CLINICAL CHEMISTRY INTERNSHIP | MODULE 2

INSTRUMENTATION

MODULE OUTCOMES

At the end of the rotation, the intern is able to:

1. Identify and familiarize correctly the different laboratory equipment’s used in clinical chemistry.
2. Operate properly automated clinical chemistry analyzers.
3. Explain accurately the principles of the different chemistry analyzers.
4. Demonstrate correctly proper pipetting techniques.
5. Practice properly quality assurance and laboratory safety.

INTRODUCTION
Instrumentation is the use or application of instruments for observation, measurement, or control. It
involves the use of or operation with instruments; especially: the use of one or more instruments in carrying out
laboratory tests. Instrumentation is the development or use of measuring instruments for observation, monitoring or
control. Laboratory instrumentation is a collection of laboratory test equipment. Such a collection of equipment
might be used to automate testing procedure. It could also include: "The design, construction, and provision of
instruments for measurement, control, etc.; the state of being equipped with or controlled by such instruments
collectively."

KEY CONCEPTS

I. Terminologies
A. Batch testing – all samples are loaded at the same time, and a single test.
B. Parallel testing – more than one test is analyzed concurrently on a given clinical specimen.
C. Random access testing – any test can be performed on any sample in any sequence.
D. Sequential testing – multiple tests analyzed one after another on a given specimen.
E. Open reagent system – a system other than manufacturer’s reagents can be utilized for measurements.
F. Closed reagent system – a system where the operator can only use the manufacturer’s reagents.
G. Pneumatic tube delivery system – it provides point to point delivery of specimens to the laboratory and
offered several advantages over specimen transport by humans.
II. Types of glassware
A. Borosilicate glass (pyrex and kimax)
1. Used for heating and sterilization process
2. Characterized by a high degree of thermal resistance, low alkali content
3. Strain point: 515 deg. Celsius (Pyrex)
B. Boron- free glassware/Soft glass
1. It has high resistance to alkali
2. Thermal resistance is less as compared to borosilicate
C. Corex (Corning)
1. Is a special alumina- silicate glass that has been strengthened chemically than thermally
D. Vycor (Corning)
1. It is utilized for high thermal, drastic heat shock and extreme chemical treatment with acids (except
hydrofluoric) and dilute alkali
2. It can be heated to 900 deg Celsius
E. Flint glass
1. Made up of soda- lime glass and a mixture of calcium, silicon and sodium oxides
2. Has poor resistance to high temperature
III. Pipet classification
A. Types according to design/calibration marks
1. TD (To Deliver)- it delivers the exact volume/amount it holds to a container
2. TC (To Contain)- it holds the particular volume but does not dispense the exact volume
B. Types according draining characteristics
1. Blow-out- has a continues etched rings on top of the pipet or two small continues rings very close
together located near the top of the pipet
- exact volume is obtained when the last drop is blown out
2. Self- draining- absence of etched rings; liquid is allowed to drain by gravity
C. Types according to purpose
1. Transfer Pipet:
a. Volumetric pipet
i. For nonviscous fluid; self- draining; small amount left in the tip should not be blown out.
ii. For aqueous fluid. Has the greatest degree of accuracy and presicion.
b. Ostwald- Folin
i. for viscous fluid; with etched ring
ii. blow-out pipet, for biological fluids with viscosity greater than that of water
c. Pasteur Pipet

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 i. transfers fluid without consideration of a specific volume
d. Automatic macro- or micropipettes
i. Most routinely used pipet in todays clin chem lab. Adv: safety, stability, ease of use, increased
precision and less cleaning required (the tips often being disposable. Pipetting capacity of less
than 1 mL is considered as microppet..more than 1ml macro..available range is from 1 uL to
5000 mL
2. Graduated or Measuring pipet
a. Serological Pipet
i. with graduations to the tip; blowout pipet
b. Mohr Pipet
i. with graduations to the tip; calibrated between 2 marks; self- draining pipet
c. Bacteriologic pipet
d. Ball, Kolmer and Kahn pipet
e. Micropipets: (< 1mL) TC pipets

i. Sahli- Hellige pipet

ii. Lang- Levy pipet
iii. RBC and WBC pipet
iv. Kirk and Overflow pipet

3. Mechanical or Automatic Pipets

a. Air Displacement pipet:

i. It relies on piston for suction creation to draw the sample into a disposable tip
ii. The piston does not come in contact with the liquid
b. Positive Displacement Pipet:
i. It operates by moving the piston in the pipet tip or barrel, much like a hypodermic syringe
ii. Does not require a different tip for each use
c. Dispenser/Dilutor Pipet:
i. It obtains liquid from a common reservoir and dispensed it easily
ii. It combines sampling and dispensing functions
IV. CENTRIFUGE
A. Used to separate substances of different mass or density
B. RCF = RPM2x r x 1.12 x 10-5
C. Types of Centrifuge:
1. Horizontal Head Centrifuge:
a. Swinging bucket type; the centrifuge tubes are held in a vertical position when not moving but are
horizontal when the centrifuge is fully in motion
b. This type is recommended for serum separator tubes
2. Angle Head Centrifuge
a. Has fixed 25-35 degree angle at which the tubes are held during centrifugation
b. Less affected by heat build- up due to air friction than horizontal centrifuge heads
3. Ultracentrifugation
a. Generates the highest speed, centrifuge head is held at a fix angle but generates tight sediment
buttons due to high speed generated
b. Ultracentrifuges are refrigerated
c. Used for lipoprotein measurements since refrigeration enhances the separation
D. Centrifuge Maintenance Procedure
1. Weekly- clean interior components with soap and water followed by freshly made 10%w/v bleach
solution, including sample buckets
2. Monthly- check for unusual vibrations, braking mechanism to ensure a smooth, gradual stop, the timer
of the centrifuge using a stopwatch
3. Quarterly(every 3 months)- check for the revolutions per minute at several commonly used speeds,
while centrifuging a balanced load using a tachometer
 Use to calibrate centrifuge:
 Stopwatch – use to calibrate time.
 Tachometer – use to calibrate speed of the centrifuge.
 Strobe light – use if tachometer is not available.

V. COLORIMETRY
A. This involves the Beer’s Law
B. In this method, the constituent is colored (it absorbs light within the visible spectrum)
C. Two types:
1. Spectrophotometry
a. most commonly used for routine chemistry, measures light intensity in a narrower wavelength
b. Measurement of intensity of light at selected wavelength
c. It uses monochromators to allow a more sensitive and precise measurement and makes the analysis
suitable for both colored and colorless solutions.
d. Kinds of spectrophotometer
i. Single beam spectrophotometer
 designed to make one measurement at a time at one specified wavelength

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  Simplest type of absorption spectrometer
ii. Double beam spectrophotometer
 Splits the monochromator light into two components
 One beam passes the sample, the other beam through a reference solution and a blank
 Additional beam corrects for variation in light source intensity
 Requires 2 or more cuvette holders
 2 types of Double- beam spectrophotometer:
- Double- beam in space:uses 2 photodetectors, for the sample beam and reference beam
- Double- beam in time:uses one photodetector and alternately passes the monochromatic
light throgh the sample cuvet and then the reference cuvet using a chopper or rotating
sector mirror
 Chopper: for light source variation
e. Principle of spectrophotometry
i. It determines the amount of light emitted by a molecule after excitation by electromagnetic
radiation.
ii. Wavelength:
 Describes a position within a spectrum, measured in nanometers
 The distance between the peaks of a light wave
 distance between two successive peaks
 The wavelength is inversely related to frequency and energy; the shorter the wavelength,
the higher the frequency and energy and vice versa.

400 -700 nm = visible spectrum

< 400 nm = ultraviolet region
> 700 nm = infrared region
iii. Light also behaves as if it is composed of discrete energy packets called photons whose
energy is inversely proportional to wavelength
 The longer the wavelength, the lower the energy, and vice versa

Spectrum of Light:
Visible light

Gamma rays X- rays UV 400 < V I B G Y O R >700 IR microwaves

radiowaves

iv. Energy
 is transmitted via electromagnetic waves that are characterized by their frequency and
wavelength
v. Planck’s formula:
E= hv
E = energy of a photon in joules
h = constant ( 6.626x10-34 erg sec)
v = frequency
vi. Frequency
 the number of vibrations of wave motion per second “the lower the wave frequency the longer
the wavelength”
vii. Beer’s Law: aka Beer Lambert’s Law
 It states that the concentration of the unknown substance is directly proportional to the
absorbed light (absorbance or optical density) or inversely proportional to the logarithm of
transmitted light (% transmittance)
A = 2-log%T
Au/As = Cu/Cs
A= abc

Where:
A=absorbance
a=absorptivity constant for a particular compound at a given wavelength
b=length of the light path
c= concentration
f. Parts of the spectrophotometer
i. Light/Radiant source
 It provides polychromatic light and must generate sufficient radiant energy or power to
measure the analyte of interest
 An intense beam of light is directed through the monochromator and the sample
 2 types:
1. continuum source
=emits radiation that changes in intensity; most commonly used
Ex:
Tungsten = most common light source in visible and near infrared region
Deuterium= routinely used to provide UV radiation

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 Xenon = covers both the UV and the visible range.

2. Line source
 emits limited radiation and wavelength
Ex: mercury and sodium vapor lamps in spectrophotometer (UV and visible region)
and the hollow cathode tube (AAS)
ii. Entrance Slit
 It minimizes any stray light and prevents the entrance of scattered light into the
monochromator system
 Stray light- refers to any wavelength outside the band transmitted by the monochromator;
 causes absorbance error most common cause of loss of linearity at high analyte
concentration
iii. Monochromator
 Isolates specific or individual wavelength of light
1. Prisms:
 Are wedge- shaped pieces of glass, quartz or sodium chloride It can be rotated,
allowing only the desired wavelength to pass through an exit slit
2. Diffraction gratings:
 Are the most commonly used; better resolution than prisms made by cutting grooves
into aluminized surface of flat piece of crown glass
 wavelength are bent as they pass a sharp corner
3. Filters
 made by placing a semi-transparent silver films on both sides of dielectric such as
magnesium fluoride
4. Holographic gratings
iv. Exit slit
 controls the width of light beam (bandpass)
 controls the light which has been isolated by monochromator
v. Cuvet
 Also called absorption cell/analytical cell/sample cell
 It holds the solution whose concentration is to be measured
 Types:
1. Alumina silica glass- most commonly used
2. Quartz/plastic- used for measurement of solution requiring visible and ultraviolet spectra
3. Borosilicate glass
4. Soft glass
vi. Photodetector
 It detects and converts transmitted light into photoelectric energy
 It detects the amount of light that passes through the sample in the cuvet
 Kinds of Photodetector:
1. Barrier Layer Cell/Photocell – simpliest detector,least expensive and temp. sensitive.
2. Phototube – contains cathode and anode enclosed in a gas case.
3. Photomultiplier tube (PMT) – most commonly used detector, measures visible and UV
regions.
4. Photodiode – measures light at a multitude of wavelengths – detects less amount of
light.
vii. Read- out/Meter device
 It displays output of the detection system
 Measures the magnitude of the current generated by the detector
 Converts electrical energy into readable numbers
 Examples: galvanometer, ammeter,light- emmiting diode (LED) display

2. Photometer
a. measurement of light intensity but of multiple wavelength
b. Measurement of the luminous intensity of light falling on a surface from a source

3. Flame emission photometry (FEP)

a. It measures the light emitted by a single atom burned in a flame
b. Principle: excitation of electrons from lower to higher energy state

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 c. Light source: Flame (also serves as the cuvet)
d. Method: Indirect Internal Standard Method
e. Internal standard: Lithium/Cesium- corrects variations in flame and atomizer
characteristics
f. Cesium: alternative if Lithium will be measured
g. Photodetector: Photocell
h. Monochromator: Filter
i. It is used for the measurement of excited ions (Na and K)
Lithium Red
Potassium Violet
Sodium Yellow
Calcium red orange
Magnesium Blue
Rubidium Red

4. Atomic Absorption Spectrophotometry (AAS)

a. It measures the light absorbed by the atoms dissociated by heat
b. Principle: Element is not excited but merely dissociated from its chemical bond and place in an
unionized, unexcited, ground state
c. Used for measurement of unexcited trace metals: Ca, Mg
d. Internal standard is not needed
e. Light source: Hollow- cathode lamp (energy source); lined with the element being measured
f. AAS chopper: or nebulizer which sprays the sample into flame
g. Lanthanum or Strontium chloride is added to samples to form stable complexes with phosphate

VI. Fluorometry
A. measures analyte which have the ability to absorb light of lower wavelength and transmit it at a higher
wavelength
B. also known as molecular luminescence spectrophotometry
C. Uses 2 monochromators (either filter, prisms or gratings)
D. Primary monochromator: selects wavelength (isolates light)
E. Secondary monochromator: prevents the incident/ stray light
F. About 1000x more effective than spectrophotometer
G. Affected by Stokes effect: the difference between the max. wavelength excitation and emitted fourescence.
Caused by quenching- due to pH and temp. changes, contaminants and UV light

VII. Nephelometry
A. Principle: Measured the amount of scattered by a particulate matter suspended in a turbid solution
B. Detection of light energy scattered towards the detector is not in the path of the transmitted light, usually at
90 or 30 degrees angle from the incident light
C. For proteins (ag-ab reaction)
D. Light scattering depends on wavelength and particle size
E. Uses photomultiplier: responsible of sensitivity of nephelometry tube
F. More sensitive than turbidimetry
G. (pic) detection of light energy scattered or reflected towards a detector is not in direct path of the
transmitted light, usually at 90 or 30 deg angle of the incident light

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VIII. Turbidimetry
A. Principle: Determines the amount of light blocked or reduced by a particulate matter in a turbid solution
B. measures abundant large particles proteins and bacterial suspensions
C. as the number of particles increase, the lesser transmittance of the incident light through the solution
D. Uses: CHON measurements in CSF and urine,detect bacterial growth in broth culture, detect clot formation.
E. Depends on specimen conc and particle size
F. The measurement of reduction of light due to particle formation, as the number of particle increases, the
lesser transmittance of the incident light through the solution

IX. Electrophoresis
A. Principle: movement of charged particles in a medium resulting to their separation based on their electrical
charges when an electrical current is applied
B. Use: for the separation of proteins in body fluids
C. Components of electrophoresis: electrical power, support medium, buffer, sample and detecting system
D. Buffer: Barbital (pH 8.6)
E. Factors affecting the rate of migration:
1. Net electric charge of the molecule
2. Size and shape of the molecule
3. Electric field strength
4. Nature of the supporting medium
a. Cellulose acetate: most commonly used, separates by molecular size
b. Agarose gel: separation based on electric charge
c. Polyacrylamide gel (PAGE): increased resolution, movement or separation is based on both
charge and size
5. Temperature of operations- 37 deg Celcius
a. Denaturation: loss of physical and chemical properties of proteins (tertiary structure of proteins)
X. Densitometry
A. measures absorbance of stain/dye- concentration of the dye and protein fraction
B. It scans and quantitates electrophoretic pattern
C. Used in electrophoretic pattern where the concentration of stained molecules is measured
XI. Chromatography
A. Separation of chemical mixture in to different component based on their physical- chemical characteristics
and the interactions of molecules with the mobile (gas or liquid) and stationary phase (solid or liquid)
through a support medium.
B. 2 forms of Chromatography
1. Planar
a. Paper chromatography
i. fractionation of sugar and amino acid Sorbent: whatman paper
b. Thin layer chromatography
i. For semiquantitative drug screening test
ii. Based on retention factor (Kf) value
iii. Kf value: relative distance of separation from the point of application
iv. Sorbent: thin plastic plates impregnated with a layer of silica gel or alumina
2. Column

a. Gas chromatography
i. Separate mixtures of compounds that are volatile
ii. Used for separation of steroids, barbiturates, blood, alcohol and lipids
iii. Samples must be vaporized (if not volatile)
iv. Sample: blood, urine, body fluid
 Mass Spectrophotemeter
i. Based on the fragmentation and ionization of molecules using a suitable source of energy
ii. Detects structural information and determination of molecular weight

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 iii. independent method; can be paired with other chromatographic method
iv. GC-Mass spectrometry =the gold std for drug testing (elicit and therapeutic)
v. Tandem mass spectroscopy=can detect 20 inborn errors of metabolism from a single blood
spot
b. Liquid chromatography
i. based on the distribution of solutes between a liquid mobile phase and stationary phase
ii.
 HPLC (High Performance Liquid Chromatography)
i. uses pressure for fast separations,controlled temperature,in line detectors and gradient elution
technique.
ii. USES:
 fractionation of drugs, hormones, lipids, carbohydrates and proteins
 quantitation of various hemoglobins associated with specific diseases
 rapid HbA1c test (within 5 minutes)
XII. Chemiluminescence
A. The chemical reaction yields an electronically excited compound that emits light as it returns to its ground
state
B. Emission of light is created from a chemical or elctrochemical reaction
C. Use: immunoassays
D. Advantages:
1. Subpicomolar detection limits
2. Speed
3. Ease of use
4. Simple instrumentation
E. Disadvantage:
1. Impurities can cause a background signal that degrades sensitivity and specificity.

XIII.Electrochemistry
A. Relationship of electricity and chemicals
B. Measures current or voltage produced by specific ions
C. Uses: for blood gases, pH, electrolytes, glucose, urea, ionized calcium, lead and chloride
1. Potentiometry:
a. Ion- selective electrode:
i. Ion selectivity depends on membrane and barrier composition
ii. Sensitive and selective for the ion it measures
iii. Used for measurement of electrolytes and ammonia
iv. Composition: Valinomycin: potassium and aluminum; silicate: sodium
v. Three basic ISE classes:
 Ion- selective glass: for hydrogen ions, sodium and ammonia
 Solid- state electrodes: Ag-AgCl for Cl determination in sweat
 Liquid ion- exchange membranes- for pH determination

2. Coulometry:
a. Measurement of the amount of electricity (in coulumbs) at a fixed potential
b. Endpoint: detected by amperometry
c. Use: chloride ion measurement in serum, CSF and sweat
d. Governed by Faraday’s Law
3. Amperometry
a. Measurement of the current flow produced by an oxidation- reduction reaction
b. Use: pO2 measurement
c. Polarography: specific for pO2, measurement of differences in current at constant voltage
Governed by Ilkovic equation
4. Voltammetry
a. Measures current, detects very low analyte levels
b. Anodic stripping voltammetry: measures heavy metals (lead)
XIV. Isoelectric Focusing
A. Separates molecules by migration through a pH gradient
B. Ideal for separating proteins of identical sizes but with different net charges
C. pH gradient is created by adding acid to the anodic area of the electrolyte cell and adding base in the
cathode area
D. Supporting media: agarose gel, polyacrylamide gel and cellulose acetate

XV. Automated Analyzers

A. Continuous flow
1. A device that automatically analyzes a series of samples of biologic materials that are continuously
pumped into tubes along with appropriate reagents.
2. Liquids are pumped through the continuous tubing
3. Resolves the major consideration of uniformity in the performance of tests because each sample
follows the same reaction vessel and pathway
4. Assist the laboratory that needs to run many samples requiring the same procedure
5. Air bubbles at regular intervals serves as separating and cleaning media

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 6. A heating bath maintains the required temperature of the reaction to allow complete color
development (reaction rate is controlled by temperature)
7. Mixing of sample and reagent: by using a glass coil inserted to the flow path
8. Example: Simultaneous Multiple Analyzer (SMA), Technicon

B. Centrifugal analyzer
1. Uses force generated by centrifugation to transfer specimen and reagents
2. Liquids are placed in separate cuvets for measurement at the perimeter of a spinning rotor
(1000rpm)
3. Uses acceleration and deceleration of the rotor to transfer the reagents and sample from one
chamber to another
4. Most capable of running multiple samples one test at a time in a batch
5. Mixing: centrifugal force (rotor) is utilized or bubbling of air
6. Ex: Cobas-Bio ( Roche Diagnostics), IL Monarch= fully integrated walk-away design

C. Discrete analyzers
1. samples travel through the instrument in its own reaction vessel
2. automated syringes/pumps are used to mix samples and color reagents together, and the resultant
color formation is analyzed by a colorimeter/photometer
3. most popular and versatile analyzer
4. requires minimum volume of the sample
5. capable of running multiple-tests-one-sample-at-a-time
6. employs a variety of syringe pipets (positive displacement)
7. for mixing: magnetic driven teflon, stirring paddle, forceful dispensing

 Roche Cobas Integra

Advantages:
 Random access capability: allows STAT samples to be easily tested
Ex: Vitros, Dimension Dade, Beckman Astra system, etc.

XVI. Notes to remember!

A. For calibration of Pipettes
 Class A pipets do not require recalibration ( made of plastic, considered disposable).
 Distilled water is the calibrating medium for TD pipets while mercury is for TC pipets

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  Gravimetric and spectrometric methods: used to verify pipet volume accuracy and precision
 0.1% phenol red soln. in dist. water: used to compare the reproducibility of brands of pipet tips
B. Volume Measurements
 1 lambda= 1 microliter (0.001 mL)
 1 microliter= 1.0 milligram
C. Pipets
 Plastic pipet tips are made primarily of polypropylene
 Pipets should be held in a vertical position during transferring to the receiving vessel, with the tips
against the side of the receptacle
 For volumetric TD pipet, it should not be shaken or hit against the wall of the container during draining

 Acid dichromate- cleaning solution for glassware

D. Advantages of automation:
 Increase number of tests to be performed in given period.
 Minimizes variation of results from one laboratorian to another.
 Eliminates the potential error in manual analyses such as pipetting, calculation and transcription.
 Less exposure to biohazard.
E. Carry Over
 Is the transport of quantity of analyte or reagent from one specimen reaction into another, and
contaminating a subsequent one. It can be prevented by frequent washing and flushing.

FURTHER READING
 Bishop, Michael L. et al (2013). Clinical Chemistry Principles, Techniques, Correlations, 7th ed., Lippincott
Williams and Wilkins, Philadelphia.
 Mcpherson, Richard A. and Matthew R. Pincus. Henry's Clinical Diagnosis and Management by Laboratory
Methods 22nd ed. Philadelphia: Elsevier Inc., 2012
 Sacher, Ronald and Richard McPherson. Widmann's Clinical Interpretation of Laboratory Tests 11th ed.
Thailand: F.A Davis,2000
 Maria Teresa Rodriguez. A Review on Clinical Chemistry.
 Lecture notes of Prof. Erlinda Villano, Ms. Jastine Camile Velayo, Mr. Ronaldo E. Puno, Mr. Rodolph C.
Lagarto and Asst. Prof. Joy G. Raso

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