radiation that gives an energy absorption of 1 × 10−2 J kg−1. A single Gy is equal to 100
rad. However, the damage caused by identical absorbed doses varies with the type of radia-
tion. Thus, to assess different radiations it is necessary to compare how much ionization they
produce when air is exposed to their ionizing effects. Some measuring devices still recorded
exposure in the pre-SI unit, the roentgen (R), which is equal to the quantity of ionizing radia-
tion that will produce one electrostatic unit of electricity in one cubic centimetre of dry air at
0°C and standard atmospheric pressure. This is equivalent to the amount of radionuclide that
produces 1.61 × 1015 ion pairs kg−1. The SI unit of exposure is the Coulomb kg−1 (C kg−1), which
again is the electric charge released when radiation comes into contact with a set volume of air.
A coulomb is a unit of electrical charge equal to the charge transferred by a current of 1 ampere
s−1. One R is equal to 258 C kg−1.
Effective dose
Additional factors need to be considered when assessing damage in biological tissues. For
example, the linear energy transfer (LET) is the rate of ionization along the path the par-
ticle takes through the tissue. The biological damage caused by α-particles is greater than a
β-particle or photon of γ-radiation of the same energies because of the relative efficiencies
with which energy is transferred to the tissue. The damage caused is related to the relative
biological effectiveness (RBE) of the radiation, which is defined as:
RBE = dose of X-rays that produces a given effect/dose of

radiation required to produce same effect
Typically, RBE values are 1 for high energy β-particles (emitted from the nuclides commonly
used in biological experiments), 2 for low energy β-particles and 20 for α-particles. The effec-
tive dose of radiation is the absorbed dose × RBE and is measured in Roentgen equivalent
man (rem) or Sieverts (Sv). The rem is the amount of radiation that gives a dose in humans
equivalent to that of 1 rad of X-rays. It is not an SI unit and has been replaced by the Sv, which
is the amount of radiation that gives a dose in humans equivalent to 1 Gy of X-rays.
Cross reference
You can read more about health See Health and Safety Box ‘Health & safety in relation to radiation’ for safety precautions.
and safety in Chapter 4.
HEALTH & SAFETY

In relation to radiation
Safety precautions
In the United Kingdom, the Ionizing Radiations Regulations 1999 (IRR99) established the reg-
ulatory framework for exposure to ionizing radiations and sets an annual limit of exposure of
20 mSv. Some organs of the body are more susceptible to radiation and, therefore, total organ
dose equivalents are used. For example, the lens of the eye is subject to a low limit of 6 mSv.
Handling of radioisotopes
Radioisotopes must normally be used only if an alternative is unavailable and the minimum quan-
tity should be used. A laboratory will establish local rules for the safe handling of radioisotopes.
A radiation safety advisor will be responsible for overseeing these. To limit exposure, contact with
radiation should be kept to a minimum.
The intensity of radiation decreases with the distance from its source. Thus, any technique that
maintains distance should be used, for example using tongs to hold samples. Shields will also
reduce exposure. Perspex will reduce exposure to a- and f-emitters. Lead shields may be required
for more penetrating types of radiation.
To limit internal exposure, the entry of the radionuclide into the body should be prevented by pro-
tecting the skin, and preventing its swallowing and inhalation. The normal laboratory practices of
wearing gloves and a laboratory coat must always be followed. A photographic film badge needs
to be worn and developed regularly at set time intervals to assess exposure. All work involving
radioisotopes must be carried out in a fume hood. If a radioactive source enters through the skin
it may pass directly into the bloodstream. If it is ingested its fate will depend on the route of entry
and the chemistry of the particular radionuclide. If inhaled the nuclide may be swallowed, trapped
in the trachea, or enter the lungs and subsequently the blood stream. Once in the body, many
radionuclides concentrate in one organ, the critical organ, for example calcium and phosphorus
isotopes accumulate in bone, others, such as 3H and 22Na distribute evenly in body tissues.
10.8 CLINICAL APPLICATIONS OF R ADIOACTIVIT Y 249
10.8Clinical applications
of radioactivity
In general, there are few routine applications of radioisotopes in the medical laboratory. There
are, however, many applications in nuclear medicine, a term used to describe the use of radio-
active compounds to perform diagnostic imaging or tests and in treating some diseases. Some
of the radionuclides and their uses in nuclear medicine are listed in Table 10.4 (see Box 10.2
for more on radiopharmaceuticals).
TABLE 10.4 Radionuclides and their applications in nuclear medicine
Radionuclide Clinical application

11
C Brain imaging
18
F Brain imaging when attached to glucose
133
Xe Cerebral blood imaging
67
Ga Tumour imaging
201
Tl Cardiac muscle
123
I Study of thyroid function
82
Rb Myocardial perfusion
Radioisotope imaging
The extent of penetration of α- and β-particles in tissues is small making them unsuitable
for imaging. However γ rays with energies greater than 50 keV are ideal for use in a number
of clinical applications. A range of radioisotope imaging techniques that rely on detecting
BOX 10.2 Radiopharmaceuticals
Radiopharmaceuticals are radionuclide-labelled agents The preferred characteristics of a radiopharmaceutical are:

that distribute in the body. They provide information on
■ It should have a half-life as close as possible to the time
physiological and metabolic functioning of organs in the
body. This is in contrast to other imaging techniques, which of the test to avoid excess exposure and to be elimi-
provide anatomical information. The distribution of radi- nated from the body within the half-life
opharmaceuticals in the body is determined by the method ■ The nuclide should be a f-emitter with an energy in the
of administration, the circulation, and the metabolism in range of 50–300 keV.
the organ. Imaging is based on detection of photons in the ■ It should accumulate only at the site of interest
form of X- or f rays emitted from the source. An image is
■ It should be simple to prepare and incorporation of
constructed from the detected photons by using a collima-
radioactivity should not alter the biological properties
tor, which only accepts photons from a narrowly defined
of the radiopharmaceutical
region of the body.
γ rays are available. Calcium does not have a radioactive isotope suitable for bone scans but
a radioactive isotope of technetium (99Tc), is a suitable replacement. If 740 MBq (20 mCi) of
99
Tc-methylene diphosphanate is administered intravenously it diffuses into the extracellular
fluid surrounding the bone and becomes fixed into the solid phase of the bone by exchang-
ing with Ca2+. The sites of incorporation of the isotope in the skeleton can be detected using a
scanning gamma camera, which detects γ rays emitted by the nuclide and builds up a three-
dimensional image of the targeted organ following repeated scans of the area. The concentra-
tion of 99Tc is highest in those areas of increased bone activity and blood flow, at, for example
the sites of bone fractures (Figure 10.5) or cancer metastases, providing a sensitive method of
identifying such regions.
Myocardial perfusion scanning is a technique for visualizing blood vessels in the wall of the
heart. It is applied to patients with angina (chest pain as a result of exercise) or with unex-
plained chest pain that may be due to partial blockage of arteries or to visualize partial block-
ages in an artery caused by plaques. Damage caused by a myocardial infarction (a heart
attack) may also be evaluated by the technique. Again, commercial preparations of 99Tc or
thallium are injected into a vein of the arm or hand. Scanning of the patient with a gamma
camera shows the areas where the radionuclide has accumulated because damaged areas
have a decreased flow of blood and do not absorb as much of the isotope. Thus, a stress myo-
cardial perfusion scan assesses the flow of blood to the heart muscle or myocardium when
it is subjected to stress by exercise or medication. The technique can identify those areas of
the myocardium that have decreased blood flow using a gamma camera to take pictures of
the heart.
Positron emission tomography (PET) utilizes nuclides that decay by positron emission. The
positron collides with an electron producing two photons of γ rays, which travel collinearly
in opposite directions. A gamma camera, which contains a scintillator connected to photo-
multiplier tubes, is used to detect the photons. The γ rays are focused and the data collected
can be used to construct a three-dimensional image of the tissue. However, the mechanisms
by which this is done are complex and need not be discussed here. Positron emission tom-
ography is particularly useful for producing images of the brain. Glucose is the major energy
source for the brain where it is degraded by glycolysis. However, the modified form of glucose,
2-deoxyglucose is retained in the brain. If this compound is labelled with a γ-emitter, such as
18
F-2-deoxyglucose or 11C-2-deoxyglucose, then areas of metabolic activity in the brain can be
visualized, as you can see for the relative activities in a normal brain and that of a sufferer of
Alzheimer’s disease in Figure 10.6 (a, b).
SELF-CHECK 10.10
Are there any potential hazards in radioisotope imaging?
FIGURE 10.5
The presence of two fractures in the pelvis detected
using radioimaging with 99Tc.
(a) (b)
FIGURE 10.6
Positron emission topography
(PET) images showing
metabolic activity in the brains
of (a) a healthy person and
(b) a patient with Alzheimer’s
disease. Courtesy of the
Alzheimer’s Disease Education
and Referral Centre, National
Institute on Aging, USA.
Applications in haematology
Cross reference
Radioisotopes have haematological roles, in particular for determining blood volume and You can read more about
erythrocyte survival times. haematology in the companion
textbook, Haematology.
The disorder polycythaemia vera (also called polycythaemia rubra vera) is a myeloproliferative
disorder in which the abnormal bone marrow produces an excess of erythrocytes. As a result,
the blood is more viscous than normal and has haemocrit values (the proportion of blood
occupied by erthrocytes) greater than 54% (reference range 40.7–50.3%) in men and 49% in
women (reference range 36.1–44.3%). In some patients, the leukocyte and platelets may also
be increased in number and morphologically abnormal. However, the spleen may also be
abnormally enlarged, leading to an apparent increase in blood volume and making the haem-
ocrit value falsely normal. Thus, a diagnosis of polycythaemia vera requires a definitive dem-
onstration of an increase in the mass of erythrocytes. This can be measured by labelling the
erythrocytes with 51Cr. The assay involves withdrawing a blood sample (20–22 cm3) from the
patient and mixing it with 51Cr-sodium chromate of known radioactivity. The sample is then
kept at room temperature for 35 minutes to equilibrate. The blood is separated by centrifu-
gation, the plasma discarded, and the erythrocytes washed with isotonic saline before being
suspended in 12 cm3 of isotonic saline. A sample of the blood is retained as a standard and the
labelled blood cells injected back into the patient. After 10 minutes, a new blood sample is
withdrawn and the packed cell volume (PCV) determined. The erythrocytes are then lysed and
the released radiation measured. The erythrocyte volume can be calculated as the total cpm
injected divided by the cpm per cm3 of erythrocytes. Erythrocyte masses greater than 36 cm3
kg−1 in men (reference range 28.3 ± 2.8 cm3 kg−1) and 32 cm3 kg−1 in women (reference range
25.4 ± 2.6 cm3 kg−1) are abnormal.
Erythrocyte survival studies are used to assess the presence and severity of haemolytic anaemia.
Haemolysis may occur by one of two mechanisms: intravascular or extravascular. Intravascular
mechanisms include complement fixation, trauma, or other extrinsic factors. It is associated
with conditions such as glucose 6-phosphate dehydrogenase deficiency, thrombotic throm-
bocytopenic purpura, disseminated intravascular coagulation, and paroxysmal nocturnal
haemoglobinuria. Extravascular mechanisms are more common and here erythrocytes are
removed from the circulation by the mononuclear phagocytic system because they are intrin-
sically defective or because of the presence of bound immunoglobulins on their surfaces.
Normally, erythrocytes circulate for about 120 days. This is much shorter in haemolytic anae-
mia and bone marrow activities cannot compensate for their increased loss. Erythrocyte or
red cell survival is tested by labelling the cells with 51Cr and determining the blood volume as
described above for polycythaemia vera using 10 cm3 samples of blood removed at 24- and
48-hour intervals for up to 14 days.
Radioimmunoassays
Radioimmunoassay (RIA) was a commonly used technique for quantifying a number of clini-
cally important biochemicals, such as hormones, drugs, and immunoglobulins, although its
use is now much more restricted. Any material that can act as an antigen and stimulate the
production of a complementary antibody can be detected using a suitable RIA. Thus, RIAs
combine specificity, due to the recognition of the antigen by the antibody, with the sensitivity
of radioisotope techniques. The technique is based on preparing a radiolabelled form of the
material to be measured, which is used as an antigen. For example, tyrosine residues in protein
antigens can be labelled with 125I. Non-protein antigens can be labelled using 3H.
You can see an outline of how RIAs are performed in Figure 10.7 (a). Increasing amounts of com-
bined radiolabelled and unlabelled antigen are added to fixed quantities of antibody. The anti-
body is unable to discriminate between the labelled and unlabelled forms of the antigen hence
the amount of labelled antigen that binds to the antibody will decrease as the amount of unla-
belled increases. The antibody–antigen complexes are then separated from the free antigen in
one of a number of ways. For example, as shown in Figure 10.7 (a) a second antibody that binds
to the first can be added to the solutions and this precipitates the antibody–antigen complexes
leaving free antigen in solution. The radioactivity of each is then determined. A calibration curve
is produced by adding the labelled antigen to a known range of concentrations of unlabelled
antigen at the same time as the test on the sample is performed. This can be used to determine
the concentration of the analyte in the unknown sample as shown in Figure 10.7 (b).
Immunoradiometric assays (IRMAs) are more sensitive and specific than RIAs in measur-
ing the concentrations of small Mr peptides, and steroid hormones. The basis of IRMAs is to
immobilize the antibody to the ligand to be detected, for example a hormone, on insoluble
polystyrene beads. The antigen binds to the immobilized bead and the unbound antigen can
be removed by washing. The bound antigen is detected and quantified by adding a second
antibody labelled with 125I to the bound antigen. The beads are washed again but this time to
remove the excess 125I-antibody. The radioactivity of the beads is counted and this is now a
measure of the amount of antigen (hormone) that was present in the sample. A standard curve
must also be produced using known quantities of the hormone, which is used to relate the
radioactivity of the hormone bound to the beads to its concentration in the sample.
Non-isotopic methods utilizing chemiluminescence immunoassays, fluorescence immu-

Cross reference noassays, and enzyme-linked immunosorbent assays (ELISAs) have largely replaced RIA for
You can read more about ELISA most tests. Enzyme-linked immunosorbent assays use an antibody or an antigen linked to an
in Chapter 16 ‘Molecular biology enzyme and so combine the specificity of the antibody–antigen reaction with the sensitivity of
techniques’. enzyme assays. The sensitivity of ELISAs may be greater than RIA but more significantly, they
are quicker to perform and do not require the use of radioisotopes. However, RIAs are still of
use in diagnosing the condition systemic lupus erythematosus (SLE). Most patients suffering
from SLE produce antibodies against double stranded DNA (dsDNA) and, at some time dur-
ing their illness, these can be detected in their sera. The most commonly used clinical tests to
detect the anti-dsDNA antibodies of SLE are the so-called Farr RIA and an ELISA. Some evalua-
tions of both methods indicate that the Farr RIA-based method is superior to the ELISA dsDNA
(a) (b)
Radioactive antigen (Ag)
Antibody (Ab)
Radioactivity of bound antigen

against the antigen
Radioactivity of free antigen

Add unlabelled antigen
Radioactive antigen
displaced by
unlabelled antigen
Ratio of
sample
[Antigen] in
Separate Ag-Ab complexes
sample
with for example a second
antibody against the first
[Unlabelled antigen]
Radioactivity of supernatent
≡ free antigen
Radioactivity of precipitate
≡ bound antigen
FIGURE 10.7
(a) Outline of the procedure for radioimmunoassay (RIA). See text for details. (b) Use of a RIA
calibration graph to determine the concentration of an antigen in a patients’ sample.
test in diagnosing (identifying) patients with SLE. For this reason, some clinical biochemistry
laboratories still use it, although in most it has been replaced by the ELISA method, because of
the general advantages given above.
SELF-CHECK 10.11
List two advantages of replacing RIA by ELISA.
Radiotherapy
Radiotherapy is the medical use of ionizing radiation as part of the treatment of certain can-
cers. Where applicable, it is used as the primary treatment to kill cancer cells, either alone or
in combination with surgery or chemotherapy. It is also used as a palliative treatment to relieve
symptoms, if the cancer is untreatable. The basis of the therapy is that radiation interacts with
oxygen or water molecules forming free radicals that damage DNA. Since tumour cells have
a higher rate of cell division than normal tissues, they are more susceptible to damage by free
radicals. However, a major problem encountered in radiotherapy, is that the poorly developed
blood systems of cancers means they are short of oxygen, which reduces its effectiveness.
Cells differ in their sensitivity to radiation. For example, 99% of bone marrow cells are killed by
a dose of 4 Gy, but a dose of 16 Gy is required to kill 99% of thyroid cells. Similarly, cancer cells
also show a range of sensitivities to radiation and so the dose has to tailored to the particular
type of tumour. Leukaemias and lymphomas are very sensitive, epithelial tumours are moder-
ately so, requiring doses in the range of 60–70 Gy. Renal tumours are insensitive. A typical dose
applied to solid epithelial tumours is in the range 60–80 Gy and for lymphomas it is 20–40 Gy.
Radiotherapy is applied externally or internally. In either case, the dose of radiation used is
limited because it also damages healthy tissues. The adverse effects of external treatment is
reduced by irradiating the tumour with several beams of X-rays focused from different direc-
tions. The total dose of X-rays applied is fractionated into multiple doses, which are given over
several weeks. Typically, doses of 2 Gy are used daily over a period of several weeks or months;
a regimen known as continuous hyperfractionated radiotherapy (CHART). The advantage of
dividing the dose in this way is that healthy tissue can repair itself but tumour cells are less
efficient at self-repair, and so recover less well between the doses. In internal therapy, the
radioisotope is placed near to or inside the tumour for a short period, a procedure known as
brachytherapy. For example, intracavitary radiotherapy involves the insertion of 137Caesium
into a body cavity using an applicator. This form of treatment is used in treating cancers of the
vagina, cervix, and uterus. Alternatively, thin radioactive wires may be inserted directly into
the tumour, as, for example, in treating prostate cancers. Internal therapy may also involve
giving the patient a radioactive liquid either orally or intravenously.
SUMMARY
■ Radiation is the energy released when an unstable atom decays to a stable form
■ Radiation is emitted as α- or β-particles or as γ-radiation
■ Radiation is detected by gaseous or liquid scintillation counters or by autoradiography
■ Radiation is potentially harmful and precautions must be taken in its handling and uses
■ Radiation has applications in clinical imaging, diagnosis and in treating some forms of
cancer, and in biomedical research
FURTHER READING
● Hendee WE, Ritenor ER. Medical Imaging Physics. John Wiley and Sons, Harlow,
2003.
Describes the methods and theory of the application of radiobiology to imaging.
10.8 CLINICAL APPLICATIONS OF R ADIOACTIVIT
QUESTIONS Y 255
● Hogle WP. The state of the art in radiation therapy. Seminars in Oncology Nursing,
2006; 22: 212–220.
This paper describes the methods used to administer radiation and the effects of
treatment.
● O’Neill P, Wardman P. Radiation chemistry comes before radiation biology.

International Journal of Radiation Biology, 2009; 85: 9–25.
This article illustrates contributions of radiation chemistry to radiation biology.
● Sharp PF, Gemmell HG, Murray AD. Practical Nuclear Medicine. Springer-Verlag,
Berlin, 2005.
A detailed study of the theory and practice of nuclear medicine.
● Steele G. (ed.) Basic Clinical Radiobiology. Oxford University Press, Oxford, 2002.
A collection of articles describing the science of radiobiology and its applications.
● Wigg D. Radiation: facts, fallacies and phobias. Australasian Radiology, 2007; 51:
21–25.
A discussion of the use of radiation in diagnosis and treatment, and the benefits and costs
of the use of radiation.
QUESTIONS
1. Indicate whether the following statements are TRUE or FALSE:
(a) In any given nuclide, the number of electrons is the same as the number of
neutrons
(b) Nuclei become more unstable the bigger the difference in numbers of protons and
neutrons
88Ra) to radon ( 86Rn) is an example of α-decay
The decay of radium (226 222
(c)
(d) The unit of absorbed energy from radiation is the gray
(e) The gray is defined as the absorbed dose when 1J is deposited per gram of tissue
(f) The linear energy transfer (LET) is the degree of ionization caused by the interac-
tion of the radionuclide with tissue
(g) Values for LET are in the range of 20 for β-particles and 1 for α-particles
2. Insert the correct term where a gap appears in the following text. Choose from the fol-
lowing list of alternatives, which may appear more than once: radioisotopes; radiation,
a-particle (x 2), b-particle, g rays, 4, helium, electron, neutron, proton, mass, half-life, 5760
years, Becquerel, 1, 2, 1 disintegration per second, energy, 1 volt, electron volt.
Unstable atomic species called ______ can decay into more stable species by the emission
of ______ . The energy emitted can be carried as an ______ or a ______ or as ______. An ______
has a mass of ______ units and is a ______ ion. Release of an α-particle decreases the atomic
mass of the radioisotope by 4 mass units and the atomic number by ______. A β-particle is
an ______ produced when a ______ decays to a ______ . In the process the ______ of the
radionuclide remains unchanged, but the atomic number increases by ______ unit. The
time taken for half of the radioactive sample to decay is called the ______ and varies from
8.04 days for 131I to ______ ______ for 14C. The SI unit of radioactivity is the ______, which
is defined as ______ ______ ______ ______. The ______ ______ is the unit used to express the
energy released during radioactive decay. It is defined as the ______ gained by an electron
accelerated through a potential of ______ ______.
3. Define the following terms; Becquerel, Curie, rad.
4. The half-life of 32P is 14.2 days. How long would it take a preparation of the isotope with
an initial activity of 21 000 dpm to decay to 1000 dpm?
5. What precautions should you take when carrying out an experiment to label a protein
with 32P?
6. In an experiment labelling a sugar with 3H the sample was counted in a liquid scintilla-
tion counter and a reading of 32 600 cpm was obtained. Using a standard source of 3H
known to contain 555 000 dpm, a reading of 425 000 cpm was obtained. (a) What is the
efficiency of counting and (b) express the activity of 3H as dpm?
7. A sample of protein Mr 64 000 was labelled with 14C. After its purification it was found to
have a specific activity of 1.7 × 107 dpm μg−1. What is the specific activity of the purified
protein in Ci mol−1?
8. Explain the cytotoxic effect of radiation. Why would you expect cell damage to increase
with the energy of radiation?
11
Spectroscopy
Qiuyu Wang, Helen Montgomery,
Nessar Ahmed, and Chris Smith
Learning objectives
■ Define the term spectroscopy

■ Describe the electromagnetic spectrum
■ Discuss the general theory of spectroscopy
■ List the major spectroscopic techniques
■ Discuss some of the uses of spectroscopy in the biomedical sciences
Introduction
Spectroscopy is the study of the interactions between electromagnetic radiation and matter.
Atoms, molecules, and ions can scatter, absorb, or emit electromagnetic radiation in ways that
depend upon the chemical composition and structure of the substance. Thus, the pattern of
absorption or emission is characteristic for a given substance. This means that spectroscopy
can be used as an analytical technique to identify, which elements are present in a sample,
determine how much of an analyte is present, and even be used to elucidate the structures of
molecules. It is a non-invasive technique and samples are not destroyed during spectroscopic
analyses. These characteristics give spectroscopic analysis a broad range of applications in the
biomedical and biological sciences.
11.1Radiation and the

electromagnetic spectrum Cross reference
radiation in Chapter 10
Radiation is the emission or transfer of energy (E) as electromagnetic waves or particles. The
‘Radioactivity and radiation’.
particles are often called photons. Electromagnetic radiation, such as light, is propagated
258 11 SPECTROSCOPY
l
E
H Dire
ct
FIGURE 11.1 prop ion of
agat
ion
Propagation of electromagnetic E
radiation. See main text for
explanation.
through space as electric and magnetic fields, which are mutually perpendicular to each other.
You can see this expressed diagrammatically in Figure 11.1.
Two characteristic features of any sort of wave are its wavelength (λ) and frequency (v). You
can see in Figure 11.1 that the wavelength is the distance between any two adjacent peaks.
The frequency is the number of peaks passing a fixed point in a given time, which is normally
measured in seconds (s). Wavelength and frequency are interrelated by the velocity of light (c)
in the following way:
λ = c/v
The energy (E) of any type of electromagnetic radiation is described as:
E = hc/ λ = hv
where h is Planck’s constant. The use of these simple equations means that the frequency,
wavelength and energy of electromagnetic waves can be easily interconverted although care
must be taken over units (see Box 11.1).
Thus, the energy of a wave is directly proportional to its frequency: the higher the frequency,
the greater the energy. However, the energy is inversely proportional to its wavelength, the
shorter the wavelength, the greater the energy. The correlation between wavelength and
BOX 11.1 Spectroscopy and units
Wavelengths (k), frequencies (v), and the Planck constant (h) can all be expressed in one
of a number of units. Wavelengths are usually given in nanometres (nm), but some older
texts use the angstrom (Å), which is equal to 10−10 m or 0.1 nm. Frequencies are normally
given in cycles per second or hertz (Hz), which is reciprocal seconds (s−1). However, fre-
quencies may also be described as a wave number (v’), which is often expressed as recip-
rocal centimetres (cm−1) simply because the numbers become easier to manage. Planck’s
constant has a value of 6.63 ë 10−34 J s−1. Again, it can be expressed in alternative units
including 6.63 ë 10−27 erg s−1 and 4.135 ë 10−15 eV s−1 (eV, an electron volt, is the energy
acquired by a single electron accelerating through a potential difference of 1 volt and is
equal to about 1.6 ë 10−19 J).
The velocity of light (c) is 2.998 ë 108 m s−1.

11.2 INTER ACTIONS BET WEEN R ADIATION AND MAT TER 259
Increasing energy
Increasing wavelength
10 nm 0.1 cm
0.0001 nm 0.01 nm 1000 nm 0.01 cm 1m 100 m
Gamma Ultra- Microwaves

X-rays Infrared Radio waves
rays violet
Visible
430
490
530
580
630
Orange
Yellow
Green
Violet
Extreme Far Near Near Middle Far

Blue
Red
UV UV UV IR IR IR
10 200 300 400 500 600 700 800 900 1000 1500 6000 40000
Wavelength/nm
FIGURE 11.2
A portion of the electromagnetic radiation spectrum.
energy resembles the energy of a spring. Think of squeezing a spring, the more you compress
it, which gives it energy, the greater its tendency to jump back to its original, resting length.
The full range of electromagnetic radiation is often called the electromagnetic radiation
spectrum. It is often subdivided or classified in to a number of types of radiations depending
upon their wavelengths (or frequencies) as shown in Figure 11.2.
Interactions between radiation

11.2
and matter
When radiation of a particular wavelength interacts with matter, the radiation may be scat-
tered or it may be absorbed. Scattering, often referred to as Rayleigh scattering, occurs when
a photon collides with an atom or molecule, and its direction of propagation is changed. Cross reference
However, its wavelength and frequency, and therefore energy, are not usually altered. Turbidity You can read about gel
and nephelometry depend on the scattering of light and are briefly described in Section 11.6. electrophoresis, analytical
Light scattering can be used to determine the Mr of macromolecules in solution or the sizes gel filtration, and analytical
of larger suspended particles, such as those of viruses. This technique has been superseded by microultracentrifuges in
simpler, more convenient methods, such as gel electrophoresis and analytical gel filtration or Chapters 8, 12, 13, and 14
respectively.
by using analytical microultracentrifuges.
The scattering of radiation is also the basis of a number of techniques used to investigate
the structures of particles of clinical and biological interest. These techniques include X-ray
diffraction, electron microscopy, and neutron scattering. A discussion of these techniques is,
however, outside the scope of this chapter.
260 11 SPECTROSCOPY
If a photon of radiation is absorbed by matter, energy is transferred from it to the atom or mol-
ecule. This means that the electronic state of the atoms or molecules is affected; their energy is
increased from a ground state to an excited one and the atom or molecule is described as being
excited. The atom, molecule, or even part of a molecule that becomes excited by absorp-
tion is often called a chromophore. The excitation energy is usually dissipated as heat when
the excited atom or molecule collides with, for example, a solvent molecule. However, some
molecules can lose the energy by emitting a photon of lower energy in a process called pho-
toluminescence. If the photon is emitted virtually instantaneously, this is called fluorescence.
Phosphorescence differs from fluorescence in that the phosphorescent material continues to
emit light after removal of the source of excitation, although this slowly diminishes. By contrast,
fluorescent materials fluoresce only while excited.
SELF-CHECK 11.1
In fluorescence, which of the photons, absorbed or emitted, has the shorter wavelength?
11.3 Radiation, particles, and quanta

Electromagnetic radiation is often difficult to describe in general everyday terms. This is
because it cannot be described solely in terms of the properties of waves, but neither does
it possess only the properties of particles. Instead it has attributes of both. Some of the ways
in which radiation interacts with matter, for example, the scattering of light, are most easily
described by assuming it is a wave. However, other aspects, such as the absorption of light, are
more easily envisaged if radiation is regarded as a particle.
The frequency of radiation is related to its energy content by Planck’s constant (6.63 × 10−34 J s−1),
as described in Section 11.1. The introduction of Planck’s constant means that a specific frequency
or wavelength has a discrete and fixed amount of associated energy. Equally, when an atom or
molecule absorb or emits energy, the amount of energy absorbed or released can only be of
discrete or fixed amounts. These ‘amounts’ are called quanta (singular quantum); a quantum is
the smallest individual quantity of energy. Hence, quanta occur in discrete, specific amounts, and
not as a continuous range of values. Furthermore, atoms and molecules can possess only specific
amounts of energy or be at certain ‘energy levels’; so they too are quantized.
For a photon to be absorbed, its energy must exactly match the difference in energy between
two energy levels in an atom or molecule. The discrete energy levels (quanta) that are associ-
ated with atoms and molecules are usually illustrated using an energy-level diagram like the
one in Figure 11.3. If you examine this figure you can see that the ground state is indicated
by the lower red line (I). However, in an excited state, the energy of an atom or molecule
has increased by a fixed amount or quantum. One such excited state is represented by the
upper red line (II). You can also see that within each of the electronic states [the ground (I)
and an excited (II)], there are different possible vibrational energies, which are represented
by the thinner blue coloured lines. Rotational energies also occur at values between those for
vibrations. Our diagram shows only a limited number of these as short green coloured lines;
otherwise it would become too cluttered!
A change between any two energy levels is called a transition. The vertical arrow in Figure 11.3
shows one of the many possible electronic transitions. You should note that a transition can
occur only if the atom or molecule absorbs an amount of energy exactly equal to that between
the two relevant states. This is indicated on the energy axis as ΔE. However, transitions
can occur not only between electronic states, but also between the various vibrational and
11.4 ABSORBANCE 261
Excited state (II)

Energy
Ground state (I)

ΔE
FIGURE 11.3
Schematic energy-level diagram from the ground state (I) to
the first excited state (II). The arrow shows a transition from the
Distance between electrons and nucleus ground state to the third vibrational level of the first excited
or between atoms in a molecule state, which requires an input of energy equal to DE.
rotational states. The sizes of the energy changes decrease from electronic through vibrational to
rotational and therefore the wavelengths of the photons required to promote them increase.
SELF-CHECK 11.2
Do the frequencies of photons (or electromagnetic radiation) increase or decrease with the
order of the following transitions: rotational, vibrational, and electronic?
When an excited molecule returns to its ground state, energy is lost and the transition has been
from a higher to lower energy level. The energy can be lost as photons, as in fluorescence or
phosphorescence (Section 11.7). When radiation is not emitted, however, the transition is called
non-radiative decay. Except in the cases of fluorescence or phosphorescence, radiation is only
emitted by materials at temperatures that are so high they would normally degrade molecules
of clinical or biological interest. Hence, in the biomedical sciences, spectrophotometry is virtu-
ally synonymous with measuring the absorption of radiation. Figure 11.2 indicates the relative
energies associated with different wavelengths. Each of these is used with different types of
spectroscopy. All of these different types of spectroscopy have different uses in clinical practice.
These include determining the absorbance, sometimes called optical density, of a substance or its
absorption spectra. We will discuss both of these analytical approaches in the following sections.
11.4 Absorbance
Absorbance is a means of quantifying how much light of a particular wavelength is absorbed
by a sample. In contrast, absorption spectra show the fraction of electromagnetic radiation
absorbed by a material over a range of different wavelengths, frequencies, or wave numbers
(see Box 11.1).
Spectroscopic techniques can be used to make quantitative measurements. For example, to
determine the amount of a substance (the analyte) that is present in a sample. The absorb-
ance of an analyte is proportional to its concentration (c) in a manner described by the Beer–
Lambert law. This law describes the absorbance of monochromatic light, which is one of a
single wavelength, as it passes through a dilute solution. The law states that the absorbance is
directly proportional to both the concentration of the solution and also to the length of the path
that the light takes through that solution.
262 11 SPECTROSCOPY
I0 (c) I
FIGURE 11.4 l/L

The absorbance of monochromatic light
in accordance with Beer–Lamberts law. A = εcl or acL
You can see an illustration of the law in Figure 11.4. Light of a specific wavelength and intensity
I0 is entering a sample of concentration c with a path length l or L. Light leaving it has an inten-
sity of I. If the sample does not absorb or scatter any of the light, then I will equal I0. If, however,
some of the light is absorbed by the sample then I will be less intense than I0, such that the
transmitted light (T) is equal to I/I0. This value is usually multiplied by 100 and expressed as a
percentage value. The logarithm of 1/T or I0/I (log (I0/I)) is the absorbance (A).
The Beer–Lambert law is summarized by the expression:
log (I0/I) = A = εcl
where ε is often referred to as the molar extinction coefficient.
Absorbances are logarithmic values and so do not have units. In traditional, non-SI units, l is
expressed in cm and the concentration in mol L−1 or M. This gives ε units of M−1 cm−1. However,
if SI units are used, then the equivalent Beer–Lambert law expression becomes:
log (I0/I) = A = acL
where the constant (a) is called the absorptivity, c is in mol m−3 and the path length (L) is in
m. This gives absorptivity units of m2 mol−1. Whatever system of units is used, the generally,
observed range of transmittance is from 0 to 100%. Absorbance values then vary from 0 to 2.
The absorbance of a given substance will vary according to the wavelength of the light to which it
is exposed. When using absorbance for quantitative analysis, however, the absorbance of a solu-
tion is normally determined at the wavelength at which the material being investigated absorbs
most strongly, that is where the absorbance is at its greatest. This wavelength is often designated
as Aλ. At a single wavelength, the absorbances of dilute solutions of a given analyte are directly
proportional to the concentration of analyte. This is illustrated in Figure 11.5: note how the graph
is only a straight line for lower concentrations in accordance with the Beer–Lambert law.
Absorbances are usually measured with the solution contained in specialized transparent
containers called cuvettes, which normally have a standard path length of 1 cm or 0.01 m.
This length can be assumed unless you are told otherwise. Disposable cuvettes made of
polymethacrylate and polystyrene are commercially available for use at wavelengths of
11.4 ABSORBANCE 263
Absorbance
FIGURE 11.5
Graphical illustration of
the Beer–Lambert Law.
Concentration See text for details.
280–800 and 350–800 nm, respectively. They therefore suffer the disadvantage that they can-
not be used to determine the absorbances of solutions of nucleic acids, which have λmax at
about 260 nm, and perform poorly with protein solutions that absorb maximally at about
280 nm. Reusable glass, quartz, and fused silica cuvettes are also manufactured. Again, glass
cuvettes cannot be used in the ultraviolet range; their lowest usable wavelength is 340 nm.
Cuvettes of quartz and fused silica are the most versatile and can be used throughout the
ultraviolet and visible ranges that is from about 200–800 nm.
Given the relationship between absorbance and concentration and because many chemi-
cals absorb light maximally at specific wavelengths, measuring the absorbance of a solution
of analyte is extremely useful, since it provide a means of determining its concentrations in
patient samples. In theory, if ε (or a) is known for a compound, then its concentration in a
sample can be determined simply by measuring its absorbance under the same conditions
for which ε (or a) was found. In practice, however, it is more usual to determine the absorb-
ance of that sample and also for a known concentration or a series of concentrations of the
analyte. The unknown concentration of an analyte can then be calculated from the ratios of
the absorbances given that the concentration of the standard is known. Alternatively, a graph
or standard curve of absorbance against the known concentration can be plotted from which
the unknown concentration can be obtained (Figure 11.6).
SELF-CHECK 11.3
Calculate the concentration of the analyte X, given that a 10-fold dilution of the specimen
gives an absorbance of 0.458 and a standard solution of the same analyte of concentration
50 μg cm−3 had an absorption of 0.625.
Spectrophotometers and colorimeters

Two types of spectrometers are typically encountered. These are single beam and double
beam instruments. Both types have a visible light source and either type may have a deute-
rium lamp that produces ultraviolet light so that absorbance at shorter wavelengths can also
be measured. You can see outline structures of each type of spectrophotometer in Figure
11.7 (a) and (b). In single beam instruments, a monochromator separates the beam into its
component wavelengths. Monochromators can be prisms or diffraction gratings [Figure 11.7 (c)],
but either type allow a monochromatic beam of relevant wavelength to pass through the
264 11 SPECTROSCOPY
2.0
1.5
B
A500
1.0
FIGURE 11.6
Standard or calibration curve, relating the concentration
of glucose to the absorbance at a wavelength of 500 nm
using a glucose oxidase-based test. Two serum samples, 0.5
A and B were analysed for glucose using the method at A
the same time. Sample A was from a healthy individual
and B from a poorly controlled diabetic. The absorbances
obtained for A and B were 0.30 and 1.40, which 0.0
correspond to concentrations of 7.0 and 35.0 mmol dm−3, 0 10 20 30 40 50
-3
respectively. [Glucose]/mmol dm
sample in a cuvette. The amount of light transmitted is measured to give the absorbance of
the solution. Single beam instruments must be ‘zeroed’ using a reference solution (‘the blank’)
in the cuvette. This solution contains all the components of the sample other than the analyte.
The reference is then replaced with the sample and its absorbance noted. The same cuvette
must be used for both solutions to avoid absorbance errors arising from variations in the trans-
parencies of different containers.
In double beam instruments, a monochromatic beam is, again, selected for use. However, it is
split into two beams of equal intensity using a half-mirrored device. One of these, called the ref-
erence beam, passes through a cuvette containing only the solvent. The other half-beam passes
through a second cuvette containing a solution of the sample compound being studied. The
intensities of these light beams are determined and electronically compared. The intensity of
the reference beam is automatically subtracted from that of the sample to give its absorbance.
Immunoassays, which we describe more fully in Chapter 15, are often performed in special-
ized containers that have 96 wells. These are generally referred to as 96-well plates. A single
assay is performed in each well. When the assays are completed and colour has developed,
the absorbance of each well in the plate is measured using a specialized spectrophotometer
called a plate reader.
The term colorimetry is often used when the absorbance of visible, and therefore coloured, light
is used. In this case, rather than using a spectrophotometer, which produces light with a lim-
ited range of wavelengths, a colorimeter is used. Colorimeters do not produce light of specific
wavelengths but use a series of filters that produce narrow bands of light in the visible spectrum.
Clinical applications of measuring absorbances

Spectrophotometers are incorporated in automated analysers in hospital laboratories and
used to measure a wide range of analytes colorimetrically. For example, serum creatinine con-
centrations are determined using the Jaffe’s reaction. Creatinine is a breakdown product of
11.4 ABSORBANCE 265
(a) Slit Slit

Output
device
Sample
Detector
Light source Monochromator
(b) Slit Slit
Light source
Sample
Detector
Monochromator Output
device
Slit Slit
Mirror
Reference
Detector
Monochromator
(c)
Slit Slit
Diffraction beam
FIGURE 11.7
(a) Single and (b) double
Incident beam beam spectrophotometers.
(c) Shows the use of a prism
and a diffraction grating as
monochromators. See text for
details.
creatine phosphate in muscle tissues and its concentration in serum is used to assess kidney
function because it is produced at a fairly constant rate in the body and is freely excreted by
the kidneys. Reference ranges for serum creatinine are typically 60–110 μmol dm−3 in men
and 45–90 μmol dm−3 in women. If the filtering mechanism in the kidneys declines, for exam-
ple, due to loss of nephrons as in renal failure, then concentrations of serum creatinine will Cross reference
increase above these ranges in proportion to the decline in function.
automation in Chapter 17
Creatinine reacts with picric acid in an alkaline solution to give an orange coloured product. After ‘Laboratory automation’.
an incubation of 15 minutes at room temperature to allow for colour development, the absorb- Chapters 1–3 of the companion
text, Clinical Biochemistry, will
ance is measured at a wavelength of 490–500 nm using the spectrophotometer. The absorbance
also provide useful material.
produced is proportional to the concentration of creatinine in the sample of serum.
266 11 SPECTROSCOPY
11.5 Absorption spectra

An absorption spectrum is a graph of absorbance against wavelength and shows the frac-
tion of incident electromagnetic radiation absorbed by a material over a range of different
wavelengths, frequencies, or wave numbers (see Box 11.1). In a relatively short time, a suitable
spectrophotometer can automatically determine absorbances, as described in the previous
section, but at a number of different wavelengths.
Different regions of the electromagnetic spectrum can be used to investigate different features
of an atom or molecule. Look at Figure 11.2. You can see that the higher energies of ultraviolet
(usually 200–400 nm) and visible lights (approximately 400–800 nm), collectively often abbre-
viated to UV–Vis or ‘standard’ absorption spectroscopy, are associated with electronic trans-
formations. Infrared spectroscopy uses radiation of 2500 to 16,000 nm, whose lower energies
promote vibrational transitions. Rotational transitions can occur with the longer wavelengths.
Microwave radiation (about 10–300 mm wavelengths) is used in nuclear magnetic resonance
spectroscopy (Section 11.8). Thus, by using electromagnetic radiation of different wavelengths
it is possible to investigate different aspects of the same substance, and gradually build up a
picture of its composition and structure. In essence, the absorption spectrum for a compound
constitutes a type of ‘fingerprint’ for it, which can be useful in identifying a specific analyte in
a sample. Thus, absorption spectra, particularly those associated with longer wavelengths of
the infrared and microwave regions, can be used to help identify specific analytes in samples.
We will now consider a number of these techniques in turn.
Ultraviolet–visible absorption spectrophotometry

Many compounds absorb electromagnetic radiation of specific wavelengths. Many colourless
compounds of clinical or biological interest, such as proteins, nucleic acids, purines, pyrimidines,
and some drugs, vitamins, and amino acids absorb non-visible, ultraviolet light (Figure 11.2). This
is of help in identifying those wavelengths most appropriate to use in measuring absorbances to
determine the concentration of an analyte (Figure 11.8).
SELF-CHECK 11.4
A partial absorption spectrum of oxyhaemoglobin is given in Figure 11.8 (a). What would be
suitable wavelength(s) to measure its concentration?
Infrared spectroscopy
Infrared (IR) spectroscopy is a useful analytical tool in identifying or partially identifying an
analyte. In principle, it is not different from UV–Vis spectroscopy. However, the energies of
photons in the IR region of the spectrum vary from approximately 0.25 to about 3.755 J mol−1.
Unlike ultraviolet and visible light this is insufficient to produce electronic transitions. Rather,
the absorption of lower energy IR light excites covalently bonded atoms and organic func-
tional groups in molecules resulting in vibrational and rotational transitions. Such excitation is
possible because the covalent bonds in molecules do not behave like rigid, non-elastic struc-
tures, but more like springs that can be stretched and bent.
Thus molecules of virtually all organic compounds can show a large number of vibrational
motions, which are characteristic of the atoms or groups of atoms present. This means infrared
11.5 ABSORPTION SPECTR A 267
(a)
Oxyhaemoglobin
Relative absorbance
Deoxyhaemoglobin
500 520 540 560 580 600 620 640 660

Wavelength/nm
(b)
Vitamin B12
Vitamin B6
Absorbance
Thiamine
Riboflavin
200 250 300 350 400

Wavelength/nm
FIGURE 11.8
Ultraviolet–visible absorption spectra of (a) deoxyhaemoglobin and oxyhaemoglobin and
(b) for a number of vitamins.
spectroscopy is especially useful for analysing organic compounds because the different func-
tional chemical groups present absorb IR at characteristic wavelengths. We have listed the char-
acteristic absorption wave number values of some functional groups in Table 11.1. Thus, many
of the functional groups in the molecules of an analyte can be relatively easily identified.
Absorption by these groups gives rise to seemingly rather complex spectra when compared
with those of the UV–Vis range. You can see what we mean if you examine Figures 11.8 and
11.9. Look at the relative simplicity of the UV–Vis spectrum of the complex protein haemo-
globin shown in Figure 11.8 (a) compared with the complex IR spectra of the structurally much
simpler compounds shown in Figure 11.9. These spectra are typical of IR absorption spectra in
that the absorption is expressed as percent transmittance and wavelengths are given as wave
numbers (Box 11.1). However, as we noted above, the very complexity of IR spectra means
268 11 SPECTROSCOPY
TABLE 11.1 Values of some infrared radiation wavelengths characteristically absorbed

by some chemical groups
Bond Wave no.r/cm−1 Chemical group/notes

Single bonds
C–H 2850–3000 Saturated alkane; presence too numerous to be

of diagnostic value
C–C variable Presence too numerous to be of diagnostic value
C–N; C–O 1000–1400 Overlap makes assigning signals difficult
C–Cl 700–800
O–H 3200–3550 Alcohols and phenols; hydrogen bonding will

broaden peaks
N–H 3100–3500 Amines; primary amines give several signals,

secondary give one peak, tertiary no peaks
P–H 2275–2440 Phosphorus compounds
Double bonds
C=C 1600–1675 Often a weak signal
C=N 1630–1690 Overlaps with other signals
C=O 1715–1750 Esters
C=O 1680–1740 Aldehydes
C=O 1665–1725 Ketones
C=O 1670–1720 Carboxylic acids
C=O 1630–1695 Amides
N=O 1300–1370 Nitro compounds

1450–1570
P=O 1140–1300 Phosphorus compounds
Triple bonds
C≡C 2120–2260 Alkynes; weak bands
C≡N 2210–2260 Nitriles
they provide a ‘fingerprint’ of an analyte that can be extremely useful in identifying, or at least
partially identifying, what it is.
Infrared spectra can be obtained from gaseous, liquid, or solid samples. Glass cuvettes can-
not be used since glass absorbs IR light. Instead, samples are usually examined as thin films
sandwiched between two polished salt plates, which are transparent to IR light. Liquids can
be applied as films directly between the plates. Solid samples can be examined following their
incorporation into thin potassium bromide disks prepared under high pressure. They can
be mixed with a solvent and ground to a fine paste, which is spread between the plates. In
some cases IR spectra can be obtained directly from finely powdered preparations of samples,
although the resolution is sometimes poorer than using salt plates (Figure 11.10 (a) and (b)).
(a) 72 CH3
H3C CH3
70 COOH
68
66
CH3
64
Transmittance/%
62
60
58
56
54
52
50
48
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber/cm−1
(b) 60
55
50
45
Transmittance/%
40
35
30
H
25
N CH3
20
15 O
HO
10 FIGURE 11.9
4000 3500 3000 2500 2000 1500 1000 500 Infrared spectra of (a) retinoic
Wavenumber/cm−1 acid and (b) paracetamol.
SELF-CHECK 11.5
Identify the chemical group that is the one most likely to have produced the trough at
1690 cm−1 in the infrared absorption spectrum of retinoic acid given in Figure 11.9. Table 11.1
will be of use!
Macromolecules, such as proteins, can dissolve in volatile solvents; the resulting solution can
then be spread as a thin film on one of the plates. Evaporation of the solvent then leaves a
thin film of protein molecules on the plate for analysis. Unfortunately, organic solvents tend
to denature proteins.
If a solvent is used to dissolve solids, care must be exercised in its choice; the solvents
chosen should not absorb IR light in regions of the spectrum that may be of analytical
use. Hence, water cannot be used because it absorbs strongly in the 1600 and 3400 cm−1
regions, also it is normally present in high concentrations in samples and would dissolve
the salt plates. Commonly used solvents include carbon tetrachloride, chloroform, and
tetrachloroethene.
270 11 SPECTROSCOPY
(a) 52 CH3
50 OH
48 CH3
46
44 H C O
3
42
Transmittance/%
40
38
36
34
32
30
28
26
24
22
20
18
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber/cm−1
(b) 103.5
103.0 CH3
OH
102.5 CH3
102.0 H3C O
Transmittance/%
101.5
101.0
100.5
100.0
FIGURE 11.10 99.5
Infrared spectra of (a) ibuprofen
in KBr plates and (b) obtained 99.0
directly using a powdered 4000 3500 3000 2500 2000 1500 1000 500
tablet. Wavenumber/cm−1
Raman spectroscopy
The difficulties associated with obtaining IR spectra in water can be overcome using Raman
spectroscopy, which also probes the vibrational energy levels of atoms and molecules. You can
see an outline of a Raman spectrophotometer in Figure 11.11.
The incident, monochromatic, light used in Raman spectroscopy is chosen so that its wave-
length is not absorbed by the sample; hence, most of it is simply transmitted. However, a
relatively small proportion of the light will be scattered and this can be viewed at an angle of
90° to the sample. It was noted in Section 11.6, that scattering does not in general alter the
frequency of the exciting light. However, if an exceedingly small proportion of the energy of an
exciting photon is transferred to a sample molecule, it can lead to a transition from one vibra-
tional state to another. Hence, a small proportion of the scattered light will have its frequency
Mirror
Laser
Curved
focusing
Sample
mirror
Mirror to reflect light

back through sample, Monochromator
effectively doubling
the intensity of the
scattered light
Detector
Output
FIGURE 11.11
device Outline of a Raman
spectrophotometer. See main
text for an explanation of its
operation.
decreased due to its involvement in the vibrational transitions. The net result of such changes
in frequencies means that a series of vibrational transitions can be observed as a collection
of spectral lines in the scattered light collected at 90° to the sample. These spectral lines form
the Raman spectrum.
Given that the information gained in Raman spectroscopy is from vibrational transitions, it is
similar to that obtained by IR spectroscopy. However, it differs in that the information is inde-
pendent of the frequency used to excite the atoms/molecules. Thus, any wavelength can be
used to produce a Raman spectrum. However, because only a small proportion of the light is
absorbed by vibrational transitions, a high intensity light source is needed; typically a laser is
used. Thus, Raman spectroscopy is carried out with a dedicated, specialized spectrophotom-
eter. The sample must also be highly concentrated. In practice, a 2% solution is usually used,
which can lead to problems with molecules aggregating or precipitating.
Clinical applications of infrared and Raman spectroscopy

Infrared and Raman spectroscopies, often coupled with gas liquid chromatography (GLC,
Chapter 13), have been used to study drugs such as the various derivatives of penicillin and
amphetamines. Amphetamines are a group of structurally similar molecules that are difficult
to differentiate. However, amphetamines in samples can be identified using differences in
their infrared spectra using GLC–IR. Infrared spectroscopy is used to monitor pollutants in the
environment, particularly harmful chemicals in the air. These include measurement of toxic
gases, such as carbon monoxide in garages and formaldehyde produced during manufacture
of plastics. There is considerable interest in the use of infrared spectroscopy as a non-invasive
272 11 SPECTROSCOPY
technique to determine the concentrations of glucose in samples of blood from diabetic

Cross reference
patients. The blood glucose is quantified by exposing a region of the skin to infrared radiation
You can read about
chromatography in Chapter 13. and analysing the resultant spectrum.
11.6 Light scattering methods

The two main experimental techniques encountered in the clinical laboratory based on the
scattering of light are turbidity and nephelometry.
Turbidity is the degree of opacity or cloudiness of a fluid caused by particles suspended in

it, which causes a beam of light passing through the suspension to scatter. An Increase in
turbidity reduces the amount of light that can penetrate the suspension, i.e. its transmittance
is decreased. The amount of light that is scattered depends on a number of factors including
the size and shape of the particles, and their concentration. Turbidimetry is the measurement
of turbidity. It relies on the use of a spectrophotometer, which can be used to determine the
concentration of the suspension of particles in a turbid sample. The particles are often bacte-
rial cells or precipitated protein. A beam of light is passed through the suspension. Some of
the light is absorbed, reflected, and scattered. However, the rest is transmitted through the
solution and measured by the spectrophotometer and used to determine the concentration
of particles. Turbidity (Tb) can be defined mathematically as:
I = I0e−lT or, more simply, Tb = 2.303 × 1/l × log (l0/l)
where I0 and I are the intensities of incident light and transmitted beams of light, and l is the
length of the light path. This relationship only holds for relatively finely dispersed suspensions
of absorbances less than 0.3–0.5.
Turbidimetric measurements have some clinical uses. For example, they can be used to deter-
mine the number of bacterial cells in microbial suspensions. In some clinical disorders, for
example nephrotic syndrome, proteins are lost in the urine. Determining their presence and
concentration therefore has medical value. The urine samples are treated with sulphosalicylic
acid, which causes any protein present to precipitate as a fine dispersion. The amount of pre-
cipitation is measured by its turbidity and is proportional to the protein concentration. The
technique is simple and rapid, but has the disadvantages of lacking sensitivity and precision.
SELF-CHECK 11.6
A suspension of Eschericia coli cells of dry weight 0.4 g dm−3 give an absorbance value of 0.8.
Calculate its corresponding turbidity (Tb) value.
Nephelometry (from the Greek nephele meaning cloud) is a technique in which the intensity
of light scattered by a suspension is measured at an angle to the incident beam of light, rather
than using the transmitted light to determine the concentration of suspended particles. Thus,
unlike turbidity, nephelometry is not measured in a spectrophotometer, but uses a nephelom-
eter (Figure 11.12). Nephelometry resembles turbidity, but has the advantage of being more
sensitive.
The amount of scattered light depends on the size of the particles and their concentration.
Thus, for example, the concentration of protein in clinical samples can be determined by com-
parison with known concentrations. One clinical application of nephelometry is to measure
the concentration of rheumatoid factor (RF) in samples of serum. Rheumatoid arthritis is an
extremely painful and debilitating chronic condition, which is characterized by symmetrical
11.7 FLUORESCENCE AND FLUORIMETRY 273
Sample
90°
Light source scattering Cross reference
angle You can read more about the
clinical uses of turbidity and
Detector nephelometry in Chapter 15
Output
device FIGURE 11.12
Schematic illustrating measurement
using nephelometry. See text for an
outline of the method.
arthritis, radiological changes to the bone and the presence of autoantibodies, the RF, in the
plasma of patients. Rheumatoid factor are immunoglobulins that recognizes and bind to anti-
genic sites at specific positions of IgG antibodies. The presence of high levels of RF in patients
is associated with a poor prognosis.
11.7 Fluorescence and fluorimetry

Fluorescence was introduced in Section 11.2. To recap briefly, when most molecules absorb
electromagnetic radiation, the electrons move to a higher energy level before falling back
to their ground state, generally releasing energy in the form of heat in doing so. However, in
some molecules only part of the energy is lost as heat and the rest is emitted as fluorescent
light. The wavelength of this light (λem) is longer than that used to excite the molecule (λex)
because as we noted above, the excited molecules lose some of the absorbed energy as heat.
The difference between the two wavelengths is known as the Stokes shift.
A fluorimeter or fluorescence spectrophotometer is an instrument used to measure the fluo-

rescence of a given substance. Its components are outlined in Figure 11.13. The source of light
produces high energy radiation of short wavelength. This passes through the first monochro-
mator, which can be used to select an excitation beam of appropriate wavelength that passes
through the cuvette. Following its excitation, the sample in the cuvette emits fluorescent radia-
tion in all directions. The second monochromator, which is placed at right angles to the cuvette,
only allows emitted light that is of the fluorescent wavelength to fall onto the detector and be
measured. The detector produces an electrical signal that is proportional to the intensity of the
fluorescent light, which in turn is proportional to the concentration of the sample.
Fluorescence has two principal advantages over the use of visible or UV spectrophotometry.
First, it is more sensitive, indeed this can be up to a 1000-fold greater. Secondly, it is also more
specific as two wavelengths are used to produce a signal not one as in spectrophotometry.
However, there are also a number of disadvantages to using fluorimetric techniques. They
are sensitive to changes in pH and the presence of contaminating molecules in the solution.
Furthermore, not all compounds generate fluorescence, although it may be possible to couple
non-fluorescent compounds with those that do exhibit fluorescence. For example, proteins
can be reacted with fluorescamine to produce a fluorescently labelled complex, which allows
274 11 SPECTROSCOPY
Mirrors
Grating
Reference
Slit Slit Sample detector
Excitation Beam
Mirrors beam divider
Emitted fluorescent
Monochromator
beam
Light
source
Monochromator
FIGURE 11.13 Output

Outline of a fluorimeter. device
The main text describes
the operational basis of the Fluorescence
instrument. detector
the concentrations of minute amounts of them to be quantified. Fluorescent groups can also
Cross reference
be coupled to immunoglobulins and this is the basis of immunofluorescence assays, which are
Chapter 15 ‘Immunological
techniques’. described in Chapter 15.
Sometimes, the emitted fluorescence is less than that expected. This phenomenon is called
quenching and occurs when substances in the sample absorb the emitted fluorescence or
when molecules such as oxygen in the sample interfere with energy transfer.
Fluorimetry can be used to measure the concentration of phenylalanine in samples of serum

from newborn infants in screening for phenylketonuria (PKU). This condition is characterized
by excessive concentrations of phenylalanine in the plasma and is associated with mental retar-
dation in untreated children. Phenylalanine is reacted with ninhydrin in the presence of the
dipeptide leucyl-alanine to give a product whose fluorescence is proportional to the concen-
tration of phenylalanine. The method is calibrated using known standards of phenylalanine.
Nuclear magnetic resonance

11.8
spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy provides information on the position of
specific atoms within a molecule by exploiting the magnetic properties of certain nuclei.
Nuclei with odd masses or odd atomic numbers, for example 1H and 13C, have a magnetic
moment, also called spin or angular moment. The chemical environment that is the sur-
rounding atoms, of these types of nuclei can be determined using NMR spectroscopy. This
information can then be used to identify chemical functional groups in a molecule, identify
some compounds in samples and even construct models of the three-dimensional structures
of compounds. Most NMR studies in the biological and biomedical sciences exploit the spin
of 1H (1H-NMR) or 13C (13C-NMR), since these elements are common in organic molecules.
11.8 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 275
However, a number of other magnetic isotopes are suitable for NMR studies. You can see TABLE 11.2 Examples
some examples in Table 11.2. of isotopes with odd
masses or odd atomic
SELF-CHECK 11.7 numbers with magnetic
One isotope of carbon, 12C, is used extensively in biological investigations despite being properties making them
nonmagnetic. Can it be used in NMR spectroscopy? amenable to analysis
by nuclear magnetic
resonance
Nuclei can be regarded as moving charged particles and so have associated magnetic fields;
in effect they resemble simple bar magnets. In the absence of an external magnetic field, the Nuclei
spins of the atoms are randomly orientated (Figure 11.14 (a)). However, when a magnetic field 1
H, 2H
is applied the atoms become orientated in a direction that is parallel to that of the applied field
13
(Figure 11.14 (b)). The spins, however, can be aligned with the field (α orientation) or ‘point’ in C
the opposite direction (β orientation). 14
N, 15N
Those nuclei with their spins aligned with the field have a lower energy than those with the 17
O
opposite orientation; they therefore predominate. However, the energy difference between
the two orientations is not large and an equilibrium distribution occurs in which the α:β ratio 19
F
is approximately 1.00001:1.00000. You can see this situation illustrated diagrammatically in 23
Na
Figure 11.15 (a). Equilibrium is disrupted if pulses of radio frequency (RF) electromagnetic
radiation of a certain frequency are absorbed by a nucleus. Specifically, the absorption of 25
Mg
the RF radiation by nuclei with low-energy spins (α) changes their orientation to that of 31
P
the higher-energy spin (β) state. The absorptions are often called resonances because the
33
S
39
(a) K
+
+
+
+
+
+
No applied magnetic field
(b)
+ +
+ +
H0
+ + FIGURE 11.14
(a) The spins of atoms are randomly
orientated unless (b) they are subjected
Magnetic field to an applied magnetic field (H0).
276 11 SPECTROSCOPY
(a)
High energy
Ground state
(b)
High energy
FIGURE 11.15
(a) Shows the non-random alignment of
nuclei in the magnetic field H0, using arrows
to represent the direction of spin. (b) Pulses Transition
Spin flip
of radiation in the RF region of the
H0
electromagnetic spectrum are used to
reverse the alignment of nuclear spins from
the low energy spin aligned state (`) to the
opposite higher energy spin state (a). The Low energy
energy required for this transition depends
on the strength of the applied magnetic field. hv
frequency of the RF radiation matches that at which the nuclei spin. The exact resonance or
frequency of any given magnetic nucleus depends upon its exact chemical environment. This
enables NMR spectroscopy to characterize the structures of chemical compounds. When the
nuclei revert back to the equilibrium distribution, that is when those nuclei that have been
excited to a high-energy spin state relax to their original low-energy state, RF radiation is emit-
ted, and can be detected and measured. Nuclear magnetic resonance spectra are graphs of
intensity against the RF applied.
Figure 11.16 shows the basic components of an NMR spectrophotometer. The sample is
placed in the magnetic field and excited by pulses in the RF input circuit. The realignments of
the magnetic nuclei in the sample induces a radio signal in the output circuit, which generates
a complex output signal. The exciting pulse is repeated as many times as necessary to allow
the signal output to be distinguished from any background noise. A mathematical (Fourier)
analysis of the output produces the NMR spectrum.
In practice, the resonance frequencies are converted to chemical shifts (δ) to allow results
obtained in different experimental conditions to be easily compared. Chemical shifts are
defined as the resonance frequency associated with the sample compared with that of a refer-
ence compound whose chemical shift is defined as 0, using the expression:
Chemical shift (δ) = (Vsignal − Vreference) × 106/Vreference
You can see from this equation that the scale of the chemical shift is unitless. The multiplica-
tion by 106 is simply for the convenience of being able to express chemical shifts as parts per
million (ppm). Tetramethylsilane [TMS, Si(CH3)4] is often the reference compound of choice
11.8 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 277
H0
Radio frequency
Radio frequency input output
FIGURE 11.16
Basic arrangement of a
nuclear magnetic resonance
(NMR) spectrophotometer.
although other substances, for examples 3-trimethylsilyl 1-propane sulphonate (DSS), are
used. Whatever the standard, its nature must be specified. You can see examples of NMR
spectra in Figure 11.17 (a) and (b).
The frequencies and duration of the applied RF pulses may be varied in an enormous number
of combinations allowing different molecular properties of the sample to be investigated.
The exact frequency emitted by each nucleus depends upon its molecular environment.
For example, protons in methyl groups (–CH3) have chemical shifts of about 1–2 ppm, while
those in aromatic rings have values of approximately 7–8 ppm. Thus, the frequencies differ
for each nucleus unless they are chemically equivalent and in identical molecular environ-
ments. One-dimensional NMR spectra, like those in Figure 11.17, give valuable informa-
tion about the structures of the molecules since each of the chemical shifts arises from a
particular chemical functional group (Figure 11.18). Also, like the spectra obtained from IR
spectroscopy (Section 11.5) the pattern of signals can act like a ‘fingerprint’ and help identify
a particular sample.
SELF-CHECK 11.8
How many peaks would you expect to see in NMR spectra arising from the hydrogen atoms of
(a) methyl bromide (CH3Br) and (b) methyl ethanoate (CH3COOCH3)? Explain your answer.
In theory, it should be possible to obtain chemical shifts for all the hydrogen atoms present
in a molecule and, therefore, obtain a one-dimensional NMR spectrum for any molecule. In
practice, however, this is not possible for complex molecules such as those of polypeptides
and nucleic acids. These molecules contain many hydrogen atoms and therefore the differ-
ences in their chemical shifts are smaller than the resolving power of the technique. In other
278 11 SPECTROSCOPY
(a) (b)
300 300
200 200
Signal
Signal
100 100
0 0
6.0 5.0 4.0 3.0 2.0 1.0 0 6.0 5.0 4.0 3.0 2.0 1.0 0
ppm ppm
FIGURE 11.17
Nuclear magnetic spectra for (a) methanol and (b) ethanol.
words, the NMR signal is obscured by the presence of so many hydrogen atoms making it
impossible to identify the signal from any one specific atom. Even using extremely strong
magnets to increase the frequencies of the radio waves required to change the spin states of
the nuclei, which increases the energy of the waves and, therefore, the resolving power of
the technique, does not improve the situation. However, since the 1980s a variety of two-
dimensional NMR spectroscopy techniques have been developed, which use a sequence of
pulses separated by different time intervals, rather than the single burst associated with one-
dimensional NMR. These techniques produce additional peaks that arise from the interac-
tions of protons that are less than 0.5 nm apart in the molecules. This sort of data, together
with other structural information, such as the lengths and angles of covalent bonds, allows
the three-dimensional structures of complex molecules, such as those of relatively large pro-
teins, to be predicted.
–OH –NH
12 11 10 9 8 7 6 5 4 3 2 1 0 ppm
CH2–Ar
RCO2H RCHO Ar–H C=C–H CH2–X CH2–N CH3
CH2–O CH2–S CH2
FIGURE 11.18
H CH
Typical values of chemical shifts for
CH2=O
protons influenced by a single chemical
functional group. CH2=C
11.9 MASS SPECTROMETRY 279
Clinical applications of nuclear magnetic

resonance spectroscopy
Magnetic resonance spectroscopy (MRS) is a non-invasive technique used to investigate
neurological disorders such as epilepsy, brain tumours, Alzheimer’s disease, and strokes. This
technique produces a spectrum or graph that identifies the types of chemicals in the brain
(or other organs) and quantifies the amounts of each. The information provided is a useful
indicator of the metabolic processes and biochemical changes occurring in diseased tissues.
In contrast, nuclear magnetic resonance imaging (MRI, Box 11.2) provides a picture of the
anatomical changes that occur in disease conditions.
11.9 Mass spectrometry

Mass spectrometry (MS) is a technique that allows the molecular masses of analytes to be
accurately determined on the basis of their mass (m) to charge (z) ratios (m/z). Analytes must
BOX 11.2 Nuclear magnetic resonance imaging
Nuclear magnetic resonance imaging (MRI) is a medical X-ray techniques may increase the likelihood of developing
imaging technique used to examine structures and func- a malignancy particularly in foetuses. Furthermore, the reso-
tions of the body. The technique uses the nuclear magnetic lution provided by MRI is as good as that provided by CT, but
resonance of protons in the water of healthy or diseased offers better contrast resolution, i.e. the ability to distinguish
portions of the body to produce a signal that reflects the between two similar but not identical tissues.
proton density. Since different tissues of the body contain
different amounts of water, a computer can interpret the
data in the signal to produce an image of the area being
examined. The images are of higher resolution than those
provided by other techniques such as X-ray photographs,
computed tomography (CT), and positron emission tom-
ography (PET) scans and the technique has the additional
advantage of being non-invasive.
Magnetic resonance imaging is particularly applicable to

viewing soft tissues areas, such as the spinal cord and the
brain (Figure 11.19) and for cardiovascular, musculoskele-
tal, and oncological investigations. Nuclear magnetic reso-
nance imaging can differentiate between pathological and
normal, healthy tissues. For example, MRI is used to pro-
duce pictures of the blood vessel supplying the brain, neck,
thoracic, and abdominal aorta, renal system, and legs. This
information can be of use when investigating arteries for
any abnormal narrowing or stenosis, or dilations of vessel
walls or aneurysms. The technique also can be used to FIGURE 11.19
accurately identify the location, shape, and orientation of a Nuclear magnetic resonance image of a head. The
tumour in a patient prior to radiation therapy. MRI scan shows much more detail than an X-ray
A major advantage of MRI is that it is harmless to the patient investigation. Courtesy of Dr G. Williams, Wolfson
because it uses strong magnetic fields and non-ionizing radi- Brain Imaging Centre, Cambridge, UK.
ation. In contrast, the ionizing radiation utilized in traditional
280 11 SPECTROSCOPY
be ionized to allow their masses and charges to be determined by passing them through elec-
tric or magnetic fields in a mass spectrometer. Molecular mass is usually expressed in daltons
(Da); one Da is approximately equivalent to the mass of a hydrogen atom, or proton, or neu-
tron. Thus, the mass in Da is numerically equal to the Mr. Mass spectrometry can be used
to determine the Mr of compounds ranging in size from a few hundred, as would occur in
pharmaceutical drugs, to several hundred thousand, such as those of macromolecular com-
plexes. Mass spectrometry can also provide information regarding chemical structures and the
primary structures of peptides and proteins. Both pure samples and complex mixtures from
organic and inorganic sources can be subjected to mass spectrometric analyses. Thus, MS is
a powerful technique, with a range of applications in the biomedical and biological sciences.
Here, we can give only an overview of the general principles of MS and describe some of the
techniques developed since the 1990s and their applications to biomedical science.
Mass spectrometers
Mass spectrometers generally consist of three parts: the ionizer or source region, the analyser,
and the detector (Figure 11.20 (a) and (b)). Each part is held at high vacuum, of between 10−2 and
(a) Recorder
Sample Ionizer Analyser Detector
Vacuum system
(b) Ion Ion Ion Ion Ion

formation acceleration focusing separation detection
Ion source Analyser Detector
Ions: each symbol

Indicates voltage Indicates focusing
represents a different
difference lens system
mass
FIGURE 11.20
(a) Schematic showing the general organization of a mass spectrometer. (b) Overview of the
operation of a mass spectrometer. An electric potential difference is used to accelerate ions
through the system.
10−10 Pa, to limit collisions between analyte and residual molecules of air, which could reduce
the accuracy of the analysis or even cause compounds to fragment. Typically subfemtomole
(<10−15) to low picomole (10−12) amounts of sample are introduced into the mass spectrom-
eter through a staged vacuum lock. Atoms or molecules in the sample need to be ionized by
adding a H+ to them to form a positive ion (M + H+) or removing one to give a negative ion
(M − H−). Ionization occurs in the source region using one of a number of different techniques.
Samples can be liquids, as with electrospray ionization (ESI), where they are dispersed into
the gas phase using high voltage electric currents. Alternatively, solid samples can be mixed
with a UV-absorbing matrix compound and irradiated using a UV laser. This forms sample ions
in a gas phase, as is the case with matrix-assisted laser desorption ionization (MALDI). Less
commonly used ionization techniques are electron ionization (EI), chemical ionization (CI),
and fast atom bombardment (FAB). In EI, an electron beam is passed through the vaporized
sample causing it to ionize. Samples can be chemically ionized by introducing a gas, such as
methane, ammonia, or isobutene, in the source region and allowing it to collide with mol-
ecules of the analyte. In FAB, the analyte is mixed with a non-volatile matrix and then ionized
by bombarding it with a beam of high-energy atoms, typically of an inert gas, for example
argon or xenon. You can see the different uses and some applications of these ionization
techniques in Table 11.3.
Whatever the method used, once ionized, the application of a potential difference is used to
transfer the sample into the analyser as you can see in Figure 11.20 (b). The analyser uses static
or dynamic electric or magnetic fields to separate the ions produced from the different com-
ponents of the initial sample. Separation of the ions occurs because each has a characteristic,
but different movement through the analyser, which depends upon its mass.
TABLE 11.3 Ionization techniques used in mass spectrometry
Ionization method Analytes Sample introduction Mass range/Da

method
Electron impact (EI) Small molecules GC 1–1000
volatile Solid
Liquid
Chemical Small molecules GC 1–1000

ionization (CI) volatile Solid
Liquid
Fast atom Carbohydrates Solid/liquid dissolved 1–6000

bombardment (FAB) Organometallics in glycerol
Peptides
Non-volatile
Electrospray Small molecules Liquid 1–2,000,000

ionization (ESI) Proteins/peptides HPLC
Nucleotides
Carbohydrates
Matrix-assisted laser Small molecules Solid/liquid mixed with 1–5,000,000

desorption ionization Proteins/peptides a solid or liquid matrix
(MALDI) Nucleotides
Carbohydrates
282 11 SPECTROSCOPY
TABLE 11.4 Common mass analysers used in mass spectroscopy
Analyser Capability
Sector (magnetic/electrostatic) High resolution, exact mass
Quadrupole (Q) Unit mass resolution, fast scan, low cost
Time-of-flight (TOF) Wide mass range, high throughput
Quadupole ion-trap (QIT) MS/MS and MSn capability
Ion cyclotron resonance (ICR) Very high resolution, exact mass
The most common types of analysers used for biological applications are listed in Table 11.4;
each type has advantages for different applications. A quadrupole (Q) MS consists of four
(hence, quad) parallel circular rods. Oscillating electric fields are applied to opposing rod pairs,
which causes the ions to separate in the quadrupole based on differences in the stabilities of
their trajectories between the rods. In time-of-flight (TOF) analysers, electric fields are used
to accelerate the ions to the same kinetic energy, so that the velocity of the ion over the flight
path is dependent on its m/z ratio. Thus, the time to reach the detector also varies with the
m/z ratio. Since all the particles travel over the same known distance to the detector, the times
required for each particle to reach it can be measured allowing the Mr of the particle to be
determined.
Quadrupole ion-trap (QIT) analysers are composed of three hyperbolic electrodes: a ring elec-
trode and two end cap electrodes. The application of a potential difference to these electrodes,
traps the ions in stable, oscillating trajectories within the cavity formed by the electrodes.
These trajectories are governed by the applied voltage and the m/z ratio of the ions. Altering
the voltage allows the ions to be ejected from the trap in order of their increasing m/z ratios,
thus allowing the Mr to be determined. Once the ions are separated by the analyser, they are
directed towards the detector again because of the applied electric field, which you can see
in Figure 11.20 (b). The current produced by the ion impacting on, or passing through the
detector surface, is recorded. This value is converted into an m/z value and a mass spectrum,
typically a graph of m/z against ion signal intensity, produced as you can see in Figure 11.21.
1570.68
100
90
80
70
Intensity/%
60
50
40
FIGURE 11.21 30
Mass spectrum of the peptide with the sequence 20
EGVNDNEEGFFSAR, where each letter stands for its 10
usual amino acid residue, and of Mr 1569.65. The 0
value for the ionized peptide (M+H)+ was found to 1500 1520 1540 1560 1580 1600 1620 1640 1660
be 1670.68. m/z
Recorder
CID
Sample Ionizer Analyser Analyser Detector
cell
FIGURE 11.22
Vacuum system Schematic showing the
general organization of a
tandem mass spectrometer.
Mass spectrometers that have a single mass analyser can only perform simple Mr determina-
tions, as described above. However, mass spectrometers can be designed with two mass ana-
lysers arranged in sequence. These instruments are referred to as tandem mass spectrometers.
If you look at Figure 11.22, you can see the layout of a tandem MS, which produces spectra
called MSMS.
The sample is introduced into the ionizer and the individual components of the mixture sepa-
rated from one other in the first analyser and a selected ion is transmitted into the collisional
dissociation (CID) cell. Here, the ion is fragmented when it collides with molecules of an inert
gas, typically helium or argon, present in the CID cell. Fragments of ions from an individual
component are analysed in the second analyser to give an MSMS spectrum that is representa-
tive of that compound. The fragmentation of the ions from any one individual component
of the sample usually occurs in a predictable manner. Thus, the sizes of the fragments pro-
duced can be pieced together like a jigsaw to give information about the structure of the
component.
In some specific cases, as with quarupole ion-trap instruments, several rounds of mass analy-
sis can be performed in sequence within the same analyser. This analysis is performed by
sequentially trapping and fragmenting the ions generated in the source. Specific fragments
are trapped and fragmented in turn. This process can be repeated, giving up to 10 consecutive
rounds of mass analysis. The resulting spectrum is commonly referred to as an MSn spectrum,
where n is equal to the number of consecutive mass analyses.
SELF-CHECK 11.9
Briefly describe the use of tandem mass spectrometry (MSMS).
The types of mass spectrometers most commonly used in biomedical sciences couple ESI or
MALDI ionization methods with Q, QIT, or time-of-flight (TOF) analysers. These machines can
determine the Mr of a wide range of materials. They have a high throughput: for example,
one sample per minute for MALDI TOF mass spectrometers. Moreover, this speed of analy-
sis is coupled to excellent accuracy, typically to 5–100 ppm of the real mass, and sensitivity.
Furthermore, the results obtained using such techniques are reproducible, which enables the
data obtained to be compared with that held in electronic databases.
Applications of mass spectrometry

Mass spectrometry can be applied to many different areas of the biomedical sciences. It is rou-
tinely used in hospital laboratories to screen newborn babies for inborn errors of metabolism, for
example PKU, and the determination of illicit drugs in biological fluids, as we describe below.
284 11 SPECTROSCOPY
Phenylketonuria is a genetic disorder caused by a deficiency of hepatic phenylalanine hydroxy-

lase (PAH). This enzyme is necessary for catalysing the conversion of phenylalanine to tyrosine.
Thus, in PKU, phenylalanine accumulates in the blood and if the condition is left untreated
causes mental retardation. Phenylketonuria cannot be cured but can be treated by restricting
the amount of dietary phenylalanine while ensuring an adequate intake of tyrosine. However,
the mental retardation caused by PKU is irreversible; hence, its early detection in newborns is
essential. Tandem mass spectrometry is routinely used to measure the molar ratio of phenyla-
lanine to tyrosine in bloods spots collected from newborn babies. This method of investigat-
ing PKU has been shown to be more effective than other types of screening. It is also used to
screen for other treatable inborn errors of metabolism.
Mass spectrometry is also routinely used to test for illicit drugs in biological fluids. These test
are normally performed on urine samples, as the concentrations of the drugs and their metab-
olites in urine are relatively high compared with other fluids. Liquid chromatography or gas
chromatography coupled to tandem mass spectrometry is a powerful technique in this area,
providing selectivity, sensitivity, and reliability. It is also used for the study of drug metabolism,
and for the investigation of the dynamics of endogenous biologically active substances, as well
as contributing to the development of new therapeutics.
The research applications of MS include the analysis of peptide/protein (proteomics), metabo-

lite studies (metabolomics), and carbohydrate analysis (glycomics). Emerging, although lesser
used areas include mass spectrometric imaging and microorganism identification.
The proteome is all the proteins present in a given cell, tissue, or organism. Proteomics is the
study of proteomes. However, the proteome changes in disease cells or tissues compared with
healthy ones, both in composition and in the amounts of specific proteins present. Mass spec-
trometry techniques involving ESI–MS and MALDI–MS can identify and quantify the proteins
present in a sample, allowing tissues from normal (reference) and diseased states (patients) to
be directly compared. For example, increases in the concentrations of proteins such as amy-
loid-A protein are associated with the inflammatory response and can be detected in samples
of plasma from patients with rheumatoid arthritis. Thus, proteins in a given tissue that change
during the disease can be identified as a potential marker for that disease and/or as targets for
drug therapy. This is often referred to as biomarker discovery.
Comparison of the proteins in clinical samples is generally done in an indirect manner

called peptide mass fingerprinting (PMF). Samples of tissues are digested using a proteolytic
enzyme, such as trypsin, to generate a set of peptides. The peptides are then analysed by
MS and MSMS and the resulting mass spectrum is visually interpreted, as shown in Figure
11.23; alternatively it is matched against electronic protein databases containing similar data
for known samples. The outcome of these types of analyses is a list of the proteins present
in each sample, which can identify those proteins that may be a specific biomarker of the
disease investigated.
In most diseases, however, it is not the presence or absence of a specific protein, but rather
the amounts of a given protein, which increase or decrease compared with a healthy state. To
identify such changes requires a quantitative technique to be used. In most cases a chemical
labelling system is used where the proteins from the two states, diseased and healthy, are
modified with tags of different masses to form heavy and lighter versions of molecules, which
still have the same chemical formula. The heavy form is produced by incorporating a number
of stable isotopes such as 2H, 13C, 15N, or 18O into the protein molecules thus forming a small,
but easily measurable mass difference. The diseased and healthy states are labelled with the
light and heavy versions, respectively. The two types are mixed, enzymatically digested, and
the resulting peptides analysed by MS.
504.33 (M+H)+
100
90
310.23
80
601.35
70 Δm=97Da Δm=97Da
Signal intensity
213.17
60
50 116.11
Δm=97Da Δm=97Da 407.25
40
Δm=97Da+H2O Δm=97Da Δm=97Da
30
FIGURE 11.23
20 MSMS spectrum of a peptide. Note the
difference in mass (Dm = 97 Da) between the
10 consecutive peaks in the series is equivalent
0 to the residue mass of the amino acid proline
0 200 400 600 Mr = 97). Thus, a valid interpretation of the
m/z data gives the sequence, PPPPPPP.
Analysis of the resulting spectrum allows the quantitative ratio of a particular protein present
in samples from patients and healthy individuals to be calculated. Analysis requires examina-
tion of the relative signal intensities of the ions from the heavy and light versions of both sets
of peptides. A PMF analysis of the peptides allows the proteins in question to be identified.
This, combined with the ion ratios allows the amounts or concentrations of any identified
protein in the diseased and healthy samples to be calculated. Those proteins that change
significantly may be potential biomarkers or indicators of disease.
In addition to the discovery of potential biomarkers, proteomics also includes the study of
protein modifications, many of which have been implicated in disease pathways. These modi-
fications occur post-translationally and may be catalysed by enzymes, for example phospho-
rylation and glycosylation, or occur non-enzymatically as is the case with glycation. Often
proteins are not active until they are enzymatically modified in the cell. Analysis of protein
modifications tends to exploit MSMS-based techniques, which are used to identify signature
ions or differences in masses characteristic of a particular modification. The fragmentation
pattern obtained by MSMS analysis can also identify the specific sequence of the modified
peptide, thus identifying the site where the modification has occurred.
The metabolome is all the metabolites, which are molecules of small Mr, present in a biologi-
cal system. These include, for example, many hormones, signalling molecules, as well as the
chemical intermediates of metabolic pathways. It is believed that about 3000 metabolites are
essential for normal human growth and development. Studying variations of these metabo-
Cross reference
lites could be helpful in determining if a patient is fighting a disease, reacting badly to a drug
You can read about the various
therapy, or responding to another form of stress. This type of knowledge could have essential forms of chromatography in
roles in optimizing therapy and management of a disease. Chapter 13.
Mass spectrometry, along with NMR, is a major technique for analysing metabolomes. How-
ever, in this role MS is usually combined with a chromatographic separation method such as
GLC or high performance liquid chromatography (HPLC), which are described in Chapter 13.
286 11 SPECTROSCOPY
Gas liquid chromatography and HPLC greatly simplify the analyses by separating the compo-
nents of complex clinical samples (urine, serum, cerebrospinal fluid for example) that are typi-
cally studied in metabolomics. This simplification means each component can be subjected
to MS analysis in turn. This separation, coupled with the speed, high throughput, accuracy,
and sensitivity of MS provides a powerful tool for the validation, evaluation, and monitoring
of drug therapies, biomarker discovery, and clinical diagnosis.
Imaging by mass spectrometry was first used in 1999 and specifically uses MALDI MS. A sec-
tion of tissue is placed directly in the source region of the MALDI mass spectrometer. The
sample is moved in two dimensions as a laser is fired at it. Specific ions are formed at each
point on the section by the action of the laser light. This procedure allows an image of the tis-
sue section surface to be generated using a computer with appropriate software. This image
can be manipulated to show the location of any given mass in the tissue section and therefore
track, for example, where a drug and its metabolites are accumulating. This is illustrated in
Figure 11.24, which shows mass spectrometric images of a mouse brain section highlighted
for three different masses.
Mass spectrometry can be used to identify species of bacteria and fungi. Traditional methods
for identifying these groups are time consuming and often relatively complicated. Using MS,
identification can be carried out using standard proteomics-type techniques, as described
above or, more elegantly, by an approach called intact-cell MALDI. The organisms are grown
on agar plates until mature colonies are visible, then a single colony is transferred into the
FIGURE 11.24
Mass spectrometric imaging of a section of tissue from a mouse brain. The upper panels
show the relative distributions of compounds with m/z ratios of 14,187, 15,035 and 6,733,
respectively. The corresponding lower panels are tissue sections to allow the sites of the
compounds identified by the imaging to be mapped to specific areas of the brain.
SUMMARY 287
MALDI source region and irradiated with a laser. As with proteomics, the analysis of these sam-
ples provides a pattern or fingerprint of the microorganism being investigated. This fingerprint
is then compared with a database of known organisms and so identified provided it has previ-
ously been subjected to analysis. This technique has obvious uses in identifying pathogenic
organisms in clinical samples.
SELF-CHECK 11.10
Which techniques are typically combined with mass spectrometry to simplify the analysis of
complex mixtures?
SUMMARY
■ Spectroscopy is concerned with the interactions of electromagnetic radiation and matter,
particularly the absorption of radiation by matter and, in some cases, its emission and can
be used to quantify molecules of interest in biomedical laboratories.
■ Many molecules absorb light of characteristic wavelengths in the visible, ultraviolet, or

infrared regions of the electromagnetic spectrum, and this is used as the basis for deter-
mining their concentrations using spectrophotometers and colorimeters.
■ The absorbance of light of a specific wavelength by a solution of biological molecules

depends on their concentration and the distance travelled by the light through the solu-
tion as described by the Beer–Lambert law.
■ Infrared and Raman spectroscopy are specialized techniques in spectroscopy with appli-
cations in identifying biological molecules.
■ Light scattering methods include turbidimetry and nephelometry. Turbidimetry can be

used to estimate concentration by measuring the amount of light scattered by a suspen-
sion, whereas the more sensitive technique of nephelometry measures the amount of
light scattered at an angle to the incident beam of light.
■ Some molecules are fluorescent and are able to absorb light of a characteristic wavelength
and then emit radiation of a longer wavelength. Fluorescence-based methods are highly
sensitive, but are limited to molecules that fluoresce or which bind to one that is.
■ Nuclear magnetic resonance (NMR) utilizes magnetic properties of nuclei within mol-
ecules and provides information on positions of atoms within their structures. Its value is
in identifying molecules of biological interest and in determining their structures.
■ Nuclear magnetic resonance imaging can distinguish between healthy and diseased tis-
sues and is of value, for example, in locating the positions of tumours in patients.
■ Mass spectrometry allows us to determine the masses of molecules based on their charge
to mass ratios. The technique has wide applications in biomedical science, ranging from
detecting phenylalanine when screening newborn babies for phenylketonuria and drugs
of abuse in urine of suspected individuals, to identifying and sequencing proteins. This
sophisticated technique is also the basis of locating the positions of molecules of interest
in patients.
288 11 SPECTROSCOPY
FURTHER READING
● Chary KVR, Govil G. NMR in Biological Systems: From Molecules to Human. Springer,
Berlin, 2008.
● Gore MG. Spectrophotometry and Spectrofluorimetry: a Practical Approach. Oxford

University Press, Oxford, 2000.
● Hoffman E, Stroobant V. Mass Spectrometry: Principles and Applications. John Wiley

& Sons, Chichester, 2007.
● Honour JW. Benchtop mass spectroscopy in clinical biochemistry. Annals of Clinical

Biochemistry, 2003; 40: 628–638.
● Huettel SA, Song AW, McCarthy G. Functional Magnetic Resonance Imaging, 2nd
edn. Sinauer Associates, Massachusetts, 2009.
● Moolenaar SH, Engelke UFH, Wevers RA. Proton nuclear magnetic resonance spec-
troscopy of body fluids in the field of inborn errors of metabolism. Annals of Clinical
Biochemistry, 2003; 40: 16–24.
● Riddle P. Principles of spectrophotometry. Biomedical Scientist, 2005; 49: 135–137.
● Smith E, Dent G. Modern Raman Spectroscopy: A Practical Approach. John Wiley &
Sons, Chichester, 2004.
● Stuart BH. Infrared Spectroscopy: Fundamentals and Applications. John Wiley &
Sons, Chichester, 2004.
● Stuart B, George B, McIntyre P. Modern Infrared Spectroscopy. John Wiley & Sons,
Chichester, 1998.
● Thomas MJK. Ultraviolet and Visible Spectroscopy: Analytical Chemistry by Open

Learning. John Wiley & Sons, Chichester, 1996.
● Watson JT, Sparkman OD. Introduction to Mass Spectrometry: Instrumentation,

Applications, and Strategies for Data Interpretation, 4th edn. John Wiley & Sons,
Chichester, 2007.
QUESTIONS
1. Which of the following requirements must be satisfied to establish a colorimetric method
to measure the concentrations of a specific analyte (X)?
(a) Pure analyte (X) is needed for the production of a standard or calibration graph.
(b) Analyte X should absorb light of a particular wavelength or it should be possible to
convert it to one that can.
(c) The wavelength of light at which maximum absorbance by X occurs needs to be
determined.
QUESTIONS
(d) The relationship between concentration and absorbance of light for X must obey
the Beer–Lambert law.
(e) All of the above criteria must be satisfied.
2. State which of the following are TRUE or FALSE?

(a) Spectrophotometers differ from colorimeters in only being able to analyse col-
oured compounds.
(b) Tungsten lamps in spectrophotometers are used to measure light in the ultraviolet
region of the spectrum.
(c) Light of the desired wavelength can be selected using a monochromator.
(d) Plastic cuvettes are used when measuring light in the ultraviolet region of the
spectrum.
(e) The relationship between concentration and absorbance is not linear when an
analyte obeys the Beer–Lambert law.
3. A 1.0 mmol dm−3 solution of a compound gives an A340 of 0.6. Calculate (a) the molar
extinction coefficient (ε) and (b) the absorptivity (a) of the compound.
4. What would be the advantage of using a standard curve formed from a range of concen-
trations of an analyte compared with estimating its value from the absorption of a single
solution of known concentration?
5. An aqueous solution of yeast DNA has an absorptivity of 25 m2 mol−1 at 260 nm. Calculate
the percentage transmission of a solution of the DNA of concentration 2.0 mol m−3 if the
absorbance is measured in a standard-sized cuvette.
6. What happens to light as it passes through a turbid suspension contained in a cuvette in

a colorimeter?
7. In 1H-NMR spectroscopy, the amino acid alanine [+NH3CH(CH3)COO−] produces two

major chemical shifts at 1.5 and 3.8 ppm, respectively, using TMS as standard. Which
hydrogens (protons) are responsible for each of these signals?
8. Describe the major components of a mass spectrometer and give an example of

each.
9. Give three examples of how mass spectrometry is used in the biomedical sciences.
12
Centrifugation
Learning objectives
■ Describe the basic features of a centrifuge

■ Explain the general principles of centrifugation
■ Calculate the speeds and forces involved when separating mixtures by centrifugation
■ Describe the major types of centrifuges, tubes, and rotors encountered in clinical laboratories
■ Discuss the principal techniques used in centrifugal separations
■ Outline the precautions to be taken in the safe use of a centrifuge
■ List some of the uses of centrifuges in a clinical laboratory
Introduction
Given sufficient time, gravity will cause many particles, such as cells, suspended in a liquid
to eventually settle to the bottom of their container. However, this is not usually a practical
method of separating or isolating the particles as the time required for them to sediment is nor-
mally too long. Indeed, particles that are extremely small in size will not separate at all unless
they are rotated at a sufficiently fast speed that the rotational or centrifugal force generated
causes the particles to move radially away from the centre of rotation. Centrifugation is the
term applied to the mechanical process of separating mixtures by applying centrifugal forces. It
is one of the most widely used techniques in biochemistry, molecular and cellular biology, and
in the biomedical sciences. Applications of centrifugation are many and may include harvesting
viruses and cells from growth media, the separation of subcellular organelles, the collection
of lipids, and the isolation of macromolecules, such as DNA, RNA, and proteins. For example,
centrifugation can be used to separate blood into plasma and blood cells, or obtain cells from
body liquids, such as the ascites fluid that accumulates in the abdomen in some pathological
conditions. Centrifugation can be applied to a great many clinical and biological materials, so
for the sake of convenience in this chapter we will generally refer to them all as particles.
Centrifugation is performed in a centrifuge. At its simplest, a centrifuge is a mechanical

device that produces a centrifugal force by spinning a sample around a central axis. They
12.1 BASICS OF CENTRIFUGATION THEORY 291
consist essentially of two compartments. One contains a motor whose drive shaft enters the
second compartment, called the rotor chamber, where it can be attached to a specialized
container called the rotor. You can read about rotors in Section 12.3. The rotor contains liquid
samples in suitable centrifugation tubes or bottles, although in some applications the samples
are centrifuged in plastic bags. The centrifugal force generated by the motor-driven rotation
of the rotor separates components of the mixture that differ in size and/or density. In general,
the mixture is separated into two fractions. One fraction is the pellet at the bottom of the
tube that contains the sedimented material; the other is the liquid supernatant that con-
tains unsedimented material. Whatever the type, all centrifuges operate in essentially similar
ways. However, different types are designed for a variety of uses, which you can read about
in Section 12.4, and so differ in design details. Centrifuges vary from relatively simple bench
top models to sophisticated floor standing instruments that must be sited with care (Figure
12.1). All types of centrifuges have a brake to rapidly slow and stop the spinning rotor follow-
ing centrifugation and incorporate a number of safety features, such as locking lids that can be
opened only when the rotor has stopped spinning.
12.1 Basics of centrifugation theory

The rate of sedimentation of a particle when spun in a centrifuge depends upon the cen-
trifugal field or acceleration (G), which is directed outwards from the centre of rotation. The
magnitude of this field is described by the equation:
G = ω2r
where ω is the angular velocity of the rotor in radians per second and the radius of the particle
from the centre of rotation is r in cm (Figure 12.2).
Rotor
(a)
Bucket
Lid
(b)
Armoured
guard ring
Drive shaft Control panel
Motor
G
Control Armoured
panel chamber
Sliding door/lid
Rotor r
FIGURE 12.2
Vacuum Motor Cooling Diagrammatic representation
pump system of the centrifugal field (G)
operating on a spinning
particle (P), which is a distance
FIGURE 12.1 of r cm from the centre of
Outline structures of (a) bench and (b) floor standing centrifuges. rotation.
292 12 CENTRIFUGATION
One revolution equals 2π radians. However, the speeds of centrifuges are always given in rev-
olutions per minute (rev min−1), although often expressed as rpm. Hence, the angular velocity
of a spinning rotor is given by:
ω = 2π (speed in rev min−1)/60
So, if G = ω2r
G = 4π2 (speed in rev min−1)2 r/602
It is usual to express G as a relative centrifugal field (RCF), that is in terms of the earth’s gravi-
tational field (g), which is an acceleration of 981 cm s−1 per s, giving:
RCF (in g) = 4π2 (speed in rev min−1)2 r/602 × 981,
which simplifies to:
RCF (in g) = 1.12 × 10−5 (speed in rev min−1)2 r
Alternatively, the RCF can be determined using a nomogram (Box 12.1).
If it is to be repeated successfully, an initial report of an experiment or procedure that involved

centrifugation must state the rotor dimensions, so that r can be determined, and the speed
of the centrifugation, or the RCF and rotor radius plus, in each case, the duration of the cen-
trifuge run.
Sedimentation velocity and sedimentation

coefficient
The value of G (that is the RCF) influences the rate of sedimentation or sedimentation velocity
(v) of a particle, i.e. how fast it is moving through the liquid when it is centrifuged. However,
v also depends upon a number of other factors, including the size of particle, its density and
frictional coefficient, and the density of the solvent, which are interrelated in the following
manner:
v = Mr(1 − −
v ρ) ω2r/Nƒ
The mass of the particle measured in grams is m0, but this is converted into its relative molec-
ular mass (Mr) by the introduction of Avogadro’s number (N, 6.02 × 1023). The densities are
accounted for by − v and ρ, which are the partial specific volume of the particle (that is the
change in volume that occurs when the particle is added to a large excess of solvent) and
the density of the solvent, respectively. The sedimentation of a particle through the solvent
is opposed by a frictional resistance that is equal to the product of its velocity and frictional
coefficient (ƒ), that is the vƒ of the particle. Thus as v and ƒ become larger, the resistance also
increases. The frictional coefficient depends upon the shape and size of the particle. It is larger
BOX 12.1 Nomograms
Nomograms consist of three scales. A straight line that connects a value on one scale to
that of another on either of the other scales gives the corresponding value on the third
scale. The use of nomograms relating the radius of rotation, speed of the rotor, and RCF
can save a lot of arithmetic! Figure 12.3 shows how a nomogram can be used to estimate
the speed needed to produce a specified RCF for a given radius of rotation.
12.1 BASICS OF CENTRIFUGATION THEORY 293
90,000 9000
80,000 8000
70,000 7000
60,000 6000
30
1,000,000 10,000
25
50,000 5000
20 500,000 5000
18 400,000 4000
16 300,000 3000
14 40,000 4000
200,000 2000
12
10
9 100,000 1000
8 30,000 3000
7
50,000 500
6
4 20,000 200
20,000 2000
3
10,000 100
2 5000 50
1
1000 10 10,000 1000
9000 900
Radius/cm RCF/g Speed/rev min-1
FIGURE 12.3
Nomogram to estimate the speed in rev min−1 needed to produce a specified RCF for a
given radius of rotation. Note the line giving the RCF of 100,000 or 1000 g for a radius of
10 cm and specified speeds of 30,000 and 3000 rev min−1, respectively.
SELF-CHECK 12.1
Use Figure 12.3 to estimate the speed required to produce an RCF of 50,000 g for a rotor of
radius 7.67 cm.
for asymmetric molecules, for example nucleic acids and fibrous proteins, than for spherical
ones, such as globular proteins, of similar Mr.
The rather complex expression given above that relates all these terms can be simplified by
introducing a new term, the sedimentation coefficient (s) of the particle, which is the ratio
of its velocity to the applied centrifugal field, that is
Mr(1 − −
v ρ)/Nƒ = v /ω2r = s
or, more simply:
s = v/ω2r
The units of the sedimentation coefficient (s) are those of time. Many biological particles have
values of s of 1 × 10−13 to several thousand × 10−13 second. For convenience, they are usually
given as Svedberg units (S), where 1 S = 1 × 10−13 s (see Box 12.2). Try not to get too confused
with all the forms of the letter s!
Bigger particles generally have larger sedimentation coefficients (whether given in s or S!),
but the sedimentation coefficients of individual particles in an aggregate cannot simply be
added together to give that of the complex, since the sedimentation velocity is also affected
by the shape of the particle. For example, the two subunits of a bacterial ribosome have Mr of
approximately 1.0 × 106 and 1.8 × 106 and sedimentation coefficients of 30 and 50 S, respec-
tively. The subunits combine to form the complete ribosome with the expected Mr of 2.8 × 106.
However, its sedimentation coefficient is only 70 S, a situation that arises because the value
of s depends also on the shape of the particle. Temperature and the nature of the solvent also
affect the value a of particle’s sedimentation coefficient, so they are normally corrected to the
defined conditions of 20oC in water, which are expressed as s20, w. Figure 12.4 lists the sedimen-
tation coefficients of some particles of biomedical interest.
BOX 12.2 Theodor Svedberg
The Svedberg (S) is a non-SI unit of time, equal to 1 × 10−13 s, used in characterizing the
behaviour of particles during centrifugation. It was named after Theodor Svedberg
(1884–1971), a Swedish physicist and chemist. Svedberg studied at the University of
Uppsala, where he obtained his doctorate in 1907. He was elected Professor of Physical
Chemistry at the University of Uppsala in 1912. Svedberg’s work largely involved the
chemistry of colloids and proteins whose macromolecules are too small to settle out of
the surrounding water by gravity alone. To better study these particles, Svedberg and
co-workers began developing the ultracentrifuge, for which he is best remembered,
to produce the centrifugal forces necessary to sediment them. This involved combin-
ing mechanics and optics in the instrumentation and using mathematics to describe
and analyse the behaviour of the particles. In 1924 they constructed an ultracentrifuge
capable of generating a centrifugal force of up to 5000 g. Svedberg was able to perform
experiments with the machine to calculate the Mr of the milk protein, casein, and eventu-
ally that of haemoglobin. Later centrifuges generated hundreds of thousands of times the
acceleration of gravity. In 1933–34 RCFs of more than half a million g could be achieved.
Studies based on the ultracentrifuge by Svedberg were instrumental in demonstrating
that the molecules of certain pure proteins are all of a single size.
Svedberg was awarded the 1926 Nobel Prize in Chemistry for his development of the
ultracentrifuge.
12.2 T YPES OF CENTRIFUGES 295
Molecules Virus/organelles/cells
0
1
Lysozyme 2
tRNA
4
rRNA
6
Proteins
8
10
Nucleic acids Catalase
20
rRNA
40 Ribosomal subunits
DNA (24 kb)
60
Ribosomes
80
100
200 Polyribosomes Viruses
400
600
800
1000
4000
8000 FIGURE 12.4
The sedimentation coefficients (s) of
10,000 some structures (particles) of biological
Bacterial cell/mitochondria
50,000 and biomedical interest.
SELF-CHECK 12.2
An enzyme has Mr 14,400 and a sedimentation coefficient (s) of 1.9 S. However, when bound
to an inhibitor of only Mr 220, the sedimentation coefficient of the complex was only 1.6 S.
How can you account for this change?
12.2 Types of centrifuges

The main types of centrifuge most likely to be encountered in biomedical or biological sci-
ences laboratories are:
• Bench centrifuges
• Continuous flow centrifuges
• High speed refrigerated centrifuges
• Ultracentrifuges
Bench centrifuges (Figure 12.1(a)) are the commonest type and so the most likely type to
be encountered. Small bench centrifuges generally have maximum speeds in the range of
4000 to 10,000 rev min−1 and generate RCFs of 3000–7000 g. They are mainly used to collect
material that sediments rapidly, such as erythrocytes, yeasts, and others cells, and relatively
granular precipitates of, for example, proteins. Larger bench centrifuges develop similar RCFs,
but can accommodate a greater variety of tubes with sample volumes of 5–250 cm3. They can
also accommodate a number of adapters that allow, for example, the 96-well plates used in
enzyme-linked immunosorbent assays you can read about in Chapter 15, to be centrifuged.
Many high throughput biochemical-based assays that require the rapid yet efficient separa-
tion of rather coarse precipitates or cells depend upon bench centrifuges.
Microfuges are small bench top centrifuges capable of reaching speeds of 12,000–15,000 rev
min−1 producing RCFs of about 12,000 g in only a few seconds. They are used to process sam-
ples of less than 1.5 cm3 contained in Eppendorf tubes. Microfuges are indispensable tools in
molecular and cell biology, being used to harvest small volumes of cells, isolate micro- and
milligram quantities of proteins and nucleic acids, and even ensure that all the reagents used
in some experiments, which may have volumes of only a few microlitres, are ‘pelleted’ to the
bottom of the tube and so mix together thoroughly.
Continuous flow centrifuges are a form of high speed centrifuge but the sample material is added
continuously throughout the separation process. The process is shown graphically in Figure 12.5.
A vertical rotor is accelerated to the desired rotational speed and the suspension to be separated
is added continuously through an input port and rotated to form a vertical liquid column. The
solvent and any unwanted smaller particles are immediately washed to the surface and are dis-
charged through an outlet pipe. The larger particles are, however, forced towards the rotor wall,
where they accumulate and can be recovered when centrifugation is completed. Alternatively,
they can be collected in an outflow, as shown in Figure 12.5. In some circumstances, continuous
Removal of solvent
and
smaller particles
Rotating liquid column
Drive
shaft
FIGURE 12.5
Outline of a continuous
flow centrifuge. See text Collection of larger particles
for details. e.g. bacterial cells
12.2 T YPES OF CENTRIFUGES 297
flow centrifuges can save up to 80% of the time required by discontinuous centrifugation and
provide a rapid and reliable means of separating large volumes of suspension that have low solid
matter content. This makes them ideal for harvesting cultures of bacterial and human or animal
cells from fermentors and bioreactors in just a single centrifugation.
High speed refrigerated centrifuges maintain a predetermined fixed temperature, often set
at 4oC, during the centrifugation. Refrigeration is necessary to counteract the heat produced
by friction between the rapidly spinning rotor and air molecules in the chamber. Low tem-
peratures help maintain the stability and activities of clinical and biological specimens. High
speed refrigerated centrifuges can accept sample volumes of up to 1.5–2 dm3 and are capable
of speeds of about 25,000 to 28,000 rev min−1, with corresponding maximum RCFs of up to
approximately 100,000 g. High speed refrigerated centrifuges are used to collect most precipi-
tates, harvest larger viruses and bacterial, animal, and plant cells, and in separating many cell
organelles from homogenized tissues.
The distinction between bench and high speed centrifuges is becoming increasingly blurred,
given some bench top types can achieve speeds of approximately 20,000 rev min−1.
Ultracentrifuges (Figure 12.1(b)) are not only refrigerated, but the rotor chamber is also evacu-
ated to prevent friction between air molecules and the rotor increasing the temperature of
the rotor and sample during centrifugation. They are capable of speeds of about 100,000 to
150,000 rev min−1, with corresponding maximum RCFs of up to approximately 800,000 g. Two
types of ultracentrifuge are used: preparative and analytical types. Preparative ultracentrifuges
are used, for example to collect membrane fractions and ribosomes, or to prepare samples of
viruses and isolate macromolecules. As with high speed refrigerated centrifuges, preparative
ultracentrifuges can use a variety of types of rotors and so a range of sample volumes can be
processed depending upon the size and type of rotor accepted by the centrifuge.
Analytical ultracentrifuges incorporate some form of optical system to observe the sedimen-
tation of the particles, which are often macromolecules or molecular aggregates. This allows
biophysical analyses to be performed on the particles during centrifugation. The use of small,
tabletop micro-ultracentrifuges has made ultracentrifugation particularly popular for investi-
gating the hydrodynamic properties of biological particles, as outlined in Box 12.3.
BOX 12.3 Analytical ultracentrifugation
Estimating the purity of samples and determining the Mr of microprocessor controlled tabletop micro-ultracentrifuges.
molecules is usually performed using chromatographic and These instruments accept small volume samples, up to about
electrophoretic methods, which are quick and convenient. 4 cm3, but can reach speeds of 100,000–150,000 rev min−1 and
You can read about these procedures in Chapters 13 and 14, generate RCFs of over 800,000 g. This allows analytical experi-
respectively. However, the Mr obtained by these methods is ments to be performed in a fraction of the time previously
estimated by comparison with standards of known size. An associated with analytical centrifugation. This, together with
advantage of analytical ultracentrifugation is that the Mr value contemporary methods of data capture and analysis means
and other biophysical properties of macromolecules are given that analytical ultracentrifugation is often the method of
from first principles. Such analytical methods and the analy- choice for determining a range of hydrodynamic properties of
ses of data were relatively time consuming. Also, while high biological molecules. These properties may include, for exam-
speed refrigerated centrifuges and preparative ultracentri- ple, determining the numbers of subunits that bind together in
fuges are bulky floor standing instruments, large by any stand- complexes, the value of the associated equilibrium constant,
ards, analytical ultracentrifuges were even bigger! However, and characterizing the conformational changes undergone by
analytical ultracentrifugation can now be performed with a macromolecule on binding with a ligand.
12.3 Tubes and rotors

During centrifugation the samples are generally contained in specialized tubes. The tubes are
made of a variety of materials, including glass, polyallomer, polycarbonate, and polypropyl-
ene, and range in volume from less than 1 cm3 to over a litre. Safe working practice means
choosing the most suitable type of tube, rotor, and centrifuge for the separation or experiment
to be performed. Most centrifuges can be used with a variety of rotors that accept tubes which
differ in volume, or by using a single rotor that has adapters for different tubes.
Rotors can be broadly classified into three common types:
• Swing out bucket rotors

• Fixed angle rotors
• Vertical tube rotors
You can see how the rotors differ from one another in Figure 12.6. In swing out bucket
rotors, the tubes are placed in pivoted buckets that move from the vertical to horizontal
(a) Axis of rotation
rmin
rav
rmax
At rest During centrifugation
(b) (c)
Axis of rotation Axis of rotation
rmin rmin
rav rav
rmax rmax
FIGURE 12.6
Schematics showing the basic structures of (a) swing out bucket (b) fixed angle and
(c) vertical tube rotors. Note that in each case, the effective radius of rotation varies
along the length of the tube from a minimum (rm) to the maximum (rmax) value. Relative
centrifugal fields are often quoted for the average radius (rav).
12.4 SEPAR ATION METHODS USING CENTRIFUGES 299
as the rotor gathers speed. As their name implies, fixed angle rotors hold the tubes at a
constant angle, which generally varies from 14o to 40o. Vertical tube rotors are a variant of
the fixed angle type, where the tubes are at right angles to the horizontal. During centrifu-
gation, rotors are subjected to considerable stress (see Section 12.5) and must be made of
appropriately strong material. Those used in bench centrifuges are generally composed of
steel. However, high speed and ultracentrifuges traditionally used rotors made of aluminium
alloy or titanium.
Aluminium is easily corroded especially by alkalis. However, aluminium rotors are given an
anodized (aluminium oxide) surface that resists corrosion. Unfortunately, it is thin and easily
scratched by sharp metal objects. Titanium is resistant to corrosion and can withstand higher
centrifugal forces than aluminium, but has the disadvantage that it is much more expensive.
Metal rotors have the further drawback of being heavy; not only to handle, but also to accel-
erate in the centrifuge. The weight of a metal rotor also contributes to the potential hazards
involved in using centrifugation (see Section 12.5), since the forces involved are the product of
the weight of the fully loaded rotor and the RCF applied. If a rotor fails during a centrifugation,
then the large forces involved mean nearby persons are in potential danger. Also, considerable
damage can be done to the centrifuge and its surroundings.
Rotors made of carbon fibre composites are available, which are lighter than metal ones in
weight. These can be accelerated and decelerated more rapidly than metal types resulting in
quicker centrifugations and reduced maintenance costs.
SELF-CHECK 12.3
What is the force (apparent weight) of a rotor of weight 13.6 kg in (a) tonnes and (b) tons, when
centrifuged at 150,000 g? Assume 1 kg is 2.2 lb.
Separation methods using

12.4
centrifuges
If you examine Table 12.1, you should notice that many materials of biomedical interest differ
in size and/or density. They can therefore be separated or studied by centrifugation. The two
commonest methods used to separate particles in centrifugation are differential centrifuga-
tion and density gradients.
TABLE 12.1 Sizes and densities of some materials of

biological and biomedical interest
Particle/structure Size Density/g cm−3

Haemoglobin ~8 nm ~1.35
Nucleic acids variable ~1.6
Viruses 20–50 nm 1.3–1.45
Ribosomes ~25 nm ~1.6
Mitochondria 0.5–2 μm 1.19
Lysosomes 0.05–1.0 μm 1.21
Peroxisomes 0.1–1.5 μm 1.23

Differential centrifugation
Differential centrifugation, as the name implies, separates particles mainly on differences
in the sizes of the particles. It is the commonest centrifugation technique and is illustrated
diagrammatically in Figure 12.7. However, to achieve an effective separation of two particles
they must differ in size by an order of magnitude, that is one must have a diameter 10-times
larger than the other.
The centrifuge tube is filled with the sample solution and then centrifuged. Depending upon
its size and the conditions applied during the centrifugation, any one component of the initial
mixture may end up in the supernatant, the pellet, or even be distributed between the two
fractions. However, since all the components were initially distributed evenly throughout the
sample, a pellet of larger particles will always be contaminated with smaller ones. It is, of course,
possible to carefully decant off the supernatant. The pellet can then be resuspended and recen-
trifuged, which will improve its purity. The supernatant can also be recentrifuged but at a higher
speed and possibly longer time to allow smaller particles to be obtained as a pellet.
Separation by differential centrifugation is commonly used in simple pelleting to isolate cells

from blood and in obtaining partially pure preparations of subcellular organelles and macro-
molecules for example.
Density gradient centrifugations

Density gradient centrifugation is performed in a centrifuge tube that contains a column of
liquid whose density increases towards the bottom of the tube. This separates particles in a
mixture with greater resolution than discontinuous centrifugation because larger particles are
not contaminated with smaller ones. The increase in density can be in discrete steps forming a
discontinuous gradient or it can be continuous as shown in Figure 12.8. Discontinuous or step
gradients are suitable for separating whole cells or subcellular organelles and or isolating some
types of viruses. Continuous gradients are the more commonly used. Like step gradients they
are used in isolating cell organelles and some viruses but also hormones, enzymes, and other
proteins, as well as nucleic acids.
Whether continuous or discontinuous, the gradient is formed by dissolving a suitable solute

in an appropriate solvent. Sucrose dissolved in water or buffer is common but, depending on
the sample, other materials can be used. The sample to be separated is generally layered on
top of the gradient and then centrifuged. Two methods of density gradient centrifugation are
routinely used: rate zonal and isopycnic as you can see in Figure 12.9.
Centrifugation
FIGURE 12.7
Outline of separation by differential centrifugation.
12.4 SEPAR ATION METHODS USING CENTRIFUGES 301
Rate zonal centrifugation (Figure 12.9(a)) separates particles because they sediment through (a) [Sucrose]
the gradient at different velocities that depend upon their sedimentation coefficients. They /% (w/v)
thus separate into discrete zones, with each consisting of one type of particle. The zones of
the separated particles are stabilized because the gradient slows diffusion and convection cur-
5
rents. The length of the gradient must be sufficient to allow the zones to separate. However,
given that the densities of the particles in the sample must exceed that of the densest part of
the gradient, the centrifuge run must be terminated before any or all of the zones reach the 10
bottom of the tube. In isopycnic density gradient separation (Figure 12.9(b)), the density gra-
dient is such that it exceeds the densities of all the particles in the sample. Thus, particles will
15
sediment in the gradient to a position that equals their own density. Once all the particles have
reached these positions, they will remain there and further centrifugation will not increase the
migration of the particles. 20
A variety of materials are used to form gradients suitable for density gradient separations in
biomedical and biological applications. For example, gradients of sucrose and CsCl are com-
monly used to separate subcellular fractions and nucleic acids, respectively. In many den-
sity gradient centrifugations, separation is really achieved by a combination of rate-zonal
and isopycnic principles. Rate zonal centrifugation is often used to purify cellular organelles (b)
(Box 12.4) and to isolate proteins, such as immunoglobulins, since all classes of immunoglobu-
lins have similar densities but differ in their masses. Rotors must be selected with care, since
the choice affects the efficiency of the separation.
Choice of rotor
In fixed angle rotors, the particles move radially outwards during centrifugation as shown in
Figure 12.10(a). Thus they travel only a short distance before hitting the tube wall. The par-
ticles then slide down the wall and form a pellet at the bottom of the tube. This makes fixed
angle rotors effective for the differential collection of precipitated materials and harvesting
cells. Swing out bucket rotors are prone to cause convection currents during deceleration
FIGURE 12.8
and the particles also have long sedimentation paths. This makes them inefficient for pel-
(a) Continuous and
leting particles by differential centrifugation but useful for rate-zonal (Figure 12.10(b)) and
(b) discontinuous density
isopycnic techniques. Vertical tube rotors are popular for isopycnic centrifugation. During the
gradients.
centrifugation the sample and gradient reorientate as shown in Figure 12.10(c). This result is
an effectively short sedimentation distance and rapid separation. However, the resolution of
the particles is less effective than when a swing out bucket rotor is used.
(a) (b)
Centrifugation Centrifugation
FIGURE 12.9
Outline of separations by (a)
rate zonal and (b) isopycnic
density gradient (vertical
rotor), centrifugations.
(a) Centrifugal force
Pellet
(b) At rest (c) At rest
Centrifugal force
Centrifugal force
Centrifugal force
Centrifugal force
At rest
Deceleration
At rest
FIGURE 12.10
The behaviour of particles
during (a) differential in a
fixed angle rotor, (b) rate-
zonal (swing out rotor) and
(c) isopycnic centrifugations.
12.5 SAFET Y AND CENTRIFUGES 303
BOX 12.4 Subcellular fractionation
If a cell homogenate is subjected to differential centrifugation, then generally larger

particles, such as organelles in this case, will sediment faster than the smaller ones. This
is the basis of obtaining fractions enriched in specific subcellular organelles. Thus, cen-
trifugation of the homogenate at 1000 g for 10–20 minutes will result in a pellet consist-
ing largely of unbroken cells and larger nuclei. The supernatant can be recentrifuged at
10,000 g for 20 minutes, which will mainly pellet mitochondria and the larger types of
lysosomes and microbodies. Centrifuging the supernatant at 105,000 g for 60–90 min-
utes will sediment smaller subcellular structures, such as ribosomes and the artificial
vesicles formed from fragments of the endomembrane system and the plasma mem-
brane that are often called microsomes. The particles remaining in solution are largely
macromolecules and are generally regarded as the soluble fraction.
Obtaining partially purified organelles by differential centrifugation is usually only a

preliminary purification step; the fraction is normally purified further using some form
of density gradient separation. FIGURE 12.11
Surface view showing
alternative arrangements for
balancing four tubes in a rotor
Balancing rotors capable of accommodating
eight. Filled tubes are indicated
Whatever the type of rotor or centrifugation procedure to be used, the rotor must be bal- by the solid surfaces. Notice
anced; that is the weight of the tubes and their contents must be distributed evenly around how the tubes are distributed
the centre of rotation. If the rotor is imbalanced, it can damage the drive shaft of the centrifuge symmetrically, with filled tubes
and even cause it to move. You can see an example of how tubes can be added to a rotor to always opposite one other.
balance it in Figure 12.11. It is necessary to attach all of the buckets when using a swing out
rotor even if you are not using all of them for sample tubes. This is because the buckets and
their lids are an integral part of the balancing of the rotor.
12.5 Safety and centrifuges

Centrifuges, especially high-speed and ultracentrifuges, are potentially dangerous pieces of
Cross reference
equipment. Care and attention to detail must be applied at all times during their use. However,
General aspects of health and
if you follow the procedures outlined below, then the associated risks will be reduced to a safety are described in Chapter 4.
minimum. Note that staff in charge of any centrifuge must read standard operating procedures
for their particular machine, taking note of any Medicines and Healthcare Products Regulatory
Agency (MHRA) or Health and Safety Executive (HSE) safety alerts.
Qualified users
Centrifuges must only be operated by suitably trained persons or under their supervision.
All staff/students must be trained by someone who is competent in their use before they use
any centrifuge. If you notice a fault, report it promptly, do not attempt repairs yourself: only
authorised and suitably trained persons may service or repair a centrifuge. Do not use the cen-
trifuge until the fault has been repaired. A log book must be kept for ultracentrifuges and their
rotors, as the number of hours used determines the life of the rotor. Excessive use can lead
to metal fatigue, where microscopic changes in the metal structure can lead to small cracks,
which eventually enlarge to the point of causing metal failure. Where necessary, the details of
the centrifugation must be entered in the centrifuge log book. Before using a centrifuge, the
rotor chamber should be checked to ensure that it is clean and free of dust or loose debris and
condensation. Check also that the drive shaft is clean and free of scratches or burrs.
Rotor faults
Faults with a rotor can lead to its failure: the greater the speed used, the more likely this is to
occur and the more extreme the results. Always examine a rotor for cleanliness and damage. Any
extraneous material including dirt in the rotor cavities will affect its balance. Dirty rotors must be
cleaned by an approved method using, for example, plastic coated brushes and mild detergents.
Soaps and many detergents are alkaline and can attack the anodized surfaces of aluminium
rotors. The body of a swing out bucket rotor should never be immersed since hanging mecha-
nisms are difficult to dry and can corrode. Only the buckets should be washed. After cleaning,
the rotor must be rinsed with deionized water and air dried with the buckets or cavities pointing
downwards. Corrosion, cracks, or dents on any part of the rotor are potentially hazardous and
even the most careful design will not protect against its misuse and abuse. When centrifuged,
rotors are subjected to stress causing them to be stretched. You can see the effects of stress
diagrammatically in Figure 12.12. The first stage of stretching constitutes the elastic limit and the
rotor is able to return to its original size when the stress, that is the centrifugation, stops. However,
above a certain level of stress, plastic strain occurs and the rotor will not regain its former dimen-
sions. Plastic damage can lead to cracks in the fabric of the rotor and cause its eventual failure.
Damaged rotors must not be used and must be reported to the appropriate supervisor.
Check that all O rings, which are gaskets consisting of a flat rubber or plastic ring used to seal a
joint against high pressure, are in place and in good condition. Rotors must never be used if their
O rings are missing or perished. Chill the rotor in a refrigerator to the desired temperature before
placing it in the rotor chamber since this minimizes the chance of it seizing to the drive shaft.
Close the rotor lid when pre-cooling it to prevent condensation in the chambers or buckets.
Tube checks
Use only correctly fitting tubes and check that they are made of a material that is compatible
with the sample solvent. Balance the rotor to within its specified limits as described in Section
12.4. Do not operate the centrifuge without the appropriate rotor lid securely fitted and its
Elastic seals in place. Never exceed the maximum stated speed for any rotor. Following centrifuga-
limit tion, do not remove the centrifuge tubes or bottles from the rotor with a metal object. Use
Stress/arbitrary units
only the tools provided by the centrifuge manufacturer for that purpose.
Fracture
point
After checks
Check the rotor chamber and the rotor for any leaks and clean if necessary. Return the rotor to
its normal storage place. Rotors must be stored inverted in a dry environment.
Strain/arbitrary units
SELF-CHECK 12.4
FIGURE 12.12 You need to isolate viral particles from a cell culture lysate, when you notice that the alumin-
Schematic showing the effects
ium rotor you were going to use has some fine hairline cracks in its surface. You do, however,
of stress on a typical rotor
have a pressing need to prepare the sample. What should you do?
alloy.
12.6 EXAMPLES OF CLINICAL CENTRIFUGATION 305
Examples of clinical
12.6
centrifugation
In addition to freestanding centrifuges, a number of automated instruments, for example DNA
extractors (see Chapter 16), encountered in clinical laboratories include ‘built-in’ centrifuges
to help process specimens. Specialist centrifuges for use in routine clinical laboratories are
uncommon. One specific type, the cytocentrifuge or slide centrifuge is described more fully in
the Cytopathology text of this series. A cytocentrifuge is essentially a low speed centrifuge with a
modified rotor that accommodates microscopic slides as well as liquid samples. The centrifugal
force separates the cells and sediments them as a monolayer onto the slides, while preserving
their integrity. This simple technique can be used, for example, to isolate cells from samples of
cerebrospinal fluid or fine needle aspirates directly for cytological examination. Haematocrits
are a type of centrifuge that were used to determine the ratio of the relative volumes of eryth-
rocytes and plasma in blood samples but they have been superseded largely by automated
blood analysis systems (see Chapter 17), which can determine all haematological indices. The
multibucket centrifuges used for serum separations are probably the commonest type found
in biochemistry, immunology, and haematology pathology laboratories; although microfuges Cross reference
are used in a range of applications as discussed in Chapter 16, and refrigerated centrifuges find You can read about
immunosorbent assays in
use in specialist complement work (Chapter 15) and some biochemistry assays. For example,
Chapter 15 ‘Immunological
adaptors can allow the 96-well plates used with enzyme-linked immunosorbent assays (ELISAs) techniques’.
to be directly centrifuged allowing a high throughput of samples (Figure 12.13).
Blood is the most frequently requested of clinical samples that require centrifugation. Once
collected, it is added to a blood tube. Most blood tubes are made from a strong plastic like
FIGURE 12.13
Centrifuge with a specialized rotor (R) for centrifuging enzyme
linked immunoassays (ELISA) plates. An ELISA plate is indicated
(P). Courtesy of Department of Clinical Biochemistry, Manchester
Royal Infirmary, UK.
polyethylene terephthalate (PET), and so the blood sample can be directly centrifuged. Many
blood tubes also have an inert gel barrier added to them during their manufacture, which aids
in the centrifugal separation. The specific gravity of the gel is between those of the blood clot
and the serum. During centrifugation the barrier gel moves upward and forms a distinct dividing
barrier between the plasma and cells or the serum and clot, respectively (Figure 12.14). Thus,
aliquots of the sample can be taken directly from the tube for analysis. Most of the analytes rou-
tinely investigated in clinical laboratories remain stable several days if the tubes are used accord-
ing to manufacturer’s instructions and if stored at 4oC. The introduction of separator gels has
enhanced the use of primary samples for clinical analyses. A primary sample is one presented to
the analyser in the same container in which it was originally collected from the patient. Aliquots
may be taken directly from the serum/plasma and directly sampled by the analyser.
Manufacturers generally colour code blood tubes depending on whether they have a gel bar-
Cross reference
rier, and whether they include an anticoagulant or not and, if so, what type of anticoagulant is
obtaining blood samples in present. For example, tubes with grey coloured caps contain oxalate and fluoride, and are used
Chapter 7, and more about blood for samples to determine blood glucose measurements. The oxalate binds Ca2+ and prevents
tubes in Chapter 17. coagulation, while fluoride is an inhibitor of hexokinase, and so prevents glycolysis. The effect
of the two additives is to maintain the concentration of blood for several days. Purple-capped
tubes also contain an anticoagulant, in this case the metal chelator, ethylenediaminetetra-
acetic acid (EDTA). A green cap indicates the tube contains the anticoagulant lithium heparin.
These tubes are available with and without gel barriers. The heparin allows the sample to be
centrifuged immediately, so whole blood or plasma can be tested. Tubes with red and yel-
low caps both contain clot activators and are used in most routine chemistry tests on serum.
Centrifugation of the former prevents analytes moving between the serum and the clot. The
yellow capped tubes have a gel barrier, which allows for primary sampling.
(a) (b)
FIGURE 12.14
(a) Blood tube containing a blood sample (B). Note the position of
the separator gel (G). (b) Blood tube with a separator gel following
centrifugation. Note how the gel (G) has moved up the tube to form
a layer between the cells and clot (C) and serum (S). Courtesy of
Department of Clinical Biochemistry, Manchester Royal Infirmary,
UK. See also Figure 7.4.
The automated chemistry analysers used to perform many of the standard clinical tests on patient
samples contain centrifuges, which may be stand alone devices or occur as discrete units on a
tracked system. The tubes are centrifuged in specialized racks. Robots can perform all the opera-
tions associated with centrifugation of the samples. Thus, they transfer the sample tubes onto a
Cross reference
balance to weigh them and then allocate each tube to an appropriate position on a centrifuge
The sampling of blood, the
rack. This ensures the final load in each centrifuge rack is balanced before they are transferred
centrifugation of clinical
to the centrifuge. Blood specimens are centrifuged at 1000 g or higher for eight to 12 min- specimens, and the use of
utes. Following centrifugation, the robot removes the racks from the centrifuge and may decap primary samples are described
the tubes before transferring the racks onto a track to transfer to other units for clinical testing. more fully in Chapters 1 and 2 of
Centrifugation is used extensively in blood banks for a number of procedures (Box 12.5). Clinical Biochemistry.
BOX 12.5 Blood banks
For a number of clinical reasons, blood that contains leu- antibodies may mediate an immediate and fatal transfu-
kocytes cannot be transfused legally in the UK. Hence, sion reaction.
usually within a few hours of collection, leukocytes are
The terms Rh positive and negative refer to the presence
removed from blood by filtering it in a process called leu-
or absence of the Rh(D) antigen. An Rh incompatibility
kodepletion. The filters used are leukocyte-specific and
between mother and foetus is a common cause of haemo-
trap them but not the smaller erythrocytes or platelets.
lytic disease of the newborn in Caucasians.
Leukodepletion does not involve centrifugation, but cen-
trifugation is used extensively in blood banks for a number The majority of transfusion laboratories now use gel tech-
of procedures. nology for blood grouping and compatibility testing, which
involves some centrifugation. Recent years have seen
To ensure safety, the blood is tested to check that it is not
increasing use of the Diamed typing system to detect hae-
contaminated with harmful microorganisms and to deter-
magglutination. This system uses typing monoclonal anti-
mine its blood group. Blood must be typed for the ABO and
bodies (mABs), which are distributed in a gel contained
Rhesus (Rh) blood group systems. The ABO system depends
in individual tubes set in plastic ‘cards’. Erythrocytes to be
on the presence of antibodies in the serum of individuals,
typed are added to the antibodies and the cards are cen-
which are complementary to the antigens on the erythro-
trifuged using a specialized rotor. Specific blood groups
cytes. It is these antibodies that determine the identity of
are recognized by the complementary mAB and agglutina-
the blood group. Thus, individuals of group A have anti-
tion occurs. In those tubes where agglutination occurs, the
bodies in their serum to blood group B antigens and those
agglutinates remain on top of the gel during centrifugation,
of blood group B possess antibodies to group A. Group AB
whereas nonagglutinated cells sediment through it to the
individuals do not have either type of antibody; however,
bottom of the tube (Figure 12.15).
group O individuals possess both. ABO blood groups are
identified for all donors and recipients to ensure that blood In addition, the production of blood products, such as eryth-
used for transfusion is of the same or a compatible type. If rocytes, platelets, and plasma, all involve the large-scale
ABO incompatible blood is administered to a patient, the centrifugation of blood using dedicated centrifuges.
(a)
(b) FIGURE 12.15
(a) A centrifuge with a
specialized rotor that
allows gel cards to be
centrifuged for blood
group typing as outlined
in the text. (b) A typical
gel card used in blood
group typing. Courtesy
of the Manchester Blood
Transfusion Service, UK.
SUMMARY
■ Centrifugation is a separation technique conducted in an instrument called a centrifuge.
A centrifuge holds a spinning rotor containing samples in tubes that are rotated around a
central axis by a motor. This spinning of the rotor generates a centrifugal force that sepa-
rates the components in the mixture on the bases of differences in size and/or density.
■ Particles sediment when subjected to a radial acceleration or centrifugal field (G). This is
normally expressed as a relative centrifugal force (RCF) as multiples of the earth’s gravita-
tional field (g).
■ A variety of centrifugation tubes, rotors, and centrifuges are available, which allows a
range of different centrifugation methods to be used as appropriate.
■ The major centrifugation techniques used are differential and density gradient centrifuga-
tion. The former separates particles on differences in size. Density gradient separation can
use rate zonal and isopycnic centrifugations. Particles that differ in their sedimentation
coefficients can be separated by rate zonal centrifugation, while isopycnic methods rely
on differences in the densities of the particles.
■ All appropriate safety considerations must be observed when using centrifuges. For exam-
ple. only clean and undamaged rotors must be used.
■ Centrifugation has applications in biomedical science where it is commonly used, for

example, in separating blood materials or isolating DNA. Clinical laboratories use spe-
cialist cytocentrifuges to isolate components onto slides prior to cytological analysis. In
research laboratories, centrifugation is used in preparing fractions of cellular constituents
such as macromolecules and organelles.
FURTHER READING
● Cole JL, Hansen JC. Analytical Ultracentrifugation as a contemporary biomolecu-
lar research tool. Journal of Biomolecular Techniques. 1999; 10: 163–176. Laue T.
Biophysical studies by ultracentrifugation. Current Opinion in Structural Biology.
2001; 115: 579–583.
These two journal articles provide a valuable synopsis of the uses of analytical ultracen-
trifugation in characterizing the behaviour of macromolecules.
● Goodman, T. Centrifuge safety and security. American Laboratory, 2007; 39: 20–21.
An interesting article detailing design features relating to safety and security that are avail-
able on a number of centrifuges.
● Goodman T. Centrifuge rotor selection and maintenance. American Laboratory, 2007;

39: 12–14.
An interesting short read that describe the different types of centrifuge rotors, their
applications, and also outlines procedures for their safe handling and maintaining
their life.
QUESTIONS
● Goodman T. Choosing the right centrifuge for your application. American Laboratory,
2008; 40: 28–29.
A short, useful article, whose title says it all.
● Graham JM. Biological Centrifugation. Bios Scientific Publishers, Oxford, 2007.

Excellent coverage of the basics of centrifugation. Also includes a number of useful cen-
trifugation protocols.
● Rickwood D. (ed.) Preparative Centrifugation – a Practical Approach. Oxford

University Press, Oxford, 1992.
A very helpful reference text.
● Rickwood D, Ford T, Steensgaard, J. Centrifugation: Essential Data. John Wiley &

Sons, Chichester, UK, 1994.
A pocket-sized (128 pp) volume that provides core data in tabulated formats for perform-
ing centrifugal separations of macromolecules and cells.
QUESTIONS
1. Calculate the RCF to the nearest 100 g, experienced by a particle 8.0 cm from the centre
of rotation when it is centrifuged at 3500 rev min−1.
2. Which of the following centrifuges:
● Microfuge
● Ultracentrifuge
● Bench centrifuge
● High speed refrigerated centrifuge
would be your first choice to:
(a) Isolate bacterial cells from liquid medium

(b) Collect a fine precipitate of DNA molecules
(c) Isolate mitochondria from a tissue homogenate
(d) Harvest the relatively small poliomyelitis viral particles (diameter 28 nm)
3. Which of the following would not affect the sedimentation velocity of a particle during
centrifugation?
(a) Solvent density

(b) Atmospheric pressure
(c) Particle density
(d) Distance of the particle from the centre of rotation
(e) Angular velocity
(f) Mr of the particle
4. A viral particle takes 10 minutes to move 1 cm when centrifuged at 20,000 rev min−1.
How long would it take to move the same distance if the speed was doubled?
5. Which would have the larger frictional ratios (ƒ) and sedimentation coefficients (s): enzyme
molecules or DNA fragments of comparable Mr?
6. Non-filamentous influenza viral particles have a sedimentation coefficient (s) of 750 S.

How far would a virus particle travel in 10 minutes, when centrifuged at 30,000 rev min−1
at the rav position of 6.12 cm?
7. Serum albumin has a sedimentation coefficient (s) of 4.6 S. Determine its Mr, given its
partial specific volume (v−) and frictional coefficient (ƒ) are 0.74 cm3 g −1 and 6.63 × 10−8 s−1,
respectively. The density of water at 20oC is 0.998 g cm−3.
8. You are going to perform a centrifugation experiment using CsCl (a corrosive salt) when
you spill a little of the solution into a rotor bucket. You do, however, have to complete
the experiment quickly, since the results are needed in a hurry. Should you ignore this
small spillage and proceed with the experiment?
13
Chromatography
Learning objectives
■ Define the term chromatography

■ Discuss the general basis of chromatographic separations
■ List some of the major chromatographic techniques
■ Describe the theoretical bases of specific chromatographic techniques
■ Discuss the relative efficiencies of some chromatographic methods
■ List some of the uses of chromatography in the clinical and biological sciences
Introduction
Chromatography was first used by the Russian botanist Mikhial Tsvet, who used it to separate
plant chlorophyll and carotenoid pigments. Tsvet first gave a written description of the method
in 1903, although he did not coin the term chromatography, which he adopted from the Greek
words chroma, colour in reference to the plant pigments, and graphein, meaning to write, until
1906. Chromatography is now the collective term for a family of analytical techniques used
to separate the components of mixtures of molecules for their identification and possible
estimation of their concentrations in the original mixture. A major difficulty that is often
encountered when investigating clinical and biological samples is the presence of substances
that can interfere or be confused with the substance or analyte under investigation. This means
it is often necessary to separate and isolate a particular analyte from other contaminants in the
mixture to allow its concentration to be determined. The principal uses of chromatographic
methods are to remove contaminating materials from the analyte of interest, to aid in the
identification of unknown analytes, and to estimate the concentration of analyte in the sample.
All chromatographic techniques are based on differences in the relative affinities of the mol-
ecules in a mixture of two different and immiscible phases, one of which is mobile and the
other stationary. Mobile phases may be liquids or gases that flow over or through the station-
ary phase, which can be a solid or an immobilized liquid. Different combinations of phases
can be used. For example, in column chromatography, particles of a hydrated gel are used as
312 13 CHROMATOGR APHY
Have greater affinity for

stationary phase
Have greater affinity for

mobile phase than
stationary phase
Chromatography
Stationary
phase
Mobile
phase
FIGURE 13.1
Schematic illustrating the separation of two
substance using differences in their relative affinities
for two phases in chromatography.
the stationary phase and a liquid moving through the gel is the mobile phase. In many cases
these have been developed into extremely high resolution techniques by forcing the liquid
through the column under high pressure. These latter methods are usually referred to as high
performance liquid chromatography or HPLC. Whatever the specific type of chromatography,
different substances in a mixture can partition differentially between the two phases and so
are separated from one another. You can see this schematically in Figure 13.1. Even com-
plex mixtures of closely related molecules can be separated if an appropriate combination of
phases is selected.
Most types of chromatography are used in biomedical science laboratories to assist in ana-
lysing and purifying analytes from a variety of clinical samples. You will be introduced to a
number of these applications in the chapter.
13.1Partition or distribution
coefficient
The basis of all forms of chromatography is the partition or distribution coefficient, (Kd),
which describes the way in which a substance distributes at equilibrium between two immis-
cible phases. For example, the Kd of a substance X, which distributes between the two immis-
cible phases A and B is given by:
Kd = The concentration of X in A/The concentration of X in B

13.1 PARTITION OR DISTRIBUTION COEFFICIENT 313
given that the distribution of X between A and B is at equilibrium. You can see an illustration A
of this in Figure 13.2.
[x]A
Partition coefficients are normally determined at 20 or 25°C. Often one of the two phases
is water and the other a hydrophobic solvent such as octanol. This gives an octanol–water
partition coefficient, KOW. Note that the KOW is not the same as the ratio of the solubility of B
the substance in octanol to that in water because the two phases are not fully immiscible! At
equilibrium, octanol contains 2.3 mol dm−3 of water and the water phase 4.5 × 10−8 mol dm−3
[x]B
of octanol. The measured values of KOW for organic compounds vary from 10−3 to 107. This is
an enormous range. Hence they are often quoted as their logarithmic value (log KOW), which
condenses the range from −3 to 7. The KOW value describes the relative hydrophobicity/
[x]A
hydrophilicity of the compound in question: the lower the value, the more hydrophilic the kd =
compound; the larger it is, the more hydrophobic. [x]B
FIGURE 13.2
SELF-CHECK 13.1 The definition of the partition
coefficient for a solute (X)
What are the units of Kd, KOW, and log KOW?
following its equilibrium
partitioning between
In chromatography, the mobile and stationary phases must be chosen such that the molecules the two immiscible phases,
to be separated differ in their partition coefficients. This, in turn, depends upon them differ- A and B.
ing in some particular physical property. If you examine Table 13.1 you will see some of the
properties upon which different types of chromatographies rely.
Polar molecules have an unequal distribution of electrons due to differences in the electro-
negativity of the atoms from which the molecule is composed. For example, water is a polar
molecule, as oxygen has a greater electronegativity than hydrogen. This means the electrons
are more attracted to the oxygen leaving less of an electron density around the hydrogens. As
a result, the oxygen acquires a slightly negative charge and the hydrogens have an equal but
positive one (Figure 13.3).
The polarity of molecules influences their solubility and adsorption on to surfaces. Thus, Adsorption is the
in chromatographic methods based on differences in solubility and polarities, the particles accumulation of molecules
partition to different extents between two immiscible liquids, and so separate. This is often from a solution on the surface
of an adjoining solid without
penetrating its interior.
TABLE 13.1 A summary of the differences in physical properties of molecules in

the mixture that can be used as a basis for their separation by various forms of
chromatography
Property Chromatographic techniques

Solubility and polarity Paper chromatography
Thin layer chromatography (TLC)
Size (mass) Gel filtration chromatography

½ δ+H
Charge Ion-exchange chromatography
δ−
Hydrophobicity Hydrophobic chromatography O
Gas–liquid chromatography (GLC) ½ δ+H
Ligand binding Affinity chromatography
FIGURE 13.3
Metal chelation chromatography
Structure of a water molecule.
described as ‘differential partitioning’. We will describe how differences between the solubili-
ties and adsorption properties of molecules are exploited in paper and thin layer chromatogra-
phies in Section 13.2. Differences in the sizes of molecules can also be exploited in gel filtration
chromatography. Molecules may become charged under certain conditions. These charges
can be used as a basis for their separation, as in ion-exchange chromatography. Hydrophobic
and gas–liquid chromatographies exploit differences in the partitioning of hydrophobic mol-
ecules between two phases. Finally, differences in how molecules bind to ligands are utilized
as the basis for their separation in affinity and metal chelation chromatographies (Sections
13.2–13.5).
The major types of chromatography likely to be encountered in biomedical science laborato-

ries are those grouped together as:
• Planar techniques
• Column chromatographies
• Gas–liquid chromatography
Each of these and some of their uses will be discussed in turn.
13.2 Planar chromatography

Planar or open bed chromatography uses a stationary phase consisting of a thin, essentially
two-dimensional, sheet. The two major types you are likely to encounter are paper and thin
layer chromatography. In paper chromatography, a sheet or a narrow strip of paper binds the
stationary phase. In thin layer chromatography (TLC), a film of solid particles, bound together
for mechanical strength with a binder such as calcium sulphate, is coated on a supporting
plate and also binds a stationary liquid phase. Both are examples of partition chromatography
because molecules distribute themselves between bound and mobile liquid phases, although
adsorptive effects to the particles in TLC also contribute to the separation.
Paper chromatography
In paper chromatography, the stationary phase consists of a layer of water or other polar sol-
vent molecules that are bound to the cellulose fibres of the paper, even though it may appear
dry. Materials to be separated are applied to the paper, generally as a series of discrete spots.
One end of the paper is then dipped into the mobile liquid phase, which moves through the
paper by capillary action. The molecules to be separated are differentially partitioned between
the stationary and mobile phases and so move along the paper at different rates and separate.
Paper chromatography is a useful method for assessing the purity of substances but is now
seldom used in clinical laboratories having been superseded by TLC.
Thin layer chromatography

Thin layer chromatography relies on the differential partitioning of substances between
stationary and moving liquid phases. In this respect, it resembles paper chromatography.
A solvent is used to suspend an adsorbent material, which is then bound to an inert glass, plas-
tic, or aluminium plate as a thin layer and allowed to dry. The thin layer of solvent remaining
in the bound adsorbent forms the stationary phase (hence the name). A range of materials are
13.2 PL ANAR CHROMATOGR APHY 315
TABLE 13.2 Suitable adsorbents for TLC and their applications
TLC adsorbent Used in separating

Aluminium oxide Steroids
Calcium phosphate Proteins
Cellulose Proteins
Magnesium carbonate Porphyrins
Silica gel Steroids, amino acids, lipids
suitable for forming the adsorbent layer and, indeed, mixtures of materials can be used. Some
of them are listed in Table 13.2; the choice depends on the material to be analysed.
Again, a mobile liquid phase migrates through the thin layer by capillary action. A wide range
of solvents may be used as mobile phase, including water, ethanoic acid, ether, hexane, meth-
anol, and benzene, which can be mixed together in different combinations and proportions
as appropriate. A general procedure for TLC is outlined in the Method Box ‘Generalized pro-
cedure for TLC’ and illustrated in Figure 13.4.
METHOD Generalized procedure

for TLC
■ The adsorbent material is prepared by mixing it with a binding agent such as calcium
sulphate or plaster of Paris (10%) and water to form a slurry. The binding agent helps
hold the adsorbent to the plate.
■ The slurry is applied to the plates as a thin uniform layer using a plate former and
allowed to set.
■ The plates are activated by heating in an oven at 100–120°C to remove free liquid,
with the remainder forming a thin layer bound to the adsorbent particles. The final
thickness of the adsorbent layer is between 0.25 and 2 mm.
■ A 1–20 μdm3 (lL) sample is applied to the plate as a concentrated spot or band using
a micropipette or microsyringe and allowed to dry.
■ The TLC plate is placed in a tank which contains the mobile phase and developed
for 30–90 minutes, as shown in Figure 13.4. The atmosphere within the tank must
be saturated with solvent vapour in equilibrium with the mobile phase to minimize
evaporation from the plate surface and prevent the composition of mobile phase
changing due to losses of its volatile components. The mobile phase travels up
the plate by capillary action. Development is stopped when the moving edge of the
mobile phase (usually referred to as the solvent front) approaches the top of the plate
(Figure 13.4).
■ Following their separation on the TLC plate, the positions of the separated compo-
nents of the sample can be determined and the presence of the analyte of interest
confirmed and its concentration in the sample possibly estimated.
TLC plate
Standards (1 to 3) and
Origin sample (4) applied as
1 2 3 4 spots (origin) in four
separate lanes
‘Spotted’ TLC plate

placed in
chromatography
tank
Solvent front
Plate developed
using mobile
phase [■]
FIGURE 13.4
General procedure for thin layer
chromatography. See text and Method box
‘Generalized procedure for TLC’. In this case, B
A
lanes 1, 2, and 3 have been used to ‘run’
substances of known identity (‘standards’).
C D
Lane 4 was used for an unknown sample. On
the basis of its migration, the unknown would 1 2 3 4
appear to contain standards 2 and 3. Lanes
As with all chromatographic techniques, the substances to be separated partition differen-

tially between two phases. Initially, the stationary phase consists of the liquid retained by the
adsorbent layer during its drying. Separation is achieved because as the mobile phase carries
sample and standards up the thin layer, the more polar molecules in them will associate with
13.2 PL ANAR CHROMATOGR APHY 317
the thin layer. In contrast, the relatively hydrophobic substances will be more soluble in the
mobile phase and be carried further along the plate. However, the mobile phase may be a
complex mixture and contain polar liquid molecules. As these move through the thin layer
by capillary action, they may bind to the adsorbent material and will form what is essentially
a new stationary phase, which may assist in the separation. Thin layer chromatography plates
have a large surface area. Hence, significant adsorption of sample molecules to the particles
can also take place. This ability is often due to the presence of hydroxyl groups (–OH) on
their surface, which can form hydrogen bonds with molecules in the sample. The more polar
the molecules in the sample, the greater the strength of the bonds and the more adsorption
occurs. Adsorption to the particles can increase the effectiveness of separation. It is apparent
that the factors that affect separation in TLC are complex!
Following its development, it is necessary to detect the separated components on the plate.
A number of methods are available to achieve this, including the use of:
• Ultraviolet (UV) light
• Specific colouring reagents
• Autoradiography
• Sulphuric acid
Examining the TLC plate under ultraviolet (UV) light will show the positions of compounds
that absorb or fluoresce in the presence of UV light. For example, nucleotides and nucleic
acids absorb UV light, and so appear as dark spots. Spraying of plates with specific colour-
ing reagents can be used to stain certain compounds. For example, ninhydrin will react with
amino acids to form purple-coloured complexes, which will indicate their location on the
Cross reference
plate. Alternatively, the TLC plates can be sprayed with sulphuric acid and then heated to
You can also read about
110°C. Most organic compounds are charred by this procedure and show up as brown-black autoradiography in Chapter 10
spots or bands. Radioactively labelled compounds will present as dark spots or bands on X-ray ‘Radioactivity and radiation’.
film after the TLC plates have been subjected to autoradiography (Box 13.1)
It is usual to calculate the Rƒ (relative to front) value, which is the distance travelled by any
component relative to that moved by the solvent using the expression:
Rƒ = migration distance of analyte/migration distance of solvent front
BOX 13.1 Autoradiography
Autoradiography is a simple and sensitive method of producing a photographic image of

a specimen containing radioactively labelled material(s). The image is formed by placing
an X-ray film directly in contact with a chromatogram, histological preparation, or electro-
phoresis gel. These are then wrapped in a material which prevents external light fogging
the film. However, radiation from the labelled materials impinges on the film causing the
deposition of silver grains in its emulsion. The length of exposure varies depending upon
the intensity of the radiation source: it can be as little as a few minutes or up to several
days. The package is often stored in a freezer at −70°C during longer exposures to reduce
the diffusion of the molecules in the sample. Once the film is developed, it forms a radio-
graph, whose patterns of deposited silver record the location(s) of radioactive substance(s) Cross reference
in the specimen. The intensity of the developed spots or bands on the film is proportional See the Histopathology book
in the same series and
to the dose of radiation and, thus, the concentration of the labelled material.
Chapter 14 ‘Electrophoresis’.
In theory, Rƒ values are identical for any given substance analysed under the same chromato-
graphic conditions. However, given experimental conditions vary, it is more usual to also ‘spot’
known reference compounds, in addition to the samples to be analysed, and subject them
all to TLC. Component(s) in a mixture can then be tentatively identified by comparing the
Rƒ value(s) to those of the standards. You can see this diagrammatically in Figure 13.4, where
the unknown compounds in the sample (Lane 4) have migrated the same distances (C,D) rela-
tive to the front (A) as the standards in Lanes 2 and 3.
SELF-CHECK 13.2
Is it possible to have an Rƒ value of 1.2 for a particular analyte? What effect would changes in
temperature have on the TLC? What difference would an increase in room temperature make
to Rƒ values?
Thin layer chromatography has a number of advantages over some other chromatographic
techniques, particularly over paper chromatography. One particular advantage is its versatil-
ity. If you examine Table 13.2 you will see it can be applied to the separation of a number of
differing types of substances. Moreover, separation is quick, often requiring as little as 30–90
minutes, it is simple to perform and is inexpensive. Hence, it is often the first method of choice,
particularly when the components of the mixture are known to be structurally similar. Its major
disadvantage is that it is not especially suitable for quantitative measurements, for example for
determining the amounts of each substance in a mixture. However, TLC is used successfully
to identify a number of substances of clinical interest, including drugs, lipids, amino acids and
vitamins. Figure 13.5 illustrates its use in separating a number of drugs of abuse.
Once separated using TLC, analytes can be located on the plate using UV light, and any of
potential interest can be extracted from the plate and identified using gas chromatography-
mass spectrometry.
CASE STUDY 13.1 Investigating

galactosaemia
1 2 3 4 5 6
Joe, a child of 7 months age, was suspected of having galactosaemia. His urine sample
FIGURE 13.5 and three known sugars, glucose, fructose, and galactose, were subjected to thin layer
Thin layer chromatographic chromatography. The following Rƒ values were obtained:
separation of morphine
(Lane 1), cocaine (2), nicotine Glucose 0.42
(3), methadone (4), heroin Galactose 0.35
(5), and a clinical sample Fructose 0.39
(6) using silica gel as the Urine sample 0.35
adsorbent material and a
QUESTIONS
mobile phase consisting of
ethanol: dioxin: benzene: (a) Which sugar is most likely to be present in Joe’s urine sample?
ammonium hydroxide (b) Is the diagnosis supported?
(4: 40: 50: 5).
13.3 COLUMN CHROMATOGR APHY 319
SELF-CHECK 13.3
Calculate the Rƒ value of the drugs and the unknown analyte used in Figure 13.5. What is the
most likely identity of the unknown analyte?
13.3 Column chromatography

In column chromatography, the stationary phase is organized into a column by packing it
into a glass or steel cylinder (Figure 13.6), which is in equilibrium with a liquid mobile phase.
Equilibrium is achieved by using a gravity-based system or motorized pump to move the
Buffer reservoir
(mobile phase)
Valve to add
sample to column
sampl
Pump Column
Colum
(stationary
(statio phase)
Recorder printing
the elution profile
Absorbance
UV monitor
Time, eluant Eluant

volume, or tube
number Fraction collector
FIGURE 13.6
Outline of a general column chromatography
system (see text for details).
mobile phase through the column. The sample is added to the top of the column as a band,
which is then flushed through the column by the mobile phase. Individual components in the
sample are separated within the column and eluted from it at different times as the mobile
phase leaves the column. The liquid leaving the column and carrying the (hopefully) separated
molecules is often called the eluant. It is normally collected in discrete fractions in a series of
tubes, either manually or automatically using a fraction collector. The different fractions can
Cross reference be analysed and an elution profile or chromatogram constructed by plotting a graph of the
See Chapter 11 ‘Spectroscopy’ amounts of substances eluted against time, eluant volume, or fraction number, as shown in
and 15 ‘Immunological Figure 13.6. Hopefully, different fractions will contain different components of the original
techniques’. mixture and so it will at least have been partially separated.
Whatever the form of column chromatography, it is necessary to be able to detect the analytes
of interest as they leave the column in the elution buffer. If the analytes are coloured then this
is not a problem, but many materials of biomedical interest are colourless. However, most pro-
teins and nucleic acids are colourless, but absorb light at about 280 and 260 nm, respectively
This is also the case for many other compounds of clinical interest. Thus they can all be detected
(a)
by monitoring the column using UV light, which will indicate when they are being eluted.
It is also possible to exploit biological activities of some substances to monitor their separa-
tion. For example, in the cases of enzymes the fractions of eluant can be analysed for the reac-
tion they catalyse. Carbohydrates are often detected by monitoring the refractive index of the
eluant, which changes when a sugar is present.
The movement of any one analyte through a column is described by its individual retention
time (t) or elution volume (Ve). The retention time for an analyte is the period between addition
of this substance to the column and its maximum concentration in the eluant. The term Ve is the
(b) volume of mobile phase required to elute a particular analyte from the column. It is used par-
ticularly when referring to separations by gel filtration (see below). The time taken for an analyte
to pass through the column without any interaction with the stationary phase is the dead time
(to) and the volume of mobile phase required for its elution is the void volume (V0).
Following column chromatography, the resulting chromatogram will show discrete peaks for
each of the separated components, which are symmetrical in nature (Figure 13.7 (a)). In some
cases, asymmetrical peaks are produced where there is a slow rise at the beginning of the peak
and a sharp fall after the peak. This is called fronting (Figure 13.7 (b)) and is due to overloading
of the column with the sample. Occasionally peaks occur where there is a normal rise before
(c) the peak but a slow fall after the peak and this is referred to as tailing (Figure 13.7 (c)). It is
usually due to retention of analytes on certain binding sites, usually hydroxyl groups, on the
stationary phase. Tailing can be eliminated by removing these hydroxyl groups by treating the
gel with a silanizing agent, such as hexamethyldisilazine.
The ability of a column to distinguish and separate two similar analytes a and b is given by
its selectivity (α), which is essentially the difference in retention times between two analytes
(a and b). It is calculated using the expression:
FIGURE 13.7 Selectivity (α) = (tb − t0)/ta − t0

(a) Shows the symmetrical
peaks following an ideal where ta and tb are retention times for a and b, respectively. The greater the value of α, the
separation of two components more selective the column.
by column chromatography. The resolution (R) of any separation technique is its ability to separate (or resolve) completely
Components isolated by one analyte (a) from another, similar one (b). In column chromatography, it can be determined
column chromatography using the expression:
showing (b) fronting and
(c) tailing. R = 2(tRb − tRa)/Wa + Wb
Va or tRa
a
Vb or tRb b
Peak Peak
height height
a b
FIGURE 13.8
Wa Wb Definitions of elution volumes or times, peak heights,
Elution volume or time and peak widths.
where Wa and Wb are the base widths of the peaks for a and b, respectively, as shown in Figure
13.8, and tRa and tRb represent their respective elution volumes. However, in the case of high
performance liquid chromatography, which we will discuss in Section 13.4, they correspond to
the retention times. Values of 1 or more for R are generally acceptable since they correspond
to a separation of 98% for two consecutive symmetrical peaks.
A number of different forms of column chromatography are routinely used in biomedical sci-
ence laboratories. These include:
• Gel filtration chromatography
• Ion-exchange chromatography
• Affinity chromatography
• Metal-chelation chromatography
• Hydrophobic chromatography
We will now consider each of these in turn.
Gel filtration
Gel filtration, also called permeation chromatography or ‘molecular sieving’, is a form of col-
umn chromatography that separates molecules according to differences in their sizes and to
some extent shapes. The column is composed of polymeric materials hydrated in the form of
microscopic porous beads. These are in equilibrium with a buffered solution suitable for use
with the molecules to be separated, which usually has a constant composition and pH. This is
an isocratic elution.
Molecules are washed out of the column in order of decreasing size. You can see this diagram-
matically in Figure 13.9. Each bead is permeated by a three-dimensional network of pores that
can only be penetrated by molecules of a specific size range. Molecules bigger than the pore
dimensions are completely excluded from the gel particles and will pass through the spaces
between the beads as they are washed through the column. The volume of liquid required
to elute such molecules from the column is its void volume (V0). Smaller molecules that can
enter the pores of the beads completely, however, will be distributed between the solvent that
is both inside and outside the beads and will therefore be eluted by a volume equal to the
total volume of the column (Vt). Molecules between these two extreme sizes will be eluted
by intermediate volumes. Hence, if a mixture of different sized molecules is applied to the
Mixture of
analytes to
be separated
Gel beads
of column
Column
bed
FIGURE 13.9
Schematic outline of
separation by gel filtration.
The separation of two
differently sized particles in a
mixture is illustrated. See text
for details. Movement of tubes
column the largest ones will pass through the column faster than the smaller ones. Thus the
larger molecule will require a lesser volume of eluant than the smaller ones to move it through
the column.
A variety of commercial materials are available for gel filtration. Sephadex is a commonly used
material and consists of particles of dextran that have been modified to give varying degrees
of cross-linking and, therefore, different pore sizes. Other substances used include agarose and
polyacrylamide. They are available in the form of dry ‘beads’ that swell in aqueous buffers to
form gels with pores of differing sizes. This allows various ranges of molecules to be separated
(Table 13.3).
SELF-CHECK 13.4
Which of the Sephadex materials listed in Table 13.3 would be most suitable to separate a
mixture of the proteins haemoglobin (Mr 64,500) and insulin (Mr 11,470)?
TABLE 13.3 Some commonly used gel filtration materials
Trade name Polymer Mr fractionation range

Sephadex Dextran G10 <700
G25 1 × 103 to 5 × 103
G50 1.5 × 103 to 3 × 104
G100 4 × 103 to 1.5 × 105
G200 5 × 103 to 6 × 105
Sephacryl Dextran S200 5 × 103 to 2.5 × 105
S300 1 × 104 to 1.5 × 106
S400 2 × 104 to 8 × 108
Sepharose Agarose 2B 1 × 104 to 4 × 106
4B 6 × 104 to 2 × 107
6B 7 × 104 to 4 × 107
Bio-Gel Polyacrylamide P2 1 × 102 to 1.8 × 103
P6 1 × 103 to 6 × 103
P30 2.5 × 103 to 4 × 104
P100 5 × 103 to 1 × 105
P300 6 × 104 to 4 × 105
Column performance
The commonest types of partition chromatography are paper and TLC chromatographies.
However, gel filtration is also a partition method, in which separations are based on differ-
ences in size. Thus, molecules to be separated by gel filtration must have different distribution
coefficient (Kd) values for their successful separation. In gel filtration, the Kd calculated for a
given type of molecule represents that fraction of the stationary phase available to it. In prac-
tice, Kd is difficult to determine in relation to gel filtration, and it is usually replaced by a Kav
value since there is a constant relationship between Kav and Kd. A value of Kav for a solute can
be determined using the equation:
Kav = (Ve − Vo)/(Vt − Vo)
where Ve is the elution volume of the solute. Molecules must have values of Kav that differ
significantly if separation is to be effective.
While components of a mixture are moving through a gel filtration column, their separation
is hindered by the diffusion of their molecules, which tends to mix them together. This can
restrict the resolution and lead to an incomplete separation, with the individual components
overlapping with one another as they leave the column. The net result is a less efficient separa-
tion, which you can see diagrammatically in Figure 13.10.
The efficiency of a column is a measure of the diffusion of the sample component during
separation. A column can be thought of as consisting of a number of adjacent zones called
FIGURE 13.10
Overlapping peaks resulting from the
incomplete separation of two components in a
mixture during column chromatography.
theoretical plates. The length in the column (L) is the height of a theoretical plate (H) multi-
plied by their number (N). The solute reaches equilibrium between the mobile and stationary
phases within each plate; thus, N is a measure of the efficiency of the column. It can be calcu-
lated using the formulae:
N = 16 (t/W)2 or 5.54 (t/Wh)2
where W is the peak base width and Wh the peak width at half the peak height as shown in
Figure 13.8, and t is the retention time. If W and Wh are expressed in units of time, then N is
unitless.
The ability of a column to separate (resolve) different substances increases with the length of
the column, L, since this increases N. Calculating the values of N for different columns can be
used to compare their relative efficiencies. Indeed, the simplest way to increase the efficiency
of a gel filtration step in purification would be to use a column of increased length. There are
obvious practical limitations to the maximum size of L that can be used!
The number of theoretical plates is related to the surface area of the particles forming the
stationary phase. Smaller particles have a relatively greater surface area than larger ones, thus
the smaller the size of the particles of the stationary phase, the more effective the resolution
of the column. If the size of N is calculated for a column of known length, the size or height
equivalent to a theoretical plate (HETP) can be calculated using the formula:
HETP = L/N
and is normally expressed in μm. The smaller the value of HETP, the better the chromato-
graphic performance. Thus, the HETP value can be used to assess changes in the efficiency of
the same column under different chromatographic conditions.
SELF-CHECK 13.5
Calculate the HETP of the column of length 64 cm that eluted a protein with a Ve of 90 cm3
using a suitable buffer at a flow rate of 45 cm3 h−1. The base width of the eluted protein peak
was equivalent to 30 cm3.
Applications of gel filtration

Gel filtration chromatography has a number of applications including:
• The purification of analytes
• Desalting samples
• Determining the Mr of biological molecules (analytical gel filtration)
Gel filtration has been used to purify molecules of biomedical interest by separating them
from larger or smaller contaminating molecules. For example, it has been applied to the puri-
fication of a large range of proteins, including enzymes, hormones, and antibodies, as well as
nucleic acids, polysaccharides, and viruses.
Desalting is the process of removing unwanted small molecules, particularly ions, from prepa-
rations of macromolecules. An example is the removal of phenol and ammonium sulphate
from partially purified nucleic acids and proteins, respectively. A column of Sephadex G-25 is
often used because the macromolecules elute in the void volume, while the smaller contami-
nant molecules are retarded on the column. This process is faster and more effective than the
alternative desalting process of dialysis.
The Mr of molecules can be estimated using analytical gel filtration to determine the Kav values (b)
of molecules of known Mr and similar type to that being investigated. A calibration curve can Mixture
be constructed by plotting the Kav values on the y axis against the logarithm values of Mr on the containing
x axis. The Mr of the unknown molecule can then be estimated from the graph once its Kav has analyte of
been determined. This method allows different columns to be compared. If this comparison interest
is not required, then a simple graph of elution volumes against the logarithm values of Mr will
Bead of
suffice to estimate that of the unknown analyte. column
Ion-exchange chromatography
Ion-exchange chromatography uses a stationary resin in the form of a charged gel that
is able to bind ions of the opposite charge, and a mobile liquid phase that is able to selec-
tively release the bound ions, as we explain below. Ion-exchange resins are insoluble porous Bound
cross-linked polymers that contain chemical groups of a particular charge. The extent of cross- analyte
linking of the resin influences the size of its pores. Polymers commonly used include those of
cellulose, polystyrene, and agaroses modified by the attachment of charged functional groups,
such as sulphonate (—SO3−) and ammonium derivatives (+NR3—). These are regarded as strong
exchangers because they are ionized over a wide range of pH values. In contrast, carboxylate
(—COO−) and diethylammonium [—H+N(CH2CH3)2] are weak exchangers because they are ion- Impurities
ized only within a narrow range of pH. washed off
column
Positively charged resins are anion exchange resins, while those with a negative charge are cation
exchangers. In both cases, ions of the opposite charge can bind to the column. Ion exchange Excess of
occurs rapidly and is an equilibrium process, which you can see in Figure 13.11 (a). The strength counterions
to analyte
(a) Column
matrix
Cation exchanger
– –
SO3 Na+ + +NH3—R SO3 +NH3—R + Na+
Anion exchanger
CH3 CH3
CH2 CH2
N+HCl− + −OOC—R N+H −OOC—R + Cl−
CH2 CH2
CH3 CH3 Release of

analyte
FIGURE 13.11
(a) Schematic illustrating the binding and release of ions from gels used in ion-exchange
chromatography. (b) Binding of an anionic analyte from a mixture of materials and its
release from an anion exchange resin.
of binding depends on the extent of ionization of the analytes in the mobile phase. The more
highly charged an ion, the tighter it will bind to oppositely charged groups on the column. The
total capacity of a column, which is the maximum number of exchangeable ions it can bind,
must not be exceeded. If it is, the ‘excess’ charged analyte molecules will simply pass through
the column without binding. Manufacturers normally give total capacities of ion-exchange res-
ins as milliequivalents of exchangeable groups per mg of dry resin.
The ion-exchange column must initially be equilibrated in a buffer whose composition and pH
will (a) stabilize the analyte of interest and (b) allow it to bind to the column. Once bound, the
components of the mixture need to be released (eluted) sequentially (Figure 13.11 (b)). This
may use an isocratic buffer system, which differs in composition and pH from the one used to
equilibrate the column. More often, a buffer of gradually changing pH or one whose concen-
tration of salt progressively increases (Figure 13.12) is used and this is referred to as gradient
elution. The former changes the charge of the bound molecules so they are released from the
column in to the mobile phase. The effect of increasing the concentration of salt is that its ions
displace the bound components from the column.
SELF-CHECK 13.6
Give the most probable order of elution of the following amino acids alanine [+NH–CH(CH3)
COO−], glutamate (+NH3CH(CH2CH2COO−)COO−) and lysine (+NH3CH(CH2CH2CH2CH2+NH3)
COO−) from a cation exchanger gel column at pH 7.
Affinity chromatography
Affinity chromatography relies on a specific binding between the analyte of interest and
some type of ligand, which you can see diagrammatically in Figure 13.13. The ligand is immo-
bilized by covalently attaching it to the column matrix (Box 13.2). Dextrans, agarose, and
polyacrylamide have all been used as matrices in affinity chromatography. The ligand is often
attached to the matrix using a chemical with an extended structure. This forms a spacer arm
that separates the ligand from the bulk of the column preventing the matrix interfering with
the specific binding of ligand and analyte.
0.8
0.60
0.6
Absorbance at 280 nm
[NaCl]/mmol dm-3
0.45
0.4
0.3
0.2
0.15
FIGURE 13.12 0.0 0.00

Sequential release of four proteins from an ion-exchange 0 100 200
column using a gradient of increasing salt concentration. Elution volume/cm3
When the sample is added to the column, only those molecules that can bind to the ligand
that is those of the analyte, will be attached to it. The column is then washed with buffer to
remove the nonbinding impurities. It is then possible to release the analyte of interest and
elute it from the column in a pure form. The release of the bound molecule requires that the
affinity between them and the ligands be reduced or that molecules be added to the eluting
buffer that have a greater affinity than the bound ligand and so can displace the analyte from
the ligand. For example, bound enzymes can be eluted from the column with solutions of
substrate, coenzyme, or an inhibitor that competes for the enzyme’s binding sites. If binding
requires the presence of a metal ion, ethylenediaminetetraacetic acid (EDTA), which chelates
and removes free metal ions, can be added. Often changing the pH of the mobile phase can
alter the conformation of bound proteins allowing their release.
Key Points
Chelation is the binding and therefore removal from solution of metal ions by sub-
stances such as some amino acids and EDTA.
Impurities Binding of Elution Isolation

analyte to of of
Analyte column impurities analyte
Ligand
Spacer
Gel (matrix)
Impurities separated Purified analyte

from analyte
FIGURE 13.13
Outline of the use of affinity chromatography in purifying an analyte.
See text for details.
BOX 13.2 Selecting a matrix for

affinity chromatography
An ideal matrix for affinity chromatography will have at least some of the following
characteristics. It must:
■ Form a stable link between the ligand to the matrix

■ Be stable during binding of the macromolecule and its subsequent elution
■ Not interact with the macromolecules generally to avoid non-specific adsorption
■ Allow the mobile phases to flow freely through the matrix readily
■ Be a hydrophilic polymer with pore sizes large enough to allow free passage of
molecules.
Affinity chromatography has been used to isolate:
• Enzymes
• Antibodies/antigen
• Lectins
• Receptor proteins
• Nucleic acids
Enzymes can be isolated and purified from complex mixtures by using their substrates, coen-
zymes, or competitive inhibitors as ligands attached to affinity columns. Antibodies and/or
antigens can be purified by immobilizing either the complementary antigen or antibody,
respectively, on the column matrix. Lectins are glycoproteins that can bind other glycoproteins
by recognizing all or part of their carbohydrate component. Affinity chromatography columns
that use immobilized lectins have been used to isolate glycoproteins, particularly those solu-
bilized from cell membranes. The receptors of a number of hormones have been isolated by
using immobilized forms of the appropriate hormone as an affinity ligand. Once bound to the
column, the receptor is released in ways that resemble the purification of enzymes with, for
example, eluting buffers containing the hormone in free solution.
Nucleic acid strands are able to bind other strands that possess a complementary sequence
of nucleotides, as explained in Chapter 16. Individual nucleotides or short single stranded oli-
gonucleotides can be immobilized for use as affinity ligands. These have been used to extract
nucleic acids with the complementary nucleotide sequence or to isolate nucleic acid binding
proteins that are capable of recognizing and binding the nucleotide or oligonucleotide.
Metal-chelation chromatography
Metal-chelation chromatography can be regarded as a form of affinity chromatography
which exploits differences in the strengths of binding of different amino acids to the immo-
bilized ions of some heavy metals. Metal ions, such as Cu2+, Zn2+, Hg2+, and Ni2+, are immobi-
lized by chelation with aminodiacetic acid, which, in turn, is covalently attached to suspended
agarose gel. The suspension of agarose is then poured into a tube to form a column. The side
chains of a number of amino acid residues found in proteins, including imidazole groups of
13.4 HIGH PERFORMANCE LIQUID CHROMATOGR APHY 329
histidine, thiol groups of cysteine, and indole groups of tryptophan, can bind to the metal
ions. When mixtures of proteins are washed through the column, some will bind to the metal
ions and be retained. The bound proteins are eluted sequentially by, for example, adding free
amino acids to the mobile phase, which can displace the bound proteins. Thus, mixtures of
proteins are at least partially separated.
Hydrophobic interaction chromatography

Hydrophobic interaction chromatography can be regarded as a form of affinity chroma-
tography that is based on differences in the binding of hydrophobic sites on the surfaces of
protein molecules to immobilized hydrophobic ligands attached to a solid support matrix in
the form of a column. Hydrophobic interaction chromatography is generally used to separate
mixtures of proteins. Proteins often have hydrophobic sites on the surfaces of their molecules
due to the presence of certain amino acid residues, such as those of valine, leucine, isoleucine,
and phenylalanine. However, the hydrophobic sites of different proteins vary in their nature
and area, so the strengths of binding of different proteins to the column vary.
The bound proteins are eluted from the column by reducing the hydrophobic interactions
between sample molecules and the stationary phase by, for example, eluting the column with
a solution of a non-ionic detergent, such as Triton X−100, or by changing the pH or tempera-
ture of the eluting mobile phase.
13.4High performance liquid

chromatography
Increasing the length of a column and its number of theoretical plates improves its ability to
resolve mixtures into their components (Section 13.3). However, long and densely packed
columns of small particles have a greater resistance to the flow of the mobile phase. To over-
come this resistance, and gain the advantages of faster and increased resolution, columns have
been developed that can withstand the relatively high pressures produced when high pressure
pumps are used to force the mobile phase through the column. The pumps used should give
continuous flows of at least 10 cm3 per minute. This group of techniques is called high per-
formance liquid chromatography (HPLC). A typical HPLC system is shown in Figure 13.14.
The columns are contained in tubes made of stainless steel that can withstand pressures up
to 5.5 × 107 Pa. Columns typically have lengths of 15–50 cm and are normally 1–4 mm in
diameter. The stationary phase consists of tightly packed small particles of material giving a
correspondingly large surface area.
Three types of material are used in forming these particles:
• Microporous
• Pellicular
• Bonded
Microporous particles have diameters of 5–10 μm and contain pores with diameters of less
than 2 nm. Pellicular particles are porous and coated onto an inert solid core, such as a glass
bead of about 40 μm diameter. Bonded particles have a stationary phase that is chemically
joined to an inert support, such as silica. Columns packed with pellicular material offer effi-
ciencies, but only accept samples of small volumes compared with those with microporous
Solvent reservoir
Solvent filter
Pump Gradient
programmer
Solvent filter
Injector port system

for adding sample
Guard column
Column
Absorbance
Detector
FIGURE 13.14 Recorder
General components of a high Time
performance liquid chromatography Waste collector
(HPLC) system.
material. Hence, the latter are generally preferred. Bonded phases give excellent chromato-
graphic separations, and are chemically and mechanically stable, giving long-lasting columns.
Partition chromatography (see below) usually uses a liquid stationary phase coated onto an
inert microporous or pellicular support. A problem with such supports is the tendency of the
stationary liquid phase to be washed away by the mobile phase. However, bonded phases
have been developed where the liquid stationary phase is chemically joined to the silica to
overcome this problem.
The choice of mobile phase depends on the nature of the analytes to be separated. The polar-
ity of the solvent chosen should be such that effective partitioning of components in the sam-
ple occurs between the two phases. All the solvents used must be of high purity because
contaminants can affect the column and also interfere with the detection system (see below).
Solvents also require degassing, not only because gas in the solvent can adversely affect the
separation of the sample components, but also because it can interfere with the continuous
monitoring of the eluant by, for example, absorbing UV light. Unfortunately, some solvents, for
example, acetone, also absorb at these wavelengths. The mobile phase should not react with
the stationary phase nor break the chemical bonds linking it to its supporting material, since
this will, of course, decrease the effectiveness of purification and/or interfere with the detec-
tion of the components leaving the column.
Detector response/arbitrary units
2 4
3
1. Oxytocin
2. Angiotensin II
3. Angiotensin I
1 4. Insulin
FIGURE 13.15
Use of reverse phase high performance liquid chromatography to
separate a mixture of proteins and protein hormones. The hormones
separated are 1, oxytocin; 2, angiotensin II; 3, angiotensin I, and
0 12
Time/min 4, insulin. Chromatogram courtesy of Varian, Inc.
The correct application of samples onto HPLC columns is necessary to obtain the best resolu-
tions. The sample is injected onto the column using a microsyringe. There are two ways of
applying the sample: stop flow and loop injection systems. In the former, the pump is switched
off so that the pressure inside the column falls. The sample is then applied and the pump then
switched back on. Loop injection systems allow the sample to be added while there is still a
high pressure in the column. Repeated additions of impure samples can cause the column 1 3
to lose its resolving power, which can be prevented by using a guard column. This is a short
column of material similar to that of the main one, sited between the sample injector system
and the analytical column. It is able to retain contaminating material, so protecting the main
column and extending its lifespan.
High performance liquid chromatography methods generally involve adsorption and partition
Absorbance
effects. Partition chromatography exploits differences in the solubilities and partition coef- 2
ficients of polar substances as the bases for separating polar substances between stationary
and mobile phases. The two usual techniques of partition chromatography are normal phase
and reverse phase liquid chromatography. In normal phase, the stationary phase is a polar
substance whereas the mobile phase is a non-polar solvent. These are reversed in reverse
phase liquid chromatography (Figure 13.15), hence the name! Here, the stationary phase is a
non-polar substance and the mobile phase is a polar solvent. This is the commonest type of
HPLC. In reverse phase liquid chromatography, non-polar molecules dissolve in the non-polar
stationary phase to varying extents, whereas polar molecules pass rapidly through column
within the mobile phase.
However, HPLC can also be used with ion-exchange (Figure 13.16), exclusion and affinity 0 1 2 3 4 5 6
chromatographies. Time/min
1 = ovalbumin
Detector systems used in HPLC 2 = cytochrome c
3 = lysozyme
Whatever the form of HPLC, a sensitive method is required to detect the separated compo- FIGURE 13.16
nents, including the analytes of interest, as they are eluted from the column. Often the eluant Separation of a mixture
passes through a spectrophotometric cuvette or flow cell, which are described in Chapter of proteins using a high
11, and its absorbance in the ultraviolet region of the spectrum is continuously monitored to performance liquid
detect proteins and nucleic acids, which absorb maximally at about 280 and 260 nm, respec- chromatography system with
tively. However, other detection systems are deployed. These include fluorescence and elec- an ion-exchange column.
trochemical detectors. Fluorescence detectors have greater sensitivity than simple ones based The proteins concerned are
on absorbance. Their applications are, however, limited due to the relatively small number of 1, ovalbumin; 2, cytochrome c,
compounds of interest that fluoresce. Electrochemical detectors are increasing in popularity. and 3, lysozyme.
Most are amperometric: they measure a current produced by oxidation–reduction reactions

Cross reference
of the analyte at an electrode.
See Chapter 9 ‘Electrochemistry’.
Whatever the method of detection, calibration of the system is necessary to compensate for
differences in the volumes of samples, variations in their injection into the system, evapora-
tion of solvent, and other irregularities. This is usually achieved using an internal standard
(IS) that is similar in structure to the analyte. The same quantity of IS is added to the sample
and to each solution of standard. You can see this in Figure 13.17. The peak height ratio
(Figure 13.17 (a)) is measured for each eluted analyte or standard concentration using the
expression:
Peak height ratio = height of analyte peak/height of internal standard peak
A graph of peak height ratio on the y axis against the corresponding concentrations of stand-
ards on the x axis will give a straight line graph as you can see in Figure 13.17 (b).
(a)
X IS
Detector
A B
0 10 20
Time/min
A
Peak height ratio =
B
(b) 2.0
1.5
Peak height ratio
1.0
0.5
0
0 20 40 60 80
Concentration/mg dm–3
FIGURE 13.17
(a) Schematic showing the separation of an analyte X (or a standard of fixed concentration)
and an internal standard (IS). The ratio of the heights of their peaks give a peak height ratio
for that concentration. Calculating peak height ratios for a number of concentrations of
the standard gives a calibration graph as shown in (b), which can be used to determine the
concentration of an analyte in a sample.
SELF-CHECK 13.7
A method to determine the concentration of drug X in clinical samples was established
using HPLC. This method was calibrated using a range of standard concentrations of X and
an internal standard, IS. A serum specimen from a patient was analysed and the chromato-
gram produced is shown in Figure 13.17 (a). Use this chromatogram and the accompanying
calibration curve (Figure 13.17 (b)) to determine the concentration of X in the serum of the
patient.
Applications of high performance liquid

chromatography
The major advantages of HPLC are its speed, wide applicability, and sensitivity. The versatility
of HPLC ensures it has many applications in biomedical science. It has been used to sepa-
rate and analyse a wide range of materials of interest including amino acids, proteins, nucle-
otides, nucleic acids, drugs, and vitamins. For example, ion-exchange resins are used in HPLC
to determine the proportion of glycated haemoglobin (HbA1c) to the total haemoglobin in the
blood of diabetic patients (Figure 13.18). In erythrocytes, glucose binds to haemoglobin non-
enzymatically and the extent of this glycation depends on the degree and duration of hyper- Cross reference
glycaemia. Measurements of HbA1c give an estimate of average glycaemia over the preceding You can read more about
two months. In non-diabetics, the proportion of HbA1c is less than 6% whereas that in diabetic diabetes in Chapter 13 ‘Diabetes
patients is often in excess of 9%. A high relative proportion of HbA1c reflects a poor control of mellitus and hypoglycaemia’,
blood glucose concentration. It is well established that such patients are more susceptible to in the companion text Clinical
Biochemistry.
long-term micro- and macrovascular complications.
20.0
17.5
15.0
12.5
A1c 1.03
A1c/%
1.65
10.0
1.55
7.5
0.11
0.32
0.21
0.78
5.0
0.60
1.95
1.77
FIGURE 13.18
2.5 Use of high performance liquid chromatography to
separate and estimate the proportion of glycated
0 haemoglobin (HbA1c) to total haemoglobin.
0 0.5 1.0 1.5 2.0 2.5 3.0 Courtesy of Department of Clinical Biochemistry,
Time/min Manchester Royal Infirmary, UK.
13.5 Gas–liquid chromatography

Gas–liquid chromatography (GLC) partitions molecules between a stationary phase consist-
ing of a microscopic layer of liquid or polymeric material contained within a glass or metal
tubing, called a column, and a mobile gas phase. The mobile phase is forced continuously
through the column and carries the components of the mixture being analysed. Samples
for analysis are injected into the system where they are vaporized and introduced into the
gas phase. The gas is heated to comparatively high temperatures to keep the components
of the sample in the gas phase, allowing them to be carried along in the mobile phase.
Those components that have a greater affinity for the stationary liquid phase have their
movement through the column retarded. Those more soluble in the gas are carried through
the column faster. The partitioning of the components of the mixture (sample) between
the stationary and mobile phases causes different compounds to elute from the column at
different retention times.
A typical GLC system is shown in Figure 13.19. The columns have capillary diameters between
0.03 and 1 mm and may be as long as 100 m. Two types of capillary columns are used: wall-
coated open tubular (WCOT) and support-coated open tubular (SCOT). In the former, a
liquid stationary phase is coated directly onto the wall of the capillary column, whereas the
latter uses a liquid stationary phase coated onto a support material which is then bonded to
the walls of the column. Stationary phases must be non-volatile and not enter the gaseous
phase at the temperatures used. They are usually high boiling point organic compounds
such as silicone greases.
Injector port
for samples
Oven
Oven
Waste vent
Oven
Column
Detector
Carrier gas Recorder
FIGURE 13.19
Typical outline of a gas-liquid chromatography (GLC) system.
13.5 GAS–LIQUID CHROMATOGR APHY 335
The mobile phase is usually an inert gas such as helium or argon or a nonreactive one, such
as nitrogen. The gas is forced through the column at a rate of 40–80 cm3 per minute. The col-
umn is contained within an oven that maintains the column at higher than ambient tempera-
ture during separation. This is necessary because, in general, the higher the temperature, the
greater the volatility of molecules. Temperature therefore influences the separation because
higher temperatures speed up the movement of more volatile molecules through the column.
Temperatures may be set at constant values between 50 to 250°C to give isothermal condi-
tions. However, for some applications it may be necessary for it to be pre-programmed to
increase by, for example, 10°C every minute.
The sample for analysis is dissolved in a solvent, such as acetone or methanol, and injected
into the GLC system using a microsyringe. The injection region of the column is usually at a
higher temperature than the column itself. This ensures the rapid and complete vaporization
of the sample. Wall-coated open tubular columns have a relatively small amount of stationary
phase and so only small volumes of samples can be analysed. The sample injection port has a
splitter system, which allows a small fraction of injected sample to reach the column and the
remainder is vented as waste. The greater capacity of SCOT columns means there is no need
for a splitter system and the sample is injected directly into the column.
In some ways, GLC resembles column chromatography. However, it differs from other forms
of chromatography in that the molecules must be volatile, that is have low boiling points, if
they are to move through the column and be separated. Gas–liquid chromatography is there-
fore particularly suitable for separating hydrophobic molecules or ones of low polarity, which
therefore have low boiling points.
Molecules that are non-volatile can, however, often be converted to volatile derivatives prior Cross reference
to their separation. For example, long chain fatty acids can be converted to their methyl- You can read about mass
esters and monosaccharides to trimethylsilyl compounds. Both of these types of derivatives spectrometry in Chapter 11
have much lower boiling points than their parent compounds and can be analysed by GLC ‘Spectroscopy‘.
(Figure 13.20).
Like thin-layer chromatography (Section 13.2), GLC can be combined with other analytical
techniques. For example, clinical samples can be subjected to GLC and the separated compo-
nents identified by tandem mass spectrometry. The peroxisomal diseases adrenoleukodystro-
phy, Zellweger syndrome, and Refsum’s disease, although rare, are extremely debilitating and
are associated with high concentrations of very long chain fatty acids (VLCFAs) in the blood of
patients. Thus, GLC–tandem mass spectrometry is used, for example, to investigate the VLCFA
composition of samples of plasma from patients suspected of having these diseases.
Gas–liquid chromatography is thus a versatile technique and can be used to analyse a wide
range of substances including lipids, alcohols, phenols, and a number of drugs. The major
advantages offered by GLC in separating such materials are:
• Excellent resolution of a wide range of materials
• Reproducibility of separations
• Excellent sensitivity of its analyte detection systems.
Detector systems used in GLC

The most commonly used detectors in GLC are flame ionization and electron capture
detectors (flame ionisation detector (FID) and electron capture detector (ECD), respectively).
In the former, the gas from the column is passed through a hydrogen flame. Most organic
3 4 5 6 7 8
Peak Identification
1 TAG (Tri-C28)
2 TAG (Tri-C30)
3 TAG (Tri-C32)
3.0 2
4 TAG (Tri-C34)
5 TAG (Tri-C36)
6 TAG (Tri-C38)
7 TAG (Tri-C40)
2.5 8 TAG (Tri-C42)
9 TAG (Tri-C44)
10 TAG (Tri-C46)
11 TAG (Tri-C48)
9 12 TAG (Tri-C50)
2.0
13 TAG (Tri-C52)
Detector response
14 TAG (Tri-C54)
1.5
10
1.0
11
1 12 13
0.5 4 14
0.0
2 3 4 5 6 7
Time/min
FIGURE 13.20
Separation of a number of triacylglycerols (TAGs) by gas–liquid chromatography (GLC).
The lengths of the fatty acids in each TAG are given as the number of carbon atoms
present. Courtesy of Varian, Inc.
compounds produce ions and electrons as they move through creating a small current, which
is amplified and used as a detection signal. The main advantages of FID are that it is extremely
sensitive, capable of detecting as little as 0.1 pg, which is 1.0 × 10−13 g of an analyte, and has
a linear response over a wide range of concentrations. Its principal disadvantage is that the
sample is destroyed during analysis.
In electron capture detectors the gas from the column is ionized by a radioactive source to
release electrons. These produce a current across two electrodes. However, when an electron-
absorbing compound is eluted from the column, it captures some of the electrons produced
from the gas. The drop in current is recorded and so signals the elution of the compound from
the column. Like FID, electron capture detectors are sensitive but their obvious disadvantage
SUMMARY 337
is that they can only be used with substances capable of capturing electrons, for example
polychlorinated compounds such as dichloro-diphenyl-trichloroethane (DDT).
In both types of systems, it is customary to incorporate a known amount of an analyte into the
test sample as an internal standard. This allows variations between samples, such as changes in
volume, to be accounted for. However detected, the amount of a compound eluted from the
column is recorded against its retention time. Retention times have a characteristic value for a
given compound under set conditions as shown in Figure 13.20, for example. Comparison of
the retention times for different compounds is equivalent to the analytical power of GLC, in a
manner similar to HPLC (Section 13.4).
SELF-CHECK 13.8
Predict the order of elution of the methylesters of the following fatty acids:
Decanoic acid (CH3 (CH2) 8COOH)

Eicosanoic acid (CH3 (CH2) 18COOH)
Hexadecanoic acid (CH3 (CH2) 14COOH)
Octadecanoic acid (CH3 (CH2) 16COOH)
Tetradecanoic acid (CH3 (CH2) 12COOH)
SUMMARY
■ Chromatography is a set of techniques that separate mixtures whose components differ in
their distribution or partition coefficients
■ Chromatographic techniques can also be useful in identifying individual components of

the mixture and in providing quantitative data in some instances
■ Planar chromatographies include paper and thin layer chromatographies. Both separate
mixtures using a mobile liquid phase moving through a bound stationary liquid. Thin layer
chromatography is a simple, quick, and inexpensive technique, suitable for qualitative but
not quantitative analyses. It is used to separate a wide range of components, which can
often be identified by their Rƒ values using those of known standards
■ Column chromatographies comprise a range of techniques in which an immobilized solid

forms a cylinder of material, through which a mobile liquid phase is allowed to perco-
late. Molecules in a mixture are applied to the column and separate due to differences in
their sizes as in gel filtration chromatography, their charges in ion-exchange chromatog-
raphy, hydrophobicities in hydrophobic chromatography, and ligand binding in affinity
and metal chelation chromatographies. Whatever the type of column chromatography,
components of the mixture differentially distribute between the stationary and mobile
phases. Thus, individual components leave the column and can be identified by their elu-
tion volumes or retention times. An effective separation of the components of a mixture
is indicated if they appear as discrete peaks on the chromatogram that do not overlap or
show tailing or fronting
■ Specialized types of column chromatography materials are used to increase the surface
area of the stationary phase, allowing the mobile liquid phase to be forced through the
column at high pressure, as is the case in high performance liquid chromatography. This
gives an improvement in resolution over low pressure systems
■ Gas–liquid chromatography separates mixtures of volatile compounds using a stationary

liquid phase and a mobile gaseous phase. Partitioning of the individual components of the
mixture between the two phases, effects their separation. Individual components leaving
the column are detected by their absorbance, fluorescence, or electrochemical proper-
ties, and identified by their retention times
FURTHER READING
● Bollag DM. Ion exchange chromatography. Methods in Molecular Biology, 1994; 36:
11–22.
● Cutler P. Size-exclusion chromatography. In: P. Cutler (ed.) Protein Purification

Protocols. Humana Press, Totowa, 2004.
● Dong M. Modern HPLC for Practising Scientists, John Wiley & Sons, Harlow, 2006.
● Ho WF, Stuart B. High performance liquid chromatography (Coordinating author

Prichard, E). Royal Society of Chemistry, Cambridge, 2003.
● Lunn G. HPLC methods for pharmaceutical analysis, Vols 2–4. Wiley, New York,
2004.
● Stuart B. Gas Chromatography. (Coordinating author Prichard, E.). Royal Society of

Chemistry, Cambridge, 2003.
● Wall PE. Thin Layer Chromatography: A Modern Practical Approach. Royal Society of
Chemistry, Cambridge, 2005.
● Zachariou M. Affinity Chromatography: Methods and Protocols, Humana Press,

Totowa, 2008.
Three Useful Journals

■ Biomedical Chromatography
■ Chromatography Today
■ Journal of Chromatography B: Biomedical Sciences and Applications
QUESTIONS
1. Chloramphenicol, fluorouracil, and paracetamol are three therapeutic drugs. Their log
KOW values are 1.14, –0.85, and 0.48, respectively. List them in order of their increasing
water solubility.
QUESTIONS 339
2. In chromatography, the separation of a mixture of molecules is determined by their dif-

ferential distributions between two different phases. Which ONE of the following terms
best describes this relationship?
(a) Retardation factor
(b) Partition coefficient
(c) Elution volume
(d) Void volume
(e) Electrophoretic front
3. Which of the following statements concerning thin layer chromatography (TLC) is

INCORRECT?
(a) The adsorbent material often used is silica gel

(b) The mobile phase moves up the thin layer by capillary action
(c) The Rƒ value is always less than 1
(d) TLC is a simple, relatively inexpensive technique
(e) The separation of molecules is based on differences in their sizes
4. Predict the probable order of elution from a column of Sephadex G-100 of the follow-
ing proteins: carbonic anhydrase (Mr 30,000), catalase (Mr 250,000), chymotrypsinogen
(Mr 23,250), fibrinogen (Mr 330,000), haemoglobin (Mr 64,500), and insulin (Mr 11,470).
You may find it helpful to consult Table 13.3 when attempting this question.
5. Gel exclusion chromatography was used to determine the Mr of catalase. The elution
volumes (Ve) of five proteins of known Mr from a column of Sephacryl were determined,
as was that of catalase. Chromatography was carried out at 4°C using a dilute buffer of
pH 7.4. Under these conditions, the structures of all the molecules concerned are pre-
served. Use the data in the following table to determine Mr of catalase.
Protein Mr Ve /cm3
Hexokinase 102, 000 24.7
Aldolase 158,000 22.7
α-amylase 199,000 21.6
Fibrinogen 330,000 19.4
RNA polymerase 450,000 18.0
Catalase ? 20.7
6. Amino acids are polar molecules and mixtures of them cannot be resolved by gas–liquid
chromatography. Explain a general strategy for overcoming this limitation.
7. Which ONE of the following properties of molecules generally forms the basis for their
separation by affinity chromatography?
(a) Size
(b) Volatility
(c) Charge
(d) Shape
(e) Polarity
8. Galactosyl transferase catalyses the reaction:
UDP-galactose + acceptor → UDP + galactose-acceptor
The enzyme binds to a UDP-agarose affinity column in the presence of Mn2+. Suggest a
plausible method of eluting it from the column.
9. Two molecules, X and Y were separated using HPLC. They had retention times of 3 min-
utes 30 seconds with a base width of 38 seconds, and 5 minutes and 30 seconds and base
width 42 seconds, respectively. A molecule Z, which was completely excluded from the
column, had a retention time of 1 minute and 30 seconds. Determine (a) the selectivity
of the column and (b) the resolution of X and Y. Comment on each value.
14
Electrophoresis
Learning objectives
■ Explain the underlying principles of electrophoresis

■ Describe the uses of the major types of electrophoretic separation techniques
■ Describe how the positions of separated components may be determined following
electrophoresis
■ Explain the underlying principles of isoelectric focusing
■ Discuss the uses of pulsed field and two-dimensional gel electrophoresis
Introduction
Electrophoresis is the movement of charged molecules or ions in an electric field (E ). Ions
that differ in their charge and/or mass will move at different rates such that, if they have a
common starting point they will separate over time as some will move faster than others. As
a result, the components of a mixture will be separated. Thus, electrophoresis is a separa-
tion technique. However, it can also be adapted to provide analytical data, such as the size
(Mr) of molecules. Many biological molecules, such as amino acids, peptides, proteins, nucle-
otides (nt), and nucleic acids, possess groups that can ionize, and give the molecules an overall
negative or positive charge. Hence, electrophoresis has many applications in the biomedical
sciences: in separating proteins and nucleic acids, in assessing purity of biological samples,
and in estimating the sizes of molecules.
14.1 Principles of electrophoresis

If a potential difference or voltage (V) is applied between two electrodes, it produces an elec-
tric field or potential gradient (E ). The value of E can be calculated by dividing the applied
voltage by the distance (d) between the electrodes:
E = V/d
342 14 ELECTROPHORESIS
Anode Cathode
+ –
Cation
+ Direction of migration
+ –
FIGURE 14.1
+
Direction of migration
– Anion
–
Schematic showing the
movements of anions and
Electric field (E)
cations in an electric field.
Values of E are normally quoted in units of V cm−1. When an E is applied to molecules carry-
ing a charge of q, the force produced is equal to Eq; this force drives the molecules towards
the electrode of opposite charge. For example, the positive electrode or anode will attract
negatively charged molecules or anions; whereas the negative electrode or cathode attracts
positively charged ones or cations (Figure 14.1).
SELF-CHECK 14.1
Calculate the value of the electric field (E) when a potential difference of 200 mV is applied
between two electrodes 20 cm apart.
During electrophoresis, the applied potential difference, usually expressed in volts (V), is nor-
mally kept at a constant value. However, in some cases, electrophoresis is performed using
a constant current, and the voltage will vary with the electrical resistance of the system.
Occasionally, electrophoresis is carried out under conditions of constant power, which is the
product of the current and voltage.
The strength of E can be increased by simply increasing the voltage applied between the elec-
trodes. An increase in voltage increases the force that the molecules experience, making them
move more quickly. As a result, an increase in voltage shortens the time needed to complete
electrophoresis experiments. However, it is not always practical to use high voltages because
of their heating effects. Heating can increase the diffusion of molecules in the sample and may
also cause convection currents, both of which decrease the efficiency of separation. Higher
temperatures can also result in the thermal denaturation of some types of biological mol-
ecules, such as proteins and nucleic acids.
Electrophoretic mobility
Cross reference
You met frictional coefficients in
The movement of ions subjected to an E is opposed by a frictional resistance, which depends,
Chapter 12 ‘Centrifugation’.
in turn, upon the frictional coefficient (ƒ) of the ions.
The velocity (v) of the ion is given by:

v = Eq/ƒ
However, the movement of an ion is often described by its electrophoretic mobility (μ) that is
its velocity per unit of electric field. Electrophoretic mobility can be obtained by dividing v by E:
μ = v/E
14.2 FACTORS AFFECTING ELECTROPHORETIC SEPAR ATIONS 343
Combining these equations, gives the expression:
μ = q/ƒ
Thus, in theory it should be possible to obtain the value for ƒ of a molecule from its mobility in
an electric field if its net charge is known, which should make it possible to obtain information
about its shape and size. In practice, this has not proved possible, because the simple equa-
tions given above do not adequately describe electrophoretic processes. For example, interac-
tions between migrating molecules and the medium in which electrophoresis is performed all
complicate the system. However, what is clear is that during electrophoresis, molecules that
differ in their values of μ will separate from one another.
Initially, electrophoresis experiments were carried out in free solution, that is, a buffer, but this
poses problems associated with diffusion of sample molecules, which opposes their move-
ment during electrophoresis. Subsequently, porous mechanical support media were intro-
duced, through which both buffer ions and sample molecules migrate during electrophoresis.
This type of technique greatly reduces the diffusion of sample molecules, allowing the differ-
ent substances in the sample to migrate as discrete zones or bands during electrophoresis.
Indeed, the separations can be made extremely efficient by performing the electrophoresis in
support media that offer greater frictional resistance to larger molecules than to smaller ones
(Section 14.2).
Factors affecting electrophoretic

14.2
separations
The equipment used for electrophoresis typically consists of an electrophoresis unit connected
to a power pack, as you can see in outline in Figure 14.2. The electrophoresis units may be
arranged to hold the supporting material in either a horizontal or a vertical arrangement. The
power pack provides a flow of direct current to the electrodes in each compartment of the unit.
Buffer solution in the compartments and in the supporting medium that links them then carries
electricity between the two electrodes to generate a flow of electricity through the system.
The electric field generated during electrophoresis is associated with the current that is car-
ried by the buffer connecting the electrodes, although a relatively small proportion of it is also
conducted by the ions of the sample. If the ionic strength of the buffer is increased, the pro-
portion of current carried by it will also increase; whereas that carried by the sample ions will
decrease. Consequently, their velocities will slow. If the ionic strength of the buffer is decreased,
the converse happens; the proportion of the current carried by the ions of the sample will
increase and their rate of migration will speed up. However, although a low ionic buffer
decreases the overall current and causes less heat production, there is greater diffusion of the
sample due to an increase in separation time and, therefore, some loss of resolution or sepa-
ration may occur. Thus, the E chosen is always a compromise between the time required to
separate the molecules and the resolution obtained.
During electrophoresis, all ions are subjected to the same E and separation occurs because
different substances have different electrophoretic mobilities. Individually, the mobility of
charged ions is influenced by a number of factors including:
• Their sizes and shapes
• The buffer in which electrophoresis is performed
• The supporting medium

(a)
Platinum wire cathode Platinum wire anode
– Direction of migration
+
Supporting
medium
Power
supply
(b) Platinum wire cathode
–
Direction
of
Power migration Supporting
supply medium
FIGURE 14.2
Schematics showing (a) horizontal and (b) vertical
electrophoresis systems. Note how in each system,
the electrodes are positioned in their own buffer +
compartments and the buffer completes the
electric circuit between them. The electrodes in
both systems are made from a platinum wire. Platinum wire anode
The sign of the charge (+ or −) of an ion will determine whether it moves towards the anode
or cathode, as described in Section 14.1. However, the mobility of an ion during electrophore-
sis at constant voltage depends solely on the ratio q/ƒ irrespective of the direction in which
it is moving. For mixtures of molecules with similar conformations, for example, globular pro-
teins or linear DNA molecules (Chapter 16), then the variables affecting their mobility are
restricted to the individual charges and ƒ values. The value of ƒ depends to a great extent upon
the mass of the molecule. Thus, each type of molecule in the mixture will migrate in a given
electric field at a velocity proportional to its mass-to-charge ratio. The shape of molecules also
influences their mobility. For example, globular proteins migrate differently to fibrous proteins
of the same mass-to-charge ratio.
During electrophoresis, a buffer is used to saturate the supporting medium. The choice of
buffer can affect the migration of particles in a number of ways. Its pH influences the ioniza-
tion of some chemical groups within the sample molecules, in particular that of amino and
carboxyl groups of polypeptides, and so determines the overall charge of the ions. pH is tem-
perature dependent, which is a major reason for performing electrophoresis under conditions
in which the temperature remains constant.
14.3 DETECTION OF SEPAR ATED COMPONENTS 345
The ionic composition of the buffer is also a factor to be considered. Ions in the buffer, such as
Na+, K+, Mg2+, Cl−, and those of phosphate, can all interact with charged groups on molecules
of the sample. This results in them becoming surrounded with ions of the opposite charge, and
decreases both their overall mass-to-charge ratio and mobilities.
In general, electrophoresis is carried out in buffers of low ionic strength that are compatible
with the solubilities of biological macromolecules. Such compatibility reduces the types of
interaction described above to minimal levels. In some cases, however, the binding of buffer
constituents to sample molecules can be exploited. For example, borate buffers are used when
separating carbohydrates as their components bind to carbohydrates. This converts neutral
carbohydrates to charged complexes, which can then be subjected to electrophoresis.
The support medium used in electrophoresis is usually inert, although it can lead to molecu-
lar sieving, adsorption and electro-osmosis, each of which can influence the rate of migra-
tion of sample ions. Some support media consist of gels that contain pores (Sections 14.6
to 14.8) that restrict the flow of ions, particularly those of large Mr, during electrophoresis.
Smaller ions experience a lower frictional resistance as they migrate through the gel and so
travel faster and therefore further than larger ones for a given time. This effect is referred to as
molecular sieving and, as we shall see in Sections 14.6 to 14.8, allows separation of molecules
based on their size alone.
Adsorption is the retention of sample molecules onto the surface of the support medium.
Adsorption slows the migration of sample molecules and causes tailing of the sample so that it Cross reference
moves in the shape of a comet, that is, in an extended area, rather than as a distinct zone. Thus, We discuss adsorption in the
context of chromatography in
the individual components of a mixture do not separate into distinct bands and the resolution
Chapter 13 ‘Chromatography’.
of separation is impaired.
Electro-osmosis occurs in support media that possess negatively charged groups on their
surface. These groups attract cations present in the buffer. However, the cations, like all ions
in solution, are hydrated and surrounded by a layer of water molecules. When an electric
field is applied, the cations and associated water are attracted towards the cathode and move
towards it in a flow that opposes the migration of anions towards the anode. The force gen-
erated by movement of the water is called electro-osmosis and may be so large that weakly
charged anions can be carried towards the cathode! Electro-osmosis is the basis of capillary
electrophoresis (CE), which we will discuss in Section 14.9.
Detection of separated
14.3
components
Following electrophoresis, the positions of the separated components on the support medium
need to be detected, other than in the case of capillary electrophoresis (Section 14.9). The two
major groups of compounds we will consider are proteins and nucleic acids.
If the substances are coloured, such as the proteins haemoglobin, myoglobin, or the cyto-
chromes, then their positions are apparent because they are directly visible on the gel. However,
most proteins are colourless and, in general, are detected by staining them with coloured or
fluorescent dyes or by reacting them with reagents that convert them to coloured complexes.
These treatments allow them to be seen as coloured bands in the gel.
Normally, staining is performed by immersing the gel in a solution of the dye. Following staining,
the gel is soaked in a solvent to remove excess stain and clear the background: a procedure usually
called destaining. Table 14.1 lists a number of dyes and staining procedures that are available.
TABLE 14.1 Common stains used in electrophoresis
Stain Substance(s) detected Comment

Acridine orange (Section 14.8) DNA single stranded RNA Use with denatured RNA
Alcian blue (Section 14.7) Glycoproteins Stains only the carbohydrate portion
Amido black Proteins Lacks sensitivity, gives high background

stain with paper and cellulose acetate,
shrinks polyacrylamide gels, generally
replaced by Coomassie dyes
Coomassie blue dyes (Section 14.7) Proteins Probably the most widely used dye
to detect proteins on native and SDS
polyacrylamide gels
Ethidium bromide (Section 14.8) DNA Sensitive for double-stranded

RNA nucleic acids, used with agarose and
polyacrylamide gel electrophoresis
Enzyme activities (Section 14.7) Enzymes Sensitive, specific for enzyme activity, can
be time consuming
Ninhydrin (Section 14.4) Amino acids, peptides, proteins Sensitive reagent, mainly used with paper
electrophoresis
Ponceau S (Section 14.5) Proteins Used with cellulose acetate and starch
gels, stains rapidly
p-anisidine/ethanol and sodium Sugars Two reagent technique for detecting

periodate/acetone (Section 14.4) sugars following paper electrophoresis
Periodic acid-Schiff (PAS) reaction Glycoproteins Stains only the carbohydrate portion,
(Section 14.7) lacks sensitivity
Pro-Q emerald 300 (Section 14.7) Glycoproteins Sensitive fluorescence stain for detecting
sugar portions of glycoproteins
Silver staining (Section 14.7) Proteins More sensitive than Coomassie dyes
but more expensive and staining can be
variable
Sypro orange and red (Section 14.7) Proteins Sensitive fluorescence dyes for detecting
proteins on polyacrylamide gels
Following staining, scanning densitometry may be used to estimate the amounts of the sepa-
rated proteins or nucleic acids present. The densitometer passes a narrow beam of light from
a laser through the gel and measures the proportion of light transmitted by each band of
protein. The central processor of the densitometer or a computer to which it is connected
can then calculate the amount of each protein present from the amount of light it absorbs:
the smaller the amount of light transmitted by the gel, the greater the amount of sample
present. These data are often presented as graphs of absorbance against the distance each
protein migrated during electrophoresis, as you can see in Figure 14.3. However, the esti-
Cross reference mated amounts of each protein must be treated with caution. Absorbance is only linear up to
You can read about the a particular concentration of protein, in accordance with the Beer–Lambert law described in
Beer–Lambert law in Chapter 11
Chapter 11. Furthermore, equal amounts of different proteins do not stain to the same extent.
‘Spectroscopy’.
Thus, estimates of the amounts of proteins present are, at best, semi-quantitative.
14.3 DETECTION OF SEPAR ATED COMPONENTS 347
Albumin
Absorbance
β FIGURE 14.3
α2 γ Densitometric scan of proteins, in this
α1
case serum proteins, separated by
Distance electrophoresis.
Gel documentation systems are now replacing densitometers. These consist of a computer
linked to a video imaging system enclosed in a small dark compartment containing a white
or ultraviolet (UV) light transilluminator. Transilluminators are instruments that project a nar-
row beam of visible or UV light through a translucent sample, such as an electrophoresis gel,
to allow the stained bands of the separated components to be observed and photographed.
Images of the gel can be stored on the computer and the intensities of the protein bands
analysed.
SELF-CHECK 14.2
Examine Figure 14.3 and deduce from the densitometric scan which protein is present in
serum in the greatest concentration.
How the separated components are detected and analysed depends upon their nature and
the type of electrophoresis performed. Although the principles and points outlined in Sections
14.1 and 14.2 underpin all types of electrophoresis, there are major differences between the
various techniques. These include the types of support media used, which in turn depends
upon the sizes and nature of the molecules to be separated, and whether the equipment has
a vertical or horizontal geometry.
The support media used in electrophoresis include:
• Paper (cellulose)
• Cellulose acetate
• Starch
• Polyacrylamide
• Agarose
Horizontal electrophoresis systems (Figure 14.2 (a)) are often used in paper, cellulose acetate,
or agarose gel electrophoresis. In contrast, polyacrylamide electrophoresis generally employs
a vertical system (Figure 14.2 (b)).
We will now review the different types of support media used in electrophoresis in more
detail, starting with paper electrophoresis.
H2C — OH H2C — OH H2C — OH

O O O
O O O O
FIGURE 14.4
HO OH HO OH HO OH
Structure of cellulose.
14.4 Paper or cellulose electrophoresis

Paper is composed of the polymer of β-glucose called cellulose (Figure 14.4). It was first used as
a support medium in the 1950s in both vertical and horizontal geometries. Paper electrophore-
sis is largely used to separate small molecules, many of which, for example, amino acids and sug-
ars, are of biomedical importance. However, the use of paper in electrophoresis has a number of
flaws. These include its fragility, particularly when soaked in buffer, making it difficult to handle.
Furthermore, a number of biological molecules, for example those of proteins, interact with the
hydroxyl groups of the cellulose retarding their mobility and resulting in tailing (Section 14.2).
Following electrophoresis, the dye ninhydrin (Table 14.1) can be used to detect amino acids
because it reacts with their amine groups to give a blue–purple coloured complex (Figure
14.5). Imino acids, proline, and hydroxyproline, are secondary amino acids and react to yield
yellow–orange coloured products.
A number of reagents are available to detect sugars following their electrophoresis. For exam-
ple, the electrophoresis paper can be immersed in a solution of p-anisidine/ethanol followed
by one of sodium periodate/acetone, which converts the sugars to coloured derivatives. This
method can detect as little as 20 μg of a variety of sugar types, including polyols, pentoses,
hexoses, sugar acids, amino sugars, and their acetyl derivatives.
Cellulose contains a relatively small number of carboxyl groups, but these can ionize in many
buffers and result in electro-osmosis. Both tailing and electro-osmosis hinder resolution and the
separation of mixtures. For these reasons, other support media have largely replaced paper.
O
R
OH
OH + +NH CHCOO–
3
O Amino acid
Ninhydrin
OH–
O O
N + R • CHO + CO2 + 3H2O
FIGURE 14.5 O O
The reaction of ninhydrin Blue—purple
with an amino acid. coloured product
14.5 CELLULOSE ACETATE ELECTROPHORESIS 349
14.5 Cellulose acetate electrophoresis

The use of cellulose acetate in the late 1950s overcame some of the disadvantages of paper
described in Section 14.4. Cellulose acetate is made by acetylating the hydroxyl groups in
cellulose (Figure 14.6). The absence of hydroxyl groups reduces interactions with sample ions
and produces a structure that is less hydrophilic and, therefore, holds less water, both of which
increase resolution. The pores inside cellulose acetate are uniform and of sufficient size to
allow passage of all except the largest molecules.
Single strips of cellulose acetate, typically 2.5 × 12 cm, are used for single samples although
wider ones are available to allow the analysis of multiple samples simultaneously. The strips are
first fully wetted in electrophoresis buffer, and placed on a glass or plastic sheet in a horizontal
geometry. Buffer-soaked filter papers are used to connect the strip to the electrode compart-
ments (Figure 14.7). Care must be exercised to ensure the strips never dry out. The pH of
the buffer used (8.6) ensures proteins have a negative charge and move towards the anode.
A small, 1 or 2 μL volume of sample is applied as a 1 cm wide band to the cathode end of
the strip.
Electrophoresis is carried out for about 45 minutes and since different proteins have different
mass-to-charge ratios, they migrate at different rates and separate. Following electrophoresis,
proteins are fixed in their positions in the cellulose acetate by immersing the strip in a solution
of trichloroacetic acid (TCA), which denatures proteins and reduces their diffusion. The strips
are stained to detect the proteins often with the dye, Ponceau S (Table 14.1 and Figure 14.8).
O O O
H2C — O — C — CH3 H2C — O — C — CH3 H2C — O — C — CH3
O O O
O O O O
O O O
O O O
O C O C O C
O C O C O C
CH3 CH3 FIGURE 14.6
CH3 CH3 CH3 CH3 Structure of cellulose acetate.
Paper wick Cover
Anode Cathode
Cellulose acetate strip
+ Glass plate –
Buffer solution Buffer solution
FIGURE 14.7
Power pack Typical apparatus for
carrying out cellulose acetate
electrophoresis.
HO SO3– Na+
SO3– Na+
N
N N
Na+ –O3S N
FIGURE 14.8
Structure of Ponceau S. SO3– Na+
The negatively charged dye binds to positively charged amino groups of the protein but it also
binds through hydrophobic interactions with non-polar parts of the protein.
Alternatively, the strips can be rendered transparent by immersing them in a mineral oil whose
Cross reference
refractive index equals that of cellulose acetate. The positions of the proteins can then be
You can read about
spectrophotometry in Chapter 11 determined spectrophotometrically.
‘Spectroscopy’.
Cellulose acetate electrophoresis lacks the resolution associated with gel-like materials, such
as starch, polyacrylamide, and agar. However, it has the advantages that it is rapid, simple, and
relatively inexpensive, and is used in pathology laboratories to analyse serum proteins, for
example, in the investigation of multiple myeloma (MM).
Multiple myeloma and paraproteins

Multiple myeloma is the cancerous proliferation of immunoglobulin-producing plasma cells
located in the bone marrow. It has an incidence of 4 in 100,000 worldwide. The condition is
more frequent after the age of 50 years, but can occur before the age of 30.
B lymphocytes arise in the bone marrow and would normally leave it and circulate to lym-
phoid tissues. Here, they can develop into plasma cells when appropriately stimulated. Plasma
cells from a single clone all synthesize and secrete an identical immunoglobulin (Ig) or mono-
clonal antibody and as these cells multiply, large amounts of a particular Ig are produced.
However, in MM, plasma cells in the bone marrow produce excessive amounts of their specific
Ig, which is released into the circulation. The large amounts of Ig are usually IgG molecules,
but can be IgA or IgD. Following electrophoresis of serum from the patient, the Ig produced
is detected as a discrete band, often called a paraprotein. Thus, the presence of paraproteins
aids in the diagnosis of MM. The large amount of the particular Ig produced suppresses the
production of other antibodies and reduces their concentrations in the blood of the patient.
Cross reference Immunoglobulins are large molecules, and the paraproteins cannot be removed from the
You can read more about blood by the kidneys and so increase the viscosity of the blood. However, in some patients,
Bence-Jones protein in Chapter 18 a defect in the plasma cells results in the production of only light chains of the Ig being pro-
‘Specific protein markers’ in the
duced, which can be filtered by the kidneys and lost in the urine. These can be detected and
book, Clinical Biochemistry.
referred to as Bence-Jones protein.
Patients suffering from MM present with a variety of symptoms that include bone pain,
coma, and retinopathy, the latter two a consequence of blood hyperviscosity. They have
an increased susceptibility to infections due to the decline of other immunmoglobulins,
present with anaemia due to a decline in other bone marrow functions, such as erythro-
cyte, white blood cell and platelet syntheses, and suffer kidney damage due to deposition of
14.6 STARCH ELECTROPHORESIS 351
FIGURE 14.9
Separation of serum and plasma proteins by cellulose acetate electrophoresis.
Lane 1 shows the separation of a sample of serum from a healthy individual.
Lane 2 is serum from a patient with multiple myeloma. The position of the
separated paraprotein is indicated. Lane 3 shows the separation of plasma
from a healthy individual. The position of fibrinogen is indicated.
Ig molecules. About one-third of patients with MM die of renal failure and the average sur-
vival time is 2–3 years.
A diagnosis of MM is assisted by demonstration of a paraprotein in the serum following elec-

trophoresis. It is essential that serum and not plasma is used for paraprotein analysis as the
latter contains fibrinogen, which can present as a discrete band and be mistaken for a para-
protein (Figure 14.9). Other clinical tests include demonstrating the presence of abnormal
malignant cells in the bone marrow following a biopsy. X-rays of bones can also be used to
aid in the diagnosis as characteristic holes are seen in affected areas of skull, pelvis, ribs, and
vertebrae in patients suffering from MM. Treatment of patients involves use of radiotherapy
and cytotoxic drugs.
14.6 Starch electrophoresis

Starch was first used in the late 1950s as a molecular sieve in gel electrophoresis to separate
proteins. The usual type of apparatus is shown in Figure 14.10. As you can see, it has a horizon-
tal geometry, which holds a slab of starch connected to the buffer compartments with wicks,
rather like the system encountered in cellulose acetate electrophoresis (Section 14.5).
Starch gels are normally prepared using hydrolysed potato starch. The starch is suspended
in the electrophoresis buffer and heated until a transparent solution forms. The solution is
then poured into a suitable mould and solidifies to form a gel as it cools. Slots are cut into
the gel into which the samples for electrophoresis are added. Following electrophoresis, Cross reference
the gel can be sliced into a number of layers using a fine wire, which provides several rep- You can read about isoenzymes
licate gels, each of which can be analysed using different detection methods. For example, in Chapter 7 ‘Clinical
different slices of gels can be examined for different enzyme activities (see Section 14.7), enzymology and biomarkers’
in the accompanying textbook,
particularly to demonstrate the presence of isoenzymes, such as those of serum lactate
dehydrogenase.
Paper wick Cover Well for sample
Anode Cathode
Starch gel
+ Glass plate –
Buffer solution Buffer solution
FIGURE 14.10 Power pack

Schematic showing typical apparatus
for starch gel electrophoresis.
The major drawbacks of using starch as a support medium in electrophoresis are that, like cel-
lulose, starch contains some negatively charged groups that can interact with sample cations,
and also lead to electro-osmosis. Also, the pore sizes of starch gels vary with the concentra-
tion used. This is a major disadvantage to its use as a sieving medium. If the concentrations of
starch are too extreme, the gel is too soft to handle or excessively stiff to use, which restricts
the range of sizes of molecules that can be analysed.
14.7Polyacrylamide gel
electrophoresis
Polyacrylamide gel electrophoresis (PAGE) separates mixtures of molecules of proteins or
nucleic acids through a gel composed of polyacrylamide. The gel is formed in situ between
two glass plates that are clamped to form a sandwich, with the plates being held apart by plas-
tic spacers that also retain the reaction mixture used to form the gel.
Polyacrylamide gels are formed by polymerizing acrylamide units into long chains. The
polymerization is initiated by free radicals generated in the mixture by a reaction between
ammonium persulphate and N,N,N’,N’-tetramethylethylenediamine (TEMED) as shown in
Figure 14.11 (a) and (b). In Figure 14.11 (a) the free radical is represented as X∙, and the acry-
lamide unit as A. The free radical X∙ reacts with A to form X–A∙, which is still reactive as it
has an unpaired electron. Thus, it will react with another molecule of A to form XAA∙ and so
polymers of acrylamide units are built up in a stepwise fashion. As the chains grow, they are
cross-linked by bisacrylamide (N,N’-methylene bisacrylamide), which consists of two acry-
lamide units linked by a methylene group. The cross-linking converts the solution to a solid
gel, whose structure is shown in Figure 14.11 (b), and consists of a three-dimensional network
of pores.
Acrylamide gels can also be formed in a photopolymerization reaction, where the ammonium
persulphate and TEMED are replaced by riboflavin. Exposure of the gel solution to bright light
for 2–3 hours leads to the photodecomposition of riboflavin, which generates free radicals
that catalyse polymerization of the acrylamide units.
Separation during PAGE is based not only on differences in the electrophoretic mobilities of
the different biological molecules, but also on the molecular sieving effects of the pore size.
The sizes of the pores in the gel can be varied by altering the concentrations of acrylamide
and bisacrylamide. Typically, gels consist of 3–30% concentrations of acrylamide. Those with
14.7 POLYACRYL AMIDE GEL ELECTROPHORESIS 353
(a) A A A A
X• X—A• X—A—A• X—A—A—A•
A Acrylamide unit
X• Free radical
(b) CH2 CH
CH2
CONH
TEMED
+ CH + (catalyst)
CH2
CONH2
CONH
Acrylamide
CH2 CH
N,N,N’,N’-methylenebisacrylamide
Ammonium persulphate or
riboflavin
(free radicals)
CH2 CH CH2 CH CH2 CH
CO CO CO
n
NH NH2 NH
CH2 CH2
NH NH2 NH
FIGURE 14.11
CO CO CO (a) Schematic illustrating the
polymerization of acrylamide.
CH2 CH CH2 CH CH2 CH
n See text for details. (b)
Polyacrylamide Structure of polyacrylamide.
a low percentage of acrylamide have larger pores and are more suitable for separating larger
proteins or nucleic acids as described in Chapter 16. Smaller molecules move through the gel
easier than larger ones and so the relative rates of migration are the smallest ones fastest and
the largest slowest.
There are a number of elementary safety precautions which should be observed for electro-
phoresis (see Health and Safety Box ‘Some elementary precautions for electrophoresis’).
HEALTH & SAFETY

Some elementary precautions for electrophoresis
■ Mouth pipetting must not be used in preparing any of the solutions used for gel formation,
staining, blotting, or detection.
■ All acrylamide solutions should be prepared in a fume cupboard. Unpolymerized acrylamide
and bisacrylamide are a skin irritant and a potential neurotoxin, respectively. Thus safety
gloves must be worn throughout all handling procedures and a face mask when weighing the
dry powder. Mercaptoethanol has a powerful and unpleasant odour, and should be handled
only in a fume cupboard.
■ Acrylamide begins to polymerize when tetramethylethylenediamine (TEMED) and ammo-
nium persulphate are added to it. The ammonium persulphate solution should be freshly
prepared and it and the TEMED added to the acrylamide solution immediately before it is
poured into the gel apparatus.
■ Do not touch the electrophoresis chamber or wires while electrophoresis is in progress.
Voltages may be routinely as high as 200–300 V and shocks may be fatal.
■ Ethidium bromide is a mutagen. Always wear gloves when handling solutions or agarose
gels containing the compound. Dispose of the final electrophoresis buffer containing
ethidium bromide according to directions from your instructor and local health and safety
procedures.
■ Avoid looking into ultraviolet (UV) light during the detection of DNA fragments on the agar-
ose gel. Wear UV protective goggles.
SELF-CHECK 14.3
What basic safety precautions must you take when you prepare and handle a polyacryla-
mide gel?
Polyacrylamide gel electrophoresis occurs in a vertical gel (Figure 14.2 (b)). A photograph of
a typical commercial vertical apparatus for carrying out vertical gel electrophoresis is shown
in Figure 14.12 (a), which is shown schematically in (b). The typical dimensions of a poly-
acrylamide gel are 12 × 14 cm with a thickness of 1–2 mm, although gel dimensions can vary
enormously.
Whichever method is used to polymerize the acrylamide, a plastic comb or well former is
inserted between the plates into the polymerization mixture before it sets. The spacers and
comb are removed following polymerization. The indents in the gel formed by the comb form
wells to which samples are subsequently added.
The buffer provides the electrical contact between the electrodes and the gel. After assembly,
the lower electrophoresis tank buffer surrounds the gel plates helping to keep them cool.
Polyacrylamide gel electrophoresis can use continuous or discontinuous buffer systems.
Continuous buffer systems

In a continuous system, the same type of buffer is used to dissolve the sample as that present
in the gel and in the compartments of the electrophoresis equipment. Sucrose or glycerol are
added to the samples, which are loaded directly into the wells. This increases their densities
ensuring they settle quickly to the bottom of the wells. The samples also contain a so-called
(a)
(b) Platinum wire cathode
Buffer
–
Sample in well
Polyacrylamide
Power
gel
supply FIGURE 14.12
(a) Photograph of a
typical commercial gel
electrophoresis system for
performing polyacrylamide
gel electrophoresis.
Buffer
+ (b) Schematic showing typical
apparatus for performing
polyacrylamide gel
Platinum wire anode electrophoresis.
tracking dye, usually bromophenol blue, which is a small molecule and passes through the
pores in the gel to act as a marker for the electrophoretic front.
Electrophoresis is performed for a defined period ranging from an hour to overnight, usually
until the tracking dye has neared the bottom of the gel. The current is then switched off.
SELF-CHECK 14.4
If 6 cm3 of a 30% combined acrylamide and bisacrylamide solution was made up to 18 cm3
with the other gel forming reagents, what is the concentration of acrylamide in the final gel?
Following electrophoresis, the gel is removed from the glass plates. The separated proteins are
fixed in position usually using a mixture of TCA and methanol; the gel is then stained to reveal
the positions of the separated proteins.
Discontinuous buffer systems

In a discontinuous buffer system, the buffers used to dissolve the samples, and those present
in the gel and reservoir compartments of the equipment differ in terms of their composi-
tion and pH. In addition, the gel is formed in two parts. The main separating or resolving gel
is made as described above. However, it occupies only part of the space between the glass
plates and does not contain wells. Once this has polymerized, a much smaller stacking gel is
formed above it, which generally has a buffer of lower pH to the resolving gel. The wells for
samples are formed in the stacking gel, which typically consists of only 4% acrylamide or even
less. The large size of its pores allows the sample molecules to move through it freely during
electrophoresis.
Samples are added to the wells of the stacking gel generally in the same type of buffer used to
form the resolving gel. When an electric field is applied to the system, the molecules of pro-
teins or nucleic acids become sandwiched between various ionic species of the buffer, which
concentrates them as a thin band within the stacking gel. Although the pH is higher in the
resolving gel, which would tend to increase the mobilities of the ions, its pore size is smaller
and separation of the various molecular species occurs as described above for continuous
systems. However, because the proteins or nucleic acids have entered the resolving gel as
narrow concentrated bands discontinuous systems have enhanced resolution over continuous
systems.
Polyacrylamide gel electrophoresis can be performed under non-denaturing conditions as

described above, in which case the technique is referred to as native PAGE. Native PAGE,
although useful for many applications, is not generally applicable for determining the Mr of
molecules or of resolving molecules that have similar sizes, because mobility under these con-
ditions is influenced by both charge and size. However, when it is unnecessary to preserve the
structures and functions of biological molecules, PAGE can also occur in denaturing condi-
tions, such as in the presence of the detergent sodium dodecyl sulphate (SDS).
Sodium dodecyl sulphate electrophoresis

The most widely used PAGE technique is known as sodium dodecyl sulphate polyacrylamide
Cross reference gel electrophoresis (SDS-PAGE). Sodium dodecyl sulphate-PAGE is particularly useful in ana-
The sequencing of nucleic lysing proteins to assess their purity or to determine their Mr and was extensively used in
acids is outlined in Chapter 16
sequencing DNA molecules, as described in Chapter 16, before being replaced by capillary
‘Molecular biology techniques’.
electrophoresis (Section 14.9).
Sodium dodecyl sulphate-PAGE, as the name implies, uses the anionic detergent SDS (Figure
14.13), which is added to samples and also incorporated in the acrylamide gel and added to
the electrophoresis buffers. Samples are boiled for several minutes or kept at 37°C overnight
in a buffer containing SDS. The SDS strongly binds to molecules and disrupts the hydrophobic
interactions that are largely responsible for maintaining their structures. In the case of protein
samples, β-mercaptoethanol is often added to reduce the disulphide bonds that help stabilize
the protein structure and which hold the monomers of some quaternary proteins together.
Urea is added to nucleic acid samples to denature them to single stranded forms, suitable for
analysis by SDS-PAGE as, for example, in the diagnosis of some genetic diseases such as cystic
fibrosis (CF), which is discussed in Box 14.1.
FIGURE 14.13
Structure of sodium dodecyl
sulphate (SDS). CH3 – CH2 – CH2 – CH2 – CH2 – CH2 – CH2 – CH2 – CH2 – CH2 – CH2 – CH2 – SO4- Na+
In general, proteins bind SDS in quantitative amounts with one SDS molecule binding to every
two amino acid residues; furthermore, the amount of SDS bound overwhelms the native
charges carried by the proteins. Since the negative charges of the sulphate groups of SDS repel
each other, the protein molecules adopt rod-like structures (Figure 14.14). This means that all
proteins in the presence of SDS and β-mercaptoethanol have a similar rod-shaped confor-
mation and possess similar mass-to-charge ratios. Thus, the negatively charged proteins will
move towards the positive electrode. Furthermore, all the protein molecules will now have the
same charge per unit length and, therefore, travel with the same mobility in a given electric
field. However, the gel through which they migrate offers frictional resistance and, therefore,
smaller molecules migrate faster than larger ones through the pores in the polyacrylamide gel.
Thus, molecules separate from each other according to differences in their sizes.
Gels are prepared with different pore sizes depending on the range of sizes of molecules to
be separated. For example, 15% polyacrylamide gels are typically used to separate molecules
with Mr ranging from 10,000 to 100,000. Gels with a lower concentration of acrylamide and,
therefore, larger pore sizes are used for larger molecules. For example, 5% acrylamide gels
separate molecules with Mr in the range of 60,000 to 350,000.
Visualizing proteins following separation

Perhaps the most commonly used dyes to stain proteins in polyacrylamide gels are the
Coomassie types (Table 14.1 and Box 14.2). Coomassie dyes bind non-specifically to vir-
tually all proteins through hydrophobic and van der Waals interactions, and because the
negative charges of their sulphonate groups make ionic interactions with arginine, lysine,
and histidine residues of the proteins. Gels are stained by immersing them for 2–4 hours in
a solution of the Coomassie brilliant blue dye in a mixture of methanol and TCA, until the
gel is a uniformly blue colour. The solvents used precipitate the proteins and so fix their
positions in the gel. Following this immersion, the gels require destaining by gently shaking
them in a mixture of methanol and acetic acid until the background is clear, which usually
takes four to 24 hours. The proteins will then appear as darkish blue bands against a light
background (Figure 14.17). The inclusion of positively charged resins speeds up the destain-
ing time considerably.
S
Native protein S S
H3+N
COO–
S
β-mercaptoethanol
+
SDS
SH SH
FIGURE 14.14
H+N
3
COO– Schematic showing the denaturation of
proteins by a-mercaptoethanol and
SH SH
Denatured protein sodium dodecyl sulphate.
BOX 14.1 Investigating cystic fibrosis
Cystic fibrosis (CF) is the commonest fatal, inherited homozygous recessive disorder of
Caucasian populations. It affects approximately 1 in 2000 people in the UK and about 1
in 20 carry a defective gene. The condition results from mutations in the gene CFTR that
encodes the CF transmembrane conductance regulator protein. Thus, there is reduced
permeability to Cl– in the apical membranes of epithelial tissues that line the lungs and
other organs, and viscous mucus accumulates in the lungs, digestive tract, and associ-
ated organs and epididymis. Patients suffer chronic respiratory disease, malabsorption,
cirrhosis, and electrolyte disturbances.
Possible cases of CF are investigated by applying pilocarpine to the skin of patients to stim-
ulate a flow of sweat, which is then sampled. Concentrations of Cl– in the sweat greater
than 70 mmol dm–3 are indicative of CF. Unfortunately, the sweat test is not always reliable
in the first 6 weeks of life or in adulthood, and some patients with CF test normally. Several
screening tests for CF based on its effects on pancreatic function are available. One meas-
ures immunoreactive trypsin (IRT) in dried spots of blood. The amount of IRT increases sig-
nificantly in infants with CF compared with healthy ones. However, after the first few weeks
of life pancreatic insufficiency develops and IRT decreases, and the test cannot be used.
Identifying mutations in CFTR of CF patients can not only confirm diagnosis, but also assist
with assessing the carrier status of their relatives. It also provides an accurate and rapid
prenatal diagnosis of CF once the parental genotypes are confirmed. Unfortunately, over
1300 mutated forms of CFTR have been reported, which makes their identification in indi-
vidual cases difficult. The commonest CF mutation is called ΔF508 and is the deletion of
the codon in CFTR that specifies a phenylalanine residue at position 508 in the amino acid
sequence of the encoded protein. Molecular techniques offer relatively simple ways to
detect this mutation. The gene is hydrolysed and the relevant portion amplified to increase
its concentration using specific enzymes. The commonest technique produces a DNA
fragment from healthy individuals that is 98 base pairs (bp) in size. However, if the DF508
mutation is present, the product is only 95 bp long. Sodium dodecyl sulphate-PAGE can
separate the appropriate gene fragments as shown in Figure 14.15 and detect the deletion.
Cross reference The test is not totally specific for DF508 given that other 3 bp deletions in this region of the
See Chapter 16 ‘Molecular
gene would also result in the same difference in size. Mutations other than DF508, can also
biology techniques’.
be identified because they produce differing banding patterns following SDS-PAGE.
98bp
95bp
1 2 3
FIGURE 14.15
Use of electrophoresis in detecting the DF508 mutation in cystic fibrosis. Lane 1
shows the position of the 95 bp fragment associated with the mutation. Lane 2 has
the two bands that occur in the heterozygote condition, the normal 98 bp fragment
and the 95 bp one. Lane 3 is the normal condition where only a single 98 bp
fragment is observable.
BOX 14.2 Coomassie dyes
Coomassie dyes (Table 14.1), which are sometimes called Coomassie dyes can be added to native (non-denatured)
Coomassie brilliant dyes, were introduced in the 1960s as proteins, rather than SDS prior to electrophoresis. The
stains to detect proteins following electrophoresis in the name of this technique is Blue Native- or BN-PAGE. Like
1960s. They are particularly used following native PAGE and SDS, Coomassie dyes impart a net negative charge to the
SDS-PAGE. The Coomassie dyes were original developed as proteins, allowing them to separate during PAGE. Only neg-
acid woollen dyes (they stain wool in acid conditions). They atively charged proteins would migrate without the pres-
were named to commemorate the 1896 British occupa- ence of the dye (or SDS) during native PAGE.
tion of Kumasi, which was formerly called Coommassie, in
The Bradford method is one of the commonest used to
Ghana, West Africa.
determine the concentrations of proteins in solution.
Coomassies dyes constitute a group of chemically related Coomassie dyes are one of the components of the Bradford
compounds. The first to be developed was Coommassie reagent. Coomassie Brilliant Blue G-250 can bind to pro-
Blue R-250; the R stands for reddish tint of the dye, while teins in acid solution and, when it does so, its wavelength
the 250 is an indicator of the strength of the dye on a scale of maximum light absorbance shifts from 465 to 595 nm.
used by the original manufacturer, Imperial Chemical Equal amounts of Bradford reagent are added to solutions
Industries. Coommassie Blue G-250, G for greenish, and containing known concentrations of proteins, as well as
Coomassie Violet R-150 later followed. The structures to the samples whose concentrations are unknown. The
of R-250 and G-250, which are the most commonly used absorbances of the resulting coloured solutions are then
for staining proteins in electrophoretic gels, are shown in measured in a spectrophotometer. The data from the
Figure 14.16 (a) and (b). Coommassie R-250 generally gives known solutions are then used to construct a calibration
better resolution and is more often used than G-250, even graph from which the concentration of protein in the sam-
though the latter has greater sensitivity. ples can be determined.
CH2CH3 CH2CH3
(a) (b)
NCH2 NCH2
SO3– SO3–
H3C CH3
C C
CH3CH3O NH NCH2 CH3CH3O NH NCH2

CH2CH3 CH2CH3
SO3– SO3–
FIGURE 14.16
Structures of Coomassie blue (a) R-250 and (b) G-250.
SELF-CHECK 14.5 Cross reference

What is the structural difference between Coommassie R-250 and G-250? You can read more about
spectrophotometry and
standard graphs in Chapter 11
Coomassie Blue R-250 has a detection limit of about 0.3–1 μg of protein per band on the gel. If ‘Spectroscopy’.
greater sensitivity is required to detect even smaller amounts of proteins, then Coomassie blue
in a colloidal form may be used. A solution of colloidal and molecularly dispersed Coomassie
blue in equilibrium is prepared by adding methanol and ammonium sulphate to a solution
of the dye in dilute phosphoric acid. During staining, only the molecular form enters the gel
Mr
14,000
18,000
24,000
FIGURE 14.17
Photograph of a polyacrylamide gel showing the separated proteins 35,000
stained with Coomassie blue R-250. Lanes 1 shows the positions of
proteins of known Mr . Lane 2–4 show the positions of a number
of proteins of differing Mr from three separate samples. Courtesy 66,000
of Dr M. S. Ahmad, School of Healthcare Science, Manchester
Metropolitan University, UK. 1 2 3 4
and stains the proteins preferentially, while the colloidal form is unable to enter the gel and so
background staining does not occur.
The fluorescent Sypro orange and red dyes (Table 14.1) have comparable sensitivities to col-
loidal Coomassie dye and can detect about 1–2 ng of protein. Stained proteins can be seen on
the gel by illuminating them with light of approximately 300 nm. Although both fluorescent
dyes have similar staining properties, Sypro orange is the more sensitive, but the red form
gives less background interference.
Silver staining is the most sensitive procedure and has a detection limit about 300 times less
than that of conventional Coomassie Blue R-250. Proteins must be fixed in the gel using TCA
prior to silver staining. The gel is stained by immersing it in a solution of silver nitrate. Silver
ions react with thiol and basic groups in the proteins, and are then reduced using formalde-
hyde, which causes the deposition of grains of metallic silver on the protein. Thus, the protein
bands can be visualized as they are stained brown or black against a light amber stained gel.
Despite being the most sensitive method of detecting protein following electrophoresis, prob-
lems can occur with silver staining. Different proteins can respond differentially to the stain
and, indeed, a number of proteins do not stain at all. Hence, the observed sensitivity varies
between proteins.
SELF-CHECK 14.6
Calculate the approximate lowest sensitivity in ng of silver staining to detect the presence of
Cross reference proteins following PAGE and SDS-PAGE.
Radioisotopes are described in
Chapter 10 ‘Radioactivity and
Proteins can also be detected by labelling them with radioisotopes such as 125I and their posi-
radiation’.
tions on the gel are detected by autoradiography (Section 10.6 and Box 13.1 Autoradiography).
Detection of glycoproteins
Glycoproteins can be detected on the gels using Alcian blue stain or the periodic acid-Schiff
(PAS) reaction (Table 14.1). The former is now little used. You can see an outline of the PAS
procedure in Figure 14.18. The periodic acid oxidizes sugars that contain hydroxyl groups on
Sugar in glycoprotein
CH2OH
O
O
O OH
OH
IO –
4
CH2OH
O
O
O
O O
H2N
R
H2N
Schiff ’s reagent
CH2OH
O
O
O
OH HO
H N N H
FIGURE 14.18
R Schematic showing the periodic acid
Pink to red coloured product Schiff reaction of sugars.
adjacent carbon atoms in the carbohydrate part of the glycoprotein to a dialdehyde product.
The aldehydes subsequently react with Schiff reagent to form a coloured product. However,
the method lacks sensitivity, being only able to detect bands with at least 2.5 ng glycoprotein.
Thus, the weakly coloured pink or light red bands are often difficult to see on the gel.
The Pro-Q emerald 300 dye (Table 14.1) also reacts with periodate-oxidized carbohydrates
Cross reference
but gives a bright green fluorescent signal when excited with light of wavelength 300 nm. This
You can read about the blotting
sensitive fluorescence stain can detect as little as 0.5 ng of glycoprotein. However, rather than of gels and the use of probes in
using these methods to detect glycoproteins, blotting techniques using lectins as sensitive and Chapter 16 ‘Molecular biology
specific probes of the carbohydrate portion of the molecule are often employed. techniques’.
If the proteins are enzymes, then their position in the gels can be detected by exploiting their
catalytic activities. This method of detection is particularly applicable to native PAGE and
starch gel electrophoresis (Section 14.6) because these support media are permeable to the
substrates, cofactors, and dyes required to detect enzyme activities. For example, hydrolases
can be detected using ρ-nitrophenylphosphate. This artificial substrate is hydrolysed by the
enzyme to phosphate and ρ-nitrophenol:
Hydrolase activity
ρ-nitrophenylphosphate + H2O ρ-nitrophenol + phosphate
The substrate is colourless, but ρ-nitrophenol is yellow coloured. Hence, the positions of
hydrolases on the gel show as bright yellow bands.
SELF-CHECK 14.7
Why is it not possible to detect the positions of enzymes following SDS-PAGE by monitoring
their catalytic activities?
Whatever staining procedure is used, it may cause hydration or dehydration of the gel, and
so alter its size. Such changes must be taken into account when measuring the mobilities of
the separated components. Thus, the relative mobility of, for example, a protein can be deter-
mined using the expression:
Relative mobility = (migration distance of protein/migration distance of tracking dye)

× (length of gel before staining/length of gel after staining)
If the Mr of an unknown protein was being estimated by SDS-PAGE, then proteins of known
size would be included in the electrophoresis as shown in Figure 14.17. A calibration graph
would then be drawn of the logarithm of size (Mr) on the y axis against relative mobility on the
x axis. The Mr of the unknown protein may be estimated by determining its relative mobility
using the calibration graph.
14.8 Agarose gel electrophoresis

Agarose is one of the components of agar, which, in turn, is a mixture of polysaccharides iso-
lated from some seaweeds. It is a linear polysaccharide consisting of units of agarobiose, that,
in turn, consist of alternating galactose and 3,6-anhydrogalactose residues linked together by
alternating α1-4 and β1-3 glycosidic bonds (Figure 14.19).
Agarose is able to gel because hydrogen bonding within and between the agarose polymers
forms a three-dimensional lattice containing a continuous system of pores. The size of the gel
pores is determined by the concentration of agarose. A low concentration of agarose gives a
gel with larger pores and vice versa. The use of an appropriate concentration allows mixtures of
different sized molecules to be resolved during electrophoresis because the smaller molecules
are able to move faster through the gel than the larger ones. Like all supporting media, the gel
also limits the diffusion of the molecules, which improves separation.
Molecules that differ in their Mr by as little as 1% can be resolved on agarose gels. In addition
to its ability to separate molecules with a wide range of Mr with high resolution, other advan-
tages of using agarose include its lack of toxicity and relative ease of use. Agarose is generally
used in electrophoresis to separate nucleic acids as we discuss below, but is also used in iso-
electric focusing (IEF) to separate proteins (Section 14.10).
HO
H2COH O O
O
O HO
HO O
FIGURE 14.19
Structure of agarose. — 3) — β — Gal — (1—4) — 3,6 — anhydro — α — Gal — (1—
14.8 AGAROSE GEL ELECTROPHORESIS 363
Agarose gel electrophoresis is often used to separate and analyse mixtures of nucleic acid
molecules, particularly fragments of DNA. These are usually of large size and their separa-
tion requires gels with larger pores. The size of double stranded DNA is often expressed as
the number of base pairs (bp) or kilobase pairs (Kbp). However, even a fragment of DNA as
small as 1 Kbp has a Mr of 620,000. In the case of RNA and single-stranded DNA, size is usually Cross reference
Chapter 16 ‘Molecular biology
given as the number of nucleotides. You can read more about the structures of nucleic acids
techniques’.
in Chapter 16.
Every nucleotide in DNA and RNA includes a negatively charged phosphate group. Thus, the
mass-to-charge ratio for all nucleic acid molecules is identical. In this respect they resemble
polypeptides in the presence of SDS (Section 14.7 and Figure 14.14). This means that DNA
and RNA molecules will move towards the anode with identical mobilities when subjected to
an electric field. Hence, in theory, they should not separate during electrophoresis. However,
in agarose electrophoresis, the gel matrix resists the movement of DNA and RNA molecules:
larger fragments have the most difficulty in getting through the pores, while the smaller frag-
ments move faster because they experience the least resistance. Indeed, the mobility of very
large nucleic acids may be blocked completely. Therefore, separation of DNA and RNA mol-
ecules by agarose gel electrophoresis depends solely on differences in their sizes, like proteins
in SDS-PAGE.
The use of appropriate concentrations of agarose with different pore sizes allows nucleic
acids of varying ranges of size to be resolved. Those most frequently used include 0.3% agar-
ose gels, which are used to separate fragments of DNA 5–60 Kbp in size, 0.8% for 0.5–10 Kbp
fragments, and 1–2% agarose gels for 0.1–3 Kbp fragments. Using an appropriate concentra-
tion of agarose means the distance moved by a limited range of nucleic acid fragments is
inversely proportional to the logarithm of their sizes, as was the case for SDS-PAGE described
in Section 14.7. Whatever the concentration of agarose to be used, the gel is formed by dis-
solving dry agarose in buffer and boiling the mixture until a clear solution forms. The solu-
tion is poured into the gel former and a plastic comb inserted in it before it sets. Once the
solution cools, it sets to form a rigid gel. Removal of the comb leaves wells into which sam-
ples can be added. The gel is placed in the electrophoresis tank and covered by the buffer
(Figure 14.20 (a) and (b)).
Samples for electrophoresis are, as in PAGE (Section 14.7), prepared by dissolving them in a
buffer that contains sucrose or glycerol to increase their densities above that of the electro-
phoresis buffer. The samples are loaded directly into the wells using a micropipette and settle
quickly to the bottom of each one. Also, as in PAGE, a dye, most commonly the small molecule
bromophenol blue, is normally included in the sample buffer. The dye moves rapidly through
the gel in advance of all but the smallest DNA fragments. It therefore acts as a marker for the
electrophoretic front allowing electrophoresis to be stopped before the samples migrate so far
that they run out of the end of the gel!
Staining nucleic acids in agarose gels

Ethidium bromide (Table 14.1 and Figure 14.21) is the stain of choice for double stranded DNA
molecules that have been separated by agarose gel electrophoresis. It is normally added to the
electrophoresis buffer and so is present throughout the electrophoresis run. Since ethidium
bromide consists of relatively small, planar molecules, it binds to all the DNA molecules present
by intercalating between their stacked base pairs (Figure 14.22 (a)). In this position, the ethid-
ium bromide enhances the normally weak fluorescence of the purine and pyrimidine bases by
(a)
(b) Power
supply
Buffer
+ –
Platinum wire Well for Platinum wire
Agarose gel
anode sample cathode
FIGURE 14.20
(a) Photograph of a typical commercial
gel electrophoresis system for performing
agarose gel electrophoresis. (b) Schematic + –
showing side and top views of typical
apparatus for performing agarose gel
electrophoresis.
NH2
N+ Br–
H2N
C2H5
FIGURE 14.21
Structure of ethidium bromide.
14.8 AGAROSE GEL ELECTROPHORESIS 365
(a)
(b) 1 2 3 4
Size/bp
Ethidium bromide (EtBr) 9416

6557
4631
2322
2027
564
FIGURE 14.22
(a) Intercalation of ethidium bromide into double stranded DNA. (b) A photograph of an
agarose gel showing the separated DNA molecules stained with ethidium bromide. Lanes 1,
2, and 3 show DNA fragments from samples stained with ethidium bromide. Lane 4 shows
stained fragments of known size. See also Figures 16.17 and 16.30.
about 25 times. Hence, the separated DNA molecules fluoresce with an intense orange colour
when the gel is irradiated with UV light. Fluorescence stains are sensitive means of detecting
the fragments: as little as 1 ng of DNA can be detected (Figure 14.22 (b)).
Following staining, it is possible to determine the relative mobility of each band from the
formula:
Relative mobility = distance moved by band from origin / distance moved

by electrophoretic front from origin
Since the ethidium bromide is usually added to the electrophoresis buffer prior to electro-
phoresis, it is not necessary to correct for changes in the size of the gel during staining. It is
possible to determine the sizes or Mr of unknown DNA fragments by including fragments
of DNA of known size in the electrophoresis (Figure 14.22 (b)). A calibration graph of the
logarithm of size (in Kbp or Mr) on the y axis against relative mobility on the x axis can then
be drawn. The values of the unknown nucleic acids can then be determined by using their
relative mobilities and the calibration graph. This is essentially the same method as used to
estimate the Mr of proteins using SDS-PAGE (Figure 14.17 and its associated text).
CH3 CH3
N N N
H3C CH2
FIGURE 14.23
Structure of acridine orange.
Agarose gel electrophoresis can be used to separate molecules of RNA, as well as DNA. Single-
stranded molecules of RNA are able to double back on themselves. Consequently, intrachain
double stranded regions can form that will bind ethidium bromide, allowing their detection.
However, determining the sizes of RNA molecules requires the electrophoresis be performed
in the presence of denaturants, such as formaldehyde, glyoxal, or methylmercuric hydroxide,
which are compatible with agarose. These denaturants bind to bases in the RNA and so pre-
vent double stranded regions forming.
The dye, acridine orange (Table 14.1 and Figure 14.23) can detect the single stranded RNA
Cross reference molecules following electrophoresis. Its binding involves a complex mixture of electrostatic
See Section 10.6 and Box 13.1 interactions and the intercalation of dye molecules between bases of the single-stranded
Autoradiography, and the polymer. When excited by blue light, RNA-acridine orange complexes fluoresce orange but
Histopathology text of this
DNA-acridine orange emits yellow-green light. However, in many experiments, RNA is radio-
series.
labelled and so can be detected by autoradiography.
SELF-CHECK 14.8
Explain why ethidium bromide is not a suitable stain for single stranded RNA molecules.
Determining the order of bases of a DNA molecule (sequencing) also involves separating sin-
gle stranded nucleic acids, in the case of DNA. Chapter 16 gives a description of sequencing,
which involves separating fragments of single stranded DNA by electrophoresis. The methods
initially developed used SDS-PAGE gels containing urea but contemporary techniques use
capillary electrophoresis (Section 14.10). Urea, like SDS, is a denaturant and together they
ensure the DNA chains remain single stranded during electrophoresis. Following their separa-
tion, the different lengths of DNA can be interpreted to give the equivalent sequences of bases
in the DNA sample. You can see examples of sequencing DNA using gel electrophoresis and
capillary electrophoresis in Figures 16.19 and 16.20 respectively.
Pulse field gel electrophoresis

Agarose gel electrophoresis is unable to resolve molecules of DNA larger than 15 Kbp as
molecules of this size and above tend to migrate as a group during electrophoresis. Pulsed
field gel electrophoresis (PFGE) is an electrophoretic technique able to separate such large
DNA molecules. It does this by periodically altering the magnitude of the applied electric
field across the agarose gel. Thus, during PFGE, two different electric fields are applied alter-
nately at different angles to the gel for a defined period of time, for example, 60 seconds
(Figure 14.24).
Each field causes the DNA molecules to become stretched and to move in one direction
through the gel. The application of the alternate field then causes the DNA molecules to
change direction. Even though all the DNA molecules are relatively large, the smaller ones are
able to realign faster when the direction of field changes. This enables the smaller molecules to
move faster through the gel than larger ones, leading to separation of the mixture.
14.9 CAPILL ARY ELECTROPHORESIS 367
Pulsed field gel electrophoresis is used in genotyping or genetic fingerprinting. It is also used
Cross reference
in epidemiological studies of pathogenic organisms, for example in identifying subtype strains
Chapter 13 ‘Chromatography’.
of Listeria monocytogenes in clinical samples.
14.9 Capillary electrophoresis DNA sample in well
Capillary electrophoresis (CE), as the name implies, separates the components of samples in
narrow bore tubes, whose internal diameters are only 25–100 μm, but may be 40–100 cm
in length. Like all electrophoresis, charged molecules move in an electric field. However,
because capillary tubes have a high surface area to volume ratio, the high voltages used and
Agarose gel
resistances encountered allow the considerable heat generated to dissipate rapidly. Hence,
convection currents and diffusion of sample components are minimized, and separation can
be carried out in solution without the need for the support media necessary in other forms of
electrophoresis.
Direction
Capillary electrophoresis combines the high resolution of electrophoresis with the speed of
of field
high performance chromatography, which you can read about in Chapter 13. For this reason,
CE is also called high performance capillary electrophoresis (HPCE).
Figure 14.25 shows a typical arrangement for CE. The capillary tubes are made of fused silica
and coated externally with a polymer to provide mechanical strength. Samples are injected
into the capillary by one of two methods:
• The inlet of the capillary tube is removed from the anode reservoir and immersed in the
solution of sample. A high voltage is then applied across the tube, which attracts sample
ions into the tube
• The sample can be forced into the capillary tube by pressurizing the sample container with Direction
of field
an inert gas
Whatever the method, a major advantage of the technique is that sample volumes as small as
5–10 nL can be analysed.
Once the sample has entered the tube, a high voltage is applied across the capillary tube
and the constituent molecules in the sample migrate along its length at different rates and so
separate. Typical voltages used range from 7 to 10 kV, but can be as high as 50 kV depending
upon the samples to be separated.
The basis of separation in CE is rather different to that encountered in other types of elec-
Direction
trophoresis. Normally, charged particles would move towards the electrode of the oppo-
of field
site charge. However, in CE, electro-osmosis causes all the charged molecules to be carried
towards the cathode because the electro-osmotic flow is greater than their electrophoretic
mobility. Electro-osmosis occurs in the capillary tubes due to a net negative charge on the
fused silica surface. This attracts cations, which migrate to the inner wall forming an electrical
double layer. The cations, in turn, are surrounded by water molecules. When a potential dif-
ference is applied to the column, the cations migrate towards the cathode. Since these cations
are solvated and clustered at the walls of the capillary tube, they drag the rest of the solution
with them including the anions (Figure 14.26).
Although all components of the sample, both charged and neutral, migrate towards the
cathode, cations reach it first because they move by a combination of electro-osmotic flow
FIGURE 14.24 Separated and stained

Schematic showing the operation of pulsed field electrophoresis. See text for details. DNA fragments
Signal to computer
Detector
Direction of migration
Light source
Platinum anode Platinum cathode
DC power
supply
+ –
Source vial Destination vial
FIGURE 14.25
Schematic showing typical apparatus for performing capillary electrophoresis.
and electrophoretic migration. Cations are followed by neutral molecules and then anions.
Cross reference
Thus, the components of the sample are all separated in typically 10–20 minutes. As they flow
Chapter 11 ‘Spectroscopy’ and
Chapter 9 ‘Electrochemistry’. towards the cathode, the individual components are detected by UV or visible spectropho-
tometry, fluorescence, or using electrochemical detection methods.
Amino acids, peptides, proteins, DNA fragments, drugs, and even metal ions can all be sepa-
rated by CE. A major use is in sequencing DNA molecules and assessing plasmid homogeneity
(Box 14.3). The main application of CE in biomedical laboratories is to analyse serum proteins
and to monitor disease. For example, CE is used in the detection of paraproteins in the sera
of patients as their presence indicates MM, a malignant proliferation of B cells in the bone
marrow (Section 14.5). Haematology laboratories use CE to separate haemoglobins for the
qualitative analysis of haemoglobin S in diagnosing sickle cell anaemia.
Wall of capillary tube
Si O 2
O Si O Si O Si O 2
O Si O Si O
O
H 2O H 2O
O 2 2 O 2
H O 2 H
H 2O
+ H 2O
+ H 2O
H H H
O– O– O– O– O– O–
H 2O
+ + +
H
+
H 2O
H 2O
H 2O
H 2O
H 2O
H 2O
– – – – + +
Electroosmotic flow + + +
+ – – – +
–
+ +
O 2 O O 2
H 2O
O 2 O 2 2
H
H 2O
O
H 2O
2
H 2O H 2O
+
H H H H
O– O– O– + O– O– O–
H 2O
+ + +
H
+
H 2O
H 2O
Anode Cathode
H 2O
H 2O
H 2O
H 2O
Si O Si O Si O Si O Si O Si O
Wall of capillary tube
O 2
H 2O
H
+ Hydrated cations
H 2O
FIGURE 14.26
The role of electro-osmosis in capillary electrophoresis. See text for details.
14.9 CAPILL ARY ELECTROPHORESIS 369
BOX 14.3 Separating plasmids using electrophoresis
Plasmids are extrachromosomal, double-stranded DNA techniques’). Thus, both purity and heterogeneity of prepa-
molecules found in bacteria, yeasts, and some other rations of plasmids must be assessed before their use as a
eukaryotic cells. A specific type of plasmid can occur in sev- vector. Pharmaceutical grade plasmid DNA is of adequate
eral forms that differ from one another in conformation and homogeneity if more than 90% of the preparation is in the
size. One conformation is the supercoiled covalently closed form of ccc molecules.
circular (ccc) structure. In this most compact form, the cir-
Agarose gel electrophoresis (AGE) is the traditional method
cular DNA double helix is interwoven, rather like a twisted
for assessing the homogeneity of plasmid DNA. However,
rubber band. Loss of this compact structure happens if one
AGE suffers some drawbacks: it cannot be automated and
of the DNA strands breaks, which allows the circular mole-
is only semi-quantitative. Furthermore, the assignment of
cule to relax with a loss of coiling producing the open circu-
bands to the different plasmid conformations is difficult,
lar (oc) or nicked conformation. If both strands of the DNA
since their mobility changes with electrophoretic condi-
are hydrolysed, then the closed circular structure is lost and
tions. However, capillary electrophoresis (CE) is a more
a linear plasmid formed. Further structural heterogeneity
useful technique for the routine analysis of plasmid prepa-
results from the ability of plasmids to dimerize.
rations (Figure 14.27). It has high resolution, sensitivity, and
Plasmids are used as vectors in the production of recom- reproducibility, in addition to being readily automated.
binant DNA molecules (Chapter 16 ‘Molecular biology
1
40
30
Detector response
20
10
2 3
7
4
0
20 25 30 35
Time / min
1. ccc monomer of pUC19 plasmid

2. ccc dimer of pUC19 plasmid
3. Linear monomer of pUC19 plasmid
4. Linear dimer of pUC19 plasmid
5. Marker of 5.7 Kbp size
6. oc monomer of pUC19 plasmid
7. oc dimer of pUC19 plasmid
FIGURE 14.27
Separation of forms of the plasmid pUC19 by capillary electrophoresis. Courtesy
of Dr. Martin Schleef, PlasmidFactory, Bielefeld, Germany. See also Figure 16.28.
14.10 Isoelectric focusing

Isoelectric focusing (IEF) is used to separate molecules usually peptides and proteins, accord-
ing to differences in their isoelectric points (pI). The pI of a molecule is the pH at which it lacks
a net charge. The charge on a protein (or amino acid or peptide) is pH dependent. If the pH of
the solvent is below the pI of the protein, then the protein will be positively charged and will
move towards the cathode. Conversely, if the pH of the buffer is above the pI, then the protein
will be negatively charged and will move towards the anode. If the pH, however, is equal to
that of the pI of the protein it will not migrate because it carries no charge. Thus, separation
occurs in a gradient of pH values formed using a mixture of ampholytes. Ampholytes are syn-
thetic low Mr polyamino-polycarboxylic or polysulphonic acids that differ from one another
in the value of their pI (Figure 14.28).
In most IEF systems, focusing is carried out in horizontal, large pore gels (for example, 4%
acrylamide, or agarose gels of low concentration) on glass or plastic plates or in horizontal
glass tubes. The gel stabilizes the pH gradient and the large pores reduce any molecular
sieving. Thus, for example, gels can be prepared as thin layers between two glass plates,
using a solution containing riboflavin and acrylamide as described in Section 14.7. The
solution also contains a range of ampholytes. Different ranges of pH, for example, 3–10,
7–8 or 2–12 are available. Naturally, a range is chosen that encompasses the pI values of
the molecules to be separated. Once polymerization of the gel has occurred, one of the
glass plates is removed and thick strips of wetted filter paper are used as wicks to connect
the gel to the electrode compartments. The anode compartment contains phosphoric acid
and the cathode chamber sodium hydroxide. At this stage, the pH is constant throughout
the gel. However, when a potential difference is applied, the ampholytes migrate and form
an appropriate pH gradient within the gel. The current is then switched off and the samples
applied to the gel as a concentrated, salt-free layer on its surface. Alternatively, the sample
can be added directly to the gel preparation solution. When the current is switched back
on, the proteins are subjected to an electric field and migrate either towards the anode or
the cathode depending upon their charge and position in the pH gradient (Figure 14.29).
Once they reach a position where the pH of the gradient is equal to their pI, they will no
longer have a net charge and will cease moving (Figure 14.29). Given that different proteins
differ in the value of their pI, they will move to different parts of the pH gradient and so
separate.
Following focusing, the gels are treated with TCA, which removes the ampholytes and fixes the
proteins in the gel. The positions of the proteins on the gel can then be found by staining them
as described in Sections 14.3 and 14.7.
CH2 N (CH2)n N CH2
(CH2)n R
R R
R = H or — (CH2)n — COOH
FIGURE 14.28
General structure of an ampholyte. n = 2 or 3
14.10 ISOELECTRIC FOCUSING 371
pH gradient
2 3 4 5 6 7 8 9 10 11 12
+ pI=2 + + –
pI=10 pI=7
Isoelectric focusing
+ pI=2 + + –
pI=7 pI=10
Isoelectric focusing
FIGURE 14.29
+ pI=2 pI=7 pI=10
– Outline of the operation of isoelectric focusing.
Isoelectric focusing can resolve proteins that differ in their pI by a pH value of as little as 0.01:
it is, therefore, a high resolution technique. It can also be used to determine the pI of a protein
if a mixture of proteins of known isoelectric points are incorporated onto the same gel as the
test protein. After staining, the distance of each protein from one electrode is measured and
a calibration graph constructed of the distance moved by each protein against its pI. The pI of
the test protein is obtained as the value corresponding to its distance on the graph. Box 14.4
illustrates the use of IEF in investigating clinical conditions.
BOX 14.4 Diagnosis of multiple

sclerosis using IEF
Multiple sclerosis (MS) is an autoimmune condition where antibodies or immunoglobu-

lins (Ig) attack nerves within the central nervous system (CNS) causing their demyelina-
tion. These antibodies are produced within the CNS and are not found in blood. The
disease is commoner in females than males and its onset is typically during young adult-
hood. Multiple sclerosis affects between 2 and 150 per 100,000 people.
In the majority of patients, MS can be diagnosed using IEF. Samples of serum and cerebro-
spinal fluid (CSF) are obtained from patients, the latter after performing a lumbar punc-
ture (see Figure 7.8), and subjected to IEF. Up to 79–90% of samples from patients with
MS show 2–5 so-called oligoclonal bands following IEF. Oligoclonal bands are molecules
of IgG or their fragments secreted by plasma cells. The presence of oligoclonal bands in
CSF together with their absence in serum indicates that they are produced in the CNS as
Cross reference
you can see in Figure 14.30. It is therefore normal to subtract bands in serum from bands
You can learn more about
in CSF when investigating CNS diseases.
immunoglobulins in Chapter 15
+
FIGURE 14.30
Use of isoelectric focusing in
diagnosing multiple sclerosis (MS).
Oligoclonal bands are absent in
cerobrospinal fluid (CSF, lane 1)
and serum (lane 2), indicating the
person concerned does not
suffer MS. Oligoclonal bands are
present in CSF (lane 3), but absent
–
in serum (lane 4), which indicates 1 2 3 4
the individual suffered from MS. Normal Oligoclonal
14.11Two-dimensional
electrophoresis
Two-dimensional electrophoresis is a high resolution technique that combines IEF and SDS-
PAGE. In the first dimension, the samples are subjected to IEF, which separates proteins on
the basis of differences in pI (charge). The IEF is carried out in an acrylamide gel containing
ampholytes for making an appropriate pH gradient and 8 mol dm−3 urea and a non-ionic
detergent. These conditions denature the protein molecules, which separate according to
their pI values.
Following IEF, the gel is equilibrated in a buffer containing the detergent SDS, which gives the
denatured protein molecules a uniform mass-to-charge ratio. The gel is then transferred to the
top of an SDS-polyacrylamide gel. The proteins are now subjected to SDS-PAGE, which forms
the second dimension by separating the proteins according to differences in their sizes. The
gels are usually silver stained to show the separated components as described in Section 14.7.
Large (20 × 20 cm) 10% acrylamide two-dimensional gels are often used in this technique and
have been reported to resolve up to 10,000 proteins in samples isolated from cells or in tissue
extracts (Figure 14.31).
Two-dimensional electrophoresis can be used to detect and compare changes in gene expres-
sion. For example, in two different tissues where one is healthy and the other is diseased.
An extract from each of these tissues is subjected to two-dimensional electrophoresis and
the gel patterns of the two can be compared. Such a comparison may reveal the presence
14.11 T WO-DIMENSIONAL ELECTROPHORESIS 373
pH
4.0 5.0 6.0 7.0 8.0 9.0
200
100
50
Mr ⫻ 10—3
25 FIGURE 14.31
Photograph of a two-
dimensional electrophoretic
separation of proteins in a
sample of cerebrospinal fluid.
Courtesy of P.R. Burkhard,
Faculty of Medicine,
University of Geneva,
5 Switzerland.
or absence of a particular protein in the diseased state, which may ultimately be identi-
fied and its gene expression investigated. Thus, this technique can increase understanding of
the disease.
Proteomics is that branch of science that studies the complete complement of proteins
encoded by a genome. It includes identifying and quantifying all the proteins produced
(expressed) by a cell, tissue, or organism during the development of a disease. Two-dimensional
electrophoresis aids proteomic studies in a number of ways, particularly when combined with
other analytical techniques. For example, separate two-dimensional ‘maps’ can be produced
using extracts from a cell or tissue from a healthy individual and from a patient, respectively.
The gels are difficult to compare because of the large amounts of data (the number of spots).
However, there is commercially available software that can compare the two different 2-D
maps, and analyse these data both quantitatively and qualitatively. Any proteins that differ
between the two maps are of clinical interest and require further analysis. Thus, they may
be subjected to peptide mass-fingerprinting. The spots of interest are cut out of the gel and
digested with trypsin, which hydrolyses the polypeptide at the carboxyl terminal of each
arginine and lysine residue to produce a number of peptides. Matrix-assisted laser desorption Cross reference
ionization-mass spectrometry (MALDI-MS) is then used to give quantitative measurements of You can read more about mass
the peptides. The set of peptides produced is highly specific for any particular protein; hence, spectrometry in Chapter 11
the pattern of peptides produced from the protein of interest can be compared with those in ‘Spectroscopy’.
an electronic database and so identified. In a similar way, it is possible to explore the effects of
exposure to a particular drug or treatment regime, or toxin.
374 ELECTROPHORESIS
14 ELECTROPHORESIS
SUMMARY
■ Electrophoresis is the movement of charged molecules or ions in an electric field.
Separation of the ions is influenced by their nature, the support media used and the
potential difference used.
■ Electrophoresis is performed using different types of support media, some that provide
only physical support such as paper and cellulose acetate, while others are gels, for exam-
ple, starch, polyacrylamide, and agarose. The latter offer improved resolution because of
their molecular sieving effect.
■ Sodium dodecyl sulphate-PAGE and agarose gel electrophoresis are the methods of choice
for separating proteins and nucleic acids, respectively. In both procedures, mixtures of
molecules have uniform negative charges and are separated on the basis of their sizes by
the molecular sieving effect of the gel.
■ Pulsed field gel electrophoresis is used to separate larger molecules of DNA by periodi-
cally altering the size and direction of the applied electric field.
■ Capillary electrophoresis combines the high resolution of electrophoresis with the speed
of high performance liquid chromatgraphy, and is used to separate analytes such as amino
acids, proteins, DNA, and drugs.
■ Isoelectric focusing is a specialized technique where molecules are separated on the basis
of differences in their isoelectric points.
■ Electrophoretic techniques have applications in biomedical science, including investiga-

tion of diseases such as MM, CF, and multiple sclerosis.
FURTHER READING
● Hames BD. Gel Electrophoresis of Proteins: a Practical Approach. Oxford University
● Harris LR, Churchward MA, Butt RH, Coorssen JR. Assessing detection methods for gel-
based proteomic analyses. Journal of Proteome, 2007; 6: 1418–1425.
● Perrett D. Capillary electrophoresis in clinical chemistry. Annals of Clinical

Biochemistry, 1999; 36: 133–150.
● Righetti PG. Electrophoresis: the march of pennies, the march of dimes. Journal of
Chromatography A. 2005; 1079: 24–40.
● Sasse J, Gallagher SR. Staining proteins in gels. Current Protocols in Molecular Biology,
2009; 10: 10.6.1–10.6.27.
● Westermeier R. Electrophoresis in Practice. Wiley-VCH, Weinheim, 2006.

QUESTIONS 375
QUESTIONS
1. You normally separate fragments of DNA by electrophoresis using a potential differ-
ence of 100 mV. The distance between the electrodes in your apparatus is 150 mm.
However, this apparatus is unavailable and the larger system available does not have
a protocol or manufacturer’s instructions. The distance between its electrodes is
200 mm. What potential difference is necessary to produce the electric field you would
normally use?
2. Why are glycerol and the dye bromophenol blue added to gel loading buffers?
3. A protein was subjected to native PAGE and centrifugation to determine its size. Native
PAGE indicated that the molecules had a Mr of 64,000, which was confirmed by the
centrifugation studies (Chapter 12). However, its Mr was found to be only 16,000 when
subjected to SDS-PAGE. Are these data compatible? If so, explain your answer.
4. When DNA molecules are separated by agarose gel electrophoresis, which of the follow-
ing statements best describes their migration?
(a) Shorter molecules move faster than longer ones because they have a greater
density
(b) Shorter molecules move faster than longer ones because they can move through
the pores in the gel more easily
(c) Longer molecules move faster than short ones because they are subjected to more
frictional resistance
(d) Longer molecules move faster than shorter ones because of their larger mass
(e) Longer molecules move faster than short ones because they have a greater mass-
to-charge ratio
5. Which of the following are used to stain proteins and which to stain nucleic acids?
(a) Acridine orange

(b) Amido black
(c) Coomassie blue R-250
(d) Ethidium bromide
(e) Silver stain
6. A number of standard proteins of known Mr were subjected to SDS-PAGE. The bromophe-

nol blue tracking dye moved a distance of 57.0 mm. The distances moved by the pro-
teins were measured. The distances and their Mr are given in the accompanying table.
A protein of unknown Mr was also included in the experiment and the distance it
migrated is also shown in the table. Staining and destaining did not affect the dimen-
sions of the gel.
376 ELECTROPHORESIS
14 ELECTROPHORESIS
Mr × 10–3 Migration distance/mm
14.3 50.0
18.4 43.0
24.0 30.0
34.7 20.0
66.0 10.0
? 38.0
Calculate the relative mobilities of each of the proteins and plot a suitable graph of these
data. Use this graph to determine the Mr of the unknown protein.
7. A number of standard proteins of known pI were subjected to isoelectric focusing.

Following staining, the distances of the proteins from the anode edge of the gel were
measured. The distances and their pI values are given in the accompanying table. Two
test proteins, A and B, of unknown pI were also included in the experiment and their
distances are also shown in the table.
Standard proteins pI Distance/mm
3.50 4.5
4.55 17.0
5.20 33.5
5.85 53.0
6.55 69.0
6.85 79.5
7.35 90.0
8.15 103.0
8.45 116.5
8.65 125.0
9.30 140.0
Test proteins
A ? 85.5
B ? 31.5
Plot a suitable graph of these data and determine the pI values of A and B.
15
Immunological
techniques
Christine Yates
Learning objectives
■ Outline the components and functions of the immune system

■ Describe the in vivo production of antibodies
■ Explain the principles of antigen–antibody reactions
■ Discuss the uses of immunological techniques in the clinical laboratory
■ Discuss the uses of monoclonal antibodies in the clinical laboratory.
Introduction
A grasp of the underlying principles of the immune system and the fundamentals of immu-
noassay are essential to the understanding of routine immunological diagnostic assays.
Immunological assays are techniques that depend upon the visualization of interactions
between antibodies and antigens to detect and measure the concentrations of a wide range of
substances in health and disease. They are relative newcomers to the clinical laboratory hav-
ing developed from basic agglutination tests (Box 15.1). The applications were revolutionized
in the mid 1970s with the development of monoclonal antibodies. The rapid growth in our
understanding of the immune system and the utility of antibodies as diagnostic markers and
reagents has led to the development of a wide range of applications that assist in diagnosis
and treatment.
Immunological techniques largely depend upon the visualization of antibody–antigen reac-

tions, detecting the formation of immune complexes or the attachment of labelled anti-
bodies to cells or an artificial surface by a variety of methods, which we describe in this
chapter. A range of immunological techniques have been developed to detect and measure
the concentrations of cellular and humoral elements of the immune system in health and
disease and, in some cases, assess their biological activities. They have also been used to raise
378 15 IMMUNOLOGICAL TECHNIQUES
BOX 15.1 Beginnings of research into

the immune system
The discovery of antigen–antibody reactions took place at the beginning of the 20th
century. Michael Heidelberger measured antibodies using a precipitation reaction in the
1930s producing a globulin fraction of 70% purity and established the existence of com-
plement (Box 15.2). In the decades that followed, electrophoretic and ultracentrifuga-
tion studies increased our knowledge of immunoglobulins and antibody activities in the
immune response. In 1972, Rodney Porter and Gerald Edelman received the Nobel Prize
in Medicine for their work on the structure of immunoglobulin molecules. These studies
were the beginning of our understanding of the immune response and led the way for
the techniques that we take for granted in clinical laboratories of the present day.
antibodies to other analytes, such as hormones and tumour markers increasing the range of
immunoassays available to biomedical scientists.
15.1 Outline of the immune system

The immune system comprises a number of physical barriers, lymphoid organs, cells, and
humoral components that work in harmony to protect the body from infection. Its primary
function is to destroy or disable microorganisms, for example, viruses, bacteria, fungi, and
protozoa, and parasites, such as helminth worms, which are present in the environment.
Lymphoid organs are divided into two types: primary and secondary organs. Primary organs
consist of the bone marrow and thymus, where the cells of the immune system are gener-
ated and undergo maturation. Secondary lymphoid organs consist of the spleen, lymph nodes,
mucosa-associated and gut-associated lymphoid tissues (MALT and GALT respectively). These
are essential for the development of the immune response and cells circulate between the tis-
sues and lymphoid organs in the blood and lymph and maintain the process known as immune
surveillance. The cells and humoral elements of the immune system are listed in Table 15.1.
Innate immunity
Innate immunity is that present at birth. It is non-specific, does not develop an immunologi-
cal memory, and is most effective against pyogenic bacteria, fungi, and multicellular parasites.
Physical barriers, such as the skin that present an impermeable surface and secretions that
wash mucosal tissues, are the first line of defence of the innate systems. Humoral components
such as complement (Box 15.2), mannan-binding lectin, and proteolytic enzymes, which are
present in blood, mucous, saliva, and other body fluids are also part of the innate response.
The cells of the innate system are neutrophils, eosinophils, mast cells, and natural killer cells.
All of these factors combine to disable and dispose of potentially pathogenic organisms.
Acquired immunity
Acquired immunity is also known as the adaptive response. Unlike innate, acquired immu-
nity is not present at birth, but develops throughout life. The response to microorganisms
15.1 OUTLINE OF THE IMMUNE SYSTEM 379
TABLE 15.1 Cells and humoral elements of the immune system
Cells Myeloid:
• Neutrophils
• Eosinophils
• Macrophages
• Mast cells
• Dendritic cells
Lymphoid cells:
• Lymphocytes
• Plasma cells
• Natural killer cells
Humoral elements Antibodies

Complement (Box 15.2)
Mannan binding lectin
Proteolytic enzymes, cytokines
Adhesion molecules
BOX 15.2 Complement
The complement system is part of the innate immune system and, as such, it is not adapt-
able and does not change over the course of an individual’s lifetime. Plasma contains
over 40 complement proteins. They are often abbreviated as C and given a number, for
example, C1q, C2, C3; although others, such as mannose-binding lectin (MBL), factors B,
D, and so on, obviously do not follow this rule. Complement proteins work in a sequen-
tial cascade fashion; individual proteins in the sequence activating several molecules of
the next component. The effect of such a cascade is to greatly magnify the final effect.
The cascade may initially follow one of three pathways:
■ classical pathway, which is activated by an antibody
■ the pathway activated by MBL
■ the alternative pathway, which is activated by direct contact with bacteria
However, all three pathways converge in a common final pathway that destroys the cells
of the pathogen. For example, complement kills bacteria by piercing their cell surface
membrane. A further function of the complement system is the disposal of immune
complexes that are produced naturally as the immune system combats infection.
An inappropriate activation of such a powerful process could lead to many potential

problems, hence control of the complement pathway is strict, and includes a number of
inhibitory proteins, for example, C1-inhibitor, factor I, and factor H.
is specific. Acquired immunity has the ability to develop an immunological memory so the
responses increase in intensity with repeated exposure to a microorganism.
The active components of the adaptive immunity are lymphocytes derived from the bone
marrow. These produce antibodies in response to protein antigens on the surfaces of viruses
and bacteria. The antibodies bind specifically to the organisms carrying their complemen-
tary antigen and interact with complement and receptors on granulocytes and mononuclear
phagocytic cells leading to the death of the target organism.
Cytokines
In both innate and acquired immunities, chemical messengers called cytokines are produced
by cells. These allow the cells to communicate with each other, and co-ordinate and control
their growth, differentiation, and functions. Chemokines and adhesion molecules are also
instrumental in the movement of cells between the different lymphoid organs.
15.2 Synthesis of antibodies in vivo

Antibodies are glycoproteins that are able to bind to antigens, usually proteins that are nor-
mally bound to cell surfaces. They belong to a group of proteins called immunoglobulins,
which are so called because they have a role in acquired immunity and appear in the globulin
fraction when serum proteins are separated by electrophoresis (see, for example, Figures 14.3 and
14.9). Immunoglobulin molecules are formed of two heavy and two light chain polypeptide
Cross reference chains joined together by disulphide bonds. The structure of their molecules can be thought
You can read about of as Y-shaped. It consists mainly of a relatively conserved constant region. However, there is a
electrophoresis in Chapter 14 region at the end of each of the two arms of the Y called the variable region, which forms the
‘Electrophoresis’. antigen binding site and which varies in different types of antibodies (Figure 15.1).
Immunoglobulins may be divided into one of five classes depending largely upon their struc-
tures and which of the five main types of heavy chain are present. There are two types of
immunoglobulin light chain that are named lambda and kappa. The different light chains
have no particular function except to contribute to the diversity of the variable region.
Immunoglobulins that exist as multiples of immunoglobulin G (IgG) molecules are joined
together by a protein called the J chain.
The five classes of immunoglobulins are:
• Immunoglobulin G
• Immunoglobulin A
Antigen Antigen
binding binding
sites sites
Variable region
Constant region
Light chain
Heavy chain
FIGURE 15.1
Structure of an immunoglobulin G (IgG)
molecule.
15.2 SYNTHESIS OF ANTIBODIES IN VIVO 381
• Immunoglobulin M
• Immunoglobulin E
• Immunoglobulin D.
Immunoglobulin G (IgG) is the most abundant immunoglobulin in samples of serum. It may

be subdivided into four subclasses. Immunoglobulin G is most effective when it associates
with its antigen. It can then bind to complement proteins, which results in the recruitment
and activation of phagocytes. An interesting aspect of IgG activities is its ability to cross the
placenta and give the newborn baby some protection against infection during the first few
months of life.
Immunoglobulin A (IgA) it found in relatively low concentrations in serum in a form that
resembles two IgG molecules joined together. It is secreted generously in saliva, tears, and
other fluids that bathe the mucosal surfaces limiting microbial entry to the body through these
vulnerable routes.
Immunoglobulin M (IgM) is the first antibody produced in the primary immune response to
infection. It is formed of the familiar four polypeptide chains but in a structure where they are
repeated five times. This gives it multiple antigen-binding sites. Immunoglobulin M is a potent
activator of complement and phagocyte recruitment.
Immunoglobulin E (IgE) occurs at low concentrations in serum samples from healthy indi-
viduals but this concentration increases in patients with parasitic infections or allergies.
Immunoglobulin E principally interacts with mast cells that produce substances capable of
deactivating parasites and which contribute to the development of allergic reactions.
Immunoglobulin D (IgD) is found on the surface of immature B lymphocytes (Section 15.2).
It is normally present in very low levels in serum and its function is not fully understood.
SELF-CHECK 15.1
(a) List the primary lymphoid organs. (b) Define innate immunity. (c) State the major activity
of an antibody.
Polyclonal and monoclonal responses

Antibodies are produced by plasma cells that are activated through receptors on the surface
of T and B lymphocytes. These cells recognize specific parts of antigens called antigenic deter-
minants or epitopes. The recognition sites of antibodies interact with the complementary
epitope surface rather like pieces of a jigsaw puzzle. Plasma cells which produce and secrete
antibodies develop from B lymphocytes. Each type of plasma cell is a clone and uniquely
produces a single, specific type or monoclonal antibody. However, antigens are complex
molecules that potentially have multiple epitopes on their surfaces. They, therefore have the
potential to stimulate the production of many different types of antibodies. Most infections
would elicit the production of multiple antibodies, which is known as a polyclonal response.
Measurable monoclonal responses are usually confined to clinical cases where a plasma cell
tumour leads to the over-production of a single clone of cells, or when plasma cells are manip-
ulated under laboratory conditions.
When an antigen is first encountered, the immune system mounts a primary response within Cross reference
5–10 days, usually producing an IgM antibody, whose concentration increases then falls to a You can read more about
low level. However, if the same antigen (epitope) is encountered again, memory plasma cells immunoglobulins in the relevant
chapter of the Immunology
respond by producing a secondary response. This is usually in the form of an IgG antibody that
book of this series.
is produced more rapidly than the initial IgM and is of a higher intensity and specificity.
Associated disease states

Diseases of the immune system are varied, but are generally categorized by an inability to
mount an effective immune response or by an inappropriate activation of the immune sys-
tem. Individuals with immunodeficiency disorders are unable to raise an appropriate immune
response. This may be due to an abnormality in the development of the cells of their immune
system, a defect in communication between the immune cells, human immunodeficiency virus
(HIV) infection, or a fault in another essential process, such as the complement pathway.
Autoimmune disorders occur when immune responses are directed against an individual’s own
tissues or specific organs. This is normally avoided by the development of tolerance mecha-
nisms during the maturation of T and B cells. Individuals may be genetically predisposed to
developing autoimmune disease, but it usually takes the added trigger of an environmental
stimulus, such as a viral infection, to start the chain of events that lead to the development of
disease. In some cases, an autoantibody can be identified as a diagnostic marker, which may
or may not have direct pathological activity. The autoimmune disorders are divided into organ
specific where a particular organ is targeted, for example the pancreas in Type I diabetes, or
non organ specific, where antibodies are directed at cell components such as nuclear proteins
as in systemic lupus erythematosus (SLE).
In multiple myeloma (see Figure 14.9 and associated text), a clone of malignant plasma cells
produces excessive amounts of a single immunoglobulin, which may or may not behave as an
antibody. This may inhibit the production of other immunoglobulins effectively creating an
immunodeficient state and making the individual particularly vulnerable to infections.
Cross reference Allergic symptoms develop in susceptible individuals when an antibody reaction is elicited by
You can read about diseases a normally harmless environmental substance or food. The presence of the allergen, which
of the immune system in the may be inhaled pollen or an ingested food substance, stimulates the production of IgE. The
Immunology volume of this molecules of IgE cross-link receptors on mast cells, leading to the release of histamine, and
series.
other factors that produce the allergic reaction.
SELF-CHECK 15.2
(a) Describe the structure of an immunoglobulin. (b) What is the main function of a plasma
cell? (c) Name the two factors that may combine to predispose an individual to develop an
autoimmune disease.
Production of polyclonal and

15.3
monoclonal antibodies in vitro
Polyclonal antibodies are heterogeneous pools of antibodies that arise from a secondary
immune response to an antigen; that is, they are directed against multiple epitopes. They are
produced by injecting subjects with antigenic material that may be from the same species or
a different species: a procedure known as immunization. A variety of animals are used to raise
antibodies such as the antihuman globulins used in immunological assay systems (Section 15.5).
Antigens are injected into the subject accompanied by a carrier called an adjuvant. It is
unclear how adjuvants work, but it is thought they slow down the release of the antigen in the
subject resulting in a series of small ‘hits’ to the immune system. Examples of adjuvants include
Freund’s adjuvant, Syntex adjuvant, Gerbu adjuvant, saponin, and aluminium hydroxide, which
is one of the few licensed for use in humans. Protocols vary depending on the response that
15.3 PRODUCTION OF POLYCLONAL AND MONOCLONAL ANTIBODIES IN VITRO 383
is required and the routes of administration, but the procedures take into account the size of
the subject, the form of the antigen, whether it is particulate, cellular, or soluble material, and
the amount of antigen available.
Polyclonal antibodies may be raised in human volunteers but the range is limited by the choice
of acceptable antigens and other ethical considerations. An example of the human antibody
production is given in Box 15.3. Animal sources (Figure 15.2) are also controlled by stringent
legal requirements, but offer a wider range of possible antigens and anti-human options in
antibody production. Polyclonal antibodies are versatile and easy to produce; they may rec-
ognize a range of epitopes on a single antigen, which is often an advantage in immunoassay.
However, drawbacks to their use include limited reproducibility, cross-reactivity, low quantity,
and the high cost of maintenance of laboratory animals.
Monoclonal antibodies are homogenous pools of antibodies that are raised from one clone of
B cells, which produce antibodies directed against one specific epitope. The technology was
developed in the 1970s by Georges Köhler and César Milstein, and is considered one of the
most significant contributions to the biological sciences. B lymphocytes from the spleen of
mice immunized with antigen are fused with myeloma cells. The resulting hybridoma forms a
single clone of cells that produces a monoclonal (pure) antibody. You can see this procedure
in outline in Figure 15.3. The hybridoma cells may be grown in culture for many years raising
large quantities of antibody to almost any antigen.
Monoclonal antibodies are specific and may be produced in unlimited supply maintaining
high quality. They are invaluable reagents for research and diagnostic purposes, and have been
developed to treat some immune disorders and cancers. However, high levels of investment
are required for their production as sophisticated methods of selection, characterization, and
purification are necessary; especially if the final product is a therapeutic agent (see Box 16.4).
BOX 15.3 The production of polyclonal

anti-D antibodies in human volunteers
Approximately 15% of the UK population have the rhesus negative blood group, which is
determined by the absence of the rhesus D antigen on their erythrocytes. When a rhesus
negative woman conceives a rhesus positive baby there is a risk that the mother may
produce anti-D antibodies to her unborn child. This may have implications when she
has a second child, as the secondary immune response will produce IgG antibodies that
can cross the placenta and enter her baby’s bloodstream. The antibodies will react with
the foetal blood cells, damaging them and causing anaemia, jaundice, and an enlarged
liver and spleen. This is known as haemolytic disease of the newborn, and potentially
could affect 70,000 babies per year in the UK. However, this life-threatening condition
can be averted by immunizing mothers with antibodies to the D antigen during their
pregnancy. Thus, the anti-D antibodies can attach to D positive cells, destroying them
before they can illicit a reaction from the mother’s immune system.
The anti-D antibodies are obtained by raising a polyclonal response in male human
volunteers who donate plasma regularly. The volunteers are injected with rhesus posi-
tive blood cells, which stimulate their immune systems to produce anti-D antibodies.
Research into producing a monoclonal source of the antibody is continuing.
Antigen
Epitopes
Serum containing antigen

is injected into rabbit as a
series of boosters
Blood is taken
from rabbit
Serum is
separated
A polyclonal mixture of
antibodies is obtained
from the serum
The required antibody may be

purified by chromatography
(Chapter 13 ‘Chromatography’)
FIGURE 15.2
Schematic showing the production of polyclonal
antibodies in a rabbit. See main text for details.
15.4 ANTIGEN–ANTIBODY INTER ACTIONS 385
Antigen
Epitopes
Antigen is injected
into mouse
Tumour cell Spleen or lymph

culture node is removed
Mouse
Myeloma B lymphocytes
B lymphocyte
Hybrid cells
Hybridoma cells are

cultured and the
resulting cells tested
for binding against
Neg Neg Pos Neg Neg
antigen of interest
Large-scale culture
FIGURE 15.3
Monoclonal antibody Schematic showing the production of monoclonal
produced antibodies in a mouse. See main text for details.
SELF-CHECK 15.3
How are adjuvants thought to work?
15.4 Antigen–antibody interactions

To appreciate the reaction between an antigen and an antibody, two points are worth empha-
sizing. First, not all antigen–antibody reactions occur with the same strength; that is antibodies
have different affinities for antigens. Secondly, antigen-antibody reactions are reversible, and
differ in that they may not have the same capacity to remain bound to each other.
In the primary response to an infection, many antibodies will be produced to the anti-
genic stimuli presented on the surface of the pathogen. Some of the antibodies fit better to
epitopes on the antigen than will others, that is their binding is stronger or of higher affinity.
As the infection progresses, further antibody production will favour those antibodies with the
best fit. If the infection is encountered again the memory B cells will be those with the high-
est affinity. Primary antibody responses are generally IgM types with relatively low affinities
but with many binding sites due to the five-fold IgG structure of IgM. Secondary responses
are largely IgG in nature with much higher affinity due to the fine tuning of immunoglobulin
production.
Antibodies vary in their number of binding sites depending on the immunoglobulin type.
Thus, IgG has two binding sites, IgM has 10. If one binding site of an IgG antibody binds to
an antigen, then the other is also likely to bind. However, if the antigen–antibody immune
complex disassociates, the two binding sites must release at the same time. In contrast, if the
antibody is an IgM, there is potential for all ten binding sites to be bound and the likelihood
of simultaneous release becomes much less. In other words IgM molecules may have lower
Affinity is an indication of the affinity but they will usually have much higher avidity.
strength of antigen–antibody
binding. Avidity is a measure Determining the affinity and avidity of an antibody is a complex procedure. It is generally
of the ability of an antibody to confined to research or commercial laboratories, where antibodies are produced as research
bind multiple binding sites. tools or commercial products.
Methods of detection based on

antigen–antibody interactions
Cross reference
You can read more about High quality, fast turnaround assays are performed in today’s clinical laboratories, largely in
black boxes and automation ‘black boxes’ where the sample goes in one end and the answer appears on a VDU screen.
in Chapter 17 ‘Laboratory However, to understand the limitations and capabilities of the analysis systems at our disposal,
automation’.
we need to understand the basic principles on which they are based.
As knowledge of the components of the immune system expanded, the need to demonstrate
their presence and activities in controlled laboratory conditions increased. The increasing
precision and sophistication of research has led to the development of assays that could be
reliably validated for diagnostic uses. Early precipitation experiments led to the purification of
immunoglobulins (Box 15.1) and the ability to measure their concentrations in serum reason-
ably accurately.
The quantitative precipitation curve

The quantitative precipitation curve was devised by Michael Heidelberger and Forrest Kendall
in the 1950s, and is the basis for all quantitative studies of antigen–antibody reactions. In
essence, increasing aliquots of antigen are added to a series of tubes containing a constant
volume of a complementary antibody. Antigen–antibody immune complexes form and pre-
cipitate in each tube. The precipitates may be separated by centrifugation and weighed or
the particles of precipitate dispersed and the absorbance of the resulting suspension may be
measured. The amount of antigen–antibody formed can then be plotted against that of anti-
gen added, which gives the type of graph you can see in Figure 15.4. The Heidelberger curve
15.4 ANTIGEN–ANTIBODY INTER ACTIONS 387
Free Free
antibody antigen
1.0
Absorbance of precipitate
Point of
equivalence
0.5
FIGURE 15.4
Precipitation curve for human serum albumin
(HSA). An increasing amount of HSA is added to a
fixed concentration of anti-HSA. Free antibody is
present in solution prior to the point of equivalence.
x 100 200 300 400 However, after the point of equivalence, antigen will
Amount of HSA added to AntiHSA / μg be in excess. See text and Box 15.4 for details.
varies according to the species from which the antibody or antigen is derived. In the initial
tubes, the amount or concentration of antibody is in excess of that of antigen. However, in the
later tubes, the antigen is in excess and so the amount of precipitation will decrease because
of the formation of soluble complexes. These arise because, although the binding sites of the
antibody molecule are blocked with antigen, they are unable to form cross-links, as explained
in Box 15.4.
This type of experiment illustrates the basic principles of all immunological test systems that
depend upon the formation of immune complexes. An equivalence of antigen and antibody
is reached just before maximum precipitation occurs (Box 15.4). There are points of equal
precipitation on both sides of the curve shown in Figures 15.4 and 15.5: calculations based
on readouts from this type of curve must take this into account. The method can be adapted
to quantify the amounts of antibody in samples of serum, to determine antigenic valency
and is, indeed, the basis of all other precipitation reactions. Light scattering methods, such
as nephelometry and turbidimetry (Section 15.5) have, however, largely replaced precipi-
tation methods, making the quantitation of immune complexes more accurate and more
sensitive.
Other immunological techniques visualize immune complexes in media such as gels or

observe the agglutination of cells or particles with antigens attached to plastic surfaces, or
are based on the attachment of antibodies to thin sections of solid organs or sheets of tissue
cultured cells.
SELF-CHECK 15.4
(a) Define the meanings of avidity and affinity in relation to antigen–antibody binding.
(b) At what point is equivalence reached on the quantitative precipitation curve?
BOX 15.4 To precipitate or not
Antigen–antibody or immune complexes can precipitate from solution or in a gel or

other formats, which are described in this chapter. Antibodies have a number of sites
per molecule that can bind to the corresponding antigens. Molecules of IgG antibodies
have two binding sites, in the case of IgM there can be ten. Thus, the immune complexes
potentially consist of large aggregates or lattices of combined molecules that form pre-
cipitates. In experiments when a comparatively small amount of antigen is added to
a constant amount of antibody, all of the antigen molecules will be bound up in the
precipitate. This is called the state of antibody excess. When more antigen is added, the
amount of precipitate formed will increase until the ratio of antigen to antibody is equal.
This is the point of equivalence. Adding more antigen at this point will cause precipita-
tion to decrease and a state of antigen excess is reached.
Sometimes the antigen or antibody that is being measured may appear to be absent or
a negative result is obtained on analysis. However, when the sample is diluted a posi-
tive result is obtained. This is known as the prozone phenomenon, when relatively high
levels of the antigen or antibody prevent complete precipitation by blocking binding
sites, without completing the formation of a lattice of immune complexes. The diagram
in Figure 15.5 illustrates this situation. At points A and B the readout for the assay is the
same but the quantity of antigen present is very different. This is the basis for the prozone
phenemenon.
Antibody Equivalence Antigen

excess excess
Immune complex formation
A B
Amount of antigen
Antibody
Antigen
FIGURE 15.5
Prozone phenomenon. The basis of the prozone phenomenon is
that at points A and B, the results for the assay are the same even
though the quantities of antigen present are different. See text
for details.
15.5 IMMUNOLOGICAL TECHNIQUES USED IN CLINICAL L ABOR ATORIES 389
15.5 Immunological techniques used

in clinical laboratories
Immunological techniques depend in some way upon visualizing and quantifying interactions
between antigens and antibodies to detect and measure the concentrations of analytes in
clinical samples. We will discuss them under the following headings:
• Immunodiffusion techniques
• Agglutination techniques
• Immunoelectrophoresis techniques
• Immunoassays
• Nephelometry and turbidimetry techniques
• Enzyme-linked immunosorbent and enzyme linked immunospot assays
• Flow cytometry and fluorescence activated cell sorting
• Multiplex techniques
• Microarrays
• Complement assays
• Indirect and direct immunofluorescence techniques
• Immunocytochemistry
Immunodiffusion techniques
Immunodiffusion techniques depend upon the diffusion of antigens and antibodies from
holes or wells cut into an agarose gel. The antibodies employed may be monoclonal or poly-
clonal, depending on the design of the testing protocol. Where antibody meets antigen in the
gel, recognition, and binding occur forming a white line of precipitated immune complexes.
The gel may be inert or may contain defined quantities of known antibody or antigen.
Polyethylene glycol may also be added to enhance precipitation or Coomassie blue may
be used to stain and clarify the lines of precipitation. The incubation time and temperature
employed depends on the type of the immunoglobulin employed in the method. Incubation
times range from 24–72 hours, although with some IgG antibodies, reactions can be observed
after only four hours if incubated at 37°C.
The use of gel diffusion was a significant development in the transfer of immunology methods
from the research area to the clinical laboratory. The techniques require smaller volumes of
reagent and have diverse applications in diagnosis.
Single radial immunodiffusion

In single radial immunodiffusion (SRID), antigen of unknown concentration is added to the
wells in a gel that contains uniformly distributed known antibody or antiserum, which is serum
that contains known antibodies and so may be used as an analytical reagent. The gel is usually
incubated at room temperature for 24–48 hours. The antigen diffuses through the gel and on
binding with its complementary antibody forms a precipitate in the shape of a ring that sur-
rounds the well and moves outwards from it. Movement of the ring stops at a diameter where
the point of equivalence is reached, that is the antigen and antibody are present in equal
quantities. The final diameter of the ring is directly proportional to the concentration of the
FIGURE 15.6
Photograph of a single radial immunodiffusion experiment, showing
precipitation rings of varying diameter relative to the quantity of
antibody in the sample. Courtesy of D. Raine, Immunology
Department, Hull Royal Infirmary, UK.
antigen present (Figure 15.6). This is measured using a calibrated eyepiece that magnifies the
ring of precipitation or an electronic reader. A standard curve can be constructed using known
concentrations of antigen in a number of wells to allow the semi-quantitation of unknown
antigen samples. The method can be reversed by incorporating antigen in the gel to quantify
the amount of antibody in a sample.
Single radial immunodiffusion has limited applications in present day clinical laboratories
Cross reference as the determinations are not sufficiently accurate and are operator dependent, being eas-
The Immunology volume of ily affected by the delivery of sample into the wells and inattention to the test protocol. The
this series has further details method is also time consuming and inappropriate for the analyses of large numbers of sam-
about complement assays and ples. This method is, however, routinely used to measure the components and inhibitors of
the practical applications of gel
the complement system in rare immunodeficiency disorders. In these cases, the number of
diffusion methods.
samples is likely to be low and few other analytical options are available.
Double diffusion and Ouchterlony double diffusion techniques

Double diffusion techniques involve placing antigen and antibody in opposing wells of gel
and observing the precipitation of bands where immune complexes form. The commonest
application is in the Ouchterlony format, where a number of antigens or antibodies can be
compared around a single well of known antigen or antiserum. In the Ouchterlony double dif-
fusion method, a number of wells are cut in the gel such that one is placed centrally and the
rest are equidistant from it. Figure 15.7 outlines the Ouchterlony double diffusion technique.
AntiRo
A
FIGURE 15.7
Schematic showing the results of an Ouchterlony double diffusion AntiRo Ro antigen
assay. Wells in the gel plate were loaded with three test sera, A, B, B
and C. The central well contained a purified nuclear antigen (Ro, and
alternate surrounding wells contained known anti-Ro. Following
incubation, the line of precipitate formed to give the results shown.
Thus, sample A is positive for anti-Ro as it shows a line of full identity
with the Ro antiserum. Sample B is negative as there is no line C
of precipitation and sample C shows some reactivity with the Ro AntiRo
antigen but cannot be fully identified as anti-Ro. This latter type of
reaction can be caused by impurities in the antigen preparation.
If a known antibody is placed in the central well and a variety of unknown antigens placed in
the surrounding ones, then precipitation lines will appear between them that may be one of
three characteristic patterns:
1. Line of full identity

2. Line of non-identity
3. Line of partial identity
A line of full identity is a continuous one and indicates that the antigens reacting with the
antibody are all identical. In contrast, a line of non-identity is cross-shaped, thus the antigens
reacting with the antibody are different. Lastly, a line of partial identity appears as a single spur
on a line indicating that the antigens reacting to the antibody have some activity in common.
The Ouchterlony double diffusion technique may be used to differentiate the different anti-
nuclear antibodies in autoimmune disease. However, it is not a quantitative method and its
use in clinical laboratories is becoming less frequent as automated methods have replaced
such time-consuming manual techniques.
SELF-CHECK 15.5
(a) What happens when an antigen meets its corresponding antibody in an agarose gel?
(b) What term is used to describe the situation when high concentrations of analyte prevent
the formation of an antigen–antibody precipitate?
Agglutination techniques
Visible agglutination, that is the binding together of particles, may be observed in a liquid
matrix on mixing antibodies complementary to an antigen carried on the surfaces of cells or
particles, as you can see in Figure 15.8. Erythrocytes and latex particles can be used to give a
semi-quantitative assay for estimating the concentrations of antibodies. This is achieved by
preparing a range of serum dilutions from high to low concentration. Equal quantities of each
dilution and appropriate cells or particles are then mixed on slides or in microwell plates. The
strength of the antibody is expressed as a titre which is the dilution at which agglutination is
still, but only just, visible; as you can see in Figure 15.9.
In blood group serology, the agglutination of erythrocytes with known antisera is used to
identify ABO and Rhesus blood groups. When donor blood is selected for transfusion into
an individual, a cross-match procedure is performed to determine the compatibility of the
donated blood with that of the recipient. Incompatibility is indicated if agglutination occurs
1 2 3
FIGURE 15.8
Schematic showing the agglutination of erythrocytes and latex
particles on a slide. On the left erythrocytes (pink) and latex
particles (blue) are clumped or agglutinated, giving a grainy
4 5 6 appearance. On the right the suspension is smooth in appearance
because the cells and particles are not agglutinated.
Reagent
control
1 2 3 4 5 6 7 8 9 10 11 12
1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1:
27 100 400 1600 6400 25600 102400 409600 1638400 6553600 26214400
(102) (202) (402) (802) (1602) (3202) (6402) (12802) (25602) (51202)
Unsensitized Sensitized
particles particles
FIGURE 15.9
Agglutination of particles in multiwell plate. Particles coated with thyroid antigen have
been mixed with varying dilutions of test sera. Non-agglutinated cells settle in a small
pellet at the bottom of the well. Agglutinated particles form a smooth layer on the well
and partially agglutinated or weak reactions settle as a ring in the well.
when the donor’s blood is mixed with the recipient’s plasma. The ABO blood group are anti-
bodies, which are usually of the IgM type. They will agglutinate erythrocytes of an opposing
ABO group within a few seconds at room temperature. However, IgG antibodies have fewer
binding sites and their agglutination reactions are more temperature dependent. They may
Cross reference attach to cells and bind complement but not achieve visual agglutination. In the case of IgG
You can read more about blood Rhesus antibodies, tests are performed at 37 ºC and antihuman globulin antibodies are used
transfusion in the accompanying that attach to the heavy chains of molecules of IgG and complete the agglutination reaction.
text of the series, Transfusion Antihuman globulin reagents are polyclonal antibodies raised in laboratory animals to specific
and Transplantation Science.
immunoglobulin isotypes and complement components.
In the past, animal erythrocytes were used fresh or preserved and coated with antigen or anti-
body in a range of assays, for example, the Rose Waaler test for rheumatoid factor; the detec-
tion of thyroid antibodies and the Paul Bunnell test for infectious mononucleosis. In some cases
sera containing antibody required heat inactivation at 56 ºC to destroy complement activity
that could potentially haemolyse the cells during incubation. Most of these tests have now
been replaced by automated analysis, nephelometry, or other forms of immunoassay. The use
of erythrocytes has largely been replaced by polystyrene or latex particles. Commercial kits
(see Box 16.1) have reduced the use of reagents obtained from animals and increased the
shelf life of products. Simple rapid slide tests may be used to detect rheumatoid factor, C
reactive protein, and a wide range of other immunological assays that are varied in quality
and utility. However, these kits are not suitable for high volume work in contemporary clinical
laboratories.
Immunoelectrophoretic techniques
Diffusion methods are confined to systems that use few reactants and where the precipitation
lines can be easily observed. However, the resolution of multiple antigens and antibodies can
be achieved by combining immunodiffusion with electrophoresis.
Proteins in serum and urine, including immunoglobulins can be separated, by electrophoresis and Cross reference
visualized by staining them with one of a number of different dyes (Table 14.1). The quantities Electrophoresis is described in
of the separated proteins can be determined by densitometry. This is the method of choice Chapter 14.
when detecting and monitoring monoclonal paraprotein, which is a measure of tumour load
and a feature of myeloma and other B cell malignancies.
The simplest application of antibodies and electrophoresis is immunofixation. Antisera to

the range of immunoglobulin isotypes present in the sample is added to proteins separated
by electrophoresis, which identifies the light chain type of the immunoglobulin paraprotein.
However, the early methods were unsatisfactory. Adding a range of antisera to immunoglobu-
lin isotypes following their electrophoretic separation formed lines of precipitation in the aga-
rose gel, where each antibody reacted with a different immunoglobulin.
Counter immunoelectrophoresis
Since immunoglobulins migrate towards the cathode during electrophoresis and most other
proteins migrate to the anode, a combination of double immunodiffusion and electrophoresis
can be used to speed up the reaction. This is particularly useful in the preparation of antisera
and antigens for use as reagents as their specificities can be assessed quickly.
Electrophoresis in gels containing antibodies

Single radial immunodiffusion, which we described above, can be combined with electro-
phoresis. The precipitate forms in rocket-shaped zones as the antigen moves through the gel
and binds with the antisera. The area under the rocket is proportional to the concentration
of antigen.
Crossed or two-dimensional immunoelectrophoresis

In crossed or two-dimensional immunoelectrophoresis, antigens may be separated once by
electrophoresis then again at right angles to the first separation, but this second time, through
a gel that contains antibody. This technique has been used historically to separate and analyse FIGURE 15.10
components of the complement system (Box 15.2). You can see a photograph showing the Photograph showing
results of this technique in Figure 15.10. the results of
immunoelectrophoresis.
Samples have been placed
Immunoassays in wells (W) and antibody
in a trough (T). Following
The term, immunoassay is broadly applied to methods of analysis that use antibodies to detect electrophoresis, arcs of
and determine the concentration of any substance present in serum and urine in health and precipitation have formed and
disease. This is made possible by attaching a detectable label, which is usually an enzyme, been stained with Coomassie
radioisotope, or a fluorescent tag to an antibody or antigen. Immunoassays are flexible and blue dye. See text for
this has led to the development of a number of manual and automated techniques, which details. Courtesy of D. Raine,
are used in clinical biochemistry, virology, histology, cytology, and immunology laboratories. Immunology Department,
Many immunoassays are now available for diagnostic purposes using a wide range of tests on Hull Royal Infirmary, UK.
large high throughput analytical platforms. Clinical tests are available to measure the concen-
trations of, for example, hormones, tumour markers, autoantibodies, allergens, and for helping
to diagnose many infectious diseases. Biomedical scientists are often challenged in validating
selected assays for clinical use, bearing in mind the wide variation in analytical and clinical
sensitivity and specificity, and the quality of technical performance.
Since it makes no difference in principle whether the concentrations of an antibody or antigen

are being measured by the assay, the unknown substance is usually referred to as the analyte
and the labelled substance, reagent.
Early immunoassays were based on the principle of saturation analysis where excess labelled
antigen (the analyte in this case) is reacted with a limiting amount of antibody (reagent).
When known aliquots of unlabelled antibody are added they will inhibit the binding of
labelled antigen; determining the amount of bound labelled antigen indicates the amount
of unlabelled antigen that has been added. In this way an inhibition standard curve can be
drawn to estimate the amount of analyte by the level of inhibition it induces. Ideally, the anti-
body concentration should be relatively low, however, this can intensify the effects of other
factors, including the accuracy of the method for detecting the labelled reagent, the speed of
reaction, and the concentrations, avidities, and affinities of the reactants. The development
of immunoassays was initially limited by the cross-reactivity of polyclonal antibodies raised
in animals, and by the difficulty in producing large quantities of reliable antisera. However,
with the advent of commercially available monoclonal antibodies (see Box 16.4) such limita-
tions have been largely overcome producing high affinity, specific, reagents of good quality
that give accurate and reproducible results. A schematic that outlines the use of radioactively
labels is given in Figure 15.11.
Immunoassays may be classified using a number of factors:
• Choice of label, whether radioisotope, enzyme, or fluorochrome
• Method of signal detection
• Competitive or non-competitive assay
• Whether the assay requires the separation of bound and unbound reactants
Cross reference
Radioisotope labels
You can read more about health Radiolabelled immunoassays with radioisotopes, such as 125I, 3H, and 32P, are rarely performed
and safety in Chapter 4. in clinical laboratories because of the stringent health and safety restrictions that apply to
their use.
Such safety regulations define the location where an experiment may be performed, the
monitoring of staff, the storing of reagents and disposal of waste, as well as requiring a trained
radioisotope safety officer to oversee all such activities. These limit the practicality of the tech-
nology in routine clinical laboratory tests.
A number of technical aspects to the use of radioisotopes also pose problems; especially
when measuring the relatively low concentrations of analytes in serum and urine. Measuring
the activities of radioisotopes requires low background levels and isotopes with a radioactive
half-life that is short enough for the assay protocol, but long enough to give a reasonable shelf
life. The radioactive signal can be counted using a γ scintillation counter or liquid scintillation
counter, which is sensitive and accurate. However, the signal of an isotope is determined
by its rate of radioactive decay; to obtain useful results the isotope must bind to millions
of reagent molecules to give a count that is statistically significantly above the background
(see KP below).
Radiolabelled antigen
Test antigen of
unknown quantity
Antigens compete to bind antibody
Separation stage
Scintillation counter
The number of radiolabelled antigens FIGURE 15.11

bound to antibody can be used to Schematic showing a competitive radioisotope labelled
calculate the concentration of test antigen immunoassay. See text for details.
Key Points
Theoretically a minimum of 600 molecules of labelled reactant (equivalent to 10−21
mole) must be detected in an analysis to obtain a statistically relevant result.
Enzyme labels
Attaching an enzyme to a reactant provides a convenient way of generating a sensitive signal.
In many cases, a chromogenic substrate is used which the enzyme can convert into a coloured
product. Since a single enzyme molecule is capable of converting many molecules of sub-
strate to product, a detectable colour signal is readily available. Unfortunately, the photom-
eters or spectrophotometers used to measure the intensity of the colour are limited by the
optical quality of the cuvettes and microtitre trays used in analyses. The commonest enzymes
used in immunoassays are alkaline phosphatase (AP) and horse radish peroxidase (HRP),
together with chromogenic substrates such as tetramethylbenzidine (TMB) and azino-

bis-ethylbenzthiazoline sulfonic acid (ABTS).
Some immunoassays combine the advantages of both radioactive and enzyme labels by using
an enzyme-labelled antibody or antigen with a radioisotope-labelled substrate. Thus, the
action of the enzyme releases the radioactive label, which can then be isolated and accurately
measured.
Cross reference Fluorescent labels

Fluorescence is described in Fluorescent molecules absorb radiant energy, but then emit energy as light of a longer wave-
Chapter 11 ‘Spectroscopy’.
length because energy is lost in the process. Using fluorescent labels in immunoassays presents
a number of problems, such as high background fluorescence and quenching of the emitted
light by other components in the assay system. The advantage of using fluorescent labels, for
example, fluorescein and rhodamine β-isothiocyanate in immunoassays is the sensitivity they
give to the system.
Whatever type of label is used, radiolabelled, enzyme, fluorescent, all can be combined with
the biotin-avidin system outlined in Box 15.5.
Competitive and non-competitive assays

Immunoassays using labelled reagents may be subclassified into homogenous and heteroge-
neous types. Homogenous assays are quicker and easier to perform. Heterogeneous assays
require an extra step in the procedure to remove unbound antigen or antibody from the test
system. They may be competitive or non-competitive.
Competitive immunoassay is when the unknown analyte competes with a similar labelled
antigen to bind with an antibody. The quantity of detectable labelled reagent is therefore
inversely proportional to the concentration of analyte because the greater the amount of
labelled reagent that is able to bind means there are fewer binding sites available for the
analyte. In non-competitive or sandwich immunoassays, the unknown analyte is bound to an
antibody that is usually attached to a solid phase. Thus, when a labelled antibody is attached
to the bound analyte it forms a type of molecular ‘sandwich’. In this case, measurements of the
signal from the labelled antibody are directly proportional to the quantity of the analyte.
BOX 15.5 Biotin and avidin
Biotin is a coenzyme that is synthesized by many plants and microorganisms, particularly

those present in the flora of the gastrointestinal tract. Avidin is found in egg white and
can also be isolated as streptavidin from Streptomyces avidinii.
Biotin can bind to antibodies, enzymes, and many other proteins without significantly
affecting their biological activities. Avidin can bind to antibodies and be linked to a wide
variety of enzymes, fluorochromes, and radioisotopes. Avidin also has an extremely
high affinity for biotin; indeed, when combined, they may be considered for practical
purposes to be linked by the equivalent of a covalent bond. Avidin has four binding sites
for biotin. If a range of proteins are biotinylated, and all four sites on the avidin molecule
are used to recognize and bind to them, this gives many possible combinations of com-
plexes. Many variations of biotin–avidin systems are available commercially for use in
immunoassays.
Separation of bound and unbound reactants

Incomplete separation of the labelled component from the other components of the immu-
noassay mixture is a potential error in the test result. Separation may be achieved in a number
of ways:
• Adsorption onto activated charcoal
• Precipitating the antibody with 50% saturated solution of ammonium sulphate
• Precipitation with polyethylene glycol
• Using a secondary binding antibody such as antihuman globulin (AHG)
• Attaching a component to a solid phase, such as plastic tubing or a microtitre plate so that
unbound components can be washed away
• Attaching a component to a solid phase in the form of beads or particles so that the bound
reactant can be separated by simple centrifugation
Cross reference
Adsorption and precipitation methods have largely been replaced in clinical laboratories by Automation is described
the effective use of antibodies in solid phase assays that can be adapted for use on automated in Chapter 17 ‘Laboratory
analyzers with the capacity for high volumes of tests. automation’.
Nephelometry and turbidimetry

Nephelometry and turbidimetry techniques are based on the absorbance or deflection of Cross reference
monochromatic light by immune complexes in suspension. They differ in that turbidimetry You can read some more about
measures the amount of light absorbed by the complex, while nephelometry measures the nephelometry and turbidimetry
amount of light scattered at an angle to the original light path. in Chapter 11 ‘Spectroscopy’.
The most advanced instruments are fully automated, incorporate laser light sources and can
detect quantities of analyte. Rate nephelometry is a frequent method of choice in clinical
immunology laboratories; this measures the amount of immune complex production against
time. As explained earlier (Section 15.4), the principles of the quantitative precipitation curve
must be taken into account. This is especially true regarding the phenomenon of antigen
excess, where the antibody reagent may be completely consumed by relatively high amounts
of antigen in the sample, leading to an inaccurate result.
The concentrations of immunoglobulins in samples of serum are normally measured by neph-

elometry or turbidimetry in the clinical laboratory. An antihuman globulin reagent is employed
and the heavy chain of the immunoglobulin that is being measured becomes the antigenic
target. A wide range of analytes, such as complement, rheumatoid factor, β,2-microglobulin,
and α,1-antitrypsin may be measured by these methods when investigating B cell malignancies,
autoimmune and liver diseases, and immunodeficiency.
SELF-CHECK 15.6
(a) List three ways in which immunoassays may be classified. (b) What is the main practical dis-
advantage of using radioisotope-based immunoassays? (c) Name three separation techniques
used in immunoassays.
Enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISAs), also known as enzyme immunoassays (EIAs),
are one of the commonest immunoassay techniques used in clinical laboratories. The gen-
eral technique has many applications in immunology, virology, and other analytical areas.
It is easy to perform manually, but there are many automated ELISA platforms. This is a highly
commercially driven area that has expanded rapidly. Clinical laboratories have a wide choice of
readily available commercial kits for a multitude of different analyses. Enzyme-linked immuno-
sorbent assays are commonly used in the diagnosis of autoimmune and infectious diseases, such
as when investigating possible human immunodeficiency virus (HIV), hepatitis B viral infections,
and a range of autoantibodies, complement components, and immunoglobulin subtypes.
The basis of ELISA technology is that unknown antigens are attached to a solid phase in the
Cross reference
form of a plastic multiwell plate. Areas of the plate that might non-specifically bind antibody
More on ELISAs is given in
Chapter 16 ‘Molecular biology are prevented from doing so by blocking them with an inert protein. Enzyme-labelled antisera
techniques’. that can detect the antigen are added to the wells. After a period of incubation the unbound
antibody is washed away. Removing the excess antibody or reagent by repeated washings at
each stage of the procedure is essential to the technique since residual reagent or analyte in the
well can potentially interfere with the next step of the process. Washings are usually performed
using an automated plate washer or as part of a fully automated procedure. Following washing,
a chromogenic substrate is then added. The enzyme catalyses the release of a coloured prod-
uct. Colour development is stopped after a defined period of incubation using substances that
prevent enzyme activity, usually by changing the pH of the solution. Colour intensity is meas-
ured as the absorbance or optical density at an appropriate wavelength on a specially adapted
spectrophotometer designed to monitor ELISA plates. The intensity of colour development is
directly proportional to the quantity of antigen bound to the plate (Figure 15.12).
Some ELISAs use a standard curve to quantify the level of antibody in patient sera. This employs
a range of solutions containing known concentrations of antibody, which are measured at the
same time as the unknown serum. The absorbances obtained on measuring these solutions
FIGURE 15.12
Photograph showing the final stage of an
ELISA with different intensities of colour
development in the wells. The colour
developed in an assay using a biotinylated
antigen with an avidin-linked peroxidase
and ABTS substrate. Courtesy of D. Raine,
Immunology Department, Hull Royal
Infirmary, UK.
are plotted against their known antibody concentrations producing a standard curve. In many
cases, there is no recognized international standard for the antibodies measured by ELISA,
so often results are expressed in arbitrary ELISA units. If a recognized standard does exist, the
reagents in commercial kits should be calibrated to it and the results may be expressed as
international units.
Fluorescent labels and other detectable probes that are used in similar ways with multi-
well plates are usually referred to as ELISAs because they use similar protocols, even though
enzymes are not involved.
Modifications to the basic ELISA protocol include:
• Sandwich ELISA
• Competitive binding ELISA
• Automated procedures
Sandwich ELISA is a sensitive and robust variation to the basic ELISA protocol, which is out-
lined in Figure 15.13. A known antibody is bound to the wells of the microtitre plate at a
predetermined concentration. Diluted sera containing unknown concentrations of the ana-
lyte is then added to the wells. The bound reagent will capture the analyte within the wells.
Following washings, a second, antibody is added. The second antibody, which has an enzyme
label attached to it is the detection antibody, and binds to the immobilized analyte. The rest
of the procedure is as described above for the basic technique. Competitive binding ELISA
is commonly used for detecting small molecules with limited numbers of epitopes. The sera
containing unknown quantities of analyte are added to wells that have been coated with a
capture antibody, along with an enzyme-labelled reagent of similar antigenic material. If the
colour change of the chromogenic substrate is less than that obtained from a well containing
only labelled reagent, then the difference is proportional to the quantity of the analyte.
Commercially developed automated techniques based on the principle of the sandwich

ELISA use individual plastic wells or nitrocellulose sponge discs as the solid phase. However,
when the enzyme comes into contact with its substrate, a light-emitting fluorescent product
is formed. The fluorescence is measured using an ultraviolet light source and its value can be
compared to a standard curve to determine the amount of analyte in the sample of serum.
This type of analysis is the basis for determining IgE values of serum and a wide range of
allergens and autoantibodies. Other adaptations, using a range of absorbent materials, attach
antibodies to the solid phase such that the colour change produced forms a line or dot if
the result is positive. This technique underlies home pregnancy kits and a range of rapid
techniques that are used in laboratories, including autoantibody typing and the diagnosis of
malaria.
Key Points
ELISA techniques depend upon the purity and quality of the immunological reagents
and robust binding to the solid phase.
Enzyme-linked immunospot assays

Enzyme-linked immunospot (ELISPOT) assays are an adaptation of the ELISA technique, which
were developed to measure antibodies produced by plasma cells. The cells are isolated from
immunized mice and added to antigen-coated microtitre tray wells where they bind to antigen
Test sample is
Well
added to
antibody-coated
well
Antigen binds to
antibody and excess
is washed away
Wash step
Detection of antibody
using a secondary
antibody with attached
enzyme ( )
Wash step
Substrate ( ) is added
and colour intensity
(absorbance of
FIGURE 15.13 product) ( ) is
Outline of the sandwich ELISA. See measured
text for details. See also Figure 16.25
and its associated text.
and are captured on the solid phase. The cells are then removed by washing and enzyme-
labelled antihuman globulin (AHG) is added to the wells, followed by a substrate suspended
in agarose gel. The appearance of coloured spots in the gel shows where the plasma cells are
located in the wells.
Modifications to the original ELISPOT method have been made to investigate the production
of cytokines by T lymphocytes. Stimulated T cells are isolated and added to wells containing
a monoclonal antibody to the cytokine of interest. The procedure is the same as the original
ELISPOT, but uses a labelled antibody directed against the cytokine. The sites of colour devel-
opment indicate the locations of cells secreting the cytokine as you can seen in Figure 15.14. If
the spots are counted, it is possible to estimate the proportion of T-cells secreting the cytokine
present, based on the number of cells originally added to the plate.
Commercial variations on the ELISPOT have been developed that have increased the repro-
ducibility of the assays and made them more suitable for use in clinical laboratories. They are
used for the detection of interferon γ secretion by T lymphocytes that have been sensitized to
Mycobacterium tuberculosis. This is of particular importance in the diagnosis of immunodefi-
cient individuals who may have latent or subclinical tuberculosis.
SELF-CHECK 15.7
(a) Why are washing steps essential in ELISAs? (b) Which components of the immune system
are detected by ELISPOTs?
Flow cytometry and fluorescence-activated

cell sorters
Flow cytometry is a method of counting and evaluating cells or particles by passing them in a
thin stream through a laser beam. A fluorescence-activated cell sorter (FACS) is able to sepa-
rate the cells as they are identified. Figure 15.15 outlines their operation.
Flow cytometers are incorporated into analysers that count and identify leukocytes in routine
haematology tests. More complex flow cytometers with multiple lasers are used to differenti-
ate subpopulations of a range of cells. This is an important tool in the immunophenotyping of
cells when classifying leukaemias and in the classification of immunodeficiency diseases.
FIGURE 15.14
(a) Positive control for an ELISPOT assay. (b) Sample of a patient showing a positive ELISPOT
result. Courtesy of D. Spradbery, Immunology Department, Hull Royal Infirmary, UK.
Lymphocytes are mixed with a range of

fluorochrome-labelled antibodies
Cells are streamed

through a flow cell
Photomultiplier
tubes detect light
scatter at various
angles and the
antigens on the cells
can be determined
Laser
Photomultiplier
tubes
FIGURE 15.15
Basic principles of flow cytometry.
Different types of leukocytes, which may superficially appear similar when stained and exam-
ined by light microscopy (see Figure 8.1 (a)), have, however, many differences in their cyto-
plasm and on their surfaces allowing them to be distinguished and identified. Some surface
markers are termed clusters of differentiation (CD). Many CD markers have been identified and
numbered. For example, we refer to CD4 and CD8 lymphocytes when monitoring cell counts
in acquired immunodeficiency syndrome (AIDS) patients.
Cells are incubated with monoclonal antibodies directed against a predetermined panel of
CD markers or intracellular components. The antibodies are labelled with fluorescent dyes
called fluorochromes that will vary in their response to lasers of differing wavelengths. As
the dilute suspension of cells is forced through a fine tube they pass through the laser beam.
Sensitive photomultiplier tubes detect the extent of light scattering, which will indicate the
size and granularity of the cells. They also measure the fluorescence emission: this indicates
which of the labelled antibodies is attached to the cells. The data are electronically stored
for analysis and can be displayed in the form of a graph or histogram if only one antibody is
103
M1 M2
77.0% 0.3%
CD8 cells labelled with phycoerythrin
102
CD8 T lymphocytes
101
M3 M4
100 3.7% 18.9%
CD4 T lymphocytes
100 101 102 103

CD4 cells labelled with fluorescein
FIGURE 15.16
A dot plot showing the separation of CD4 and CD8 T lymphocytes in a sample from
a patient with HIV. Courtesy of D. Spradbery, Immunology Department, Hull Royal
Infirmary, UK.
used and as a two-dimensional dot plots if two or more antibodies have been used as you
can see in Figure 15.16.
The use of complex analysers with multiple lasers, and the wide variety of fluorochromes and
monoclonal antibodies have increased the use of this versatile technology. Flow cytometers
are able to process large numbers of cells and calculate the number of cells carrying a particu- Cross reference
lar marker. This is necessary in the routine testing of patients who are being treated for HIV Immunology in this series gives
more information on flow
infection. Along with FACS analysis, flow cytometry has added enormously to our knowledge
cytometry.
of the cells of the immune system.
Multiplex technology
Multiplex technology is an advanced development in the clinical laboratory, which makes
it possible to measure multiple analytes in one assay. There are a number of variations on
the basic principle, but all allow complex panels of antibodies and antigens to be evaluated
simultaneously.
Microscopic polystyrene beads or microspheres of approximately 5 μm diameter with differ-

ing ratios of fluorescent dyes incorporated into them form the solid phase of the assay. The
combination of the dyes can be varied to produce up to a hundred different coloured beads;
each bead can then have a different antigen or antibody attached to it. The beads are added
to a sample of serum that potentially contains antibodies or antigens that will attach to an
appropriate bead. A secondary fluorochrome-labelled antibody, which recognizes and binds
to the primary one, is then added. The beads are then analysed using flow cytometry, which
Beads of varying
colours and antigens
bind antibodies
Unbound antibodies are

removed by washing and
fluorescent antihuman
globulin added
Laser 1
Laser 2
Laser 1 Detects type of bead

by their colour
Detects the quantity of
Laser 2 antibody by measuring
FIGURE 15.17 intensity of fluoresence
Basic principles of multiplex analysis. See text for details.
we described above. As the beads pass through a flow cell, they pass through two laser beams:
one detects the bead and the other excites the fluorochrome. The data obtained will identify
the bead and determine the type and quantity of antibody or antigen bound to the surface of
the bead (Figure 15.17).
The potential of this technology to assist clinical laboratories and research facilities is impres-
sive, providing extensive data on a wide range of analytes.
Microarrays
Typically, microarrays are used for DNA analysis where they are known as ‘DNA chips’. However,
their use has widened with the introduction of microarrays to measure cytokines. Glass slides
with minute wells etched into their surface are loaded with a range of monoclonal antibod-
ies that are used to bind (‘capture’) the cytokine of interest. When the serum to be analysed
is added to the microarray, the capture antibodies will bind to some of the proteins in the
serum. The analysis is performed very much like an ELISA, a further antibody that has an
attached fluorescent label is added. The slides are read using an array reader with compatible
image-analysis software. The presence of a fluorescence signal indicates the positions of the
Cross reference
bound cytokines.
For more information on
microarrays see Chapter 16
SELF-CHECK 15.8 ‘Molecular biology techniques’.
(a) What name is given to the cell surface markers that are detected by flow cytometry?
(b) What is the main advantage of multiplex technology over other antibody-based methods
of analysis?
Complement assays
Severe or chronic infections will cause a continuous activation of complement (Box 15.2)
and increased concentrations of circulating immune complexes, which are a feature of some
autoimmune diseases. Both types of condition increase consumption of complement proteins
and therefore may lower their concentrations in the blood. Complement proteins are also
labile and care must be taken to prevent their activation in the sample tube prior to testing.
This means samples should be tested promptly or frozen immediately until required for test-
ing. Some procedures may require the use of the anticoagulant ethylenediamintetra-acetic
acid (EDTA) in samples or the addition of a protease inhibitor.
Complement components C3 and C4 are present in low, but relatively stable and measurable
quantities in normal sera. Both are routinely analysed by nephelometry and other automated
immunoassay systems as a first line complement screen. If a complement immunodeficiency
is suspected, many of the other proteins can be determined. A number of complement pro-
teins can be measured by single radial immunodiffusion (SRID).
Haemolytic assays are sometimes used when investigating complement deficiencies to deter-
mine which activation pathway is affected. Erythrocytes are used to demonstrate the presence
of complement by creating an environment in which the complement system pierces the
erythrocyte membrane and releases haemoglobin into solution. The procedure was referred
to as the total haemolytic complement test or CH100. However, it is difficult to determine the
final end point of haemolysis, and therefore the working end point is usually accepted as
occurring when 50% of the erythrocytes are lysed (CH50).
The classical pathway is investigated using sheep cells that have IgG anti-sheep antibodies
attached to their surfaces. They are incubated at 37°C and observed for haemolysis at regular
intervals until 50% haemolysis is reached. You can see an example of this in Figure 15.18. This
test may be performed in test tubes, multiwell plates or on SRID gels containing erythrocytes.
The time and detection systems vary with method of choice. A number of in-house protocols
and commercial kits are available.
If 50% lysis is achieved under comparable conditions to a ‘normal’ serum control, the comple-
ment activity of that pathway is normal.
The alternative pathway is tested in the same way as the classical, but antibody is not required
to activate complement. Rabbit, guinea pig, or chicken cells that are susceptible to direct
haemolysis by human complement may all be used.
In virology laboratories, complement fixation tests are used to detect the presence of micro-
organisms such as mycoplasma, those causing Q fever and psittacosis, and a range of influ-
enza viruses. The antibody to be detected is incubated with an appropriate antigen in the
presence of complement. Sheep erythrocytes that have been coated with anti-sheep IgG are
FIGURE 15.18
The CH50 test (to determine where 50% of
the erythrocytes are lysed) for haemolytic
complement activity. The photograph shows
erythrocytes in the gel. Circles of haemolysis
can be observed where complement activity
is present. Courtesy of D. Raine, Immunology
Department, Hull Royal Infirmary, UK.
then added and after a further incubation, the suspension is centrifuged. If there is still free
complement in the mixture the erythrocytes will be haemolysed, that is the complement will
have interacted with the IgG and pierced the cell surface membrane. This means that the test
is negative as the complement has not been bound by an antigen–antibody reaction during
the first incubation.
Indirect immunofluorescence and

immunocytochemistry
Indirect immunofluorescence and immunocytochemistry techniques differ from those already
described in that the solid phases necessary to detect the analytes are sections of solid organs,
or cells grown in tissue culture that have been fixed to a glass or plastic slide.
Indirect immunofluorescence
Indirect immunofluorescence (IIF) is a commonly used technique in clinical immunology lab-
oratories to detect and identify antibodies in the serum of individuals with autoimmune dis-
eases. Many autoantibodies may be detected using this technique by employing a wide range
of substrates that may be sections of animal tissues or cultured cell lines. Table 15.2 shows you
examples of antibodies and tissues used in investigating autoimmune diseases. The AHG used
is normally directed at IgG, however, anti-IgA is used for some cases.
The principle of the technique is outlined in Figure 15.19. A range of animal specimens are
prepared by cutting sections of frozen tissue and fixing them in an alcohol–acetone mixture.
TABLE 15.2 Tissue substrates used in the detection of autoantibodies by indirect

immunofluorescence
Cell/tissue Source Target antigen Disease state

Hep2 cells Human epithelial cells Nuclear components, SLE
e.g. nucleolus, centromere
Liver Rat, mouse Mitochondria, nuclear components PBC
Oesophagus Primate Tissue transglutaminase Coeliac disease
Neutrophils Human ANCA Vasculitis

Test serum
Hep2 slide
Wash step
Add fluorescein-
labelled antihuman
globulin
FIGURE 15.19
Outline of indirect
Examine microscopically using immunofluorescence. See text
ultraviolet light for details.
Cultured cell lines used include Hep2, which are human epithelial-type cells derived from a carci-
noma, that have large nuclei and a rapid rate of division. This allows observation of many nuclear
antibody patterns. The tissue or cultured cell substrate is fixed to a glass slide, which is then flooded
with dilute serum, which may contain antibody. Following a period of incubation, the serum is
washed away leaving antibodies attached to their target antigens. Fluorescently-labelled polyclo-
nal AHG is then added to the slide and will bind to the antibodies attached to the antigens. After
further incubation, the excess labelled antibody is removed by washing. As with ELISAs (see above)
the washing steps are essential to ensure excess unbound components are removed (Figure 15.20).
The sections or cells are then immersed in mounting medium and a cover slip added.
(b)
(a)
FIGURE 15.20
(a) Photograph showing commercial indirect immunofluorescence slides that have tissue
sections attached to them and diluted serum placed in the wells prior to incubation.
(b) Photograph showing the careful washing of the indirect immunofluorescence slides.
Courtesy of D. Raine, Immunology Department, Hull Royal Infirmary, UK.
The slides are then examined using fluorescent microscopy and the patterns formed by the
Cross reference
location of antibody in the tissues or cells recorded. Figure 15.21 shows examples of the types
Chapter 8 ‘Microscopy’
has further information on of staining patterns. Depending on the results obtained in the IIF panel of tests, immunology
fluorescence microscopy. laboratories will follow up positive results with more specific assays such as ELISA for subtypes
of antinuclear antibodies, liver antibodies, and vasculitis-related antibodies.
The amount of antibody present can be estimated by testing a range of dilutions of serum on
the slide and expressing the result as a titre, that is the dilution at which fluorescence can still
be observed in the cells.
The commonest fluorochromes employed in IIF are fluorescein, rhodamine, and phycoerythrin.
Many antibodies can be identified by indirect immunofluorescence. Antinuclear antibod-

ies include a number of antigenic targets and create a variety of patterns on the nuclei of
(a)
(b)
FIGURE 15.21
Photographs of indirect
immunofluorescence patterns.
(a) Nucleoli of Hep2 cells are indicated
by the presence of an anti-nucleolar
antibody. (b) Diffuse, granular staining
of kidney tubules indicates the presence
of mitochondrial antibodies. Courtesy of
D. Raine, Immunology Department, Hull
Royal Infirmary, UK.
SUMMARY 409
Hep2 cells, for example smooth homogenous staining of the nucleus is seen in systemic lupus
erythamatosis (SLE). Rat or mouse kidney and liver sections will reveal the presence of mito-
chondrial antibodies as seen in primary biliary cirrhosis (PBC). Pancreatic islet cells and sec-
tions of adrenal gland, ovary, testes, and oesophagus are commonly used when investigating
for autoantibodies such as those for anti-tissue transglutaminase, which occur in coeliac dis-
ease. Human neutrophils are used to demonstrate the antineutrophil cytoplasmic antibodies
(ANCA) that are a feature of some forms of vasculitis.
The drawback of IIF in routine clinical laboratory testing is that it is extremely operator depend-
ent and time consuming. The method relies on the expertise of individuals in slide preparation Cross reference
and microscopy. Higher than normally expected numbers of staff are also required to achieve The Immunology volume of this
an appropriate throughput of samples. This has been alleviated to some extent by using auto- series covers an extensive range
of autoimmune disorders and
mated slide processors, but eventually many of the high volume antibody tests are likely to be
their associated antibodies.
performed by multiplex technology-based assays (see above).
Direct immunofluorescence
Direct immunofluorescence is used to detect substances that may be deposited in tissues and
organs during some disease states, for example, some skin disorders can be diagnosed when
antibodies and complement can be demonstrated in all layers of the epidermis. A biopsy of
the patient’s tissue is removed from a site adjacent to a skin lesion and fixed on a glass slide.
Antihuman globulin with a fluorescent marker is added to the slide, which will bind to antibod-
ies present in the tissue sample. The procedure is the same as indirect immunofluorescence
in that excess antihuman globulin is removed by washing, and then the tissue is examined for
the fluorescence that will demonstrate the presence of autoantibodies.
Immunocytochemistry
Immunocytochemistry is similar to the fluorescence methods described above, in that direct Cross references
and indirect methods can be used. The techniques were developed to identify tumours by You can read more about
accurately targeting tumour cell surface markers. This technology has been automated and cytological and histological
is a growing area in laboratories that specialize in histological and cytological investigations. investigations in Chapter 7
‘Samples and sample collection’
Although fluorescent labels can be used, the methods are often based on colour development
and Chapter 8 ‘Microscopy’.
using enzyme-labelled antibodies and substrates similar to those employed in ELISAs.
You can learn more about
SELF-CHECK 15.9 direct immunofluorescence
and immunocytochemistry in
(a) What is the difference between direct and indirect immunofluorescence? (b) Name the Histopathology book of this
two examples of fluorochromes that may be used when detecting antibodies by indirect series.
immunofluorescence.
SUMMARY
■ The immune system consists of the interaction of specialized organs, cells and humoral
components that protect the body from infection.
■ Innate immunity is present at birth, is immediate acting, but non-specific. Acquired immu-
nity is slower to develop, more specific and has immunological memory.
■ Immunoglobulins, in the form of antibodies, are essential components of the immune

system, which have been adapted as diagnostic tools.
■ The formation of antibody–antigen immune complexes and their behaviour form the
bases of immunoassays.
■ The use of radioisotopes, enzymes, and fluorochromes to label antibodies and antigens
was an important advance in the development of immunoassay.
■ The use of monoclonal antibodies in immunoassays has revolutionized laboratory

medicine.
■ Advances in areas, such as flow cytometry and multiplex technology, will have a lasting
impact on clinical laboratories that were previously dominated by manual methodology;
small-scale manual immunoassays are being overtaken by automated analytical methods
in all specialities in clinical laboratories.
FURTHER READING
● Cruse JM, Lewis RE. Historical Atlas of Immunology. Informa Healthcare, 2005.
An interesting and well illustrated account of the development of our knowledge of
immunology.
● Hay FC, Westwood OMR. Practical Immunology, 4th edn. Blackwell Science, Oxford,
2002.
Practical Immunology is a useful bench textbook for research scientists and gives insight
into the origins of immunological techniques and increases the understanding of bio-
medical scientists.
● Murphy KM, Travers P, Walport M. Janeway’s Immunobiology, 7th edn. Garland

Science, 2007.
A comprehensive text that gives a detailed, well written account of the immune system.
Excellent diagrams and a CD-ROM with animations and videos. Although a little advanced
for undergraduates, it is an invaluable resource for studying all aspects of immunology.
● Peakman M, Vergani D. Basic and Clinical Immunology, 2nd edn. Churchill Livingstone,
London, 2009.
This excellent book describes the immune system and associated disease states. It is well
laid out and relevant to clinical laboratory practice.
● Price CP, Newman DJ. Principles and Practice of Immunoassay, 2nd edn. Macmillan,
Oxford, 1997.
Comprehensive account of the development and practice of immunoassay in the clinical
laboratory. Covers all aspects of methodology, standardization, and quality.
● Sompayrac LM. How the Immune System Works, 3rd edn. Wiley-Blackwell, Harlow,
2008.
Brilliant, readable account of how our immune system works; recommended to anyone
who wants to learn about immunology. Well written, humorous, and explained in simple
terms; difficult concepts are presented in a highly imaginative way making them easier to
remember.
15.5 IMMUNOLOGICAL TECHNIQUES USED IN CLINICAL L ABOR
QUESTIONS
ATORIES 411
QUESTIONS
1. Select three characteristics of a high affinity antibody from:
(a) An antibody with more than two binding sites

(b) An antibody produced in the secondary immune response
(c) Is most likely to be IgG
(d) Forms immune complexes that do not easily dissociate
(e) Produced by memory B cells that have refined ‘best fit’ antibodies
2. Which of the following statement(s) is/are TRUE.
(a) Acquired immunity is present at birth

(b) Complement can kill pathogens without the involvement of antibodies
(c) Antibodies are produced by T lymphocytes
(d) Genetic predisposition alone commonly causes autoimmune diseases
(e) The precipitation curve is not affected by the source of the antibody or antigen
3. Discuss the difference between a polyclonal antibody response and a monoclonal

response.
4. What are the drawbacks of using polyclonal antibodies?
5. Give a short description of the three types of antibody labels that may be used in immu-
noassays with examples of assays that use them.
16
Molecular biology
techniques
Learning objectives
■ Outline the structures of nucleic acids and proteins

■ Describe the general functions of biological macromolecules
■ Explain the basic molecular biology encountered in clinical laboratories
■ Discuss the principal techniques of molecular biology used in clinical tests
■ Outline the uses of molecular biology techniques in clinical laboratories
Introduction
The term, molecular biology is thought to have been first used by Warren Weaver in a 1938
report to the Rockefeller Foundation. Molecular biology is the study of biological phenomena
in terms of the physical and chemical properties of the molecules of organisms. It can be
described generally as the study of the structures, functions, and interactions of biological
macromolecules. Two major types of macromolecules common to all cells are the nucleic
acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and proteins. DNA and RNA
carry biological information. Deoxyribonucleic acid contains the genetic information of all
cells and some viruses, that is to say it is the material of genes. Ribonucleic acid only holds the
genetic information of some viruses. However, it is present in all cells in a number of forms,
including messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules
that are necessary to allow the genes to be expressed and form proteins. Proteins perform
most of the activities in cells. They catalyse metabolic reactions, perform mechanical work in
muscles, cilia, and flagella, act as sensors, perform regulatory, and control functions, and also
constitute some of the major structural features of the cell.
The definition of molecular biology given above is rather limited in that it fails to describe
how the subject overlaps with other areas of biology, chemistry, microbiology, and compu-
tational biology, in particular biochemistry, genetics, immunology, genomics, proteomics,
16.1 STRUCTURE AND PROPERTIES OF MACROMOLECULES 413
and bioinformatics. The techniques of molecular biology have reached sophisticated levels,
and are used extensively in the biological and biomedical sciences. Many of these techniques
Cross reference
have been automated and are performed by robots, which makes them particularly suitable
You can read about automation
in the diagnosis of clinical conditions and monitoring treatments. This chapter will provide an in Chapter 17 ‘Laboratory
overview of the scope of molecular biology and introduce you to the structures, properties, automation’.
and functions of some of the molecules involved. It will describe some of the experimental
techniques of molecular biology and outline a number of their applications in the biomedical
sciences.
16.1Structure and properties R — OH + HO — R’

of macromolecules
H
Macromolecules are ones that are large, that is have Mr in excess of approximately 5,000.
O
Biological macromolecules are polymers, formed by covalently linking much smaller units
H
together in condensation reactions (Figure 16.1). Within the polymer, such units are often
referred to as residues. Linking two units gives a dimer, three a trimer, and so on to eventually R — O — R’
give a polymer, which may contain hundreds or thousands or millions of residues.
FIGURE 16.1
A general condensation
reaction.
Nucleic acids
The complete hydrolysis (see Section 16.4) of any nucleic acid gives:
• Pentose (five carbon) sugars
• Pyrimidine and purine bases
• Phosphate groups
The sugars are ribose (Rib) in the case of RNA and deoxyribose (dRib) in DNA. Deoxyribose
nucleic acid contains the purine bases adenine (A) and guanine (G), and the pyrimidines, cyto-
sine (C) and thymine (T). Ribonucleic acid also contains A, G, and C, but the primidine uracil
(U), rather than T (Figure 16.2). Minor amounts of other, chemically related, bases are also
released. The phosphate groups are negatively charged at physiological values of pH.
The combination of a base and sugar forms a nucleoside (Figure 16.3 (a)). Note that the
atomic positions in sugars and bases need to be distinguished, and this is achieved by adding
a prime (′) to the sugar positions as you can see in Figure 16.3 (a). Bases are attached to the
1′ carbon atoms of the sugars. The addition of a phosphate(s) to a nucleoside gives a nucle-
otide (Figure 16.3 (b)). Phosphates may be bonded to the 5′ or 3′ positions giving nucleoside
5′ phosphate or nucleoside 3′ phosphate respectively. Table 16.1 summarizes the nomenclature
of the major bases, nucleosides, and nucleotides.
Both DNA and RNA are polymers of nucleotides; hence, their alternative name of polynucle-
otides and both are similar in structure. Phosphodiester bonds link the nucleotides together. If
you examine Figure 16.4, you will see these bonds link the 3′ and 5′ carbons of adjacent sugar
residues giving the link directionality: the convention when writing or drawing them is to place
the 5′ to the left and the 3′ to the right (as in Figure 16.5).
This means the sequence of bases also runs in a 5′ to 3′ direction forming the primary structure
of the nucleic acid, which differs in nucleic acids from different sources. Primary structures
of nucleic acids can be described by one of several conventions, which you can see in Figure
16.5 (a) and (b).
414 16 MOLECUL AR BIOLOGY TECHNIQUES
Ribose Deoxyribose
5 O 5 O
HOCH2 OH HOCH2 OH
4 1 4 1
H H H H
H H H H
3 2 3 2
HO OH HO H
NH2
Adenine (A) Guanine (G) O
C C
6 N 6 N
N1 5C 7
H N1 5C 7
8 C H 8 C H
2 4
H C 3
C 9
H2N C
2 4
C 9
N 3 N
N N
H H
Thymine (T) O Cytosine (C) NH2

C C
4 4
H N3 5 C CH3 N3 5 C H
2 6 2 6
O C 1
C H O C C H
1
N N
H H
Uracil (U) O
C
4
H N3 5C H
2 6
O C 1
C H
FIGURE 16.2 N
Structures of the sugars and bases
of nucleic acids. H
(b) NH2
(a) O
Uracil C
6 N
C N1 5C 7
4
H N3 5C H 8 C H
2 4
H C 3
C 9
Ribose O C
2 6
C H N
1 O O O N
5’ N 5’
O −O O
HOCH2 P O P O P OCH2
4’ 1’ −O −O −O 4’ 1’
H H H H
H H H H
3’ 2’ 3’ 2’
HO OH HO H
FIGURE 16.3
Structures of (a) the ribonucleoside, uridine, and (b) the nucleotide, deoxyadenosine
5′-triphosphate (dATP). See also Table 16.1.
TABLE 16.1 Standard nomenclature of the major nucleic acid bases, nucleosides,
and nucleotides
Bases Ribonucleoside Ribonucleotide* †

Adenine (A) adenosine adenosine 5‘-monophosphate (AMP)
Guanine (G) guanosine guanosine 5‘-monophosphate (GMP)
Uracil (U) uridine uridine 5‘-monophosphate (UMP)
Cytosine (C) cytidine cytidine 5‘-monophosphate (CTP)
Bases Deoxyribonucleoside Deoxyribonucleotide (as 5’-monophosphate)
Adenine (A) deoxyadenosine deoxyadenosine 5’-monophosphate (dAMP)
Guanine (G) deoxyguanosine deoxyguanosine 5’-monophosphate (dGMP)
Thymine (T) deoxythymidine deoxythymidine 5’-monophosphate (dTMP)
Cytosine (C) deoxycytidine deoxycytidine 5’-monophosphate (dCTP)
* Where the attachment for the phosphate is not specified, the 5’ can be assumed by default. Other sites, for
example C-3’, must be denoted.
†
Other nucleotides with two (di), three (tri), and, rarely four (tetra) phosphates are known. For example,
adenosine 5′-triphosphate (ATP), deoxythymidine 5′-diphosphate (dTDP).
–O P O
O
O Basen
CH2
H H
3’
H H
O H
–O P O Phosphodiester bond
O
5’ O
CH2 Basen+1
H H
H H
O H
–O P O
FIGURE 16.4
Structure of a phosphodiester link.
(a) U
G OH
O
A OH
O O
O
C OH
O O P OCH2
O
OH –O
O O P OCH2
O
–O
O O P OCH2
–O
P OCH2
–O
C A G U
P P P P
. . . pCpApGpU . . .
. . . pC – A – G – U . . .
. . . pC A G U . . .
(b) T
G H
O
A H
O O
O
C H
O O P OCH2
O
H –O
O O P OCH2
O
–O
O O P OCH2
–O
P OCH2
–O
C A G T
P P P P O
. . . pdCpdApdGpdT . . .
. . . pdC – dA – dG – dT . . .
. . . pdCdAdGdT . . .
FIGURE 16.5
Various conventions used in showing the primary structures of (a) RNA
and (b) DNA.
Native DNA exists as two complementary strands held together by hydrogen bonds. The
strands form the famous double helical or duplex structure first described by Crick and Watson,
and constitute the secondary structure of DNA. You can see this in Figure 16.6. The strands run
in opposite directions, an arrangement described as antiparallel. The bases occur as comple-
mentary pairs, with A hydrogen bonded to T and G with C. In each case, a purine base is com-
plementary to a pyrimidine meaning the lengths of each base pair (bp) are approximately the
same and the strands can line up as a double helix of regular diameter. Two hydrogen bonds
hold each A-T pair together unlike the G–C combination that has three, making it the more
stable of the two pairs. The sequence of bases of one strand automatically gives that of the
other. Ribonucleic acid is single stranded. However, it can form double helical regions, when
the strand double backs on itself. Its base pairing rules are similar to those of DNA, but A pairs
with U and G with C. It is also possible to form heteroduplexes that consist of complementary
strands of DNA and RNA.
Complementary base pairing is the basis for replicating genes and for allowing their expres-
sion. During replication, each strand of DNA acts as a separate template for the synthesis of a
new complementary strand. Deoxyribonucleic acid dependent DNA polymerases (usually Cross reference
abbreviated to DNA polymerase or DNA pol), assisted by a variety of other proteins, copy You can learn more about
each template to give a new complementary strand. Thus, the two new DNA molecules each replication and transcription of
consist of one old (parental) and one newly synthesized strand, hence, the process is called DNA in Elliott & Elliott (2009)
semi-conservative replication. Molecules of RNA are formed when DNA dependent RNA and Craig et al. (2010), which
polymerases use a single strand of DNA as a template to catalyse the formation of a comple- are listed in the Further reading
section.
mentary RNA copy. This process is called transcription.
SELF-CHECK 16.1
A strand of DNA has the sequence ATGCCGTTAGGAGTA. Give the complementary sequence
to it, but written in the 5′ to 3′ direction.
10 bp = 3.4 nm
FIGURE 16.6
Diameter Computer generated model of
2 nm double-stranded DNA.
All chromosomes contain a single molecule of DNA. In prokaryotic cells, the genetic material
usually occurs as a single chromosome containing a single circular DNA molecule. However,
mutual repulsion between the negative charges of the phosphate groups opposes the bending
of the molecule. In addition, bacterial chromosomes have Mr of at least 2 × 109, and are over
1 mm in circumference and need to be folded to fit into bacterial cells that are only approxi-
mately 1 μm diameter. The charges on the phosphate groups are neutralized by binding of basic,
that is positively charged, proteins to the DNA. This makes the bending and elaborate folding of
DNA possible. Bacterial, and some yeasts, which are eukaryotes, also contain extrachromosomal
DNA structures called plasmids. Like the bacterial chromosome, plasmid DNA is circular and
double stranded. However, it is much smaller with Mr of 2 × 106 to 20 × 106, which corresponds
to 3000–30,000 bps. Bacterial plasmids normally contain genetic information for proteins that
confer a specialized and sometimes protective phenotype on the organism often, for example,
imparting antibiotic resistance. Plasmids are self-replicating and their DNA is duplicated before
every cell division and copies of the plasmid DNA segregate to each daughter cell, assuring con-
tinued propagation of the plasmid through successive generations of the host cell.
The paired chromosomes of eukaryotes are much larger than bacterial ones. For example,
a human haploid cell contains DNA molecules of total length approximately 3 × 109 nucle-
otides divided between 23 chromosomes (22 autosomes, and a sex chromosome, X or Y).
Each eukaryotic chromosome contains a linear DNA molecule many millimetres long. These
can only fit into a nucleus by being even more elaborately folded than bacterial types, follow-
ing their combination with a variety of chromosomal proteins.
SELF-CHECK 16.2
Calculate the total length of DNA in a diploid human cell. You may find it helpful to consult
Figure 16.6.
The sequences of bases in the nucleotides of a gene that encodes a protein are transcribed
to eventually form a mRNA molecule. The mRNA is then used in the synthesis of the cor-
responding polypeptide in a process called translation; the sequence of bases in the mRNA
encodes the sequence of amino acid residues in the polypeptide. Translation of mRNA occurs
on ribosomes and requires the activities of many other nucleic acids and proteins. Each of the
groups of three bases called codons in the mRNA specify each of the 20 types of amino acids
used in translation. Hence, the sequence of bases in the gene (DNA) determines the sequence
of amino acid residues in the polypeptide.
Ultraviolet absorption by nucleic acids

The presence of the purine and pyrimidine bases (A, T, G, C, and U) means nucleic acids absorb
significant amounts of light in the ultraviolet (UV) region of the spectrum (see Chapter 11), with
maximal absorbance occurring at about 260 nm (Figure 16.7). Hence, solutions of pure dou-
ble and single stranded DNA and RNA of concentrations 50 and 40 μg cm−3, respectively, have
absorbances of 1.0 at 260 nm. Proteins, the major contaminants in isolated DNA samples, also
Cross reference absorb in the UV region, but maximally at 280 nm (Figure 16.7). Thus, the ratio of the absorbances,
Chapter 11 ‘Spectroscopy’, gives A260/A280, is often used to assess the purity of isolated samples of nucleic acids. The typical A260/A280
much more information on
for isolated DNA with little contaminating protein is about 1.8. Lower ratios indicate increased
measuring absorbances.
contamination with proteins. The A260/A280 ratio of extracted RNA is generally 1.8–2.0.
SELF-CHECK 16.3
A 10 μL sample of a purified DNA solution was diluted to a final volume of 1 cm3. The A260 of
the diluted solution was 0.55. What is the concentration of DNA in mg cm−3?
DNA
Relative absorbance
Protein
240 260 280 300 FIGURE 16.7

Wavelength/nm Ultraviolet absorption maxima of DNA and proteins.
Separating the double helical structure of purified DNA with denaturing agents, such as heat,
alkali, organic solvents, to form two single strands causes its absorption at 260 nm to increase
markedly. This hyperchromic effect arises because the transition from an ordered double-
helical structure to unpaired single stranded DNA means base–base interactions are now at
a minimum. The resulting change in the properties of the rings of the bases means they can
absorb more UV light. If the strands are allowed to reassociate, or reanneal, to give the origi-
nal double helical structure, the UV absorption returns to its initial value. Thus, the processes
of DNA disassociation and reannealing under various experimental conditions can be fol-
lowed by monitoring the absorbance at 260 nm.
SELF-CHECK 16.4
Would you expect RNA molecules to show a hyperchromic effect?
Proteins
Proteins are polymers of amino acid residues, which all have a common structure, but differ
from one another in the nature of their side chains, which you can see in Figure 16.8.
Individually, proteins have more complex structures than nucleic acids because they are
formed from 20 different types of amino acids, which are linked together by peptide bonds
(Figure 16.9).
The structures of most proteins are subdivided arbitrarily into hierarchical levels. The four
main levels are the primary, secondary, tertiary, and quaternary structures, which you can see
illustrated in Figure 16.10, although other structural levels have also been described. Note, like
any classification, this is an artificial division. The primary structure of a protein is the sequence
of its amino acid residues or side chains. The secondary structure is the regions of local folding
of the polypeptide chain. The famous α helix and β sheet are examples of secondary struc-
tures. Tertiary structure specifies the positions of all the atoms in the polypeptide. Quaternary
structures occur only in proteins composed of more than one polypeptide chain. For example,
COO–
+
H3N C H
R=
Glycine —H
Alanine — CH3
Serine — CH2OH
Aspartic acid — CH2COO–
+
Lysine — CH2 — CH2 — CH2 — CH2 — NH3
CH3
Leucine — CH2 — CH
CH3
Phenylalanine — CH2—
Cysteine — CH2SH
O
Glutamine — CH2 — CH2 — C
NH2
Histidine — CH2
+
FIGURE 16.8 HN NH
Structures of some α amino acids.
the quaternary structure of adult haemoglobin is α2β2, meaning its molecules are composed of
two so-called α polypeptides and two β polypeptides. Individual polypeptides of a quaternary
structure are usually called subunits.
Protein molecules are only marginally stable. The energy of a folded (native) protein is only
relatively slightly below that of an unfolded or denatured protein; hence, samples containing
COO– COO–
+ +
H 3N C H + H 3N C H
R R’
H2O
R O COO–
+
H3N C C N C H
H H R’
FIGURE 16.9
Formation of a peptide bond by Peptide
condensing two amino acids. bond
(a) G-I-V-E-E-C-C-A-S-V-C-S-L-Y-E-L-E-D-Y-C-D
(b) (c)
(d)
FIGURE 16.10
Examples of the four main structural levels of proteins. (a) Primary structure of the A chain
of insulin denoted using the standard one-letter abbreviations for each of the amino acid
residues. (b) Secondary structure (largely α-helical) of a myoglobin molecule. The red
portion is a haem group. (c) Tertiary structure of myoglobin. (d) Quaternary structure
(α2β2) of haemoglobin.
proteins have to be treated with care. The structure of any protein is determined largely by
its primary structure that is the sequence of its side chains. In an aqueous environment, the
polypeptide folds so that side chains that cannot interact favourably with water molecules
segregate to the interior of the molecule. This is referred to as the hydrophobic effect. The
hydrophobic effect ensures each type of protein has a unique conformation that determines
the properties of that protein and the types of molecules with which it can interact.
Proteins generally function by recognizing and specifically interacting with other molecules
called ligands. Ligands are normally small, that is, they are not macromolecules, but proteins
can also bind to relatively small areas of other proteins and nucleic acids. Protein molecules
have discrete sites on their surface that are complementary to the ligand, which allows the
protein to recognize and specifically bind to it in a reversible manner. The protein–ligand
complex can dissociate back to the free molecules or it can induce a change in the conforma-
tion of the protein, which induces some biological event to occur. For example, in the case of
enzymes, the ligand would be a substrate and the event would be the catalysis of a reaction
that converts it to product. If the protein were a receptor and the ligand a hormone, then
changes would be stimulated in target tissues.
16.2Molecular biology-based
techniques
The complementary binding of pairs of bases in nucleic acids offers a convenient way of rec-
ognizing and isolating specific base sequences within a fragment of a DNA or RNA molecule or
of isolated nucleic acid molecules. Similarly, proteins specifically recognize and bind to a vari-
ety of different types of molecules. The abilities of macromolecules to bind complementary
substances is the basis of many routine clinical techniques, for example fluorescence in situ
hybridization (FISH) and enzyme-linked immunosorbent assays (ELISAs), as we will describe
later (Section 16.8).
Throughout this chapter you will encounter many molecular biology techniques where the
specific binding properties of macromolecules has been exploited in recognizing, isolating,
and analysing molecules in biological and clinical samples. In all cases, the specificity of rec-
ognition is such, that the techniques are extremely sensitive. They therefore require relatively
small amounts of sample compared with more traditional chemical techniques and can be
performed on volumes as small as a few microlitres, hence minimal amounts of reagents are
required saving the health services’ money. Experimental molecular biology kits are available
(Box 16.1).
16.3 Isolation of nucleic acids

The study of nucleic acids and their analysis or use in clinically related procedures, for example,
preparing the probes (Box 16.2) used in fluorescence in situ hybridization (FISH) described in
Section 16.8, often involves their isolation from cells and tissue samples. The nucleic acid puri-
fied for clinical purposes is usually DNA, rather than RNA. Chromosomal DNA can be purified
from bacterial or eukaryotic cells and this is generally described as total DNA. However, plas-
mids (Section 16.1) can also be isolated from bacterial and some types of eukaryotic cells, such
as yeasts. In general, the separation of plasmids follows the same basic protocols as those used
to obtain chromosomal DNA, although at some stage, procedures must be used to separate
the plasmid from the bulk of the cellular DNA. This section will discuss general methods for
isolation and purification of chromosomal DNA from bacteria and eukaryotic cells; the isola-
tion of plasmids is described in Section 16.9.
Chromosomal DNA is difficult to isolate in an intact and undamaged form because of the large
size and fragile nature. However, several isolation procedures have been developed to purify
it from cells. Indeed, a number of them are commercially available in kit form. Used correctly,
these procedures produce preparations of DNA that are stable and of large Mr, and relatively
16.3 ISOL ATION OF NUCLEIC ACIDS 423
BOX 16.1 Experimental molecular

biology kits
Many of the techniques of molecular biology are well established and can be performed
with experimental kits that can be purchased from commercial suppliers. Indeed, exper-
imental kits are central to research in molecular biology and a number of the protocols
described in this chapter make use of them. For example, you can buy kits to extract
DNA or RNA from tissues (Section 16.3) and synthesize complementary DNA (cDNA)
from the isolated mRNA (Section 16.9). Such kits provide all the reagents and other
materials needed for the particular experiment, descriptions of the underlying method,
and easy to read and follow protocols. Reasonably ‘good’ results can usually be obtained
using a kit. Indeed, in many cases, experimental results are easier to obtain than with
the traditional method of purchasing the materials separately and preparing individ-
ual solutions of the reagents. This saves time, but kits are generally the more expensive
option. They are also inflexible in that their protocols are difficult to modify for dif-
ferent circumstances. Furthermore, their extensive use carries the danger of lowering
one’s general laboratory skills. You will never fully understand an experimental method
if your depth of knowledge is that 3 μL of the reagent in the red tube must be added to
5 μL of sample and then heated to 30°C for 5 minutes! A thorough understanding of any
experimental procedure will enable you to use it to its full effect, know what to do when
it goes wrong, as it will on some occasion, and appreciate when a result is aberrant or
outside the experimental limits, artefactual, or simply incorrect. The best advice is to
learn and understand the technique before using the corresponding kit.
free of contaminating RNA and proteins. Whatever, its source, a number of points must be
considered in the isolation of DNA. These include:
• pH
• Temperature
• Ionic strength
• Nuclease activities
• Mechanical stress
The isolation of DNA requires it be released into a medium of appropriate pH. This is nec-
essary to preserve the interactions and chemical bonds that stabilize the molecules. For
example, hydrogen bonding between the complementary strands is stable at pH 4–10, while
the phosphodiester linkages become unstable below pH 3 and above 12. Furthermore, the
N-glycosidic bonds joining purine bases (adenine and guanine) to the deoxyribose sugars are
hydrolysed at pH values of 3 or less. Phosphodiester linkages and N-glycosidic bonds are sta-
ble at temperatures up to 100ºC. However, temperatures of 80–90ºC destabilize the hydrogen
bonds holding the double helix intact causing it to unwind. Care must be taken in choosing
an appropriate ionic strength for solutions used in DNA extractions. Deoxyribonucleic acid
is most stable and soluble in salt solutions. However, concentrations less than 0.05 mol dm−3
weakens the hydrogen bonding between complementary strands. Enzymes, such as deoxy-
ribonucleases or DNases (see Section 16.4) are present in all cells and can degrade DNA during
its purification if their action is not inhibited.
BOX 16.2 Probes
Probes are labelled molecules that are complementary to the molecules one is studying.
For example, if a solution of DNA is heated at 100°C or exposed to pH values above 13
the complementary base pairs are disrupted and the double helix dissociates into its two
single strands. Complementary single strands of DNA, however, readily reform double
helices by a process called annealing, hybridization, or DNA renaturation, if they are
allowed sufficient time at temperature at or below 65ºC. Hybridization reactions can also
occur between any two single–stranded complementary polynucleotides forming DNA–
DNA, RNA–RNA homoduplexes, or RNA–DNA heteroduplexes. These specific hybridiza-
tion reactions are the basis of sensitive methods widely used to detect and characterize
specific nucleotide sequences in both RNA and DNA in samples. In such cases, the probe
could be a single-stranded DNA molecule that has been labelled in some way to identify
and quantify the resulting hybrid (sample nucleic acid – DNA probe).
Native DNA molecules are highly asymmetric, being relatively long (mm to cm), but only
2 nm in diameter. The extraction of DNA involves removing the stabilizing basic proteins,
which leaves the DNA prone to breakage by shearing forces generated by routine laboratory
procedures, such as grinding, shaking, stirring, and pipetting. Hence, all extraction procedures
must be performed with care. It is not, however, possible to isolate chromosomal DNA with-
out fragmenting the molecules: isolation procedures always result in preparations containing
DNA molecules whose lengths are much shorter than those found in chromosomes. However,
damage to the secondary (double helical) structure does not usually occur.
The isolation of DNA and purification from cells and tissues are essential for many other
molecular biological techniques. A number of methods have been developed for extracting
and purifying DNA from different cells, tissues, and organisms, which are often modified by
different laboratories to accommodate different types of samples. Here, we will give simplified
protocols used in isolating DNA from bacteria and animals samples.
DNA extraction from bacteria

Deoxyribonucleic acids can be extracted from bacteria following the generalized procedure
outlined below. Its isolation from specific species of microorganisms may require modifica-
tions to the procedures.
The cell wall and cell surface membrane of the bacterial cells must be disrupted, often using
lysozyme and sodium dodecyl sulphate (SDS), to release the DNA. Lysozyme catalyses the
hydrolysis of glycosidic bonds of cell wall peptidoglycans and lyses the cell wall, while the SDS
disrupts the hydrophobic interactions that stabilize the membrane. Hence, DNA, together
with other cellular components, is released into a medium in which it is soluble and protected
from degradation. The medium of choice is usually a saline solution, buffered to pH 8 that
contains ethylenediaminetetraacetic acid (EDTA). The DNA is soluble in the salt solution. The
EDTA chelates divalent metal ions, such as Ca2+, Mg2+, Mn2+, removing them from free solu-
tion so they cannot form salts with the phosphate groups of DNA and so inhibits the DNases
that require their presence for activity. The relatively high pH also reduces nuclease activities.
A secondary action of SDS is to act as a denaturant of DNases and other proteins.
Basic proteins bound to native DNA must be dissociated from it during the extraction. The
presence of detergent, the high concentration of salt, or the addition of sodium perchlorate
Aqueus phase
to the medium, and its mildly alkaline (pH 8) nature all reduce electrostatic interaction between
containing
the two. The DNA can now be isolated from other soluble cellular components by removing the nucleic acid
proteins and then precipitating the DNA in a relatively pure form. The solution is deprotein-
ized by adding a mixture of chloroform–isoamyl alcohol followed by centrifugation. Following White precipitate of
centrifugation, three layers are produced as shown in Figure 16.11: an upper aqueous phase that protein at interface
contains the DNA, a lower organic layer, and a compact band of denatured protein at the
interface between the aqueous and organic phases. Chloroform causes surface denaturation Organic phase
of proteins. Isoamyl alcohol is used because it reduces foaming and stabilizes the interface
between the aqueous phase and the organic phase where the protein collects. The upper
aqueous phase containing nucleic acids is removed from the centrifugation tube. The ionic
nature of DNA is exploited in its purification because the addition of an organic solvent to the FIGURE 16.11
aqueous medium reduces its polarity so it is no longer a solvent for nucleic acids. Thus, the Final centrifugation stage in
addition of ethanol precipitates the DNA as threadlike material that can easily be collected the extraction of DNA.
from the medium. Any remaining proteins contaminating the isolated DNA are removed by
dissolving it in a saline medium and repeating the chloroform–isoamyl alcohol treatment until
denatured protein no longer collects at the interface. While RNA does not normally copre-
cipitate with DNA during these procedures, it could still be present as a minor contaminant.
Ribonucleases (RNases) may be added to the preparations during the procedure after the first
or second deproteinization step to digest any RNA present. Its removal sometimes makes it
possible to remove additional proteins using chloroform–isoamyl alcohol.
If highly purified DNA is required, several deproteinization and alcohol precipitation steps
may be necessary. In general, up to 50% of cellular DNA can be recovered by this procedure,
with an average yield of 1–2 mg DNA per gram of wet packed cells.
Phenol and chloroform are potentially dangerous materials and their use in extracting DNA
from samples is often time consuming. Extraction procedures based on the reversable binding
of DNA to columns of silica overcome these disadvantages because phenol and chloroform are
not used. Thus, these methods are safer than the organic solvent extraction methods described
above. They are also easier to perform because there is less handling and manipulation of the
sample. Such methods rely on the property of DNA to bind to silica in the presence of high
concentrations of chaotropic salts, such as sodium iodide, guanidine hydrochloride, or sodium
perchlorate, but to be released when their concentrations are reduced. The procedure is out-
lined in Figure 16.12. The column is contained within a small tube that can be centrifuged in a
microfuge to drive the various solutions through it, speeding up the purification considerably.
High concentrations of salts dehydrate the DNA and favour the formation of hydrogen bonds
between it and the silica. Thus, if the DNA is extracted in a medium of high salt concentration,
the sample can be added directly to the column. The column is then extensively washed with Cross reference
alcohol-based solutions to remove the impurities. The DNA can then be recovered in a pure Centrifuges, including
microfuges, are described in
form by washing the column with a buffer containing a low concentration of salt, for example a
Chapter 12 ‘Centrifugation’.
2-amino-2-hydroxymethylpropane-1,3-diol-(Tris)-EDTA buffer or even nuclease-free water.
DNA extraction from animal cells

The major steps for isolating DNA from animal cells, such as clinical samples, resemble those
used for bacteria described above. The tissue must be disrupted, the cellular components
solubilized and the DNA separated from the other released cellular components. Care in the
initial collection of animal tissue samples greatly reduces the problems encountered during
DNA isolation. Ideally, samples should be immediately preserved in a freezer at −80oC or in
liquid nitrogen. Excellent samples of DNA can be prepared from blood, heart, liver, kidney,
stomach, intestine, cerebrospinal fluid, and skeletal muscle. Clinical samples are often blood
Sample
containing DNA
and its cells should be separated from the plasma prior to freezing.
The simplest means of disrupting cells to isolate DNA is to boil them in water or to use an
alkaline extraction medium. High temperatures (100ºC) or pH values above 9 disrupt cell
membranes, releasing the DNA into solution. However, the DNA obtained by these methods
is denatured, but active degradation in low ionic strength solutions is stimulated by metal
Binding ions also released from the cells. Chelating resins have been used to remove metal ions
from solutions, thus making feasible the rapid isolation of DNA. It should also be noted that
Column because of the high alkalinity (pH 10–11) of the chelating resin, samples prepared with it may
degrade much more rapidly than those prepared in other ways such as isolation using protei-
nase K with detergent SDS or with formamide. However, denatured DNA is amenable to some
Centrifuge molecular biology techniques, for example polymerase chain reaction, but not others such as
recombinant DNA cloning (Section 16.9), Southern blotting, or restriction fragment analysis
DNA now bound (Section 16.7).
to column
Extraction buffers used to isolate animal DNA are usually based on Tris and contain EDTA to
chelate divalent cations such as Ca2+ and Mg2+ and thus inhibit nuclease activity, and a Na+ or
Discard K+ salt to stabilize the nucleic acids in an isotonic medium. A protease, usually proteinase K, is
often added to digest cellular proteins. An anionic detergent, usually SDS, is normally included
to solubilize cellular membranes and denature proteins, as is formamide, an ionizing solvent
that dissociates DNA–protein complexes and denatures the released proteins. However, its
presence significantly reduces the activity of proteinase K. Both the pH of the isolation buffer
Washing buffer
and the specific protective agents or detergents included may need to be modified to give
optimal conditions for specific samples. Once released into solution, the DNA can then be
purified by the methods outlined above for bacterial samples.
Centrifuge Two washes to Robotic workstations are available that automatically purify genomic DNA from tissue sam-
remove impurities ples. Figure 16.13 shows a typical instrument that can, for example, simultaneously process
8 or 16 of 5–10 cm3 samples of whole blood with yields of up to 350–500 μg of DNA per
sample. The DNA obtained is sufficiently pure for its immediate use in many of the techniques
described in the remainder of this chapter.
Discard
Isolation and purification of RNA
Typical mammalian cells contain approximately 10−5 μg of RNA, of which 80–85% is rRNA,
the remaining 15–20% is largely low Mr tRNA and small nuclear RNA molecules, which can
Elution of DNA be isolated by density gradient centrifugation, anion-exchange or high-performance liquid
chromatography, or gel electrophoresis. Messenger RNA constitutes 1–5% of the total cel-
lular RNA and is heterogeneous in both size, from several hundred bases to many kilobases in
Centrifuge length, and sequence. However, most eukaryotic mRNAs have a tract of several 100 adenylate
residues at their 3′ termini, the so-called poly(A) tail, that is exploited to allow their purifica-
tion by affinity chromatography with a column of oligo(dT) cellulose. The yield of total RNA
depends on the tissue or cell source.
Ribonucleic acids are chemically more reactive than DNA because they have hydroxyl groups
Purified DNA at both the 2′ and 3′ positions. They are, therefore, more susceptible to hydrolysis by con-
taminating nucleases. Ribonucleases are released from cells during purification procedures
FIGURE 16.12 and are also present on the skin. Unlike many DNases, RNases do not require divalent cations
Column-based purification of for activity and are not inactivated by EDTA or other chelators in buffer solutions, and are
DNA. See text for details. often resistant to prolonged boiling and mild denaturants. Thus, glassware and material are
often autoclaved to denature any RNases present. Disposable gloves must always be worn.
FIGURE 16.13
Robotic workstation capable of extracting DNA from
numerous samples simultaneously. Courtesy of S. Smith,
Regional Genetics Services, St Mary’s Hospital,
Manchester, UK.
To avoid contamination with RNases during work with RNA, instruments, such as automatic
Cross reference
pipettes should be reserved for that specific use and all procedures carried out in restricted
You can read about
areas or fume cupboards. centrifugation, chromatography,
When isolating RNA from tissues such as pancreas or the gastrointestinal tract, that are rich in and gel electrophoresis in
chapters 12, 13, and 14,
digestive enzymes, it is best to cut the dissected tissue into small pieces and add them quickly
respectively.
to liquid nitrogen. These fragments are stored in a freezer at about −70ºC or used immediately
for RNA extraction. Extraction requires the cells or tissues to be disrupted in denaturing solu-
tions that contain phenol, and guanidinium thiocyanate or guanidinium isothiocyanate, that
help disrupt the cells while solubilizing their components, and denature endogenous RNases
simultaneously. The addition of chloroform followed by centrifugation separates the sam-
ple into a colourless upper aqueous phase, an interphase, and a lower organic phase. RNA
remains exclusively in the aqueous phase and can be recovered by precipitating it with alcohol
and then washing the precipitate with 75% ethanol before redissolving it in RNase-free water
or 0.5% SDS solution.
The Health & Safety Box ‘Precautions when isolating DNA’ highlights some of the safety pro-
cedures necessary when using the harsh chemicals used in extracting DNA. Naturally, similar
precautions are essential when isolating RNA from samples because for example guanidinium
thiocyanate and isopropanol are harmful if ingested or absorbed through the skin or if the
alcoholic fumes are inhaled.
HEALTH & SAFETY

Precautions when isolating DNA
■ Experimental work with bacterial cells presents a potential biohazard. Some bacteria recom-
mended for use, for example strains of Salmonella, Haemophilus, are pathogenic and can
cause disease. Eukaryotic tissues carry the danger of harbouring pathogens.
■ Benches and other work surfaces must be washed with a 10% solution of bleach before and
after DNA extractions. Do not pipette any solutions by mouth. Always wear a suitable labora-
tory coat, protective goggles, and disposable gloves, and wash hands well with hot water and
soap when leaving the laboratory, and before eating or drinking.
■ Chloroform is a volatile liquid that irritates the skin, eyes, mucous membranes, and respiratory
tract. It is also carcinogenic and can cause liver and kidney damage. Work involving chloro-
form must always be performed in a fume cupboard.
■ Ethanol is volatile and flammable. There must be no naked flames in the laboratory during
its use.
■ Isoamyl alcohol can cause tissue damage following inhalation, ingestion, or topical
absorption.
■ Lysozyme is an hydrolytic enzyme that can damage mucous membranes.
■ Sodium dodecyl sulphate (SDS) is an irritating toxic detergent that can severely damage eyes.
It can also cause tissue damage following inhalation of its dry powder form or ingestion of
the powder or solution.
16.4Hydrolysis of nucleic acids and

restriction endonucleases
If you re-examine the structures of Rib and dRib in Figure 16.2 you will see that the only
difference in their structures is the presence of the 2′-OH on the former but only a 2′-H on
dRib. The name, in fact, says this: deoxy means without oxygen. The presence of this additional
hydroxyl group means RNA is much more susceptible to alkaline hydrolysis than DNA. Mild
acid hydrolysis releases purine bases from DNA.
The alkaline or acid hydrolyses of nucleic acids degrades susceptible bonds in a random fash-
ion. However, enzymes are available that can catalyse the hydrolysis of nucleic acids in a more
specific manner. These enzymes are called nucleases or phosphodiesterases and they catalyse
the hydrolysis of phophodiester bonds, although they differ considerably in their specificities.
Exonucleases catalyse the sequential removal of terminal nucleotides; endonucleases hydro-
lyse internal phosphodiester bonds. Some nucleases can only use DNA or RNA, respectively,
as substrates, while others can degrade both. Some nucleases are only active against single
stranded nucleic acids, but others can catalyse the hydrolysis of double stranded structures.
Look again at the structure of the phosphodiester link shown in Figure 16.4. Note that it can
be cut on one of two sides. If the hydrolysis occurs on the 3′ or a side then the product is a
5′-phosphate derivative; hydrolysis on the 5′ or b side gives a 3′ prime product. Figure 16.14
summarizes both hydrolyses. Again, different nucleases differ in a or b specificity. Some nucle-
ases can only catalyse the hydrolysis of phosphodiester bonds associated with nucleotides
that contain only purine or only pyrimidine bases. Restriction endonucleases (REs) are even
more specific and only catalyse the hydrolysis of specific phosphodiester bonds within or
near to short specific base sequences in double-stranded DNA. Restriction endonucleases
are classified into three different groups: I, II, and III, type II REs are the most widely used in
molecular biology.
Restriction enzymes
Type II REs are endonucleases produced by bacteria that typically recognize specific 4–8 bp
sequences, called restriction sites, and then hydrolyse both strands of the DNA at this site
at the same position. For example, the RE, EcoRI recognizes the sequence GAATTC and
hydrolyses it at the positions indicated in Figure 16.15. If you examine Table 16.2, which lists
a number of REs, notice how the sites of hydrolysis occur at corresponding bonds within
16.4 HYDROLYSIS OF NUCLEIC ACIDS AND RESTRICTION ENDONUCLEASES 429
O
HOCH2 B’
H 3’ H
a or 3’ hydrolysis
P H
b or 5’ hydrolysis
O
5’ CH
2
B
H H
HO H
a or 3’ hydrolysis b or 5’ hydrolysis
H2O H2O
5’ O O
P CH2 B HOCH2 B’
OR
H H H 3’ H
HO H P H
FIGURE 16.14
Definition of a and b hydrolyses of a nucleic acid. Note how each type of hydrolysis form a
different product.
identical sequences of bases in both of the strands when they are read in the 5′ → 3′ direc-
tion. Restriction sites are therefore inverted repeats and are called palindromes because the A palindrome is a word or
sequence of the site is the same on each DNA strand. Different REs recognize and hydrolyse phrase that has the same order
of letters when read in the
different palindromes. Different species of bacteria synthesize different REs, which protect
reverse direction, for example
them from viruses by degrading viral DNA. The bacteria DNA is not degraded because each madam, tit.
RE has a corresponding enzyme that recognizes the same palindrome and modifies it, so it is
no longer recognized by the RE (Box 16.3).
EcoRI
. . . G A A T T C . . .
5’ 3’
. . . C T T A A G . . .
3’ 5’
EcoRI
Hydrolysis 2H2O
FIGURE 16.15
5’
. . . G 3’ 5’
A A T T C . . .3’ Palindrome recognized and
+ G . . . hydrolysed by the restriction
. . . C T T A A 3’ 5’
3’ 5’ enzyme, EcoRI.
BOX 16.3 Restriction endonuclease:

sources and nomenclature
Restriction endonucleases are produced by bacterial cells. They function in protecting the
bacterium from attack by viruses by degrading the viral DNA. The host bacterial DNA is
protected from hydrolysis because each RE has a corresponding methyl transferase with
an identical palindrome specificity. Thus, the bacterial palindromes, but not the viral, have
methyl (-CH3) groups attached to bases near to the palindrome that prevents their recog-
nition by the REs. Several hundred REs have been isolated from different bacterial species
and characterized. The nomenclature adopted for them consists of a three-letter abbre-
viation that identifies the bacterial source, followed by a letter representing the strain of
the species (if necessary) and, lastly, a Roman numeral designating the order of discovery
of that enzyme when more than one RE has been isolated from that strain. Thus EcoRI was
the first RE to be isolated and characterized from the R strain of Escherichia coli.
Many restriction enzymes make staggered cuts in the two DNA strands at their recognition
site and produce products that have a single stranded portion at each end, as is the case with
EcoRI. Look carefully at the products of the reaction. Note how they have overlapping strands
that are often called ‘sticky ends’. Such REs are used extensively in recombinant DNA technol-
ogy, which we will describe in Section 16.9. However, some REs have actions that form prod-
ucts with blunt cut ends, as you can see is the case with EcoRV (Figure 16.16).
SELF-CHECK 16.5
Which REs in Table 16.2, besides EcoRI, produce products with sticky ends?
Uses of restriction endonucleases

The abilities of REs to recognize palindromes in DNA and restrict hydrolytic events to those sites
means they can be used to degrade molecules of DNA into a specific number of fragments in a
reproducible manner. Thus, REs are invaluable tools in molecular biology and used extensively to:
• Specifically hydrolyse large DNA molecules
• Produce physical maps
• Convert circular plasmids in to linear DNA molecules
• Produce recombinant DNA molecules
• Investigate restriction fragment length polymorphisms (RFLPs)
Restriction endonucleases can be used to hydrolyse large DNA molecules into smaller fragments
that are more amenable to analysis. The fragments are readily separated and their size estimated
by agarose gel electrophoresis as you can see in Figure 16.17 (Section 16.5 and Chapter 14, you
may also wish to look at Figure 14.22 (b)). It is unlikely that the sets of fragments produced from
any two different DNA molecules using the same RE will be the same. Hence, the pattern of DNA
products is likely to be unique and so can be considered a ‘fingerprint’ of the DNA substrate.
Restriction endonucleases can be used to produce physical maps of DNA molecules. An

understanding of the genetics, metabolism, and regulation of an organism requires knowledge
TABLE 16.2 Examples of restriction endonucleases and their palindrome specificities.

The red arrows indicate the sites of hydrolysis
Restriction endonuclease Palindrome recognized and

(bacterial species) sites of hydrolysis
AluI ↓
(Arthrobacter luteus) . . . AGCT. . .
. . . TCGA. . .
↑
BamHI ↓
(Bacillus amyloliquefaciens H) . . . GGATCC. . .
. . . CCTAGG. . .
↑
EcoRI ↓
(Escherichia coli RY 13) . . . GAATTC. . .
. . . CTTAAG. . .
↑
EcoRV ↓
(Escherichia coli J62 pLG74) . . . GATATC. . .
. . . CTATAG. . .
↑
HaeIII ↓
(Haemophilus aegptius) . . . GGCC. . .
. . . CCGG. . .
↑
HindIII ↓
(Haemophilus influenzae Rd) . . . AAGCTT. . .
. . . TTCGAA. . .
↑
KpnI ↓
(Klebsiella pneumonia) . . . GGTACC. . .
. . . CCATGG. . .
↑
MspI ↓
(Moraxella species) . . . CCGG. . .
. . . GGCC. . .
↑
PstI ↓
(Providencia stuartii 164) . . . CTGCAG. . .
. . . GACGTC. . .
↑
PvuII ↓
(Proteus vulgaris) . . . CAGCTG. . .
. . . GTCGAC. . .
↑
TaqI ↓
(Thermus aquaticus) . . . TCGA. . .
. . . AGCT. . .
↑
EcoRV
. . . G A T A T C . . .
5’ 3’
. . . C T A T A G . . .
3’ 5’
EcoRV
2H2O
. . . G A T 3’
5’ 5’
A T C . . . 3’
FIGURE 16.16 +
. . . C T A T A G . . .
Reaction catalysed by EcoRV. 3’ 5’ 3’ 5’
of the precise arrangement of its genetic material. The construction of a RE map for a DNA mol-
ecule provides some of this information since it will show the sites that can be cut by different REs
and the number of fragments obtained after each digestion. Two characteristics for every DNA
fragment produced by an RE digest are known: the nature of the fragment ends (from the
known specificity of the individual RE) and its approximate Mr (from electrophoresis separation).
1 2 3 4 5 Size / kbp
11.0
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.6
1.0
FIGURE 16.17
Separation of DNA fragments by agarose gel electrophoresis. Lane
1 shows a stained sample of DNA isolated from λ bacteriophage.
Lanes 2, 3, and 4 show the fragments obtained by digesting the 0.5
bacteriophage DNA with the restriction endonucleases HindIII, TaqI,
and EcoRV, respectively. Lane 5 shows the positions of fragments
of DNA of known sizes (‘ladder’). Note how each of the enzymes
has produced a different set of products by hydrolysing the DNA
at different palindrome sites. Courtesy of N. Shaheen, School of
Healthcare Science, Manchester Metropolitan University, UK.
See also Figure 14.22 (b).
Other information about the fragments, for example the sequence of their bases (Section 16.6)
can be obtained using other experimental methods. (See the Method Box ‘Experimental proce-
dure for performing a restriction endonuclease digest’).
The circular DNA molecules present in many plasmids can be converted into linear molecules by
the action of some REs. Note that it is necessary that the plasmid have only a single recognition
site for the RE being used, otherwise digestion will fragment it! Restriction endonucleases are
valuable tools in the construction of recombinant DNA molecules. Thus, once a circular plasmid
has been ‘linearized’, it is capable of binding to other fragments of DNA produced by the actions
of the same RE on different samples irrespective of the source of the DNA. This gives molecular
biologists a way to cut and splice different pieces of DNA together to produce recombinant DNA
molecules, which can be transferred into bacterial cells and cloned as described in Section 16.9.
The use of REs not only opens plasmids for insertion of a DNA fragment, they can also destroy a
phenotype. For example, if they cleave at a site that specifies resistance to a particular antibiotic,
then when the recombinant plasmid is added back to a suspension of bacteria, those that are
transformed by taking it up can be identified by their susceptibility to the antibiotic.
The digestion of DNA using REs and the separation of the digest fragments by electrophoresis
can be used for clinical purposes, such as the diagnosis of inherited disorders for example
sickle cell anaemia using RFLPs, which we describe in Section 16.7.
METHOD Experimental procedure

for performing a restriction
endonuclease digest
■ Commercially available REs are usually dissolved in an appropriate buffer and posted
in vials surrounded by dry ice. Disposable gloves must be worn when removing the
enzyme container. All REs are heat-labile and should quickly be stored at −20ºC in a
buffer containing 50% glycerol. The solution of enzyme should be removed from the
freezer just before use and stored on ice.
■ A typical RE digest is performed in a microcentrifuge tube (Chapter 12). The mixture
normally contains approximately 1 μg or less of DNA and about 1 unit of RE activity
in the appropriate incubation buffer.
■ Restriction nucleases have optimal reaction conditions for activity, especially regard-
ing the composition and pH of the reaction buffer, and incubation temperature. The
information for commercially available enzymes is readily available from the sup-
plier or from published literature. The total reaction volume is usually between 20
and 50 μL. The contents of the tube are mixed by gently tapping it or briefly centri-
fuging the mixture in a microcentrifuge.
■ Incubations are most often carried out at the recommended temperature, usually
37°C for about 1 hour.
■ The reaction is then stopped by adding EDTA, which removes the divalent metal ions
essential for nuclease activity from solution.
■ The products of digestion are often analysed by agarose gel electrophoresis (Section
16.5 and Chapter 14). They can usually be temporarily stored at 0–4°C until sub-
jected to electrophoresis.
16.5 Gel electrophoresis

Electrophoresis is the movement of ions, for example nucleic acids and proteins, in an elec-
tric field. You can read more about electrophoresis in Chapter 14. Here, we will briefly high-
light the main points. Particles that differ in their mass-to-charge ratios will move at different
speeds in the field and thus separate from one another. However, because phosphate groups
are regularly spaced along DNA and RNA molecules, they all have the same mass-to-charge
ratio and will all move at the same speed for a given electric field and so would not separate.
However, performing electrophoresis in agarose or polyacrylamide gels means plasmids and
smaller DNA and RNA molecules, and proteins can all be separated. The concentration of
agarose or polyacrylamide in the gel determines the sizes of its pores. Using appropriate
concentrations therefore allows mixtures of different sized molecules to be resolved during
Cross reference
electrophoresis because the smaller molecules are able to move faster through the gel than
larger ones.
In many types of gels, the distance moved by a limited range of nucleic acid fragments is inversely
proportional to the logarithm of their sizes. The size of DNA fragments is usually expressed in
bp or kilobase pairs (kbp), but can be given in Mr. In general, the lower the concentration of
agarose in the gel, the larger the DNA molecules that can be separated. However, gels with
concentrations of agarose below 0.3% are too fragile for ordinary use and this therefore forms
a practical lower limit. Gels of this concentration allow the analysis of linear double-stranded
DNA fragments of 5–60 kbp (Mr up to 150 × 106). Gels of 0.8% agarose can separate DNA in the
range of 0.5–10 kbp; and 2% agarose is used to separate smaller DNA fragments of 0.1–3 kbp. In
addition to the DNA molecules of analytical interest, fragments of DNA of known size are also
subjected to electrophoresis, forming a so-called ‘DNA ladder’. The sizes of unknown fragments
can then be estimated by comparing their mobilities with those of known size in the ladder.
Once stained, separated fragments of DNA in the gel are usually detected using ethidium bro-
mide as a stain (Figure 14.21). Ethidium bromide consists of relatively small, planar molecules,
which can bind to DNA by intercalating between the stacked base pairs, which enhances the
normally weak fluorescence of the purine and pyrimidine bases by about 25 times (Figure
14.22 (a)). Thus, when irradiated with UV light the separated fragments fluoresce with an
intense orange colour giving a sensitive means of detecting the fragments: as little as 1 ng of
DNA can be seen (Figure 16.17).
DNA fragments containing 1000 or fewer bp can be separated by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE). Such gels are also widely used to separate mix-
tures of proteins, as you can see in Figure 14.17. Normally, proteins are separated following
their denaturation using β-mercaptoethanol and SDS. β-mercaptoethanol reduces the disulfide
bonds that help stabilize protein structure and which hold the subunits of some quaternary
proteins together, while the negative charges of the SDS bound to the proteins repel each other.
In these conditions, protein molecules adopt rod-like structures. Proteins bind SDS in a size-
dependent manner and since SDS overwhelms the native charges of the proteins, all proteins
in the presence of SDS have similar mass-to-charge ratios. Polyacrylamide, like agarose, forms
gels whose pore size is determined by the concentration used. Hence, if the electrophoresis is
carried out in a gel also saturated with SDS (hence SDS-PAGE), smaller proteins or very small
DNA fragments will migrate faster through it than larger ones and separate from one another.
The higher the concentration of acrylamide, the greater the resolution of proteins of lower
Mr; the lower the concentration the better the resolution of larger ones. Following separation,
proteins can be detected using one of a variety of different staining procedures (Table 14.1).
16.6 DNA SEQUENCING 435
As with agarose electrophoresis of nucleic acids, it is normal to treat and separate a mixture of
proteins of known Mr to allow the sizes of unknown proteins to be estimated.
16.6 DNA sequencing

Sequencing of DNA, that is determining the order of its bases, is mainly performed using the
chain termination or ‘dideoxy’ method first devised by Sanger in the 1970s. Sequencing meth-
ods are largely automated and performed by robotic machines whose results are analysed by
computers. However, the basic procedure is relatively straightforward. The DNA must be pure
and hydrolysed to give a series of suitably sized overlapping pieces. Each fragment is amplified
(Section 16.9) before being independently sequenced. Since the fragments have overlapping
regions of bases, it is possible to assemble them in the appropriate order to give the complete
sequence.
The dideoxy method has two key features. One is that it exploits the specificity of DNA
polymerase, the enzyme that copies DNA strands to produce a new complementary strand.
This enzyme catalyses the extension of a polynucleotide chain by forming new 3′-5′ phospho-
diester bonds. Bonds are formed when the enzyme adds a new nucleotide to the terminal
3′-OH. The base sequence of the new strand is determined by that of the template. The sec-
ond feature exploited in the method is the use of dideoxynucleoside triphosphates (ddNTPs).
These lack a 3′-OH and, so once a ddNTP is added to the end of a newly forming chain, its
growth ceases because the absence of a 3′-OH prevents the addition of a further nucleotide.
You can see this diagrammatically in Figure 16.18.
Dideoxy sequencing occurs in a reaction mixture that contains a sample of the single stranded
DNA to be sequenced (the template), an oligonucleotide primer that is complementary to
part of the template, DNA polymerase, the four deoxynucleotides, dATP, dTTP, dGTP, and dCTP,
3’. . . G A T A C C A T G A T C . . . 5’ DNA to be sequenced
Primer 5’ C T A T G 3’
DNA Polymerase
dATP : ddATP
dTTP : ddTTP
dGTP : ddGTP
dCTP : ddCTP
C T A T G G
Products
C T A T G G T separated by
C T A T G G T A capillary
electrophoresis
C T A T G G T A C
(Figure 16.20)
C T A T G G T A C T or SDS-PAGE
FIGURE 16.18
C T A T G G T A C T A (Figure 16.19)
Outline of the dideoxy method for sequencing
C T A T G G T A C T A G DNA. See text for details.
and relatively small amounts of each of the four ddNTPs. The DNA polymerase then catalyses
the addition of nucleotides and extends the length of the DNA primer. The newly formed
extension is complementary to the template. However, during extension of the primer the
enzyme will sometimes use a ddNTP, rather than the normal dNTP. When this happens, exten-
sion of the growing chain will cease to occur. If the mixture contains an appropriate ratio of the
respective ddNTPs to dNTPs, then when the reaction is completed, the mixture will contain a
set of new but incomplete DNA chains complementary to the template. Extension of each will
have started at the same point, but because the ddNTP was selected in a random fashion by
the enzyme, they will differ in length by a single base.
The newly formed fragments in the reaction mixture can then be separated on a SDS-PAGE
gel containing urea (Section 16.5) or by capillary electrophoresis. Each technique can sepa-
rate DNA molecules that differ by only a single base in length. The denaturants SDS and urea
ensure the DNA chains remain separate during electrophoresis. Following separation, the dif-
Cross reference ferent lengths of DNA form a densely packed ‘ladder’ of bands on the gel, as you can see in
Chapter 14 ‘Electrophoresis’. Figure 16.19. These ladders can be interpreted to give the equivalent sequences of bases in
the template. Each of the four ddNTPs is tagged with a different dye. A laser light passes along
the gel and each dye fluoresces with a different coloured light that identifies the particular
base used in terminating that length, for example, yellow for G(uanine), red for T(hymine).
The length of the DNA fragments and their colours, which identify each terminal base, are
analysed by a computer to give the base sequence of the template.
Sodium dodecyl sulphate-PAGE gels cannot resolve more than 500–800 bases in a single
electrophoresis run, which limits sequencing experiments. However, modern DNA sequenc-
ers separate the fragments using capillary electrophoresis (Chapter 14) and automatically
detect dye fluorescence and record the data output as a fluorescent peak trace (Figure 16.20).
The use of such automation means many thousands to millions of bases can be sequenced
in an hour.
FIGURE 16.19
Portion of a sequencing gel. The colours identifying each base
are green for adenine (A), blue, cytosine (C); yellow, guanine
(G), and red, thymine (T). See text for details. Courtesy of
Wellcome Trust Sanger Institute, Cambridge, UK.
16.6 DNA SEQUENCING 437
3.5
...
3.0
2.5
Flourescence
2.0
1.5
1.0
0.5
0.0
800 900 1000 1100 1200 1300 1400 1500 1600 1700
Sequence
FIGURE 16.20
Trace showing the sequencing output (capillary electrophoresis) from a Beckman Coulter
CEQ 8000 automated sequencer. Part of the corresponding base sequence is shown.
Courtesy of Dr Patricia Linton, School of Healthcare Science, Manchester Metropolitan
University, UK.
SELF-CHECK 16.6
Complete the sequence of bases shown in Figure 16.20.
The application of automated methods means that DNA sequencing is largely a routine and
rapid procedure. Indeed, the genomes of many of the model organisms studied routinely in
the laboratory have been sequenced. These include a number of different yeasts, the nema-
tode Caenorhabditis elegans, the Drosophila fruit fly, the mouse, dog, and chimpanzee and,
finally, yet of great clinical importance, that of humans. The complete sequences of many
100s of parasitic viruses, for example, the smallpox and Epstein–Barr viruses, large numbers of
pathogenic bacteria, such as those responsible for cholera, tuberculosis, syphilis, gonorrhoea,
Lyme disease, and stomach ulcers, as well as parasitic protozoa like Plasmodium falciparum,
have also been fully determined (Table 16.3). Knowing these sequences should help produce
more rapid and effective diagnostic tools, such as in designing specific probes (Box 16.2) for
use in FISH (Section 16.8) and PCR (Section 16.9) methods. It is anticipated that analysis of
their genomes will also provide useful information about their virulence and indicate more
effective treatments for the clinical conditions they cause.
Sequencing specific genes can also be used to determine if a person is a carrier of a genetic
disease or to confirm a provisional diagnosis, particularly in those conditions where the inher-
itance does not follow simple Mendelian rules, for example, Fragile X syndrome. This is
TABLE 16.3 Examples of pathogens whose genomes have been completely sequenced
Group Virus or organism disease

Viruses Enterovirus 100, Meningitis
Immunodeficiency viruses 1 and 2 Acquired immunodeficiency

syndrome (AIDS)
Papillomavirus16 Cervical cancers
Herpes virus Herpes
Influenza A Influenza
Poliovirus Polio
Bacteria Bacillus anthracis Anthrax
Bordetella pertussis Whooping cough
Campylobacter jejuni Gastroenteritis
Clostridium botulinum Botulism
Clostridium tetani Tetanus
Corynebacterium diphtheriae Diphtheria
Escherichia coli Food poisoning
Helicobacter pylori Stomach ulcers
Klebsiella pneumoniae Pneumonia
Legionella pneumophila Legionnaires disease
Mycobacterium leprae Leprosy
Mycobacterium tuberculosis Tuberculosis
Neisseria gonorrhoeae Gonorrhoea
Neisseria meningitidis Meningitis
Salmonella typhimurium Gastroenteritis
Shigella dysenteriae Dysentery
Staphylococcus aureus Skin infections
Streptococcus mutans Dental plaque
Treponema pallidum Syphilis
Vibrio cholerae Cholera
Yersinia pestis Bubonic plague
Protozoa Plasmodium falciparum Malaria
Trypanosoma brucei Sleeping sickness

16.7 BLOT TING TECHNIQUES 439
the commonest cause of mental retardation in the UK, affecting one in about 4100 males,
but is less common in females, at approximately one in 8000, and who are generally less
severely affected. The condition is associated with repeated copies of a trinucleotide, CGG,
which occurs in the FMR1 gene. Most people have a stable copy of FMR1 containing about
30 repeats. Those with 45 to 55 copies have an increased chance of passing on an even larger
number to their children. Individuals with 55–200 are said to possess a premutation because
their children may have more than 200 CGG repeats, which is the full mutation associated with
Fragile X syndrome. Sequencing FMR1 or relevant portions of it will directly determine the
number of repeats present and confirm the status of the patient.
16.7 Blotting techniques

Electrophoresis is a convenient way of separating nucleic acid and protein molecules.
Unfortunately, it is difficult to identify specific molecules in the gel because probes (Box 16.2) do
not readily diffuse into gels. Also, gel matrix materials, like agarose or polymerized polyacryl-
amide, are fragile and can be difficult to handle. These difficulties can be overcome by the com-
mon laboratory procedure of blotting, in which nucleic acids or proteins in the gel matrix are
transferred from the gel to, for example, a piece of nitrocellulose paper or a nylon membrane.
The positions of the molecules of interest on the paper/membrane can be identified and their
analysis facilitated because they are bound to the surface of the paper or membrane.
Three major types of blotting techniques are used, namely:
• Southern blotting
• Northern blotting
• Western blotting.
The first blotting technique devised was Southern blotting by E. M. Southern, hence its name.
The nomenclature of Northern and Western blotting were given with reference to it. These
techniques allow the detection of specific DNA fragments, RNA molecules, and individual
proteins, respectively. Thus, while genes or fragments of DNA can be detected by Southern
blotting, the expression of specific genes is usually monitored by Northern or Western blot-
ting, which detects the specific mRNA or protein molecules produced in the cell. In general,
Southern and Northern blotting methods are insufficiently sensitive to detect the amount of
nucleic acids obtainable from a single cell. However, the polymerase chain reaction or PCR,
which we describe in Section 16.9, can be used to amplify the nucleic acid in the sample to
amounts that are detectable.
Southern blotting
Southern blotting follows gel electrophoresis of double stranded fragments of DNA, which
are separated on the basis of differences in their sizes. The DNA fragments present in the gel
are then denatured with alkali and the resulting single stranded molecules transferred using a
buffer onto a nitrocellulose filter or nylon membrane by blotting, as shown in Figure 16.21.
This procedure preserves the pattern of distribution of the DNA fragments in the gel, by
forming a replica of the gel on the filter paper or membrane. The filter is then incubated
under hybridization conditions with a specific radiolabelled DNA probe. The DNA restric-
tion fragment that is complementary to the probe hybridizes with it, and its location on
the filter can then be revealed, often by autoradiography (Box 13.1 ‘Autoradiography’).
DNA
Cleavage with
restriction enzymes
Gel
Electrophoresis
Weight
Filter papers
Nitrocellulose
membrane
Alkaline solution Gel
DNA transferred from gel to

nitrocellulose membrane by
capillary action
Nitrocellulose
membrane
Hybridization with, for example,

radiolabelled DNA probe to DNA
indicated on gel by
Autoradiogram
FIGURE 16.21
Outline of Southern blotting, which is explained in the general text.
However, autoradiography suffers from a number of disadvantages, including possible health

hazards, the inconvenience of handling a radioisotope and the relatively short half-life of 32P.
The digoxygenin (DIG) system provides a sensitive alternative to autoradiography for analysing
blots and is simple to use. Analysis involves adding a probe labelled with DIG to the nitrocel-
lulose filter or membrane. An antiDIG-antibody (see Western blotting below) tagged with an
enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) is added to the
reaction mixture. The addition of substrates that are broken down by the enzyme to give
a chemiluminescence product means that even the binding of a single enzyme molecule is
amplified because catalysis produces many molecules of product. The chemiluminescence
can be detected using a high sensitivity detection device, such as a cooled charge-coupled
device (CCD) camera system.
Digoxygenin-labelled DNA or RNA probes can be used to analyse the products of all types of
hybridization reactions, including in situ hybridization and ELISAs (Section 16.8), microarrays
(Section 16.10), and Northern blots (see below).
Restriction fragment length polymorphisms

Southern blotting is sufficiently sensitive to detect a single specific product in a complex mix-
ture of DNA fragments as is formed when the entire human genome is hydrolysed with a
restriction endonuclease. Southern blotting can detect and identify the genomes of specific
viruses and microorganisms and the presence of mutations, or polymorphisms, in samples
of DNA from patients produced for example by the deletion or the insertion of short DNA
sequences; most single-base changes, however, cannot be detected in this way unless they
form or break a palindrome that can be recognized by a RE. Sickle cell anaemia is a common
disorder among some populations inhabiting or originating from malarial infested regions.
Patients typically present with severe joint pain and a microscopic examination of their blood
will show the presence of sickle-shaped erythrocytes. The disorder arises from a single point
mutation in the gene for β globin, one of the two types of polypeptide that make up haemo-
globin. A thymine substitutes for an adenine base resulting in a valine residue being incorpo-
rated at position 6 in the globin molecule rather than a glutamate, forming haemoglobin S.
β globin genes are inherited from each parent in a Mendelian fashion. Hence, individuals can
be homozygous for adult haemoglobin (HbA/HbA) or for haemoglobin S (HbS/HbS), or het-
erozygous (HbS/HbA) and form a mixture of adult and S haemoglobins. The condition can be
diagnosed using foetal material obtained by amniocentesis using the restriction endonuclease
(Section 16.4), MstII. The normal globin gene has three restriction sites that can be hydrolysed
by MstII. However, the sickle mutation destroys one of these sites, resulting in a product that
is larger in size. Thus, electrophoresis of the globin gene fragments followed by blotting pro-
duces different patterns for the hetero- and homozygotes as shown in Figure 16.22.
Northern blotting
The expression of a particular gene can be followed by detecting its corresponding mRNA by
Northern blotting. This procedure is similar to Southern blotting, but electrophoresis is per-
formed on a sample of mRNA. The RNA sample is often the total RNA purified from cells or
tissues. It is denatured by treatment with agents, for example, formaldehyde, that disrupt the
hydrogen bonds between any intrastrand base pairs, which ensures that all the RNA molecules
have an unfolded, linear conformation. The sample is then subjected to gel electrophoresis
and RNA molecules separated on the basis of differences in their sizes. As in Southern blotting,
the denatured RNA molecules are transferred to a sheet of nitrocellulose or nylon membrane
MstII restriction sites

. . .C C T N A G G. . .
. . .G G A N T C C . . .
Restriction site
lost in HbS
5' 3'
3' 5'
5' 3' 1.4 kbp

3' 5' 1.2 kbp
DNA fragments
Normal Sickle cell Sickle cell

trait
FIGURE 16.22
Application of restriction fragment length 1.4 kbp
polymorphism (RFLP) analysis using the
restriction endonuclease MstII and Southern
blotting to diagnose sickle cell anaemia. 1.2 kbp
See text for explanation.
which is then probed with a labelled DNA that is complementary to the RNA of interest.
Molecules of RNA that hybridize to the probe on the paper/membrane are located by detect-
ing the bound probe by autoradiography or by the DIG system described above. The sizes of
the hybridized RNA molecules can be estimated by reference to RNA standards of known size
that are subjected to electrophoresis side by side with the experimental sample.
Northern blotting is widely used to compare the amounts of a particular mRNA molecule
in different cells grown in varying conditions. For example, it can be used to show if a muta-
tion in a gene increases or reduces the amount of normal-sized mRNA produced. It can also
be adapted to demonstrate if the mutation has resulted in the transcription of abnormally
short mRNA molecules by testing the blotted paper/membrane with a series of shorter DNA
probes, each complementary to short portions of the mRNA; this would also demonstrate
which part of the normal RNA molecule is missing.
Western blotting
Western blotting (immunoblotting) combines several techniques to detect specific proteins in
a sample. Like other blotting techniques, Western blotting gives information about the size of
the protein or the relative amounts of protein produced in different cells, or the same cell in
different conditions.
You can see an outline of this procedure in Figure 16.23. As with Southern and Northern blots,
gel electrophoresis is used to separate native or denatured proteins, which are then trans-
ferred to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane. Nylon membranes
are not used in Western blotting because they are cationic and strongly bind acidic proteins.
SDS-polyacrylamide gel
Proteins
separated by
electrophoresis
Proteins transferred
to porous membrane
SDS-polyacrylamide gel
Membrane
Incubate with antibody (Ab)
S
S
S
S
S
S
S
S
S
S
S
S
Binding of Ab to
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
corresponding
S
S
S
S
S
S
protein (antigen)
S
S
S
S
S
S
Incubate with enzyme-

linked secondary antibody
S
S
S
S
S
S
S
S
Binding of secondary
S
S
S
S
S
S
S
S
S S
S S
S
S S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S S
S
S
S
Ab to first Ab
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S S
S
S
S
S
S
S
S
S
S
S
S
Incubate membrane
with substrate
Reaction with
substrate to form
precipitate
FIGURE 16.23
Outline of Western blotting.
Thus, under many conditions they bind so much protein that detection procedures colour the
membrane strongly and it is difficult to identify the proteins of interest. Polyvinylidene difluo-
ride also binds proteins strongly, but gives light background staining after analysis. However,
nitrocellulose membranes are the commonest choice for general use. They are the cheapest
alternative and bind adequate amounts of protein, largely due to hydrophobic interactions.
However, they are relatively fragile and do not stand up well to repeated probings and do not
bind proteins of Mr less than 14,000 strongly. Following protein transfer, the uniformity and
overall effectiveness of transfer of protein from the gel to the membrane can be checked by
staining the membrane with a general protein stain such as Coomassie blue or Ponceau S dyes
(Table 14.1).
In Western blotting, the membrane is probed with antibodies, which we describe in Chapter 15.
To briefly recap, antibodies are glycoproteins synthesized when a foreign, that is non-self,
Cross reference agent such as an infectious microorganism, stimulates the immune system. Each type of anti-
Chapter 15 ‘Immunological body is produced in response to the presence of a macromolecular antigen, for example a
techniques’. protein or lipopolysaccharide. Individual antibodies recognize specific sites on antigens called
epitopes. Thus, antibodies can be used in Western blotting because they can specifically bind
to the target protein(s) and so locate their positions on the membrane. However, given that
the membrane also binds proteins, but in a non-specific fashion, steps must be taken to pre-
vent any interactions between it and the antibody, otherwise detection of the target protein
would be hindered. This is achieved simply by adding an inexpensive protein, for example
bovine serum albumin (BSA) or non-fat dry milk in a dilute solution of detergent such as
Tween 20, to the membrane. These proteins attach to the membrane and block nonspecific
binding sites. Hence, when the antibody is added, it can only bind to epitopes of the target
protein. The excess BSA or dried milk is then removed by washing with a suitable buffer. The
bound protein–antibody complex must now be detected on the membrane to pinpoint its
position. Traditional detection uses a two-step process, in which the antibody used to detect
the protein is called the primary antibody. The membrane is rinsed to remove unbound pri-
mary antibody, and a secondary antibody, which is species-specific to a portion of the primary
antibody is added. This secondary antibody is usually linked to biotin or to a reporter enzyme
such as ALP or HRP whose activities produce a coloured signal allowing the position of the
bound protein to be identified (Figure 16.24). Commonly, a HRP-linked secondary antibody is
used in conjunction with a chemiluminescent agent. The reaction product produces a lumi-
nescent signal, whose intensity is proportional to the amount of protein. The light may be
(a)
Samples
1 2 3 4 5
p27
Tubulin
(b) 40
Relative amounts of p27
FIGURE 16.24
(a) Picture of a Western blot showing the presence of the
30
protein, p27 in five different samples. In each case, the
product of the housekeeping gene, tubulin has also been 20
analysed as a control. Since each sample contains the
same quantity of tubulin, any experimental variation in 10
the treatments of the samples can be eliminated.
(b) Densitometry to show the relative expression of 0
p27 in each of the samples. Samples
detected on photographic film or by using a CDD camera that captures a digital image of the
Western blot.
Images of Western blots can be analysed by densitometry, which is explained in Chapter 14.
This allows us to compare the amounts of a protein present in different samples, if known quan-
tities of a so-called house keeping protein are analysed at the same time (Figure 16.24). The Mr
of the protein of interest can also be estimated if proteins of known sizes are also included in
the analysis. In some applications, detection can be achieved by a one-step process, where the
antibody probe, in addition to recognizing the target protein, also carries the detectable label
(Box 16.4). When using primary antibodies or the one-step approach, incubation times vary
from 30 minutes to overnight depending on the temperature. Temperatures can be varied
between approximately 4–25oC. Higher temperatures are associated with enhanced specific
binding of the antibody to the target protein, which increases the signal from the target on the
Cross reference
membrane. Unfortunately, it can also produce more non-specific binding, which increases the
background staining.
Western blotting is used in biochemical and clinical investigations because it is one of the
most specific means of identifying the presence of individual proteins in complex biologi-
cal samples, such as blood, serum, saliva, urine, cells, and tissues; it is thus the basis of many
diagnostic assays. For example, the serodiagnosis of human immunodeficiency virus type 1
(HIV-1) infections relies on detecting antibodies to the virus in the sera of potential patients.
Serum proteins from potential patients are isolated and blotted onto a membrane. Antihuman
immunoglobulin G conjugated to an enzyme that produces a coloured product with an
appropriate substrate is used to detect the presence of antiviral antibodies. As with all clinical
tests, positive and negative control sera must be analysed simultaneously with that of the test
serum. Western blotting also provides improved serological diagnoses for conditions such as
Lyme disease, some forms of spongiform encephalopathy, congenital toxoplasmosis, syphilis,
and in patients with rheumatic disease, and is the basis for many home diagnostic tests, for
example for pregnancy testing kits.
BOX 16.4 Antibodies for Western

blotting
The results of Western blotting depend on the quality of antibody probe used, in par-
ticular how specific it is for the target protein.
Both monoclonal and polyclonal antibodies (see Box 15.4) against a large number of
target proteins are readily obtainable from commercial sources. If, however, the protein
is a novel one, the antibody must be produced in-house, although commercial com-
panies are available that will also do this for a fee! In either case, small amounts of the
target protein must be purified to stimulate antibody production. A large number of
species, such as rats, mice, rabbits, guinea pigs, chickens, goats, sheep, and donkeys, are
available in which to raise antibodies. Generally, monoclonal antibodies take longer to
develop than polyclonal ones and are therefore more expensive (Figures 15.2 and 15.3).
Monoclonal antibodies are, however, usually more specific to their targets and bind more
strongly to their epitope than polyclonal types. This means that fewer nonspecific bands
are detected on the membrane and the background staining is of a lower intensity.
SELF-CHECK 16.7
List the principal uses of Southern, Northern, and Western blottings? State the major use of
each technique.
16.8Enzyme-linked immunosorbent
assays and fluorescence in situ
hybridization
Enzyme-linked immunosorbent assays (ELISAs) and fluorescence in situ hybridization (FISH)
are two sensitive groups of techniques that rely upon the respective abilities of proteins
(antibodies) and nucleic acids to specifically recognize and bind to complementary mole-
cules, as we described in Section 16.1. Each of the techniques forms the basis of many clinical
assays.
Enzyme-linked immunosorbent assays

Like Western blotting, enzyme-linked immunosorbent assays, or ELISAs, rely upon the ability
of antibodies to recognize and bind to their complementary antigens. This makes them pow-
erful reagents for detecting and quantifying analytes of clinical interest. The use of antibodies
in this respect is called immunoassay. Immunoassays are used in all branches of the biomedi-
cal sciences. Thus, ELISAs use an antibody to which an enzyme has been attached to measure
the amount of a specific antigen in a sample. The enzyme used is one that will convert a
colourless substrate into, for example, a coloured soluble product that can be measured spec-
trophotometrically. Horseradish peroxidase and ALP are the most frequently used enzymes.
The simplest ELISA format (Figure 16.25 (a) and (b)) is to allow a protein antigen to absorb onto
the wells of a plastic microtitre plate. Aliquots of known concentrations of the antigen are also
applied to other wells of the plate. An enzyme-labelled antibody that binds to the antigen is
then added to the wells. Wells containing a large amount of antigen will bind more antibody
and therefore more enzyme. The substrate for the enzyme is then added and, after a limited
Cross reference period, the reaction is stopped and the amount of product formed determined. The amount
See Chapter 15 ‘Immunological of product formed with the known concentrations of antigen can be used to form a stand-
techniques’. ard curve from which the concentration of the unknown sample can be determined. Given
each enzyme molecule forms many thousands of molecules of product, even relatively tiny
amounts of antigen can be detected. Other labels that can be used in immunoassays include
fluorescent labels and bio- and chemiluminescent labels.
The development of ELISAs in 1971 by Engvall and Perlmann revolutionized medicine by

replacing many awkward to perform and time-consuming diagnostic tests. Since then,
the basic protocol has been subjected to many different adaptions that allow non-protein
antigens to be measured or which have increased sensitivity. The specificity of antibodies
Cross reference allows biomedical scientists to measure the concentration of an analyte, such as a steroid
You can read about mutations in hormone or autoantigens in patients with autoimmune diseases, in biological fluids such as
Chapter 20 ‘Inherited metabolic plasma, which contain hundreds of other biomolecules, some of which are similar in struc-
disorders and newborn
ture to the analyte being measured. Enzyme-linked immunosorbent assays are widely used,
screening’ in the companion
for example, in diagnosing cases of malaria in Africa to home pregnancy testing kits in the
volume, Clinical Biochemistry.
developed world.
16.8 ENZ YME-LINKED IMMUNOSORBENT ASSAYS AND FLUORESCENCE IN SITU HYBRIDIZATION 447
(a)
Well Antigen (Ag)
binding to
well
Wash
Antibodies (Ab)
binding to antigen
S
S
S
S
S
S
S
S
S
S
Wash
E E
S S
S S
Enzyme (E)-linked
S S
S S
S S
S S
antibody binding to
Ab-Ag complex
S
S
S
S
S
S
S
S
S
S
Wash (b)
S P S P
E E
S S
S S
S S
S
S
S S
S
S
Substrate (S)
converted to
coloured
S
S
S
S
S
S
S
S
S
S
product (P)
by enzyme
Absorbances measured in
spectrophotometer
FIGURE 16.25
(a) Schematic outline of an enzyme-linked immunosorbent assay (ELISA). See text for details.
(b) The outcome of a typical ELISA assay. See also Figure 15.13 and associated text.
Fluorescence in situ hybridization

In FISH, single-stranded DNA molecules chemically linked to a fluorochrome are used
as probes to detect DNA samples that have sequences complementary to them; you can see
this diagrammatically in Figure 16.26 (a). Fluorochromes are molecules or parts of molecules
that absorb light of one wavelength, often in the UV range and re-emit most of the energy,
Cross reference but at a longer wavelength. Thus, the fluorochrome appears bright against a dark background
Fluorescence is described when viewed with a fluorescence microscope, giving a technique that can be sufficiently
more fully in Chapter 11
sensitive to detect single DNA molecules in microscopic sections of karyotypes, cells, or
‘Spectroscopy’.
tissues.
Fluorescence in situ hybridization techniques are used for diagnosis. For example, the analysis
of metaphase chromosomes is a major tool in cancer cytogenetics. Thin sections of the tumour
obtained from biopsies are treated to separate the DNA strands, which are then hybridized
in situ with fluorochrome labelled probes (Box 16.2) for known mutated forms of cancer-
associated genes. When the slides are viewed using a fluorescence microscope, any fluorescent
areas in chromosomes from cells in the metaphase indicate the presence of a mutation. For
example, patients with DiGeorge anomaly (DGA) exhibit a range of signs including cardiac
malformations, hypoparathyroidism, and hypocalcaemia. The majority of patients have a
deletion or partial monosomy of chromosome 22. These genetic lesions are detectable in
utero if cells obtained by amniocentesis are analysed using FISH with an appropriate probe
(Figure 16.26 (b)).
The Quadruple Test for Down’s syndrome calculates the potential risk of a pregnant woman
carrying a Down’s syndrome foetus. When the test indicates a higher risk of Down’s syndrome,
FISH, or polymerase chain reaction (PCR, Section 16.9) tests are available from many laboratories
in the UK to help clarify the situation.
(a)
Chromosome
analysed by FISH, (b)
showing presence of
bound fluorescent Excitation
probe light
Fluorescent
light
DNA probe with

fluorescent label
Chromosomal DNA
FIGURE 16.26
(a) Schematic to outline the binding of a fluorescently labelled DNA probe to target specific
chromosomal DNA sequences. (b) Picture of a fluorescence in situ hybridization (FISH)
analysis confirming DiGeorge anomaly (DGA). The TUPLE probe fluorescing red shows the
22q11 region that is deleted in DGA. The ARSA green signal is diagnostic for a region at the
end of the q arms of chromosome 22, confirming the deletion to this chromosome.
Courtesy of Paul Virgo, Department of Immunology, Southmead Hospital, Bristol, UK.
16.9 DNA CLONING 449
16.9 DNA cloning

Gene or DNA cloning is the preparation of many identical copies of a DNA molecule, that
is the amplification of a particular piece of DNA. Cloning can be accomplished by preparing
recombinant DNA in a cloning vector or by using the polymerase chain reaction.
Recombinant DNA
DNA recombination is the combining of a piece of the DNA one is interested in into another
type of DNA to form a recombinant DNA molecule. A recipient cell is then induced to assimi-
late and replicate the recombinant DNA. Many copies of the recombinant DNA may be pro-
duced by the progeny of the recipient cell; thus, the DNA of interest is cloned. If the inserted
fragment is a functional gene that encodes a specific protein then the protein could be pro-
duced in the host cell when the gene is transcribed and translated. This process is used for
the large-scale production of proteins valuable in the clinical sciences, for example insulin,
somatostatin, and other hormones, but which are difficult or expensive to prepare by other
methods. You can see an overview of the steps involved in cloning DNA by recombination in
Figure 16.27.
The carriers of the DNA to be cloned, often called vehicles or vectors, are usually plasmids
(Section 16.1) or bacteriophage DNA (Boxes 16.5 and 16.6). The recombinant DNA mole-
cule is formed by hydrolysing the vector with a RE to break both its strand at a specific point
(as you can see in Figure 16.27). The DNA of interest is prepared as a fragment using the
same RE as used to cleave the vector. The fragment and the vector are allowed to anneal
at their complementary overlapping ends. DNA ligase is then used to catalyse the cova-
lent joining of the ends of the restriction fragment and vector DNA together to form the
recombinant DNA. Ligation is most efficient when the ends overlap, but the DNA ligase
from bacteriophage T4 can join fragments and vectors that have been produced using REs
that produce blunt ended DNA, although this is a less efficient means of ligating the DNA
together.
A major breakthrough in recombinant DNA technology was the isolation of mutant strains of
Escherichia coli that are not able to digest foreign DNA. These strains are suitable as host organ-
isms for replicating recombinant DNA. The recombinant DNA must now be taken up by such a
host cell in a process called transformation. For example, E. coli cells can be made transiently
permeable to DNA and then mixed with recombinant vector DNA. Under certain conditions,
a small fraction, about one in about 10,000, of the cells will take up the recombinant plasmid.
The transformed cells can be distinguished from the others by using, for instance, a plasmid
containing a gene that confers resistance to the antibiotic ampicillin. Thus, if the cells are
grown in an ampicillin-containing medium, then only the transformed cells will be viable and
can be selected. Replication of the plasmid during the growth of the transformed cells ensures
that the inserted DNA is cloned.
It is sometimes necessary to isolate plasmid DNA from cells to allow it to be characterized

and manipulated. We introduced general methods for purifying DNA earlier (Section 16.3).
However, several methods for isolating plasmid DNA have been developed to separate plasmid
from chromosomal DNA once the cells have been disrupted. Plasmid DNA differs from chro-
mosomal DNA in having a tight circular conformation, whereas the chromosomal DNA usually
consists of linear fragments formed when the chromosome is sheared. The plasmid DNA is still
Plasmid
(vector) with
antibiotic
resistant gene
Antibiotic resistant gene
Plasmid hydrolysed with same

restriction endonuclease used in
producing DNA fragment
Plasmid
DNA fragment inserted
into plasmid vector
Recombinant
DNA
molecule
FIGURE 16.27
Overview of the steps involved
in a typical recombinant DNA Host eg
Escherichia
cloning experiment. A plasmid
coli cell
is used in this case as a vector.
The plasmid and the piece of Plasmid inserted
DNA of interest are spliced into host cell Cells cultured on nutrient
to form a recombinant DNA agar containing antibiotic
molecule. Bacterial cells are
then transformed when they
take up the recombinant
DNA. Only transformed cells
can grow in an antibioticrich Only those cells carrying
medium because of the recombinant DNA are
antibiotic resistance gene on resistant to antibiotic and
the plasmid. Growth of the grow to form colonies
bacteria amplifies the DNA of
interest.
much smaller than these fragments. These structural differences can be exploited to separate
the two types of DNA. For example, plasmid DNA can be specifically adsorbed on to nitrocel-
lulose microfilters and hydroxyapatite columns or the chromosomal DNA can be selectively
precipitated by using buffers of extreme pH, high temperature or other denaturing conditions.
BOX 16.5 Ideal characteristics of a

cloning vector
The ideal cloning vector should have a number of properties, including the following:
■ The vector should possess only a single site for a specific RE.
■ The vector should contain identifiable markers so that it is possible to screen trans-
formed cells for uptake. At least two selective markers are desirable; one to confirm
the insertion of foreign DNA into the plasmid, the second to allow the presence of
the plasmid in the cell to be confirmed.
■ The vector should generally be small relative to the fragments of chromosomal DNA,
making its isolation and purification easier and reducing the number of potential
sites for attack by REs.
■ The vector should be capable of being replicated rapidly by transformed cells to
ensure an efficient cloning of the recombinant DNA.
Finally, centrifugation can be applied to separate them because their size difference means
they differentially sediment.
Plasmid vectors based on the naturally occurring F plasmid of E. coli are used to clone DNA
fragments of 300,000 to 1 million nucleotide pairs. Unlike smaller bacterial plasmids, the
F plasmid and its derivative, the bacterial artificial chromosome (BAC, Box 16.6) is present
in only one or two copies per E. coli cell. The fact that BACs are kept in such low numbers in
bacterial cells may contribute to their ability to maintain large cloned DNA sequences stably.
Because of their stability, ability to accept large DNA inserts, and ease of handling, BACs are
now the preferred vector for building DNA libraries of complex organisms, including those
representing the human and mouse genomes.
cDNA cloning
An alternative cloning strategy is to begin the process by selecting only those DNA sequences that
are transcribed to give an mRNA molecule. These sequences are presumed to correspond to pro-
tein-encoding genes. This is done by extracting the mRNA from cells as outlined earlier (Section
16.3). A complementary DNA or cDNA is then made of each mRNA using reverse transcriptase,
an enzyme prepared from retroviruses, which synthesizes a DNA molecule complementary to an
RNA template. The single-stranded cDNA is converted into a double stranded molecule using
DNA polymerase. Each double stranded DNA molecule is then inserted into a vector and cloned
in the normal manner. Each clone prepared in this way is called a cDNA clone and the entire col-
lection of clones derived from one mRNA preparation constitutes a cDNA library.
The major advantage of using cDNA molecules is that uninterrupted coding sequences of a
gene are cloned. Eukaryotic genes usually consist of short coding sequences of DNA called
exons that are separated by normally much longer non-coding sequences called introns.
During the production of a specific mRNA, those portions of it that were formed by tran-
scribing the introns are removed and the coding sequences (from the transcribed exons) are
spliced together to give a continuous sequence. However, the mRNA transcripts of many genes
are subject to alternative splicing, so one gene can encode a number of alternatively spliced
and therefore different mRNAs. Thus, a cDNA library often contains many of the alternatively
spliced mRNAs produced from a given cell line or tissue.
BOX 16.6 Vectors
Plasmids are small, only 3–20% the size of the bacterial chromosome, meaning they
can carry inserts only 1–20 kbp in size. Their relatively small size also makes them com-
paratively easy to isolate from the bulk of the DNA present in the cell (Sections 16.3 and
16.9). Also, they have high multiple copy numbers, up to 500 plasmids per cell in some
cases, and so the inserted DNA of the recombinant plasmid may be greatly amplified.
The presence of selectable markers, such as the genes for antibiotic resistance or the
lac Z gene (see below), means that bacterial cells transformed by the recombinant DNA
can be easily detected because those cells that do not take up the vector will be unable
to grow in a medium containing the antibiotic. Thus, a number of cloning vectors have
been derived from plasmids.
The pUC vectors were introduced in the early 1980s and are based on fragments of DNA
derived from naturally occurring Escherichia coli plasmids. Figure 16.28 shows the struc-
ture of the pUC18 plasmid. The pUC19 vector is identical except its genes are organized in
the opposite direction. The pUC vectors have an origin of replication allowing them to be
replicated in high copy numbers. They contain the gene for β-lactamase, which confers
resistance to the antibiotic, ampicillin (AmpR) and allows transformed cells, which have
acquired the plasmid, to be identified. Additionally, pUC18/19 also contains a portion of
the E. coli lac operon, which allows cells containing the plasmid to synthesize the amino
terminal portion of β-galactosidase. pUC vectors may be used with appropriate host cells
that have been modified to express the gene for only the carboxyl terminal portion of the
enzyme. In the presence of a suitable inducer, for example, isopropylthio-β-galactoside
(IPTG), those cells containing a pUC plasmid will produce the two fragments of enzyme
and possess β-galactosidase activity. Such cells are able to hydrolyse the artificial sub-
strate 5-bromo-4-chloro-3-indolyl-β-galactoside (X-gal) to form a blue-coloured prod-
uct. Thus, colonies of the cells will appear blue when grown on nutrient agar containing
IPTG and X-gal. However, pUC18 also contains a multiple cloning site or polylinker region
near the lac gene, which contains the recognition sites for a number of different REs
(Figure 16.28). The formation of a recombinant plasmid means the inserted fragment
of DNA in this region prevents the formation of an active β-galactosidase. Hence, trans-
formed cells possessing the recombinant DNA will be unable to hydrolyse X-gal and their
colonies will appear white and so are easily identified.
The F plasmid or factor contains ‘fertility genes’, which enables a bacterium possessing
it (F+ cells) to produce sex pili during bacterial conjugation and act as genetic donors to
those cells lacking the factor (F− cells). Thus, the plasmid and its genetic information is
transferred between bacterial strains. Unlike smaller plasmids, only one or two copies of
the F plasmid are present in each bacterial cell. Plasmid vectors based on the F plasmid
of Escherichia coli, called bacterial artificial chromosomes (BACs), can be used to clone
pieces of DNA as large as 300 to 103 kbp. The low numbers of BACs in bacterial cells are
thought to contribute to the stability of the recombined BAC, despite the large sizes of
the DNA inserts. This stability and their relative ease of handling, means BACs are useful
vectors for building DNA libraries of, for example, the human genome.
The ability of bacteriophages or phages to replicate inside bacterial cells has allowed a
number of them to be developed as cloning vectors. Examples of phages used include
the Enterobacteria phage λ or lambda phage, the filamentous bacteriophage M13,
EcoRI
SacI
KpnI
Multiple cloning site of pUC18
SmaI XmaI
BamHI AflIII HaeII
HaeII
XbaI lacI
Origin of AlwNI
SalI AccI HincII replication
PSTI site
Operator
SphI Promotor
HindIII lac Z
NarI
pUC18
HaeII
NdeI
Eco0109I
AatII
CfrI
SspI
AmpR gene
XmnI ScaI
FIGURE 16.28
Outline of the structure of the pUC plasmid.
bacteriophage Mu (phage Mu) and phage P1. Phage P1 has been modified to form
phage artificial chromosomes (PACs) that, like BACs, can be used to clone relatively large
pieces of DNA. Cosmids are a type of hybrid plasmid that contain part of the DNA from
the lambda phage to which suitable origin of replication from other phages or plasmids
and a gene for selection, such as antibiotic resistance, have been added. Thus, they can
be used to clone DNA inserts as large as 30–50 kbp of DNA and so can be used to build
genomic libraries.
Polymerase chain reaction

Mullis devised the polymerase chain reaction (PCR) in 1983 and was awarded the Nobel Prize
in Chemistry in 1993 for this work. The PCR is an elegantly simple in vitro method to increase or
amplify the number of relatively short strands of specific DNA fragments in a sample. The frag-
ments are normally 0.5 to 5 kbp in length, although longer ones up to 40 kbp can also be ampli-
fied. The DNA fragments may be complete genes, although they more usually comprise only a
portion of one. Thus, the technique complements the recombinant DNA cloning strategy.
The PCR is used to replicate a DNA sample (template DNA) using a DNA polymerase. This
enzyme copies the template to produce new complementary DNA strands. DNA polymer-
ases cannot begin to synthesize a strand de novo, but only extend an existing piece of DNA.
Thus, two primer DNA strands are needed to initiate the copying process. Primers are artificial
oligodeoxynucleotides less than 50 nucleotides long that are complementary to sequences
that flank the region of the template DNA of interest. Hence, the primer determines the begin-
ning of the region to be amplified. Primers are usually made to order by commercial suppliers
who must be supplied with the required sequence. Some DNA polymerases can proofread,
that is correct any mistakes in the newly formed strand, which ensures that the fidelity of the
sequence is preserved. Other polymerases do not, however, have this property.
The PCR consists of a series of cycles, each of which doubles the number of molecules of the
DNA template. Prior to the first cycle, the DNA is often heated to 90–96 ºC for 5–10 minutes in
a ‘hot start’ designed to break the hydrogen bonds connecting the two strands and ensure the
template and primer DNA molecules are fully separated. This separation is called melting. Each
of the subsequent cycles consists of three identical steps, as shown in Figure 16.29. In the first
step, double-stranded DNA is heated for 0.5–3 minutes at 94–96ºC, which is normally sufficient
for melting. In the second step, the temperature is reduced to 50–65ºC for 0.5–6 minutes dur-
ing which the primers bind or anneal to their complementary sequence of the templates. The
primer must be present in amounts that are in excess of the target DNA, otherwise its strands
will simply rejoin. The design of the length of the primer requires careful consideration. Primer
melting temperature increases with the length of the primer. The optimum length for a primer
is generally 20–40 nucleotides, which will have melting temperatures of 56–75 ºC. If primers are
too short they will anneal at random positions on the relatively long template and result in a
non-specific amplification. However, if the primer is excessively long, the corresponding melt-
ing temperature would be above 80ºC and this could reduce the activity of the polymerase. The
third step is the extension of the primer by the polymerase. This usually requires 0.75–2 minutes
at 72ºC. The extended portions of DNA are complementary to the template strand.
The high temperatures used in PCR mean that DNA polymerases from thermophilic organisms
are preferred. The Taq polymerase from Thermus aquaticus, often abbreviated to Taq pol, or
simply Taq, is widely used although it has the disadvantage of lacking proofreading capabilities
and therefore can introduce errors (mutations) of 1 in 400–500 nucleotides in the newly formed
DNA. Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms
that significantly reduce mutations and are used in ‘long-range’ PCR of up to about 30 kbp.
The first cycle results in two double stranded DNA molecules, which are usually overexten-
sions of the target sequences. Each is composed of one of the original strands plus the newly
formed complementary strand and associated primer. Thus, the amount of DNA present has
been doubled. Cycles of PCR are usually repeated 20–30 times, with each cycle doubling the
amount of DNA present (Figure 16.30). Thus, the yield of DNA increases exponentially with
each cycle: after 30 cycles the original amount is amplified over 230 or 109-fold. A PCR experi-
ment normally terminates with a 10 minute incubation at 72ºC to ensure that all of the new
DNA molecules are fully extended by the polymerase.
Cloning by PCR is extremely sensitive to contamination and extremely stringent conditions

are necessary to prevent unwanted template DNA being coamplified with the sample. The
products of PCR are identified by determining their base sequences (Section 16.6) and/or
their size using agarose or polyacrylamide gel electrophoresis against standards of known size
(Section 16.5).
The great advantages of PCR are the increase in sensitivity it provides by amplifying the amount
of DNA present in a sample and the ease with which its step can be automated in a thermo-
cycler. These are instruments that can be programmed to heat and cool the reaction tubes
to the appropriate temperatures, for the desired times and the required numbers of cycles.
Samples of DNA isolated from even a single cell or that present in samples many years old can
be amplified and then analysed.
5' 3'
3' 5'
94−96
5' 3'
+ +
3' 5'
+
+
60−75
Temperature/°C
Key
DNA
Primer strands
50−65
Step 1 Step 2 Step 3

Melting Annealing Extension Melting
FIGURE 16.29
Three steps of the polymerase
30 s−3 min 30 s−1 min 45 s−2 min 30 s−3 min
chain reaction. See text for
Duration details.
In addition to its application in many areas of molecular biological research, PCR has four
major clinical uses:
1. The identification of infectious disease organisms for diagnostic purposes
2. The detection of variations and mutations in hereditary diseases
3. Detecting acquired mutations that lead to cancers
4. Tissue typing.
FIGURE 16.30
Agarose gel electrophoresis separation of a polymerase chain reaction
amplification. Lane I shows the positions of fragments of DNA of known sizes.
Lane 2 shows a 390 bp fragment of DNA, which forms the positive control. Lanes
3 to 7 show the results of amplifying this DNA by 10, 20, 25, 30, and 35 cycles
of polymerase chain reaction. Lane 8 shows the results of the negative control,
1 2 3 4 5 6 7 8 where the DNA was replaced by distilled water in the amplification.
Polymerase chain reaction is especially useful in diagnosing diseases caused by organisms that
are difficult or impossible to culture. Its ability to amplify small amounts of DNA means that PCR-
based tests can identify sources of infection more accurately, reliably, rapidly, and cheaply than
previous methods. For example, PCR is the basis of sensitive and specific tests for Helicobacter
pylori, the main causative agent of stomach ulcers. Three different sexually transmitted disease
agents, Herpes, papilloma viruses, and Chlamydia, can be detected on a single swab using PCR
tests. Indeed, even specific strains of papilloma viruses that predispose individuals to cervical
cancer can be identified. Diagnostic tests are also available for the viruses involved in AIDS, viral
hepatitis, and viral meningitis. In 2002, 47% of meningococcal infections in England and Wales
were diagnosed using PCR tests for meningococcal DNA in clinical samples. Bacterial infections
in middle ear fluid from children suffering otitis media are detectable by PCR, indicating an
active infection, even when standard culture methods for the bacterium fail. The Lyme disease
bacterium, Borrelia burgdorferi, is often difficult to diagnose accurately using its general symp-
toms but PCR can amplify, and therefore identify, its DNA in samples of body fluids. Cloning by
PCR has largely replaced Southern blotting for diagnosing genetic diseases.
Reverse transcriptase PCR

Reverse transcriptase-PCR (RT-PCR) is a technique that allows an RNA molecule to be ampli-
fied but as complementary DNA copies, whose preparation was described above. The first
step is to hybridize an oligodeoxynucleotide primer to the target RNA molecule. This primer
may be specifically designed to hybridize to the mRNA from a specific target gene. However,
oligo(dT) can be used as a universal primer for RT-PCR of mRNA molecules because it binds to
the endogenous poly(A) tail they all possess. An RNA-dependent DNA polymerase or reverse
transcriptase, obtainable from retroviruses, forms a cDNA to the target RNA molecule by
extending the primer. The cDNA can be amplified by PCR, generally using gene-specific prim-
ers to generate copies of it. The amplification of contaminating genomic DNA can be mini-
mized by treating the RNA preparation with RNase-free DNase, which will degrade it.
Reverse transcriptase-PCR is a sensitive and versatile procedure. It is used to:
• Clone the 5′ and 3′ termini of mRNA molecules
• Generate large cDNA libraries, that is copies of the mRNA molecules present in cell or
tissue extracts
• Identify mutations (polymorphisms) by analysing mRNA molecules transcribed from the

defective gene.
Real-time polymerase chain reaction

Real-time polymerase chain reaction is also called real time PCR, quantitative real-time PCR
Cross reference (qPCR), and kinetic PCR. Real-time PCR is a technique based on the PCR that not only amplifies
The automated use of PCR the target DNA, but also simultaneously quantifies the amount present. The amount can be
is discussed in Chapter 17 expressed as the absolute number of copies or as the relative amount of target DNA compared
‘Laboratory automation’.
to a so-called normalizing gene. Real-time PCR follows the general principles of PCR outlined
above. However, the amount of amplified DNA is determined as it accumulates in the reac-
tion in ‘real time’ following each cycle by one of two common methods. One method uses a
fluorescent dye that intercalates into double-stranded DNA. The other method uses modified
deoxyoligonucleotide probes that fluoresce when hybridized with their complementary DNA.
In both cases, the amount of fluorescence is proportional to the amount of DNA formed.
Real-time PCR is often used in combination with RT-PCR to determine how many molecules
of a specific mRNA are present in a sample. This gives the relative expression of its gene at a
particular time, or in a particular cell or tissue type.
16.10 DNA MICROARR AYS 457
16.10 DNA microarrays

DNA microarrays are glass microscope slides, nylon filters, or silicon chips to which large num-
bers of DNA fragments have been attached (Box 16.7). Each fragment is an oligodeoxynucle-
otide whose sequence is a probe for a specific gene. The densest DNA microarrays contain
probes for all of the approximately 21,000 human genes packaged into an area smaller than
a postage stamp! Thus, despite their small size, microarrays allow thousands of hybridization
reactions to be performed simultaneously. Hybridization reactions use mRNA molecules iso-
lated from the cell and used directly or more stable cDNA copies (Section 16.9) of them. In
either case, their sequences are complementary to the probes of the microarray. Each sample
mRNA or cDNA is radiolabelled with 32P (or 33P) or tagged with a fluorescent dye. The labelled
samples are allowed to hybridize to the probes on the microarray. Note how the probes differ
from those we described for other techniques, such as FISH and blotting, in not carrying the
label, which in this case is attached to the sample molecules. The microarray is thoroughly
washed to remove any unbound (that is non-hydridized) mRNA or cDNA molecules. The tar-
gets can be identified by autoradiography or by its fluorescent label. Usually, the fluorescent
cDNA from the experimental samples is labelled, for example with a dye that fluoresceces red
when excited, and is mixed with a reference sample of cDNA fragments labelled with a dye that
shows green fluorescence. Thus, if the amount of a specific mRNA expressed from a particular
gene is increased relative to that of the reference sample, the resulting spot on the microarray
is red. Conversely, if the expression of the gene is decreased in comparison, the spot is green.
However, If there is no change compared to the reference sample, the spot is yellow (Figure
16.31). These initial hybridization intensities require sophisticated computerized analyses to
normalize, summarize, and analyse the differential gene expression patterns. However, using
such an internal reference, allows the expression of many genes to be accurately assessed.
Since their introduction in 1995, DNA microarrays have proved useful in analysing the expres-
sion of numerous genes by allowing the concentrations of all the mRNA molecules produced
by a cell to be simultaneously monitored. Hence, we can now identify and study the patterns
of gene expression that underlie both normal and pathological cell physiology and investigate
the differential expression of genes as cells grow, divide, and differentiate. For example, micro-
arrays are used to identify and classify tumours based on the patterns of gene expression they
exhibit. This should help identify new targets in tumours for chemotherapy and the develop-
ment of novel anticancer drugs. Microarrays can also be used to quickly identify disease-causing
microorganisms by hybridizing the DNA isolated from infected tissues to a microarray of
genomic DNA sequences from potential pathogens. At the time of writing, microarrays require
rigorous operating procedures making them difficult to use in every day clinical applications.
BOX 16.7 Preparation of DNA

microarrays
DNA microarrays are prepared using longer fragments of DNA produced by PCR (Section
16.9) and then spotted onto the slides by a robot. Shorter artificially synthesized oligode-
oxynucleotides are attached to the surface using techniques similar to those that are used
to etch circuits onto computer chips. In both cases, the sequence and position of every
probe on the chip is known. Thus, any nucleotide fragment that hybridizes to a probe can
be identified as the product of a specific gene by detecting its location on the microarray.
Cross reference FIGURE 16.31

You can read about antibody Photograph showing a portion of a microarray
microchips in Chapter 15 following an experiment on gene expression.
‘Immunological techniques’. Genes whose expressions have been increased
during the experiment (up-regulated) compared
to the control are indicated by red spots.
In contrast, those genes whose expression
decreased (down-regulated) appear as green
spots. Yellow spots show genes whose expression
was unchanged during the experiment.
Furthermore, they are expensive to use; both in the cost of the microarrays themselves and in
their need for highly trained personnel. It is generally expected that as their costs diminishes,
microarray analyses will be powerful diagnostic tools in the clinical sciences. However, as the
costs of DNA sequencing continues to fall, this more robust technique is likely to be the method
of choice given that the two techniques have many similar biomedical applications.
SELF-CHECK 16.8
Huntington disease, myotonic dystrophy, and spinobulbar muscular dystrophy are all asso-
ciated with increased numbers of trinucleotide repeats in their associated genes. Which
method(s) could conveniently be used to confirm the status of potential carriers or patients?
SUMMARY
■ Molecular biology is generally concerned with the structures, functions, and interactions
of the two major groups of macromolecules, the nucleic acids deoxyribonucleic acid
(DNA), and ribonucleic acid (RNA), and proteins. Deoxyribonucleic acid and RNA carry
biological information. Proteins perform many of the activities in cells and form some of
their structural features
■ Nucleic acid molecules or fragments of molecules are able to interact and recognize
one another: adenine hydrogen bonds to its complementary bases of thymine in DNA
and uracil in RNA, and guanine hydrogen bonds with its complement, cytosine. Protein
molecules have discrete sites on their surfaces that are complementary to specific small
ligands or to relatively small areas on the surfaces of other proteins and nucleic acid mol-
ecules. Thus, proteins also generally function by recognizing and specifically interacting
with other biological molecules
■ Nucleic acids can be purified from organisms using a variety of techniques. Restriction
endonucleases can digest the isolated DNA into smaller-sized fragments suitable for anal-
ysis and for use in a number of techniques of clinical interest
■ Molecular biology techniques used in biomedical science that rely on the complementary
binding nucleic acids to provide a convenient way of recognizing and isolating specific
FURTHER READING 459
base sequences within fragments of DNA or RNA molecules include the sequencing of iso-
lated DNA, Southern and Northern blotting, fluorescence in situ hybridization (FISH), the
cloning of DNA by recombination and PCR technologies, and DNA microarray analysis
■ One group of proteins used extensively in clinical tests are the immunoglobulins or anti-
bodies. The binding of antibodies to their complementary antigens forms the basis of
Western blotting and immunoassay techniques such as enzyme-linked immunosorbent
assays (ELISAs), which are used in all branches of the biomedical sciences
■ The chapter discusses the underlying basic principles behind these techniques and pro-
vides general descriptions of their associated experimental protocols. In addition, we also
highlight a number of essential precautions that must be observed if the reagents are to
be handled and the procedures carried out safely
FURTHER READING
● Brown TA. Gene cloning and DNA analysis: an introduction, 5th edn. Blackwell,
Oxford, 2006.
● Craig N, Cohen-Fix O, Green R, Greider C, Storz G, Wolberger C. Molecular Biology:
Principles of Genome Function. Oxford University Press, 2010.
● Dreier J, Störmer M, Kleesiek K. Real-time polymerase chain reaction in transfusion
medicine: applications for detection of bacterial contamination in blood products.
Transfusion Medicine Reviews, 2007; 21: 237–254.
● Elliott W, Elliott D. Biochemistry & Molecular Biology, 4th edn. Oxford University
● Kricka LJ. Stains, labels and detection strategies for nucleic acid assays. Annals of
Clinical Biochemistry, 2002; 39: 114–129.
● Lo YMD. (Ed.) Clinical Applications of PCR, 2nd edn. Humana Press, NJ, 2006.
● Maraqa L, Donnellan CF, Peter MB, Speirs V. Clinicians’ guide to microarrays. Surgical
Oncology, 2006; 15: 5–10.
● Mikhailovich V, Gryadunov D, Kolchinsky A, Makarov AA, Zasedatelev A. DNA micro-
arrays in the clinic: infectious diseases. BioEssays, 2008; 30: 673–682.
● Nicholl DST. An Introduction to Genetic Engineering, 3rd edn. Cambridge University
Press, Cambridge, 2008.
● Sambrook J. Molecular Cloning: A Laboratory Manual (3 volume set), Cold Spring
Harbor Laboratory Press, Cold Harbor, 2001.
● Speers DJ. Clinical applications of molecular biology for infectious diseases. Clinical
Biochemist Reviews, 2006; 27: 39–51.
● Volpi EV, Bridger JM. FISH glossary: an overview of the fluorescence in situ hybridiza-
tion technique. Biotechniques, 2008; 45: 385–386.
● Williams SA, Slatko BE, McCarrey JR. Laboratory Investigations in Molecular Biology.
Jones and Bartlett, Sudbury, 2007.
● Wiltgen M, Tilz GP. DNA microarray analysis: principles and clinical impact.
Hematology, 2007; 12: 271–287.
QUESTIONS
1. Which of the following statements is/are UNTRUE?
(a) RNA contains ribose residues
(b) RNA contains deoxyribose resides
(c) RNA contains the bases adenine, guanine, uracil, and cytosine
(d) RNA molecules do not possess any double stranded structure
(e) RNA contains the bases adenine, guanine, uracil, and thymine
2. Which of the following statements is/are UNTRUE?
(a) Proteins are largely concerned with the storage and transmission of genetic
information
(b) Proteins can form structural elements of cells and tissues
(c) Proteins form when amino acid residues are joined by phosphodiester bonds
(d) Proteins can act as catalysts
(e) A protein composed of 336 amino acid residues would contain 337 peptide bonds
3. How does SDS help disrupt the plasma membrane?
4. How is contaminating RNA removed from a DNA preparation?
5. What is the major use of ethanol in the preparation of samples of DNA?
6. Pancreatic ribonuclease is an endonuclease that shows b-type specificity at nucleotide
residues on the 5’ side of pyrimidine nucleotides. List the possible terminal nucleotides
of its products.
7. Which of the following base sequences would not be potential recognition sites for
restriction endonucleases? Explain your choice.
5′
(a) . . . GAATTC. . . 3′ (b) 5′
. . . CATATG. . . 3′
3′
. . . CTTAAG. . . 5′ 3′
. . . GTATAC. . . 5′
5′
(c) . . . GACGGAT. . . 3′ (d) 5′
. . . CAATTG. . . 3′
3′ 5′ 3′
. . . CTGCCTA. . . . . . GTTAAC. . . 5′
5′
(e) . . . CATTAG. . . 3′
3′
. . . GTAATC. . . 5′
8. Why is polyacrylamide gel electrophoresis unsuitable for analysing plasmids?

9. If the proteins you wished to analyse by Western blotting are (a) acidic (anionic) and of
relatively low Mr, and (b) relatively hydrophobic and of Mr 65,000, respectively, what type
of membrane would give optimal binding?
10. Outline a test based on Western blotting that would help in the diagnosis of infection
with the AIDS virus. Assume that a sample of serum is available from the patient.
17
Laboratory
automation
Tim James
Learning objectives
■ Identify the major benefits of automation

■ List the types of specimens that are processed in a high volume automated laboratory
■ Describe the common automation units including those associated with the pre- and post-
analytical processes
■ Recognize the role of automation in the efficiency and productivity of all biomedical
disciplines
Introduction
Laboratory automation has been defined by the Association for Laboratory Automation, the
leading organization for laboratory automation, as ‘a multi-disciplinary strategy to research,
develop, optimize and capitalize on technologies in the laboratory that enable new and
improved processes and products’. All clinical laboratories utilize automation albeit to a vari-
able extent, dependent on the discipline and requirements of the service. The application of
this automation has produced improvements in efficiency and effectiveness of testing and
improved consistency of analysis.
The investigations and analyses undertaken by hospital laboratories can be categorized in a

number of ways. Traditionally this has been based on the association of any given laboratory test
with a defined biomedical science discipline, and conventionally these are haematology, clinical
biochemistry, immunology, microbiology, histology, and cytology. Automation is evident in each
of these disciplines and you can find discipline-specific descriptions of automation throughout
the accompanying volumes in this book series. However, the availability of analytical systems
with increased sophistication in terms of both robotics and underlying information technology
has yielded a model of laboratory operation that crosses the traditional discipline boundaries.
462 17 L ABOR ATORY AUTOMATION
Specimen reception
Sample receipt and registration
Sample preparation
Sample presentation to analysers
Sample analysis
Sample archiving
FIGURE 17.1 Sample storage

The basic steps undertaken for most samples in
clinical laboratories utilize a common process path.
Sample disposal
Most of these steps can be automated.
Figure 17.1 outlines the basic pathway that a sample may undergo within most clinical labo-
ratories. The sample will arrive in the laboratory and be registered, usually by booking the
specimen into the laboratory computer system. The sample is then prepared for analysis, pre-
sented to analysers, and undergoes analysis. Following analysis the sample will be archived,
stored and will eventually, after a defined period of retention, be disposed in an appropriate
and safe manner.
Automation of the central analytical step usually using instrumentation of varying complexity
is apparent in all hospital laboratories. However, the preceding steps and those following anal-
ysis can also be automated. Whilst the timescale and complexity of steps may vary between
each of the individual disciplines, the underlying process is the same. Consequently, there
has been a convergence of automation and technology across the conventional disciplines
resulting in the development of what is termed the core automated or the blood sciences
laboratory. This chapter will review automation in the context of general automation features
and their associated benefits.
17.1 Benefits of automation

Cross reference The most apparent benefits of full automation compared with manual or semi-automated
Audit is discussed in Chapter 19
analysis are the improved speed and efficiency in processing samples to produce test results
‘Quality assurance and for clinical management. It has been estimated that growth in workload of clinical labora-
management’. tories is between 5 and 10% per annum. The management of this expanding workload has
only been possible through greater utilization of automation of increasing sophistication and
capacity. However, the benefits are much broader than this, as detailed in Table 17.1. The
ability of automation to process samples using a common procedure has resulted in simpler,
more consistent sample handling and this has been associated with a reduction in process-
ing errors. A consistent finding in laboratory audits is that the majority of errors, in some
cases it has been found that greater than 80%, occur in the pre-analytical phase. This is the
step most associated with manual sample handling. Equally, manual laboratory procedures
may involve several steps, where the sample may need to be manipulated and analysed.
17.1 BENEFITS OF AUTOMATION 463
TABLE 17.1 General benefits of automation
Benefit Impacts
Efficiency benefits Improved sample throughput
Reduced sample splitting
Reduced sample retrieval requirements
Reduced staff time required for manual sample handling
Improved health Reduced exposure of staff to clinical specimens and therefore

and safety clinical infection
Reduced risk of repetitive strain injury
Error reduction Reduced risk of samples being mis-aliquoted into the wrong
secondary containers
Reduced risk of samples being misplaced as the sample location
can be monitored at all stages of the laboratory process
Improved patient care Reduced volume of blood and number of tubes collected
Reduced and more consistent turnaround times for test results
resulting in improved patient management
Misidentification of samples and errors can occur at each of these steps even when there are
significant quality measures in place. Evidence suggests that automated processes, whether
they are pre-analytical, analytical, or post-analytical, are systematic and associated with lower
error rates.
Cross reference
Automation has produced a number of improvements with respect to health and safety. You can read more about health
and safety in Chapter 4.
Manual handling of uncapped, open samples can expose staff to a range of hazardous
organisms. Automating laboratory processes reduces the number of occasions an individual
may need to handle specimens and thereby reduces the risk of exposure to these hazards.
Equally, in a laboratory setting where several thousand specimens are manipulated, there is
a risk of repetitive strain injury. Replacing the manual manipulation of samples by an equiva-
lent automated process will remove this risk. For example, many automation systems can
uncap and recap tubes, steps that if undertaken repeatedly by the same member of staff
carry the risk of straining hand, wrist, and shoulders, and temporary or permanent muscu-
loskeletal injury.
A consideration discussed widely in laboratories is the time taken for a test result to be avail-
able to the clinician after the specimen is taken from the patient. This is commonly referred to
as the turnaround time (TAT). Most laboratories use some measure of TAT as a quality indica-
tor of efficiency. The TAT can be measured in two ways, the total TAT and the within laboratory
TAT. Both can be influenced by laboratory automation. The total TAT is the time taken from
the collection of the sample to the time the report is available to the clinician to interpret the
result. The within laboratory TAT is the time taken from the receipt of the specimen in the
laboratory to the laboratory authorization of the result.
All TAT requirements vary with clinical setting and the particular investigation, for example, the
TAT requirement for glucose analysis in an unconscious patient with diabetes will need to be
rapid and is measured in minutes. In comparison, the TAT requirement for cholesterol testing
undertaken to assess cardiovascular risk in a healthy, ambulatory patient can be longer and
will be measured in hours. Reduced and more consistent TATs are apparent with increased
levels of automation and this may improve the timeliness of clinical decisions and patient
treatment.
Cross reference SELF-CHECK 17.1

You can read about accuracy How might a reduced test turnaround time help patient care?
and precision in Chapters 5
‘Statistics and handling data’
and 19 ‘Quality assurance and Sequential generations of automated analysers have produced more consistent and repro-
management’. ducible results, as evidenced by reduced imprecision. They are more robust, as measured by
the proportion of time the analyser is in operation. The related changes and benefits have
included:
• A reduction in the specimen volume required for individual assays
• A reduced usage of reagent
• Increased analyser capacity relative to physical size
The lower specimen volumes required by newer automated analysers compared with manual
techniques and older analysers translates into the advantage that a greater number of tests
may be undertaken from a single clinical sample. This is particularly important in children and
neonates where blood collection may be difficult, but is also beneficial in other patient groups
where blood collection is challenging, such as patients with learning difficulties or those with
fragile veins, such as the very elderly.
The reduced reagent volume has a great number of associated advantages. Total reagent costs
will be reduced if less is used and less chemical waste is generated thereby reducing the envi-
ronmental impact. Reagent storage requirements, both on the analyser itself and within labo-
ratory storage facilities are reduced if lower volumes are required. This further reduces the
space requirements for both room temperature and refrigerator storage.
Modern automated instrumentation is often considered physically large, but the analytical
capacity, as measured either as samples or tests per hour, relative to their size has increased
significantly. Therefore, whilst most laboratories have been modified in design and layout to
accommodate automation, the total laboratory space requirement has often remained the
same. The components utilized within analysers have become increasingly sophisticated, and
particularly with respect to liquid handling, the accuracy and precision of sample and rea-
gent delivery is both highly precise and robust. The consequences of these improvements are
that total assay imprecision has improved and methods are highly reproducible. Most instru-
ment manufacturers have a programme of monitoring to identify patterns of analyser com-
ponent failure. This informs component supplier requirements and subsequent instrument
design. Increasingly remote diagnostics, where a manufacturer will monitor critical instrument
parameters electronically using the internet, may even predict analyser deterioration before
it actually impacts on test results. The best design features utilized within automation systems
are maintained and copied by competitors, and therefore it is possible to see common fea-
tures in a range of different suppliers’ instrumentation. The overall analyser component failure
rate has reduced and this has produced automated instrumentation that is operational for a
greater proportion of time. This reduces demands on both laboratory operators and service
engineers.
Automation allows flexible use of staff time as large automated systems require less human
intervention for operation and a greater number and proportion of laboratory investigations
can be undertaken by automated systems. This could be considered to be an opportunity to
reduce overall operating costs, but the majority of laboratories will deploy any staff released
by automation and redirect them to analytically more demanding laboratory areas or to
17.2 COLLECTION OF SUITABLE SAMPLES AND DELIVERY TO THE L ABOR ATORY 465
improving laboratory quality. However, as automation hardware is expensive, its introduction

into clinical laboratories requires careful consideration of the clinical necessity and staff saving
against capital expenditure.
Key Points
Test turnaround times (TATs) are key indicators of laboratory performance. Turnaround
time requirements vary with respect to investigation/test and clinical context. Auto-
mation can lead to significantly reduced and more consistent TATs.
Collection of suitable samples

17.2
and delivery to the laboratory
The majority of the samples processed in a core automated laboratory will be blood speci-
mens taken from a vein by phlebotomists. A smaller proportion, usually less than 10%, of
the laboratory investigations are undertaken on other clinical specimens, for example urine.
Clinicians often require a number of assays to be undertaken simultaneously and therefore,
multiple assays may be required on a patient at the same time. In the UK the average test
to request ratio of a typical hospital laboratory is between 6 and 8, however this will vary
depending on the local arrangements and requirements as described in Box 17.1.
Cross reference
Key Points Blood sampling is described in
A laboratory test may be considered an individual investigation or assay, for example, Chapter 7 ‘Samples and sample
collection’.
measuring blood glucose concentrations. A request is regarded as one or more tests
asked for by the clinician at a single point in time, for example, a patient who is tired all
the time may require a number of different tests to assess possible causes. A request is
not the same as a profile, which is usually a number of clinically or metabolically related
tests grouped together, for example, a liver function profile, which will include several
tests associated with this organ’s functions.
It would be convenient if all blood tests could be processed using a single specimen tube.
However, many blood constituents require specific preservation conditions to stabilize the
test component. Consequently, a number of different tube types are required depending on
BOX 17.1 How to calculate the

test to request ratio
The average test to request ratio is simply the number of tests divided by the number of
requests. A laboratory that undertakes 750,555 requests and 4,742,200 tests per annum
therefore has a test to request ratio of 6.31.
TABLE 17.2 Commonly used collection tubes and associated colour coding. See also
Table 7.2.
Tube type Additive Commonly used tube

cap colours
Serum Clot activator Red, brown
Heparinized plasma Lithium heparin Green, orange
EDTA blood Sodium EDTA* Lavender, pink
Citrated plasma Sodium citrate Blue, green
Fluoride oxalate plasma Sodium fluoride, potassium oxalate Grey, yellow
*EDTA = ethylenediaminetetraacetic acid.
the combinations of tests required. Commonly available specimen tubes used in routine diag-
nostic laboratories on automated systems are presented in Table 17.2 together with com-
monly observed colour coding systems used to identify the different additives.
A serum collection tube contains no anticoagulant and the sample will form a clot: this sample
can be centrifuged to yield serum. Serum requires time, usually about 30 minutes at room
temperature, for the clot to form. To reduce the clotting time, many serum tubes are manu-
factured containing an additive to accelerate clotting so that the sample may be centrifuged
more rapidly. Serum is suitable for a wide range of tests including the vast majority of bio-
chemistry, immunology, and virology assays. The tube often contains a gel that has a density
that is greater than that of serum, but lower than that of the clot. Therefore, when the sample
tube is centrifuged, the contents are distributed into three components: a clot at the bottom
of the tube; the gel in the middle, forming a barrier, and the serum on the top (see Figure
12.14). The majority of automated instrumentation analyses this serum directly from the origi-
nal sample tube, a process called ‘primary sampling’ as distinct from transferring the serum
into a secondary container. Many automation systems can produce a secondary aliquot of the
specimen, but it is more convenient in terms of efficiency not to do this. Most automation
systems benefit significantly from primary sampling.
SELF-CHECK 17.2
Why is it necessary to use a number of different specimen tubes when undertaking laboratory
investigations?
Sodium ethylenediaminetetraacetic acid (EDTA) is an anticoagulant by virtue of its ability to

chelate Ca2+, which is essential for the clotting process. Blood collected into a tube containing
EDTA will not clot and a whole blood sample is produced that preserves the cellular con-
stituents. This tube type is most frequently used for the analysis of standard haematology
investigations such as the full blood count and erythrocyte sedimentation rate. It is also a
suitable specimen tube for a range of other investigations including the analysis of glycated
haemoglobin, a common test used for the monitoring of diabetes mellitus.
Sodium citrate, like sodium EDTA, binds Ca2+ and prevents coagulation. This tube may be cen-
trifuged to yield citrated plasma for coagulation studies. A critical factor for this tube type is
the ratio of blood to anticoagulant, which is essential for the accurate analysis of coagulation
parameters. To ensure the correct volume of blood is added to each collection tube an indicator
line is marked onto the side of the tube as a guide.
17.3 SPECIMEN RECEPTION CONSIDER ATIONS IN THE CORE AUTOMATED L ABOR ATORY 467
Lithium heparin is an effective anticoagulant as it inhibits the pro-thrombin to thrombin

step of clotting. Centrifugation of this sample type produces heparinized plasma which
is used for many investigations undertaken in clinical biochemistry. The advantage of this
sample type over serum is the ability to centrifuge almost immediately after collection. An
additional advantage of lithium heparin blood is that the plasma volume obtained is greater
than the comparative yield from an equivalent volume of blood converted into serum.
Whilst only a small effect, this can be beneficial for paediatric specimen and their naturally
smaller sample sizes. It is common for lithium heparin tubes to contain a gel to enable pri-
mary sampling in a similar manner to that described for serum above.
The fluoride–oxalate blood collection tube contains two additives: sodium fluoride which
binds Mg2+, a co-factor of several enzymes associated with the glycolytic pathway thereby
inhibiting glycolysis, and potassium oxalate which binds Ca2+ and prevents coagulation. This
tube is usually associated with the analysis of glucose and lactate, assays that are adversely
affected by continued glycolysis, which will occur in the other specimen tubes.
The development of laboratory automation systems has been helped considerably through
standardization of specimen container shape and size and manufacturers produce the range
of tubes described above in a standard format with respect to their physical dimensions and
capping mechanism. The improved standardization of tube shape and size has improved sam-
ple presentation to analysers, the manipulations needed to remove and replace caps and their
transfer by robotic arms. The two commonest diameters of specimen tube are 13 and 15 mm,
and the commonly encountered tube heights are 75 and 110 mm. Many laboratories attempt
to standardize the size of tube used by the automated system so, for example, all the labora-
tory users are supplied with tubes of one size, for example, 13 × 75 mm.
When the sample tubes have been collected from the patient they may be transported to the
laboratory. The process of conveying the specimen from clinical to laboratory area within a
hospital has been automated through the use of pneumatic air tubes. These tubes connect the
clinical areas to the laboratory, and convey the specimen safely and promptly to enable faster
total turnaround times.
Key Points
The application of automation to clinical laboratories was greatly enhanced with the
introduction of separator gels that are incorporated into the blood collection tubes
during manufacture. These gel-containing tubes allow processing of the plasma and
serum samples through all steps of the analytical process, from collection through to
disposal, in a single vessel, removing the requirement for specimen transfers.
Specimen reception
17.3
considerations in the core
automated laboratory
Most samples still arrive in the clinical laboratory with their paperwork in the form of a
request card that contains information about the patient, the specimen, and the investiga-
tions required. This information is conventionally transcribed by manual data entry into the
laboratory computer system. Improved efficiency and accuracy in this process is achieved if
the data entry process is partly or completely electronic. Whatever method of sample reg-
istration is utilized, the interface between the specimen reception area and the automated
instrumentation is a critical factor in overall laboratory efficiency. Automation alone does not
produce the benefits detailed in Section 20.2; it is its utilization in the overall laboratory proc-
ess that is critical.
Specimen reception is the usual location for an adhesive sample bar code to be applied to
each sample tube thereby allowing its unique identification and routing through the labora-
tory automation. An alternative process exists in which the bar code is applied at the point
of collection in the clinical or phlebotomy area. The accurate reading of the bar code label is
required at many points in the automation process from the specimen reception workstations,
as well as all of the interconnected devices and analysers. Consequently, the bar code quality
in terms of clarity to bar code readers needs to be high enough to allow consistent reading
and identification at each step. The format may be a simple number (numeric) or may allow
inclusion of one or more letters into the specimen number (alpha-numeric).
The interface between specimen reception and automated systems can be described using
three basic models presented in Figures 17.2 (a–c):
1. A traditional interface model

2. A semi-automated interface model
3. A totally automated interface model
Model 1, Figure 17.2a, represents the traditional laboratory in which the specimen reception
process includes the step of sorting specimens into a range of racks appropriate and suitable
for presentation to different analysers or laboratory areas. The disadvantage of this approach is
that the specimen reception work station may be relatively cluttered and that multiple manual
steps may be required to present samples to analysers. Model 2, Figure 17.2b, a model in
which the specimen reception work station is simplified by using a single sample rack into
which the bar coded specimens are placed. The single rack is then presented to a stand-alone
automated sample management system that will then distribute the specimens into different
Cross reference analyser racks. The subsequent presentation to the analysers is manual. This model may be
Automation is also explored
particularly beneficial if a laboratory utilizes analysers from more than one manufacturer that
in Chapter 2 ‘Automation’ in cannot be connected through a single tracking system. The system is also beneficial if labora-
the companion text, Clinical tory space and/or layout is constrained and analysers are in different laboratory rooms and
Biochemistry. areas. Stand-alone automation can be utilized to improve the efficiency of multi-disciplinary
laboratories but are most often found in clinical biochemistry laboratories. Figure 17.2c, rep-
resents a system in which both the sorting and conveyance of samples to analysers is achieved
with a track system. Again, the work of the specimen reception is simplified by having only one
rack in which to place all the specimens. In reality, specimen reception areas usually operate
a mix of these models depending on the automation available (primarily whether the labo-
ratory uses stand-alone sample management systems as opposed to tracks, as described in
Section 17.4), the testing repertoire, and laboratory organization. Most automated labora-
tories review their specimen reception processes when new or different automation is intro-
duced, with the primary aim of streamlining all the steps, reducing variation, and removing
unnecessary steps.
SELF-CHECK 17.3
Specimen reception is the only place where bar code labels are applied to specimen tubes.
True or false?
17.3 SPECIMEN RECEPTION CONSIDER ATIONS IN THE CORE AUTOMATED L ABOR ATORY 469
(a) Specimen reception
Manual sort to multiple racks
Haematology Biochemistry Coagulation Other investigations

sample rack sample rack sample rack sample rack
Manual transfer Manual transfer Manual transfer Manual transfer

and presentation and presentation and presentation and presentation
to haematology to biochemistry to coagulation to required
analyser(s) analyser(s) analyser(s) analyser(s)
(b) Specimen reception
Single rack for manual

presentation to automated
sample sorter
Automated
distribution to
analyser racks
Manual transfer Manual transfer Manual transfer Manual transfer FIGURE 17.2
and presentation and presentation and presentation and presentation Specimen reception,
to haematology to biochemistry to coagulation to required automation interface models.
(a) Samples are sorted and
presented to individual
analysers manually. This
is complex with respect to
the processes on specimen
(c) Specimen reception reception. (b) Samples are
placed into a single rack that
is presented to a standalone
Single rack for manual
automated sample manager
presentation to automated
sample sorter that distributes the samples
to analyser racks. Racks are
removed and presented to
Automated loading the analysers manually. (c)
onto a track
Samples are placed into a
single rack that is loaded
onto the input rack of an
Automated transfer Automated transfer Automated transfer Manual transfer automated tracking system
and presentation to and presentation to and presentation to and presentation and automated sorting,
haematology biochemistry coagulation to required
distribution, and presentation
to analysers.
17.4Tracked automation systems

and the core automated
laboratory
A tracked automation system in a core automated laboratory can be considered as a group of
modules or units each with a specific function. The number and type of units/modules within
the automation system is dependent on the repertoire of assays and the number of samples/
tests requiring analysis. There is also a general requirement for instrumentation back-up, par-
ticularly if continuous analysis is required. Since no two clinical laboratories are identical, a
range of different combinations of instrument can be found connected to tracking systems to
meet the requirements of the specific laboratory. You can see an outline design of an auto-
mated tracked system in Figure 17.3.
The modules/units of an automated tracking system can be categorized as:
• Modules that prepare samples for analysis
• Modules that analyse
• Modules that undertake post-analytical processes
Preparation modules include sample input systems, centrifugation units, and tube decappers.
The introduction of samples into an automated system requires the specimens to be placed
into a rack that can then be loaded onto a sample manager or input module connected to
the track. The rack capacity varies depending on the design of the system and may be as low
as 10 or as high as 100. Most systems also offer the facility to introduce one or a group of
samples that can be given priority to ensure they have a faster turnaround time. The subse-
quent movement of the samples around the automation system may use pushing and pulling
mechanisms to position small racks of samples or more commonly robotic arms (Figure 17.4)
that can lift and convey individual specimens. The robotic arm usually moves the sample into
an individual specimen holder that is positioned on the track and is referred to as a carrier or
a puck. This carrier or puck moves around the track and will be directed to the modules and
analysers as required for each particular sample. As the specimen progresses around the track
the position of the tube may be monitored by re-reading the bar code at bar code reading sta-
tions at set intervals around the track. This enables a constant check to be made on the most
Sample input Sample output

storage
Centrifuge unit
Centrifuge unit Analyser 3
FIGURE 17.3 Analyser 1 Analyser 4

A schematic of an example tracking system
and layout. The modules and units for any Analyser 2 Analyser 5
given system will vary depending on local
laboratory requirements.
17.4 TR ACKED AUTOMATION SYSTEMS AND THE CORE AUTOMATED L ABOR ATORY 471
FIGURE 17.4
An example of a robotic arm placing a
sample onto a tracking system.
appropriate route for the specimen tube to take. Alternatively, each of the pucks/carriers can
be identified with a radio frequency tag that is monitored as it progresses around the track.
When the sample is placed into each puck an association is established between them and this
will be maintained until the sample is removed.
The majority of investigations on blood specimens require the plasma or serum component
of blood to be obtained for which a centrifugation step is required. This can be achieved with Cross reference
a centrifuge unit integrated into the automation system. The centrifuge unit will weigh speci-
centrifugation in Chapter 12.
men tubes and load them into centrifuge buckets in an even manner to achieve a balanced
load. The buckets are then placed into the centrifuge and spun for a defined period of time,
usually set between 8 and 10 minutes, with application of a force of approximately 1000g.
SELF-CHECK 17.4
How can the progress of a specimen tube on an automation system be followed?
The effect of the centrifugation is to bring the serum or plasma to the upper part of the col-
lection tube where it can be sampled. Following centrifugation, the buckets are lifted from
the centrifuge and the spun samples removed. Whilst some analysers have probes that can
penetrate the rubber caps of the tubes to remove the plasma/serum/blood samples, most
require the caps to be removed. Automated decappers provide this function and these can be
found in three general formats:
1. As part of a stand alone pre-analytical system
2. As part of an integrated centrifuge-decapper module
3. As a distinct device fitted onto a track system
The general movement of a decapper is a hold, twist, and lift movement. Once removed, the
caps are dropped into a waste receptacle. The waste receptacle may have varying levels of
sophistication to alert the operator to the requirement to periodically empty the waste con-
tainer. Once the cap has been removed the tube proceeds to the next stage of the automation
process. In some instances, it may be necessary to produce aliquots of the primary sample as
part of the standard automated process on the particular instrument, or because the speci-
men requires splitting for analysis across several analytical platforms. Sample aliquoting may
also be required for referral to another laboratory.
SELF-CHECK 17.5
How do automated decappers reduce the risk of musculoskeletal injury to staff ?
Each sample will be routed to one or more of the analysers connected to the track. In many
systems the sample remains on the track and the analyser sample probe will extend out and
over the track to a pre-set, defined position. The sampling probe will then be lowered until
it detects the surface of the liquid sample, it will progress beneath the surface to a preset
depth and liquid will be sampled and transferred into the analyser. This sample presentation
is sometimes called ‘point in space’ sampling. Alternatively, a robotic device can remove the
sample from the track and place it within a distinct and separate sampling area from which the
analyser can sample.
The organization of the analytical modules reflects the balance of testing across each of
the standard automated chemistry, immunoassay, haematology, coagulation, and urinalysis
analysers. Table 17.3 illustrates some common testing areas undertaken on such systems.
TABLE 17.3 Typical automated analyser units and examples of the associated test repertoire
Traditional Analyser unit Assays for:

discipline
Clinical General chemistry Sodium, potassium, chloride, bicarbonate, urea, creatinine, calcium, phosphate, albumin,
biochemistry total bilirubin, alanine transaminase, alkaline phosphatase, C-reactive protein, glucose, total
cholesterol, HDL cholesterol, triacylglycerol, uric acid, magnesium, total protein, creatine
kinase, aspartate transaminase, lactate dehydrogenase, gamma-glutamyl transferase,
amylase, lipase, iron, transferrin, conjugated bilirubin, lactate, paracetamol, salicylate,
ethanol, zinc, ammonia, bile acids, angiotensin converting enzyme
Immunoassay Thyroid stimulating hormone, total and free thyroxine, total and free tri-iodothyronine,
prostate specific antigen, alpha-feta protein, carcinoembryonic antigen, β–human
chorionic gonadotrophin, Ca-125, Ca 19-9, cortisol, testosterone, oestradiol, progesterone,
androstendione, follicle stimulating hormone, adrenocorticotrophin hormone, growth
hormone, insulin-like growth factor-1, drugs of abuse assays
Haematology Haematology
analyser Full blood count, red cell count, white cell count, haematocrit, haemoglobin
ESR analyser ESR
Slide maker Preparation of blood films
Immunoassay Vitamin B12, serum folate, red blood cell folate, erythropoetin
Coagulation Pro-thrombin time, activated pro-thrombin time, international normalized ratio, fibrinogen
Immunology Immunoassay Autoantibodies, allergens, complements C3 and C4, immunoglobulins
Virology Immunoassay Hepatitis A markers, hepatitis B markers, hepatitis C markers, HIV, CMV, rubella, syphillis
17.5 AUTOMATION IN WIDER L ABOR ATORY SET TINGS 473
Each of these individual analysers will generally have a large test capacity ranging from a few
hundred tests per hour through to several thousand. In Figure 17.3 the analysers may be the
same or any combination of analyser type. Further detailed descriptions of the attributes of
the individual analyser types can be found in the discipline-specific books in this series.
Post-analysis samples may be presented to a storage system. This may hold a variable number
of specimens, usually in numbered racks within which the specimen is allocated to a specific,
x y co-ordinate based position. The storage capacity may be as high as 15,000 samples and it
is sometimes refrigerated to reduce specimen evaporation and deterioration. Samples may be
re-accessed for further analysis if re-runs or additional tests are required and in these circum-
stances the samples may be retrieved manually or automatically reloaded onto the track, and
re-routed to the designated analyser.
Practical considerations for large automation systems include the requirements for services.
These include:
• Suitability of space, including the structural integrity of flooring as the systems may be
very heavy, walking space around the instrumentation, and access for routine use and
maintenance
• Water, which may be both a normal tap water quality requirement and/or de-ionized from
a deionization system
• Power supply – large tracking systems will require a specific, dedicated power supply. It is
standard practice to protect the power supply to each unit with an uninterruptible power
supply (UPS) in case of power failures
• Drainage of waste – the waste drainage is best achieved with floor drainage rather than pumps
• Information interchange – most automated systems have a complex array of computers

controlling different aspects of the system operation
Key Points
The majority of modules connected to a track will be conventional automated analysers
that utilize a particular technology suitable for the analyses being undertaken. The other
modules undertake sample processing functions, such as centrifugation, archiving, and
storage.
17.5Automation in wider
laboratory settings
The biomedical science disciplines most often associated with automation are clinical
biochemistry and laboratory haematology. However, all of the biomedical science disci-
plines, even those that have traditionally been associated with the manual skills and dexterity
of biomedical scientists, such as histology, cytology, and microbiology, now have significant
automation in use within clinical laboratories. The benefits of automation in any of the labo-
ratory areas mirror those detailed in Section 17.1. Many laboratory processes require a series
of independent manual steps and at each one there is potential for sample misidentification.
Automation of the more traditionally manual areas of biomedical science is often associated
with a reduction in the number of these manual steps at which this misidentification may occur.
This can therefore be seen as a significant improvement in patient safety, as it reduces labo-
ratory errors. The introduction of automation in any of the laboratory areas also requires a
re-assessment of laboratory operation. This often involves moving towards a laboratory work
flow that is continuous, rather than as a batch mode and may also involve laboratory opening
for longer periods of the day to maximize the benefits of automation.
In histology, several discrete stages of the preparation of histology slides have been auto-
mated. Automated tissue processors for preparation of tissues for sectioning, automated
Cross reference staining instruments, and automated cover slippers are all used routinely. The benefits of
You can read more about liquid these individual automated devices are consistency and efficiency. Linking each of these indi-
based cytology in the companion vidual discrete automated elements together into integrated single systems using robotics
volume Cytopathology in this
is developing. This type of automation is also an integral part of automated liquid based
book series.
cytology.
While virology has utilized immunoassay technology and analysers (as described earlier) since
the 1980s, the automation of bacteriology processes have only been evident since around 2005.
The application of clinical samples to growth media within plates, the allocation of the plates
to appropriate incubation conditions, and subsequent retrieval for visual assessment of colony
growth has conventionally been undertaken manually. Modular instrumentation (Figure 17.5)
can be used to automate these processes. Staff work at designated work stations where they
are presented with the pre-labelled plate sets for innoculation and sample application. Once
prepared the plates can then be conveyed automatically to incubators. After defined incuba-
tion periods the plates are automatically re-presented for review at which time point they will
also be photographed. This enables comparison of plate growth at different time points and
provides greater flexibility to when and where plates are read and interpreted.
FIGURE 17.5
The Kiestra system: an example of advanced automation in the bacteriology laboratory.
SUMMARY 475
Automated systems in blood transfusion laboratories can be used for the majority of rou-
tine testing including blood grouping and sub-grouping, antibody screening as well as cross-
matching. These systems can be highly reliable, sensitive, and specific.
In molecular diagnostics, the polymerase chain reaction (PCR) is a powerful analytical tech- Cross reference
nique with a wide range of clinical applications in infectious diseases, cancer and genetic dis- The PCR is described in
orders. The instrumentation used for PCR has a range of designs related to the thermocycler, Chapter 16 ‘Molecular biology
the assay features, the detection system and an increasing sophistication with respect to auto- techniques’.
mation. Real-time PCR, in which the amplified genetic material is quantified after each cycle,
is becoming a standard application. The automated instrument platforms have a growing rep-
ertoire of applications.
In several clinical laboratory areas there has been a high degree of professional expertise in
pattern recognition, for example in reviewing histology and blood slides or identification of
colony formation on a microbiological plate. The use of digital images in such disciplines can
be considered an aspect of automation and allows remote review of images. It is also possible
to develop pattern recognition software and this is an emerging field.
SELF-CHECK 17.6
Which of the following laboratory areas can benefit from automation?
1) Coagulation
2) Immunology
3) Microbiology
4) Virology
5) Haematology
SUMMARY
■ All laboratory disciplines utilize laboratory automation to improve efficiency of
processing.
■ The extent of laboratory automation use will depend on the size and complexity of the
clinical laboratory service.
■ Key aims of automation are process efficiency and consolidation.
■ Typical clinical laboratory workload growth of between 5 and 10% per annum has been
managed by increasing levels of automation, rather than by increasing staff numbers.
■ The design of modern automation systems aims to minimize human intervention in the
total analytical process.
■ The test turnaround times for large automated track systems are generally considered to
be faster and more consistent than separate analytical units.
■ Good design features within automation systems are maintained and copied by competi-
tors and, therefore, it is possible to see common features in a range of different suppliers’
instrumentation.
476 17 L ABORATORY AUTOMATION
■ Pre- and post-analytical processes such as centrifugation, decapping, and recapping of

specimen tubes, aliquoting, archiving, and storage of samples can be integrated into a
single automated process by using tracking systems.
■ Automation is prevalent well beyond the highly automated biomedical science areas, and
many microbiology, histology, cytology, and blood transfusion laboratories have a range
of automated instrumentation.
FURTHER READING
● Hawker CD. Laboratory automation: total and subtotal. Clinical and Laboratory
Medicine 2007; 27: 749–770.
● Hubl W, Zogbaum M, Boyd JC. et al. Modular analytics: A new approach to automa-
tion in the clinical laboratory. Journal of Automation Methods and Management in
Chemistry, 2005; 2005: 8–25.
● Melanson SEF, Lindeman NI, Jarolim P. Selecting automation for the clinical chemistry
laboratory. Archives of Pathology and Laboratory Medicine. 2007; 131: 1063–1069.
● Wheeler M. Overview of robotics in the laboratory. Annals of Clinical Biochemistry.

2007; 44: 209–218.
● Sarkkozi L, Simson E, Ramanathan L. The effects of total laboratory automation.

Clinical Chemistry, 2000; 46: 751–756.
The Journal of Automated Methods and Management in Chemistry is a publication that pro-
duces articles of relevance and can be found on the link: http://www.hindawi.com/journals/
jammc/ (accessed 20 February 2009).
Useful Website
■ www.labautomation.org/about/index.cfm
QUESTIONS
1. Why do blood specimens require centrifugation on an automated system?
2. Sodium fluoride is a suitable preservative for glucose because:
(a) It inhibits coagulation

(b) It promotes glycolysis
(c) It inhibits the formation of thrombin from pro-thrombin
(d) It inhibits glycolysis
(e) It promotes coagulation
3. What is the average test to request ratio for a laboratory that undertakes 606,531 requests
and 5,365,852 tests per annum.
QUESTIONS 477
4. How do automation units reduce the risk of infection to laboratory staff?
5. TRUE or FALSE? If a re-analysis of a test is required samples may be retrieved from the
automation system’s sample storage area.
6. TRUE or FALSE? The test turnaround time may be measured in two ways, the total turna-
round time and the within laboratory turnaround time.
7. The gels added to blood collection tubes to enable primary sampling have:
(a) A density higher than serum

(b) A density higher than plasma
(c) A density higher than red blood cells
(d) A density higher than a blood clot
(e) A density between serum and plasma
18
Point of care testing
Jan Still and Lynda Petley
Learning objectives
■ Describe the basis and regulations that underpin POCT
■ Discuss the requirements for POCT
■ Discuss the differences between internal quality control, external quality assessment, and
quality assurance in POCT
■ Outline the equipment procurement process
■ Describe the audit information required to minimize risk in the provision of a POCT
■ Discuss the provision of a patient focused POCT service
Introduction
Point of care testing (POCT) is the provision of a diagnostic pathology testing service outside
the traditional clinical laboratory setting and physically closer to the patient. This type of test-
ing may be carried out by healthcare professionals in the ward, clinic, or GP surgery, by high
street pharmacists, or by the patients themselves. This chapter describes point of care testing
and its impact on the provision of pathology services, and outlines those aspects that must be
considered when providing an effective POCT service.
Point of care testing is not a new concept, but earlier technologies paved the way for new
devices in response to the demand for a more patient centred health service. Thus, urine dipstick
testing has been an accepted practice in healthcare for many years and blood glucose meters
were first introduced in the 1970s. Technological advances over the years have widened both
the test repertoire and software capabilities available to POCT devices, with blood gas analyser
manufacturers leading the marketplace in terms of quality standards for POCT. Indeed, POCT
blood gas analysis is a well-established practice, having been developed in response to clinical
requirements. Modern POCT now consists of a variety of devices from simple hand-held meters
and test kits, some available for purchase and use by patients, to more sophisticated portable
and bench top analysers, which provide a variety of tests in all disciplines of pathology.
This chapter introduces the basic concept of rapid testing for the benefit of the patient at the
point of care. It illustrates how POCT is governed and regulated, and describes the requirements
18.1 STANDARDS AND GUIDELINES 479
for a well-managed, reliable POCT service, and how such a service can be established and
maintained. The training and competence of POCT practitioners is discussed, and the dangers
associated with a lack of or poor quality training are highlighted.
18.1 Standards and guidelines

Every area of pathology laboratory testing is subject to regulation, inspection, and accredita-
tion, including the POCT service. The aim of regulation is to ensure a safe and reliable serv-
ice, offering quality assured, consistent, and accurate results from in vitro diagnostic devices
(IVD) that are fit for purpose and used by suitably trained, competent individuals. The organi-
zation, management, and documentation of the POCT services are defined to ensure best
practice and the competency of users. Point of care testing requires separate or additional
requirements to the traditional types of analytical investigations because most or all of the
analytical processes take place away from the direct overview of the clinical laboratory. Both
standards and guidelines are necessary to ensure that POCT is performed to an appropriate
level of quality, thereby ensuring the safety of the patient. Standards define what must be
complied with, whereas guidelines are recommendations for best practice.
The European-wide directive, Guidance notes on In-vitro diagnostic medical devices directive
98/79/EC (applicable to member states of the EU and now also part of UK law) regulates all
POCT IVDs, including analysers, meters, single use kits, associated reagents, and consumables.
This directive was amended in February 2006 and deals specifically with the safety, quality,
and performance of IVDs on the European market. All manufacturers must meet the essential
requirements listed in Annex 1 of the directive to ensure that IVDs do not compromise the
health and safety of patients or users, and perform at the levels claimed by their manufactur-
ers. In vitro diagnostic devices that meet the requirements of the directive are denoted by the
display of the CE mark (Figure 18.1). It is a requirement for all POCT IVD devices used in com-
munity or clinic settings to have this mark.
Pathology laboratories are subject to inspection and subsequent accreditation to standards

produced by the International Organization for Standardization (ISO); its regulatory document
for clinical laboratories is ISO 15189:2003 Medical laboratories – Particular requirements for
quality and competence. A POCT specific adjunct to this document is ISO 22870:2006 enti-
tled Point of Care Testing Requirements for Quality and Competence, which is a comprehensive
guide to all aspects of POCT and how it should be organized as a quality assured, well man-
aged, and accurately documented service.
Key Points
ISO 22870:2006 is a standard applicable to clinical laboratories and is an adjunct to ISO
15189:2003. It is not possible to assess against ISO 22870 alone and it is not suitable for
use in community or clinic situations either.
FIGURE 18.1
The CE mark consists simply of the initials in the form shown. The marking on the device must
be clearly visible, legible, and indelible throughout the expected life of the device. For complex
products, the initials are followed by the identification number of the body involved in the control
of its production. All the letters and numbers must have substantially the same vertical dimensions
of not less than 5 mm. They may be in any colour provided it is legible and unlikely to be confused
with any other markings.
480 18 POINT OF CARE TESTING
Accreditation bodies incorporate the ISO requirements into their own standards for medical
laboratories. All UK laboratories are inspected to these standards every 4 years by the Clinical
Pathology Accreditation (CPA) UK Ltd now merged with United Kingdom Accreditation Service
(UKAS). If its standards are met, the laboratory will be issued with a Certificate of Accreditation.
This is a mark of quality, safety, and performance for that laboratory.
Any failure to achieve the necessary standards through deficiencies or non-compliances found
during a laboratory inspection will result in the laboratory being given a period of time in which to
rectify them. Continuing unresolved failures may result in the loss of or non-award of a Certificate
of Accreditation. This, in turn, may have an adverse effect on the laboratory’s ability to recruit and
retain professional staff or to provide its analytical services to users or other health care provid-
ers. A number of other professional organizations have produced their own guidelines, including:
the Institute for Biomedical Science (IBMS) Point of Care Testing Guidance on the Involvement of
the Clinical Laboratory (2004) and the Medicines and Healthcare products Regulatory Agency
(MHRA) Management and Use of In-vitro Diagnostic Point of Care Testing Devices DB 2002 (03).
Cross reference These guidelines, like the ISO standards, are intended for laboratory use but the MHRA has
More detail about accreditation also produced more informal leaflets to guide POCT users, such as MHRA Point of Care Testing
and international standards can Top Ten Tips. These cover tests such as those to estimate the concentrations of glucose and
be found in Chapter 19 ‘Quality cholesterol in blood samples and for urinalysis, and are useful for practice nurses and other
non-laboratory based users.
18.2Advantages and limitations

of point of care testing
From the advent of medicine, successful clinical outcomes have relied upon accurate diag-
nosis followed by appropriate intervention. Consequently, laboratory services have become
more centralized in recent years to help increase efficiency by economies of scale. This has
resulted in clinical laboratory testing often being remote from the clinician and patients, which
may lengthen the turn around times for some important or urgent tests.
Advantages of POCT
Point of care testing can redress many of the problems associated with traditional testing in the
clinical laboratory. For example, the clinician can obtain pathology results during the consulta-
tion. Having access to the information required to make a diagnosis, or change the manage-
ment of the patient during the consultation offers many benefits, which we will discuss in the
rest of this section.
Cost benefits
The cost per test in POCT is often higher than when the same analysis is conducted in a labo-
ratory. However, the impact of a rapid result may offset this extra unit cost. The following list
describes some of the financial benefits that can be achieved by POCT:
• Timely interventions improve patient outcomes, potentially reducing the length of stay as
an inpatient
• It can reduce the inappropriate administration of antibiotics and other prescription drugs,
and the use of blood products
18.2 ADVANTAGES AND LIMITATIONS OF POINT OF CARE TESTING 481
• Point of care testing can determine a patient’s immune state and help decide whether
expensive drugs require purchasing. The efficient and timely management of chemother-
apy drugs eliminates wastage
• Individuals who are less experienced can competently perform POCT, enhancing their role
and releasing other professional staff for more appropriate duties
• The enhanced disease management benefits the patient and reduces costly complications
• Over the counter testing kits, for example, fertility monitoring kits and pregnancy testing
kits cut costs associated with their provision by the health service
• Diagnostic testing carried out on finger prick capillary blood samples provides a cost saving
as trained phlebotomists, syringes, sample tubes, and pathology request forms are not
needed
• The use of POCT devices in, for example, the prison service may reduce the need for costly
secure transport of prisoners to the local hospital for treatment, ensuring that only those
with a genuine need are referred
• Travel and parking costs for patients are reduced. With POCT both the test and the visit to
the clinician to review results can be completed in a single visit
Estate management
The use of POCT has implications for the management of hospital and human resources, and
affects the strategic management of many hospital departments. Point of care testing can affect
the whole patient experience where pathology results may be a rate-limiting factor. This contrib-
utes to the efficient use of bed space and clinician time, and can help shorten waiting lists.
In outpatient departments, the use of a ‘One Stop Shop’ approach reduces repeat visits, mini-
mizes the flow of people visiting the hospital site (see Case Study 18.1). Fewer visits may also
reduce cross-infections as patient contact is reduced.
Point of care devices sited within the hospital reduce the requirements for transportation of
samples to the laboratory. They also reduce pressure on the clinical laboratory through work-
load reductions and can provide some temporary ‘back up’ service in the event of labora-
tory analyser failure. The introduction of local units that use POCT in satellite laboratories can
reduce Accident and Emergency (A&E) Department admissions. This enables patient triage
and observation to be performed in a community hospital or ‘polyclinic’ setting.
CASE STUDY 18.1 Appointments
An audit of average turnaround times at a district general Prior to the implementation of POCT, the patient was
hospital showed that the time taken from a patient booking required to make two visits to the Outpatient dept – one for
in at the Outpatient Haematology Clinic to his or her full the blood test, and a second follow-up appointment with
blood count result being available from the POCT analyser the consultant for the results.
was eight minutes. This included taking the blood sample
(a) What impact could this have for the patient?
from the patient and delivering the result to the consultant.
Had this test been carried out in the laboratory, the turna- (b) What are the possible benefits to hospital staff?
round time would include transport of the specimen to the
laboratory, booking it in, processing the sample, testing the
sample, and authorizing the result.
Management of acute conditions

The management of rapid onset or acute conditions often involves the use of ‘bench top’ POCT
analysers in a ‘rapid response laboratory’ setting. In these situations, the test results give a base
line from which the clinician can monitor, refer, or discharge the patient. Diagnostic testing is
also performed by the healthcare professional on the sample that they obtained, reducing the
frequency of samples becoming lost or the result being attributed to the wrong patient. This
assumes correct, relevant information on the sample is correctly entered into the analyser.
Smaller sample volumes are required for POCT compared with conventional laboratory sam-
ples. This is better for the patient as it minimizes blood loss related to phlebotomy. Furthermore,
the analysis of fresh samples reduces inaccuracies caused by sample deterioration.
Management of chronic conditions

The management of longer-term or chronic conditions does not require the same level of com-
plexity of devices as required in the acute setting. In chronic conditions, both diagnosis and
prognosis are already established, and so only appropriate tests required to manage the patient’s
condition are necessary. However, the accurate monitoring of chronic diseases enables better
Cross reference
treatment and reduces the burden of complications caused by poor patient management.
The management of the chronic
disease, diabetes mellitus is The patients may be able to monitor themselves at home using a POCT device, increasing their
described in Chapter 13 of the understanding of their own disease and giving them more responsibility in their own disease
accompanying volume, Clinical
management.
Biochemistry.
Chronic conditions can also be monitored in a GP surgery, health centre, or even local phar-
macy, releasing hospital resources for the diagnosis and treatment of acute conditions or
patients.
Patient satisfaction
Patient satisfaction may be often overlooked in the design of health service provision but must
always be a major consideration. The provision of an efficient POCT service, delivered at the
right time in an appropriate setting improves the patient experience. Test results provided dur-
ing the consultation reduce worry and stress of patients who are likely to be concerned about
their condition by allowing them to ask about the implications of their results and to make
better informed lifestyle choices. Patient experiences are illustrated in Case Study 18.2.
Meeting government directives

The NHS throughout the UK is expected to be cost-effective and may be required to achieve
government-defined health promotion or prevention targets. For example, in England, patients
presenting at A&E departments must be treated within 4 hours of arrival. Point of care testing
is of obvious help in this situation by offering a rapid pathology diagnostic service within the
department. Other targets include the 18-week pathway, where the patient can expect to wait
no longer than 18 weeks from referral to the start of their treatment. While the laboratory
may provide an efficient service, any requirement for laboratory testing will result in repeat
appointments with the clinician guiding the treatment plan. The use of POCT may eliminate
the need for repeat appointments and so speed the patient progress.
Recent trends require the primary care sector to offer outpatient services to relieve the bur-
den on secondary care (hospital) departments. This has necessitated a different approach in
the provision of pathology services and POCT may prove a more effective way of providing
test results.
18.2 ADVANTAGES AND LIMITATIONS OF POINT OF CARE TESTING 483
CASE STUDY 18.2 POCT: the patients’ Cross reference
perspective Arterial blood sampling is

described in Chapter 7 ‘Samples
and sample collection’.
The use of portable blood gas analysers allows respiratory practitioner nurses to meas-
ure blood gases in a patient’s home. The following comments are from two patients who
experienced this form of POCT.
Patient Mrs P. I am really grateful that I don’t have to have the needle in my wrist for
arterial blood. The nurses take a small sample of blood from my ear, which is not at all
painful, and are able to tell me the result in 2 minutes. If I am unlucky enough to need to
be admitted to hospital I dread the arterial stab – it is very traumatic for me.
Patient Mr R. It would be very difficult for me to get to the hospital to the oxygen clinic.
I used to attend, but found that the worry about being on time made my breathlessness
worse. The nurses always told me not to worry, but I did. They now visit me at home
with the portable analyser and can let me know the result at once without the need to
attend the hospital.
SELF-CHECK 18.1
Indicate which of the following statements are true or are false.
(a) POCT is useful in managing chronic and acute conditions.
(b) Patients do not like point of care testing.
(c) Patients must not perform their own self-checks.
Limitations of point of care testing

Analysis using POCT is often seen as a quick and easy alternative to standard clinical laboratory
testing. However, a report from the British In-Vitro Diagnostics Agency (BIVDA) in 1997 found
that hospital managers were twice as confident in the usefulness of POCT as clinicians. This
may be because managers concentrate on factors such as rapid turn around times and lower
costs, whilst clinicians have a much better understanding of what can go wrong in practice.
A POCT solution may be sought when the problem is really about issues such as transport to
the laboratory, or speed in obtaining test results. If these difficulties are addressed, then the
laboratory could, in fact, be the most cost efficient, as well as reliable option. There are many
potential and actual limitations in POCT, of which some are readily apparent, but others may
only become evident in practice. Where POCT devices are in use, safeguards and protocols
must be rigorously adhered to in order to maintain the reliability of results and ensure patient
safety. The clinical laboratory must always be used for the confirmation of unexpected or
spurious results.
If a particular POCT test becomes commercially available, there must be a clear clinical need
that its use, rather than continuing with the laboratory-based analysis, will benefit the patient.
There is little advantage in producing a test result in minutes if a doctor will not see it in hours
or even days. Easy availability may result in ‘over-testing’ and duplication of effort by continu-
ing requests to the laboratory for the same test, resulting in overall increased costs. Infrequent
use of a POCT device will result in increased user error and wastage.
Before introducing any POCT device, consideration must be given as to who is going to do
the testing, where and when. Nursing or clinical staff must have the necessary time to do the
analysis and documentation, or patient care may be compromised. Staff will require training,
competency checks, and annual update training. This all takes time and resources. Siting of
the equipment must be considered from a health and safety perspective and for the conven-
ience of the user. Maintenance and troubleshooting of equipment may divert laboratory staff
from other duties, while poor maintenance may result in breakdowns, inaccurate results, and
increase the risks of infections. Equipment may be damaged by misuse, especially if unauthor-
ized users can gain access to it.
Unless staff complete written logbooks or data is automatically downloaded from the equip-
ment, information may be lost. Thus, there may be a lack of a permanent record of POCT
results. Users may not be correctly identified due to personal passwords being stolen or simply
passed on to others. Errors in transcription may occur when test results are written into the
patient’s notes, and printouts may become damaged, lost, or degrade with time. There is often
a lack of traceability: staff in a hurry or pressed for time may omit entering patient identifiers
into the analyser so that reports cannot be traced to the correct patient. Reports and printouts
that are identifiable may be left in view, leading to issues of patient confidentiality.
When a POCT result is obtained, there may be a lack of ‘thinking time’ for consideration of that
result and for reflection on further action, which may lead to rushed and inappropriate deci-
sions. This can have serious consequences for the patient, such as a misdiagnosis, which may
result in litigation (see Section 18.11).
The main source of error with any item of POCT equipment is, quite simply, the user. Table 18.1
shows some of the most frequently encountered errors in POCT. Constant observation and
the monitoring of all POCT devices is necessary to avoid these errors and ensure that POCT
provides a safe and cost effective addition to the laboratory service. Further information on
the use of standard operating procedures (SOPs) for POCT may be found in Section 18.9, but
Case Study 18.3 describes what may happen when an SOP is not followed and the potential
outcomes for a patient.
SELF-CHECK 18.2
State the major advantage(s) of using POCT.
POCT committee and

18.3
POCT policy
The POCT committee and POCT policy provide the cornerstones for the POCT service.
Establishing a multi-disciplinary POCT committee will help ensure that all POCT processes
are compliant with the Trust’s own POCT policy and with national guidelines, and ensures
good clinical governance. The committee also ensures effective management in control-
ling POCT staff training and the use of resources, and so contributes to the production of
reliable results that will benefit the patient. The committee must be accountable to the
Trust Board, and maintain links with other relevant committees. Indeed, there may already
be an established committee, such as a Medical Devices Committee, or Medical Equipment
and Procurement Committee, whose aims and interests are broadly similar and relevant.
It may prove effective to incorporate POCT into this existing structure, cutting down on
18.3 POCT COMMIT TEE AND POCT POLICY 485
TABLE 18.1 Sources of error in point of care testing
Stage Error
• Pre-analytical • Wrong patient
• Wrong type of sample container
• Wrong type of sample (urine or blood?)
• Wrong time for sampling (fasting? pre- or post-dose for drugs)
• Poor sampling techniques resulting in poor quality samples affected
by factors such as haemolysis or the introduction of air bubbles in
blood gas samples
• Delays in analysis and failure to mix samples
• Analytical • Failure to follow instructions or standard operating procedure (SOP)

• Lack of training
• Lack of maintenance or servicing
• Failure to carry out quality control (QC) or appreciate when
it is incorrect
• Failure of calibration
• Failure to identify the patient on the analyser
• Failure to understand error codes or warning messages
• Post-analytical • Failure to check unexpected results with the laboratory

• Failure to understand ‘out of range’ flags or implausible results
• Failure to consult reference ranges or making inappropriate
assumptions about them
• Entering results into the wrong patient’s notes
• Transcription errors when entering results into patient’s notes
• Clinical decisions made on poor quality or incorrect results
• Delay in acting on results
• Loss of results
CASE STUDY 18.3 POCT and SOPs
A 27-year-old female presented to an A&E department The nurse carrying out the pregnancy test had not received
with severe abdominal pain. She was not taking any form training and had not followed the SOP. A false negative
of contraception. However, she stated her periods were result was obtained because too much urine was used and
often irregular. A urine pregnancy test was negative. She had flooded the test kit.
was treated for mild food poisoning and discharged. The
(a) Could this situation have been avoided?
patient was readmitted as an emergency the following day
with a ruptured ectopic pregnancy. She underwent sur- (b) Is this case an example of a pre-analytical error?
gery, received massive blood transfusions, and spent weeks
in intensive care. She later sued for damages.
the number of separate meetings and combining established committee members with
new recruits. A typical organizational chart for POCT clinical governance resembles that
shown in Figure 18.2.
Clinical laboratories have key functions in the promotion, management, and regulation of a POCT
service. The initial impetus for setting up a POCT committee may come with the appointment
of a POCT manager or co-ordinator, or quality manager. This person is usually a senior labora-
tory scientist, and will be the driving force behind the service. The committee should meet on
a regular basis and must be as multi-disciplinary as possible. This allows for shared knowledge
and experience, and a wide cross-section of opinions. The committee will need a chairperson
who is committed to the concept of POCT and willing to be its figurehead. This person must
have authority, respect, and drive. A senior clinician or pathologist may be ideally suited for this
role. Other members must include the POCT co-ordinator, quality manager, and representatives
from each of the pathology disciplines. Clinical representatives from surgical and medical direc-
torates, as well as nursing and midwifery staff will involve those most likely to be active users of
POCT devices in wards and departments. Given that the committee may make decisions regard-
ing the purchase or replacement of POCT devices, a representative from Finance to give advice
on economic considerations and funding of the service can be helpful. A committee member
from the NHS Logistics Department to supply information when selecting and acquiring devices
is also useful. Pharmacists who are involved in the purchase of relevant items, such as blood
glucose strips, pregnancy tests, and also in their storage and issue to wards should also be invited
members.
Clinical governance and clinical risk departments should also be represented on the POCT
committee and an advisor on training is desirable. Devices may require repair and mainte-
nance, and laboratory staff and biomedical and clinical engineering departments will have
relevant input and experience. Valuable insights may come from patient representatives, and
if possible, general practitioners, and community nurses. The committee should aim to reflect
Trust executive
board
Finance Risk management Clinical governance Quality management

committee committee committee committee
Clinical audit Medical devices

committee committee
Multi-disciplinary
POCT committee
POCT co-ordinator
FIGURE 18.2
Clinical governance and
organizational chart for Local POCT team Local POCT team Local POCT team
point of care testing.
18.3 POCT COMMIT TEE AND POCT POLICY 487
the interests of all POCT service users. Secretarial support will be required for taking minutes
and preparing agenda items.
Once the committee is appointed, it must agree and develop draft terms of reference. These
will define the clinical governance, in terms of scope and responsibility of the committee; how
it is constituted; accountability and direction; and how the committee relates to the healthcare
trust structure.
Once formed, the committee must now develop and publish a trust-wide POCT policy with
regard to the elements contained in the standards and guidelines, which we discussed in
Section 18.1. The policy must be robust, concise and comprehensive, and provide clear guid-
ance for users. It is recommended that the policy and the minutes be published electronically
and be easily accessible. The policy should be subject to an annual review, with an updated
version produced at least every three years. The policy must be ratified by the trust board and
be accessible both in hard copy and electronically by all staff. The policy should define the
organization, management, and funding for POCT, and include the extent to which patient
self-testing is supported. The document should refer to European and national legislation, and
to the accreditation standards of the laboratory and the trust.
The procedures and required documentation for selecting and procuring equipment, including the
submission of a detailed business case, must be clear and relevant (Figure 18.3). These documents
should show that consideration has been given to the processes involved in the ordering, supply
Week 1 Establish project plan, prepare and

post advertisement on OJEU
(open for 37 days)
Produce tender document and

specification, include means of
evaluation
Prepare and issue pre-qualification

questionnaire (PQQ)
Receive PQQ shortlist and issue

invitation to tender documents
(out for minimum of 40 days)
Tenders received,
round 1 short-listing processed
Carry out evaluations before final

short-listing
Issue lease tender

(allow 40 days)
Receive leasing tenders

FIGURE 18.3
Issue intent to award Flow chart summarizing the
(10 day standstill period applies) steps involved in approving the
commissioning of a new point
Completion of award, including of care testing device. OJEU, Official
Week 36 posting of award into OJEU Journal of the European Union.
and storage of reagents and other consumables, and the cost of both internal quality control (IQC)
and external quality assessment (EQA). The POCT committee should ensure that procedures are in
place for the safe location and decontamination of equipment. Point of care testing devices must
not be purchased, loaned, or installed without prior approval by the POCT committee.
Quality objectives are necessary to ensure correct performance and to maintain safety stand-
ards, and these must be regularly evaluated and reviewed. The POCT committee must have
the authority to act in the event of poor performance or similar risk issues, including unau-
thorized use, password theft, or wilful misuse. Action may include withdrawal of the service, or
the recommendation of disciplinary action for serious or repeated breaches.
Training objectives and methods of assessing competency must include the provision of stand-
ard operating procedures (SOPs) for each POCT IVD or test, including risk assessments. You
can read more about some aspects of training in Section 18.8. The policy must define the vari-
ous staff grades authorized to carry out the POCT procedure, and detail their responsibilities.
The policy must define how records of all POCT results are kept and stored. This may include
electronic transmission from POCT sites direct to an electronic patient record. Such connectiv-
ity must be available for all POCT IVDs. Connectivity is discussed in more detail in Section 18.7.
Finally, mechanisms for peer review and shared experience must be defined, with an annual
review report of the POCT service provided for the trust board.
SELF-CHECK 18.3
Which of the following records would be essential when preparing for a forthcoming accredi-
tation inspection of the POCT service by CPA?
(a) Staff sickness records

(b) POCT Policy
(c) POCT Committee minutes
(d) Laboratory SOPs
(e) POCT SOPs
18.4 Procurement
The procurement of POCT devices is best managed by the clinical laboratory in co-ordination with
the supplies department, once the source of funding has been agreed. Funding could be from a
specified budget or from specific capital funds, charitable funds, or as part of a defined project.
Procurement must be undertaken in accordance with the hospital trust policy and procedures for
the procurement of equipment and services, and the process must also comply with the hospital
trust standing financial instructions, public procurement law, and European Union legislation.
Compliance with all the procedures and legislation ensures that best value for money is
achieved and procurement is in line with the trust’s strategic and business plans.
The guidelines for purchasing equipment were discussed in Section 18.3. Before the decision
to procure any POCT equipment is taken, the POCT manager must undertake the following:
• A cost benefit analysis
• A patient benefit analysis

• An equipment evaluation
• A connectivity evaluation
• Site evaluation.
18.5 EVALUATING A POCT DEVICE 489
Cost benefit analysis is a technique for deciding the economical outcome of a change of prac-
tice. It is derived from the sum of the costs of an action subtracted from a sum of the benefits.
While the costs are usually simple to identify, the benefits of moving a test from the clinical
laboratory to the point of care can be subjective and difficult to forecast, as the impact of such
changes are often far reaching. Patient benefit analysis, as the term implies, is an assessment of
the impact that a change has in practice on patient outcomes. The analysis must consider the
effect on the whole patient experience, as small alterations in the patient pathway may have
unforeseen negative effects. Speeding the patient through one area may cause a bottleneck in
another, if this has not also been improved by similar efficiencies.
An evaluation of equipment for POCT use is described in Section 18.5. However, in addi-
tion to assessing the POCT device itself, the stability, and credibility of the supplier must also
be examined to ensure the long-term viability of the support, maintenance, and availability
of consumables throughout its life. Connectivity evaluation requires the involvement of the
hospital information technology (IT) department from the outset in the purchase of any POCT
device. This is necessary to ensure that the hospital network can support any IT solutions
offered by the manufacturers. Health and safety aspects must be included in the site evalua-
tion (see Section 18.6), hence, it can be useful to invite the laboratory health and safety officer
to visit and offer advice prior to any purchase.
The results gathered from all the evaluations must answer the following questions before pro-
ceeding with the purchase of the POCT device:
• How much extra cost will be incurred during the lifetime of the new device?
• What will be the effects on the laboratory, the POCT service provision, and the staff involved?
• What will be its impact on the patient experience and outcome?
• Will the new technology offer a safe and reliable service?
• Is a complete audit trail in place that provides information when required?
• Is there an appropriate infrastructure and back-up from the provider?
The results from these analyses and evaluations can be used to formulate a business case to iden-
tify the requirement for the goods. A robust business case must include a service level agreement
that outlines the support offered by the clinical laboratory. This will vary according to resources
and service needs, but should include a series of core requirements so that the evaluation of the
whole business case can be considered. The roles of the laboratory will include the maintenance,
quality control (QC), and assessment of the device, and include the training and competency
assessment of staff expected to use it. The financial information to be considered should include
the funding stream for the device(s) including delivery, the costs of consumables over its lifetime,
maintenance and service contracts (see Section 18.6), expenses associated with training staff, the
disposal of clinical waste, and a depreciation and replacement strategy.
Case Study 18.4 shows what may happen if POCT devices are procured without taking into
account all of the factors that a POCT committee or co-ordinator may consider.
All contracts from the public sector, which are valued above a defined threshold for the whole
lifetime costs, need to be advertised in the Official Journal of the European Union (OJEU) to
comply with EU legislation. Information on the process can be accessed from the website
http://www.ojec.com.
18.5 Evaluating a POCT device

Evaluations are required to ensure that any equipment purchased for POCT is fit for purpose
and suitable for safe and effective use at the location for which it is intended. All medical IVD
CASE STUDY 18.4 Grateful relatives
A female patient’s relatives wished to thank the ward for the care she has received
during a long hospital stay. The ward Sister asked them if they could raise the money
to purchase a full blood count analyser, as delays in receiving results from the clini-
cal laboratory had sometimes resulted in postponed treatments. The relatives managed
to raise the full cost of the instrument. No one discussed this purchase with the POCT
co-ordinator, clinical laboratory, or the consultant haematologists.
What are the potential disadvantages of purchasing a POCT instrument by this

procedure?
(a) The above scenario could result in which of the following:

i) An unsuitable instrument could be purchased
ii) Running costs could exceed budget
iii) Untrained staff could use the instrument
iv) Quality control and EQA could be overlooked
(b) How could the problem of slow turnaround time have been approached more
fruitfully?
kits and reagents in use within member states of the European Union now carry a CE mark
(Section 18.1) that indicates their performance has been tested and verified in accordance
with the claims of the manufacturer. This does not imply that a particular device will meet
the needs and requirements of day-to-day clinical use. It must be borne in mind that what
works beautifully in a manufacturer’s demonstration in a well-ordered laboratory may well be
unsuitable for a busy critical care area. It is essential to carry out a thorough evaluation of the
device prior to its purchase, to ensure that the users will have confidence in their ability to use
it in a safe and competent manner.
An initial evaluation of an IVD may have been undertaken by a national device evaluation
centre, such as the Centre for Evidence-based Purchasing (CEP), which is now part of the NHS
Purchasing and Supply Agency (PASA). Reports are free to NHS sites, and details can be found
at: http://www.pasa.nhs.uk/cep. If there has not been such a national evaluation of a device
one is interested in, then information on its performance may be found in a literature search
of peer-reviewed articles.
Evaluating the operation of POCT equipment

In a competitive tender situation, each proposed device should be placed in the POCT site
for a set period. This will enable users to familiarize themselves with the device, comment
upon its design, performance, and how well it fits their requirements. Planning is essential to
ensure that such trials take place with a minimum of delay, so that if more than one device is
under consideration, users can make valid comparisons between them. Staff must be trained by
the manufacturer to an agreed set of training objectives and allowed free access to the equip-
ment. Results obtained during trials should not, however, be used for clinical decision-making.
18.5 EVALUATING A POCT DEVICE 491
If the equipment under trial requires an additional blood sample to be taken from the patient,
then approval from the hospital ethics committee is required with informed patient consent.
This procedure is required because the additional duplicate sample will not be used for the
direct benefit of the patient, and he or she has the right not to have their blood used in such
a way.
Users should have access to evaluation forms that they can complete after using each piece
of equipment. They should be asked to score each separate device for overall ease of use.
This may include what sample types are required and how they are handled, and how pass-
words and patient identifiers are entered. The speed of use and time from obtaining a sample
to acquiring results can be assessed, as can the quality of reports and ease of data recall.
Instructions help menus and manuals must be clear and easily understandable. Quality con-
trol and maintenance procedures should be evaluated by staff undertaking them, and trouble-
shooting interventions should be concise and relevant.
Evaluation forms should be identical for each item and collated at the end of the trial. Laboratory
staff should also have access to the equipment, or a separate trial should be arranged within
the clinical laboratory. The preferences and comments of all concerned should be carefully
considered, to help avoid reaching unsound conclusions. It would be unwise to purchase any
item that none of the users liked, on the basis of other criteria, such as economy. Evaluations
can avoid expensive mistakes.
Diagnostic kits
Reports indicating the relative performances and costs may also be available for specific diag-
nostic kits; comparative reports are particularly useful in product selection. Having provision-
ally chosen a kit, the views, and preferences of their users must be sought with regard to
its ease of use and suitability for purpose. Potential sources of user error must be identified.
Considerations to take into account include the conditions for kit storage, the sizes of the
packs, possible wastage, and inevitably costs. It may be financially beneficial to consolidate Cross reference
the purchasing of diagnostic kits to a single supplier if possible. Both IQC and EQA are neces- Statistical analysis is covered
sary to ensure batch-to-batch conformity. Precision must be assessed by performing at least in more detail in Chapter 5
‘Statistics and handling data’.
20 replicate tests and calculating the coefficient of variation (CV) for the results, which should
be less than 5%.
Acceptance testing
Once delivered, the device must be checked by the appropriate department to ensure it
is electrically safe. Its acquisition must be entered into the asset register of the trust and it
requires a clear label. The supplier should install the device at its working site (Section 18.6)
and thoroughly check it to ensure its performance is as specified. Manufacturer defined qual-
ity control checks must be carried out and replicate tests performed on laboratory samples as
described above for diagnostic kits. An appropriate statistical analysis of the data is essential
to ensure that the results concur with those of the clinical laboratory instrument routinely
used in testing such samples. The acceptance forms must only be signed when everything is
satisfactory.
Evaluation is not a one-off procedure. Continuous evaluation through IQC, EQA, and audit is
necessary to ensure that the product performs satisfactorily throughout its life.
SELF-CHECK 18.4
You have just taken delivery of a new POCT analyser. Which of the following points would you
check to ensure that it was ready for placement in the ward?
(a) The electrical acceptance testing was acceptable.

(b) The ward clerk was aware that it was ready to be installed.
(c) All spares, consumables, and peripheral items associated with the analyser have been
delivered.
18.6 Use of POCT devices

The servicing, acquisition, and storage of spare parts for any POCT device should have been
considered during its procurement process in consultation with the POCT committee. Without
proper forethought and planning, additional problems may become apparent when the
device is implemented. Insufficient space, lack of adequate facilities such as light, ventilation,
and power, or an inability to store and provide spares can hamper the POCT service, cause
inefficiencies, and lead to waste.
Siting of equipment
Point of care testing equipment should be located in safe, designated sites, close to the clinical
area, and where the risks to the health and safety of patients and staff are minimal. The recom-
mendations of the manufacturer should act as a guide to the siting of all POCT appliances.
The site must conform to all Health and Safety, and Fire Safety regulations. There must be suf-
ficient space for the equipment and consumables, and room to carry out maintenance proce-
dures. Work surfaces should be at a convenient height and capable of supporting the weight of
the instrument. If it produces a significant output of heat, then air conditioning and ventilation
may be required. The equipment should be arranged in an effective manner with consumables,
documents including user manuals, spares, centrifuge, refrigerators or freezers readily to hand.
Adequate lighting, power supplies, and network ports must be provided. The mains supply should
be on a protected circuit and the equipment provided with an integral uninterruptible power
supply (UPS). Facilities for the disposal of wastes, including sharps, clinical, and non-clinical waste
are necessary. Hand washing and possibly eye washing provisions should be readily accessible.
These points must be considered prior to purchase, and included in any plans for new or
modified premises to prevent problems only being discovered later, when they may prove
costly and difficult to remedy.
Stock control
Appropriate stock control ensures that the POCT service does not fail through a lack of essen-
tial supplies, which are used economically preventing wastage. Commercial solutions are
available that use barcodes or chip recognition systems to track supplies and to re-order them
automatically. Supplies should be delivered to a secure central point for collection.
Immediately on arrival, new stock should be checked to ensure that it is as ordered, has been
delivered in the right quantity, and has a long shelf-life. Supplies must be stored in accordance
with the manufacturer’s instructions to prevent wastage. Thus, for example, there must be
18.6 USE OF POCT DEVICES 493
adequate freezer or refrigerator space for the volume of stock ordered; regular temperature
checks must be carried out and logged to ensure supplies are kept in optimum conditions.
Batch and lot numbers of stock should be recorded, together with details of when these are
brought into use. Stock rotation is necessary to ensure that reagents and consumables are
used before their expiry dates. Out-of-date stock, particularly reagents and diagnostic kits,
may deteriorate and produce invalid results. Never write on unopened packs because manu-
facturers will not accept the return of incorrect stock if the packages or boxes are marked. If
they must be marked, it is better to use removable stickers. Regular inventories are necessary
to ensure that the recorded levels of stock are accurate. It is all too easy to return an empty box
to the store and confuse the system!
Maintenance and servicing

All maintenance and servicing of POCT devices must be carried out in accordance with their
manufacturer’s instructions to ensure they remain in good working order. Lack of or poor
maintenance may result in incorrect results, safety risks, equipment failure, and invalidation
of the manufacturer’s warranty. All equipment should be entered into an asset register that
should detail all maintenance procedures and how frequently they must be carried out. The
manufacturer, supplier, laboratory staff, or clinical engineering staff may carry out mainte-
nance, and responsibility for it should be noted in the register. Maintenance logbooks should
be provided. These must be updated at each service or this information recorded electroni-
cally in the analyser itself. The logbook provides information about what has been done and
when, and thus helps pinpoint any recurrent problems. Any POCT device that does not per-
form to its specification must not be used until the problem has been rectified. Serious faults
may require the return of the equipment to the manufacturer for repair or replacement. It may
be necessary to withdraw the POCT service while such repairs are in progress, with the clinical
laboratory providing the necessary back-up service.
Users should know who to contact and how to report breakdowns. They should be aware of
alternative arrangements for dealing with samples whilst analysers are ‘down’. Manufacturers
should specify response times and frequency of service calls.
All newly purchased equipment has a warranty period, which is usually its first year of use. The
manufacturer must carry out servicing during the period of the warranty. Care must therefore be
taken not to invalidate the warranty by misuse, adaptation, or unauthorized interference with
the equipment. Service agreements should include provision of a backup instrument if required.
If equipment is required urgently, it may be possible to arrange an immediate exchange policy.
For small items, such as blood glucose meters, it is sensible to have a supply of spares in stock
for their rotation or for immediate use to replace defective instruments. This arrangement
can be stipulated in their procurement specification. However, it is not sensible to have spare
parts for large and expensive analysers, so any repairs must be swift and efficient. Returning
equipment to the manufacturer for repair may be expensive once the costs of the inspection
fee, spare parts, labour, and delivery are included. It is often economical to take out a service
contract for a fixed fee, which covers the costs of several repairs.
Items that are returned for repair require a decontamination certificate stating whether the Cross reference
equipment has been exposed to body fluids, infected material, or other biological or radio- Aspects of health and safety are
active hazards. It must also describe the decontamination process used to render it safe. covered in Chapter 4.
In-house repairs also require a decontamination certificate.
‘Repair and return’ requests must state the equipment type, its serial number, the date, the
site where it is used, the reason it requires repair, and a name and address to which it must
be returned when working. Unless the equipment is under warranty, the manufacturer may
require an order number to guarantee payment. Items must be securely packaged and dis-
patched as soon as possible. Valuable items should be sent via recorded delivery.
SELF-CHECK 18.5
If you find that reagents are not being stored appropriately on a regular basis, what could be
done to solve this recurring problem?
18.7 Connectivity
Connectivity facilitates data capture without the requirement for manual transcription. Hence,
data from POCT systems may be saved in the laboratory information system (LIS), in the Hospital
Information System (HIS) as part of the patient’s electronic record, or to ‘stand alone’ servers with
a robust back up system. However, prior to 2000, POCT connectivity had been unregulated and,
consequently, of variable quality. In January of that year, the Connectivity Industry Consortium
(CIC) was formed and devised a document describing the standards required for POCT connec-
tivity. This document formed the basis for the subsequent development of POCT connectivity
requirements, including plug-and-play facilities, which allow the bi-directional control of mul-
tiple devices using a single data capture platform. Bi-directionality is the transfer of information
from the analyser to the laboratory and back again. This allows the POCT co-ordinator to send
the same information to many devices, for example to update the set-up, or to a single device to
order a specific procedure, or lock a specific analyser that has developed a problem to prevent
its use until the fault has been rectified. The management of POCT devices is improved by access
to a bi-directional link to the devices, which allows the POCT co-ordinator to view the analyser
status and patient results in ‘real time’, and enables remote recalibration and QC. The ability to
view remotely the status of an analyser, including its ‘on board’ consumables and maintenance
schedules, reduces the time spent visiting POCT sites. Communication is central to the safe and
effective use of pathology results, particularly in the case of a remotely generated result. The
ability of a biomedical scientist to view and verify a result in real time offers the opportunity to
respond quickly to abnormal results, give advice to the clinician, and reduce risk to the patient
in the event of an error in analysis. Linking into the whole patient record allows for a continuous
and chronologically accurate record of any given test analysis. As we move towards a true elec-
tronic record of patient data, this collation of test results will be necessary for continued patient
care by those who access the records. Hospital specific networks, the NHS central system, the
Internet or other commercial packages all provide connectivity depending on the capabilities
of POCT devices. Manufacturers that currently offer connectivity for POCT devices design spe-
cific software that may allow access to devices from other manufacturers, but with limited func-
tionality. This is likely to change, as connectivity becomes more a requirement than an option.
Whichever solution is available, the hospital IT department will be involved in the maintenance
of the connection. It must also ensure that any connectivity package is compatible with any HIS
or LIS upgrades to guard against it becoming obsolete.
The cost of connectivity for POCT devices is often significant and, indeed, may be more than
the initial cost of the device. There may also be ongoing expenses for the maintenance or
licence of such links and this should always be identified in the ‘revenue costs’ part of the busi-
ness case (Section 18.3). However, the benefits of connectivity for any POCT device outweigh
the costs when the total service provided is considered.
Connectivity also provides an audit (Section 18.10) trail allowing the recall of patient results,
identification of the user, and verification of performance, lot numbers and expiry dates of
18.8 TR AINING FOR POCT 495
consumables, as well as continuing analyser status and intervention history. All of these data
are requirements of CPA and the NHS Litigation Authority (NHSLA). Audit data is required to
minimize risks to the patient. In addition, such data protects the hospital trust as patient mis-
management can lead to expensive litigation cases (Section 18.11). Although failures in IT and
information corruption may occur, the biggest challenge from connectivity to any POCT co-
ordinator is ensuring that correct patient details are entered by the user. Accurate identifiers are
essential to integrate POCT results with laboratory records for a given patient since the correct
patient information is necessary to recall results and demonstrate a quality assured patient
management system in a legal environment. However, incorrect or incomplete information
may enter the system because the patient details are simply unknown. This may happen in
a major incident, on the admission of an unconscious patient, or where senility or confusion
results in incorrect information being given by the patient. In can also happen when healthcare
staff obtain a sample and simply forget to take the patient’s details with them to the analyser.
Unauthorized users may also enter made-up patient identifiers in mandatory fields to obtain
results, bypassing the safeguards.
18.8 Training for POCT

Training and competency certification is the key to the success of any POCT service. The intro-
duction of any point of care test needs to be accompanied by a SOP. This SOP must be plainly
written and explain any scientific terminology and acronyms. The POCT co-ordinator, in con-
junction with the ward sister, practice development nurse, or hospital training department
must offer a comprehensive training package that covers:
• Pre-analytical techniques
• Analytical techniques
• Post-analytical actions
• Preventative actions
Pre-analytical techniques include assessing reagent requirements, such as their storage, stabil-
ity, and handling, the need to label samples and how to obtain samples of the necessary quality.
Substances that interfere with the analysis and invalidate the results should also be described. Cross reference
How to recognize device failure, and health and safety issues should also be included in the You can read more about
training package. Information about how the POCT device functions is an essential require- quality and quality assurance
in Chapter 19 ‘Quality assurance
ment of any training on analytical techniques. Quality issues should include QC and external
and management’.
quality assessment requirements (Section 18.9).
The operational ranges, device and analyte reference ranges, together with common user
errors must be highlighted. It may be pertinent to include examples of serious errors or the harm
caused by poor practices. Post-analytical instruction includes training in recording results, valida-
tion, and verification of results, and recognition of error codes. This is essential since corrective
actions are necessary if analytical results fall outside the range of the analyser or outside the refer-
ence limits set by the laboratory manager or clinician. Descriptions of the risk reduction aspects
of good record keeping are useful. The importance of compliance, an aspect of POC testing often
disregarded, is apparent when an audit trail is requested during, for example, litigation.
Preventative action in the event of an analytical error is the users responsibility, including
removing the device from use and alerting the laboratory. It is essential to emphasize to train-
ees that the results they generate are their responsibility. Results from any POCT device that
are inconsistent with the clinical signs and symptoms of the patient require checking by the
clinical laboratory before management or treatment of the patient is initiated.
Feedback information on a training session is a constructive method of auditing quality

and effectiveness. Unless trainees are observed performing POCT tests in a clinical setting,
it is not possible to regard them as competent. Competency can also be lost over time if
the test or technique is only performed irregularly. Competency is always the responsibil-
ity of the user. Although training update intervals may be fixed, the requirement for such
training is particular to the individual, and depends on how often he or she performs the
procedure and their ability to recall instructions. The use of eye-catching posters or short
form guides placed near the POCT analyser can be effective aide-mémoire. The over-use of
this resource, however, can result in the ‘wallpaper syndrome’ where users become blind
to information provided in this way. When designing a training programme it is useful to be
aware of the learning styles of the audience and include interactive practical demonstra-
tions wherever possible.
Monitoring the error logs of analysers can assist in pinpointing users who may require further
training. The POCT co-ordinator, when aware of an individual with questionable competency
(as illustrated in Case Study 18.5) is responsible for alerting the ward sister of the situation.
Incompetent users must be prevented from using the equipment until they are retrained and
are fully competent. However well trained the POCT team is in the use of their equipment, a
significant part of their duties is to be out and about in wards and departments, to help main-
tain contact with staff, answer questions and occasionally spot something untoward.
18.9 Quality and POCT

Quality assurance (QA) and its associated processes, quality control (QC), internal quality con-
trol (IQC), and external quality assessment (EQA) are all essential aspects of laboratory qual-
Cross reference ity management. They are perhaps even more important when considering POCT, as testing
You can read more about activities may take place outside of a laboratory and be conducted by other healthcare profes-
quality assurance and quality sionals or even the patient themselves. All these quality measures are designed to help ensure
management in Chapter 19 the accuracy of testing and to be certain that appropriate clinical action is taken on the basis
‘Quality assurance and of test results.
management’.
Effective quality assurance poses a challenge to the POCT co-ordinator, and is a principal
aspect of the role. The implementation of a system underpinned by adequate documentation
will cover:
• Standardization of methods
• Monitoring and maintenance of equipment
CASE STUDY 18.5 Incompetency
The POCT co-ordinator received a phone call from a trained nurse who complained that
a pregnancy testing kit was not working correctly. On investigation, it was found that the
nurse had filled the sample well with urine, rather than the specified amount as detailed
in the SOP.
What actions should be taken to assist someone whose competence is in doubt, as is the
case with this nurse?
18.9 QUALIT Y AND POCT 497
• Equipment downtime
• Equipment repair
• Audit of consumables
• Training and competency
• Quality control
• External quality assessment
The ISO 22870:2006 guidelines mentioned in Section 18.1 outlines a comprehensive approach
to QA.
All POCT devices must be subjected to QC as determined by the manufacturer. This must be
performed at regular device-specific intervals in order to monitor the basic functioning of the
device. There may be several levels of QC material available for devices, to assess performance
at low, normal, and high confidence levels. Quality control is designed to produce a result on
an instrument that can be compared with the recommended acceptable range. This involves
testing a solution or sample produced by the manufacturer of the instrument, stored accord-
ing to their instructions. The recommended ranges set by the manufacturers are often wide
to allow for batch-to-batch variations of consumables. An individual result falling within this
range may offer no indication of instrument bias or precision.
Analysers such as blood gas analysers, can be programmed to perform this type of QC auto-
matically from solutions stored in the analyser and can ‘lock out’ the device should it fail to
achieve the expected performance. This prevents any inappropriate clinical management of
the patient caused by inaccurate results and highlights the need for corrective maintenance of
the analyser. For devices where automatic QC is not available, the operator must perform QC
testing manually and take action in the event of its failure. These procedures must be recorded
as evidence of a robust response designed to prevent use of the device prior to corrective
maintenance.
Electronic quality control

Electronic QC involves challenging the POCT device (analyser) with an electronic stimulus
designed to produce a known measurable response from the detector. This method of QC has
the advantage of being integral in the performance of a measurement and is not user depend-
ent. Where available, the POCT device should be ‘quality controlled’ using this procedure prior
to use. The disadvantage of this method is that it may not mimic the analysis of a real sample Cross references
and so may not replicate the mechanical aspects of sample flow through the testing system. You can find out more about
Thus, the resulting QC data are qualitative, rather than quantitative and give the user no guid- the different types of data
ance as to the performance of the device in clinical use. in Chapter 5 ‘Statistics and
handling data’.
Accreditation is more fully

External quality assessment explained in Chapter 19 ‘Quality
External quality assessment (EQA) involves the analyses of identical samples, usually supplied by
an EQA scheme provider, to various sites that use the same POCT device. Collating the results
means the performances of individual analysers may be assessed. Information regarding the
accuracy and precision of a device within the distribution of results is a basis for any necessary
corrective actions. An EQA report is a retrospective ‘snapshot’ of device performance, which can
be used to proactively manage the device and highlight problems, or to indicate adverse trends.
Registration of POCT devices on a suitable EQA scheme is mandatory for CPA accreditation.
18.10 Audit
Regular audits are essential to assess the performance and clinical usefulness of all POCT
devices. CPA requires horizontal and vertical audit information (see, for example, Figure 19.4).
A vertical audit is a ‘single process, all aspects’ audit, such as the selection of a single test and
examining a range of parameters as listed in Table 18.2. In contrast, a horizontal audit selects
one element of the vertical audit, and expands the information relevant to it. Hence, it is a one
process, all aspects procedure. An example of a horizontal audit surrounding a maintenance
log may contain those points given in Table 18.2.
Accreditation will require the POCT co-ordinator to collate analyser workload figures and cost
per test, registers of training and equipment, logbooks listing any equipment replaced or peri-
ods when it was not functional (downtime logbook), and stock control information.
SELF-CHECK 18.6
Information about a POC test, such as how to analyse for blood glucose, could be provided
by which of the following?
a) Asking a user
b) Watch a user perform the test
c) Examine the record book of the device
d) Ask a patient
e) Check the patient records against the logbook
TABLE 18.2 Points to note when performing vertical and horizontal audits
Vertical audit Horizontal audit

Patient ID Device-specific training records
Date and time of test performance Competency records
Job description
Identity of user performing the test Individual’s contract
Analyser serial number Evidence of continuing professional

development (CPD) if appropriate to the
grade
Observe a test being performed
Lot number of consumables
Evidence of correct storage of consumables
Training record of user performing the test
Competency record of user performing the test
Analyser error log
Analyser maintenance log
Quality control records
External quality control records

18.11 PROBLEMS, INCIDENTS, AND LITIGATION 499
18.11Problems, incidents,
and litigation
A well-managed POCT service should ensure that competently trained users obtain high qual-
ity results that benefit the patient. However, errors and incidents will inevitably occur from
time to time. Such events must be investigated, remedied, documented thoroughly, and steps
taken to avoid any repetition. This enables users to learn from such mistakes or malfunctions
and to notify others of potential and actual risks.
Error logbooks
Fully completed error logbooks are used to record incidents, their seriousness, any remedial
actions taken, and when such actions have been completed. Each item of POCT equipment or
diagnostic kit must have its own logbook. Records must define the type of error, whether pre-
analytical, analytical, or post-analytical, and if attempts at corrective action were effective. Case
Study 18.6 shows an example of an error and the corrective action taken to prevent it happening
again. For serious errors, an incident form is required. A record must be made of notification to
the POCT committee, the Risk Management committee, or an external body, such as the MHRA.
The error logbook of a POCT analyser can be stored with its maintenance logbook but it may
be better to keep them centrally with the POCT co-ordinator. This enables recurrent errors to
be recognized and possible patterns of incidents or faults detected. These may identify a par-
ticular individual causing repeated errors, who requires retraining, or indicate that the siting or
practical use of the equipment is causing unforeseen problems.
Reporting of incidents
All NHS Trusts will have their own incident reporting forms, and ward staff may complete
these following incidents involving patients. Serious incidents that involve harm may require
CASE STUDY 18.6 When is total

haemoglobin, really total haemoglobin?
External quality assessment results from an analyser used to measure total haemoglobin
were found to be increasingly inaccurate when compared with results from the clinical
laboratory. An incident was reported where cross-matched blood had been requested
on a patient whose tHb was determined as 79 g dm–3 (below critical limits) using a POCT
analyser on the ward, but was found to be 141 g dm–3 (the reference range for men is
129–165 and for women 123–147 g dm–3) by the laboratory. An investigation showed
that the cuvettes used with the POCT instrument were placed on a shelf in direct sun-
light. The adversely high temperatures had caused them to deteriorate. Moving the
instrument and cuvettes away from the window to a site with a constant room tempera-
ture cured the problem.
(a) Could the patients have been harmed by this incident?

(b) How could this problem have been detected earlier?
a Reporting of Injuries, Diseases, and Dangerous Occurrences Regulations (RIDDOR) report,

Cross reference
a notification to the MHRA if the device was suspected to or known to be faulty, and to the
More information about health
and safety is found in Chapter 4. National Patient Safety Agency (NPSA). All such incidents must be recorded in the trust risk
register.
If the incident involved POCT as described in Case Study 18.7, then a copy of the report must
be forwarded to the POCT co-ordinator, and investigated to establish what went wrong and
how it might have been prevented. If equipment malfunctions and results were found to have
a clinical impact, then other users of similar POCT devices require immediate notification. The
manufacturer, MHRA, and NPSA must also be notified. If further investigation showed a risk of
harm or danger, then the MHRA may issue a Safety, Alert or Hazard notice to warn all possible
users of the problem. Each trust has a Safety Alert Broadcast System (SABS) which cascades the
safety notice to all possible users.
SELF-CHECK 18.7
(a) In the incident described in Case Study 18.7, whose responsibility was it to ensure this
nurse was competent? (b) Would this incident have been subject to a RIDDOR report?
Litigation
The primary concern of healthcare is always the safety of the patient. A robust and effective
system of error and incident reporting is necessary to minimize risk and maximize patient
protection. The risk of litigation is particularly relevant with regard to POCT, where unau-
thorized users may not be fully trained or competent, may fail to follow instructions, or may
adapt procedures without authorization. Trusts are insured against any adverse consequences
arising from litigation. Any that are able to demonstrate good practices in training, documen-
tation, and competence can obtain a reduction in their insurance premiums. This scheme is
the NHSLA for Risk Management Standards. It has three levels, each requiring the trust to
submit to a rigorous assessment. If the Trust meets these standards, it receives a considerable
reduction in its payments, with satisfying each level securing a greater reduction. The POCT
service can help the trust obtain these reductions by ensuring its policies and guidelines are
adhered to.
CASE STUDY 18.7 Whose fault?
An agency nurse was asked to monitor a diabetic patient who has been placed on an
insulin infusion prior to surgery. This routine procedure is necessary to stabilize the
concentration of glucose in the patient’s blood. The concentration of glucose in capil-
lary blood is measured using a glucose meter at hourly intervals. Although the nurse
was unfamiliar with the meter provided, she had used a number of different types
of glucose meters previously. For each test, she pressed the button to start the meter
and recorded the result. All results were found to be stable and within the reference
range. Unfortunately, the patient was later found in a hypoglycaemic coma and died
of complications.
Suggest a plausible explanation as to why the patient died.

18.11 PROBLEMS, INCIDENTS, AND LITIGATION 501
Human errors
User error is the major cause of incidents, which often arise from lack of training, misinterpre-
tation of instructions, or a failure to follow the relevant SOP. Making ill-considered assump-
tions on the capabilities of POCT devices, a poor understanding of the chemistry underlying
the test, or simply not thinking sufficiently about the procedures associated with it can all lead
to serious accidents. For example, blood gas analysers give a measurement of the pH of blood
samples. Some users extrapolate this to mean that the analyser is in fact a pH meter and is
able to determine the pH in almost any fluid. Even if the user manages to obtain a result on a
non-blood fluid, they will not have any idea of reference ranges, effects of dilutions, the limi-
tations of the analysis, or the presence of interfering substances. False assumptions about the
underlying chemistry of an analytical test may lead to incorrect conclusions.
The unauthorized purchases of POCT equipment can by-pass all the carefully constructed
safeguards, increase the risk of harming the patient and incur unforeseen costs. You can read
about some of the problems such actions may cause in Case Study 18.4. Constant watchful-
ness and awareness is necessary to pick up incorrect and appropriate practices, as illustrated in
Case Study 18.8. Quality assurance is unlikely to identify random errors, but regular, thorough
training and competency testing may eliminate most of them.
CASE STUDY 18.8 Blood in vomit?
Urinalysis strips are universal, simple, and easy to use. A consultant haematologist was
called to a patient who was alleged to be vomiting blood. He was shown the contents of
the vomit bowl and was unable to see any blood. The nurse was asked why she thought
the contents contained blood. She has used a urine dipstick and it showed positive for
blood. The patient was really suffering from an obstructed gastrointestinal tract (GIT)
and vomiting bile, which had stained the strip green. The positive colour for blood in
urinalysis is also green.
(a) Can urine strips be used to test other biological fluids?

(b) Could this error have led to an incorrect diagnosis?
SELF-CHECK 18.8
A POCT co-ordinator noticed three used urine drugs of abuse screening kits in a ward sluice
room. Such kits detect drug metabolites, rather than the drugs themselves. It appeared that
there were suspicions among the ward staff that a mother was adulterating her children’s milk,
orange juice, and water with her prescribed methadone. The screening kits had been used
to test the drinks.
Suggest the most plausible of the following as to why methadone would not have been
detected.
(a) There was insufficient methadone in the drinks.

(b) The drinks would interfere with the test.
(c) The nature of the kits.
SUMMARY
■ Point of care testing is the use of an analytical device for clinical testing outside the labora-
tory and close to the patient
■ Point of care testing is governed and regulated by European legislation and national
guidelines. An accreditation body conducts inspections according to these standards and
its own guidelines every four years. An accreditation certificate is awarded to POCT serv-
ices that comply fully with these standards
■ To achieve a well-managed and reliable POCT service, NHS Trusts must have their own
multi-disciplinary POCT committee, responsible for devising its own local POCT policy.
This defines the service, and how it is to be managed and organized, and how all matters
related to its services are to be documented
■ Training and competence are key elements in ensuring the safety of patients by performing
quality controls and external quality assessments of every IVD. Regular audit and monitor-
ing is necessary to ensure compliance and good performance
■ Point of care testing may be seen as a rapid process providing results almost immediately
and so speeding the diagnosis and treatment, it does have potential pitfalls in terms of
pre-analytical, analytical, and post analytical errors
■ Procuring a new IVD must be based on clinical need. Care is necessary in examining cost
benefits to the whole patient episode and not merely cost per test. Evaluations of new
devices, their acceptance testing, maintenance, and audit are part of the role of the POCT
co-ordinator to ensure the safety and continuing satisfactory performance of the device
■ Connectivity enables various devices to be monitored from a remote location, and cor-
rective actions to be initiated through a computer link. Connectivity benefits the POCT
service by allowing results to be downloaded to the electronic patient and laboratory
computer records
■ Errors and incidents must be recorded and reported to appropriate outside bodies where
necessary. Human error may result in patient harm and litigation
FURTHER READING
● Becker KM, Whyte JJ. Clinical Evaluation of Medical Devices: Principles and Case
Studies, 2nd edn. Humana, New Jersey, 2005.
All that is necessary to know to evaluate a POCT device. The case studies are particularly
useful in avoiding pitfalls and unexpected outcomes.
● Bubner TK, Laurence CO, Gialamas A, Yelland LN, Ryan P, Willson KJ, Tideman P,
Worley P, Beilby JJ. Effectiveness of point-of-care testing for therapeutic control of
chronic conditions; results from the POCT in General Practice Trial. Medical Journal
of Australia, 2009; 190: 624–626.
This short paper summarizes the first full randomized controlled trial of the clinical effec-
tiveness of POCT in Australian general practice. It is an encouraging endorsement of some
POCT tests with an interesting approach.
QUESTIONS 503
● Nichols JH, Christenson RH, Clarke W, Gronowski A, Hammett-Stabler CA, Jacobs

E, Kazmierczak S, Lewandrowski K, Price C, Sacks DB, Sautter RL, Shipp G, Sokoll L,
Watson ID, Winter W, Zucker ML. Executive Summary. The National Academy of
Clinical Biochemistry Laboratory Medicine Practice Guidelines: Evidence based
practice for Point of Care Testing. Clinica Chimica Acta, 2007; 379: 14–28.
Excellent discussion of what makes an effective application of POCT, some more US based
but a good overview.
● Price CP, Hicks JM. (eds) Point-of-Care Testing, 2nd edition. AACC Press, Washington,
2004.
This detailed and comprehensive work includes chapters on instrumentation, methodol-
ogy, and POCT in primary care. It requires some prior knowledge but is an excellent source
on all aspects for those wishing to enquire further.
● Price CP, St John A. (eds) Point-of-Care Testing for Managers and Policymakers: From
Rapid Testing to Better Outcomes. AACC Press, Washington, 2006.
A work intended for those in management and government and not an easy read but a
useful overview from a manager’s perspective.
● Westgard JO. Basic QC Practices, 2nd edn. AACC Press, Washington, 2002.
Written by the foremost authority on quality control and the originator of ‘Westgard Rules’,
this work takes the reader from simple basics to more complex statistics.
Useful Websites
■ http://www.wales.nhs.uk/documents/PFSD-August-2008-Ebook.pdf
■ http://www.acbi.ie/downloads/guidelines-for-point-of-care-testing.pdf
■ The Institute for Biomedical Science (2000) Point of Care Testing Guidance on the
Involvement of the Clinical Laboratory. Document Ref PR/07.04. Available at: http://www.
ibms.org
■ The Medicines and Healthcare Products Regulatory Agency (2002) Management and Use
of In-vitro Diagnostic Point of Care Testing Devices DB 2002 (03). Available at http://www.
mhra.gov.uk
■ Guidelines on Point of Care Testing (2004) Royal College of Pathologists. Available at
http://www.rcpath.org
■ ISO 15189: 2003 Medical Laboratories – Particular requirements for quality and competence
■ ISO 22870: 2006 Point of Care Testing (POCT) – Requirements of quality and competence.
QUESTIONS
1. Which of the following records is/are necessary to prepare for an accreditation inspection
of a POCT service by CPA?
(a) Staff sickness records

(b) POCT policy
(c) POCT Committee minutes
(d) Laboratory SOPs

(e) POCT SOPs
2. You have just taken delivery of a new POCT analyser. Which of the following may be
involved in installing the device?
(a) Porters
(b) Clinical engineering
(c) Infection control
(d) Risk management
(e) Cleaners
3. You find several boxes of reagents have been delivered. Although marked Store at 2 to 4 oC,
they have been left in a warm room for at least a day. Which of the following is the most
appropriate action?
(a) Discard all the reagents immediately

(b) Perform a quality control check on a sample of the reagent, having refrigerated the
remainder
(c) Inform the supplier and request them to replace the reagents
4. (a) How would you establish which POCT devices are in use in your hospital?
(b) In which areas would you be most likely to find such devices?
5. How would you deal with any unauthorized or unexpected devices found? What actions
could you consider?
6. You find that a particular user has been responsible for several instances of damage to
devices, has a poor record of training, and refuses to take part in the EQA scheme. What
actions do you take?
7. A Consultant surgeon has asked for a POCT device to be placed in a theatre. The same
test can be done in the laboratory in less than 30 minutes. How would you establish
whether there was a case of clinical need and what other factors should you consider
before procurement?
19
Quality assurance
and management
Elaine Moore
Learning objectives
■ Describe the features of a quality management system

■ Explain the general principles underlying quality assurance and quality control
■ Discuss the differences between clinical sensitivity and specificity
■ Outline the different types of documentation found in the medical laboratory
■ Outline the standards and regulatory requirements that affect the work of the laboratory
■ Define and use tools and techniques required for the continual improvement of quality
assurance and management
Introduction
Quality and quality assurance (QA) are the processes and practices undertaken by a labora-
tory that are necessary to ensure a test on a clinical sample gives the correct result and that
this finding is delivered to the appropriate clinician in a timely and efficient manner. Quality
is essentially about consistency; that is getting procedures right first time and on every subse-
quent occasion. Quality assurance is all the planned and systematic actions necessary to give
adequate confidence that a product or service satisfies the necessary level of quality.
There are a number of QA activities (Figure 19.1) that a laboratory can undertake to assure
its service users that the level of quality it offers is of an adequate standard. These include
implementing a quality management system (QMS), training staff, showing compliance with
current regulations and standards, as well as activities such as internal and external quality
controls (QCs), and training and competency assessments and audits. The clinical laboratory
also needs to ensure that associated clinicians, doctors, and patients are involved in the labora-
tory service.
506 19 QUALIT Y ASSUR ANCE AND MANAGEMENT
Quality assurance
FIGURE 19.1
Diagram showing many of the Health and Quality Clinical Training and Quality
facets of quality assurance. safety management governance competency control
SELF-CHECK 19.1
List three of the facets of quality assurance (QA)?
This chapter will provide you with an overview of how these types of activities lead to QA and
quality management relevant to the clinical laboratory.
19.1 Quality control

In addition to QA, quality control (QC) is necessary to ensure accurate and consistent clinical
results. Quality control is the operational techniques, activities, and requirements necessary to
reach the desired level of quality. In QC, the emphasis is on testing and stopping the release of
incorrect results from clinical tests. Examples of QC include the positive control and the known
negative control which are tested alongside a patient sample to ensure the accuracy of the test
results. Another example of QC is the checking of histological slides to ensure that the quality
of the preparation and the staining of the tissue elements are of an acceptable standard for
diagnostic purposes.
Quality assurance does not necessarily eliminate the need for QC, as this is only one aspect
of QA and some products are so critical that testing and checking as undertaken in QC is still
necessary, just in case other QA activities fail. Quality testing can be classed as either internal
or external.
Internal quality control

Internal QC is essentially performed in the clinical laboratory that is it is ‘in house’. Internal
QC is concerned with sampling and testing to ensure that the necessary and relevant tests
on a clinical sample are, indeed carried out, and that the test results are not released prior
to their quality being judged as satisfactory. Thus, the QC ensures that the results supplied by
the laboratory are within acceptable limits. The clinical laboratory also performs the tests on
control samples alongside clinical samples from patients. Given the composition and concen-
Cross references
trations of the controls are known, they act as built-in checks on the process of analysis. Some
assays require both high and low concentrations of the analyte to be determined to ensure,
immunocytochemistry in
Chapter 15 ‘Immunological for example, that a chemical analyser or a procedure is operating correctly across the range
techniques’ and the of concentrations that may be encountered in the samples from patients. If the results for the
accompanying volumes, controls vary from their known values, laboratory staff will immediately know that there is a
Histopathology and problem that must be rectified before further testing of clinical samples can proceed.
Cytopathology.
Internal QC is used in manual techniques, such as special staining procedures of tissue sections
Chapter 3 ‘Communications in and immunocytochemistry analyses in histopathology and cytopathology laboratories. Known
laboratory medicine’.
positive and negative controls are performed simultaneously with clinical samples to ensure
19.1 QUALIT Y CONTROL 507
that the method is both working appropriately and being performed correctly by the staff.
Quality control can also be used to determine the specificity and sensitivity (see below) of
an individual test. Laboratory assessors from bodies such as Clinical Pathology Accreditation
(CPA), which we introduced in Chapter 3, will usually examine QC records as part of the inspec-
tion or accreditation process.
External quality assessment

External quality assessment schemes (EQA) assess a laboratory’s performance against a defined
set of criteria. Laboratory accreditation assessors will look for evidence that a laboratory par-
ticipates in relevant EQA schemes and that the laboratory’s performance is acceptable. All lab-
oratories that perform tests on clinical samples are expected to participate in EQA schemes.
In EQA schemes, an outside agency, commonly known as a scheme organizer, checks the
accuracy of a laboratory’s test results. This is achieved by the scheme organizer sending known
test samples to the participating laboratory for analysis. The laboratory then must test the EQA
samples in exactly the same way it would test specimens from patients. The results of these
EQA test samples are then sent to the scheme organizer by the laboratory. The data from
all the participating laboratories are then collated and presented as a report. These reports
show the performance of a laboratory in two ways, by comparison with peer laboratories and
by performance against a minimum standard. These reports will also endeavour to highlight
areas of good or poor practice and indicate any trends identified when using certain reagents,
kits, machines, or methods.
Each laboratory is given a unique code to ensure anonymity and scheme organizers will offer
help, advice, or assistance to those laboratories identified as performing poorly by either
measure.
Laboratories can participate in a variety of EQA schemes (Table 19.1). In the United Kingdom,
many of these schemes are organized by the UK National External Quality Assessment Service
(NEQAS).
In addition to QC issues relating to a clinical test or investigation, it is necessary to be aware

of their limitations, which are described by the clinical sensitivity and clinical specificity of the
test in question.
TABLE 19.1 Examples of UK national external quality assessment schemes
Scheme organizer Scheme Comment

UK NEQAS Blood coagulation Assesses samples of plasma for blood clotting disorders.
UK NEQAS Foeto-maternal haemorrhage Examination of slides to identify the presence of foetal cells in the
mother’s blood.
UK NEQAS Biochemistry and sweat testing Assesses Cl− and Na+ in samples.
UK NEQAS AAFB microscopy Examination by microscopy for the presence of acid and alcohol fast bacilli.
UK NEQAS Immunocytochemistry in Assesses immunocytochemistry in formalin-fixed, paraffin-processed

histopathology sections of human tissue and alcohol-fixed cytosporin samples.
UK NEQAS Histopathology technical Assesses the quality of staining methods.

external quality assessment
(HTEQA)
Clinical sensitivity and specificity

To be effective, a medical test is expected to detect abnormalities with a high degree of con-
fidence. How likely is it that an individual who has a positive test has the disease? What are
the chances that an individual has a certain disorder even though the test for it was negative?
Sensitivity is a measure of the ability of a test to identify those individuals who have a disorder
after testing positive. Specificity is a measure of the ability of a test to identify individuals who
are healthy after testing negative. Unfortunately, clinical tests are neither 100% specific nor
100% sensitive. Indeed, the two factors show an inverse relationship to each other.
Clinical sensitivity
Clinical sensitivity is the ability of a test to correctly identify individuals who have a given dis-
ease or condition. For example, a certain test may have been shown to be 90% sensitive. If 100
people are known to have a certain disease, the test that identifies that disease will correctly
do so for 90 of those 100 cases. The other 10 people tested will not show the expected positive
result for the test. For that 10%, the finding of a ‘normal’ result is a misleading false negative
result. The sensitivity of a test is particularly important when seeking to exclude the presence of
a disease. For example, a patient can be reassured they do not suffer from acquired immuno-
deficiency syndrome (AIDS) by demonstrating that antibodies to human immunodeficiency
virus (HIV) are not present in blood samples. Screening for HIV antibodies often involves the
use of an enzyme-linked immunoassay (ELISA) test, which has greater than 99% sensitivity.
Cross reference However, a sample from a person may give a false negative result if the test is performed too
Chapter 15 ‘Immunological soon (less than 6 weeks) after the initial infection. Such a result would give the person involved
techniques’. a false sense of being disease-free when in fact they are not. The more sensitive a test, the
fewer false negative results will be produced.
Clinical specificity
Clinical specificity is the ability of a test to correctly exclude individuals who do not have
a given disease or condition. For example, a certain test may be 90% specific. Thus, if 100
healthy individuals are tested, only 90 of those 100 healthy people will be found to be disease-
Cross reference free. The other 10 people (who do not have the disease) will appear to be positive for that test.
You can read more about For that 10%, their ‘abnormal’ findings are a misleading false positive result. Patients who have
clinical sensitivity and specificity been told they are suffering a particular illness when they are not may be subjected to unnec-
in Chapter 5 ‘Statistics and essary treatment, which may be potentially painful, and additional expense, and unwarranted
handling data’.
anxiety. The more specific a test, the fewer false positive results it produces.
19.2 Quality management

Quality management is any system where the management has direct control of an organiza-
tion with regard to quality. Quality is not a series of events and therefore is not something with
defined start and end points, but is a never ending cycle of events that requires the support
and co-operation of all the people in the organization. Thus, QM is an approach, attitude, or
culture within an organization. All organizations must ensure that the quality of services they
provide are maintained and continually improved.
Every stage of a process, system, or project is a possible source of poor quality. The potential to
provide poor quality is greatest in complicated systems such as a hospital. However, learning
organizations have systems, mechanisms, and processes that enhance the services they provide.
19.2 QUALIT Y MANAGEMENT 509
They are able to adapt to their environments and apply the results of their learning to achieve
better results. Hence, they continually evolve and enhance their capabilities. In 2000, the
Department of Health published the document An Organization with a Memory, which advo-
cated the NHS needs to learn from its mistakes and also from the mistakes of others. The rec-
ommendations that an expert group outlined in this document are designed to ensure that
lessons from the past are used to reduce the risk to patients in the future. Thus, the NHS has
recognized it needs to learn from errors and mistakes, and to continually refine and improve its
systems and processes; in short to become a learning organization. Naturally, all adverse events
cannot be eliminated from complex modern healthcare, but by continually learning from past
errors, hospitals will be better able to achieve their stated objectives in a sustained manner, not
only for the benefit of the hospital, but also for patients and the community they serve.
The International Standards Organization (ISO) develops and publishes standards for interna-
tional organizations to adopt. One such document, ISO 9001:2008 outlines requirements of a
QMS. A QMS is a model that may be employed to give guidance to the selection of appropri-
ate QC activities. It is possible to apply a QMS to most areas, stages, and processes involved in
the provision of a product or service. The aim of a QMS is to prevent non-conformity or a non-
conformance to a set standard. Non-conformity is any deviation from that which is required.
An effective QMS will try to satisfy customers or consumers by concentrating on the service
or process. One way of achieving this is by customer surveys and monitoring their complaints.
A QMS also documents processes designed to achieve consistency, providing a goal for system
control and maintaining the required level of quality at each stage in the process.
There are eight requirements of a quality management system, as defined in ISO 9001:2008.
These are:
1. Customer focus
2. Leadership
3. Involvement of people
4. Process approach
5. Systems approach
6. Continual improvement
7. Actual approach to decision making
8. Mutually beneficial supplier relationships
This list forms the basis for any QMS in the clinical laboratory; they all contribute to improving
the quality of care for patients.
Customer focused QM meets the needs of the clinicians who treat the patients. Clinical labo-
ratories achieve this through user surveys, monitoring complaints and compliments, and by
analysing incidents.
Leadership is provided by management commitment from consultants and senior laboratory

managers. If senior management are not involved in the quality process, then it will not work.
Leadership is undertaken through the provision of quality policy and health and safety state-
ments, and also through the actions and attitudes of senior managers. Within a pathology
laboratory, the senior team may consist of a clinical director, a pathology service manager, and
a lead or head biomedical scientist or clinical scientist for each department.
High quality service provision only arises from the commitment of everyone in the organi-
zation. It needs everyone to be involved and aware of the systems in place. Indeed, it is
often the case that the people performing specific tasks have a better understanding of prob-
lems than the manager. The organization needs a well motivated work force who feel valued
and who take pride in their work: excellence in the service provided is the responsibility of
everybody concerned.
A process approach means individual areas must not be looked at in isolation; rather, the whole
process must be examined. Often changing something in one area will have a knock-on effect in
another area. For example, changing the patient request form to suit one department may mean
vital information is not provided for another department. Once the process has been analysed it is
necessary to consider the whole system (that is, a systems approach). For example, a large project
set out in the 2004 NHS (England) improvement plan was the 18-week pathway, which had the
aim of ensuring that by 2008 all patients are seen and treated by a hospital within 18 weeks of
referral by their general practitioner. This approach requires the collaboration of a number of dif-
ferent services such as pathology, radiology, and pharmacy, and was to a great extent successful.
Continual efforts must be made to improve the clinical laboratory service and to improve the
accuracy and timeliness of results as an aid to the diagnostic process. Many tools can be used
when continually improving the laboratory service; these include audit (Section 19.5), Lean
thinking, six sigma, cause and effect, and process redesign. However, no single improvement
tool provides all the answers to improve any one situation and it is essential to select the tool
that best fits the one in hand.
Factual approach to decision making means making decisions based on facts and the analysis
of data. This allows the clinical laboratory to match the needs of the service users with business
planning. For example, to select equipment that not only deals with the current number of tests,
but also takes into account predicted future increases in workload. The use of audit, incident
reporting, and statistical analysis to predict future trends all help in making the right decision.
Mutually beneficial supplier relationships improve services in a number of ways. Thus, if the labo-
ratory and the suppliers of laboratory equipment, and its maintenance and service contracts, rea-
gents and consumables work together this will improve the quality of the laboratory provision.
Quality management must be led by senior management who usually employ personnel to
implement and maintain the QMS and the Clinical Governance framework. These personnel
are usually called a quality manager or clinical governance manager. Thus, alongside the eight
principles listed above, it is necessary to have a clear clinical governance framework in place
in a hospital or clinical laboratory. Clinical governance is a form of QM that has been imple-
mented in some organizations to improve the quality of care for patients. It is the system by
which NHS organizations, such as hospital trusts, are accountable to continuously improve
the quality of the services they provide and to safeguard high standards of care. The organiza-
tion can achieve an environment in which clinical excellence flourishes. Clinical governance
requires the creation of a culture, as well as systems and methods of working that ensure
opportunities for quality improvement are identified in all the organization’s services and
that over time there is a major increase in the quality of care provided throughout the NHS.
It therefore provides a better experience for carers and staff.
The cost of adverse events, errors, and incidents is increasing in the NHS, and clinical govern-
ance provides an opportunity to focus upon these problems. Specific types of adverse events
are repeated in the NHS, which demonstrates that although the NHS is committed to becom-
ing a learning organization, it has yet to fully achieve that goal.
Improving the quality of medical laboratories

Adverse healthcare events, such as mistakes made in hospitals and in the clinical labora-
tory can lead to serious harm to a person or even cost lives. How many times have you seen
19.2 QUALIT Y MANAGEMENT 511
stories in newspapers or reported on the television similar to those listed in Figure 19.2? When
dealing with hundreds of samples a day it is easy to forget that there is a person who is wait-
ing to receive treatment as a result of a laboratory investigation. A pathology laboratory that
has efficient processes in place is one that strives to deliver the correct test result to the right
patient in a timely and efficient manner. This ensures that an appropriate diagnosis is made
and effective treatment can be given.
FIGURE 19.2
Newspaper articles relating to the breakdown of
healthcare and the harm caused to patients.
Once effective processes have been established, the focus is to continually improve the operation
of the laboratory. This could mean establishing more efficient ways of working, reducing costs, or
improving training opportunities. To improve we must learn; learn from mistakes, from analyses
of data, and from others. The process of continual improvement is a never ending cycle.
19.3Documentation in the
clinical laboratory
Laboratory standards, such as ISO 15189:2007 Medical laboratories – Particular Requirements
for Quality and Competence and ISO 22870:2006 Point of Care Testing Requirements for Quality
and Competence outline a set of criteria that need to be fulfilled by clinical laboratories
(Section 19.4). These standards also include the requirement for a QMS, as outlined in ISO
9001 – Quality Management Systems – Requirements. When implementing such standards in
a laboratory, written, that is documented, evidence is needed to show that the laboratory is
meeting the requirements specified in the standards. The minimum requirements of a QMS
for a clinical laboratory are a quality policy (QP), quality manual, and standard operating pro-
cedures (SOPs) for the processes in place (see Figure 19.3). There are also requirements to have
procedures to control the documents and to control changes made to any processes.
Quality policies (QPs) are short, one page, documents that outline the scope of the service
the laboratory intends to provide. A QP contains details of the laboratory's commitment to its
users and staff, and outlines the organization's quality objectives. It is usually signed by one
of the organization's senior managers such as its clinical director. The QP is communicated and
available to all staff in the laboratory, usually by displaying it on a notice board and including
it in the laboratory quality manual.
A quality manual is a document that outlines how the objectives stated in the QP are to
be achieved. Documented sections within the quality manual are linked or referenced to
the standard(s) with which the organization is trying to comply. A quality manual contains
organizational-specific information, including its organizational structure, processes, and
procedures, and the resources needed to implement quality and meet the objectives
described in the QP.
Standard operating procedures (SOPs) are written instructions that describe in a clear and
concise manner how procedures or methods are to be performed. They include such details
as to how individual tasks are to be carried out, that is what is to be done, by whom and when.
Quality
policy and
quality manual
Policies
FIGURE 19.3
Triangle showing the
possible hierarchy of Standard operating procedures
documentation in a quality
management system.
19.4 STANDARDS AND REGUL ATORY REQUIREMENTS IN THE MEDICAL L ABOR ATORY 513
They accurately describe the process and methods that are in use in the laboratory and con-
Cross reference
tain essential information such as health and safety instructions and the correct use of equip-
Chapter 4 ‘Health and safety’,
ment and controls. It is necessary to have up-to-date SOPs, that correctly describe what is discusses risks, risk assessment,
done to ensure that no step is missed and that the quality of the results is not compromised. and COSHH.
SELF-CHECK 19.2
What is a standard operating procedure and why is it needed?
Document control is an essential part of any effective QMS. Documents associated with the
QMS will change with organizational changes, changes in practices, technology, and improve-
ments in processes. Control of these changes is necessary to ensure that the procedures cor-
rectly describe the processes, are readily available, and are up to date. For example, if the
procedure to issue blood changes, these changes need to be documented so that all staff are
aware of the new procedure to follow. If out-of-date procedures are followed, mistakes, such
as using incorrect times to incubate microbiology samples may occur, and lead to an incorrect
diagnosis.
Change control in the clinical laboratory aims to ensure all changes are assessed and approved
by management before they are implemented. For example updating a computer program
without considering the effect across the network may render the system unusable in some
areas. Thus, changes to procedures or to equipment must be introduced in a controlled and
co-ordinated manner to prevent time, effort, and resources being wasted.
19.4Standards and regulatory

requirements in the medical
laboratory
Clinical laboratories are subjected to rigorous inspections by a number of different assessment and
accrediting bodies. Accreditation is the process by which an organization gains recognition that
its activities and products have attained a certain level of compliance against a set of defined Cross reference
standards. In the United Kingdom, the United Kingdom Accreditation Service (UKAS) is the sole We introduced UKAS in
national accreditation body recognized by government to assess against national and international Chapter 18 ‘Point of care testing’
standards as published by the British Standards Institute (BSI) and the International Organization and the CPA in both Chapter 3
for Standardization (ISO). The British Standards Institute develops, publishes, and markets stand- ‘Communicatios in laboratory
ards in the UK. Standards are codes of best practice that improve safety and efficiency. The medicine' and Chapter 18.
ISO is the world’s largest developer and publisher of international standards such as ISO 9001,
Quality management systems – requirements. The Clinical Pathology Accreditation Ltd (CPA
UK Ltd), which is part of UKAS, works to accredit medical laboratories to a set of standards that
incorporate ISO 15189, Medical laboratories – Particular requirements for quality and compe-
tence and ISO 9001 mentioned above. The Human Tissue Authority (HTA) and the Medicines
and Healthcare products Regulatory Agency (MHRA) are two other assessment bodies that
undertake inspections of clinical laboratories.
Clinical Pathology Accreditation standards

Organizations that declare their adherence to a defined standard of practice and have their ability
to attain these standards independently confirmed, that is they are ‘accredited’ can reassure their
users of the quality of service they provide. Clinical Pathology Accreditation provides a means by
which this can be achieved both by clinical laboratories and also by providers of external quality
assessment (EQA) schemes. The CPA has the authority to grant accreditation status to clinical labo-
ratories that meet its standards. The standards adhered to by CPA are based on ISO 15189 Medical
laboratories – Particular Requirements for Quality and Competence, which is an international stand-
ard that incorporates some elements of ISO 9001. Theses standards cover eight major areas:
1. Organization and quality management system (A standards)

2. Personnel (B standards)
3. Premises and environment (C standards)
4. Equipment, information systems, and materials (D standards)
5. Pre-examination process (E standards)
6. Examination process (F standards)
7. Post-examination process (G standards)
8. Evaluation and quality assurance (H standards)
Organization and QMS encompasses areas, such as the needs and requirements of users, the
QP document, the quality objectives and plans of the laboratory, the quality manual, docu-
ment control procedures, and control of process and quality records.
Personnel relates to areas concerned with personnel management, staff induction, records
and meetings, staff training, and education. In contrast, premises and environment, as the
name implies, encompass the work areas, staff and patient facilities, and also health and safety
in the laboratory. Equipment, information systems and materials covers the procurement and
management of equipment, materials, data, and information.
The pre-examination process encompasses information for users, specimen collection, and
handling, requirements for specimen request forms and transport of specimens, while the
examination process sets standards relating to the selection and validation of the examination
and the quality of the examination that is performed on the specimens. Post-examination
process covers areas, such as the final report and how results are reported.
Evaluation and QA investigates how the laboratory assesses user satisfaction, the laboratory
audit process, and quality improvement processes in place in the laboratory.
CPA assessment process

The assessment process involves the laboratory enrolling with the CPA, which then inspects the
laboratory every four years. The inspection process is undertaken by trained assessors who per-
form an external audit (Section 19.5) of the laboratory over 1 or 2 days. The inspection team con-
sists of a regional assessor and at least two peer assessors. The regional assessors are employed
full time by UKAS and co-ordinate the assessment visits. Peer assessors are drawn from practising
consultant pathologists, clinical scientists, and biomedical scientists. They accompany the full
time assessors and act as experts in specific subjects such as immunology or biochemistry.
Blood safety and quality regulations

The Blood Safety and Quality Regulations (BSQR) changed two European Blood Directives
(Directives 2002/98/EC and 2004/33/EC) into UK law. The UK has a duty to translate the
European Directives into national legislation. The BSQR require hospital blood banks and
blood establishments to maintain a quality system based on the principles of good laboratory
practice (GLP), which is a set of principles that provides a framework within which laboratory
19.4 STANDARDS AND REGUL ATORY REQUIREMENTS IN THE MEDICAL L ABOR ATORY 515
work is performed. The Medicines and Healthcare Products Regulatory Agency (MHRA)
assesses the compliance of hospital blood banks, and blood establishments to the BSQR.
The laboratory is required to submit an online compliance report to the MHRA, which then assesses
the online report against the BSQR. The MHRA may decide to visit the laboratory and undertake
an onsite assessment. This visit is usually conducted over one day by a single assessor. The asses-
sor checks that there is accurate and complete traceability of blood and blood products. He or
she will also examine the records kept by the hospital laboratory and wards, and check records
and documents of satellite sites that receive and store blood and blood components.
One of the BSQR requirements is that the laboratory notifies the MHRA and serious hazards
of transfusion (SHOT) of any serious adverse blood reactions and events (SABRE). This is under-
taken through an online reporting system that can be accessed through the MHRA website.
The reporting of SABREs allows the MHRA and SHOT teams to identify any trends in blood
transfusion incidents.
Human tissue act

The Human Tissue Act (England) came into force in 2004. It arose from concerns raised by
events such as those at Bristol Royal Infirmary (BRI, 1984–1995) and the Alder Hey Children’s
Hospital Liverpool (prior to 1999) where it was established that organs and tissues from children
had been removed, stored, and used without proper consent from next of kin. Subsequent
government reports showed that the storage and use of organs and tissues from both adults
and children had been widespread in the past. It became clear that current law in this area was
not as clear and as consistent as it might have been for professionals or families. The purpose Cross reference
of the Human Tissue Act is therefore to provide a legal framework for issues relating to the You can read more about
taking, storage, and use of human tissue and organs and whole body donation. The Act made the Human Tissue Act
consent the foundation for the lawful storage and use of human bodies, body parts, organs, and related issues in
and tissue, and the removal of tissue from the deceased, and regulates the use of tissues and Chapter 3 ‘Communications
in laboratory medicine’.
cells that are to be used for transplant and stem cell research from living donors.
The Act outlined the need for a regulatory authority to be appointed: the Human Tissue
Authority (HTA) is the regulatory body appointed in the UK. The HTA ensure that the activities
it regulates are undertaken in a proper manner, and provides advice and guidance related to
the Human Tissue Act and also to the EU Tissue and Cells Directives, which is also part of the Cross reference
Quality and Safety Regulations (see Chapter 4). These laws ensure that human tissue is used Chapter 4 ‘Health and safety’.
safely and ethically, and with proper consent.
The HTA ensures these laws are followed by setting standards that are clear and reasonable,
helping people to understand the legal requirements, and providing codes of practice, advice,
and support in nine different areas. These are anatomy, human application, stem cells and
cord blood, public display, research, post-mortem, coroners, transplants, and DNA.
The HTA issues codes of practice that give guidance to each of the different areas. The codes
differ for each, but all broadly cover the need for consent, implementation of governance,
and quality systems including control of documentation and records. For example, the post-
mortem standards cover consent for a hospital post-mortem, governance and quality systems,
premises, facilities and equipment, and disposal of clinical material.
The laboratory or mortuary is required to complete an online compliance form which is sent
to the HTA. The HTA then assess the online report against the appropriate set of standards. If
the report is deemed satisfactory, the HTA will issue the laboratory or mortuary with an HTA
Licence. It is unlawful to undertake certain activities, such as post-mortems, storage of the
deceased, and storage of body parts without this licence. As part of its role to ensure compli-
ance with the Human Tissue Act, the HTA will inspect the laboratories, mortuaries, and other
establishments that fall under its remit. An inspection team consisting of two or more people
will usually spend 1 or 2 days in the laboratory conducting an audit against the HTA standards.
The HTA require that a named individual, known as the designated individual, takes responsi-
bility for ensuring that suitable practices are used when undertaking licensed activities such as
post-mortems. Following an inspection, a report is written and any areas that need improving
are identified together with a timescale for their rectification.
The Human Tissue (Scotland) Act was introduced in Scotland in 2006. The HTA Codes of Practice
also cover the Human Tissue (Scotland) Act in most, but not all areas. Where the HTA refers to a
coroner in England and Wales, the equivalent person in Scotland is the Procurator Fiscal.
Point of care testing, requirements for

quality and competence
Point of care testing (POCT) can be defined as testing at or near the site of patient care, which
increases the likelihood that the patient will receive the result of a clinical test in a timely man-
Cross reference ner. Points of care testing is often used in A&E departments, neonatal care units, GP surgeries,
Chapter 18 ‘Point of care testing’. or even in the home of a patient (see Chapter 18).
The ISO standard, ISO 22870:2006 Point of Care Testing (POCT), Requirements for Quality and
Competence outlines the requirements for POCT. The clinical laboratory must ensure that
POCT is co-ordinated effectively and efficiently in the hospital environment. Thus, POCT poli-
cies and procedures need to be in place that outline how the equipment must be used. Staff
must be trained on use of the equipment and records of this training maintained. Medico-
legal considerations and the requirements of the Data Protection Act must be borne in mind
when establishing these procedures. The laboratory must be involved in any purchases of new
equipment for use outside of the laboratory, as well as being involved in training staff to use
it correctly. Most clinical laboratories have a dedicated person to look after POCT equipment,
and liaise with ward and departmental staff about its correct use and cleaning. The laboratory
also has a duty to ensure that the equipment is serviced in accordance with manufacturer’s
requirements and recommended local and/or national QA schemes (NEQAS).
Point of care testing is not assessed or accredited in isolation, but as part of the overall laboratory
accreditation process.
Key Points
Currently, POCT that takes place in the community, such as at GP surgeries or patient
homes, is neither assessed nor accredited.
Additional standards affecting clinical laboratories

Clinical laboratories are likely to be part of a larger organization, such as a hospital. In such
cases they may be subject to hospital regulatory requirements such as:
• Standards for Better Health
• Clinical negligence scheme for trusts

19.5 TOOLS AND TECHNIQUES FOR CONTINUAL IMPROVEMENT 517
Standards for Better Health came into being from April 2005 as a new performance framework
for the NHS.
This framework outlined the level of quality all organizations providing NHS care are expected
to meet or aspire to in England. These standards provide a common set of requirements that are
applied across all healthcare organizations to ensure that health services are both safe and of
an acceptable quality, and provide a framework for continuous improvement in the overall
quality of care patients receive. Thus, the standards describe the level of quality that health-
care organizations, including NHS Foundation Trusts, and private and voluntary providers of
NHS care, are expected to meet in terms of safety, clinical and cost effectiveness, governance,
patient focus, accessible, and responsive care, care environment and amenities, and public
health. Standards for Better Health form a key part of the performance assessment by the care
quality commission.
Clinical negligence scheme for trusts (CNST) standards are assessed by the NHS Litigation
Authority (NHS LA). CNST is an insurance scheme covering litigation against a hospital. It
requires the development and implementation of clinical incident reporting systems. NHS
Trusts must have basic systems in place across some of the organization to attain even the
most basic CNST standards (level 1). There must be a widespread comprehensive system in
place to reach the highest level (3). Attainment of the highest level of the standard indicates
the most sophisticated processes for incident reporting and risk detection and prevention and
thereby results in the smallest premiums payable by the hospital.
SELF-CHECK 19.3
What are the main bodies that inspect clinical laboratories?
Tools and techniques for

19.5
continual improvement
We will now outline some of the tools used to check or assure that the processes and methods
used in a clinical laboratory comply with the required standards. These tools include audit,
incident reporting, and root cause analysis (RCA).
Audit
ISO9001 defines an audit as ‘A systematic and independent examination to determine whether
quality activities and related results comply with planned arrangements and whether these
arrangements are implemented effectively and are suitable to achieve objectives’. Hence, audit
is an integral part of any QMS and is a requirement for accreditation by the CPA (Section 19.4)
and other clinical laboratory and hospital-wide standards. All audits are a means for continually
improving the service provided by the laboratory.
The concept of audit is to gather information by means of observation, interview, and sam-
pling to identify areas where improvements can be made. This is essential if a process is to
continually improve. An audit will also identify those processes that are working well, and so
give an opportunity to learn from good practice and to allow that knowledge to be transferred
to other processes. Thus, audit enables organizations to evaluate their processes, determine
deficiencies, and generate cost effective and efficient solutions to those problems.
Key Points
Audit is used to measure an organization’s ability ‘to do what it says it is going to do’.
All audits are performed to check practices against procedures and to thoroughly document
any differences. However, audits are only sampling exercises and so they cannot confirm that
all aspects of a process are being complied with at all times.
Audits fall into four main types:

1. Vertical audit
2. Horizontal audit
3. Witness or examination audit
4. Self-assessment audit
You can see an illustration of some audit methods in Figure 19.4.
A vertical audit examines a single item, following it from start to finish. For example, this could
be the journey a single clinical sample follows once it is received by the laboratory to the
result of the tests performed on it being issued to the relevant clinicians. In contrast, a hori-
zontal audit examines one element in a process that is performed on more than one item.
Start of process Vertical audit

Skin lesion removed from One sample followed
patient from start to finish
through the process
Sample transported to
laboratory and received by
sample reception
Examination audit Sample labelled, details added

Looks at a person to computer
following an SOP whilst
undertaking a task
(e.g. labelling samples)
Sample suitably processed
in laboratory
Slides made from sample
Horizontal audit Slides stained, cover slips added,

Examines many slides and slides checked for problems
for one element
(e.g. section quality)
Slides examined by consultant

and report typed
FIGURE 19.4
Diagram showing the different
methods used when undertaking Report issued
an audit. See also Table 18.2. End of process
This could, for example, be an examination of 20 request forms to ensure that details of all 20
patients have been entered correctly on the computer system. A witness audit examines a per-
son undertaking a task. This may be observing a person labelling samples to check that he or
she have read and are following the relevant SOP. Self-assessment audit is a careful evaluation
that is usually performed by the organizations own management, which results in an opinion
or judgement as to its effectiveness and efficiency.
Audits are classified as internal, external and cooperative types. Internal audits are conducted
by clinical laboratory staff themselves. For example, a biomedical scientist in haematology
could conduct an internal audit of the microbiology laboratory. An internal vertical audit
would be one conducted by laboratory staff on a clinical sample subjected to the labora-
tory process. The purpose of an internal audit is to confirm that everything is in order and as
expected and to identify any non-compliance with the standards that are applicable to the
laboratory. It will identify any deficiencies in the QMS and make recommendations of any
actions that need to be taken to improve the laboratory. Naturally, the type and content of the
internal audit will vary according to the size and activities of the laboratory concerned.
An external audit is conducted by an outside person or body, such as the CPA, who have an
interest in accrediting the laboratory. An external horizontal audit is conducted by an external
person(s) on a specific element such as staff training records. A co-operative audit is con-
ducted between the laboratory and another party for mutual benefit, such as clinical audits,
user satisfaction surveys, or benchmarking activities.
Before any audit can be undertaken, it is necessary to prepare an audit plan. The plan is called
an audit schedule and contains information about what audits are to be undertaken, when
they will be undertaken and who will perform the audit. The audit schedule is discussed and
agreed by senior management at the beginning of the year. You can see an example of an
audit schedule in Table 19.2.
SELF-CHECK 19.4
What methods of audit do external inspectors undertake when visiting the laboratory?
Performing an audit
The four stages in an audit are:
1. Prepare the audit checklist

2. Undertake the audit
3. Write a summary of the audit
4. Undertake corrective action to improve the laboratory process
TABLE 19.2 Audit schedule
Audit area January February March

Audit of laboratory Horizontal audit on equipment such
equipment as analysers, centrifuges, microscopes
used in the laboratory.
Staff records Horizontal audit on

CPD records of staff.
Transport of samples Vertical audit on receipt of samples

from general practioners to the issuing
of results of clinical tests.
An audit checklist is a list of the questions to be asked relating to the set standard(s). Hence,
if the set standard was: ‘There shall be up to date information for users, a suitable checklist
question is, ‘Is there up to date information available for users?’ The audit checklist must
also contain the names of the auditors (the person undertaking the audit), the proposed
date of the audit, a unique reference number and the area(s) being audited. The depart-
ment will appoint an auditee; a person who will assist the auditor(s), accompanying them
around the department to verify factual information. An audit is the process of going into a
department and asking the questions in the checklist. Essentially, these questions will be of
the type: WHAT …? WHERE …? WHY …? WHO …? HOW …? SHOW ME …? Evidence to support
the answers to these questions must be examined; the auditor(s) must see the evidence. If it
is not seen, it does not exist! Audit is all about seeing the evidence of compliance with the
standard being audited.
After an audit has been completed, a summary of all the findings are documented. The sum-
mary outlines areas of compliance, that is where the laboratory meets the standard, partial
compliance, where the laboratory has some evidence of meeting the standard, and points of
non-compliance, where evidence of meeting the standard was not provided. This is an essen-
tial stage in an audit and should not be missed out. It gives an opportunity to highlight areas of
good practice, what the laboratory is doing well, in addition to identifying areas for improve-
ment. Areas that need to be improved are issued with a non-compliance form, which outlines
the area of non-compliance against the standard, what corrective actions need to be taken, by
whom and when. When all identified non-compliances against the standards are corrected,
the audit can be said to be closed. However, the audit process is a never ending cycle that
leads to continual improvement in the laboratory provision and in the service it provides to
laboratory users. You can see an illustration of the cycle in Figure 19.5.
Plan
The audit schedule
and checklists
Check Do
That the actions Undertake the
are in place and audit, report and
are working identify
non-compliance
Act
Put in place
changes/actions to
FIGURE 19.5 close out the
The audit cycle is a never ending non-compliance
process of improvement.
Incident reporting and root cause analysis

Incident reporting is a major tool for examining what happened when an error or problem
occurred. Adequate and appropriate incident reporting systems are necessary to provide a
core of sound, representative information on which to base any analysis of errors or problems
and to make recommendations to prevent their repetition. Experience in other sectors, such
as the airline industry (Figure 19.6), has demonstrated the value of systematic approaches to
recording and reporting adverse events.
It is possible to identify a number of barriers that can prevent active learning, but the NHS can
draw valuable lessons from particular approaches used in other sectors. The first approach is
one of ‘safety cultures’, where open reporting and balanced analyses are encouraged in prin-
ciple and by example. This can have a positive and quantifiable impact on the performance of
organizations. In contrast, ‘blame cultures’ may encourage people to cover up errors for fear
of retribution. This acts against identifying the true causes of failure, because they focus heavily
on individual actions and largely ignore the role of underlying systems. Thus, organizational
culture is central to every stage of the learning process: from ensuring that incidents are identi-
fied and reported through to embedding the necessary changes deeply into practice. The sec-
ond approach is ‘local risk reporting systems’, which provide the bedrock for onward reporting
to regional or national systems. These are now more widely used in the NHS. Incident report-
ing systems are now in place across most NHS organizations.
The National Patient Safety Agency (NPSA) helps to improve the quality of safe patient
care by informing, supporting, and influencing healthcare organizations and individuals
working in health. It analyses national reports on patient safety incidents, identifies risks,
and recommends actions. The NPSA collates incident data and issues guidance to the NHS
on how to improve processes and systems, thereby reducing risks and harm to patients
and staff alike.
Adverse incidents involving medical devices, including laboratory equipment, are reported
to the MHRA. Information is logged on a central database, which contains details of over
48,000 incidents. Incidents are assigned to a specific level of investigation depending on the
risks involved. The outcomes of these investigations are subject to formal reviews. Patterns or
clusters of incidents can thus be identified, subjected to further risk assessment procedures,
and investigated where necessary.
When an incident reveals equipment-related safety problems, the MHRA will produce a haz-
ard or safety notice for distribution to appropriate users so that action can be taken to with-
draw or modify the device.
NHS told to learn from its mistakes

YOU WOULD NOT want to learn from Sir Liam Donaldson
just decide to forget every accident in speaking on patient
about recovering the healthcare?’ safety to Health
black box after an air Service non-executive
crash. So why should it directors in Bristol,
be thought so strange to 20 November 2001
FIGURE 19.6
Newspaper article that emphasizes the need to learn
from incidents involving patient safety.
Detecting and accurately recording errors is a fundamental step in learning from experience.
It is common sense to recognize that knowledge of what is wrong is required before steps can
be taken to put it right.
Reporting of accidents and incidents must be seen as professional behaviour rather than
as disloyalty to other staff or the NHS. All organizations, including the NHS must operate a
genuine two-way communication system, not just a ‘top down’ system from management.
Communication systems need to be in place to allow people to see what has changed as a
Cross reference result of an incident or the reporting of a near miss. An organization needs to recognize staff
concerns. The emphasis of management should be on personal and service benefits, rather
professional behaviour and than on threats. Within the NHS, more emphasis needs to be placed on creating an informed
duties in Chapter 2 ‘Fitness to culture; to raise the awareness of the costs of not taking risk seriously. This includes the human
practise’. costs, to staff and patients, as well as financial ones.
Not all unsafe systems produce bad outcomes all the time. However, more attention must
be focused on healthcare near misses, where an accident is just avoided, in addition to the
incidents themselves. This approach can remove the emotion associated with an incident and
allow learning to take place more effectively. It is also easier to keep near miss data anony-
mous, itself a factor that encourages reporting. It is essential that concerns, near misses, and
incidents can be reported without fear.
The potential for disasters may exist but for any number of reasons those may not occur, or
occur very rarely. Most accidents have the potential to produce serious injury but do not do so
in practice; either because of some intervention or compensation or simply through good for-
tune. Confining analysis to and learning from only those events that resulted in serious harm
Cross reference risks skewing learning towards a small proportion of accidents and missing important lessons
Chapter 4 ‘Health and safety’. that could prevent future adverse events. These points were also discussed in Chapter 4.
Industry has estimated that for every major injury, 29 minor ones and 300 accidents with no injury
occur. Thus, to some extent the effectiveness of a reporting system can be judged by the relative
proportion of minor incidents to more serious accidents: the greater the proportion of minor
incidents reported, the ‘better’ the reporting system is working. This is illustrated in Figure 19.7.
Root cause analysis

Root cause analysis (RCA) is a powerful tool that identifies, records and visualizes the possible
causes of a problem, error, or incident. It is undertaken as an investigation into the error to
1
major
incident
29 minor injuries
FIGURE 19.7
Triangle of incident relationships
showing the proportion of near 300 non injuries
misses to harm to or death of a
patient. See also Figure 4.6.
determine its basis that is its root cause. When an incident has occurred it is imperative that
an investigation identifies its root cause to ensure that improvements can be made and to
prevent the same incident reoccurring. The basis of RCA is to break down the problem into
smaller, more manageable tasks, which allows the problem to be identified and described.
This approach will assist in finding a solution or corrective action that will prevent the same
problem reoccurring.
Many methods can be used when undertaking RCA, including a cause and effect diagram.
Cause and effect diagrams provide a logical means to analyse a problem to get at its root
cause. Look at Figure 19.8. This show you an example of how a cause and effect diagram can
be used to determine the cause of why a laboratory analyser has failed and is giving inaccu-
rate results for tests on patient samples. Although the cause of the failure is initially unknown,
various possibilities need to be examined in detail to determine why the equipment failed.
These possible causes can be mapped on the cause and effect diagram as shown and each one
analysed until the root of the problem is discovered. An action plan can then be put in place
to ensure the same problem does not occur again.
Maintenance
and cleaning of
equipment not
undertaken
Lack of
laboratory
staff leading to
incomplete or
absence of QC
Causes of checks
equipment
failure
Lack of staff
training in
recognizing
problems
Reagents
passed their
expiry dates
Effect is to
give FIGURE 19.8
inaccurate Cause and effect diagram illustrating
results to how an equipment failure should be
patients investigated.
SUMMARY
■ The necessity to provide services of appropriate quality in clinical laboratories, as well as the
need to satisfy the users of laboratory services has led to increasing numbers of regulations
governing their operations and assessment
■ Standards against which clinical laboratories are assessed include ISO 15189 Medical
Laboratories, Particular requirements for quality and competence, which includes the stand-
ards contained in ISO 9001 – Quality Management Systems – Requirements
■ Clinical laboratories are subjected to inspection and assessment by a number of different
bodies, namely the Clinical Pathology Accreditation, The Human Tissue Authority and the
Medicines and Healthcare Products Regulatory Authority
■ Quality assurance, quality control, and the implementation of quality management sys-
tems has allowed clinical laboratories to continually evaluate, monitor, and improve their
processes and systems, and deliver a high quality laboratory service to their users
■ A variety of techniques, such as audit, can be used by the laboratory to internally assess
their compliance to set standards
■ Following good laboratory practices ensures the integrity of the sample audit trail in
the laboratory and allows the cycle of continual improvement to be embedded into the
laboratory culture
FURTHER READING
● Burnett D. A Practical Guide to Accreditation in Laboratory Medicine. ACB Venture
Publications, London, 2002.
This book clearly explains the accreditation process and the accompanying documenta-
tion needed by clinical laboratories for the inspection process.
● Department of Health An organization with a memory. Department of Health,
London, 2000.
This is a report on learning in the NHS from adverse events.
● MHRA Rules and Guidance for Pharmaceutical Manufacturers and Distributors.
Pharmaceutical Press, London, 2007.
Section II: Guidance on good manufacturing practice is worth dipping into.
● Vorley G, Tickle F. Quality Management, Tools and Techniques, 5th edn. Quality
Management & Training (publications) Limited, London, 2002.
Sections 3 and 4 give accounts of quality management systems, ISO9000, and audit.
Useful Websites
■ www.cpa.org
■ www.hta.gov.uk
■ www.iso.org
■ www.ukneqas.org.uk
■ www.ibms.org
QUESTIONS
■ www.mhra.gov.uk/index.htm
■ www.npsa.nhs.uk
■ www.nhsla.com
■ www.dh.gov.uk
■ www.ukas.com
■ www.thecqi.org
■ www.bsi-global.com
QUESTIONS
1. Which, if any, of the following has a responsibility to control quality in a medical laboratory?
(a) The quality manager
(b) Management
(c) The staff
(d) Laboratory manager
(e) All of the above
2. Indicate which of the following statement(s) is(are) TRUE?
(a) Quality Assurance, Quality Control, and the implementation of Quality Management
Systems has allowed medical laboratories to continually evaluate, monitor, and
improve their processes and systems thereby delivering a high quality laboratory
service to their users.
(b) Quality Assurance, Quality Control, and the implementation of Quality Management
Systems has allowed the medical laboratory to eliminate all errors thereby deliver-
ing a high quality laboratory service to their users.
(c) Quality Assurance, Quality Control, and the implementation of Quality Management
Systems has allowed medical laboratories to employ more staff to undertake audits
in order to monitor their processes and systems and improve patient care.
(d) Quality control means clinical laboratories do not have to check their test results
before releasing them to the clinicians.
(e) External Quality Assessment involves outside agencies checking the clinical test
results before they are sent to the requester.
3. Outline the process of quality control as performed in the laboratory. Indicate how it
differs from external quality assessment?
4. What is meant by document control? State why document control is necessary?
5. What techniques can be employed to improve the laboratory service?
6. Outline the structure of a learning organization?
7. Why is it necessary to report concerns about incidents that occur in the laboratory?
8. Give an example of how a cause and effect diagram (Figure 19.8) can be used to investi-
gate an error?
9. Why is it necessary to have an audit trail outlining the path a clinical sample takes through
a laboratory?
20
Personal development
Georgina Lavender
Learning objectives
■ Describe what is meant by personal development and continuing professional development

■ Discuss opportunities and events that in everyday life contribute to personal development
■ Discuss the types of continuing professional development needed to satisfy the require-
ments for registration with the Health Professions Council
■ Describe the opportunities provided by the Institute of Biomedical Science for professional
development
■ Identify opportunities within the workplace to extend personal development opportunities
■ List the mandatory training that must be provided by an employer
■ Select and utilize appropriate study skills for different situations
■ Describe the structure of a generic performance appraisal scheme
■ Explain how the performance appraisal and development structure within the NHS is of
benefit to a biomedical scientist
Introduction
Personal development is any means by which an individual is able to expand on his or her
existing knowledge, skills, and talents in all aspects of their lives. Newly qualified biomedical
scientists leaving behind the academic environment (Figure 20.1) and entering professional
life must not imagine that this is the end of their learning. Personal development is ongoing
and can be applied to anything that he or she chooses to do.
Anyone with a degree even remotely connected with science is aware that science is con-
stantly evolving, and that today’s innovations and discoveries are often old news and out-
dated tomorrow. Anybody listed on a professional register is required to continually reaffirm
Cross reference and review their knowledge base within their chosen profession; biomedical scientists are no
The HPC CPD requirements exception. Indeed, biomedical scientists are expected to be able to demonstrate a designated
were discussed in Chapter 2
level of continuing professional development (CPD) to remain on the Health Professions
Council (HPC) register and every 2 years at the renewal of their registration must declare they
INTRODUCTION 527
FIGURE 20.1
Undergraduate biomedical
science students learning basic
laboratory skills in a university
laboratory.
have been undertaking CPD to a predefined standard and crucially must be able to provide
evidence to support this claim.
There is a saying that it is never too soon or too late for learning and this applies to both pro-
fessional study and development and to that done for pleasure. Lifelong learning is often a
concept that many people adopt and embrace without ever realizing that they are doing so.
Lifelong learning is associated with many unexpected places, a newspaper article, a television
documentary, even helping children with homework or trying out a new recipe in the kitchen!
This learning can be carried across to biomedical science also, and a keen biomedical scientist
will easily be able to meet professional development targets, almost without realizing that
they are doing so, as there are so many opportunities within the working environment and
elsewhere for the lifelong learning that will constitute CPD.
A framework for higher education qualifications exists across Europe, the European Qualifications
Framework (EQF), which is being adopted and has been referenced to the different systems
within England, Wales, and Northern Ireland by the Qualifications and Curriculum Development
Agency (QCDA). Scotland is undertaking a similar referencing exercise to its qualifications too.
These systems describe the educational level of an award or describes the expected achieve-
ment of knowledge and skills of the student at the end of a course of study (see also Box 20.1).
Of the eight EQF levels, the principal ones of interest to biomedical scientists are:
• EQF Level 5 (Intermediate (I) level or levels 8/9 in Scotland: Foundation degree, HND)
• EQF Level 6 (Honours (H) level or level 10 in Scotland: Bachelors degree)
BOX 20.1 Qualification levels and

biomedical science training
A biomedical science undergraduate would be described as working towards an EQF

Level 6 qualification, whereas a trainee biomedical scientist with an honours degree,
but not yet HPC registered would be described as already being in possession of an EQF
Level 6 qualification and being able to work towards an EQF level 7 qualification. These
are indicators of what you are already able to achieve and what you would be able to
work towards. These factors are important when choosing your study options.
528 20 PERSONAL DEVELOPMENT
• EQF Level 7 (Masters (M) level or level 11 in Scotland: Masters degree)
• EQF Level 8 (Doctoral (D) level or level 12 in Scotland: Doctoral degree)
This chapter will examine the personal and professional development of biomedical scientists
(Figure 20.2) and, hopefully, help you to find and utilize a range of information resources that
are there for the taking. All healthcare professionals have a responsibility to participate, alone or
with work colleagues, to improve their knowledge and skills as a practising biomedical scientist.
Continuing professional
20.1
development
Continuing professional development may be defined as the ongoing learning that is car-
ried out throughout your professional career. It is needed in order to remain up to date in
all aspects of a chosen profession. It is also necessary for promotion and is often a manda-
tory requirement to remain on a professional register. The two bodies with responsibilities
for regulating biomedical science, the Institute of Biomedical Science (IBMS) and the Health
Professions Council (HPC), have their own definitions for CPD.
Key Points
The IBMS defines CPD as a process of lifelong learning, which enables you to expand and
fulfil your personal and professional potential, as well as meet the present and future needs
of patients, and deliver health outcomes and priorities. It assures that you meet the requi-
site knowledge and skills levels that relate to your evolving scope of professional practice.
The definition of CPD by the HPC is the way health professionals continue to learn and
develop throughout their careers so they keep their skills and knowledge up to date and
are able to work safely, legally, and effectively.
FIGURE 20.2
A group of professional biomedical
scientists working in a clinical
laboratory.
20.2 OPPORTUNITIES FOR PERSONAL DEVELOPMENT IN THE WORKPL ACE 529
What exactly do these definitions mean to a newly qualified and registered biomedical scien-
tist? They mean that, as a professional person, on a professional register, a biomedical scientist
has a legal responsibility to meet the standards of the HPC and also to remain abreast of cur-
rent trends and developments within his or her working environment.
Requirements of the Health Professions Council

It is a requirement of the HPC that all registrants including biomedical scientists must undertake
CPD to remain on their register. Whenever he or she is asked to renew their registration, they
must declare that they are complying with the HPC standards relating to CPD. The HPC is able to
ask for evidence from registrants selected at random, for factual support that they are meeting
the set standards. The standards of CPD state that a registrant on the HPC register must:
• Maintain a continuous, up-to-date and accurate record of CPD activities
• Demonstrate that CPD activities are a mixture of learning activities relevant to current or
future practice
• Seek to ensure that CPD has contributed to the quality of their practice and service delivery
• Seek to ensure that CPD benefits the service user
• Present a written profile containing evidence of CPD upon request
The HPC has extensively listed activities that constitute CPD (Table 20.1), together with exam-
ples of suitable supporting documents. Of the total registrants, 2.5% will be asked to submit
their personal CPD records at the biennial point of renewal of registration.
SELF-CHECK 20.1
Why is continuing professional development necessary for biomedical scientists?
Opportunities for personal

20.2
development in the workplace
An induction period is normally provided with any new post or even a change in workplace.
Information regarding the new employer is often given within the first few weeks of employ-
ment to all new employees to introduce them to specific organizational philosophies and poli-
cies. Induction should always be treated as an opportunity for personal development. Indeed, a
formal induction programme needs to be given at the start of working at a new organization.
Induction courses are also an introduction to other employees. Presentations are ideal
opportunities to find out more about your place of work, and ask seeking questions of a new
employer. Frequently, presentations are given by senior members of staff and are often an
excellent way to find out who does what within a large organization and to find out what they
look like. It can save embarrassment at a later date if you can identify your senior managers!
Certain aspects of induction training have common themes and have to comply with legal
employment and health and safety issues:
• Introduction to health and safety
• Safe manual handling
• Fire policy and evacuation of the premises

• Child protection
• Cardiopulmonary resuscitation
Introduction to health and safety

Any introduction to health and safety will include an outline of the Health and Safety at Work
Act, which was described in Chapter 4. This act places legal obligations on both employers
and employees. Employers will outline the local policies and procedures that are in place to
comply with this legislation and highlight the responsibilities of the employee to uphold these
local rules. All employees should also be given the information needed to research their own
health and safety concerns, such as access to health and safety publications (Figure 4.2).
TABLE 20.1 Examples of opportunities for professional development in the workplace
Work-based learning
Learning by doing
Case studies
Reflective practice
Audit of patients
Coaching from others
Discussions with colleagues
Peer review
Gaining and learning from experience
Involvement in the wider work of your employer (for example, being a

representative on a committee)
Work shadowing
Secondments
Job rotation
Journal club
In-service training
Supervising staff or students
Visiting other departments and reporting back
Expanding your role
Significant analysis of events
Filling in self-assessment questionnaires
Project work
Evidence from learning activities undertaken as part of your progression on the NHS
Knowledge and Skills framework (KSF)
20.2 OPPORTUNITIES FOR PERSONAL DEVELOPMENT IN THE WORKPL ACE 531
Manual handling
Local policies will define what manual handling training is necessary for employees as part of
their legal obligations. All manual handling must be carried out safely to protect the employee
against serious injury. Manual handling is often subdivided into lifting people and lifting inani-
mate objects such as heavy boxes, awkward items of furniture, rubbish containers, or com-
puter equipment. However, as laboratory staff are rarely required to lift patients, the manual
handling in regard to direct patient care rarely applies.
Fire policy and evacuation of premises

It is essential that all employees are aware of the local procedure to be followed if they detect a
fire and need to raise the alarm or if they have to evacuate the premises in response to a fire
alarm. Among the first tasks of a new employee is to become familiar with fire exit routes, fire
alarm points, and the fire fighting equipment available in the workplace.
Child protection
Local policies to protect both children and adults working with children against the physical,
mental, and emotional abuse of children are in place. Naturally, all local policies, like health
and safety policies, must comply with the wider legal framework.
Cardiopulmonary resuscitation
Training in cardiopulmonary resuscitation techniques is often a requirement for anyone
employed in healthcare professions. All employees within healthcare must be aware of their
individual responsibilities should they find a patient requiring urgent medical assistance.
Within the NHS, different levels of training apply to different groups of staff.
Human resource issues

Issues related to human resources are also opportunities for employers to introduce policies
that may relate to:
• Sickness and absence procedures

• Payroll queries
• Occupational health and vaccination procedures
• Holidays
• Dealing with the press
• Disciplinary and grievance procedures
Sickness and absence procedures

Sickness and absence procedures give clear instructions about the behaviour expected if
employees are too ill to attend work or if there is an unforeseen emergency that prevents
them from attending work. It is essential that employees follow such policies and are made
aware of the consequences to the department if they fail to comply. Staff sickness is monitored
by human resource and occupational health departments, and also may affect the pay of the
individual concerned.
Payroll queries
Pay is always an emotive subject and new employees must understand the information given
on their payslip. They must also be aware of the procedures in place to clarify pay issues, for
example, what should be done if there is a discrepancy between pay given and that expected.
Most employers will explain taxation and national insurance procedures and their own system
for giving extra payments, for example, when overtime has been worked.
Occupational health
Occupational health is the department that is devoted to the health and wellbeing of employ-
ees. Naturally, they are involved in health and safety aspects of the workplace, set policies for
preventative medicine (health screening and vaccinations), and take a non-biased approach
when employees may become unfit for their duties or return to work following prolonged
illness.
Holidays
Holiday entitlement is usually written into a contract of employment, but employers and
departments are likely to have their own individual policies regarding booking holidays in
advance and to record any leave taken. Annual leave is the predominant reason for taking
holiday from work, but there are other reasons that an employee may be able to take time
off work without encroaching on their holiday entitlement. It is therefore necessary to be
aware of one’s full holiday entitlement. Other discretionary leave may include jury service,
carer leave, and bereavement leave.
Dealing with the press

Most employers have a policy in place that employees should follow when dealing with the
press. Frequently, employees are not able to talk to the press without appropriate permission
and to do so is a disciplinary matter. This also applies to all organizational human resource
issues.
Disciplinary and grievance procedures

All employers have their own disciplinary and grievance procedures, which must be consistent
with current employment law. Employees must be made aware of basic employment law but
also of their employers’ individual policies, as there are certain channels to follow if they are
unfortunately embroiled in a difficult situation. Cases that go to court or to an employment
tribunal are frequently won or lost on procedural issues, rather than on what has actually
happened; it is therefore desirable to resolve disciplinary and grievance issues at the earliest
possible stage.
20.3 Training and development

Most employers have a training and development department that provides access for all
staff to a variety of generic training courses, appropriate to their roles within the organization.
It is always worthwhile to be aware what training may be on offer from the employer outside
the immediate laboratory environment. Many courses are available on request, and it may be
stating the obvious, but often you will only get what you ask for. When agreeing a personal
development programme, using training facilities that are readily available within the organi-
zation can reduce cost and is more likely to be agreed.
20.3 TR AINING AND DEVELOPMENT 533
Following the initial broad induction given to a new employee that was described in the previ-
ous section, a specific induction programme is usually provided by the individual’s department.
This covers all of the statutory requirements, health and safety, visitors to the department,
confidentiality, dealing with telephone calls, as well as specific human resource issues that
relate to the department. These points would include who to contact when telephoning the
department to say you will be late, who to liaise with when arranging holidays. All of these Cross reference
more informal pieces of information are specific to a place of work and are opportunities for Chapter 3 ‘Communications in
personal development, as not only are employers never exactly the same, it is unlikely that laboratory medicine’.
two departments within a single organization will be identical.
Leading on from what might seem an initial overload of information, it is now time to get
down to applying yourself to your professional work!
To satisfy the requirements of Clinical Pathology Accreditation UK Ltd (CPA) standards associ-
ated with training, it is necessary for every department to keep training records that provide
evidence that an individual has been trained and has had their competence assessed. The
proof of training and evidence of its success will form a large part of any biomedical scientist’s Cross reference
personal development portfolio. Reading the background of many of the more interesting You can read more about CPA
issues and finding information about many of the daily laboratory procedures and workload in Chapter 19 ‘Quality assurance
also gives many opportunities for reflective learning (Section 20.7). and management’.
It is clear that personal development is an ongoing process that arises naturally during the
course of a professional person simply doing his or her job at work. This is an age of evidence
collection: it is essential to document all learning and development as they arise and to keep
appropriate records to satisfy statutory legislation, the needs of the employer, and those of the
individual for either personal interest or professional registration (Section 20.1).
Additional employer-based CPD activities

Many employers offer a variety of other opportunities for professional development and
any available should be sought out. Many clinical laboratories provide access to presenta-
tions, lectures of interest by visiting speakers, opportunities to visit other departments in the
organization to find out more about its wider activities in a more informal setting than that
presented at initial induction. Again, just because something is not immediately offered, it
does not mean that it is not possible or that it does not already exist. Medical staff are usually
happy to explain interesting clinical cases and biomedical scientists are often able to accom-
pany them on visits to the wards. However, it is often up to the biomedical scientist to take
the initiative and ask!
Tutorial programmes
If an ongoing tutorial programme is not available in the workplace, the obvious question is,
why not? Is it simply because no one has bothered to organize one? It may be that several
individuals would be prepared to speak to their peers on their favourite subject. It need not
be too difficult to find a venue and put together a rotation of several lectures covering the
most recent advances in the pathology disciplines. Newly qualified staff could discuss their
degree projects, those currently studying postgraduate qualifications could pass on knowl-
edge of a particularly exciting area of research. Often a project such as tutorial organization
only requires a leader, who could be any member of staff with the right amount of enthusiasm,
participation is likely to follow.
Journal clubs
Journal clubs involve an analysis of an appropriate article that has been published in a relevant
journal. A particular journal article is selected in advance and all participants read this article
and enter into a (usually) lively debate about it, or a single participant reads an article and then
presents a review of it to the other members of the club for discussion. Members of a journal club
usually participate equally. Again, you do not have to be a senior member of staff to instigate such
activities. The skills required often have nothing to do with laboratory management and much
more to do with gentle persuasion and being able to stimulate enthusiasm in colleagues.
20.4Professional bodies and

personal development
All biomedical scientists in employment will have many opportunities to develop professional
contacts with others in the field. These contacts present opportunities for a variety of ways for
personal development, a number of which you can see listed in Table 20.2.
Naturally, the majority of biomedical scientists belong to their own professional body, the
IBMS (Figure 1.2), which offers a variety of services to its members (Figure 20.3). The IBMS
TABLE 20.2 A number of the personal development activities associated

with professional contacts
Professional activities
Involvement in a professional body
Membership of a specialist interest group
Lecturing or teaching
Mentoring
Being an examiner
Being a tutor
Branch meetings
Organizing journal clubs or other specialist groups
Maintaining or developing specialist skills (for example, musical skills)
Being an expert witness
Membership of other professional bodies or groups
Giving presentations at conferences
Organizing an accredited course
Supervizing research
Acting as a national assessor
Promotion
20.4 PROFESSIONAL BODIES AND PERSONAL DEVELOPMENT 535
FIGURE 20.3
Membership files of the Institute
of Biomedical Science.
(see also Box 20.2) recognizes the crucial role of CPD and has many resources available to its
Cross reference
members in all membership classes.
You can read more about CPD
The areas where the IBMS most directly contributes to CPD are education, journal-based learn- activities in Chapter 2 ‘Fitness to
practise’.
ing ( JBL), structured reading schemes, and by organizing regional and national meetings.
Education and the Institute of Biomedical Science

The IBMS is active in a range of educational activities (but see also Box 20.3) including
accrediting a number of postgraduate professional qualifications. These were introduced in
Chapter 2.
These qualifications are widely recognized by both employers and by higher educational insti-
tutions. Success in studying for any of them indicates that a biomedical scientist is willing to
further their personal development. If you want to progress to the higher levels of the pro-
fession, such further study is increasingly necessary to demonstrate knowledge and skills at
high levels of competence. Thus, participation in postgraduate learning is advisable, although
BOX 20.2 Biomedical scientists

and professional bodies
The IBMS is not the only professional organization to which a biomedical scientist can
belong. There are common interest organizations that are not necessarily professional
bodies. Amongst these are the British Blood Transfusion Society and the British Society
for Immunology. All these specialist societies and groups organize series of lectures
and meetings for like-minded individuals, and attendance at any such event is an oppor-
tunity for CPD.
BOX 20.3 Non-IBMS providers of

education in the biomedical sciences
Any opportunity to learn new skills or learning about other disciplines constitute CPD.
Of course, even taking a postgraduate qualification in the same area of practice, such
as a biomedical scientist choosing to study for a Masters Degree in their own subject,
constitutes PD.
Many providers of education for biomedical scientists provide courses other than the
established graduate and postgraduate degree courses. There are also specialist courses
available that are appropriate for the non-science based areas of biomedical science.
Many universities provide a series of short courses for special interest groups or to allow
biomedical scientists to venture out of their particular disciplines. Thus, for example,
haematologists may take short courses in clinical chemistry. There are also opportuni-
ties to attend courses in management topics. These may be short courses in specific
areas such as audit, finance, or tutoring, or an extended postgraduate management
course lasting several years.
Many colleges and universities offer not only taught courses but also opportunities for
distance and on-line learning in areas such as quality management, point of care testing
and training and development of others.
not yet mandatory. However, completion of the registration portfolio is mandatory for ini-
tial registration with the HPC. Once registered, there are many opportunities for professional
education.
The IBMS as a professional body has developed a comprehensive set of professional qualifica-
tions. The Specialist Diploma is awarded for the successful completion and assessment of the
specialist portfolio, which is based on detailed knowledge and the presentation of evidence
collected to meet a set of standards in one of the following subjects:
• Cellular pathology
• Cytopathology
• Clinical biochemistry
• Clinical immunology
• Haematology and hospital-based transfusion practice
• Histocompatibility and immunogenetics
• Medical microbiology
• Transfusion science
• Virology
Building on from the Specialist Diploma is the Higher Specialist Diploma. This is aimed at prac-
tising biomedical scientists who have skills and theoretical knowledge at EQF level 7. They
are normally actively performing in a complex role, and are continuously developing a clini-
cal, scientific, or management responsibility for a laboratory service. The Higher Specialist
Diploma is open to suitable candidates who already have an accredited MSc or an approved
M-level (EQF level 7) qualification and who are able to pass a series of written examination
papers following a period of self-directed study. The Higher Specialist Diploma is available in a
series of clinical laboratory specialities and, in addition, Management and Leadership.
The IBMS also offers a series of Certificates of Expert Practice, which enable biomedical scien-
tists with highly specific sets of skills and knowledge within their own specialist area to dem-
onstrate their command of a sub-speciality.
Moving forward from these certificates are further specific IBMS qualifications in sub-
specialities at differing educational levels. These include Diplomas of Expert Practice and
Advanced Specialist Diplomas. These diplomas are designed to provide evidence of the highest
level of professional knowledge and skills.
Differing levels of knowledge and skills may be required within a particular job description
or person specification for a specific post. However, the only mandatory qualification for a
biomedical scientist is the completion of an HPC approved degree or the registration port-
folio alongside an IBMS accredited degree to meet the threshold standards of proficiency for
registration with the HPC. All other qualifications and educational activities in the wider sense
(Table 20.3), while desirable in terms of proof of personal development and as aids for promo-
tion are not mandatory and the decision to complete further studies after gaining a BSc degree
is the personal choice of the individual.
SELF-CHECK 20.2
What does the IBMS suggest a new biomedical science graduate do next by way of profes-
sional qualifications if they are already in full-time employment?
Journal-based learning
In JBL, the participant is expected to read a current paper published in a recognized scientific journal
in a named area of biomedical science. The reference to the paper is supplied by the CPD provider
to the participant. However, unless the paper is in an open-access journal or in a journal belonging
TABLE 20.3 Educational activities constituting

Formal and educational qualifications
Additional attendance at courses
Further education
Research
Attending conferences
Writing articles or papers
Going to seminars
Distance learning
Attending courses accredited by a professional body
Planning or running a course

FIGURE 20.4
Front cover of a past issue of The Biomedical
Scientist. Copyright © Institute of Biomedical
Science.
to the CPD provider as in Figures 2.4 and 20.4, the participant must obtain their own copy of the
relevant paper (see Section 20.9). Any such papers provided by a CPD provider would be an expen-
sive process and may be a breach of the copyright. The chosen paper may be either scientific or
relate to a topical management issue. Typically, a CPD provider such as the IBMS then gives a list
of 20 questions related to the article, which are usually printed in The Biomedical Scientist, British
Journal of Biomedical Science, or on the IBMS website. Each question can be answered as either
true or as false. The answers to all the questions can be found in the information given in the paper.
The participant can submit his or her answers to the IBMS by post or via its website (Figure 2.5).
The answers are marked, with 17 correct answers constituting the minimum pass mark.
Structured reading
Structured reading, another activity instigated by many CPD schemes including the IBMS, is an
essay writing programme organized on a defined timeline (see also Section 20.8). A series of
essay titles are published quarterly in The Biomedical Scientist. The essay titles cross all pathol-
ogy disciplines, including management topics. The questions are searching and require up-to-
date research of current literature, some of which is supplied with the title.
The participant is invited to attempt any of the titles, so may decide to take on the extra chal-
lenge of detailed learning in a discipline that may not necessarily be their specialist subject and
will be expected to write a postgraduate level, properly referenced essay. The essay is marked
by an IBMS-appointed peer assessor who will grade it as a pass, borderline, or fail. A pass grad-
ing is proof that the topic has been studied in detail and the subject matter understood.
Key Points
Structured reading is also good preparation for professional qualifications such as a
Higher Specialist Diploma.
Professional meetings
The IBMS organizes and hosts both regional and national meetings (Figure 20.5). These meet-
ings may be small events for a specialist interest group in a local area or, at the other end of
the spectrum, may be a full multidiscipline conference at a central venue lasting several days.
The former types of events are often arranged by a willing volunteer for a small group of peo-
ple who may have a common interest. Larger meetings are usually sponsored by commercial
companies, and attendance gives opportunities to see the latest products and technologies
and to network with fellow biomedical scientists from other hospitals, as well as listening to a
formal lecture, usually by experts in their own field, or to develop ones expertise by talking to
senior, more experienced participants.
FIGURE 20.5
Scientific meetings such
as those organized by the
Institute of Biomedical
Science, provide excellent
opportunities to discuss all
aspects of the subject.
20.5Commercial concerns and

Many of the commercial concerns that supply equipment, consumables, and services provide
both short and long courses. It is possible to learn presentational and telephone skills, time
management, and many other skills that may not be specific to biomedical science or even
employees in broader healthcare organizations of the public or private sectors. Many com-
panies will offer generic courses on business premises, or many even come to a workplace
and give courses to several members of staff in the same department. This is a cost-effective
method for the employer to train several members of staff simultaneously, so again, if there
is nothing already in place where you work, it might be worth making a suggestion to your
managers.
Companies that supply scientific instruments and laboratory computer systems also offer
training. Often the courses come free of charge with the installation of newly purchased
equipment, but additional courses can usually be purchased as separate items. When a new
instrument is installed, the person who attends the training will usually become its ‘key opera-
tor’, and be the link between the laboratory and the company that supplied the instrument.
This arrangement usually works well until the key operator leaves to work elsewhere. The
remaining staff are then left to muddle through; their success in this depends on how much
knowledge was given to them before the key operator left. This situation presents an oppor-
tunity for another member of staff to receive full training from the company and again, this
is an excellent opportunity to gain in-depth knowledge into the inner workings of laboratory
equipment and thus for personal development.
Many of the major instrument and laboratory information technology (IT) suppliers also
organize meetings and user groups to update the knowledge of laboratory workers. Not only
is this an opportunity to network with colleagues, it brings together users of common equip-
ment from different sites and so is often an excellent opportunity to assess the different ways
in which one’s own instrument can be utilized.
Trade unions
Trade unions have a commitment to personal development and lifelong learning. Many offer
courses for their own members that focus on basic skills for lay persons and the development
of union representatives. Funding is available through the trade unions to develop basic skills
such as numeracy and literacy, and where English is not the first spoken language, opportu-
nities to improve spoken English. All employees should be able to attain a basic standard of
education to help their progress in the workplace; trade unions are able to act as vehicles to
help achieve this.
Many professionals are not only members of a professional body, but also of an appropriate
trade union. The professional activities undertaken by the trade unions often support and
complement those undertaken by professional bodies, and are rarely in complete contradic-
tion to one another. Trade unions offer many excellent courses in, for example, employment
and health and safety law, presentation, and negotiating skills.
The trade union philosophy is that their members in the workplace should have access to the
same level of information that is available to the employer. All management skills, regardless
of how they were acquired, are opportunities for personal development. The government fre-
quently asks for the support of trade unions to disseminate information, knowledge, and skills
20.7 REFLECTIVE LEARNING 541
to their members. Indeed, many of the lower level educational courses run by trade unions for
their membership are able to take place only because of initial government sponsorship.
20.6Opportunities for self-guided

Opportunities for personal development do not just occur in the workplace or a formal edu-
cation situation. Watching television, reading a newspaper or a magazine, or surfing the inter-
net will present professional information, even if you are not immediately aware of it. Learning
in this way is equally important and a brief paragraph of writing after reading an article in a
Sunday newspaper or watching a television documentary is perfectly acceptable as a record
for additional or unstructured CPD.
20.7 Reflective learning

Reflective learning is a mechanism of critically examining an event, situation, or experience
after it has taken place, thinking about what has happened, the context in which it happened,
and what can be learnt from the whole experience. Personal development constitutes some-
what more than listening to lectures and attending training courses. The most important
aspect of personal development happens after the learning event when there is an oppor-
tunity to really use what has just been learnt. Reflective learning is an excellent mechanism
to really think about the new skills and knowledge acquired and identify where they can be
applied in everyday procedures.
Several templates are available to help with reflective learning but, in essence, an individual
should start by asking themselves a series of questions, as shown in Table 20.4, and then try to
use their new knowledge to answer them.
TABLE 20.4 Question relevant to reflective learning
Possible questions after an incident Possible questions after a period Possible questions after a conference
in the laboratory of training
What actually happened? What was the purpose of the training? What was the purpose of the conference?
Were all policies and standard operating What did I learn? What did I learn?
procedures (SOPs) followed?
If not, what was the deviation? Was the learning useful? Was the learning useful to me?
Was the deviation avoidable? Can I change my current practices to Will the learning be useful in my own place
incorporate what I have learned? of work?
Was the outcome better, worse, or the Will my future practices benefit from Do I need to look at any of the issues in more
same as the expected outcome? these changes? detail to identify where they apply in my own
place of work?
What lessons have been learned Do I need to inform anyone else of my new
from the event? knowledge in order to be able to make
changes?
Should the SOP be changed? Will the changes be of benefit to the service?
Clearly, each period of personal development may be equally important in terms of acquiring
new knowledge and skills that may enhance the activities of the department or allow changes
to the service it provides to be carried out more smoothly. However, the most beneficial devel-
opment to health services occurs when the personal development of employees brings about
changes that provide real benefits to the treatment and experiences of patients. Reflective
practice is a means of exploring how new personal knowledge and skills can be used for the
enhancement of the patient pathway. Thus, reflective practice can run alongside and enhance
many of the other ways of CPD already mentioned; so attending a conference is not only an
opportunity to learn, for example, cutting edge science, but also to reflect afterwards as to how
the newly acquired knowledge can be used to enhance the workplace for the benefit of all.
20.8 Study skills

Study skills can be put into practice informally and formally. Skills for general study and per-
sonal development are used almost all of the time and in everyday learning. For formal assess-
ments of study, when an individual is asked to provide evidence of study such as submitting
coursework or sitting a written examination, more structured skills are needed. Four basic
groups of study skills are outlined in Table 20.5; each of course overlaps considerably.
General study skills are necessary for even the most basic academic study and are taught as part
of the school curriculum. For those wanting to return to study in later life, the lack of general study
skills is an enormous disadvantage over their younger peers. Younger biomedical scientists and
laboratory employees often have an advantage over some of their more mature colleagues.
The basic study skills listed in Table 20.5 should be acquired easily by anyone working towards
or in possession of an H-level (EQF level 6) qualification (see Introduction). Indeed, if you are
working towards an honours degree in biomedical science, or are already a graduate with
an approved degree for registering with the HPC, or a graduate needing to obtain additional
units for HPC registration, you will most likely be familiar with them. The first consideration is
the basic skills that should be familiar to anyone who has recently been part of any education
system, regardless of the level of academic achievement. These are the skills needed for any
learning, academic or vocational qualifications, and even for leisure pursuits and hobbies. We
will now expand on a number of them.
TABLE 20.5 Basic study skills
Basic study skills for General study skills Study skills for producing Revision and exams
everyday life for learning coursework
Reading Literary skills Producing coursework Assessing objectives
Writing Collecting data Gathering information Revision
Verbal communication Essay writing Research Exam technique
Using a library Case studies
Information technology skills Report writing

Citing references
Working as a team member Using the web
Numeracy Mathematical skills

20.8 STUDY SKILLS 543
Literary skills
Literary skills include the abilities to read, use language in listening and speaking, and to write.
Good communication skills are essential when working in a laboratory. Appropriate levels of
English language skills, written, and verbal, are needed for the safe communication of results,
instructions, and information. Basic literacy is essential for biomedical scientists at every level. It is
unlikely that anyone could maintain a laboratory position without literary skills equivalent to that
taught at secondary school. However, a higher level of English language and grammar is needed
to study for an H-level qualification (see Introduction), so if you are working towards or are in pos-
session of a biomedical science degree, this will give others an indication of your ability to com-
municate in English. H-level qualifications require more than the ability to communicate in the
required standard of English, you must also be able to undertake academic work, such as taking
notes, writing essays and projects, present data, and produce reports at an appropriate level.
Staff in a potentially dangerous working environment, such as a laboratory, must be able to read
health and safety notices, SOPs, and be able to differentiate between identification of samples,
chemicals, record sheets, and manuals. Writing skills are necessary to enter data at numerous
points and write safety notices, and policy documents must be read and understood.
Reading
Academic reading is a different activity to reading for pleasure: it requires an effective use of
time to obtain the maximum benefit from a book, article, or webpage in the shortest possible
time. The five steps to effective reading are:
1. Survey
2. Question
3. Read
4. Recall
5. Review
Academic reading is a means of enhancing ones understanding of or gaining basic knowl-

edge about a new topic, or finding alternative views about a particular topic and to help with
further studies. The reading, however, needs to be selective. Thus, skim reading the first few
paragraphs of a book, studying its index or the summaries of chapters will all give clues as to
the relevance of the contents. Something written in complicated English with indecipherable
jargon is not likely to be worth the time spent trying to unravel it. It should be easy to grasp the
key facts and to differentiate between these and the author’s own ideas.
It is also pertinent to look at the date of publication and the identity of the author; scientific
writing can quickly become outdated and authors can become discredited by their peers.
Academic publications may be expensive and are therefore frequently borrowed, rather than
purchased unless it is a key book or journal. Workplaces, friends, and libraries (Figure 20.6) are
inexpensive sources of academic books, particularly if you are not going to use them frequently.
This does mean that they have to be returned so there must be a maximum reading benefit in a
fixed amount of time. While it is useful to make notes of key points while reading, it is counter-
productive to copy out vast chunks of text with limited understanding of the topic.
Note taking
Notes may have to be taken from books, during demonstrations in the laboratory, and of
course, in lectures and tutorials (Figure 20.7). This is an excellent way of staying focused on
FIGURE 20.6
Undergraduate students
reading and working in a
university library.
(a)
(b)
FIGURE 20.7
Photographs of undergraduate
students studying in (a) a
formal lecture and (b) the
more informal setting of a
tutorial.
20.8 STUDY SKILLS 545
what you are supposed to be learning, except when you are expected to write reams of dic-
tated text! Notes should be concise and be an aid to self study. There is no need to write
verbatim from a lecturer or speaker.
Many ways of taking notes have been suggested; it is best to try different techniques to find
which one works for you. Notes may be linear, in sentences, or expressed only as key words.
They may take on more of a picture form, with a central theme in the middle of a page and
ideas and topics radiating outwards. You may wish to develop your own form of shorthand. In
essence, the only person who has to comprehend and use your notes is you, so it should not
matter how illegible they are to others as long as they aid your understanding of a topic, help
you to remember the points, and act as a revision prompt.
Essay writing
Essays are not simply collections of written facts; they need structure and to be a response to
a specific title or theme, and demonstrate a depth and breadth of knowledge on a specific
subject. They should be written in English prose with correct spelling and grammar.
Essays should be planned from the outset and not be a collection of summary paragraphs.
They require a clear introduction in the opening paragraph, followed by the main portion or
body of the essay, and to end with a summary or concluding last paragraph. The body of the
essay should be a collection of related paragraphs, all linked to the title of the essay. Wherever
possible, use examples to illustrate the points of the essay. Care should be taken to avoid
merely listing facts. A ‘good’ essay will demonstrate knowledge of a subject from more than
one viewpoint and contain a critical appraisal of a variety of concepts or arguments.
SELF-CHECK 20.3
What features would be shown by an essay that gained high marks?
Case studies and writing reports

Case studies and report writing are one step on from essay writing. Case studies are commonplace
in biomedical science and the success of a well presented case study is demonstrated by being able
to present the key facts of the topic in an interesting and understandable manner, while removing
much of the peripheral and often irrelevant material. A case study will include some mention of the
theory behind the case and there should be a blend of theoretical knowledge with factual data.
Report writing is also commonplace in biomedical science and many organizations ask for
them to be written in a particular format or house style. Most reports require an executive
summary or overview of the report presented as short, concise, bulleted points. The body
of the report should include an introduction (including terms of reference if appropriate),
the findings, the conclusions, and any recommendations leading on from the conclusion.
References and appendices should be at the end, and are best kept separate so as not to
detract from the main text. Many reports in science rely heavily on the presentation of data,
and this should be presented clearly with the appropriate statistical analysis and conclusions
for any non-mathematicians who may be required to read the report.
Numeracy
Basic numeracy is an essential skill for anyone working within an area of science, and biomedi-
cal science in no exception. The mathematics that is required for biomedical science centres
on manipulation of numbers in basic calculations, and a reasonable knowledge of the col-

Cross reference lection and interpretation of statistical data. The fact that you are already in possession of, or
Chapter 5 ‘Statistics and
working towards, a biomedical science degree gives others a good indication of your current
handling data’.
mathematical abilities.
Using libraries
Libraries are essential to anyone studying for their CPD. It is worth getting to know the
library and its librarians, particularly if you use it regularly, either for study or for pleasure.
Understanding the layout of a library and the services available to you will make using the
library more effective and less stressful. Contrary to the myths and stereotypes surrounding
librarians, the reality is that most are knowledgeable and willing to help when approached for
advice. In other words, it will pay dividends to make friends with yours!
Libraries are usually calm, quiet places and often offer a better study environment than
home or the immediate workplace. They are not only a source from which books can be
borrowed, but also have reference material, journals, and periodicals that can be used on the
premises. If a particular book or journal is not readily available, they can usually be obtained
through the inter-library services, so that if one library is unable to provide a copy of some-
thing in particular another can usually be contacted to supply the required book or article.
Libraries are able to supply copies of articles from scientific journals and if you want to take
part in the CPD activity of JBL (Section 20.4) you will have to source the journals needed to
participate.
SELF-CHECK 20.4
What facilities are likely to be provided by local authority libraries?
Information technology
The ability to use IT is now as important as basic numeracy and literacy. Children are taught IT
skills as part of everyday school life and it features high on the national curriculum from the age
of 4 years upwards. Some students of biomedical science may find themselves out of their depth
compared to others, as IT skills have only been taught as a compulsory part of education for a
relatively short time. Information technology is an essential aid to biomedical science. Databases
are part of everyday life and the use of spreadsheets and word processing for study is virtually
universal. Anyone who has not received training in the use of IT to store, retrieve, and present
information is extremely disadvantaged. The electronic transfer of information and the use of
electronic methods of communication are central to the operations of the NHS. Information
technology skills are frequently offered by employers as part of staff development and there
are many providers of adult education in the community that offer a variety of courses for the
beginner to the advanced student.
Team work
In the past, traditional teaching focused on the individual, but with the introduction of key
skills in compulsory education, working as an active member of a team has become a skill that
employers seek in their employees. Thus, further and higher educational institutions promote
20.9 EVIDENCE SUPPORTING PERSONAL DEVELOPMENT 547
working with others as a key skill. The advantages of teamwork are that while everyone has
clear and common objectives, an appropriate mix of people will ensure that everyone will be able
to make unique contributions to the efforts of the team as a whole. Teamwork is not confined
to the everyday functioning of the workplace but may extend into study and common interest
groups, multidisciplinary case conferences, and leisure activities.
Academic study skills

Academic study skills require assessment of course work and examination papers to monitor
progress, and all students must spend some time finding out how to get the best results based
on the requirements of the awarding body. Remember academic institutions are there to pass
students, rather than fail them, as a high pass rate reflects well on the teaching of the college
or university. Lecturers and tutors are in the business of helping their students to achieve the
highest possible standards. All help and advice given by tutors should be gratefully received
and stored away for future good use.
Under- and postgraduate courses usually rely on two types of assessment: coursework and a
written examination. Coursework must have defined learning criteria in the form of aims and
assessed outcomes, and students should be familiar with the principles by which the course-
work will be assessed. The content of coursework sometimes allows the student a degree of
freedom in choosing the topic and when this is the case, it is obviously better to base it on an
area of personal interest wherever possible. Written examinations assess knowledge and skills
at the end of a block of learning. Examinations should demonstrate what the student knows;
they are not designed to highlight what is not known. They do, however, require careful revi-
sion and preparation. Taught courses follow a specified syllabus or curriculum; knowledge of
its contents will give an indication of what material will be examined and the form the examin-
ation will take.
Assessment by portfolio is becoming increasingly popular, as this involves collecting evidence

to show that you have met specific learning objectives. Most biomedical scientists who wish
to register with the HPC should have had experience in completing the registration portfolio
themselves. More experienced members of the profession have probably been involved in
helping others to collect evidence and construct a portfolio in a clear and logical way. If you
have not yet completed your initial portfolio for professional registration, you will become
familiar with this method of assessment during your time as an undergraduate. Formative
assessment is the regular and on-going assessment during a course of study. Summative
assessment is the final or end-point assessment stage.
20.9Evidence supporting
It is essential to keep records of all activities that can be offered as proof of personal develop-
ment when asked. Table 20.6 lists many activities that provide evidence that a biomedical sci-
entist has been able to learn new skills or improve him or herself in some way. It is advisable to
keep copies of all documents that you have prepared or have been involved in the preparation
of, and to retain attendance certificates, copies of presentation handouts, notes made during
seminars, or summary documents following reading or research.
TABLE 20.6 Types of evidence showing the ability to learn new skills
Materials you may Materials obtained Materials showing reflection

have produced from others and evaluation of your learning
and work
Information leaflets Testimonies, Adapted documents arising
testimonials from appraisals, supervision
reviews
Case studies Letters from service Documents produced

users, carers, students, following local or national
or colleagues schemes related to CPD
Critical reviews Course certificates Evaluations of courses and

conferences attended
Adaptations of student Personal development

notes plans (PDPs)
Policies or position Approved claims for credit

statements for previous learning or
experience
Discussion, procedural,
and course programme
documents
Documents about national

or local processes
Recent job applications
Reports on project work,

clinical audits, reviews
Business plans
Guidelines for dealing with

patients
Course assignments
Action plans
Presentations, journals
articles, and research papers
Questionnaires
Funding applications
Induction materials for new

members of staff
Learning contracts
Contributions to the work

of a professional body or of
a special interest group
20.10 PERFORMANCE APPR AISAL AND PERSONAL DEVELOPMENT PL ANS 549
It is clear from Table 20.6 that not all types of professional development are accessible to all
grades of staff, but individuals should be aware and take advantage of, every available oppor-
tunity to professionally develop themselves.
Performance appraisal and

20.10
personal development plans
Many employers, both in the public sector and in the commercial world, integrate the per-
sonal development of employees into their organizational culture. They ensure that employ-
ees have a framework that they can work within to formally develop themselves in ways that
are of benefit to both the employer and the individual.
There is an organizational structure for targets to be set and performance against the targets is
monitored and reviewed. Usually a structured mechanism is established where an employee
is able to meet with his or her manager (or a representative of the employer) on a regular basis
to assess their past performance. The first of these meetings normally occurs soon after the
employee begins working in their new post and then usually at regular six-monthly or annual
intervals, in accordance with employment policy at the particular place of work. These targets
must be non-discriminatory and achievable, so that you (as an employee) are able to pass
comment and agree to them.
Performance appraisal is not an opportunity for employers to set unreasonable targets or

unfair additional tasks onto an individual that are not appropriate to an existing job descrip-
tion for a post. Nor should it be used as part of a disciplinary process. While it is an opportu-
nity to identify poor performance, it should be done so with the intention of offering suitable
training and support to enable the individual concerned to improve and work towards meet-
ing targets.
The NHS has introduced a programme or national strategy, called the Knowledge and Skills
Framework (KSF), to standardize knowledge and skills across all the staff groups that it cur-
rently employs. This scheme does not apply to biomedical scientists working within the pri-
vate health sector or commercial world, but many of these employers already have similar
schemes in place. The principles of KSF are that staff in different departments and even in
different organizations, but who essentially have the same role, are measured against a series
of generic standards appropriate to their grade of employment, with the aim of ensuring con-
sistency throughout the NHS in terms of expectations and rewards.
All NHS posts have a series of KSF standards that employees have the opportunity to work
towards completing. Performance appraisals are usually carried out by an immediate line
manager. This should be a two-way process in which both the appraiser and appraisee par-
ticipate equally. Performance appraisal is a structured and formal period where the NHS
employee has the opportunity to discuss how he or she is performing in line with the set KSF
standards, and other local targets specific to the position in the laboratory. It is also the time
to identify where extra help may be needed or to highlight an area that the employee may
wish to develop further. This is then formally written in to a personal development plan
(PDP). Having made reasonable requests in the PDP, the employer, both the NHS Trust and the
line managers, then have a duty to help the person concerned achieve the areas of personal
development highlighted. Performance appraisals are also conducted to ensure that requests
from employees for opportunities for personal development are being met.
The system of performance appraisal and PDP is therefore a two-way process: it should benefit
both the employer and employee. It should be an opportunity for the employer to encourage
and support staff to develop themselves as individuals, while giving added value to the service
that the member of staff is able to provide; in short it is part of the cycle of continuing profes-
sional development of the individual (Figure 20.8).
Reflect on current
state of professional
development
Continue professional
Recognise:
development to enhance
strengths
strengths, develop new
and
abilities and rectify
weaknesses
weaknesses
FIGURE 20.8
Overview that emphasizes
Specify actions that
the never ending process produce and develop
of continuing professional new strengths and
development. eliminate weaknesses
SUMMARY
■ Personal development is an ongoing process
■ The Institute of Biomedical Science has a variety of schemes and qualifications tailored to
the needs of biomedical scientists and, while not mandatory, it is advisable to take part in
a variety of continuing professional development activities
■ The Health Professions Council has the mandatory requirement that all its registrants must
be continually involved in professional development activities and be able to provide evi-
dence to support this when asked
■ There are always opportunities for personal development wherever you choose to seek
them: at work, home, within organizations and academic institutions
■ The skills needed to develop both professionally and personally are varied and extensive,
and can be adapted to suit any style of learning
QUESTIONS
20.10 PERFORMANCE APPR AISAL AND PERSONAL DEVELOPMENT PL ANS 551
FURTHER READING
● Dryden G, Vos J. The Learning Revolution. The Learning Web, Torrance, CA, 2001.
An inspirational guide for anyone involved in learning, which explores various aspects to
effective learning in an innovative and thought provoking manner. Suitable for teachers
and students alike.
● Institute of Biomedical Science. Good Professional Practice for Biomedical Scientists,
2nd edn. Institute of Biomedical Science, London, 2005.
● Wood J. The roles, duties and responsibilities of technologists in the clinical laboratory.
Clinica Chimica Acta, 2002; 319: 127–132.
● Pitt SJ, Cunningham JM. An introduction to biomedical science in professional and
clinical practice. Wiley-Blackwell, Chichester, 2009.
Chapters 1 and 8 ‘Introduction to a career as a biomedical scientist’ and ‘Development of
knowledge and competency for biomedical scientists’, respectively, are worth browsing
through.
● Wilson G. Continuing professional development: current understanding and practice.
Biomedical Scientist, 2010; 54: 168–169.
Useful short review.
Useful Websites
■ www.sussex.ac.uk/academicoffice/documents/qaafram
■ www.ibms.org
■ www.bized.co.uk/reference/studyskills
■ www.dh.gov.uk/en/Publicationsandstatistics/Publications/PublicationsPolicyAnd
Guidance
■ www.hpc-uk.org. This is the official website of the Health Professions Council. Publications
detailing the latest professional documents relating to CPD requirements are available to
download.
The following documents are particularly useful:
■ Continuing Professional Development and your Registration.
■ Continuing Professional Development and your Registration: Appendix 1
■ Continuing Professional Development and your Registration: Appendix 2
■ Demonstrating Competence through CPD
■ Standards of Conduct, Performance and Ethics
QUESTIONS
1. The Institute of Biomedical Science does NOT offer:
(a) journal-based learning

(b) specialist M-level academic qualifications directly related to aspects of biomedical
science
(c) access to a full-time doctoral programme

(d) assessment of essays based on structured reading
(e) information regarding suitable courses leading to accredited biomedical science
degrees
2. Performance appraisal is an opportunity to discuss one’s progress with a line manager.

Which of the following is NOT suitable for discussion at this type of meeting?
(a) Aspects of your everyday work that you enjoy and wish to develop
(b) Aspects of your everyday work that you find particularly difficult
(c) Funding for a postgraduate course at a local university
(d) Introduction of a new method that you have read about in a journal
(e) The behaviour of another member of staff that particularly irritates you
3. Which of the following is NOT true of the Knowledge and Skills Framework?
(a) It applies to professional and non professional groups of people

(b) Is linked to targets set in a performance development plan
(c) Applies to all employees in the NHS
(d) Has been promoted by the Department of Education of the UK
(e) It has been published by the Department of Health
4. Indicate whether the following statements are TRUE or FALSE
(a) The highest academic qualification that a biomedical scientist can achieve is at
M-level
(b) Libraries that are under the control of local authorities only provide a lending
service to the local community and do not routinely offer internet access
(c) The Health Professions Council has a clear policy regarding activities for continuing
professional development that are acceptable if you wish your name to remain on
the Biomedical Scientist register
(d) Only activities that are approved and regulated by an employer are considered to
be professional development
(e) Biomedical scientists are not required to meet minimum standards for numeracy
and literacy
5. List the formal qualifications that are offered by the Institute of Biomedical Science that
are appropriate to a single, specialist discipline.
Glossary
Absorption spectrum graph illustrating the fractions of inci- Anions negatively charged ions that are attracted to the
dent electromagnetic radiation absorbed by a material over anode.
a range of different wavelengths. Anode plate disc in the electron gun of an electron micro-
Acceleration voltage the potential difference in volts neces- scope that is maintained at earth potential and causes the
sary to produce electrons from the tungsten filament. cloud of electrons produced by the filament of the gun to be
Accreditation an inspection and examination procedure to accelerated into the microscope column.
ensure participants comply with published practice and Anode the negatively charged electrode.
documentation standards. Anthropometry study of human body measurement related
Achromatic a lens corrected for two colours only. to capabilities.
Acquired immunity or adaptive response one that produces Antibody immunoglobulin with the ability to bind to a spe-
antibodies specific to antigens on pathogens and develops cific antigen.
an immunological memory. Antigen molecule that can elicit the production of and bind
Activity a measure of an ion’s ability to affect chemical equi- to an antibody.
libria and reaction rates. Antigenic determinant or epitope site on an antigen that is
Activity the rate of decay of a radioactive nuclide. recognized by an antibody.
Adhesion molecules mediate the binding of cells to each Antihuman globulin polyclonal antibodies produced against
other or to other biological surfaces. an epitope on a human immunoglobulin molecule.
Adjuvant substance that enhances the immune response to Antinuclear antibodies antibodies to nuclear proteins.
an antigen. Antiserum one that contains antibodies and may be used as
Adsorption accumulation of molecules from a solution on an analytical reagent.
to the surface of an adjoining solid without penetrating its Apochromatic a lens corrected for three colours and which is
interior. the best quality type of lens available.
Adverse healthcare event incident or omission arising during Approved Codes of Practice explain legal regulations and
clinical care that causes physical or psychological injury to a the detailed requirements necessary to comply with them.
patient. Astigmatism the inability of a lens to bring light or electrons
Adverse incident any event that may affect the reliability of a passing through different parts of the lens to a common
result issued by the laboratory or any delay in the diagnosis focus.
and treatment of a patient following laboratory investigations. Atomic number the number of protons in the nucleus.
Affinity chromatography form of column chromatography Autoimmune disease condition in which the immune system
that relies on the specific binding of the analyte of interest of an individual reacts against self antigens.
to an immobilized ligand.
Autoradiography sensitive method of producing a photo-
Affinity measure of the strength of antibody–antigen binding. graphic image of a specimen containing radioactively
Agglutination clumping together of cells or particles by anti- labelled material(s).
body binding to antigens on their surfaces. Avidin-biotin system Avidin is found in egg white and has a
Allergy inappropriate immune response to normally harm- high affinity for biotin which is a co-enzyme. Together they
less environmental antigens. can form complexes with antibodies, enzymes, radioiso-
Amperometry a form of voltammetry that involves the topes and fluorochromes.
reduction or oxidation of an electroactive molecular species Avidity ability of an antibody to bind multiple antigens.
at a constant applied potential. B lymphocyte type of white blood cell that can form a plasma
Ampholytes synthetic polyamino-polycarboxylic or polysul- cell.
phonic acids of low Mr that differ from one another in the Balancing of a rotor consists of distributing the centrifuge
values of their isoelectric points (pI). tubes in the rotor, i.e. the weight, evenly around the centre
Analyser polarizing filter placed in the light path fixed within of rotation.
the body of the microscope. It is used together with a sub- Barrier filter a filter used in the fluorescence microscope,
stage polarizer. which is only capable of transmitting light in the visible
Analyte specific substance in a sample under investigation to spectrum and which prevents the transmission of ultraviolet
be identified and/or quantified. light.
554 GLOSSARY
Becquerel the SI unit that has superseded the Curie, and is Centrifuges mechanical devices used to separate or isolate
the amount of a radioactive nuclide that gives 1 disintegra- substances suspended in a fluid by spinning them about a
tion per second. central axis to produce a suitable centrifugal force.
Bile green coloured product of the liver stored in the gall Certificate of Competence an award made to individuals who
bladder. have completed an Institute of Biomedical Science accredited
Binding site variable part of an antibody molecule that can honours degree and their registration portfolio. This award
bind to an epitope on an antigen. allows an individual to apply to the Health Professions
Council to be admitted to the biomedical scientist register.
Biomedical science is the application of the natural sciences,
to medicine, in the study of the causes, consequences, diag- Chemokine protein that stimulate the migration and activa-
nosis, and treatment of human diseases. tion of cells.
Biomedical scientist an individual who practises biomedical Chromatic aberration the inability of a lens to bring light or
science and is entitled to use the legally protected job title. electrons of different wavelengths to a common focal point.
Only those individuals who have met the threshold require- Chromatography collective term for a family of analytical
ments of the Health Professions Council may use this term to techniques used to separate the components of mixtures of
describe themselves within the UK. molecules for their identification and possible estimation of
Biosensor an analytical device that incorporates a biologi- their concentrations in the original mixture based on differ-
cal component, such as an enzyme, and an electrochemical ences in their partitioning between two immiscible phases.
transducer. Chromophore atom, molecule, or part of a molecule that
Birefringence the ability of a substance to split a ray of light becomes excited by absorption of electromagnetic radiation.
into two components, the ordinary and extraordinary ray. Chronic ill health a long-term illness or condition that may
Blood sciences laboratory a commonly used term for an not be curable, but can be managed using laboratory tests
automated laboratory in which the predominant analyses and their results.
are blood investigations and which encompasses the tradi- Clinical governance a method by which a procedure can be
tional disciplines of biochemistry, haematology, coagulation, controlled, documented, and audited.
immunology, and virology. Sometimes called a core auto- Clinical risk a measure of anything that harms, or potentially
mated laboratory. harms, a patient.
Blotting transfer of the molecule of interest from an electro- Clone population of cells derived from a single stem cell.
phoretic agarose or polyacrylamide gel to a piece of nitro- Coherent light rays of the same amplitude and wavelength,
cellulose, paper, or a nylon membrane. which are in phase with one another.
Calibration a process using calibrants of known concen- Combination electrode a pH electrode in which the external
tration or value to set the performance parameters of a reference electrode is built into the glass electrode in a con-
device. centric double barrel arrangement.
Cannula flexible plastic tube carried on a needle that is Commensals a term used to describe all the natural bacte-
inserted into the body to obtain a sample of fluid or intro- ria that live on and in a healthy person. The main areas for
duce medication. commensal organisms are the skin, mouth, upper respira-
Capacity is the weighing range of a balance, for example up tory, gastrointestinal, and urogenital systems.
to 200 g, 1000 g, or more, respectively. Competent ability of an individual to perform a task repeat-
Cathode the positively charged electrode. edly to an agreed level of quality.
Cations positively charged ions that are attracted to the Competent having the ability to undertake a procedure cor-
cathode. rectly and according to the SOP.
cDNA library collection of clones (transformed cells) each Competitive tender where companies bid for a specified
containing a recombinant DNA prepared using mRNA mol- business contract.
ecules extracted from a single source (organism). Complement system group of proteins that act directly or
CE mark a mark placed on an IVD to show that it is compliant with antibodies to kill pathogens.
with European Directives, and is fit for the purpose stated by Continuing professional development any learning associ-
the manufacturer. ated with your chosen profession, rather than your personal
Centrifugal field an acceleration directed outwards from the development. The two are not exclusive.
centre of rotation. Continuous polyacrylamide gel electrophoresis uses the
Centrifugation tubes containers for samples used in the rotor same type of buffer to dissolve the sample as that present
during centrifugation. in the gel and in the compartments of the electrophoresis
Centrifugation term applied to the mechanical process of equipment. Compare discontinuous polyacrylamide gel
separating mixtures by applying centrifugal forces. electrophoresis.
GLOSSARY 555
Core automated laboratory an alternative name for the DNA polymerase abbreviation of DNA dependent DNA
blood sciences laboratory. polymerase.
Corrective action that taken to eliminate the cause of a Double junction reference electrode an external reference
detected nonconformity or other undesirable situation. electrode that has an additional chamber between the refer-
Corrosive substance that will destroy or irreversibly damage ence electrode and the external solution.
another that it comes into contact with. Dress code a clear set of written instructions that specifies
Control of substances hazardous to health (COSHH) set of what can and cannot be worn in a place of work.
laws that require employer control materials that are hazardous Dusts fine particles of solid substances or pellets that may be
Coulomb kg −1
the SI unit of exposure to radiation. harmful or flammable.
Coulomb is a unit of electrical charge equal to the charge Ectopic pregnancy implantation of the foetus outside the
transferred by a current of 1ampere s−1. uterus, often within the Fallopian tubes, and a life-threat-
ening condition.
Council for Professions Supplementary to Medicine pred-
ecessor of the Health Professions Council. Effective dose of radiation the product of the absorbed dose
and the relative biological effectiveness.
Critical illumination achieved when a substage condenser is
adjusted so that the light it emits comes to a focal point at Electric field the potential difference or voltage applied
the level of the specimen. between two electrodes. In electrophoresis it is normally
quoted in units of V cm−1.
Curettings scrapings of tissues.
Electroactive a molecular species that can undergo oxidation
Curie a pre SI unit, which is the amount of a radioactive
and reduction.
nuclide that gives 3.7 × 1010 disintegrations per second.
Electrode potential a spontaneous potential difference that is
Cuvettes specialized transparent container in which the
established across an electrode/electrolyte interface due to
absorbance of a solution is measured.
oxidation or reduction events occurring within the half-cell.
Cytocentrifuge or slide centrifuge a low-speed centrifuge
Electrolytic cell an electrochemical cell in which reactions
with a modified rotor used to separate cells and deposit
are not spontaneous, only occurring if an external voltage is
them as a monolayer onto the slides for cytological exami-
applied across the two electrodes.
nation.
Electromagnetic radiation spectrum the full range of elec-
Cytokine protein that directs the behaviour of other cells.
tromagnetic radiation.
Dalton a unit equal to one twelfth of that for 12C, which is
Electroosmosis occurs in support media that possess nega-
sometimes used to express the mass of an atom or molecule.
tively charged groups on their surface and so attract hydrated
Decalcifiying agents solutions of, for example ethylenedi- cations present in the buffer. When an electric field (E) is
aminetetraacetic acid (EDTA), formic, or hydrochloric acid applied the movement of cations and associated water mol-
that reduce the mineral content of samples of tissues. ecules is towards the cathode. Electro-osmosis may be suf-
Delta check a facility that allows a current result to be com- ficiently strong that weakly charged anions can be carried
pared with previous results from an individual. towards the cathode.
Destaining the removal of excess stain following staining to Electrophoretic mobility velocity of an ion per unit of elec-
give a gel of clear background. tric field.
Differential centrifugation technique that separates par- Eosinophils granulocytes that are associated with combating
ticles during sedimentation mainly on differences in their parasitic infections and causing allergic reactions.
sizes. Epitope or antigenic determinant site on an antigen that is
Discontinuous polyacrylamide gel electrophoresis uses recognized by an antibody.
a buffer that differs in composition and pH to dissolve the Ergonomics study of designing a job, equipment, and work-
sample to that present in the gel and in the compartments place to fit the worker.
of the electrophoresis equipment. Compare continuous
Error failure to complete a planned action as intended or the
polyacrylamide gel electrophoresis.
use of an inappropriate plan of action to achieve a given
Dissertation involves a literature review and a written pres- aim.
entation of the findings of that review. It is a component of Exciter filter one used in a fluorescence microscope which
an honours degree programme. transmits ultaviolet light of the required wavelength and
DNA cloning the preparation of many identical copies of a prevents transmission of unwanted visible light.
DNA molecule. External reference electrode a separate electrode that gen-
DNA dependent DNA polymerase enzyme that replicates erates a stable electrochemical potential in potentiometric
DNA. Usually abbreviated to DNA polymerase or DNA pol. analysis. Requires electrical continuity with the working
DNA pol abbreviation of DNA dependent DNA polymerase. electrode.
556 GLOSSARY
First aid provision of initial care for an illness or injury. Half-cell an electrochemical system comprising an electrode
First voided urine the initial urination of any given day. surrounded by an electrolyte.
Flammable substance that will easily ignite causing fire. Half-life the time required for half of the atoms in a radioac-
Fluorescence virtually instantaneous emission of radiant tive sample to decay.
energy by a chromophore following the absorption of radia- Harmful any chemical that causes or is likely to cause harm.
tion of a higher energy. Hazard substance, activity or process that may cause harm.
Fluorochrome fluorescent dye used to label or stain a bio- Health and safety guidance (HSG) documents written
logical substance. materials concerned with legal and best practices relating to
Fluors chemicals that give off fluorescent energy when they health and safety produced by the HSE.
interact with radiation. Health Professions Council (HPC) one of the statutory reg-
Focal length the distance between the centre of the lens and ulatory bodies operating within the UK health services. Its
its focal point. principal function is to protect the public by regulating a
Focal point the point at which a lens brings all of the light number of healthcare professionals.
passing through it to a common focus. Health state of well being in body and mind.
Frictional coefficient property of a particle sediment- Healthcare near miss situation in which an event or omis-
ing through a solvent and equal to the frictional resistance sion, or a sequence of events or omissions arising during
opposing its movement divided by its sedimentation velocity. clinical care fails to develop further and so injury to a patient
Fronting production of an asymmetrical peak during column does not occur.
chromatography by a slow rise at the beginning of the peak Hep2 cells human epithelial cells derived from tumours and
and a sharp fall after the peak. which are cultured for use in research and diagnostic tests.
Full Blood Count number of haematological measurements High performance liquid chromatography any of a variety
of blood indicating the numbers and sizes of various types of column chromatography methods that use the relatively
of blood cell. high pressures produced by pumps to force the mobile
Fume mist or vapour containing very small metallic particles. phase through the column to increase the resolution and
gain faster separation times.
Galvanic cell an electrochemical cell in which reactions occur
spontaneously at the electrodes when they are connected HPC register list of all those individuals who have met the
externally by a conductor. threshold requirements of the HPC and have been admitted
to the register.
Gel electrode a pH or ion selective electrode in which the
electrolyte of the reference electrode is retained within a gel Hybrid DNA molecules comprised of portions from two or
layer, enhancing portability, but meaning that the electrolyte more different sources. Also called recombinant DNA.
cannot be refilled as in conventional electrodes. Hybridoma hybrid cell formed by fusing two different types of
Gel filtration is a form of column chromatography that sepa- cells, for example a specific antibody-producing B lymphocyte
rates molecules according to differences in their sizes and to with a myeloma cell to produce monoclonal antibodies.
some extent shapes. Hydrophobic effect the tendency of molecules in an aque-
Gradient elution use of a buffer of gradually changing pH or ous environment to fold so that their hydrophobic portions
one whose concentration of salt progressively increases to are buried in the interior of the molecule.
wash materials out of a chromatography column. Compare Hydrophobic interaction chromatography form of column
isocratic elution. chromatography that relies on the specific binding of
Granulocyte type of leukocyte that has granules in its cyto- the analyte of interest to an immobilized hydrophobic
plasm containing enzymes that can digest microorganisms. ligand.
Gray the SI unit equal to the absorption of 1.0 J of radiant Hyperkalaemia refers to a K+ concentration that is higher
energy per kilogram of tissue. than the quoted laboratory reference range.
Ground state the lowest energy and therefore the most stable Hypoglycaemic coma a state of unconsciousness caused
state of a subatomic particle, atom, or molecule. when blood glucose falls to a dangerously low level.
Group III pathogens one of the four categories of pathogenic Immiscible phases ones that do not mix.
microorganisms recognized by the Advisory Committee for Immobilised enzyme an enzyme that is either bound to a
Dangerous Pathogens. surface or entrapped within a structure.
Haematoma a localized swelling consisting of blood that has Immune complex consists of an antibody-antigen.
leaked from veins and capillaries into the tissues. Immune surveillance capacity of the immune system to con-
Haemolysis breakdown of erythrocytes leading to release of tinually monitor for and detect foreign antigens.
free haemoglobin into the plasma. Immune tolerance failure of the immune system to respond
Haemolysis breakdown of erythrocytes affecting analysis. to an antigen.
GLOSSARY 557
Immunoassay any analytical method that uses an antibody Journal club an arrangement between a group of like-minded
to detect and/or quantity an analyte in a sample. individuals who agree that one member should be read a
Immunodeficiency situation in which the immune response particular journal paper and then present the contents to
is reduced or absent. the other members of the group.
Immunofluorescence methods employing the fluorescence Köhler illumination achieved where the substage condenser
microscope to identify the presence of specific antigens is used to focus the maximum amount of light onto the
within tissue samples using fluorescently labelled antibodies. specimen.
Immunoglobulins group of large protein molecules that Laboratory information management systems computer
includes all types of antibodies and the membrane-bound databases that hold complete patient records of incoming
receptors on B lymphocytes. requests and outgoing results and allow for the electronic
Immunophenotyping technique used to categorize cells exchange of laboratory data between computers.
by the proteins expressed on the surfaces of their plasma Lens piece of glass ground into a spherical or ovoid shape
membranes. that produces a magnified image. Two or more lenses may
Improvement notice legal order to improve practice issued be used in combination to produce higher magnifications.
by the HSE. Lifelong learning term used to encompass any new skill or
In vitro literally “in glass”: occurring in the laboratory. Com- knowledge that you may acquire at any time during your life.
pare in vivo. Linear energy transfer is the rate of ionization along the path
In vivo within the body. Compare in vitro. a radiant particle takes through body tissues.
Incident individual occurrence or event. Lipaemia high levels of fats such as cholesterol and triacyl-
Innate immunity that which is present at birth. glyrerols, especially found after eating a meal with high fat
Institute of Biomedical Science a professional body whose content resulting in cloudy or cream coloured plasma.
principal aims are to promote biomedical science and its Litigation legal action against an individual or organization.
practitioners. Lymphocyte leukocyte that mediate the activities of anti-
Internal reference electrode an electrode found within the bodies and cellular elements of the immune response.
glass bulb of a pH electrode. Macromolecules those with Mr greater than 5,000.
Ion pair consists of a positively charged ion and a free elec- Magnification the process of enlarging something only in
tron, which are formed during ionization events. appearance, not in physical size.
Ion selective field effect transistor a field effect transistor Mass number is numerically equal to the sum of the num-
with the gate modified to respond to specific ions. bers of neutrons and protons.
Ion-exchange chromatography use of a charged resin to sep- Mass is a measure of the amount of matter present in an entity.
arate ions on the basis of differences in their charge densities.
Mast cells cells found in connective tissue that play a major
Ionic strength adjustor a solution of known high ionic con- role in allergic reactions.
centration. Proprietary adjustors may also contain pH adjus-
Mediator a soluble electroactive molecule that can accept
tors and decomplexing agents or agents to remove species
electrons from an enzyme and then diffuse to an electrode
that interfere with the measurement of the ion of interest.
surface and donate the electrons to that electrode.
Ions atoms or molecules that possess an electrical charge.
Melting separation of the two strands of DNA by breaking
Irritant substance that causes irritation. the intrachain hydrogen bonds.
Isocratic elution use of a buffer of constant composition and
Metal oxide semiconductor field effect transistor the elec-
pH to progressively wash materials out of a chromatography
trical transistor found in nearly all electrical devices.
column. Compare Gradient elution.
Metal-chelation chromatography form of column chroma-
Isopotential point the pH at which a pH electrode, or the ion
tography that relies on the specific binding of the analyte of
concentration at which an ion selective electrode, gener-
interest to an immobilized metal ion.
ates zero potential, and where temperature has no effect on
potential. Methadone a heroin substitute used to wean addicts off the
drug.
Isopycnic density gradient centrifugation technique to
separate particles based mainly on differences in densi- Michaelis–Menten equation one description of the kinetics
ties during their sedimentation through a column of liquid of an enzyme-catalysed reaction.
whose density increases from the top of the centrifuge tube Microscope an instrument that forms an enlarged image of
towards its bottom. an object that would often be too small to be seen with the
Isotopes the forms of an atomic species of an element that naked eye.
have the characteristic atomic number of the element, but Microscopy the use of a microscope to examine and analyse
a varying numbers of neutrons and therefore different mass objects that would often be too small to be seen with the
numbers. naked eye.
558 GLOSSARY
Mobile phase the fluid phase used in chromatography, can Noncompliance non-fulfilment of a requirement or standard.
be a gas or liquid. Non-verbal communication any form of communication
Modified glass electrode a type of ion selective electrode that does not involve the spoken or written word.
that relies on a glass bulb to generate a potential. Nucleoside chemical combination of a base and sugar.
Molality is way of expressing concentration where the mass Nucleotide chemical combination of a phosphate(s) and a
of the solvent is used rather than the total volume of solu- nucleoside.
tion. A 1.0 molal solution would contain 1.0 mole of solute Nuclide an alternative name for the nucleus.
per kilogram of solvent.
Numerical aperture quantitative measure of the light gath-
Molar mass is the mass of all the elementary entities in one ering capacity of a lens. The greater the numerical aperture,
mole of a substance. the more efficient the lens.
Molarity a way of expressing concentration as the number O rings gaskets formed of a flat ring of rubber or plastic used
of moles of solute in a total volume of solution of one dm3 to make joints air and water tight.
or litre. Occult blood small amounts of blood in faeces that are indi-
Mole the amount of a substance that contains as many ele- cations of possible disease, but whilst invisible to the naked
mentary entities, such as photons, atoms, or molecules, as eye may be detected using a biochemical test.
there are atoms in 0.012 kg (12 g) of 12C. This value is called Ohm’s law a description of the relationship between the cur-
Avogadro’s number and is equal to 6.022 × 1023. rent passing between any two points of a conductor, the
Molecular biology study of biology in terms of the structure potential difference between the two points, and the resist-
and functions of the atoms and molecules concerned. ance of the conductor.
Molecular sieving the separation of ions during electro- Orbitals the specific energy levels to which subatomic parti-
phoresis by porous support media that restrict the flow of cles such as electrons are restricted.
ions. Smaller ions experience a lower frictional resistance Oxidation a reaction in which there is a loss of electrons.
and so migrate through the gel faster and travel further than
Palindrome word, phrase or sequence that reads the same in
larger ones for a given time.
both directions.
Monochromatic radiation electromagnetic radiation of a Paraprotein identical immunoglobulin or monoclonal anti-
single wavelength. body molecules produced by plasma cells in the bone
Monochromator a device for separating a beam of electro- marrow in excessive amounts in the condition multiple
magnetic radiation into its component parts on the basis of myeloma.
their wavelengths. Partial specific volume (of a particle) change in volume that
Monoclonal antibody single type of antibody that binds occurs when the particle is added to a large volume, that is,
to a single epitope and is produced by a single clone of excess of solvent.
B lymphocytes. Partition or distribution coefficient numerical descriptor
Monocyte type of leukocyte that are the precursors of mac- of the way in which a substance distributes at equilibrium
rophages or mononuclear phagocytes, which phagocytose between two immiscible phases.
pathogenic organisms. Pathobiology is the study of disease processes and their
Natural killer cell lymphocytes that are part of the innate effects on humans. It is a principal subject component of an
immune response and have a role in the destruction of honours degree programme in biomedical science.
some tumour cells. Pathogen a disease-causing organism.
Near miss unplanned event that did not result in injury, ill- Pathology the study of disease (pathological, relating to
ness, or damage, but had the potential to do so. disease).
Nephelometry analytical technique in which the intensity of Pellet sedimented material found at the bottom of centrifuge
light scattered by a suspension is measured at an angle to tubes or bottles following centrifugation.
the incident beam. Performance appraisal a formal interview held between an
Nernst equation an equation that describes the potential employee and their line manager to assess the workplace
developed by a potentiometric electrode. performance of the individual, both retrospectively and
Neutrophils phagocytic granulocytes that engulf and destroy looking to the future.
pathogenic organisms. Personal development plan a strategy agreed at a perform-
Nomograms arrangements of three scales, such that a line ance appraisal that includes the future training and educa-
connecting a value on one scale to that on one of the others tional needs of the employee.
gives the required value on the third scale. Nomograms Personal development a strategy by which an individual is
relating the radius of rotation, speed of the rotor, and RCF able to expand on their existing knowledge and skills, in
are useful in centrifugation. their personal life and in their professional life.
GLOSSARY 559
Personal protective equipment a series of protective cloth- Primary immune response response of the immune system
ing or devices designed to minimise or completely remove on first encountering an antigen, usually this results in the
the potential for an individual to be exposed to harm during production of an IgM antibody.
work activities. Primary sampling the analysis of a sample using aliquots
Phagocytic cells see monocyte and neutrophil. from the initial sampling tube.
Phase plate device within the substage condenser of a phase Probe labelled single-stranded polynucleotide that is com-
contrast microscope that causes light to be retarded by ¼ of plementary to, and used to detect and characterize specific
its wavelength. nucleotide sequences in RNA and DNA samples by forming
Phlebotomist a person trained to collect blood samples in a DNA–DNA, RNA–RNA homoduplexes, or RNA–DNA heter-
safe and appropriate manner. oduplexes.
Phosphor coating on the viewing screen of an electron Professional body organization of like-minded individuals
microscope that emits visible light when excited by electrons who have specialised knowledge which sets standards of
to produce an image of the sample on the viewing screen. practice and examinations on that knowledge.
Photons ‘particles’ or discrete packages of electromagnetic Prohibition notice legal order to cease activity issued by
radiation. the HSE.
Plasma cells dedicated antibody producing cells derived Project a piece of individual work involving a literature
from B lymphocytes. review, research, and the written and oral presentation of the
Plasma the bodily fluid obtained following centrifugation of findings. It is a principal component of an honours degree
a blood sample that has been collected and preserved using programme.
an anticoagulant such as lithium heparin. Proteomics the study of the complete complement of pro-
Plasmid extrachromosomal circular and double stranded teins encoded by a genome.
DNA molecule found in bacteria and some yeasts. Prozone phenomenon occurs when high concentrations of
Point of care testing (POCT) is the provision of a diagnos- antigen or antibody produce a false positive result due to
tic pathology testing service outside the traditional clinical saturation of binding sites.
laboratory and physically closer to the patient. Pyogenic bacteria Are bacteria that in the process of infec-
Polarizer filter placed in the light path of the microscope tion form pus, which is composed of dead neutrophils.
beneath the substage condenser which only allows the Quality Assurance Agency for Higher Education body (QAA)
transmission of plane polarized light. in the UK that helps ensure HE qualifications are of a suitable
Polarography a form of voltammetry that employs a drop- standard.
ping mercury electrode as the working electrode. Quality assurance the planned and systematic actions neces-
Polyclonal antibodies mixture of different types of antibodies sary to give adequate confidence that a product or service
produced when a number of B lymphocyte clones respond to satisfies the necessary level of quality.
different epitopes on the same antigen or a mixture of antigens. Quality control charts graphical representations that clearly
Polymer membrane electrode a type of ion selective elec- show how laboratory analytes or instruments are perform-
trode that incorporates an ion exchange material in an inert ing.
matrix such as PVC, polyethylene, or silicone rubber to gen- Quality control procedures internal laboratory checks carried
erate a potential. out to ensure that a scientific method or analytical instru-
Polymorphisms occurrence of two or more distinct forms of ment is working properly and producing results that are reli-
the same kind of structure, be it organism or molecule. able and valid.
Polynucleotide polymers of nucleotides therefore alterna- Quality control the techniques and activities used to reach
tive name for nucleic acid. the desired level of quality.
Potential gradient (E) the electric field found by dividing the Quality management system establishment of relevant
potential difference between two electrodes by the distance quality objectives and the policy necessary to achieve those
separating them. objectives.
Potentiometry electrochemical techniques that employ gal- Quality management activities of the overall management
vanic cells and measure the spontaneous potential of the that determine the quality policy objectives and responsi-
electrode with minimal current flow. bilities and implement them by means such as quality plan-
Precision a measure of the closeness of a series of measure- ning, control, assurance and improvement within the quality
ments of the same substance. system.
Primary fluorescence substances or tissue elements that Quality objective anything sought or aimed for related to
naturally exhibit fluorescence under UV light (also called quality.
autofluorescence). Quality consistently obtaining the correct answer.
560 GLOSSARY
Quenching phenomenon where the emitted fluorescence is Relative biological effectiveness (RBE) is defined as equal
less than expected because of the presence of contaminat- to a dose of X-rays that produces a given effect divided
ing molecules in the solution. by the dose of a specific radiation that produces the same
Rad the pre-SI unit (from radiation absorbed dose) of effect.
absorbed dose of radiation that gives an energy absorption Relative centrifugal field one expressed as multiples of
of 1 × 10−2 J kg−1. the earth’s gravitational field (g), which is an acceleration
Radiation the emission or transfer of particles or electromag- of 981 cm s−1 per s.
netic waves. Reports issued by a laboratory are the permanent record of
Radioactivity the spontaneous emission of particles or elec- the results and conclusions for a single or series of labora-
tromagnetic waves from the unstable nuclei of some atomic tory investigations.
species. Requests the investigations that a laboratory is asked to carry
Radioisotopes the unstable nuclei of some atomic species out on patient samples.
that spontaneously emit radiation. Resolution ability to separate (or resolve) completely one
Radiopharmaceuticals radioactively labelled chemical agents analyte or one point from another.
that distribute in the body. Resonance absorption of radio frequency (RF) radiation by
Radiotherapy the medical use of ionizing radiation given as nuclei with low-energy spins that changes their orientation
part of the treatment of certain cancers. to that of a higher-energy spin state.
Radius of rotation distance of a particle from the centre of Restriction endonuclease endonuclease that catalyses the
rotation. hydrolysis of phosphodiester bonds at identical positions
Rate-zonal density gradient centrifugation technique to within a palindromic site of DNA.
separate particles based mainly on differences in their Rƒ (relative to front value) distance travelled by any compo-
sedimentation coefficients during sedimentation through a nent relative to that moved by the solvent during chroma-
column of liquid whose density increases from the top of the tographic analyses.
centrifuge tube towards its bottom. Rhesus blood group blood group system defined by the
Readability the number of decimal places to which a balance presence of the D antigen on erythrocytes.
weighs accurately. Risk likelihood, high or low, that somebody or something will
Reagent books a means to record the reagent changes and lot be harmed by a hazard.
numbers that are associated with a clinical test or instrument. Roentgen non-SI unit of exposure to radiation equal to the
Real image a representation of an object (source) in which quantity of ionizing radiation that produces one electro-
the perceived location is actually a point of convergence of static unit of electricity in one cubic centimetre of dry air at
the rays of light that make up the image. If a screen is placed 0°C and standard atmospheric pressure. This is equivalent
in the plane of a real image the image will generally become to the amount of radionuclide that produces 1.61 × 1015 ion
visible on the screen. pairs kg−1.
Reanneal reassociation of two complementary DNA strands Roentgen equivalent man a non-SI unit of effective dose of
to form the original double helical structure. radiation equal to the amount of radiation that gives a dose
Recombinant DNA one whose molecules are comprised of in humans equivalent to that of 1 rad of X-rays.
strands from two different sources. Rotors specialized containers that hold liquid samples in cen-
Reduction a reaction in which there is a gain of electrons. trifuge tubes and which are rotated during centrifugation.
Reference electrode within potentiometric devices, an elec- Safety prevention of physical injury.
trode that generates a stable electrochemical potential and Salt bridge a solution of ions, often saturated potassium
thereby enables measurement of the potential difference chloride, which enables electrical continuity between two
with the working electrode. half cells.
Reference range a range of values found within the 95th per- Scintillant a liquid containing fluors that produce flashes of
centile of the population for a given substance. Sometimes light when the fluors interact with β-particles.
incorrectly referred to as a “normal” range. Scope of practice range of tasks that a registrant has been
Reflective learning assessing an event, its strengths and trained to undertake and been assessed as competent to
weaknesses, what has been learned from it, and how this perform.
could be put to future use. Secondary fluorescence the binding of fluorochromes to
Refractive index a numerical measure of the ability of a sub- substances or tissue elements in order to make them visible
stance to deviate light rays. under UV light.
Registrant a biomedical scientist whose name appears on the Secondary immune response response of memory plasma
HPC register. cells to rapidly produce specific antibodies at a higher
GLOSSARY 561
intensity against an antigen the immune system has previ- Tailing production of an asymmetric peak during column
ously encountered. chromatography characterized by a normal rise before the
Sedimentation coefficient (of a particle) ratio of its veloc- peak but a slow fall after the peak.
ity to the applied centrifugal field (v/ω2r ) when sedimenting Tare the operation used to set the readout of a balance to
during centrifugation. 0 g (zero).
Selectivity ability of a chromatography column to distinguish Teamwork a means by which a group of individuals all work
and separate two similar analytes. together to provide a common outcome.
Sensing electrode the electrode that generates a variable Terms of reference rules governing how a committee is
potential at its surface with the analyte, dependent on ana- appointed, how it acts, and how this is documented.
lyte concentration. Also called a working electrode. Thermocycler automated instrument for performing poly-
Serum the fluid derived following centrifugation of a blood merase chain reaction (PCR).
sample that has been allowed to clot. Titre Is a measure of antibody concentration based on serial
Sievert an SI unit of effective dose of radiation equal to the dilutions to a visible end point e.g. colour intensity, aggluti-
amount of radiation that gives a dose in humans equivalent nation or fluorescence.
to 1 Gy of X-rays. Toxic substance capable of causing injury or damage to an
Sodium dodecyl sulphate polyacrylamide gel electrophore- organism.
sis (SDS-PAGE) perhaps the most widely used form of poly- Traceability value or measurement of a standard in relation
acrylamide gel electrophoresis. It is particularly useful in to stated references, which allows comparisons through an
assessing the purity of proteins, to determine their Mr and unbroken chain.
was used extensively in sequencing DNA molecules. Tracking dye small coloured substances that pass through
Solid-state electrode a type of ion selective electrode that the pores in an electrophoresis gel and so act as a marker
incorporates one or more salt crystals to generate a for the electrophoretic front. Bromophenol blue is often
potential. used.
Solute the substance dissolved in a solvent to form a solution. Trainee someone who is receiving instruction, and is not yet
Solution a liquid consisting of a solute dissolved in a solvent. deemed competent to work alone.
Solvent the liquid used to dissolve a solute when making a Transcription synthesis of a complementary RNA molecule
solution. using a single strand of DNA as template.
Specific activity the amount of radioactivity per unit mass. Transcription transfer of results from a print-out or direct
Spectroscopy the study of the interactions between electro- from a device into the patient’s notes.
magnetic radiation and matter. Transducer within a biosensor, the electrochemical device
Staining the detection of separated components following that converts a biological or biochemical signal or response
electrophoresis by converting them to visible, coloured into a quantifiable electrical signal.
complexes. Compare destaining. Transformation process of a cell taking up recombinant
Standard curve method of plotting the measurements from DNA.
a known series of concentrations to determine that of an Transition change between any two energy levels that occurs
unknown sample. when an atom or molecule absorbs an amount of energy
Standard operating procedure clear set of written instruc- exactly equal to that between the two relevant states.
tions to ensure that all staff act in a consistent manner when Transport emergence card written instructions carried by
carrying out all aspects of laboratory work. drivers when transporting dangerous goods that give details
Standards codes of best practice that improve safety and of the substance, its category name, UN number and class,
efficiency. what to do and who to contact in an emergency, and first aid
Stationary phase the non-mobile phase used in chromatog- information in case of spillage.
raphy; can be a solid or an immobilized liquid. Troubleshooting sheets a means to record problems expe-
Supernatant liquid found above the pellet that contains rienced with clinical tests or instruments and to record how
unsedimented material following centrifugation. particular problems are solved.
Svedberg units unit of sedimentation coefficient (s) equal to Turbidity degree of opacity or cloudiness of a fluid caused by
1 × 10−13 seconds. suspended particles.
System set of interdependent elements that interact to Turnaround time that taken for a test result to be available
achieve a common aim. These elements may be human, to a clinician after the sample has been obtained from the
equipment and technologies. patient.
T lymphocyte type of lymphocyte that develops in the thymus Ultracentrifuge one capable of producing an RCF in excess
and has a variety of functions in the immune response. of 100 000 g.
562 GLOSSARY
Vacutainer tubes containers used for blood sampling via Voltammetry electrochemical techniques that employ elec-
venepuncture in which a vacuum has been created prior to trolytic cells and measure the current passing through an
sealing. Thus, blood enters the tube from the vein by gentle electrode when an external potential is applied.
suction. Wavelength the distance (measured in the direction of prop-
Valency of an antibody the number of other molecules it can agation) between two points in the same phase of any two
bind to simultaneously. consecutive cycles of a wave.
Vapour phase between the liquid and gaseous states of matter. Weight the force (measured for example in Newtons, grams,
Vectors carrier of the DNA into a recipient cell for cloning. or pounds) reflecting the effect of gravity at a particular loca-
Vectors are usually plasmids or bacteriophage DNA. tion upon a mass.
Vernier scale a device used on a microscope to accurately Welfare condition of well being, happiness, and comfort.
identify the position of specific points within a specimen. Wired enzyme electrode an electrode that incorporates an
VIR triangle Ohm’s Law relates voltage (V) to current (I) and immobilized enzyme within a network of fixed electroactive
resistance (R). The equation that describes Ohm’s Law may be centres that are able to shuttle electrons from the enzyme to
written in three distinct ways and this is best remembered using the electrode surface.
the VIR triangle, so that when any two of the values are known, Working electrode the electrode that generates a variable
the third unknown may be calculated using this triangle. potential at its surface with the analyte, dependent on ana-
Virtual image is an image in which the outgoing rays from lyte concentration. Also called a sensing electrode.
a point on the object always intersect at a point. A simple Workplace inspection audit of a workplace in terms of its
example is a flat mirror where the image of oneself is per- health and safety.
ceived at twice the distance from oneself to the mirror. That Workplaces exposure limit extent to which a person may be
is, if one is half a metre in front of the mirror, one’s image will safely exposed to a hazardous substance without endanger-
appear to be at a distance of 1 metre away (or half a metre ing health.
inside or behind the mirror).
Index
balances 126–31
A
Alzheimer’s disease 251
amino acids 420 calibration 129–30
ABO blood groups 391–2 amperometry 226 choice of 129
absence procedures 531 amphetamines 271 correct use of 130
absorbance 261–4 ampholytes 370 installation 128–9
glucose 263 amyloid 207 liquid volume measurement 129
measurement 262–3 analysis of variance (ANOVA) 115, 117 tare mode 129, 130
clinical applications 264–5 two-way 119–20 types of 126–8
nucleic acids 418–19 analytical balance 127–8 uses of 128
see also spectroscopy analytical ultracentrifuges 297 barrier filter 196–7
absorption spectra 266–72 anion 215, 342 beakers 133–4
infrared spectroscopy 266–70 anion exchange resins 325 beam balance 127
Raman spectroscopy 270–2 anode 215, 228, 341–2 Beer–Lambert law 262, 263
ultraviolet-visible absorption anthropometry 74 bench centrifuges 295–6
spectrophotometry 266, 267 antibodies 380–1 beta radiation 239–40
acceptance testing 491 antigen–antibody interactions 385–8 bi-directionality 494
accident reporting 79–80 detection methods 386–7 biological hazards 67–8
accreditation in vitro production 382–5 routes of entry into the body 70
degree programmes 35–6 in vivo synthesis 380–2 biomedical science 1–2
point of care testing 479–80, 498 monoclonal 381, 383, 385 definition 2
accuracy 132–3 polyclonal 381, 382–3, 384 degree programmes 3–4
achromatic lenses 191 for Western blotting 445 HPC approval 23–4
acidometer 220 anticoagulants 157–9 IBMS accreditation 35–6
acridine orange 366 apochromatic lenses 191 subject areas 3
activity 216 arterial blood sampling 161 biomedical scientists 1–3, 4–5
acute conditions 80 arthrocentesis 166 definition 4
adhesion molecules 380 ascites 172 initial employment 10–12
adjuvants 382–3 astigmatism 187, 203–4 professional skills 6–8
adsorbents 314–15 atomic structure 236–8 work undertaken 12–18
adsorption 313, 345 audit 517–20 biopsies 168
adverse healthcare events 510–12 cycle 520 trephine biopsy 166
incident reporting 79–80, 499–500, point of care testing devices 498 biosensors 228–33
521–2 schedule 519 continuous measuring
Advisory Committee on Dangerous autoimmune disorders 382 devices 232–3
Pathogens (ACDP) 68, 176 indirect immunofluorescence 406–9 single measurement devices 232
affinity chromatography 326–8 automated decappers 471–2 biotin 396
applications 328 automation see laboratory automation Bird’s Accident Triangle 79, 80
matrix selection 328 autopipettes see pipettors birefringence 195
agarose 362 autoradiography 245–7, 317, 439–41 blood banks 307
agarose gel electrophoresis 362–7, 369, avidin 396 blood cells 17, 180
432, 434, 455 Avogadro’s number 142 white 16
nucleic acid staining 363–6 blood donations 16
blood gas analysis 161
pulse field gel electrophoresis
366–7 B carbon dioxide measurement 222
agar plate 17, 18 bacteria 68 blood glucose measurement 228–32,
agglutination techniques 391–2 bacteriology laboratory 272, 500
air-displacement pipettors 136, automation 474 self monitoring devices 230–2
137, 138 DNA isolation 424–5 blood group serology 391–2
allergies 382 mass spectrometry 286–7 Blood Safety and Quality Regulations
alpha radiation 238–9 see also microbiology (BSQR) 514–15
564 INDEX
blood samples 151–61 catecholamines in urine 162 hydrophobic interaction

centrifugation 160, 305–7, 471 categorical data 97 chromatography 329
containers 154, 157–9 data analysis 114–15 ion-exchange
anticoagulants 157–9 multiple groups 117–18 chromatography 325–6
labelling 159 cathode 215, 342 metal-chelation
factors affecting accuracy of test cation 215, 342 chromatography 328–9
results 160 cation exchange resins 325 gas-liquid chromatography 334–7
microbiology testing 170 cellulose acetate high performance liquid
microscopical examination 180 electrophoresis 349–51 chromatography 329–33
primary sampling 466 cellulose electrophoresis 348 partition or distribution
sampling procedures 152–7, 465–7 CE mark 479, 490 coefficient 312–14
arterial blood sampling 161 Center for Evidence-based planar chromatography 314–19
collection tubes 466 Purchasing (CEP) 490 paper chromatography 314
precautions 154, 157 centrifugal field 291–2 thin layer chromatography 314–18
using a needle and syringe 156 centrifugation 290–1 chronic conditions 80
using vacutainers 154–6 blood samples 160, 305–7, 471 chronic inflammatory disease 207
venous blood sampling 154–6 examples of use 305–8 Clark oxygen electrode 215, 227–8
tests performed 153 rotors 298–9 clinical biochemistry 13, 14
blood sciences laboratory 462 balancing 303 clinical governance 484–7
blood transfusion 16, 17 choice of 301–2 clinical laboratory 6
blood group serology 391–2 faults 304 Clinical Negligence Scheme for Trusts
laboratory automation 475 safety issues 303–4 (CNST) 517
leukodepletion 307–8 sedimentation velocity 292–5 Clinical Pathology Accreditation (UK)
record keeping 61 separation methods 299–303 Ltd (CPA) 62, 480, 513
blotting techniques 439 density gradient assessment process 514
Northern blotting 441–2 centrifugation 300–1 standards 513–14
Southern blotting 439–41 differential centrifugation 300 clinical sensitivity 508
Western blotting 442–5 theory 291–5 clinical specificity 508
body protection 85 tubes 298 cloning see DNA (deoxyribonucleic
bone marrow sampling 166–7 checks 304 acid)
bone scans 250 centrifuges 290–1 clusters of differentiation
breast tissue 14 qualified users 303–4 (CD) 402–3
breast tumour 199 types of 295–7 colorimeters 264
bronchial fluid sampling 164–5 see also centrifugation combination electrode 217
bronchoscopy 164–5 cerebrospinal fluid (CSF) commercial concerns 540–1
burettes 134 sampling 164, 171 communication 42–3
cervical smear 15, 167, 180 confidentiality issues 48–9
C CH50 test 405–6

chelation 327
data protection 55–61
dress code 43–4
Caldicott Report 56–7 metal-chelation methods 49–55
calibration chromatography 328–9 electronic data 54–5
balances 129–30 chemical hazards 68–9, 81–2 paper reports 53–4
linearity calibration 131 routes of entry into the body 70 telephone 49–53
pH meter 220–1 Chemicals (Hazard, Information and record keeping 61–3
pipettors 140 Packaging for Supply) (CHIP) with service users 46–8
span calibration 131 Regulations 81 within the clinical laboratory 44–6
cancer chemokines 380 competitive binding ELISA 399
radiotherapy 253–4 child protection 531 competitive immunoassays 396
tumour diagnosis 206 chi-squared test 109, 117–18 competitive tender 490
cannula 153 Chlamydia trachomatis 171 complementary DNA (cDNA) 451
capillary electrophoresis 367–8 chloroform 428 complement system 379, 405
plasmid separation 369 chromatic aberration 186, 203 complement assays 405–6
carbon dioxide measurement 222 chromatography 311–12 concentration 142, 143, 144–5
cardiopulmonary resuscitation 531 column chromatography 319–29 confidentiality issues 48–9
carriers 470–1 affinity chromatography 326–8 see also data protection
case studies 545 gel filtration 321–5 confocal microscopy 198–9
INDEX 565
conical flasks 133–4 dark field microscopy 192–3 cDNA cloning 451
conjunctival swabs 174 data polymerase chain reaction
connectivity 494–5 central point 97–9 (PCR) 453–6
evaluation 489 distribution 102–4 recombinant DNA 449–51
Connectivity Industry Consortium qualitative 96–7, 104–5 fluorescence in situ hybridization
(CIC) 494 quantitative 96, 97 (FISH) 447–8
continual improvement 510–12, variance 99–102 hydrolysis 428
517–23 data handling 95–6, 105–20 restriction enzymes 428–33
audit 517–20 categorical data 114–15 isolation 422–6
continuing professional development multiple groups 117–18 from animal cells 425–6
(CPD) 526, 528–9 continuously variable data 109–14 from bacteria 424–5
IBMS scheme 23 at two time points 112–14 gel electrophoresis 432,
lifelong learning 527, 528 data from one individual 107–8 434–5, 436
standards 22, 30 data from three or more sets microarrays 457–8
see also personal development; 115–17, 119–20, 121 preparation of 457
training data from two groups of polymorphisms 441
continuous data 97 individuals 108–9 sequencing 435–9
data analysis 109–14 probability 106–7 Southern blotting 439–41
continuous flow centrifuges 296–7 statistical packages 120–2 DNA polymerase 417
continuous hyperfractionated data protection 55–61 documentation 512–13
radiotherapy (CHART) 254 Data Protection Act 1998 double diffusion techniques 390–1
Control of Substances Hazardous to (DPA) 55–6 double junction reference
Health (COSHH) regulations 68, Freedom of Information Act 2000 electrode 218
69, 80–3 (FOI) 57–8 Down’s syndrome 448
classification of hazards 81–2 Human Tissue Acts (HTA) 59–61 dress code 43–4, 90–1
compliance with 82–3 informed consent 58–9 dusts 69
Coomassie dyes 357–60 degree programmes 3–4
core automated laboratory 462
correlation 109, 109–12
HPC approval 23–4
IBMS accreditation 35–6 E
correlation coefficient 110–12 delta check 61 ear protection 87
cost benefit analysis 489 densitometer 345–7 electrical current 212–13
Council for Professions Supplementary density gradient centrifugation 300–1 electrical hazards 69
to Medicine (CPSM) 20–1 department 6 electrochemistry 211–12
counter immunoelectrophoresis 393 desalting 325 biosensors 228–33
creatinine concentration 264–5 destaining 345 classification of techniques 215
critical illumination 189 diabetes 228, 333 definition 212
crossed immunoelectrophoresis 393 blood glucose measurement electrochemical cell
curettings 151 228–32, 272, 500 representation 214–15
cuvettes 262–3 self-monitoring devices 230–2 principles 213–15
cystic fibrosis 358 diagnostic tests 12, 13 see also potentiometric techniques;
cytocentrifuge 305 differential centrifugation 300, voltammetric techniques
cytokines 380 302, 303 electrode potential 214
measurement 404–5 DiGeorge anomaly (DGA) 448 electrolytic cells 215
cytology 167 digoxygenin (DIG) 441 electromagnetic lenses 202–4
sampling 167–8 dilutions 145–8 aberrations 203–4
storage of samples 169 stock solution 145–7 electromagnetic radiation 257–8
transport of samples 169 types of 147–8 nature of 260–1
cytopathology 15, 167 disciplinary procedures 532 spectrum 258–9
Display Screen Equipment (DSE) see also light
D Regulations 73–4
dissertation 4
electronic data 54–5
back-up mechanisms 54–5
daltons 143 distribution 102–4 electronic quality control 497
Dangerous Substances and Explosive distribution coefficient 312–14 electronic transitions 261
Atmosphere Regulations DNA (deoxyribonucleic acid) 412, electron microscopes 179, 200–8
(DSEAR) 83 413–18 applications 206–8
Daniell cell 214–15 cloning 449–56 components of 201–5
566 INDEX
electron microscopes (Cont.) ergonomics 74 Freedom of Information Act 2000

cooling system 205 error (FOI) 57–8
electromagnetic lenses 202–4 adverse healthcare events 510–12 frequency 257–8, 260
electron gun 202 human 501 frictional coefficient 292
vacuum systems 204 learning from 509 fronting 320
viewing screen 205 logbooks 499 fumes 69
recording images 205 sources of 484, 485, 501 fungi 68
sample preparation 206 essay writing 545 mass spectrometry 286–7
transmission electron ethanol 278, 428 mycology testing 175
microscopes 201, 206–8 ethidium bromide 363–5 see also microbiology
electrons 236–7 European health and safety
electro-osmosis 345
in capillary electrophoresis 367–8
law 72–6
European Qualifications Framework
G
galactosaemia 318
electrophoresis 341 (EQF) 527–8
galvanic cells 215
agarose gel electrophoresis 362–7, excitation 244, 259–61
gamma rays 240–1
369, 432, 434, 455 exciter filter 196
gaseous radioactivity detectors 244
capillary electrophoresis 367–8 external quality assessment
gas-liquid chromatography
cellulose acetate (EQA) 488, 496, 507
(GLC) 334–7
electrophoresis 349–51 national schemes 507
column types 334
detection of separated point of care testing 497
detector systems 335–7
components 345–7 eyepiece lens 191–2
gas-sensing electrode 222
factors affecting eye protection 87
Geiger counter 244–5
separations 343–5
gel electrodes 218
health and safety
precautions 353–4 F gel filtration 321–5
applications 324–5
immunoelectrophoretic faeces collection 172–3
column performance 323–4
techniques 393 fatty acid separation 335, 336
glass electrode 216–17, 218–19
isoelectric focusing 370–2 fine needle aspirates 168
glomerular basement membrane
paper or cellulose fire extinguishers 84
changes 206, 207
electrophoresis 348 fire hazards 69
glucose measurement 228–32,
polyacrylamide gel fire policy 531
272, 500
electrophoresis 352–62 fire regulations 83–4
absorbance 263
principles 341–3 first aid equipment 88
self-monitoring devices 230–2
starch electrophoresis 351–2 fitness to practice (FTP) 20
glycated haemoglobin 333
two-dimensional FTP hearings 28–30
glycoprotein detection 360–2
electrophoresis 372–3 flammable substances 81, 82
gradient elution 326
electrophoretic mobility 342–3 flow cytometry 401–3
grievance procedures 532
email 54 fluorescence 195, 260, 273
group III pathogens 175–6
environmental hazards 69 fluorescent labels in
enzyme electrode 229
enzyme labels in immunoassays 395
immunoassays 396
fluorescence-activated cell sorters H
enzyme-linked immunosorbent (FACS) 401–3 haematology 16, 17
assays (ELISAs) 252–3, 305, fluorescence in situ hybridization radioisotope applications 251–2
397–401, 446–7 (FISH) 447–8 see also blood cells; blood samples
automated procedures 399 fluorescence microscopy 16, 188, haemolysis 154, 160
competitive binding 195–8 haemolytic anaemia 251–2
ELISA 399 incident light fluorescence haemolytic disease of the
enzyme-linked immunospot assays microscopes 195, 197, 198, 199 newborn 383
(ELISPOT) 399–401 transmitted light fluorescence half-life 241–2
sandwich ELISA 399 microscopes 195, 196–7 hand protection 85–7
epitopes 381 uses of 197 hand washing 90, 91
equipment evaluation 489–92 fluoride–oxalate blood 467 harmful substances 81, 82
acceptance testing 491 fluorimetry 273–4 hazards 66, 67–9, 76
diagnostic kits 491 fluorochromes 447–8 biological hazards 67–8
operation 490–1 fluors 245, 246 microbiological samples 175–6
equipment hazards 68 foot protection 85–7 chemical hazards 68–9, 81–2
INDEX 567
classification of 81–2 Health and Safety at Work Act 1974 immunodiffusion techniques 389–91
electrical hazards 69 (HASAWA) 70–2 double diffusion techniques 390–1
environmental hazards 69 duties of employees 71–2 single radial immunodiffusion
fire hazards 69 duties of employers 71 (SRID) 389–90, 393
physical, mechanical, and equipment Health and Safety Commission immunofluorescence 197, 409
hazards 68 (HSC) 68, 70 indirect (IIF) 406–9
see also Control of Substances Health and Safety (Display Screen immunoglobulins 380–1
Hazardous to Health (COSHH) Equipment) Regulations 73–4 see also antibodies
regulations Health and Safety Executive immunological techniques
head protection 87 (HSE) 62–3, 66, 67, 70–1 agglutination techniques 391–2
health 66 Approved Codes of Practice complement assays 405–6
see also health and safety (ACOP) 71 enzyme-linked immunosorbent
healthcare regulation 20–1 health and safety guidance (HSG) assays (ELISAs) 252–3, 305,
Health Professions Council (HPC) 2–3, documents 71 397–401, 446–7
21–30, 528 Health and Safety (First Aid) flow cytometry 401–3
continuing professional development Regulations 88 fluorescence-activated cell sorters
requirements 529 high performance capillary (FACS) 401–3
professionals regulated by 22 electrophoresis (HPCE) 367 immunoassays 393–7, 446
registration portfolio 5 high performance liquid competitive and non-competitive
registration renewal 30, 526, 529 chromatography (HPLC) 329–33 assays 396
sanctions on practice 28–30 applications 333 enzyme labels 395–6
case studies 29–30 detector systems 331–2 fluorescent labels 396
Standards of Conduct, Performance injection systems 331 immunoradiometric assays
and Ethics 22, 24–5, 30 normal phase liquid (IRMAs) 252
Standards of Continuing Professional chromatography 331 radioimmunoassays (RAI) 252–3
Development (CPD) 22, 30 reverse phase liquid radioisotope labels 394–5
Standards of Education and Training chromatography 331 separation of bound and unbound
(SETS) 22 high speed refrigerated reactants 397
degree course approval 23–4 centrifuges 297 immunocytochemistry 409
Standards of Proficiency 4–5, 11, histology 167 immunodiffusion
22, 25–8 laboratory automation 474 techniques 389–91
health and safety 66–7 sampling 168–9 immunoelectrophoretic
centrifuges and 303–4 storage of samples 169 techniques 393
electrophoresis and 353–4 transport of samples 169 indirect immunofluorescence 406–9
fire regulations 83–4 thyroid gland 181 microarrays 404–5
hazards 67–70 histopathology 13, 14, 167 multiplex technology 403–4
routes of entry into the body 70 holidays 532 nephelometry 397
see also Control of Substances human errors 501 turbidimetry 397
Hazardous to Health (COSHH) human immunodeficiency virus immunology 15, 16, 377–89
regulations; specific hazards (HIV) 207–8, 445 antibody production in vitro 382–5
induction training 530 human resource issues 531–2 antibody synthesis in vivo 380–2
personal health and safety 89–92 human serum albumin (HSA) 387 antigen–antibody interactions 385–8
personal protective equipment 85–7 Human Tissue Acts (HTA) 59–61, detection methods 386–7
radiation and 247–8 515–16 immune system
absorbed dose 247–8 hydrogen peroxide acquired immunity 378–80
effective dose 248 measurement 227–8, 231 associated disease states 382
reporting regulations 87–8 hydrolase detection 361–2 cytokines 380
risk assessment 76–80 hydrophobic effect 421 innate immunity 378
RNA isolation and 427–8 hydrophobic interaction polyclonal and monoclonal
statutory framework 70–6 chromatography 329 responses 381
European law 72–6 hypothesis 105–6 see also immunological techniques
Health and Safety at Work Act impact 76
1974 (HASAWA) 70–2
transport regulations 88–9 I incident light fluorescence
microscopy 195, 197, 198, 199
universal or standard precautions 92 ibuprofen 270 incident reporting 79–80, 521–2
workplace inspection 66 immunocytochemistry 409 point of care testing 499–500
568 INDEX
indirect immunofluorescence isoelectric focusing (IEF) 370–2 lipaemia 160

(IIF) 406–9 two-dimensional literary skills 543–5
induction courses 529–32 electrophoresis 372–3 lithium heparin 467
infection control 90 isopotential point 221 litigation 500
infinity corrected lenses 190 isopycnic density gradient logarithmic transformation 117
inflammation 207 centrifugation 301, 302 lumbar puncture 164, 171
influenza virus 208 isotopes 237 lysozyme 424, 428
information see data
information technology 546
informed consent 58–9
J M
Human Tissue Acts (HTA) 59–61 journal-based learning ( JBL) 537–8 macromolecules 412, 413
infrared spectroscopy 266–70 journal clubs 534 structure and properties
Institute of Biomedical Science of 413–22
(IBMS) 5, 6, 21, 30–9, 528, K see also nucleic acids; proteins
534–5 Köhler illumination 189 magnetic resonance spectroscopy
Certificate of Competence 5, 7 Kruskall-Wallis test 115, 116–17 (MRS) see nuclear magnetic
classes of membership 31, 32, 36 resonance (NMR) spectroscopy
Code of Conduct 31–3
Continuing Professional L magnification 192
Management of Health and Safety at
Development Scheme 23 labelling 89 Work Regulations 73, 76
degree course accreditation 35–6 blood samples 159 Mann–Whitney U-test 108, 109
good professional practice 34–5 laboratory automation 461–2 manual handling 531
professional qualifications awarded benefits of 462–5 manual handling operations
by 37–9, 535–7 enzyme-linked immunosorbent (MHO) 75
Advanced Specialist assays (ELISAs) 399 mass 127
Diploma 39, 537 sample collection and mass spectrometry 280–7
Certificate of Expert delivery 465–7 analysers 282–3
Practice 38, 537 specimen reception 457–9 applications 283–7
Diploma of Expert tracked automation systems 470–3 mass spectrometers 280, 281–3
Practice 39, 537 test repertoires 472–3 Material Safety Data Sheet
Higher Specialist Diploma wider laboratory settings 473–5 (MSDS) 81
(HSD) 38–9, 536–7 laboratory coats 85 mean value 98–9, 104
Specialist Diploma 37–8, 536 laboratory information management measuring cylinders 134
publications 33 systems (LIMSs) 51, 54 mechanical hazards 68
registration portfolio 5 delta check 61 median value 98–9, 104
Institute of Occupational Safety and laboratory placements 8 Medicines and Healthcare Products
Health (IOSH) 66, 67 case study 9–10 Regulatory Agency (MHRA) 62
internal quality control (IQC) 488, 496, laboratory tests 12–13 meetings 539
506–7 request form 152 melting, DNA molecules 454
International Standards Organization leadership 509 membranous glomerular
(ISO) 479–80, 509 learning organizations 509 nephritis 207
inter-quartile range (IQR) 99–102 leukocytes 401–2 meniscus 134
inverted microscopes 199–200 leukodepletion 307–8 mercury vapour lamps 188
in vitro diagnostic devices (IVDs) 479 library use 546 metabolome 285–6
see also point of care testing (POCT) lifelong learning 527, 528 metal-chelation
ion-exchange chromatography 325–6 light 185 chromatography 328–9
ionic strength adjustor 224 absorbance 261–4 metal oxide semiconductor field effect
ionization 244 measurement 262–3 transistors (MOSFETs) 224–5
in mass spectrometry 281 monochromatic 261 methadone 501
ion selective electrodes (ISEs) 216, scattering 259 methanol 278
223–4 methods 272–3 methicillin-resistant Staphylococcus
ion selective field effect transistors see also radiation aureus see MRSA
(ISFETs) 224–5 light microscopes 179 Michaelis-Menten equation 229
irritant substances 81–2 sample preparation 182–3 microarrays 404–5
isoamyl alcohol 428 linearity calibration 131 DNA 457–8
INDEX 569
microbiology 17–18 macromolecular structure and nucleus 236–7, 273

testing 169–76 properties 413–22 stability 237–8
blood 170 nucleic acids 413–19 see also radioactivity
faeces 172–3 proteins 419–22 numeracy 545–6
sputum 171 nucleic acid hydrolysis 428 nursing stations 49
urine 170–1 restriction enzymes 428–33
microfuges 296
micropipettes see pipettors
nucleic acid isolation 422–8
molecular sieving 345 O
microscopes 179 monitoring 12–13 objective lens 190–1
components of 187–92 monochromators 264, 265 occult blood 173
electron microscopes 201–5 monoclonal antibodies 381, occupational health (OH) 90, 532
eyepiece 191–2 383, 385 Ohm’s Law 213
light source 187–9 moulds 68 oil diffusion pumps 204
objective lens 190–1 MRSA 13, 18, 174 oil rotary pumps 204
specimen stage 190 multiple myeloma 350–1, 382 Ouchterlony double diffusion
stand and tube 190 multiple sclerosis 371–2 technique 390–1
inverted microscopes 199–200 multiplex technology 403–4 oxygen measurement 227
lens defects 185–7 mutually beneficial supplier
see also electron microscopes
microscopy 179–80
relationships 510
mycology testing 175 P
confocal microscopy 198–9 myoblasts 200 paired tests 109
dark field microscopy 192–3 myocardial perfusion scanning 250 paired t-test 112–14
electron microscopy 179, 200–8 palindromes 429
fluorescence microscopy 188, 195–8
illumination of specimens 189 N paper chromatography 314
paper electrophoresis 348
image formation 181–7 nasal swabs 174 paper reports 53–4
contrast 181 National Patient Safety Agency paracetamol 269
magnification 181 (NPSA) 521 paraprotein 350–1
resolution 184 near misses 79–80, 522 partition chromatography 330, 331
magnification 192 nephelometry 272–3, 397 partition coefficient 312–14
phase contrast microscopy 193–4 Nernst equation 215–16 parts per million (ppm) 145
polarization microscopy 195 NHS number 57 Pasteur pipettes 133
sample preparation 182–3 ninhydrin 348 pathobiology 3
modified glass electrodes 223 nomograms 292, 293 patient benefit analysis 489
molality 142, 144 non-competitive immunoassays 396 patients
molarity 142, 143 non-normal distribution 102–3, 104 communication with 47
molar mass 142–3 normal distribution 102, 104, 107–8 satisfaction 482, 483
mole 142, 143 normal phase liquid pattern recognition 475
molecular biology 412–13 chromatography 331 payroll queries 532
blotting techniques 439–46 Northern blotting 441–2 Pearson’s correlation method 112
Northern blotting 441–2 note taking 543–5 peptide mass fingerprinting (PMF) 284–5
Southern blotting 439–41 nuclear magnetic resonance (NMR) per cent concentration 144
Western blotting 442–5 spectroscopy 274–9 performance appraisal 549
DNA cloning 449–56 clinical applications 279 periodic acid-Schiff (PAS)
cDNA cloning 451 nucleic acids 413–19 reaction 360–1
polymerase chain DNA (deoxyribonucleic acid) 412, personal appearance 90–2
reaction 453–6 413–18 personal development 526–8
recombinant DNA 449–51 hydrolysis 428 commercial concerns 540–1
DNA microarrays 457–8 restriction enzymes 428–33 journal-based learning 537–8
DNA sequencing 435–9 isolation 422–8 opportunities for 529–32, 541
enzyme-linked immunosorbent RNA (ribonucleic acid) 412, 416 performance appraisal and 549–50
assays (ELISAs) 446–7 staining 363–6 plan (PDP) 549–50
fluorescence in situ hybridization ultraviolet absorption 418–19 professional bodies and 534–7
(FISH) 447–8 nucleosides 413 professional meetings 539
laboratory automation 475 nucleotides 413 reflective learning 541–2
570 INDEX
personal development (Cont.) limitations of 483–4 power 106

structured reading 538–9 sources of error 484, 485, 501 precision 133
study skills 542–7 litigation 500 press, dealing with 532
supporting evidence 547–9 POCT committee 484–7 prions 68
training and 532–4 POCT policy 484–8 probability 106–7
see also continuing professional procurement of devices 488–9 probes 422, 424
development (CPD); training quality issues 488, 496–7, 516 process approach 510
personal protective equipment 85–7 electronic quality control 497 professional meetings 539
Personal Protective Equipment at Work regulatory requirements 516 professional skills 6–8
Regulations 74–5 standards and guidelines 479–80 case study 8
phase contrast microscopy 193–4 training 488, 495–6 project 4
pH electrode 216–19 polarization microscopy 195 Pro-Q emerald 300 dye 361
phenylketonuria 284 polarized light 195 protective equipment 85–7
pH meter 219–21 polarography 226 proteins 412, 419–22
calibration 220–1 polyacrylamide gel electrophoresis Western blotting 442–5
phosphorescence 260 (PAGE) 352–62 proteomics 284, 373
photons 257, 259–61 continuous buffer systems 354–5 Provision and Use of Work Equipment
physical hazards 68 discontinuous buffer systems 356 Regulations (PUWER) 75–6
pipettes 135 glycoprotein detection 360–2 prozone phenomenon 388
Pasteur 133 protein visualization following pucks 470–1
pipettors 135–40 separation 357–60 pulse field gel electrophoresis
calibration 140 sodium dodecyl sulphate (SDS-PAGE) (PFGE) 366–7
features of 136–8 electrophoresis 356–7
obtaining optimal
performance 139–40
DNA fragment separation 434, 436
two-dimensional Q
types of 138 electrophoresis 372–3 Quadruple Test for Down’s
use of 138–9 polyclonal antibodies 381, 382–3, 384 syndrome 448
Planck’s constant 258, 260 production in human volunteers 383 quadrupole ion-trap (QIT)
plasma 158, 466 polycythaemia vera 251 analysers 282
plasma cells 381 polymerase chain reaction qualitative data 96–7, 104–5
plasmids 369, 418, 433, 449–53 (PCR) 453–6, 475 Quality Assurance Agency
pleural fluid sampling 165–6 automation 475 (QAA) 3, 35
point of care testing (POCT) 10, 46, real-time polymerase chain quality assurance (QA) 496, 505
478–9 reaction 456 point of care testing 488, 496–7
advantages of 480–3 reverse transcriptase PCR 456 quality control (QC) 496, 505, 506–8
acute condition management 482 polymer membrane electrodes 223 communication and 46
chronic condition polynucleotides 413 external quality assessment
management 482 Ponceau S 349–50 (EQA) 488, 496, 497, 507
cost benefits 480–1 positive-displacement pipettors 136, internal quality control (IQC) 488,
estate management 481 137, 138 496, 506–7
meeting government positron emission tomography point of care testing 488, 496–7, 516
directives 482 (PET) 250, 251 electronic quality control 497
patient satisfaction 482 potential difference 213 quality management 508–12
audit 498 electrode potential 214 documentation 512–13
connectivity 494–5 potential gradient 341 quality management system
device evaluation 489–92 potentiometric techniques 215–25 (QMS) 505, 509–10
acceptance testing 491 electrode sensitivity and requirements of 509–10
diagnostic kits 491 interference 218–19 see also standards
operation 490–1 electrode storage and cleaning 219 quality manual 512
device use 492–4 ion selective electrodes 223–4 quality policy (QP) 512
maintenance and servicing 493–4 ion selective field effect quantitative data 96, 97
siting of equipment 492 transistors 224–5 quantitative precipitation
stock control 492–3 pH electrode 216–18 curve 386–7
error logbooks 499 pH meter 219–21 quenching 245, 274
incident reporting 499–500 Severinghaus CO2 electrode 222 questionnaires 105
INDEX 571
R S
reference electrode 215
double junction 218
radiation 236, 257 external 217 safety 66
alpha radiation 238–9 internal 217 see also health and safety
beta radiation 239–40 reference range 103–4, 107 Safety Alert Broadcast System
electromagnetic radiation 257–8 reflective learning 533, 541–2 (SABS) 500
nature of 260–1 refraction 185 samples and sampling 151, 163–4, 465–7
spectrum 258–9 Regulatory Reform (Fire Safety) blood 152–61, 170, 465–7
gamma rays 240–1 Order 83 bone marrow 166–7
health and safety issues 247–8 relative centrifugal field 292 bronchial fluid 164–5
absorbed dose 247–8 relatives, communication with 47 cerebrospinal fluid 164, 171
effective dose 248 renal dipstick 163 cytology samples 167–8
interaction with matter 243–4, renal disease, glomerular basement faeces collection 172–3
259–60 membrane changes 206, 207 hazardous samples 175–6
scattering 259 Reporting of Injuries, Diseases and histology samples 168–9
methods 272–3 Dangerous Occurrences preparation for electron
radioactivity 236 Regulations (RIDDOR) microscopy 206
clinical applications 249–54 87–8, 500 preparation for light
haematology 251–2 report writing 545 microscopy 182–3
radioimmunoassays (RAI) 252–3 request form 152 microbiology testing 169–76
radioisotope imaging resolution packaging and labelling 89, 159
techniques 249–50 microscopy 184 pleural fluid 165–6
radiotherapy 253–4 separation techniques 320 sputum collection 171
detection and measurement resonances 275–6 sterile swabs 173–4
of 244–7 respiratory protection 87 synovial fluid 166
autoradiography 245–7, 317 restriction endonucleases test to request ratio 465
gaseous detectors 244 (REs) 428–33 transport 169
limitations 247 RE digest procedure 433 regulations 88
scintillation counters 245, 246 sources of 430 urine 161–3, 170–1
discovery of 240 uses of 430–3 sanctions on practice 28–30
rate of decay 241–2 restriction fragment length case studies 29–30
types of 238–41 polymorphisms sandwich ELISA 399
units 242–3 (RFLPs) 441, 442 sandwich immunoassays 396
see also radiation retinoic acid 269 scanning densitometry 345–7
radioimmunoassays (RAI) 252–3 reverse phase liquid scintillation counters 245, 246
radioisotopes 236 chromatography 331 scope of practice 28
imaging techniques 249–50 reverse transcriptase PCR 456 screening tests 12, 13
immunoassays 394–5 rhesus negative blood group 383 sedimentation coefficient 294, 295
safety precautions 247–8 risk 76, 509 semiachromatic lenses 191
see also radiation; radioactivity hierarchy of risk control 78 sensing electrode 215
radiopharmaceuticals 249 incident reporting 79–80 Sephadex 322–3
radiotherapy 253–4 risk assessment 76–80 serious adverse blood reactions and
Raman spectroscopy 270–2 legal interpretation 78 events (SABRE) 515
clinical applications 271–2 process 77–8 serious hazards of transfusion
Rank oxygen electrode 227 specialist risk assessments 79 (SHOT) 515
rate zonal centrifugation 301, 302 types of 77 serum 158, 466
reading 543 risk awareness 90 service users 46–7
reagent preparation 141–8 RNA (ribonucleic acid) 412, 416 communications with 46–8
real-time polymerase chain hydrolysis 428 Severinghaus CO2 electrode 222
reaction 456 isolation 426–8 sickle cell anaemia 441
recombinant DNA 449–51 health and safety sickness procedures 431
record keeping 61–3 precautions 427–8 silver staining 360
external regulatory bodies Northern blotting 441–2 single radial immunodiffusion
and 62–3 robotic arms 470, 471 (SRID) 389–90, 393
staff records 62 root cause analysis (RCA) 80, 522–3 SI units 126
572 INDEX
six-pack regulations 72–6 Standards for Better Health 517 tracked automation systems 470–3
sodium citrate 466 Staphylococcus aureus 180 tracking dye 355
sodium dodecyl sulphate (SDS) 428 see also MRSA trade unions 540–1
DNA extraction 424, 426 starch electrophoresis 351–2 training 10–12, 532–3
SDS-PAGE electrophoresis 356–7 statistical packages 120–2 induction courses 529–32
DNA fragment separation 434, 436 statistics see data; data handling point of care testing 488, 495–6
two-dimensional stem cells 200 standards 22
electrophoresis 372–3 sterile swabs 173–4 transcription 417
sodium ethylenediaminetetraacetic sticky ends 430 transducers 225
acid (EDTA) 466 stock control 492–3 transformation 449
solid-state electrodes 223–4 stock solution 145 transfusion science 16, 17
solute 141 dilution 145–7 translation 418
solutions 141 structured reading 538–9 transmission electron
concentration 142, 143, 144–5 Student’s t-test 108, 109 microscopes 201
dilutions 145–8 study skills 542–7 applications 206–8
stock solution 145–7 academic study skills 547 sample preparation 206
preparation of 141, 144 information technology 546 transmitted light fluorescence
solvent 141 library use 546 microscopy 195, 196–7
Southern blotting 439–41 literary skills 543–5 transport
span calibration 131 numeracy 545–6 regulations 88–9
Spearman’s correlation method 112 team work 546–7 samples 88, 169
spectroscopy 257 supplier relationships 510 packaging and labelling 89
absorbance measurement 262–3 Svedberg units 294 Transport Emergence (TREM) card 88
clinical applications 264–5 swabbing 173–4 trephine biopsy 166
absorption spectra 266–72 sweat testing 172 triacylglycerol separation 336
fluorimetry 273–4 synovial fluid sampling 166 Tukey’s post-hoc test 115, 116, 117
infrared spectroscopy 266–70 Sypro orange 360 tumour diagnosis 206
mass spectrometry 280–7 Sypro red 360 turbidimetry 397
nuclear magnetic resonance (NMR) systemic lupus erythematosus turbidity 272
spectroscopy 274–9 (SLE) 252–3 turnaround time (TAT) 463–4, 465
Raman spectroscopy 270–2 systems approach 510 tutorial programmes 533
spectrophotometers 264, 265 two-dimensional
ultraviolet-visible absorption
spectrophotometry 266, 267 T electrophoresis 372–3, 393
two-fold dilution 147
units 258 tailing 320 two-way analysis of variance
see also light; radiation teamwork 44–5, 546–7 (ANOVA) 119–20
spherical aberration 186–7, 203 telephone communications 49–53
sputum collection 171
staff records 62
incoming requests 51–2
outgoing general information 53
U
ultracentrifuges 294, 297
staining, in electrophoresis 345, 346, outgoing results 52
analytical 297
357–62 returning specific calls 53
ultraviolet-visible absorption
nucleic acid staining 363–6 ten-fold dilution 148
spectrophotometry 266, 267
standard deviation (SD) 99–102, 104 test to request ratio 465
United Kingdom Accreditation Service
standard error (SE) 100 thermocycler 454
(UKAS) 480
standard operating procedures thin layer chromatography 314–18
universal precaution systems 92
(SOPs) 44, 151, 488, 512–13 adsorbents 315
urine dipstick 163
failure to follow 484, 485 procedure 315, 316
urine samples 161–3
standards 513–17 throat swabs 174
catecholamine concentrations 162
conduct, performance and thyroid gland
collection of 24-hour samples 162
ethics 22, 24–5, 30 histology 181
collection with a catheter 171
continuing professional development time-of-flight (TOF) analysers 282
microbiology testing 170–1
(CPD) 22, 30 tissue sampling 168–9
education and training (SETS) 22
degree course approval 23–4
top pan balance 127–8
total haemoglobin 499 V
point of care testing 479–80 total haemolytic complement test 405 vacutainers 153, 154–6
proficiency 4–5, 11, 22, 25–8 toxic substances 81, 82 vaginal swabs 174
INDEX 573
variance 99–102 volume measurements 131–5 weight 127

vectors 449–51, 452–3 accuracy 132–3 welfare 66
venous blood sampling 154–6 choice of method 131–3 Western blotting 442–5
using a needle and syringe 156 liquids antibodies for 445
using vacutainers 154–6 precautions 133 white blood cells 16
Vernier scales 190 using a balance 129 Wilcoxon’s test 114
virology 17 precision 133 working electrode 215
viruses 68 volumetric flasks 134 workplace exposure limits (WELs) 83
electron microscopy 207–8 Workplace (Health, Safety and Welfare)
see also microbiology
voltammetric techniques 215, 225–8 W Regulations 73
workplace inspection 66
Clark oxygen electrode 227–8 wavelength 257–8 wound swabs 174
voltammogram 226 of light 184, 185 written reports 53–4

4 5784891476752204819

Uploaded by

Copyright:

Available Formats

4 5784891476752204819

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

4 5784891476752204819

Uploaded by

Copyright:

Available Formats

Biomedical Science Practice

5.1 Types of information and how to 8.2 Components of a microscope 187

6 Preparing and measuring 8.9 Inverted microscopes 199

Introduction 125 Further reading 209

7.3 Fluids other than blood and urine 163

10.7 Safety precautions when using 13 Chromatography 311

Further reading 288 14.6 Starch electrophoresis 351

Further reading 308 15.1 Outline of the immune system 378

15.5 Immunological techniques used in clinical Summary 475

16 Molecular biology techniques 412 Introduction 478

16.4 Hydrolysis of nucleic acids 18.6 Use of POCT devices 492

17 Laboratory automation 461 Introduction 505

Summary 524 20.6 Opportunities for self-guided

Questions 525 20.7 Reflective learning 541

Introduction 526 20.10 Performance appraisal and

20.5 Commercial concerns and personal

Produced in collaboration with the Institute of Biomedical Science, the series

• Understands the development needs of employers and the Profession.

• Places the theoretical aspects of biomedical science in their practical context.

Learning from this series

BOX 9.1 Ohm’s Law

students’ understanding of the subject

The Severinghaus electrode is a gas-sensing electrode and as it is based

Online learning materials

Guides to key experimental skills and

Biomedical science in practice

The Digital Microscope is used under licence from the Open

‘Check your understanding’ learning modules

We extend our grateful thanks to colleagues in the School of

Answers to self-check, case study,

Lecturer support materials

To register as an adopter visit www.oxfordtextbooks.co.uk/orc/ papilla of hair

glencross and follow the on-screen instructions. nerve fiber

Nessar Ahmed Tony Sims

Andrew Blann Mark Slevin

Ian Graham Alison Taylor

ABTS azino-bis-ethylbenzthiazoline sulfonic acid CF cystic fibrosis

ACOP Approved Codes of Practice CHART continuous hyperfractionated radiotherapy

BAC bacterial artificial chromosome CSF cerebrospinal fluid

BIVDA British In-Vitro Diagnostics Agency CSU catheter sample of urine

BJBS British Journal of Biomedical Science CT computed tomography

bp base pairs CTP cytidine 5’-monophosphate

BRI Bristol Royal Infirmary CV coefficient of variation

BSA bovine serum albumin d distance

BSE bovine spongiform encephalopathy dAMP deoxyadenosine 5’-monophosphate

BSHI British Society of Histocompatibility and dATP deoxythymidine 5’-monophosphate

DGSA Dangerous Goods Safety Advisor G centrifugal field or acceleration

dRib deoxyribose GMP guanosine 5’-monophosphate

DSE display screen equipment H&E haematoxylin and eosin

DSEAR Dangerous Substances and Explosive Atmosphere h Planck’s constant

EDTA ethylenediamintetraacetic acid HETP height equivalent to a theoretical plate

EI electron ionization HIS Hospital Information System

EIA enzyme immunoassay HIV human immunodeficiency virus

EIm electron impact HPC Health Professions Council

ELISA enzyme-linked immunosorbent assay HPCE high performance capillary

IOSH Institute of Occupational Safety and Health MM multiple myeloma

MALT mucosa associated lymphoid tissue PCV packed cell volume

MBL mannose-binding lectin PDP personal development plan

MHO manual handling operations PET positron emission tomography