MDU4303 - Clinical Biochemistry II

FACULTY OF HEALTH SCIENCES
DEPARTMENT OF MLS
THE OPEN
UNIVERSITY BACHELOR OF HONOURS IN MEDICAL LABORATORY
OF SRI LANKA SCIENCES: LEVEL 4
MDU4303: CLINICAL BIOCHEMISTRY II
CLINICAL BIOCHEMISTRY II
EXPERIMENTAL COPY
BLOCK I
CLINICAL BIOCHEMISTRY II
Published by
The Open University of Sri Lanka
Clinical Biochemistry 11 - Block 1
Course Team
Course Team Chair Educational Technologist

Dr. A.M.B. Priyadarshani Dr. B. G. Jayathilake
Authors Desktop Publisher
Ms. N.D. Withanage Ms. B. Y. Alvitigala

Dr. D.I. Uluwaduge Ms. R.G.L. Rathnayake
Dr. A.M.B. Priyadarshani
Ms. B. Yasassri Alvitigala Cover Designer
Ms. R.G.L. Rathnayake Ms. R.G.L. Rathnayake
Content Editor
Professor. R. Shivakaneshan
The Open University of Sri Lanka

Nawala, Nugegoda, Sri Lanka
© 2019 The Open University of Sri Lanka
All rights reserved. No part of this course book may be reproduced or transmitted
in any form or by any means, electronic or mechanical, including photocopy and
recording or from any information stored in a retrieval system, without permission
in writing from the Open University of Sri Lanka.
ii
Acknowledgements
The course team members of MDU4303 – Clinical Biochemistry II is

acknowledged.
iii
Introduction to the Course
Welcome to MDU4303 Clinical Biochemistry. This level 4 course is one of

the courses that make up the Bachelor of Medical Laboratory Sciences
(BMLS) Honours degree. This is a 3 Credit course which will require about
150 hours of Study.
Course Outline
This course is designed to provide you with basic knowledge on
biochemical aspects of blood and other body fluids, how they change in
different disease conditions and to provide opportunity to develop required
practical skills in performing biochemical investigations.
Learning outcomes
At the completion of this Course you will be able to;
▪ Explain the principles and perform biochemical investigations of blood
and other body fluids.
▪ Evaluate the results of biochemical investigations and comment on their
disease conditions.
▪ Identify and minimize pre – analytical, analytical and post analytical
errors.
▪ Discuss the characteristics of common metabolic disorders.
▪ Analyse clinical specimens to identify main metabolic disorders.
Pre-requisites
There are no any pre-requisites to learn this course.
Structure of the Course

This Course consists of one Block
Block 1 - Unit I to Unit VI
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Teaching Strategies
Online Component
Supplementary materials and learning activities relevant to each session will
be uploaded into your online portal.
Day Schools
There will be 03 days schools during which you will be able to clarify
problems you may encounter.
Day Schools 1 − Session 01− 04

Day Schools 2 − Session 05 - 08
Day Schools 3 − Session 09 - 13
As you would notice, day schools are planned to gain face-to-face teaching
and learning experiences. In order to make maximum use of your day
schools you are advised to work through the course material prior to come
for the day school.
Assessment
You will have one No Book Tests (NBTs) and a practical test as a means of
assessment of your progress. Marks obtained from the continuous
assessments will be contributed for your overall continuous assessment
marks (OCAM) as follows and will be used to determine your eligibility to
sit for the final examination.
NBT - 50%
Practical test - 50%
Total - 100%
Minimum 50 marks compulsory for PT
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Your overall performance during the course will be determined by your

scores gain from continuous assessment and final examination as specified
bellow.
Continuous Assessment - 40%

Final Examination - 60%
Total - 100%
We hope that you will find the course material interesting and that you will
enjoy your learning experience at the Open University of Sri Lanka.
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Contents
Introduction to the Course iv
Introduction to the Units 1
Unit 1: Disorders of carbohydrate metabolism and laboratory diagnosis
Session 1 Disorders of carbohydrate metabolism 5
Session 2 Diabetes Mellitus 23
Session 3 Blood Glucose determination and diagnosis of Diabetes 33
Session 4 Complications of Diabetes and laboratory diagnosis 43
Unit 2: Disorders of Lipid metabolism and laboratory diagnosis
Session 5 Disorders of Lipid and Lipoprotein metabolism 51

Session 6 Analysis of Lipids and Lipoproteins 61
Unit 3: Serum / Plasma Protein
Session 7 Changes of protein levels in disorders 69

Session 8 Estimation of serum /Plasma proteins 81
Unit 4: Other body fluids
Session 9 Analysis of Cerebrospinal fluid (CSF) 91
Session 10 Analysis body cavity fluids 113
Session 11 Stool analysis 141
Unit 5: Disorders of reproduction and laboratory diagnosis
Session 12 Disorders of reproduction in male and laboratory diagnosis 159
Session 13 Disorders of reproduction in female and laboratory diagnosis 177

187
Contributors: Session Authors and Editors
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Clinical Biochemistry 11 – Block 1
Introduction to the Units

Unit I
Disorders of carbohydrate metabolism and laboratory diagnosis will be discussed

in Unit I. Different types of disorders of carbohydrate metabolism including
diabetes mellitus, pathogenesis, signs and symptoms and related laboratory
investigations will be discussed.
At the end of Unit I, you should be able to;
 Understand the biochemical basis for the inherited disorders of
carbohydrate metabolism
 Describe defective enzymes in galactose, fructose, pyruvate and glycogen
metabolism
 Describe the role of urinary analysis in the diagnosis of disorders of
 Differentiate type 1 and type 2 diabetes.
 Describe metabolic changes occurring in diabetes
 Explain the physiological basis of signs and symptoms of diabetes.
 Describe the investigations related to screening, diagnosis and monitoring
of diabetes mellitus and their importance.
 Describe the diagnostic criteria for diabetes mellitus.
 Describe the macrovascular complications and microvascular
complications of diabetes
 Identify the markers of diabetes nephropathy
 Describe the importance of maintenance of normal glucose level among
diabetic subjects.
 Educate the public regarding diabetic complications and protection.
Unit II
The main topic which will be discussed under unit II is Disorders of Lipid
metabolism and laboratory diagnosis. You will get a thorough knowledge on
disorders of Lipid and Lipoprotein metabolism. Apart from that analysis of Lipids
and Lipoproteins also will be discussed.
At the end of Unit II you should be able to;
1
 State the clinical significance of disorders of lipid and lipoprotein

metabolism.
 Explain what is meant by "atherosclerosis", plaque formation and causes of
atherosclerotic diseases.
 Explain how atherosclerotic plaque contributes in myocardial infarction.
 Explain how genetic disorders affects the lipid and lipoprotein metabolism.
 Explain how deficiency in lipoprotein lipase activity causes
hyperchylomicronemia with elevated triglyceride concentration.
 Discuss the causes of familial hypertriglyceridemia & familial
hypercholesterolemia and the biochemical events that take place in the
body in above 2 conditions.
 Discuss how following condition are diagnosed in the laboratory; familial
combined hyperlipidemia, hyperapobetalipoproteinemia and type V
hyperlipoproteinemia.
 Explain the enzymatic methods used in determination of cholesterol and
triglycerides levels.
 Describe the precipitation method and direct HDL cholesterol assay used
in HDL cholesterol determination.
 State the Friedewald equation and β-quantification method.
 List the methods available in measurement of apolipoproteins,
apolipoproteins A-1 and B-100 and lipoprotein (a).
Unit III
Unit III is focused on Serum / Plasma Protein. Changes of protein levels in

disorders and Estimation of serum /Plasma proteins will be discussed under this
unit.
At the end of Unit III you should be able to;
 Draw and label the normal serum electrophoretic and densitometry

patterns.
 Describe the serum electrophoretic and densitometry patterns in following
diseases; cirrhosis of the liver, α1-antitrypsin deficiency (emphysema),
polyclonal gammopathy, monoclonal gammopathy, chronic inflammatory
2
states, acute phase protein response pattern, hyperlipoproteinemia, chronic

hepatitis and nephrotic syndrome.
 State why the serum/plasma protein estimation is important in
diagnosis of disease.
 State the principle of methods used in serum/plasma protein analysis.
 Discuss the advantages and limitations/disadvantages of methods of
serum/plasma protein analysis
Unit IV
Laboratory procedures including specimen collection and investigation methods of

urine have been discussed in MDU3303- Clinical Biochemistry I. in this unit it will
be discussed laboratory investigations related to other body fluids including
Cerebrospinal fluid (CSF), body cavity fluid and Stool
At the end of Unit IV you should be able to;
 State the physiology behind CSF formation, functions of CSF
 Describe the CSF specimen collection procedure, specimen handling
guidelines
 State the CSF composition
 Explain traumatic and cerebral hemorrhage
 Describe the conditions interpreted by CSF appearance
 Describe the CSF cell counting techniques and calculations
 Describe the chemical analysis of CSF and their importance
 State the identification parameters of meningitis
 Describe the blood collection procedures
 Describe the influence of blood collection
 Describe the composition of blood
 State the anticoagulants used in blood collection
 Explain the differences between transudate and exudate
 Explain the factors affecting body fluid composition
 Describe the specimen collection procedure of synovial fluid
 Explain the laboratory diagnosis of synovial fluid
 State the types of serous fluid and sample collection
 Describe the laboratory diagnosis of pleural fluid
 Describe the laboratory diagnosis of peritoneal fluid
3

 State the pathophysiology behind stool formation
 Describe the pathological conditions related to stool examination
 State the constituents of normal stool
 State the laboratory tests available in stool examination
 Describe the macroscopic examination of stool
 Describe the microscopic examination of stool
 Explain the principles, procedures and importance of chemical tests
available in stool examination
Unit V
Disorders of reproduction and laboratory diagnosis will be discussed in unit V. The

content includes disorders of reproduction and laboratory diagnosis.
At the end of Unit IV you should be able to;
 State the pathophysiology behind semen formation
 Describe the structure and function of sperm
 State the constituents of normal semen
 Describe the abnormal morphology of sperm
 Describe the specimen collection procedure of semen
 State the WHO guidelines on semen analysis
 Describe the laboratory test, their procedures and expected diagnosis
 State the sperm separation methods
 Explain the female factors of disorders in reproduction.
 List and explain the laboratory diagnosis related to disorders of
reproduction in females.
 Explain the analytical methods of steroid and peptide reproductive
hormones.
We hope four Units of the Block I will support you to improve your knowledge,
develop skills and acquire positive attitudes to serve for the people to promote their
health status.
4
Session [1]: Disorders of Carbohydrate Metabolism
Session 1
Disorders of Carbohydrate Metabolism

Content
Introduction, p5
1.1 Disorders of galactose metabolism, p6
1.2 Disorders of fructose metabolism, p8
1.3 Disorders of pyruvate metabolism, p10
1.4 Glycogen Storage diseases, p12
1.5 Laboratory diagnosis of carbohydrates, p15
Summary, p21
Learning outcomes, p21
References, p22
Introduction
Disorders of carbohydrate metabolism occur in many forms. The

commonest is diabetes mellitus. Apart from diabetes mellitus, other
disorders of carbohydrate metabolism are rare and belong to the category of
inborn errors of metabolism (i.e., genetic defects).
Inborn Errors of Metabolism (IEM) comprise a group of disorders in which

a single gene defect causes a clinically significant block in a metabolic
pathway, resulting either in accumulation of substrate behind the block or
deficiency of the product. Almost all defects of carbohydrate metabolism
are recognized as autosomal recessive traits and these are due to point
mutations.
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Unit [1]: Disorders of Carbohydrate Metabolism and Laboratory Diagnosis
1.1 Disorders of galactose metabolism
Galactosaemia is a disorder in which the body cannot catabolize galactose

which results from hydrolysis of lactose. In galactose metabolism (Figure 1)
galactose and uridine diphospho glucose are converted to glucose-1-
phosphate and uridine diphospho galactose through the action of three
sequential enzymes: galactokinase (GALK, EC 2.7.1.6), galactose-1-
phosphate uridyltransferase (GALT, EC 2.7.7.12), and uridine
phosphogalactose 4′-epimerase (GALE, EC 5.1.3.2). Inborn errors of
galactose metabolism occur with impaired activity for each of the enzymes.
There are three separate disorders of galactose metabolism of clinical

importance.
1. Galactokinase deficiency
2. Galactose-1-phosphate uridyl transferase deficiency
3. UDP galactose-4-epimerase deficiency
Recent evidence of low erythrocyte and tissue UDP gal levels, associated
with ovarian dysfunction, may indicate impaired galactoside synthesis.
Administration of uridine corrects the UDP galactose depletion and trials in
which it is added to the galactose-restricted diet have begun.
1.1.1 Galactokinase deficiency
This deficiency causes the elevation of galactose in blood and subsequent

excretion in urine (galactosaemia and galactosuria). Excess galactose is
converted to galactitol and it could lead to cataract formation. This is a
benign condition.
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1.1.2 Galactose-1-phosphate uridyl transferase deficiency
Classical galactosaemia is caused by this deficiency and it is the most

common and the most severe of these diseases, affecting from ~1 in 10,000
to 1 in 30,000 live births.
Here the trapping of inorganic phosphate as galactose 1 P leads to a

reduction in inorganic phosphate pool affecting various other metabolic
pathways (Figure 1.1). The liver is severely affected. Children and young
adults with galactosaemia can have problems with speech, language,
hearing, stunted growth and certain learning disabilities.
Figure 1.1 Galactose metabolism
(Source: https://kitchendecor.club/files/non-meter-glucose-prick.html )
7
1.1.3 UDP galactose-4-epimerase deficiency
Deficiency of GALE is the rarest condition among above discussed 3

disorders of carbohydrate metabolism. Following laboratory investigations
are important.
 Assays for galactitol and galactose-1-phosphate
 Assessment of enzyme activities of GALT, GALK and GALE
1.2 Disorders of fructose metabolism
Fructose is a monosaccharide that is present in high concentrations in fruits

and honey and is a constituent of sucrose and sorbitol. There are three
inherited disorders of fructose metabolism.
1. Fructokinase deficiency
Fructokinase deficiency leads to essential fructosuria. This is a mild

condition and does not require treatment. However hereditary fructose
intolerance (HFI) and hereditary fructose-1,6-biphosphatase deficiency
(HFBP) should be considered as serious conditions. And these two
conditions are treatable and controllable.
2. Aldolase B deficiency
Aldolase B deficiency leads to Hereditary Fructose Intolerance (HFI).

Phosphorylated intermediates get accumulated in this condition. Fructose-1-
phosphate is an example for a phosphorylated intermediate and this product
get accumulated in liver, kidney and small intestine. This leads to severe
hypoglycaemia, vomiting, jaundice, bleeding, hepatomegaly and
hyperuricaemia. This condition can be life threatening among infants. Older
children and adults may show mild to severe outcomes. One is death due to
hepatic failure. Since fructose is commonly used as a food sweetener it is
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important to identify the conditions in people in order to remove fructose

from the diet.
3. Deficiency of fructose-1,6-bisphosphatase
This deficiency compromises gluconeogenesis and results in fasting

hypoglycaemia, ketosis and acidosis (Figure 1.2).
Figure 1.2 Fructose – Glycogen metabolism
(Source: https://commons.wikimedia.org/wiki/File:Fructose-glycogen.jpg)
Subjects with hereditary fructose intolerance generally develop a strong

aversion to fructose containing food including sweets and fruits. These
subjects may develop severe abdominal pain, vomiting and low blood sugar
when they consume food with fructose. People with HFI can adopt a normal
life with fructose free diet. However early diagnosis is important since
untreated subjects can develop liver and kidney damage.
In muscle and most other tissues of the body, conversion of glucose to

glucose-6-phosphate and fructose to fructose-6-phosphate is done by the
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same hexokinase. Using the enzymes of the glycolysis this generated

fructose-6-phoshphate is metabolized to pyruvate or glycogen.
In the liver, fructose is phosphorylated largely to the 1-phosphate form and

converted to glyceraldehyde and dihydroxyacetone phosphate. Free
glyceraldehyde can be phosphorylated by a specific kinase; this and
dihydroxyacetone are metabolized further by the reactions of glycolysis.
Activity 1.1
1. Explain why Aldolase B deficiency is more severe than fructokinase deficiency?
1.3 Disorders of pyruvate metabolism
Pyruvate metabolism (Figure 1.3) disorders result when there is a lack of

ability to metabolize pyruvate. As a result, lactic acid can be elevated and
causes different types of neurologic abnormalities as below.
Figure 1.3 Metabolic relations of pyruvate
(Source: https://images.app.goo.gl/KBdwAjsi4jzafakn8)
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1.3.1 Pyruvate dehydrogenase complex deficiency
Defect in any of the 3 enzymes of the pyruvate dehydrogenase complex lead

to the condition called pyruvate dehydrogenase complex deficiency. This
condition has an autosomal-recessive or sex-linked inheritance. In this
condition the link between glycolysis and Kreb’s cycle is interrupted and
lead to elevated levels of lactate. In this condition the process of energy
production in the body is severely affected.
If the serum lactate and pyruvate levels are elevated and a lactate-to-
pyruvate ratio is below 25 in a neonate, pyruvate dehydrogenase complex
deficiency can be suspected. If normal level of lactate is reported in
cerebrospinal fluid, this condition can be excluded.
Diagnosis can be established by the detection of the reduced activity of

pyruvate dehydrogenase complex in cultured fibroblasts, liver tissues,
skeletal muscle, lymphocytes or brain tissues. It is important to do the
investigations in multiple systems since only one system may not show the
deficiency.
1.3.2 Pyruvate carboxylase deficiency
Pyruvate carboxylase deficiency affects gluconeogenesis. This disorder is

characterized by high serum ketones, pyruvate, and lactate, and a lactate-to-
pyruvate ratio above 35. The complex metabolic findings in a patient with
pyruvate carboxylase are due to high levels of acetyl-CoA and low levels of
oxaloacetate. High levels of acetyl-CoA produce ketosis. Low levels of
oxaloacetate cause low levels of aspartate. Low aspartate prevents
nicotinamide adenine dinucleotide from entering the mitochondria. An
excess of nicotinamide adenine dinucleotide in the cytosol increases the
conversion of pyruvate to lactic acid. It is this increase in the conversion of
pyruvate to lactic acid that leads to a lactate-to-pyruvate ratio above 35.
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Low oxaloacetate also impairs the citric acid cycle, glycine cleavage system,
and the urea cycle.
The diagnosis is established by finding decreased pyruvate carboxylase

activity in cultured fibroblasts. Treatment consists of metabolic support and
a diet low in fat and high in carbohydrates and protein. Aspartic acid and
biotin should be added.
Activity 1.2
1. Discuss the consequences of a deficiency in the pyruvate dehydrogenase

complex?
1.4 Glycogen Storage diseases
Figure 1.4 Structure of the glycogen molecule
(Source: http://homepage.ufp.pt/pedros/bq/glycogen.htm )
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The glycogen molecule consists of many glucose moieties. These are linked
by an α - 1,4 glycosidic bond between carbon-1 of one moiety and carbon-4
of the next. At branch points α- 1, 6 glycosidic bond links the glucose
moieties. As the name implies glycogen storage diseases are caused by an
accumulation of glycogen.
During glycogenolysis glycogen phosphorylase catalyses the phophorolysis

of glucose residues from the non-reducing end of glycogen releasing
glucose-1-phosphate. Phosphoglucomutase converts the glucose-1-
phosphate to glucose-6-phosphate. Glucose-6-phosphate can then be
metabolized, e.g., by glycolysis. The 1-6 linkages are broken by the
glycogen debranching enzyme.
When there is a defect in phosphorylase enzyme in a tissue, glycogen cannot

be broken down. This condition leads to the accumulation of large amounts
of glycogen in tissues. Further breakdown of glycogen ceases when 4-5
glucose units are remaining near the branch point of the glycogen molecule
in any direction when there is a defect in debranching enzymes. This leads
to accumulation of short branched glycogen molecules.
There are many different glycogen storage diseases (also called

glycogenoses), identified by a roman numeral. These diseases are caused by
a hereditary lack of one of the enzymes that is essential in glycogenesis or
glycogenolysis (Table 1.1). About 1 in 20,000 infants has some form of
glycogen storage disease.
The diagnosis is made by examining a biopsy. Treatment depends on the

type of glycogen storage disease and usually involves regulating the intake
of carbohydrates.
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Table 1.1 Types and Characteristics of common Glycogen Storage Diseases
Affected Organs,
Name Symptoms
Tissues, or Cell
Type O Liver or muscle Episodes of low blood glucose
levels (hypoglycaemia) during
fasting if the liver is affected
von Gierke's Liver and kidney Glucose 6 phosphatase deficiency.
disease (type Enlarged liver and kidney, slowed
IA) growth, very low blood glucose
levels, and abnormally high levels
of acid, fats, and uric acid in blood
Type IB Liver and white Intracellular G 6 P transporter
blood cells deficiency
Same as in von Gierke's disease but
may be less severe
Pompe's disease All organs Lysosomal acid maltase deficiency
(type II) Enlarged liver and heart and muscle
weakness
McArdle Muscle Muscle phosphorylase deficiency
disease (type V) Muscle cramps or weakness during
physical activity
1.4.1 Symptoms of glycogen storage diseases
Some of these diseases cause mild symptoms. Others are fatal. The specific
symptoms, age at which symptoms start and their severity varies
considerably among these diseases. Low levels of glucose in the blood and
protrusion of the abdomen (because excess or abnormal glycogen may
enlarge the liver) are some of the features. Hypoglycaemia results in
weakness, sweating, confusion, and sometimes seizures and coma. Other
consequences for children may include stunted growth, frequent infections
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or sores in the mouth and intestines. Accumulation of G 6 P in glycogen

storage diseases leads to hyperuricaemia and gout.
Urinary sugars as laboratory markers in hereditary carbohydrate disorders.

Urinary sugars of clinical interest are glucose and galactose. But urine of
infants and children must be examined with glucose oxidase strips and
copper reduction test to identify individuals of inborn errors of carbohydrate
metabolism. This identification is enhanced by paper chromatographic
procedures.
Detection of urinary sugars is an early important laboratory step in inborn

carbohydrate disorders. The Benedict’s reducing sugar test is the
commonest test that detects aldo- hexoses, pentoses as well as many
reducing substances.
The glucose oxidase test strips help to detect urinary glucose specifically.
Activity 1.3
1. List and classify the Benedict’s positive substances found in human urine?
1.5 Laboratory diagnosis of carbohydrates
The laboratory tests we discuss under this topic are; Benedict’s test for
glucose, Seliwanoff’s test for fructose, test for lactose, test for galactose and
chromatography test.
1.5.1 Benedict’s test for urine glucose
Presence of reducing substances can be detected in urine using the

Benedict’s test. This test is named after an American chemist Stanley
Rossiter Benedict. He was instrumental in developing the reagent which is
used for this test.
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1.5.1.1 Benedict’s reagent
Benedict’s reagent is used to test the presence of all monosaccharides and

also reducing sugars. It includes glucose, galactose, mannose, lactose, and
maltose.
1.5.1.2 Procedure
 Pipette 5.0ml of Benedict’s reagent in to a boiling test tube.
 Add 0.5ml (8 drops) of urine in to that tube
 Include a positive and negative control
 Boil at 100oC for 2 minutes
 Cool and observe the results
1.5.1.3 Observations/Results
Following table (Table 1.2) gives the possible observations and inference of
Benedicts test.
Table 1.2 Interpretation of Benedicts test
Observation Inference
No change in the original colour of Benedict’s solution Negative

Solution appears pale green and slightly cloudy Trace
Definite cloudy green 1+ (0.5g/dl)
Yellow to orange precipitate 2+ (1 g/dl)
Orange to red precipitate 3+ (1.5 g/dl)
Brick red precipitate & clear supernatant 4+ (>2 g/dl)
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1.5.1.4 Results interpretation
The presence of glucose in urine is called as glycosuria.
False positive reactions are known to occur due to the presence of non-
carbohydrate substances like ascorbic acid, homogentisic acid, creatinine
and uric acid.
1.5.2 Determination of fructose (Seliwanoff’s test)
Fructose appears in the urine of patients with hepatic disorders. It can be

detected by Seliwanoff’s test.
1.5.2.1 Reagent
Seliwanoff’’s reagent – prepared by dissolving 50mg of resorcinol in 33ml

of conc. HCl and diluting to 100ml with distilled water.
1.5.2.2 Principle
Hydrochloric acid acts on fructose to form a derivative of furfural which

gives a red coloured complex when linked with resorcinol.
1.5.2.3 Procedure
 Add 5.0ml of Seliwanoff’s reagent in to a test tube
 Add 0.5 ml of urine.
 Heat to boil
 Observe the colour
1.5.2.4 Observations
If no colour change – Negative for fructose
IF red colour- Urine fructose positive
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1.5.3 Determination of Galactose (orthotoluidine test)
Galactose test or orthotoludine test is qualitative test which is described

below.
1.5.3.1 Procedure
 Add 5.0ml of orthotoluidine reagent in to a test tube (20x150mm)
 Add 0.05 ml of urine.
 Heat to boil in a boiling water bath for 10 minutes
 Observe the colour
1.5.3.2 Observations
No green colour ( orginal colour of pale yellow) – galactose absent
Green colour ( if lactose and glucose absent) – Galactose positive
1.5.4 Determination of Lactose: (Osazone test)

Lactose test is qualitative test which is described below.
1.5.4.1 Principle
Monosaccharaides and two disaccharides, lactose and maltose react with

phenylhydrazine hydrochloride in acidic medium and after placing in a
boiling water bath, to form characteristic phenylhydrazone crystals. Lactose
gives lactosazone crystals which are specific and can be identified by
microscopic observation. This crystal differs from the other osazone
crystals.
18
1.5.4.2 Procedure
 Add 5.0ml of urine to a test tube and make just acidic by adding few
drops of glacial acetic acid.
 Add 1.0 g of sodium acetate powder and phenyl hydrazine hydrochloride

( 2:1)
 Heat to boil ( boiling water bath- 30 minutes)
 Cool by placing in a beaker of tap water.
 Observe the deposit under microscope by making a cover slip

preparation.
1.5.5 Identification of Urinary Sugars By paper chromatography

The following tests is not routinely done in a laboratory.
1.5.5.1 Principle
Sugars can be separated by ascending or descending chromatography on

paper and located after colour development with dinitrosalicylic acid. The
variable rates of migration depend on the solubility of sugars in the
particular solvent. The comparison rate of migrations of known and
unknown is the basis for the identification of sugars.
1.5.5.2 Reagents
Solvent (Mix 60ml n-butanol, 40ml pyridine and 30ml distilled water)
Spray reagent (Dissolve 0.5g of 3, 4-dinitrosalicylic (DNS) acid in 100 ml

of 4g/dl NaOH)
Reference sugar solutions (1.75g of each sugar in 100 ml of 2g/L benzoic

acid)
Glucose, Fructose, Galactose, Maltose, Lactose and Xylose
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1.5.5.3 Procedure
 Conduct a copper reduction test, if heavily positive dilute the sample

equivalent to 1.0g% if its 0.5g% use double the volume of urine
 Draw a pencil line 2.5 cm from and parallel to the 25cm side of a
25x35cm Whatman No 01 filter paper.
 Apply 10 l of each solution to it’s respective point( pre labelled)
 Insert the sheet into a 35cm high cylinder so that the line of application is
at the bottom. Insert the paper into a chromatographic jar. Tape the cover
and run for 16 hours( Overnight at room temperature)
 Remove the paper and mark the solvent front. Allow to air dry
 Spray the sheet using an atomizer.
 Heat the paper at 100oC for 10 minutes in a drying oven.
1.5.5.4 Observations and measurements
The reducing sugars appear as brown spots against a yellow background.
Measure the distance from the starting line to the edge of the solvent front
and calculate the Rf (Table 1.3).
Rf = Distance travelled by the solute spot/ distance travelled by the

solvent front
Table 1.3 Rf value for certain carbohydrates
Sugar Rf value
Lactose 0.22
Maltose 0.28
Galactose 0.36
Glucose 0.41
Fructose 0.46
Xylose 0.52
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Summary
Defects in the enzymes in metabolic pathways of sugars give rise to

conditions such as galactosaemia, galactose intolerance, fructosuria and
fructose intolerance, PDH deficiency disorders and glycogen storage
diseases. Some features of the inherited diseases are due to excessive
storage of abnormal substrates or to normal substrates that cells cannot
degrade normally because they lack a specific enzymatic activity. In some
diseases, abnormal metabolites (intermediates of pathways that use
carbohydrates) block the normal function of the pathway or of related
pathways. Defects of the enzymes of the pentose shunt interfere with the
normal production of nucleic acids and nucleotides, which are needed by
cells as second messengers and as coenzymes of intermediary metabolism,
as well as components of RNA and DNA.
The detection of many sugars in the urine is an important laboratory

parameter in the diagnosis of carbohydrate disorders. The tests include
Benedict’s test for reducing substances, GOD strip test for glucose,
Seliwanoff’s test for fructose, orthotoluidine test for galactose , osazone test
for lactose and paper chromatography for Glucose, Fructose, Galactose,
Maltose, Lactose and Xylose .
Learning outcomes
After reading this session students should be able to understand,
 Describe the biochemical basis for the inherited disorders of carbohydrate

metabolism
 Identify defective enzymes in galactose, fructose, pyruvate and glycogen

metabolism
 Explain the role of urinary analysis in the diagnosis of disorders of

21
 Explain the paper chromatographic technique for urinary sugars
Review questions
1. Explain the principle and observations of Benedict’s test.
2. State the principle of the test to determine galactose.
3. Describe how a single gene defect which causes a clinically significant block in a
metabolic pathway lead to subsequent development of a metabolic disorder using
an example.
References
Burtis, C. A., Ashwood, E. R., & Bruns, D. E. (2012). Tietz textbook of clinical
chemistry and molecular diagnostics-e-book. Elsevier Health Sciences.
Lehninger, A. L., Nelson, D. L., Cox, M. M., & Cox, M. M. (2005). Lehninger
principles of biochemistry. Macmillan.
Champe, P. C., Harvey, R. A., & Ferrier, D. R. (2008). Lippincott's illustrated

reviews: Biochemistry. Philadelphia: Wolters Kluwer/Lippincott Williams &
Wilkins.
Sophian, L. H., & Connolly, V. J. (1952). Chromatographic identification of

reducing sugars in urine. American journal of clinical pathology, 22(1), 41-45.
Rutishauser, S. (1994). Physiology and anatomy: A basis for nursing and health
care. Churchill Livingstone.
22
Session [2]: Diabetes Mellitus
Session 2
Diabetes Mellitus
Content
Introduction, p23
2.1 Types of diabetes, p23
2.2 Signs and symptoms of diabetes, p27
2.3 Metabolic changes in diabetes mellitus, p27
Learning Outcomes, p31
References, p32
Introduction
Diabetes mellitus is a chronic disease caused by inherited and/or acquired

deficiency in production of insulin by the pancreas, or by the ineffectiveness
of the insulin produced. Plasma glucose is maintained at a constant level by
insulin and glucagon secreted by the pancreas. As you can remember these
two hormones act in an opposing manner in the body. Normal plasma
glucose concentration is 4.5-5.6 mmol/L (70-110 mg/dl). Factors that
determine the plasma glucose level are dietary intake, rate of entry of
glucose into cells and the glucostatic activity of the liver (glucostatic
activity of the liver is described under section 2.3 in this session).
Diabetes mellitus is characterized by an elevation of fasting blood glucose.

Failure to maintain normal blood glucose level leads to long term
complications in diabetic subjects. These complications affect most of the
organs in the body including eyes, kidneys, nervous system and circulatory
system.
2.1 Types of diabetes
There are three main types of diabetes:
23
2.1.1 Type 1 diabetes (insulin-dependent)
This form is characterized by an absolute deficiency of insulin due to an

autoimmune destruction of the β- cells of the pancreas. Type 1 diabetes is
most commonly seen among children and adolescents (age group less than
30 years) (Table 2.1).
Table 2.1 Comparison of type 1 and type 2 diabetes
Feature Type 1 diabetes Type 2 diabetes

Onset Sudden Gradual
Age of onset Mostly in children Mostly in adults
Body size Thin or normal Often obese
Defect or β-cells of pancreas are Insulin resistance combined

deficiency destroyed, eliminating with inability of β-cells to
production of insulin produce appropriate
quantities of insulin
Ketoacidosis Common Rare
Auto antibodies Usually present Absent
Endogenous Low or absent Normal, decreased or

insulin increased
Prevalence ~10% ~90%
Treatment Insulin is always Diet, exercise, oral

necessary hypoglyaemic drugs
Insulin may or may not be
necessary
24
2.1.2 Type 2 diabetes (non-insulin-dependent)
This is characterized by insulin resistance, which may be combined with

relatively reduced insulin secretion (due to dysfunctional β-cells). The
defective responsiveness of body tissues to insulin is due to the less
responsiveness of insulin receptors. Type 2 diabetes is much more common
and accounts for around 90% of all diabetes cases worldwide. It occurs most
frequently in adults (age group of more than 30 years).
Figure 2.1 Type I and Type II diabetes mellitus
(Source: https://images.app.goo.gl/PbwvEqtxTKy9Y1Uo7)
2.1.2.1 Insulin resistance
This refers to the decreased ability of target tissues (liver, muscles and
adipose tissues) to respond properly to normal concentrations of circulating
insulin. Insulin resistance increases with weight gain and therefore type 2
diabetics are often obese (Figure 2.2).
25
Figure 2.2 Insulin signal transduction pathway
(Source: https://images.app.goo.gl/P3wcv7zSA1ac5RKfA)
2.1.2.2 Dysfunction of cells β- cells
In type 2 diabetics there is a gradual destruction of β- cells due to the

formation of autoantibodies against them. Affected β- cells fail to secrete
enough insulin to cope up with hyperglycaemia.
2.1.3 Gestational diabetes
This occurs when pregnant woman without a previous history of diabetes

develop high blood glucose levels.
Activity 2.1
1. Explain why type 1 diabetes mellitus shows sudden onest and type ii diabetes
mellitus shows gradual onset.
26
2.2 Signs and symptoms of diabetes
The classic symptoms of diabetes are weight loss, polyuria (increased

urination), polydipsia (increased thirst), and polyphagia (increased hunger).
Several other signs and symptoms may occur in diabetes which includes
blurry vision, headache, fatigue, slow healing of wounds and itchy skin.
Figure 2.3 Main symptoms of diabetes
(Source: https://images.app.goo.gl/LXuu58Fe5VyT31UN8)
Activity 2.2
1. List 5 common signs and symptoms of diabetes mellitus and breifly explain the
reason for each.
2.3 Metabolic changes in diabetes mellitus
The main metabolic change in diabetes mellitus is Hyperglycaemia
2.3.1 Hyperglycaemia
Insulin is the principal hormone which regulates the uptake of glucose from
blood in to insulin responsive cells of the body (liver, adipose tissue and
27
muscles) via GLUT-4 (glucose transporter). There are several glucose

transporters in the body. However only the GLUT-4 is stimulated by insulin.
GLUT-4 is present in skeletal muscles, cardiac muscles and adipose tissues.
Therefore, deficiency of insulin or the insensitivity of its receptors plays a
central role in diabetes mellitus. When insulin is deficient, the counter
acting hormone, glucagon is elevated.
In the liver, relative or absolute deficiency in insulin inhibits glycogen

synthase, the enzyme responsible for the synthesis of glycogen
(glycogenesis). Therefore glucose is not deposited as glycogen in the liver
hepatocytes, but tends to elevate in the blood.
A high level of glucagon (which acts opposite manner of insulin) stimulates

glycogen phosphorylase, the enzyme responsible for the breakdown of
glycogen. This causes increased hepatic output of glucose elevating blood
glucose levels.
High levels of glucagon stimulate lipolysis (break down of lipids) releasing

fatty acids and glycerol. Proteolysis (breakdown of proteins) is also elevated
producing amino acids. When the gluconeogenic substrates (amino acids
and glycerol) are abundant, gluconeogenesis (formation of glucose)
accelerates producing more glucose. Reduced peripheral utilization and
increased production of glucose contributes to elevated blood glucose levels
(hyperglycaemia) which is characteristic of uncontrolled diabetes.
2.3.1.1 Consequences of hyperglycaemia
Polyuria
In diabetes, excess glucose accumulates in blood and when this exceeds the
renal threshold of 180 mg/dl, glucose reabsorption by renal tubules is
reduced. Hence glucose remains in the glomerular filtrate. Since glucose is
an osmotically active solute it draws water into the lumen of the tubule and
increases the urine volume which is termed as “polyuria” (Figure 2.4).
28
Hyperglycaemia
Increase filtering of glucose into renal tubules
Glucose molecules exceed the renal threshold
Amount of glucose in the glomerular filtrate increases
Osmotic diuresis
Loss of water Na+ and K+ Glycosuria
Figure 2.4 Osmotic diuresis following hyperglycaemia
Polydipsia
This refers to excessive drinking of water. Polyuria leads to loss of

extracellular fluid causing hypovolaemia. Due to high glucose levels in
blood, tonicity of the blood increases. When the tonicity increases the osmo
receptors in the hypothalamus are stimulated and act on the thirst receptors
of hypothalamus causing polydipsia (Figure 2.5).
Excessive loss of water in urine Dehydration, hypovolaemia &

secretion of Angiotensin-II
Hypertonicity of ECF
Stimulation of osmoreceptors in the hypothalamus
Polydipsia
Thirst
Figure 2.4 Polydipsia following heperglycaemia
29
2.3.2 Formation of glycated haemoglobin
Glycated haemoglobin is formed in a non-enzymatic glycation reaction

between the amino acids of beta chain of haemoglobin and glucose. Once a
hemoglobin molecule is glycated, it remains that way throughout the life
span of the RBC. A build-up of glycated hemoglobin within the red cell,
therefore, reflects the average level of glucose to which the cell has been
exposed during its life-span. Half-life of glycated haemoglobin is related to
the life span of red cells (120 days). Glycated haemoglobin concentration is
propionate to the glycaemic control and hence the measurement of HbA1c
indicates the glycaemic control over past 2-3 months. The longer the
hyperglycemia occurs in blood, more glucose binds to hemoglobin in the red
blood cells and the higher the glycated haemoglobin (Figure 2.6).
Figure 2.4 Accumulation of Advanced glycation end products
(Source : https://images.app.goo.gl/8NErMmcnk3Eoi5sY9)
2.3.3 Hypertriglyceridemia
Excess fatty acids generated by lipolysis (due to high glucagon) are

converted to triacylglycerol. These triacylglycerol are packaged in liver and
secreted in very low density lipoproteins (VLDL). Chylomicrons are
synthesized from dietary lipids by the intestine after a meal. Due to the
defective action of lipoprotein lipase in insulin deficiency, catabolism of
30
these lipoproteins is impaired. This contributes to the hypertriglyceridemia

in patients with diabetes.
2.3.4 Ketoacidosis
Deficiency of insulin accelerates the catabolism of lipids (β-oxidation of

fatty acids) releasing fatty acids from adipose tissues and liver. Acetyl Co
enzyme A, the product of β -oxidation of fatty acids is the substrate for
ketone body synthesis. This favours the formation of ketone bodies,
acetoacetate and β –hydroxyl butyrate in excess amounts in patients with
uncontrolled diabetes. This leads to acidosis which can cause coma
(unarousable unresponsiveness) and death. Due to this fact diabetic
ketoacidosis is considered as a medical emergency and immediate hospital
admission is needed to manage effectively.
Summary
Diabetes is a most common non-communicable disease found among the

population. There are three types of diabetes. They are Type I which is
insulin dependent, Type II non-insulin dependent diabetes and gestational
diabetes mellitus. The main metabolic change associated with diabetes is
hyperglycaemia. Polyuria and polydipsia are consequences of diabetes.
Learning Outcomes
At the end of the session the students should be able to,
 Differentiate type 1 and type 2 diabetes.
 Describe metabolic changes occuring in diabetes
 Explain the physiological basis of signs and symptoms of

diabetes.
31
Review questions
1. Briefly describe the pathophysiology of type-II diabetes.
2. Explain the role of insulin and glucagon in glucose homeostasis.
3. Briefly describe the consequences of hyperglycaemia.
4. Briefly explain what is meant by diabetic ketoacidosis.
References
American Diabetes Association. (2010). Diagnosis and classification of

diabetes mellitus. Diabetes care, 33(Supplement 1), S62-S69.
Burtis, C. A., Ashwood, E. R., & Bruns, D. E. (2008). Tietz textbook of

clinical chemistry and molecular diagnostics-e-book. Elsevier Health
Sciences.
Marshall, W., Bangert, S. K., & Lapsley, M. (2012). Clinical Chemistry.

Elsevier.
World Health Organization. (2006). Definition and diagnosis of diabetes

mellitus and intermediate hyperglycaemia: report of a WHO/IDF
consultation.
32
Session [3]: Blood Glucose Determination and Diagnosis of Diabetes
Session 3
Blood glucose determination and

Diagnosis of Diabetes
Content
Introduction, p33
3.1 Specimen collection for laboratory analysis, p34
3.3 Diagnosis of diabetes mellitus, p36
3.4 World Health Organization diagnostic criteria for diabetes, p37
3.5 Oral Glucose Tolerance Test (OGTT), p37
3.6 Glycated haemoglobin (HbA1C), p39
References, p42
Introduction
One of the main features of diabetes mellitus is elevated levels of blood

glucose. Because of this fact, the determination of fasting blood glucose
level was commonly used as a method of screening, diagnosis and
monitoring of diabetes. However, in some cases hyperglycaemia is not
present at the time of the investigation. And also, some subjects tend to
manipulate their glycaemic level by changing some lifestyle patterns a few
days before investigations. To overcome these problems some other
methods investigations are introduced.
Proper sample collection is an important aspect in investigations related to

diabetes mellitus. This include proper patient identification, proper subject
preparation, correct sample collection devices and many other things.
33
Unit [1]: Disorders of carbohydrate metabolism and laboratory diagnosis
Under this sessions specimen collection and types of investigations related

to diabetes mellitus will be discussed.
3.1 Specimen collection for laboratory analysis
Blood glucose determination is one of the most common clinical diagnostic

tests. Accurate and precise measurement of blood glucose is of great
importance in the diagnosis and management of diabetes. Once the blood is
drawn, the concentration of glucose will continue to decrease because of
glycolysis, which will occur in erythrocytes, white blood cells (WBCs), and
platelets. Therefore, blood for glucose analysis is collected to tubes
containing preservatives.
Container
Preservative is Sodium fluoride. Fluoride ions prevent glycolysis by

inhibiting enolase enzyme of the glycolytic pathway.
3.1.1 Reasons for delayed analysis of blood glucose
Blood needs to be transported from the site where it is drawn to a remote

laboratory for analysis.
If automated analysis of blood is done, there may be a waiting period before

the analysis is carried out.
In clinics, immediate measurement of blood glucose is not done for many

reasons: often quantity and frequency of blood samples limits the possibility
of immediate analysis. Under such conditions, it is important to recognize
that the blood glucose values can be lower.
34
3.2 Methods of blood glucose determination
Two main methods are used to measure glucose in blood.
3.2.1 Hexokinase method
This method is the highly accurate, precise and reference method for
glucose analysis in blood.
Principle
Glucose is phosphorylated by ATP in the presence of hexokinase and Mg

2+. The glucose 6 –phosphate formed is oxidized by glucose-6-phosphate
dehydrogenase (G6PD) to 6-phopsphogluconate in the presence of
nicotinamide –adenine dinucleotide phosphate (NADP+). The amount of
NADPH produced is directly proportional to the amount of glucose in the
sample and is measured at 340nm.
Glucose + ATP Hexokinase glucose-6-phosphate +ADP
Glucose-6-phosphate G-6-PD 6-phopsphogluconate
NADP + NADPH + H+
3.2.2 Glucose oxidase method
The enzyme glucose oxidase catalyses the oxidation of glucose to gluconic

acid and hydrogen peroxide (H2O2.). Addition of the enzyme peroxidase
and chromogenic oxygen acceptor, such as O-dianisidine result in the
formation of a colored compound that can be measured
spetrophotometrically at 500nm (Figure 3.1).
35
O2
Glucose
Glucose oxidase
Gluconic acid H2O2

Chromogen
Peroxidase
H2O + 1/2O2
Oxidized chromogen
Figure 3.1 Reactions involved in glucose oxidase reaction.
3.3 Diagnosis of diabetes mellitus
Diabetes could be diagnosed when overt symptoms are present and a

random blood glucose measurement of >11mmol/L confirms the diagnosis.
In the absence of clear symptoms, diabetes can be diagnosed by any of three
measures of glucose metabolism;
1. The oral glucose tolerance test ( OGTT)
2. Fasting plasma glucose
3. Haemoglobin A1C
36
3.4 World Health Organization diagnostic criteria for

diabetes
 Fasting plasma glucose>7.0 mmol/L (126 mg/dL)

 Random plasma glucose> 11mmol/L (200mg/dL)
One abnormal laboratory value is diagnostic in symptomatic individuals.

Two values are needed in asymptomatic people. The oral glucose tolerance
test is only required for borderline cases and for diagnosis of gestational
diabetes.
 HbA1C ≥ 6.5%
3.5 Oral Glucose Tolerance Test (OGTT)
The OGTT evaluates glucose clearance from the blood circulation after oral
glucose loading under standard conditions.
3.5.1 Standard conditions
1. The patient is instructed not to restrict carbohydrate intake in the days or

weeks before the test.
2. The subject should be on 8-10 hour fast before the test (water allowed).
3. Should avoid exercise and smoking during the test.
4. Should be free from illness.
5. Timing of the test: usually morning is preferable.
6. The drugs such as oral contraceptives, hypoglycaemic agents

(sulphonylurease and insulin) must be stopped prior to the test.
37
3.5.2 Procedure
1. After an overnight fast collect blood for fasting blood glucose (base line
value).
2. Dose of the glucose to be given to adults.
50 grams of glucose in 300ml of water to be drunk within 15 minutes.
Blood and urine samples are collected at 2 hour after the intake of glucose
load.
3.5.3 Interpretation
The following table interprets the results for OGTT based on WHO criteria
(Table 3.1). The change in blood glucose per time is expressed in an OGTT
graph (Figure 3.2).
Table 3.1 The OGTT: World Health Organization criteria.
Impaired glucose
Timing of test Normal Diabetes mellitus
tolerance
Fasting <6.1 mmol/L <7 mmol/L >7 mmol/L (126

(110 mg/dl) (126 mg/dl) mg/dl)
2 hour after <7.8 mmol/L 7.8-11.1 mmol/L > 11.1 mmol/L

glucose (140 mg/dL) (140- 200 mg/dL) (200 mg/dL)
38
Figure 3.2 Interpretation of OGTT
(Source:https://diabetestalk.net/blood-sugar/oral-glucose-tolerance-test-
procedure )
Activity 3.1
1. Briefly describe importance of glucose oxidase method and hexokinase method in

diagnosis of diabetes mellitus.
3.6 Glycated haemoglobin (HbA1C)
HbA1C has been firmly established as an index of long term blood glucose
concentration and as a measure of the risk for the development of
complications in patients with diabetes mellitus. An HbA1C of >6.5% is
considered diagnostic for diabetes and a level of 5.7-6.4% would denote
increased risk of diabetes.
39
3.6.1 Methods for the determination of HbA1C
Different methods are available for the determination of glycated

haemoglobin. These methods separate haemoglobin from glycated
haemoglobin using techniques based on (Figure 3.3),
 Charge differences
Ion-exchange chromatography, HPLC, electrophoresis and iso-electric
focusing
 Structural differences
Affinity chromatography and immunoassay
 Chemical analysis
Spectrophotometry (rarely used)
Regardless of the method, the result is expressed as a percentage of total

haemoglobin.
Figure 3.3 HbA1C test results for diagnosis of diabetes
(Source: https://en.redsearch.org/images/44855679 )
40
Activity 3.2
1. Explain the importance of determination of hba1c level in a chronic diabetic subject.
Summary
Diabetes mellitus is a metabolic disease results from defects in insulin

secretion, action or both. Long term hyperglycaemia affects many vital
organs and functions in the body. Mainly blood and urine specimens are
used to determine the glycemic levels in the body. Sodium fluoride
anticoagulant is used to collect blood. Glucose can be determined mainly by
two methods; hexokinase method and glucose oxidase method, which are
photometric analysis techniques. Fasting of diabetes mellitus include three
measures. They are fasting blood glucose, OGTT and HbA1c. fasting blood
glucose, HbA1c and random blood glucose ranges determine the diabetes
mellitus according to WHO criteria.
Learning Outcomes
At the end of the session the students should be able to
 Explain the correct specimen collction containers for

investigations related to diabetes mellitus.
 Describe the investigations related to screening, diagnosis and

monitoring of diabetes mellitus and their importance.
 Instruct patients for the preparation for laboratory investigations.
 Describe the Diagnosic criteria for diabetes mellitus.
41
Review questions
1. Explain the role of insulin in glucose homeostasis.
2. Outline the physiological basis for the development of polydipsia and polyuria in
patients with diabetics.
References
American Diabetes Association. (2010). Diagnosis and classification of diabetes

mellitus. Diabetes care, 33(Supplement 1), S62-S69.
Burtis, C. A., Ashwood, E. R., & Bruns, D. E. (2008). Tietz textbook of clinical
chemistry and molecular diagnostics-e-book. Elsevier Health Sciences.
Marshall, W., Bangert, S. K., & Lapsley, M. (2012). Clinical Chemistry. Elsevier.
World Health Organization. (2006). Definition and diagnosis of diabetes mellitus

and intermediate hyperglycaemia: report of a WHO/IDF consultation.
42
Session [4]: Complications of Diabetes Mellitus and Diagnosis
Session 4
Complications of Diabetes Mellitus

and Diagnosis
Content
Introduction, p43
4.1 Pathophysiology of complications of diabetes., p44
4.2 Major complications of diabetes, p45
4.3 Other markers of diabetic nephropathy, p48
References, p50
Introduction
As described in previous sessions, many organ systems can be affected due

to diabetes. The complications arise due to diabetes can be categorize in to
two major groups as microvascular and macrovascular complications.
According to literature the prevalence of microvascular complications is
higher than macrovascular complications. These complications can reduce
the productivity and quality of life of the subject and can lead even to death.
In order to minimize these complications and to improve the quality of life it

is important to identify what are the diabetic complications and how to
diagnose them.
43
4.1 Pathophysiology of complications of diabetes.
The following are consequences of hyper glycaemia and may play a role in
pathological consequences in diabetes mellitus.
 Non-enzymatic glycosylation of a wide variety of proteins such as

haemoglobin, collagen, low- density lipoprotein (LDL) and tubulin in
peripheral nerves. This leads to an accumulation of advanced
glycosylation end- products causing injury and inflammation via
stimulatory factors (cytokines).
 Polyol pathway - The metabolism of glucose by increased intracellular

aldose reductase leads to accumulation of sorbitol in cells. This can
cause osmotic damage in the lens of the eye (Figure 4.1).
Figure 4.1 Polyol metabolic pathway
(Source: https://images.app.goo.gl/GkJNq8yevshKuURM6)
 Abnormal micro vascular blood flow. This impairs supply of nutrients

and oxygen. Micro vascular occlusion is due to vasoconstrictors and
leads to endothelial damage.
 Haemodynamic changes. Theses take place especially in the kidney.
44
Activity 4.1
1. Describe how the “polyol pathway” leads to diabetic eye complicatios.
4.2 Major complications of diabetes
Progressive damage to the eyes, kidneys, nerves and arteries represents a

major threat to the health and life of patients with diabetes mellitus (Figure
4.2).
4.2.1 Macro vascular complications
Diabetes is a risk factor for the development of atherosclerosis. They are in

a risk of stroke, myocardial infarction and amputation of foot due to
gangrene.
4.2.2 Micro vascular complications
Small blood vessels throughout the body are affected but the disease process
is of particular danger for three sites (Figure 4.3):
1. Retina
2. Renal glomerulus
3. Nerve sheaths
45
Figure 4.2 Micro and Macro vascular complications of diabetes
(Source:http://sharingknowledge.world.edu/what-is-diabetes-types-
symptoms-complications-of-diabetes/)
4.1.2.1 Diabetic eye disease
Diabetes is still the most common cause of blindness among diabetics. It

affect the eye in various ways.
4.1.2.2 Cataracts
Diabetic retinopathy; most commonly diagnosed diabetes related

complication. Its prevalence is related to the duration of diabetes.
Blindness can be prevented if retinopathy is detected early and treated with

laser photocoagulation.
46
Figure 4.3 Complications of diabetes
(Source: https://www.gonutre.com/blog/5b6085e388acbd41d06ee712)
4.1.2.3 The diabetic kidney
The kidney may be damaged by diabetes in three main ways.
1. Glomerular damage
2. Ischemia resulting from hypertrophy of afferent and efferent arterioles
3. Ascending infection
Pathophysiology:
The earliest functional abnormality in the diabetic kidney is renal

hypertrophy associated with a raised glomerular filtration rate. As the
kidney becomes damaged by diabetes, the afferent arterioles (leading to the
glomerulus) become dilated than the efferent arterioles. This increases the
intragolemular filtration pressure, further damaging the glomerular
capillaries. The initial structural lesion in the glomerulus is the thickening of
the basement membrane. Additionally, changes result in disruption of the
protein cross- linkages that normally make the membrane an effective filter.
As a result there is a progressive leak of large molecules (particularly
proteins) into the urine.
47
4.1.2.4 Albuminuria
The earliest evidence of diabetic nephropathy is micro albuminuria

(amounts of urinary albumin so small as to be undetectable by standard dip
sticks). It is a predictive marker for progression to nephropathy in diabetes.
Normal urinary albumin excretion is <30mg/24h (10-20 mg/dL is the

minimal detection limit of protein in urine and such amounts are
undetectable by dip sticks). Micro albuminuria, may after some years,
progress to persistent clinical proteinuria (> 300mg/ 24h), with gradually
declining filtration rate and increasing plasma creatinine concentration.
Screening for micro albuminuria:
Done by measuring albumin/ creatinine ratio on early morning urine. Less

than 30 is normal; a ratio of 30-300 signifies micro albuminuria and values
above 300 are considered as macro albuminuria.
4.3 Other markers of diabetic nephropathy
The main markers of diabetic nephropathy are eGFR. The diabetic foot and
neuropathy are clinical conditions that can be used as markers in diabetic
nephropathy.
4.3.1 Estimated glomerular filtration rate (eGFR)
Early detection of kidney dysfunction can help to minimize the damage.

This is important as symptoms of kidney disease may not be noticeable until
as much as 30-40% of kidney function is lost.
Estimated glomerular filtration rate (eGFR) may progressively fall from a

normal of over 90 ml/min/1.73m2 to less than 15, at which point the patient
is said to have end-stage kidney disease (ESKD).
48
4.3.2 The diabetic foot
Ischemia, infection and neuropathy combine to produce tissue necrosis in

foot causing ulcers and gangrene.
4.2.3 Neuropathy
Neuropathy can affect the sensory, motor and autonomic nervous systems.
Activity 4.2
1. Describe how the glycaemic control can help to minimize the diabetic complications
giving examples.
Summary
Consequences of hyper glycaemia may affects the pathology of diabetes

mellitus. This includes non-enzymatic glycosylation of proteins, polyol
pathway, and abnormal micro vascular pathway and haemodynamic
changes. Major complications of diabetes affects vital organs affecting the
life of patient. Patients with macro vascular complications are in a risk of
developing stroke and amputation of foot. Patients with micro vascular
complications have complications in small blood vessels. Markers of
diabetes includes albuminuria, eGFR and diabetic foot.
Learning Outcomes
At the end of the session student should be able to,
 Describe the macrovascular complications of diabetes
 Describe the microvascular complications of diabetes
49
 State the markers of diabetes nephropathy
 Describe the importance of maintenance of normal glucose level

among diabetic subjects.
 Explain the public regarding diabetic complications and

protection.
Review questions
1. Explain the consequences of hyperglycemia?
2. What do you mean by macrovascular and microvascular complications?
3. Explain the markers of diabetes nephropathy?
References


Burtis, C. A., Ashwood, E. R., & Bruns, D. E. (2008). Tietz textbook of

clinical chemistry and molecular diagnostics-e-book. Elsevier Health
Sciences.
Marshall, W., Bangert, S. K., & Lapsley, M. (2012). Clinical Chemistry.

Elsevier.
World Health Organization. (2006). Definition and diagnosis of diabetes

mellitus and intermediate hyperglycaemia: report of a WHO/IDF
consultation.
50
Session [5]: Disorders of Lipids and Lipoprotein Metabolism
Session 5
Disorders of lipid and lipoprotein
metabolism
Content
Introduction, p51
5.1 Atherosclerosis and coronary heart disease, p52
5.2 Genetic disorders of lipoprotein metabolism, p54
5.3 Deficiency in lipoprotein lipase activity, p55
5.4 Familial combined hyperlipidemia, p55
5.5 Hyperapobetalipoproteinemia, p56
5.6 Familial hypertriglyceridemia, p57
5.7 Familial hypercholesterolemia, p58
Summary, p58
References, p60
Introduction
During metabolism, body synthesizes energy from the foods which have
been ingested. During digestion, constituents in the food such as
carbohydrate, protein and lipid are broken down in to monosaccharides,
amino acids and fatty acids & glycerol, respectively and eventually energy
will be produced. With the presence of metabolic disorders the pathways
involved with metabolism get affected.
Lipids or fats can be either absorbed from the food or synthesized by the
liver. Though all lipids are physiologically essential, the elevated levels of
triglycerides and cholesterol contribute to a number of diseases.
Lipids are hydrophobic and transported as lipoproteins in blood.
Lipoproteins are categorized based on the size and density and are
51
Unit [2]: Disorders of Lipid Metabolism and Laboratory Diagnosis
clinically important because increased levels of low-density lipoproteins

(LDL) and low levels of high-density lipoproteins (HDL) are key risk
factors for atherosclerotic heart diseases.
Defects in the pathways of lipoprotein synthesis, processing and clearance
lead to gathering of atherogenic lipids in plasma and endothelium.
The clinical importance of disorders of lipid and lipoprotein metabolism is
mostly correlated with their role in atherogenesis which is associated with
coronary heart disease (CHD) and peripheral vascular diseases. Acute
pancreatitis is associated with severe hypertriglyceridemia. Disorder of lipid
metabolism is also a critical feature in nonalcoholic fatty liver disease.
Categorization of dyslipidemia is vital for selection of appropriate treatment
and may provide clues to underlying primary clinical disorders.
5.1 Atherosclerosis and coronary heart disease
Atherosclerosis is defined as “a condition where the arteries become

narrowed and hardened due to plaque formation around the artery wall”.
Atherosclerosis is the underlying cause of heart attack, stroke and peripheral
vascular diseases.
On occasion, growth of the plaque is stopped after a certain size causing

without problems to the individual. But, sometimes, the plaque obstructs the
artery, obstructing the blood flow resulting in life-threatening conditions.
In some cases, there is a possibility for the plaque to break and gather
platelets in the affected area forming blood clots. This condition may be end
up with life-threatening complications, such as stroke and heart attack.
Risk factors of atherosclerosis mainly include hyperlipidemia. Atheromas

are complex lesions and contain cellular elements, collagen, and lipids. The
development of the lesion is primarily attributes to the content of
unesterified cholesterol and cholesteryl esters. Cholesterol found in the
atheroma originates from circulating lipoproteins. These lipoproteins
include low-density (LDL), intermediate density (IDL), very low density
52
lipoproteins (VLDL) and Lp(a) species containing B-100 apolipoprotein

(Apo B-100). Further, atherogenic agents include chylomicron remnants
containing apoB-48.
All the above mentioned constituents are subjected to oxidation in the

tissues by reactive oxygen species and in addition to that, by lipoxygenases
secreted by macrophages in atheromas. Oxidized lipoproteins play a role in
impairment of endothelial cell-mediated vasodilation and it stimulates
endothelium to secrete monocyte chemoattractant protein-1 (MCP-1) and
adhesion molecules that recruit monocytes to the lesion.
Coronary heart disease is a disease in which plaque builds up inside the

coronary arteries. Atherosclerotic diseases result from increased cholesterol
level. It has been reported that the incidence and prevalence of coronary
heart disease have a positive correlation with elevated total cholesterol and
LDL cholesterol. On the other hand increased HDL cholesterol has a
protective effect for coronary heart disease. The atherosclerotic plaque may
break and form blood clots known as "thrombi" within the vessel which may
completely block the blood flow to the heart muscle. This can cause
myocardial infarction. Myocardial infarction normally begins at the
endocardium of the heart and spread towards the epicardium (Figure 5.1).
Figure 5.1 Coronary heart disease-atherosclerosis

(Source: https://images.app.goo.gl/VvADr7ZNH5kLZT6D7)
53
Activity 5.1
1. Describe the connection between coronary heart diseases and disorders of lipid
metabolism?
5.2 Genetic disorders of lipoprotein metabolism
Many genes are involved with lipids transport in the blood which is a
complex process. Because lipids are water insoluble, they are complexed
with “apolipoproteins” and transported as lipoproteins in the blood.
Apolipoproteins have other functions as well;
1. Cofactors for enzymes (apoA-I, apoC-II)
2. Modulators of enzyme activity or substrate accessibility (apoC-III)
3. Ligands for lipoprotein receptors.
Lipoproteins synthesized in the liver parenchymal cells and duodenal
enterocytes. Then they are secreted into the lymph and enter the
bloodstream. Within the vessel they undergo modifications; which include,
1. Hydrolysis and removal of core lipids
2. Exchange of lipids and surface apolipoproteins between lipoproteins.
These processes are mediated by following enzymes;

 Lipoprotein lipase (LPL)
 Hepatic triglyceride lipase (HTGL)
 Lecithin cholesterol acyltransferas (LCAT)
 Transfer proteins (cholesterolester transfer protein (CETP)
Finally lipoproteins from circulation are taken up by the tissues. Other than
lipid transport, some lipoproteins and apolipoproteins contribute to tissue
regeneration and immunoregulation. All the major genes of human
apolipoproteins, enzymes and transfer proteins which control the
intravascular metabolism of plasma lipoproteins and receptors that remove
lipoproteins from the circulation have been characterized. Mutations of
54
these genes cause definite type of dyslipoproteinemia and lipoprotein

deficiency syndromes and some result in hyperlipidemia.
Dyslipidemia refers to abnormal amount of lipids such as triglycerides and
cholesterol in the blood. Most patients with dyslipidemia have more than a
single genetic factor or a gene mutation. It is known that diet, exercise
frequency and obesity play role in hypercholesterolemia. Further, common
genetic polymorphism of enzymes, structural protein and receptors of
lipoprotein metabolism collectively has an impact on the development of
dyslipidemia.
5.3 Deficiency in lipoprotein lipase activity
Lipoprotein lipase deficiency is a genetic disorder caused by the mutation of

lipoprotein lipase (LPL) gene. It is an autosomal recessive disorder
characterized by hyperchylomicronemia with increased triglyceride
concentration as a result of deficient lipoprotein lipase activity. LPL is
needed in hydrolysis of triglycerides of chylomicrons and their conversion
into chylomicron remnants. There is a possibility to increase the VLDL
cholesterol level as well. But, the levels of HDL and LDL cholesterol are
low. The affected individuals have abdominal pain, pancreatitis,
hepatosplenomegaly and the development of skin lesions known as eruptive
xanthomas.
5.4 Familial combined hyperlipidemia
Familial combined hyperlipidemia is the most common familial form of

hyperlipidemia and is passed down through the families.
It is the most prevalent primary dyslipidemia with a prevalence of 1 in 100
and frequently remains undiagnosed. It is the most common genetically
determined dyslipoproteinemia and usually shows an autosomal mode of
inheritance. But, the genetics of this disorder are complex. This condition
can occur due to mutations along with environmental factors.
55
Familial combined hyperlipidemia is characterized by fluctuations in serum

lipid levels. Further, it may present as;
 Mixed hyperlipidemia
 Isolated hypercholesterolemia
 Hypertriglyceridemia
 Normal serum lipid profile in combination with abnormally elevated

levels of apolipoprotein B.
Families with this condition have increased plasma total and LDL
cholesterol or triglyceride or both in at least two members of the same
family, with intra-individual and intra-familial variability of the lipid
phenotype. Familial combined hyperlipidemia carries a significantly
increased risk of coronary atherosclerosis; 10-15% of patients with
premature coronary heart disease have familial combined hyperlipidemia,
including acute myocardial infarction. Patients with familial combined
hyperlipidemia have a high frequency of co-morbidity with other conditions
such as type 2 diabetes, non-alcoholic fatty liver disease, steatohepatitis, and
the metabolic syndrome.
Family history of early coronary artery disease in one or more first-degree
relatives and the family history for increased triglycerides with or without
increased level of LDL cholesterol are important in diagnosis of the disorder.
It is necessary that lipid-lowering therapy directed toward reducing
cholesterol and triglyceride along with cardiovascular risk protection to
manage this condition.
5.5 Hyperapobetalipoproteinemia
Hyperapobetalipoproteinemia is a widespread familial lipoprotein disorder

linked with premature coronary artery disease.
Hyperapobetalipoproteinemia is characterized by increased concentration of
apo-B-100 with normal or moderately elevated LDL cholesterol. Total
56
cholesterol and triglyceride concentrations may be normal or increased and

HDL cholesterol and apo A-1 concentrations are decreased.
There are two metabolic defects reported in hyperapobetalipoproteinemic
patients. Firstly, an increased synthesis of apo-B in the liver. This results in
an overproduction of VLDL particles and later the overproduction of LDL
particles. Secondly, hyperapobetalipoproteinemia develops due to delayed
clearance of postprandial TG-rich lipoproteins (chylomicrons and
chylomicron remnants).
5.5.1 Type V hyperlipoproteinemia
Type V hyperlipoproteinemia is characterized by increased chylomicrons

and VLDL which may be due to increased production and/or decreased
removal of VLDL. The LPL activity may be either normal or low. The
plasma Apo-C-II level is normal.
5.6 Familial hypertriglyceridemia

This disease is transmitted as an autosomal dominant disorder occurring in
approximately 1% of the population. It is passed down through the families.
Familial hypertriglyceridemia is characterized by an increased production of
VLDL by the liver. The cholesterol level of VLDL is also increased. But,
plasma LDL cholesterol level is within the reference range. This indicates
delayed conversion of VLDL to LDL.
This condition is linked with early coronary disease, although treatment
sometimes is different from hypercholesterolemia. Persons suffering from
this condition are at the risk for chylomicronemia syndrome, which is
characterized by increased level of chylomicrons in the blood. They are also
at the risk of pancreatitis, particularly when triglyceride levels go beyond
1000mg/dL.
In majority of the cases, familial hypertriglyceridemia is noticeable at
puberty or early adulthood. Obesity, hyperglycemia and high levels of
57
insulin are frequently present as well. These factors may contribute to even
higher triglyceride levels. Alcohol, high carbohydrates diets and using of
estrogen make the condition worse.
5.7 Familial hypercholesterolemia
This disease is transmitted as an autosomal dominant disorder and is caused

by a defect on chromosome 19. Mutations affect synthesis of LDL receptor
or its proper function as well as mutation of the Apo B-100 gene. This
causes decreased binding of LDL with Apo B-100. This results in
accumulation of LDL in plasma and increased deposition in the skin,
tendons and in arteries causing atherosclerosis. Total cholesterol and LDL
cholesterol levels are highly elevated making the individuals susceptible to
myocardial infarction. Triglyceride level may be normal or sometimes slight
elevation can be seen. HDL cholesterol concentration is slightly decreased.
Review questions
1. Discuss the pathophysiology of coronary heart diseases.

2. Explain how deficiency in lipoprotein lipase activity is diagnosed.
3. Discuss the causes and biochemical events that take place in following
conditions/disorders;
Familial defective Apolipoprotein B-100
Dysbetalipoproteinermia
Hypoalphalipoprotenemia
Summary
The disorders of lipid and lipoprotein metabolism are mostly correlated with
atherogenesis.
Atherosclerosis is defined as “a condition where the arteries become

narrowed and hardened due to plaque formation around the artery wall”.
Atherosclerosis is the primary cause of heart attack, stroke and peripheral
58
vascular diseases. The risk factors of atherosclerosis mainly include

hyperlipidemia with elevated total cholesterol and LDL cholesterol. HDL
cholesterol has a protective effect for coronary heart disease. Many genes
are involved with the lipids transportation in the blood. Mutations of these
genes cause definite type of dyslipoproteinemia and lipoprotein deficiency
syndromes and some result in hyperlipidemia. Lipoprotein lipase
deficiency is a genetic disorder which is characterized by
hyperchylomicronemia with increased triglyceride concentration as a result
of deficient lipoprotein lipase activity. Familial combined hyperlipidemia is
the most common familial form of hyperlipidemia. Families with familial
combined hyperlipidemia have elevated plasma total and LDL cholesterol or
triglyceride or both in at least two members of the same family.
Hyperapobetalipoproteinemia is linked with premature coronary artery

disease. Hyperapobetalipoproteinemia is characterized by increased level of
apo-B-100 with normal or moderately elevated LDL cholesterol.
In hyperapobetalipoproteinemia total cholesterol and triglyceride

concentrations may be normal or increased and HDL cholesterol and apo A-
1 concentrations are decreased.
Type V hyperlipoproteinemia is characterized by increased chylomicrons

and VLDL with normal plasma Apo-C-II level. Familial
hypertriglyceridemia is an autosomal dominant disorder. Familial
hypertriglyceridemia is characterized by an increased production of VLDL
by the liver and elevated levels of cholesterol and VLDL.
Learning outcome
At the end of this session, the students should be able to;
 State the clinical significance of disorders of lipid and lipoprotein
metabolism.
 Explain what is meant by "atherosclerosis".
59
 Describe plaque formation.
 Discuss the causes of atherosclerotic diseases.
 Explain how atherosclerotic plaque contributes in myocardial infarction.
 Explain how genetic disorders affects the lipid and lipoprotein

metabolism.
 Explain how deficiency in lipoprotein lipase activity causes

hyperchylomicronemia with elevated triglyceride concentration.
 Discuss the causes of familial hypertriglyceridemia & familial

hypercholesterolemia and the biochemical events that take place in the
body in above 2 conditions.
 Discuss how following condition are diagnosed in the laboratory;

familial combined hyperlipidemia, hyperapobetalipoproteinemia and
type V hyperlipoproteinemia.
References
Burtis CA, Ashwood ER, Bruns DE. 2007. Tietz Fundamentals of Clinical
Chemistry. 6th Edition. Elsevier.
Juo SH, Beaty TH and KwiterovichJr PO, Etiologic heterogeneity of

hyperapobetalipoproteinemia (HyperapoB): Results from segregation
analysis in families with premature coronary artery disease,
arteriosclerosis, thrombosis, and vascular biology. 1997; 17:2729–2736.
Genetic disorders of lipoprotein metabolism. Principles and treatment of

lipoprotein disorders. G. Utermann H. J. Menzel, pp 89-138.
60
Session [6]: Analysis of Lipids and Lipoproteins
Session 6
Analysis of Lipids and Lipoproteins
Content
Introduction, p61
6.1 Cholesterol estimation, p61
6.2 Triglycerides, p62
6.3 High density lipoprotein cholesterol, p63
6.4 Low density lipoprotein cholesterol, p64
6.5 Measurement of Apolipoproteins, p66
Summary, p67
Learning outcome, p68
References, p68
Introduction
Routine lipid and lipoprotein determinations are carried out by most clinical
laboratories. Reference methods also have been developed and these
methods are important in the standardization of lipid and lipoprotein assays.
Basic lipids measured in the clinical laboratories include cholesterol,
triglycerides, HDL cholesterol and LDL cholesterol.
6.1 Cholesterol estimation
Enzymatic methods are widely used in medical laboratories in the

determination of cholesterol level. In this procedure, cholesterol reagent is
mixed with serum/plasma and incubated under specified conditions for the
development of color. Then the absorbance is measured at 500 nm.
61
Unit [2]: Disorders of Lipid metabolism and laboratory diagnosis
Cholesterol reagent contains all the enzymes and other requirements of the
reactions.
Following reactions are involved with the colour development.
Cholesteryl ester
hydrolase
Cholesteryl ester + H2O Cholesterol + Fatty
acid
Cholesterol
oxidase
Cholesterol + O2 Cholest-4-en-3-one +H2O2 BN
Peroxidase
H2O2 + Phenol + 4-Aminoantipyrine Quinoneimine dye
+2H2O
6.2 Triglycerides
Enzyme reagents are widely used in the determination of triglycerides.

Following reactions are involved with the enzymatic method.
Lipase
Triglyceride + 3H2O Glycerol + 3 Fatty acids
Glycerokinase
Glycerol + ATP Glycerophosphate + ADP
Glycerophosphate
Glycerophosphate + O2 oxidase Dihydroxyacetone +
H2O2
H2O2 is then measured as in the cholesterol assay.
62
Otherwise glycerophosphate can be measured according to the following

reactions.
Glycerophosphate
Glycerophosphate + NAD dehydrogenase Dihydroxyacetone
phosphate + NADH + H+
NADH is then measured at 340 nm.
Diaphorase
NADH + Tetrazolium dye Formazan + NAD+
Diaphorase catalyzed reaction is measured at the wavelength of 500 nm.
Alternatively, ADP produced by the reaction catalyzed by glycerokinase is

Pyruvate
measured according to the following reactions.
kinase
ADP + Phosphoenol pyruvate ATP + Pyruvate
Lactate
Pyruvate + NADH + H + dehydrogenase Lactate + NAD+
The loss of absorbance as NADH is consumed in the reaction is measured at

340 nm.
Activity 6.1
1. Briefly explain the principle cholesterol enzymatic essay?
6.3 High density lipoprotein cholesterol
Plasma HDL concentration is measured by determining the concentration of

cholesterol associated with HDL. In precipitation methods, non-HDL
63
lipoproteins ie., VLDL, IDL, Lp(a) and chylomicrons are precipitated.

When polyanions are added to the plasma/serum non-HDL lipoproteins
precipitate is formed within 10-15 min at room temperature. The precipitate
is removed by centrifugation and HDL cholesterol in the supernatant is
measured by enzymatic methods. HDL cholesterol assays are known to be
inaccurate with the serum/plasma samples with high triglyceride
concentrations above 400 mg/dL.
The principal of direct HDL cholesterol assay which is known as

homogeneous assay is similar to the HDL cholesterol assay. But, in this
method there is no physical separation of HDL from non-HDL lipoproteins.
HDL cholesterol is measured by masking the cholesterol from non-HDL
cholesterol. Then they do not react with enzymes used in the determination
of HDL-cholesterol .
6.4 Low density lipoprotein cholesterol

There are two methods of analysis; direct method and indirect method.
6.4.1 Indirect methods
In indirect method, assumption is made that total cholesterol is composed of

VLDL, LDL and HDL. LDL cholesterol is measured by indirectly by
Friedewald equation or by β-quantification method.
6.4.1.1 Friedewald equation

Total cholesterol, triglyceride and HDL cholesterol are measured and LDL
cholesterol is measured according to the following equation.
LDL cholesterol = [Total cholesterol]-[HDL cholesterol] - [Triglyceride]/5
64
In the above equation concentrations are given in milligrams per deciliter.

When the concentration is given in millimoles per liter Triglyceride/2.22 is
used.
6.4.1.2 β-Quantification
β-Quantification is used where Friedewald equation is inappropriate. The
technique involves preparative ultracentrifugation and polynomic
precipitation. The plasma (density [d] = 1.006 g/mL) is ultracentrifuge at
105,000 x g for 18 hours at 10 °C. Then VLDL, chylomicrons and β-VLDL
are found in the floating layer and it is removed by tube slicer. The
infranatant contains LDL and HDL in that case density is greater than 1.006
g/mL. Cholesterol level in the infranatant is measured. VLDL and LDL
cholesterol levels are measured according to the following equations.
[VLDL cholesterol] = [Total cholesterol]- [d >1.006 g/mL cholesterol]
[LDL cholesterol] = [d >1.006 g/mL cholesterol] - [HDL cholesterol]
6.4.2 Direct methods
In direct methods LDL cholesterol is selectively precipitated with polyvinyl

sulfate or heparin at low pH. Alternatively, VLDL, IDL and HDL are
removed by a pretreatment method that uses a mixture of polyclonal
antibodies to apo A-1 and apo E linked to resin. LDL cholesterol is
measured enzymatically.
Activity 6.2
1. Briefly explain the indirect methods of LDL estimation?
65
6.5 Measurement of Apolipoproteins
Apoproteins are measured by;

1. Radioimmunoassay (RIA)
2. Enzyme-linked immunosorbant assay (ELISA)
3. Radial immunodiffusion (RID)
4. Immunoturbidimetric assay
5. Immunonephelometric assay
6.5.1 Apolipoproteins A-1 and B-100
Immunoturbidimetric, immunonephelometric assays, ELISA and RIA are

used to measure apo A-1 and apo B-100.
6.5.2 Lipoprotein (a)
Turbidimetric, nephelometric, radiometric and enzymatic methods are

currently used in Lp (a) determination.
6.5.3 Lipoprotein subfraction assays
Nuclear magnetic resonance spectroscopy detects lipoprotein associated

fatty acyl, methyl and methylene groups and the signals from a number of
subfractions of VLDL, LDL and HDL are resolved mathematically.
6.5.4 Oxidized LDL
ELISA method is currently available in determination of oxidized LDL.
66
Summary
Enzymatic methods are widely used in determination of cholesterol level

and triglycerides.
In precipitation method of HDL cholesterol determination, non-HDL

lipoproteins are precipitated and HDL cholesterol in the supernatant is
measured by enzymatic methods.
In the direct HDL cholesterol assay no physical separation of HDL from

non-HDL lipoproteins.
Low density lipoprotein cholesterol can be measured by Friedewald

equation or β-quantification method.
Apoproteins are measured by radioimmunoassay (RIA), enzyme-linked

immunosorbant assay (ELISA), and radial immunodiffusion (RID),
immunoturbidimetric assay and immunonephelometric assay.
Apolipoproteins A-1 and B-100 are measured by immunoturbidimetric,

immunonephelometric, ELISA and RIA methods.
Lipoprotein (a) is measured by turbidimetric, nephelometric, radiometric

and enzymatic methods.
Lipoprotein subfraction assay is performed by nuclear magnetic resonance

spectroscopy which detects lipoprotein associated fatty acyl, methyl and
methylene groups and the signals from a number of subfractions of VLDL,
LDL and HDL are resolved mathematically.
Oxidized LDL can be measured by ELISA method.
67
Review questions
1. List the factors that can interfere with the cholesterol enzymatic assay.
2. Discuss the precautions that you would take to minimize the effects of the factors
that you mentioned above.
3. Discuss the conditions that can give overestimations in endogenous glycerol

concentration affecting triglyceride assay.
4. Name polyanion-divalent cation combinations that can be used in HDL cholesterol

determination.
5. Discuss the methods that can be used to minimize the effect of high triglyceride
concentration in HDL cholesterol determination.
6. State the conditions/ disease where Friedewald equation cannot be used in LDL
cholesterol determination.
7. Discuss the sources of variation and bias in lipids and lipoproteins measurement.
Learning outcome

 Explain the enzymatic methods used in determination of cholesterol and
triglycerides levels.
 Describe the precipitation method and direct HDL cholesterol assay

used in HDL cholesterol determination.
 State the Friedewald equation and β-quantification method.
 List the methods available in measurement of apolipoproteins,

apolipoproteins A-1 and B-100 and lipoprotein (a).
References
Burtis CA, Ashwood ER, Bruns DE. 2007. Tietz Fundamentals of Clinical
Chemistry. 6th Edition. Elsevier.
68
Session [7]: Changes of Protein Levels in Disorders
Session 7
Changes of Protein Levels in

Disorders
Content
Introduction, p69
7.1 Normal serum protein, p69
7.2 Electrophoretic and densitometry patterns in diseases, p72
References, p80
Introduction
Serum proteins can be separated by electrophoresis. Most of the serum

proteins are negatively charged at pH>8.4 and they migrate toward the
positive electrode. After the separation, the electrophoretic pattern can be
scanned by a densitometer and the quantification of each protein fraction
can be done by densitometry.
7.1 Normal serum protein

Five peaks can be seen in a normal resolution pattern as in Figure 7.1. They
are;
1. Albumin
2. α1-Globulin
3. α2-Globulin
4. β-Globulin
69
Unit [3]: Serum/ Plasma Protein
5. γ-Globulin
Figure 7.1 Normal serum protein densitometric pattern

(Source: https://en.wikipedia.org/wiki/Serum_protein_electrophoresis )
Pre albumin and β1 & β2 fractions appear in a high resolution pattern.
Normal resolution serum proteins and their percentages are as follows.
7.1.1 Albumin
Concentration: 3.63-4.91 g/dL

Percentage: 47-71%
Usually a single protein
Makes up the most obvious band
7.1.2 α1-Globulin

Percentage: 2.7-5.8%
Consist of α1-antitrypsin, α1-acidglycoprotein and α1-
fetoglobulin
70
7.1.3 α2-Globulin

Consist of haptoglobulins, α2-macroglobulin,
ceruloplasmin and HDL
7.1.4 β-Globulin

Often separate into two; β1 consists transferrin with a
contribution from LDL and β2 consists of the C3
component of complement
7.1.5 γ-Globulin

Consists of immunoglobulins eg., Ig G, Ig A, Ig M,
IgD and Ig E. Some immunoglobulins are found in
the α2 and β regions
Plasma proteins include 3 major groups;

1. Albumin
2. Fibrinogen and globulins (α, β, γ)
3. Conjugated proteins (glyco and lipoproteins)
Total plasma proteins vary from 7 to 7.5 g/dL. In plasma, fibrinogen

fraction appears as a distinct band in the β-γ region (see Figure 7.2).
71
Figure 7.2 Normal plasma protein pattern

(Source: https://images.app.goo.gl/mivSrYKevFECE8mx7 )
Activity 7.1
1. Explain the normal densitometric pattern of serum proteins with the help of graph?
7.2 Electrophoretic and densitometry patterns in

diseases
7.2.1 Cirrhosis of the liver
In liver cirrhosis there is a marked increase in γ-globulin. But Albumin and

often α1-globulin concentrations are reduced. The fusion of the β and γ
bands can be seen as a result of the increased plasma IgA concentrations
(see Figure 7.3).
72
Figure 7.3 Cirrhosis of the liver

(Source: https://www.cram.com/flashcards/cp-chemistry-i-osler-6096824 )
7.2.3 α1-Antitrypsin deficiency (Emphysema)
The α1-band consists mainly of α1-antitrypsin. Therefore, in α1-antitrypsin

deficiency absence or an obvious reduction in α1-globulin band can be seen
(see Figure 7.4).
Figure 7.4 α1-Antitrypsin deficiency

(Source: https://images.app.goo.gl/Kyh7mvDGFYzL2MdM8)
73
7.2.3 Monoclonal gammopathy
The M protein (paraprotein, monoclonal protein or M-component) is

characterized by the presence of a single, sharp, well-defined peak in the β-γ
region of the densitometer tracing (see Figure 7.5), or as a thick, distinct
band on the agarose gel. M protein is composed of a single class of
immunoglobulin secreted by an abnormally expanded clone of plasma
cells.
Figure 7.5 Monoclonal gammopathy

(Source: https://images.app.goo.gl/miWHtH4QYFeeBFAM9 )
7.2.4 Polyclonal gammopathy
In most instances, polyclonal gammopathy is associated with infections,

inflammations or various reactive processes. A wide-based γ-globulin peak
appear (see Figure 7.6) as a result of antigenic stimulation of numerous
clones of plasma cells and this pattern suggests that polyclonal increase in
immunoglobulin. Chronic viral or bacterial infections, liver disease, a
variety of malignancies and autoimmune disease may cause a polyclonal
rise in the gamma fraction.
74
Figure 7.6 Polyclonal gammopathy

(Source: https://images.app.goo.gl/XzzCSvdgyDpCQ1DW9)
7.2.4 Chronic inflammatory states
In chronic inflammation, a broad peak in γ-globulin region can be seen (see

Figure 7.7) because of the increased immunoglobulin synthesis.
Figure 7.7 Chronic inflammatory states

(Source: http://ucsdlabmed.wikidot.com/chapter-7-laboratory-diagnosis-of-
protein-abnormalities )
75
7.2.5 Acute phase protein response pattern
In acute illnesses acute phase proteins synthesis is increased with a rise in

the α1 and α2-globulin fractions (see Figure 7.8).
Figure 7.8 Acute phase protein response pattern

(Source: http://ucsdlabmed.wikidot.com/chapter-7-laboratory-diagnosis-of-
protein-abnormalities )
7.2.6 Hyperlipoproteinemia
Increased concentration of α2 and β-globulins can be seen with the

appearance of another peak in between α2 and β, which is β-lipoprotein (see
Figure 7.9).
Figure 7.9 Hyperlipoproteinemia

(Source:
https://www.sciencedirect.com/science/article/abs/pii/S1933287415000392
)
76
7.2.7 Chronic hepatitis
There is an overall reduction in albumin and α-globulin with a marked

increase in β-globulin (see Figure 7.10).
Figure 7.10 Chronic hepatitis

(Source: https://slideplayer.com/slide/10187995/ )
7.2.8 Nephrotic syndrome
Plasma protein changes depend on the severity of the nephrotic syndrome.

In early stages only abnormality may be the low plasma albumin
concentration, but the typical pattern in chronic cases is reduction in
albumin, α1-globulin and sometimes γ-globulin and an increase in α2-
globulin. If the syndrome is due to systemic lupus erythematous the γ-
globulin may be normal or raised.
77
Figure 7.11 Nephrotic syndrome

(Source:
http://www.clinbiochemtopics.com/Topics/Winter/Proteins/protein.htm )
Summary
In serum protein electrophoresis five peaks can be seen in a normal
resolution pattern. They are; Albumin, α1-Globulin, α2-Globulin, β-Globulin
and γ-Globulin. In different disease conditions obvious deviations can be
seen compared to normal serum electrophoretic and densitometry pattern
which is important in disease diagnosis. A marked increase in γ-globulin
and β-globulin can be seen in liver cirrhosis. In α1-Antitrypsin deficiency
(Emphysema) absence or an obvious reduction in α1-globulin band can be
seen. In monoclonal gammopathy, the M protein is characterized by the
presence of a single, sharp peak in the β-γ region. A wide-based γ-globulin
peak is seen in polyclonal gammopathy. Chronic inflammatory state
indicates a broad peak in γ-globulin region. Acute phase protein response
pattern involves a rise in the α1 and α2-globulin fractions. In
hyperlipoproteinemia an increased concentration of α2 and β-globulins can
be seen with the appearance of another peak in between α2 and β globulins.
In chronic hepatitis there is an overall reduction in albumin and α-globulin
with a marked increase in β-globulin. In nephrotic syndrome plasma protein
are changed depending on the severity of the syndrome (Figure 7.12).
78
Figure 7.12 Interpreting serum protein electrophoresis (SPE) patterns
(Sources: https://images.app.goo.gl/LiSzuYo1Rit5UPUS9)
79
Review questions
1. Discuss the biochemical basis for the deviations observed in following disease
conditions compared to normal serum electrophoretic and densitometry patterns.
Cirrhosis of the liver, α1-antitrypsin deficiency (emphysema), polyclonal
gammopathy, monoclonal gammopathy, chronic inflammatory states, acute phase
protein response pattern, hyperlipoproteinemia, chronic hepatitis and nephrotic
syndrome.
Learning outcome

 Draw and label the normal serum electrophoretic and densitometry
patterns.
 Describe the serum electrophoretic and densitometry patterns in

following diseases; cirrhosis of the liver, α1-antitrypsin deficiency
(emphysema), polyclonal gammopathy, monoclonal gammopathy,
chronic inflammatory states, acute phase protein response pattern,
hyperlipoproteinemia, chronic hepatitis and nephrotic syndrome.
References
Priyadarshani, A.M.B. and Athiththan, L.V. (2013). Advanced Clinical

Biochemistry Techniques for Medical Laboratory Sciences. 1st ed. ISBN
No: 978-955-9054-99-3.
Burtis, C.A., Ashwood, E.R. and Bruns, D.E. (2007). Tietz Fundamentals of
Clinical Chemistry. 6th ed, Elsevier.
80
Session [8]: Estimation of Serum/ Plasma Protein
Session 8
Estimation of serum/plasma proteins
Content
Introduction, p81
8.1 Electrophoresis, p82
8.2 Immunochemical methods, p84
8.3 Quantification of total protein, p85
Summary, p88
References, p90
Introduction
Serum/plasma proteins are mainly synthesized by the liver. Plasma contains
at least 125 individual proteins. Serum is lacking with coagulation protein
which is used in blood coagulation. Functions of the proteins include
control of oncotic pressure, transportation of substances, and complement
cascade pathways.
However, changes of normal serum protein pattern provide information with
regard to individual’s general status of the body. Fractionation of total
protein is more clinically useful in diagnosis of diseases such as cirrhosis of
the liver, monoclonal gammopathy, Polyclonal gammopathy, chronic
hepatitis, and nephrotic syndrome.
Possible causes of high serum protein include;

 Amyloidosis
 Dehydration
 Hepatitis B
 Hepatitis C
81
 Monoclonal gammopathy
 Multiple myeloma
The total protein level can be lowered in liver or kidney problems or if the
protein is not been digested or absorbed properly. However, if the total
protein level is abnormal further laboratory tests are needed in order to
identify which protein concentration is high or low. This is extremely
important in accurate diagnosis of the disease.
Depending on the required accuracy different methods are used for the
determination of protein concentration of serum/plasma.
8.1 Electrophoresis
Electrophoresis is widely used in the separation and estimation of serum
protein. In electrophoresis the separation of protein is done in an electric
field.
Different types of serum protein electrophoresis are available. They have
been categorized depending on the method used in separation and
differentiation of serum components.
The net charge on a protein is determined by the sum charge of its amino
acids and the pH of the buffer where protein is dissolved. At pH 8.6 all the
proteins are negatively charged and they move toward the positively
charged anode, whilst the positively charged particles migrate towards the
cathode under an electric field. The rate of movement mainly depends on
the charge to mass ratio of the protein molecule
Albumin and globulin are present as the major protein components in the
serum. Albumin represents the largest peak which lies closest to the positive
electrode. Globulins consist of a much smaller portion of the total serum
protein but characterize the primary focus of analysis of serum protein
electrophoresis. Five globulin categories are found; alpha-1, alpha-2, beta-1,
82
beta-2, and gamma, with the gamma globulin being closest to the negative
electrode.
In zone electrophoresis, various protein subtypes are placed in separate
locations on a gel made from agar, cellulose or other plant material.
In serum protein analysis cellulose acetate, agarose gel or capillary
electrophoresis is commonly used. Special techniques include Western
blotting, two-dimensional electrophoresis and immunofixation.
In cellulose acetate or gel electrophoresis proteins are applied to a solid
matrix such as an agarose gel or a cellulose acetate membrane in the buffer
solution which will act as the inert support. Albumin has the most negative
charge, and it will migrate farthest towards the anode.
In clinical application serum is preferred compared to plasma to avoid the
fibrinogen fraction in a region in which monoclonal immunoglobulins often
migrate. But, in some instances, plasma is preferred where fibrinogen
quantification is needed such as evidence for acute inflammation and
fibrinolysis.
Separated proteins are stained by Coomassie brilliant blue which is more
sensitive compared to Amido Black and Ponceau S. Further, separation of
protein subtypes is obtained by staining with immunologically active agents,
which results in immunofluorescence and immunofixation. Lipoprotein
visualization is done with the application of special fat stains. Carbohydrate
side chains’ staining is required in visualization of α1-acid glycoprotein.
Immunofixation electrophoresis is important in detection of paraproteins or
M-protein because it produces distinct band in monoclonal gammopathy.
However, concentrations of many of the serum proteins are too low to
separate as individual bands or they are over-shadowed by more
concentrated proteins. Further, some proteins are weakly stained with the
existence of lipid and carbohydrate.
The densities of each separated fractions could be electronically calculated
to get graphical data on various protein. Quantification of individual band is
done by densitometry.
83
In capillary electrophoresis, solid matrix is not used. The sample is

introduced into a capillary with a negative surface charge. A high current is
applied and negatively charged proteins move towards the anode. Liquid
buffer flows towards the cathode and drags proteins with a weaker charge.
Activity 8.1
1. Briefly describe the working principle behind the serum protein electrophoresis.
8.2 Immunochemical methods
In clinical application, nephelometric and turbidimetric techniques are

widely used because of its speed and easiness. In principle, the reaction
between any substances with a specific reagent which results in particle
formation can be analyzed by these two techniques. When a light beam is
exposed to a fluid with suspended particles, light is absorbed, scattered,
reflected and transmitted. In turbidimetry, the quantification of the residual
transmitted light is used while the measurement of scattered light is the basis
in nephelometry.
These techniques are based on equilibrium method or kinetic method. In
equilibrium method amount of antigen-antibody complex formation is
measured where as in kinetic method the rate of antigen-antibody complex
formation is measured. Lipemic serum samples should not be assessed by
this method because extreme light scattering is done by the lipid particles.
Lipemic samples should be centrifuged prior to analyze to obtain clear
serum and thereafter the sample can be analyzed by the immunochemical
method. Further, samples, control and calibrators should be free from the
dust and dirt particles otherwise this may lead to erroneous results.
Protein precipitation is done by addition of sulfosalicylic acid alone, with
sulfosalicylic acid in combination with sodium sulphate or trichloroacetic
acid. In precipitation method fine precipitate of uniform, insoluble protein
particles is formed which scatter incident light in suspension.
84
8.3 Quantification of total protein
Total serum protein can be measured by the following methods;

1. Biuret
2. Direct photometric
3. Dye-binding
4. Folin-Ciocalteu (Lowry)
5. Kjeldahl
6. Refractometric
7. Turbidimetric
8. Nephelometric
8.3.1 Biuret method
Sodium potassium tartrate present in Biuret reagent forms a complex with

cupric ions. In Biuret method, peptides bonds of protein react with Cu2+ in
alkaline solution and a colour product is formed. The formation of a Cu2+-
protein complex involves two peptide bonds. This complex produces a
violet-colored chelate. The absorbance of this product is measured at 540
nm. In a given concentration range, the absorption at 540 nm is linear with
regard to the concentration of total protein. Therefore, a standard curve can
be used to calculate the concentration of an unknown sample.
The colour intensity of the product is therefore proportionate to the number
of peptide bonds present in the protein. Therefore, it determines the amount
of proteins present in the reaction solution. Serum is preferred in Biuret
assay compared to plasma.
85
8.3.2 Direct photometric methods
Serum proteins absorb UV light at 200 to 228 nm and 270 to 290 nm.
Aromatic rings of tyrosin and tryptophan results in absorption of UV light at
280 nm under pH of 8.
Determination of protein concentration by photometric methods takes
advantage that more a sample contain light-absorbing substances, less the
light transmit through it. Therefore, the association between concentration
and absorption is linear. This concept can be used to measure the protein
concentration of an unknown protein sample.
8.3.3 Dye-binding method
Proteins bind dyes such as Amido black 10B and Coomassie Brilliant Blue.
In the clinical application this method has a limitation as a result of different
affinities and binding capacities of individual proteins.
The Bradford assay, is based on the shift of absorbance of the
dye Coomassie Brilliant Blue G-250. The Coomassie Brilliant Blue G-250
dye exists in three forms; anionic (blue), neutral (green) and cationic
(red). In the acidic medium, the red form of the dye is converted into its blue
form, upon binding to the protein. The solution remains brown in the
absence of protein.
The Bradford protein assay is less prone to meddling with a variety of
chemical compounds such as sodium, potassium, sucrose. But, an exception
has been reported from increased concentrations of the detergent sodium
dodecyl sulfate (SDS). The interference of SDS exhibits two different
modes depending on the concentration. When SDS concentrations are less
than critical micelle concentration (known as CMC, 0.00333%W/V to
0.0667%) in a Coomassie dye solution, the detergent binds strongly with the
protein. This inhibits the binding sites of the protein to the dye reagent and
results in underestimations of protein concentration. When SDS
concentrations are above CMC, the detergent links powerfully with the
86
green type of the Coomassie dye, producing more blue form. This increases
the absorbance at 595 nm irrespective to the protein present.
High buffer concentrations overestimate protein concentration. This will not
be a problem if a low concentration of protein is used.
8.3.4 Folin-Ciocalteu (Lowery) method
The amino acids tyrosine and tryptophan either free or in an unfolded

polypeptide chain reduce phosphotungstic-phosphomolybdic acid in Folin-
Ciocalteu reagent and results in a blue colour with maximum absorption in
the region of 660 nm wavelength which can then be measured
by colorimetric methods.
The intensity of color depends on the quantity of these aromatic amino acids
present. Usually, Bovine Serum Albumin (BSA) is used as the standard
protein. The incubation time is very important in reproducibility of the assay.
8.3.5 Kjeldahl method
In this method, the sample is digested with acid to convert nitrogen present
in the protein to ammonium ion. Ammonia nitrogen concentration is
evaluated by titration or nesslerization. The protein amount is calculated
from the nitrogen concentration of the sample analyzed. The correction
factor is applied for the nitrogen present in serum non-protein compounds.
The ammonia nitrogen content is multiplied by a factor of 6.25 to obtain
total protein value.
8.3.6 Refractometry
Refractometry is the analytical method used to measure substances'

refractive index. The refractive index is measured by the refractometer.
When rapid total protein estimation is needed refractometry is a quick
alternative to chemical analysis. A refractometer can be used in
determination of total proteins in a serum or a plasma sample. At a protein
87
concentration greater than 11.0 g/dL, dilution of serum sample with equal
parts of water is needed in obtaining valid result.
Activity 8.2
1. Compare the methods measurement of serum proteins.
Summary
Changes in normal serum protein pattern provide valuable clue regarding
individual’s general status of the body. Fractionation of total protein is
clinically useful in disease diagnosis. Serum protein electrophoresis is
commonly done in the clinical laboratories to identify disorders of
serum/plasma proteins. In electrophoresis the separation of protein is based
on the charge and the mass of the protein molecule and separation is done in
an electric field. Different types of serum protein electrophoresis are
available based on the method used in separation and differentiation of
serum components. In clinical application, nephelometric and turbidimetric
techniques are commonly used because of its speed and easiness. Total
protein quantification could be achieved by following methods; Biuret,
Direct photometric, -Dye-binding, Folin-Ciocalteu (Lowry), Kjeldahl,
Refractometric, Turbidimetric and Nephelometric. In Biuret method,
peptides bonds in protein react with Cu2+ in alkaline solution which is in
Biuret reagent. This complex produces a violet-colored complex and its
absorbance is measured at 540 nm by spectrophotometry. Serum proteins
absorb UV light at 200 to 228 nm and 270 to 290 nm.
In dye binding methods Coomassie Brilliant Blue and Amido black 10B are
widely used. But, this method has limitations as the affinities and binding
capacities of individual proteins towards the dye agent differ. The amino
acids tyrosine and tryptophan reduce phosphotungstic-phosphomolybdic
acid in Folin-Ciocalteu reagent and results in a blue colour with maximum
88
absorption at 660 nm. In Kjeldahl method the sample is digested with acid
to convert nitrogen present in the protein to ammonium ion. Ammonia
nitrogen concentration is evaluated by titration or nesslerization. The
ammonia nitrogen content is multiplied by a factor of 6.25 to obtain total
protein value. When rapid total protein estimation is needed refractometry is
a quick alternative to chemical analysis.
Review questions
1. Write a short note on reference intervals of proteins.

2. State the principle of electrophoresis.
3. Discuss the procedural techniques involved with serum protein electrophoresis and
immunochemical methods.
4. Discuss the limitations of the dye binding methods used in serum/plasma protein
determination.
5. Discuss the advantages and disadvantages of different methods used in
determination of serum protein/total protein level.
Learning outcome
 State why the serum/plasma protein estimation is important in diagnosis
of disease.
 State the principle of following methods used in serum/plasma protein

analysis.
Electrophoresis
Immunochemical methods
Quantification of total protein
Biuret method
Direct photometric methods
Dye-binding method
Folin-Ciocalteu (Lowery) method
Kjeldahl method
89
Refractometry
 Discuss the advantages and limitations/disadvantages of each method

mentioned above.
References
Burtis, C. A., Ashwood, E. R., & Bruns, D. E. (2007). Tietz Fundamentals
of Clinical Chemistry 6th Edition (ISBN: 978-0-7216-3865-2), Saunders
Elsevier, St. Louis, MO.
O’Connell, T. X., Horita, T. J., & Kasravi, B. (2005). Understanding and

interpreting serum protein electrophoresis. Am Fam Physician, 71(1),
105-112.
90
Session [9]: Analysis of cerebrospinal fluid
Session 9
Analysis of cerebrospinal fluid
Content
Introduction, p91
9.1 Formation of CSF, p92
9.2 Functions of CSF, p93
9.3 Specimen collection and handling, p94
9.4 Composition of CSF, p98
9.5 Guidelines in processing, handling and transport of CSF, p99
9.6 Deterioration of CSF on standing, p100
9.7 Traumatic and cerebral haemorrhage, p100
9.8 Appearance of CSF, p101
9.9 Cell count, p103
9.7 Meningitis, p110
References, p112
Introduction
Cerebrospinal fluid (CSF) is the most precious sample in the body. The
analysis aids in the diagnosis of diseases of the central nervous system.
Lumbar cerebrospinal fluid is obtained by a procedure known as lumbar
puncture which is a risky procedure. Variations of the CSF composition and
/ or the presence of abnormal constituents including organisms in the CSF
91
Unit [04]: Other body fluids
provide clues to underlying disease. There are specific guidelines to adhere

to in handling and transport of CSF specimens. It is mandatory that the
specimen transporters and the testing staff adhere to the set guidelines for
the reasons that (a) tapping of CSF involves an invasive procedure (b) CSF
is often obtained from critically ill patients and (c) CSF analysis is a
necessity in obtaining diagnostic information.
9.1 Formation of CSF

Meninges are linings composed of 3 layers which cover the spinal cord and
brain. They are dura mater, pia mater and arachnoid mater. Choroid plexus
is situated at the two lumber ventricles and 3rd and 4th ventricles. Choroid
plexus involve in CSF production, roughly 20ml every hour. Fluid flowing
into subarachnoid space in between arachnoid and pia mater will be
reabsorbed back into blood capillaries via arachnoid granulations to
maintain a CSF fluid volume of 90-150ml in adults and 10-60ml in
neonates.
CSF is the ultrafiltration of plasma through a capillary network known as

choroid plexus which involves selective filtration under hydrostatic pressure
and active transport secretion mechanisms. Endothelial cells in the choroid
plexus have tight fitting structures known as blood—brain barrier. This
barrier helps to prevent the exchange of antibodies, medications and
chemicals to pass from blood to CSF. Damage to the barrier will cause
meningitis and multiple sclerosis (Figure 9.1).
92
Figure 9.1 Brain anatomy – Cross section

(Source:
https://commons.wikimedia.org/wiki/File:131_Meningeal_LayersN.jpg)
9.2 Functions of CSF
 Supply nutrients and oxygen to nervous tissue

 Removal of metabolic waste
 Act as a mechanical barrier or shock absorber to brain and spinal cord
against trauma.
 CSF provides buoyancy to the brain, supporting its weight; literally, the
brain floats in the CSF
There are four major categories of disease:
 Meningeal infections – such as bacterial or viral meningitis
 Subarachnoid hemorrhage – commonly referred to as a stroke.
 Central Nervous System malignancy
93
 Demyelinating disease - such a multiple sclerosis
9.3 Specimen collection and handling
CSF is collected via a special procedure known as lumber puncture. The

procedure is usually done by a qualified medical officer along with a nurse
and a MLT. The procedure of lumber puncture is briefly described below.
9.3.1 Specimen collection

Collected via a lumber puncture between the 3rd, 4th or 5th lumber vertebrae.
Procedure needs extreme care and precautions needed to be taken. Usually
performed by a skilled physician.
Collection volume depends on the volume available in patient and opening

pressure created when the needle enters the subarachnoid space. Prior to
collection, intracranial pressure should be known. During puncture extra
care should be given to prevent the damage to neural tissue and introduction
of infection to surrounding tissues.
9.3.2 Indications for lumbar puncture

Lumbar puncture is indicated under the following conditions:
1. Suspected CNS infection

2. Suspected subarachnoid hemorrhage
3. Therapeutic reduction of cerebrospinal fluid pressure
4. Sampling of CSF for any other reason.
9.3.3 Contraindications for lumbar puncture

Lumbar puncture is not undertaken if the following condition or conditions
prevail:
94
1. Local skin infections over the proposed puncture site – this is an

absolute contraindication
2. Raised intracranial pressure
3. Suspected spinal cord mass lesion or intracranial mass lesion
4. Uncontrolled bleeding diathesis
5. Spinal column deformities
6. Lack of patient cooperation.
9.3.4 Materials requirement for lumbar puncture
The materials required for lumbar puncture:
Lumbar puncture tray and Universal precautions materials are to be kept

ready by the nurse, in advance. The lumbar puncture tray contains; 20-gauge
or 22-gauge Quinke needle with stylet, prep solution, manometer, drapes,
and the local anesthetic. The laboratory should provide in advance to the
patient site sterile screw-cap CSF specimen containers appropriately
numbered: Specimen container Number-1 for appearance and chemistry
(e.g. glucose, protein, protein electrophoresis), Specimen container Number-
2 for Microbiology (e.g. Gram’s stain, bacterial and viral cultures),
Specimen container Number-3 second CSF specimen for Microbiology and
Specimen container Number-4 for microscopy (e.g. cell count and
differential count).
9.3.5 Lumbar puncture to collect CSF

The following procedural steps, as they are shown in numerical order, are
undertaken in lumbar puncture (Figure 9.2).
1. Reassess indications for the lumbar puncture procedure and obtain

informed consent.
2. Provide necessary analgesia and /or sedation as required.
95
3. Position the patient: (a) lateral decubitus position with “fetal ball”
curling up or (b) seated and leaning over a table top. Both these
positions will open up the interspinous spaces.
4. Locate landmarks: between spinous processes at L4-5, L3-4, or L2-3
levels.
5. Prepare and drape the area after identifying the landmarks. Use
lidocaine (1%) to anaesthetise the skin and the deeper tissues under the
insertion site.
Figure 9.2 Lumber Puncture procedure
(Source: https://www.oxfordmedicaleducation.com/clinical-
skills/procedures/lumber-puncture/)
6. Assemble the needle and manometer and attach the 3-way stopcock to
the manometer.
7. Insert Quinke needle bevel-up through the skin and advance it through
the deeper tissues. A slight ‘pop or give’ is felt when the dura is
96
punctured. Slightly angle the needle at insertion, between the vertebrae.

If the needle hits bone, partially withdraw the needle, reposition and re-
advance.
8. When CSF flows, attach the 3-way stopcock and manometer. Measure
the intracranial pressure (ICP); which should be 20 cm or less (the
pressure reading may not be reliable if the patient is in the sitting
position).
9. If CSF does not flow or if the needle hits bone, withdraw needle partially,
recheck landmarks, and re-advance.
10. Once the ICP has been recorded, remove the 3-way stopcock and begin
filling CSF collection tubes 1 through 4 with 1-2 ml of CSF in each.
11. After the tapping is over, remove needle, place a bandage over the
puncture site.
12. Instruct patient to remain lying down for 1-2 hours before getting up.
9.3.6 Order of collection

Tube 1 - Biochemical and serologic tests
Tube 2 - Microbiology
Tube 3 - Cell count and Haematology
Tube 4 - Microbiology. If recommended only.
9.3.7 Specimen storage

Haematology tube - Can be refrigerated
Microbiology tube - Should be at room temperature
Biochemistry tube - Can be frozen.
If CSF fluid is remaining after analysis, do not discard. Keep it frozen

until there is no further use of the sample.
97
Activity 9.1
1. Discuss the reason for different storage conditions for CSF specimens.
9.4 Composition of CSF
CSF is a protein-poor plasma filtrate secreted by choroid plexus cells in

each of the four ventricles of the brain, circulating through the ventricles of
the brain, the central canal of the spinal cord, the subarachnoid space and
returns to the blood stream at the arachnoid villi.
CSF is a clear, colorless, sterile watery body fluid. The total solid found
dissolved in CSF is little, amounting to 0.85 – 1.70 g/dL. Hence, the specific
gravity of CSF is also low, being 1.006 – 1.008. The pH of CSF is close
upon that of the blood plasma, with values 7.35 – 7.40. The cellular
elements of CSF are confined to lymphocytes, being around 0 – 8
lymphocytes per cubic millimeter. CSF is devoid of erythrocytes and
neutrophils. Normal WBC count is 0-5 WBCs/µl.
Total protein concentration of CSF is rather low, being around 80 – 320

mg/L. Of total protein, albumin is the predominant protein amounting to 50
– 70% (100 – 300 mg/L). CSF does not contain fibrinogen; hence there is no
clotting activity in CSF unlike in blood plasma. CSF has all the globulin
types the blood plasma has; 1 globulin = 3 – 9%, 2 globulin = 4 – 10%, 
globulin = 10 – 18% and  globulin = 3 – 9%.
CSF is a nutritive medium containing electrolytes (Na+ = 136 – 150 mmol/L,

K+ = 2.5 – 3.2 mmol/L, Cl- = 118 – 132 mmol/L), glucose (= 2.22 – 3.89
mmol/L), amino acids - of which glutamine predominates (= 6 – 16 mg/dL)
and minerals (e.g. Fe, Mg). Further, the CSF has small amounts of excretory
98
compounds such as urea, uric acid and metabolic waste (e.g. lactate) and
other compounds of importance such as immunoglobulins, hormones.
9.5 Guidelines in processing, handling and transport of

CSF
The CSF specimens tapped from patient must be so treated, for it is from a
critically ill patient, drawn with an invasive procedure and CSF testing is the
ultimate measure towards arriving at a definitive diagnosis. Hence, the
guideline to follow is to rush the CSF specimen containers from the patient
sit to the testing laboratory and hand deliver to the duty medical laboratory
technician. The duty laboratory technician who received prior notification of
the CSF for testing must commence the tests on CSF straight away. If the
CSF is to be tested for photo-labile chromogenic substances such as
bilirubin then the CSF specimen container (container number 1) must be
wrapped in a dark paper to keep the light out. Like any other patient sample,
CSF sample too has to be considered as potentially bio-hazardous with a
likelihood of infective organisms, requiring safe procedures in handling,
pipetting and disposing.
CSF is difficult to obtain and only a small volume is available for laboratory
testing, therefore the medical laboratory technician has to work extremely
carefully and economically with the CSF provided to the laboratory. CSF
specimens do not require pre-processing such as blood plasma or blood
serum.
99
9.6 Deterioration of CSF on standing
The facts to base avoidance of delaying of testing are to prevent;
1. Lowering of the CSF glucose concentration which would decrease on

standing,
2. Disintegration of cells such as leukocytes,
3. Lyses of bacteria such as meningococci
4. Death of Neisseria meningitides and Neisseria gonorrhoea when CSF is
in contact with air.
9.7 Traumatic and cerebral haemorrhage
The following figure (Figure 9.3) shows the difference between samples
collected under traumatic haemorrhage and cerebral haemorrhage. The
difference are further compared and explained in table 9.1.
Cerebral Hemorrhage Traumatic hemorrhage
Figure 9.3 CSF distribution in collection tubes in a Cerebral and Traumatic

hemorrhage
(Source: https://link.springer.com/chapter/10.1007/978-3-319-01225-4_22)
100
Table 9.1 Comparison of traumatic and cerebral haemorrhage
Traumatic
Cerebral hemorrhage
hemorrhage
Puncture of blood Bursting of a brain

Reason vessel during lumber artery due to trauma or
puncture malignancy etc.
Distribution of blood Uneven. More in tube

Evenly distributed
in 3 tubes 1 and less in tube 3
Form clots due to

Clot formation No fibrinogen to clot
fibrinogen release
Colour of
supernatant upon Xanthrochromic Clear
centrifuge
9.8 Appearance of CSF
Appearance of CS can be changed due to the presence or absence of certain

constituents like blood, proteins, lipids and drugs. Figure 9.4 shows the
change of color in CSF samples and Table 9.2 describes the reasons behind
the appearance of CSF.
101
Figure 9.4 CSF Appearance. Left to right, Normal CSF, mildly

xanthochromic CSF, moderately xanthochromic CSF, red-tinged turbid CSF
caused by hemorrhage, and cloudy red-tinged fluid from a horse with
bacterial meningitis.
(Source: https://veteriankey.com/cerebrospinal-fluid/)
Table 9.2 Reasons behind the appearance of CSF.
Appearance Possible causes
Crystal clear Normal
High protein/ lipid content

Cloudy/ Turbid/
Infection due to microorganisms
Milky
Cloudy due to WBCs
Hemoglobin due to hemorrhage
Billirubin – RBC degradation/ high serum levels

Xanthochromia
(pink/ orange/ Carotene – High serum levels
yellow) Proteins
Melanin in meningeal melanosarcoma
102
Oily Radiographic contrast media
Clotted Clotting factors – Traumatic tap
Protein and clotting factors due to disruption in

Pellicle
blood brain barrier and tubercular meningitis
Activity 9.2
1. Briefly discuss the importance of assessment of appearance of CSF in laboratory

investigations.
9.9 Cell count
Total CSF count, WBC count, RBC count and differential count are done
mainly under CSF analysis.
9.9.1 Total count

Undiluted specimens are used for counting if the specimen appearance is
clear. But if the specimen is turbid, dilution is done with normal saline. 4
corner squares and the center square of neubaeur chamber is counted for
cells.
Number of cells counted × Dilution × 1µl = cells/ µl

1µl (0.1 × 10)
103
9.9.2 WBC count

 Specimen should be analyzed immediately within 1 hour as WBCs start
to lyse. In case of delay, specimens should be refrigerated. In newborns,
they have 30 mononuclear cells/µl.
 Neubauer counting chamber is used for counting of WBCs.
 Dilution is done with 3% glacial acetic acid and allowed to stand for 1
minute to lyse the red cells and then load the neubaeur chamber.
9.9.3 RBC count

RBC count can be obtained from the values of total cell count and WBC
count.
Total cell count – Total WBC count = Total RBC
9.9.4 Contamination correction

In case of traumatic tap, CSF may be contaminated with proteins.
Number of artificially added WBCs can be calculated using,
WBC (added) = WBC (blood) × RBC (CSF)

RBC (blood)
≈ WBC (CSF) = Actual count - Added WBC
9.9.5 Differential cell count

To ensure that the maximum number of cells are available for calculation,
specimen should be concentrated. Concentration techniques include;
1. Sedimentation
2. Filtration
104
3. Centrifugation
4. Cytocentrifugation
Lymphocytes are high during viral, tubercular and fungal meningitis and
multiple sclerosis. Neutrophil count increases during bacterial meningitis
and cerebral hemorrhage. Blast cells can be seen during acute leukemia.
Malignant cells are seen in malignant conditions.
9.9.6 Non-pathological cells

 Choroid cells – Epithelial lining of choroid plexus
 Ependymal cells – Lining of ventricles and neural canal
 Spindle shaped cells – Lining of arachnoid.
9.10 Chemical analysis of CSF

Protein, glucose, lactate and glutamine are main chemical constituents
analyzed in CSF sample.
9.10.1 Protein
 Most prominent CSF chemical analysis is protein.
 Normal range : 15-45 mg/dl
Elevated in conditions:
Meningitis, hemorrhage and multiple sclerosis, CNS tumors, Guillain-Barre
syndrome, Neurosyphilis, Cushing syndrome, Polyneuritis, Diabetes
Decreased in conditions:
CSF leakage / trauma, recent puncture, rapid production of CSF and water
intoxication.
105
Protein fractions present in CSF

1. Albumin – Major protein fraction.
2. Prealbumin – 2nd major fraction
3. Alpha globulins – Haptoglobin and ceruloplasmin
4. Beta globulin – Major fraction is transferrin
5. CDT which is a carbohydrate deficient transferrin fraction
6. Gamma globulin – Mainly IgG and IgA in less. IgM, fibrinogen and beta
lipoprotein are absent in CSF.
7. Artificially added plasma proteins during a traumatic tap
8. Myelin basic protein - Estimate recent disruption of myelin sheath
around axons of neurons. Used in multiple sclerosis diagnosis. Technique
used is Immunoassay.
 It is important to determine the protein fractions present in CSF to

examine the associated condition. Increase in protein fraction is CSF will be
mainly due to the disruption of blood brain barrier.
Eg:- Multiple sclerosis – High IgG fraction
Calculations in CSF protein analysis
1) Added protein correction

While doing the WBC correction due to added proteins, the same correction
has to be done during protein analysis.
Protein, Globulin and Albumin estimation can be done by,
 Turbidity production/ dye binding method via automated
technique.
 Automated chemistry analysis
Protein = CSF protein - (Serum protein x 1000 x (1 – Hematocrit) / 100)
x CSF RBC / (Blood RBC x 106))
106
Where:
CSF protein is in mg/dL CSF RBC is in /mm3
Serum protein is in g/dL Blood RBC is in mil/mm3
Hematocrit is in %
2) IgG calculation
IgG may be added to CSF in two ways;
i) Produced in CSF
ii) Added as a result of disrupted blood brain barrier
Two calculation methods are available.
i) CSF/ serum albumin index – Estimate the integrity of blood brain

barrier
CSF/ serum albumin = CSF albumin (mg/dl)

Serum albumin (g/dl)
Interpretation: Index > 9 – Undamaged blood brain barrier. Index increases

relative to the damage to the barrier.
ii) CSF IgG index – Measure IgG levels within CSF
IgG index = CSF IgG (mg/dl) / Serum IgG (g/dl)

CSF albumin (mg/dl) / Serum albumin (g/dl)
Interpretation: Index > 0.70
107
3) Electrophoresis technique
 Agarose gel method with brilliant blue stain.
 Higher resolution can be achieved by using immunofixation
electrophoresis and isoelectric focusing with silver stain.
 Detect oligoclonal bands to diagnose CNS inflammation.
 If bands are visible in gamma region, it indicates the production of
immunoglobulin in CSF
 Moreover, systemic and neurologic effect of HIV to CSF and serum can
be determined by the banding pattern
 Multiple sclerosis has 2 or more bands which are not present in serum
electrophoresis pattern, with high IgG index.
 Encephalitis, neurosyphilis, Guillain-Barre syndrome also have bands
which can be seen only in CSF electrophoresis, but not in serum.
9.10.2 CSF Glucose

 Glucose enter CSF by selective transport through the barrier.
CSF Glucose = 2/3 × Plasma glucose
 Therefore, along with CSF glucose, plasma glucose should also be

analyzed.
Important:
 Blood for plasma glucose should be taken 2 hours before the lumber
puncture. This allows the blood and CSF glucose to reach the equilibrium
state. Specimens should be analyzed immediately to prevent glycolysis and
same analytical technique and instrument should be used for both blood and
CSF glucose analysis.
Decreased levels: Bacterial, tubercular and fungal meningitis
Increased levels: Due to plasma elevation of glucose only.
108
9.10.3 CSF Lactate

 Used to identify causative agent for meningitis. Higher levels are seen
during initial stages of treatment but rapidly fall while continuing a
successful treatment.
 A sensitive chemical parameter to determine the patient response to
therapy
 Lactic acid is produced in CSF when there is a hypoxic condition.
Therefore, other than meningitis, diseases associated with hypoxia and
severe head injuries can be diagnosed.
 Falsely high results may be obtained from xanthochromic or hemolysed
samples as lactate is present highly within red cells.
9.10.4 CSF Glutamine

 Produced in the brain cells from ammonia and α-ketoglutarate in the
process of removing metabolic toxic waste ammonia from CNS.
 Normal level: 8-18 mg/dl
 Increased levels of ammonia can be seen in liver diseases.
 When ammonia levels increases, synthesis of glutamine too increases. So
glutamine is considered as an indirect measure of ammonia. However,
this technique is more suitable than ammonia direct measure because
glutamine concentration in CSF sample is more stable than ammonia,
 Moreover, not like ammonia, glutamine is highly correlated with the
stages of clinical symptoms.
 Highly recommended to diagnose coma patients of unknown origin.
Increased levels
>35 mg/dl in disturbance of consciousness.
 Reye syndrome – (75% of children with disease show high glutamine

levels)
109
9.7 Meningitis
Meningitis can be caused either by bacterial, viral, fungal or tubercular

infection. The differences in the above categories are explained in Table 9.2.
Table 9.2 Comparison of meningitis

Bacterial Viral Tubercular Fungal
Appearance Opalescent, Normal Same as Slightly

cloudy, bacterial. cloudy or
purulent, + Fibrin web clear
clotting
WBC count Elevated Elevated Elevated Elevated
Cells/mm3 500-2000 10-2000 50-500+ 50-5000
Predominant Neutrophil Lymphocyte Lymphocyte Lymphocyte

cell & Monocyte & Monocyte
Glucose Markedly Normal Decreased Normal to

decrease decreased
(mmol.L-1) 2.5 - 5.5 0- 2.5
0- 2.5
Protein Markedly Moderately Moderate to Moderate to

increase increased marked marked
(mg.dL-1)
increase increase
50-1000+
45-500+
Lactate >35 mg/dl Normal >25 mg/dl >25 mg/dl
Pellicle +’ve india

form ink with
Cryptococcus
neoformans
110
Summary
CSF is a clear and colorless fluid found in the brain and spinal cord which
mainly functions as a shock-absorber against trauma. About 125ml of CSF
is circulated daily with a composition of 0.3% plasma proteins, few white
cells and electrolytes; mainly sodium and chloride. CSF is mainly used to
diagnose subarachnoid hemorrhages, meningeal infections, CNS
malignancy and demyelinating disease. CSF is the most precious sample in
the body because it is collected via a complex and risky process known as
lumber puncture. In CSF analysis, its appearance, cell counts and chemical
constituents are reported. CSF analysis is essential to determine the
prognosis of the patient and to differentiate the types of meningitis.
Review Questions
1. Briefly describe the lumber puncture procedure?

2. State the order of sample collection and storage of sample?
3. State the differences between traumatic and cerebral hemorrhage?
4. How do you correct the cell counting errors due to contaminations?
5. Describe the CSF IgG measurement and calculation?
6. What is the importance of CSF glutamine levels?
7. Contrast and compare the types of meningitis?
Learning outcome
 State the physiology behind CSF formation
 State the functions of CSF
 Describe the CSF specimen collection procedure
 Describe the CSF specimen handling guidelines
 State the CSF composition
 Explain traumatic and cerebral haemorrhage
 Describe the conditions interpreted by CSF appearance
 Describe the CSF cell counting techniques and calculations
 Describe the chemical analysis of CSF and their importance
 State the identification parameters of meningitis
111
References
Bishop, M.L., Fody, E.P. and Schoeff, L.E. (2010). Clinical chemistry:
Techniques, principles and correlations (6th ed). Lippincott Williams &
Wilkins, Philadelphia.
Chawla R. (2006). Practical Clinical Biochemistry- Methods and

interpretations (3rd ed.). Jaypee brothers, India.
Strasinger, S.K. and Di Lorenzo, M.S. (2008). Urinalysis and body fluids
(5th ed.). F.A. Davis company, Philadelphia, USA.
112
Session [10]: Analysis of body cavity fluids
Session 10
Analysis of body cavity fluids
Contents
Introduction, p113
10.1 Analysis of blood, p114
10.2 Transudates and Exudates, p120
10.3 Factors affecting fluid composition, p123
10.4 Synovial fluid analysis, p127
10.5 Laboratory analysis of synovial fluid, p129
10.6 Serous fluid, p132
10.7 Pleural fluid, p133
10.8 Pericardial fluid, p136
10.9 Peritoneal fluid, p137
References, p140
Introduction
The clinical biochemistry laboratory is familiar with the use of blood and
urine analysis as aids in the diagnosis of disease. Urine analysis was
described under MDU 3303 Clinical Biochemistry I module. In fact these
two body fluids are the major biological fluids that are routinely analysed in
the laboratory and they help to diagnose and assess the severity of diseases.
Yet there are other body fluids which have their own use in aiding diagnosis
of disease.
113
These body fluids are tested instead of blood because they can give more
direct answers to what may be going on in a particular part of the body than
blood or urine samples. Some other body fluids which are analysed in the
clinical chemistry laboratory include Cerebro-Spinal fluid (CSF), Synovial
fluid, Amniotic fluid, Pleural Fluid, Pericardial Fluid, Peritoneal Fluid,
Saliva and Sweat.
When analyzing these body fluids in disease conditions, the concentration of

the analyte in the disease state should be compared to what is observed in
healthy individuals and the presence or absence of an analyte would further
help in the diagnosis depending on the disease state.
10.1 Analysis of blood
Blood may be collected as whole blood from arteries, capillaries or veins,

cord blood from umbilical cord at the time of delivery or peripheral blood
from earlobe, fingertip or heel.
10.1.1 Collection of blood
Venepuncture
The median cubital vein in the antecubital fossa of the hand is the preferred
site for venepuncture. If there is difficulty in collecting blood from this site,
the ankle or the veins on the back of the hands may be used. The blood is
collected after applying a tourniquet 10-15cm above the intended site of
blood collection or by using a blood pressure cuff to occlude the veins.
Blood is drawn by using a needle with a syringe big enough to

accommodate the volume of blood required for all the analytes to be
measured. The preferred needle sizes are gauges 19-22 (Note: larger gauge
114
size number indicates a smaller bore of the needle). The tourniquet should
be withdrawn when the required volume of blood is drawn and then the
needle should be removed from the puncture site. A sterile gauze pad should
be kept over the puncture site after the needle is removed to prevent
excessive bleeding and bandaged or plastered and kept for around 15
minutes.
Skin puncture
Depending on the sample volume and the necessity in some patients a skin
puncture may be preferred over a venipuncture e.g. if the sample volume
required is less. In adults and older children blood may be obtained by
performing a skin puncture on the tip of a finger or ear lobe. In infants the
lateral or medial plantar surfaces are used for skin puncture. Although skin
puncture could be used as a method of colleting blood samples it has its own
inherent disadvantages. The greater risk of infection due to difficulties in
sterilizing these areas compared to the antecubital fossa and the longer time
necessary to draw the required volume are some of the disadvantages.
Different devices are available for collection of capillary blood specimens

and for pricking skin to obtain blood samples e.g. capillary tubes which
draw blood into it by capillary action from the skin puncture site, and spring
loaded lancets whose depth of penetration of skin could be changed.
Neonatal screening for certain diseases does not require more than one drop
of blood. In these instances blood specimen could be collected on filter
paper by a skin puncture, usually by pricking the foot.
Arterial puncture
Arterial blood may be required for certain clinical chemistry analyses like
blood gas analysis. The main sites of arterial puncture are the radial artery at
the wrist, the brachial artery at the elbow and the femoral artery in the groin.
115
Arterial puncture should be performed only by a physician or a technician

who has been specially trained in the process because of the increased risk
of complications associated if the procedure is not performed correctly. It
should also be noted that firm pressure at the puncture site for at least 5
minutes may be necessary after the arterial puncture to minimize bleeding
and hematoma formation.
10.1.2 Influence of site and hemolysis on blood composition
Depending on the site from which blood was collected the composition of
blood may vary. Blood obtained from skin puncture and arterial puncture
are alike compared to venous blood. Blood glucose concentration in venous
blood is lower compared to capillary blood as the glucose is utilized by the
tissues. This difference in glucose concentration may be as much as
7mg/dL. Blood obtained by skin puncture of the earlobe or non-warmed
skin may show an increase in serum protein (thus an increase in protein
bound constituents) and serum potassium. The composition of certain
constituents of blood may also vary depending on whether the blood was
collected from a central venous catheter or a peripheral vein.
All blood samples should be visually inspected to see whether haemolysis

has occurred as this may have an influence on some constituents. It should
be noted that visual evidence of haemolysis is shown only when the
haemoglobin concentration exceeds 20mg/dL. Although slight haemolysis
usually do not have a major effect on most constituents severe haemolysis
will dilute the plasma and this may give rise to a change in concentration of
analytes which are found in lower concentrations in the erythrocytes
compared to plasma. Yet a marked effect will be there in analytes which are
normally found in higher concentrations in erythrocytes as they will enter
the plasma in large quantities when there is haemolysis. Some of the
constituents which will increase on haemolysis are potassium, phosphate,
116
total acid phosphatase, aspartate aminotransferase, and lactate

dehydrogenase.
10.1.3 Variations in composition of blood in relation to

venepuncture
During the process of venepuncture the composition of blood may change,

especially if a tourniquet is applied for a prolonged period of time.
A tourniquet should not be left in place for more than 1 minute. If it remains
longer some constituents may increase (total protein, iron, total lipids,
cholesterol, aspartate transaminase and bilirubin for example) and some may
decrease (e.g. potassium). This is because of the increase in filtration
pressure across the capillary wall leading to fluid and low molecular weight
compounds to pass through the capillary wall i.e. fluid leaks out of blood
vessels leading to an increase in compounds which have a high molecular
weight.
Composition of blood drawn first may differ slightly in composition to

blood drawn afterwards. First drawn blood sample, which is the blood lying
closest to the tourniquet is the one which is most similar to circulating
blood. This sample is thus usually used for tests which are essential for
diagnosis of critical medical conditions e.g. calcium analysis (Note: a
tourniquet should not be used when drawing blood for serum calcium
analysis). Later samples may show an increase in constituents like protein
and protein bound products because of venous stasis in the small veins and
capillaries.
Other factors which could have an effect on the composition (hence should
be avoided) are additional trauma at the venepuncture site (may increase
creatine kinase, aspartate aminotransferase levels) and pumping of fists
117
before venepuncture (may increase plasma potassium, phosphate and lactate

levels).
10.1.4 Anticoagulants
1. Heparin
This is the most widely used anticoagulant for clinical chemistry analysis.
Heparin is available as sodium, lithium, potassium and ammonium salts.
2. Ethylenediaminetetraacetic acid (EDTA)

EDTA is a chelating agent which is used in haematological examinations
like full blood count, ESR, etc.
3. Sodium fluoride
Sodium fluoride is a preservative for glucose and a weak anticoagulant.
Sodium fluoride acts by inhibiting the glycolytic enzymes (and other
enzymes in the blood).
4. Citrate
Sodium citrate acts as an anticoagulant by chelating calcium. Thus it is
widely used in coagulation studies on blood.
5. Oxalates
Oxalates act as anticoagulants by forming insoluble complexes with calcium
ions. Sodium, potassium, ammonium and lithium salts of oxalates are used
as anticoagulants and the most widely used oxalate is potassium oxalate at a
concentration of 1-3mg/mL of blood.
The following table (Table 1.1) gives the color codes used for different
anticoagulants and their modes of action.
118
Table 1.1 Types of blood specimen collection tubes
Colour
Additives used Mode of action Uses
code
No additive Blood clots and the serum is Clinical chemistry,
(may have separated by centrifugation Immunology,
Red
silicone-coated in the lab Serology, Cross
interior) matching
Serum separator tube, Clinical chemistry,
contains a gel at the bottom immunology and
Gold No additive
to separate blood from serum serology
on centrifugation
Plasma Lithium heparin prevents Clinical chemistry
Light separating tube coagulation. Plasma is
green (PST) with separated with the PST gel at
lithium heparin the bottom of the tube.
Removes calcium by Haematology, cross
Purple EDTA
forming calcium salts matching
Light Removes calcium by Coagulation tests
Sodium citrate
blue top forming calcium salts
Inactivates thrombin and Lithium analysis (use
Sodium heparin
thromboplastin sodium heparin),
Green or lithium
sodium analysis (use
heparin
lithium heparin)
Removes calcium by Trace element testing
Dark forming calcium salts. Tube (zinc, copper, lead,
EDTA
blue designed to contain no mercury), toxicology.
contaminating metals
Sodium fluoride Anti-glycolytic agent which Glucose analysis
Light
and potassium preserves glucose (up to 5
gray
oxalate days)
Acid citrate Complement activation HLA tissue typing,
Yellow
dextrose paternity testing,
119
DNA studies
Yellow Preserves viability of Microbiology
Broth mixture
and black microorganisms
Buffered Forms calcium citrate to Erythrocyte
Black
sodium citrate remove calcium sedimentation rate
Orange Thrombin Clots blood quickly Clinical chemistry
Inactivates thrombin and Serum lead analysis
Light
Sodium heparin thromboplastin, contains
brown
virtually no lead
Potassium Forms calcium salts to Immunohematology
Pink
EDTA remove calcium
Forms calcium salts to Molecular/OCR and
Potassium
White remove calcium DNA testing
EDTA
Activity 10.1
1. Discuss what are the points that should be considered when a blood sample is
collected for the assessment of following parameters
Plasma glucose level
Serum iron level
Prothrombin time
10.2 Transudates and Exudates
Transudates are fluid formed due to systemic disorders as a result of an

imbalance between fluid filtration and reabsorption processes.
Eg:- Congestive heart failure due to alterations in hydrostatic pressure.
Nephrotic syndrome – Hypoproteinemia
120
Exudates are fluid produced as result of disruption of membranes around

the cavities. Contains a high content of protein and cellular debris which has
escaped from blood vessels and been deposited in tissues. Cellular material
may be tumor cells or foreign materials such as bacteria, viruses, parasites,
and fungi.
Eg:- Infections and Malignancy
10.2.1 Laboratory tests done to differentiate transudates and

exudates
Classifying a fluid as a transudate or an exudate can help clinicians

determine the disease process that is responsible for the accumulation of
fluid. This would help them in treating the disease with the idea of curing or
minimizing complications depending on the disease involved.
1. Appearance
Transudates have a clear and pale yellow appearance while exudates are
cloudy due to high protein and cellular debris content. Exudates may appear
in green, brown or reddish colour if red cells are present.
2. Total protein
Usually fluid: serum protein ratio is considered which is <0.5 for transudate
and >0.5 for exudate.
3. Lactic dehydrogenase
Lactate dehydrogenase catalyses the reversible reaction between pyruvate
and lactic acid and is involved in energy metabolism in cells. Where ever
there is accumulation of cells and cell death as observed in infections and
inflammation, the concentration of LDH in the area increases. As exudates
have a lot of cells associated with it, an exudate will have LDH
121
concentrations higher than 200 units/ L while transudates will have LDH
levels lower than 200 units/ L.
The ratio between LDH concentration in the fluid and the serum is lower
than 0.6 in a transudate where as in exudates the ratio exceeds 0.6.
4. Cell counts
WBC count is usually <1000/µl for transudate and >1000/µl for an exudate.
Monocytes and lymphocytes are major cells found in a transudate. Exudates
may include neutrophils, lymphocytes, monocytes, eosinophils and even
basophils depending on the infective agent.
5. Spontaneous clotting
No spontaneous clotting is seen in transudate. However, clotting is present
in exudates due to high fibrinogen content.
6. Glucose
Glucose concentrations may be used to determine the cause for an exudate
although it may not be of value in differentiating between an exudate and a
transudate. Bacterial infections, malignancies, rheumatoid arthritis, and
tuberculosis will have a decreased concentration of glucose in an
exudatecompared to plasma .
7. Amylase
Amylase is also analyzed to find out the reason for the formation of an
exudate. Conditions involving the upper gastrointestinal tract like
pancreatitis, pancreatic malignancies and rupture of the oesophagus all
could give rise to an increase in amylase concentrations in the fluid.
122
10.3 Factors affecting fluid composition
There are two types of factors that affect fluid composition. They are
controllable factors and uncontrollable factors.
10.3.1 Controllable physiological factors
1. Posture
The blood volume of an adult in an upright position is 600-700mL less

compared to an adult in a recumbent position. This has an influence on the
plasma volume as only protein free fluid passes through capillaries to the
tissues and thus there is a greater reduction in plasma volume compared to
blood volume. Fluid reduction in plasma is associated with an increase in
plasma protein concentration and this has an effect on proteins as well as
analytes which are protein bound. There are associated changes in blood
volume when a person changes form a lying to a sitting position.
2. Immobilization
The plasma and extracellular fluid volumes decrease within a few days of a
person undergoing bed rest and thus the hematocrit may increase up to 10%
within four days. With prolonged bed rest serum protein and albumin
concentration may decrease by 5g/L and 3g/L respectively along with a
reduction on protein bound constituents. Prolonged bed rest gives rise to an
increase in urinary nitrogen, calcium, sodium, potassium and phosphate and
sulfate excretion.
123
3. Exercise
Moderate exercise increase the blood glucose concentrations, along with

pyruvate and lactate levels in plasma and reduces the arterial pH and pCO2 as
well as serum creatinine. Furthermore there is an increase in enzymes like
aspartate transaminase, lactate dehydrogenase and creatine kinase. In urine
there may be an increase in urates. Strenuous exercise will give rise to a
marked exaggeration of one’s listed above for moderate exercise as well as
an increase in concentration of plasma proteins. Hematuria and proteinuria
may occur in strenuous exercise and this is proportionate to the extent of
exercise.
4. Physical training
Serum concentrations of urea, urate, creatinine and thyroxine levels are
higher in athletes compared to other individuals. The total lipids are
decreased and high density lipoprotein cholesterol is increased – both being
desirable factors.
5. Circadian variation
Circadian variation refers to the cyclical variations that occur throughout the
day. Various factors like being awake or asleep, posture, activity, daylight
or darkness have an influence in circadian variation of blood constituents.
Some examples which should be kept in mind when drawing blood samples
for analysis include serum iron concentrations changing as much as 50%
between 0800 and 1200 hours and serum cortisol levels by the same
percentage between 0800 and 1600 hours. Some hormones are secreted as
bursts and this makes it very difficult to interpret their serum concentrations.
Even in urine there are differences in composition depending on diurnal
variation. For example urinary excretion of catecholamines and their
metabolites is less at night than during daytime.
124
6. Food
Serum concentrations of some plasma constituents are affected by food with
glucose being the classic example. Glucose concentrations will rise after a
meal along with iron, total lipids, and alkaline phosphatase. Foods can also
influence other constituents/ parameters such as hormones, pH, pCO2 etc.,
and these changes are dependent on the composition of the meal.
10.3.2 Other controllable factors
These include smoking, alcohol ingestion, ingestion of specific foods and

beverages (like caffeine, bran), drug administration and certain underlying
medical conditions.
10.3.4 Uncontrollable factors
1. Age
The total body water and the volumes of fluid in various body compartments
differ from infancy to adulthood and later. These will have an influence on
the concentrations of various analytes. For example blood glucose levels are
low in newborns because of their small glycogen reserves and serum
creatinine levels increase steadily from infancy to puberty. Adult values of
constituents in body fluids are usually taken as reference with which those
of young and elderly persons are compared in diagnosis of disease. In the
elderly for example, the renal concentration ability is reduced and creatinine
clearance may decline by as much as 50% between the third and the ninth
decade. Thus it is important to note the age of the persons whose samples
are being investigated and have age dependent reference values where
possible.
125
2. Gender
Boys and girls until puberty show little differences in laboratory analytes.
Yet after puberty, due to influences of the sex hormones certain constituents
like serum alkaline phospahatse, and aminotransferase are greater in women.
Other constituents like albumin, calcium and magnesium are higher in males
and hemoglobin is less in females.
3. Race
Certain changes in body fluid constituents are observed in different races.
For example serum protein levels are higher in blacks compared to whites.
4. Environmental factors
The hemoglobin levels are markedly increased in people living at high
altitude. An acute increase in environmental temperature cause plasma
volume to expand and this would lead to a decrease in plasma protein
concentration which could be by as much as 10%. On the other hand, if
there is excessive sweating associated with heat, hemoconcentration would
occur and the plasma protein concentration would increase along with some
other constituents.
5. Menstrual cycle
There are physiological changes associated with the menstrual cycle
accompanying changes in sex hormone and other hormone levels. For
example, plasma corticosterone levels are about 50% higher in the luteal
phase of the menstrual cycle compared to follicular phase. Some other
factors which may show changes in concentration with the menstrual cycle
include cholesterol (which shows the lowest levels at ovulation), total
protein, albumin and fibrinogen (which decrease at the time of ovulation).
126
6. Body habitus
In people who are obese there is usually an increase in the concentrations of
cholesterol, triglycerides, and β-lipoproteins. Similarly serum lactate
dehydrogenase and glucose concentrations have been found to be high in
obese people.
Activity 10.2
1. Describe the factors which can lead to variations of measured biochemical

parameters.
10.4 Synovial fluid analysis
Several pathological conditions are associated with synovial fluid can be

diagnosed by synovial fluid analysis.
10.4.1 Pathological conditions associated with Synovial fluid
Synovial fluid is present in the cavities of movable or synovial joints,

providing the nutrition to the vascular deficient cartilage. It is a viscous
liquid formed from ultra-filtrate of plasma. Except high molecular weight
proteins, concentration of most of the other chemical constituents is similar
to plasma concentration.
Diseases associated with synovial fluid are,
1. Non-inflammatory – Osteoarthritis
2. Inflammatory – Rheumatoid arthritis, Gout, Pseudo-gout
3. Septic – Microbial infection
4. Haemorrhagic - Traumatic injuries, Hemophilia
127
10.4.2 Specimen collection
 Collected via needle aspiration. Technique is referred as arthrocentesis.
 Normal synovial fluid volume is 3.5ml. It may be increased more than 25

ml during an inflammation.
 No clotting is seen unless there is a leakage of fibrinogen due to a

pathological condition.
 Synovial fluid is collected for,
Gram stain and culture - Sterile Heparin tube

Cell count - Heparin / EDTA
Biochemistry - Plain tube
Glucose - Sodium fluoride tube
10.4.3 Normal synovial fluid parameters
Volume <3.5ml
Colour Colourless to pale yellow
Clarity Clear
Viscosity Form 4-6cm string
WBC <200 cells/µl
Neutrophil <25% of differential count
Lymphocytes <15% of differential count
Crystal Absent
Glucose: plasma difference <10mg/dl less than blood
Total protein <3g/dl
128
10.4.4 Synovial fluid laboratory parameters in pathological

conditions
Following table (Table 1.2) describes the different pathological conditions

associated with synovial fluid.
Table 1.2. Pathological conditions associated with synovial fluid

Condition Appearance Cell count Glucose
Non- Clear WBC <1000µl Normal
inflammatory Yellow Neutrophil
<30%
Viscosity -
good
Inflammatory Cloudy WBC 2000- Decreased
75000 µl
Yellow
Neutrophil
Viscosity -
>50%
Poor
Gout Cloudy, Milky WBC up to Decreased
100000 µl
Viscosity -
Low Neutrophil
<70%
Septic Cloudy WBC 50000- Decreased
100000 µl
Yellow-green
Neutrophil
Viscosity-vary
>75%
Haemorrhage Cloudy WBC and Normal

Neutrophils –
Red
same as blood
Viscosity-Low
10.5 Laboratory analysis of synovial fluid

The appearance, microscopic analysis and chemical constituent analysis of
synovial fluid diagnosing the pathological conditions are discussed below.
129
10.5.1 Appearance
 Dark yellow – Non-inflammatory or Inflammatory conditions
 Greenish – Bacterial infection
 Reddish – Haemorrhage or trauma
 Turbidity – Due to WBC in an infection
 Milky – Due to crystals
10.5.2 Viscosity
 Due to polymerization of hyaluronic acid.
Techniques used to measure viscosity are:

1. String test – Measure the length of the string made at the syringe tip.
2. Measure hyaluronate polymerization with a mucin clot. In normal
synovial fluid, upon addition of 2-5% acetic acid, clotting occurs
surrounding a clear fluid.
10.5.3 Cell counts
WBC count
 Most frequently done on synovial fluid which uses Neubauer chamber

for counting
 Viscous fluid should be pre-treated by adding hyaluronidase or 0.05%

hyaluronidase in phosphate buffer to 0.5ml of fluid following incubation
at 370C for 5 minutes.
 RBC should be lysed using 0.3% hypotonic saline with saponin.
 If methylene blue is added, WBC will stain in blue allowing

differentiation of RBC from WBC.
130
Differential count
 Done on thin smears made from cytocentrifugation.
 Normal fluid consist of monocytes, macrophages, synovial tissue cells

and primary cells
 Pathological conditions may show bacterial cells, fungi, malignant cells

and cellular debris
Crystals
 Monosodium urate - Gout
 Calcium pyrophosphate - Psuedogout
 Cholesterol - Extracellular
 Corticosteroid - Injections
 Calcium oxalate - Renal dialysis
 Apatite (Ca phosphate) - Osteoarthritis
10.5.4 Chemical analysis of synovial fluid
Glucose
 Both blood and synovial fluid samples has to be taken after 8 hours of
fasting. This is to allow the equilibrium of glucose between two fluids.
 Analysis should be done within 1 hour from fluid collected to sodium

fluoride tube. This will prevent glycolysis and false negative values.
Protein
 High molecular proteins cannot be filtered from plasma into the synovial
fluid unless in case of a pathological condition. Therefore presence of
protein in synovial fluid should be <3g/dl. Increased values represent
inflammatory and haemorrhagic disorders.
131
Uric acid determinations

 Uric acid levels increases in gout and used especially in diagnosis of gout
in the absence of crystals.
10.6 Serous fluid
Serous fluid is the ultra-filtrate of plasma as a result of hydrostatic and

colloidal pressures exerted from the capillaries around the cavities. Due to
alterations in the above mechanisms, excess fluid fills in between the
membranes. This fluid is called as an effusion.
10.6.1 Underlying causes of forming an effusion
 High hydrostatic pressure - Congestive heart failure
 Decreased oncotic pressure - Hypoproteinemia
 Increased capillary permeability - Inflammation and infection
 Lymphatic obstruction - Tumours
10.6.2 Types of serous fluid found in the body
 Pericardial fluid
 Peritoneal fluid
 Pleural fluid
10.6.3 Specimen collection of serous fluids
 Fluid is collected from the cavities via needle aspiration.
 Process of collecting pleural fluid is known as thoracentesis.
 Pericardial fluid collecting procedure is known as pericardiocentesis
132
 Peritoneal fluid collection is known as paracentesis.
 Usually about 100ml of fluid is collected.
10.6.4 Specimen collection containers
 EDTA tube – For cell counts
 Heparin tube - Cytology, Microbiology, Biochemistry
 Plain tube - Biochemistry
Specimens analysed for pH must be maintained anaerobically in ice
10.7 Pleural fluid

Pleural fluid is found in the cavity between the parietal membrane covering
the chest wall and visceral membrane covering the lungs.
10.7.1 Laboratory diagnosis – Appearance
Normal pleural fluid – Clear and pale yellow
Infection - Turbid and white
Haemorrhagic Trauma, Malignancy - Bloody
Chylous/ Pseudochylous material - Milky
Rupture of amoebic liver abscess - Brown
Aspergillous - Black
Malignant mesothelioma - Viscous
10.7.2 Differentiation of chylous and pseudochylous effusions
The table 1.3 compare the differences between chylous and psudochylous
effusions in pleural fluid.
133
Table 1.3 Differentiation of chylous and pseudochylous effusions

Chylous Pseudochylous
Presence of material due Due to chronic

Occurrence
to thoracic duct leakage inflammations
Appearance Milky/ white Milky/ green
Cholesterol Low High content
High Low
Triglycerides
>110mg/dl <50 mg/dl
Leukocytes Mainly lymphocytes Mixed cells
10.7.3 Differentiation of pleural transudate and exudate
 Pleural fluid cholesterol >60mg/dl
 Fluid: serum cholesterol >0.3
 Fluid: serum total bilirubin>0.6 indicates that the pleural fluid is an

exudate.
10.7.4 Differential count
 Eosinophil >10% - Indicates trauma
 Neutrophils are significant in pneumonia, pancreatitis and pulmonary

infarction
 Lymphocytes are seen during tuberculosis, malignancy and viral

infections
 Mesothelial cells are normal and reactive forms have no clinical

significance.
 Plasma cells can be detected in tuberculosis.
134
 Malignant cells in metastatic carcinoma and other types of malignant

conditions
10.7.5 Biochemical analysis
Common chemical constituents are;
 Glucose
Since pleural fluid is an ultra-filtrate of plasma, both plasma and

fluid glucose should be measured parallel. If fluid glucose is <60mg/dl
when compared with plasma glucose, condition can be interpreted as
decreased glucose state associated with either tuberculosis, or rheumatoid
inflammation or purulent infections.
 pH
If pH<7.0, it indicates that patient needs a chest-tube drainage along with

administration of antibiotics in case of pneumonia. All the cases with pleural
fluid pH at least 0.3 less than blood pH is considered significant. pH<0.6
indicates oesophageal rupture due to influx of gastric juice.
 Lactate
Increased during bacterial infections
 Adenosine deaminase (ADA)
Normally high levels are shown in malignancy. In tuberculosis, levels

increase >40U/L.
 Amylase
Higher levels are seen during pancreatitis and amylase.
 Triglycerides
Differentiate chylous and pseudo-chylous effusions
135
10.8 Pericardial fluid
Pericardial fluid (10-50ml) is found in between pericardial serous

membranes.
Pericardial transudate is formed due to uraemia, hypothyroidism and

autoimmune disorders. Effusions are formed during pericarditis and
malignancy.
WBC counts have no significance on pericardial fluid analysis. However,

counts >1000 WBC/µL with increase in neutrophil count indicates a
bacterial endocarditis.
10.8.1 Laboratory analysis of pericardial fluid

Appearance
Clear and pale yellow - Normal fluid
Turbid - Infection
Turbid and bloody - Malignancy
Grossly bloody - Accidental cardiac puncture
Misuse of anticoagulation therapy
Milky - Chylous and psedo-chylous fluid
10.8.2 Chemical analysis of pericardial fluid
 Fluid: serum protein
 Lactic dehydrogenase is used to differentiate whether the pericardial

fluid is an exudate or transudate.
Other chemical constituents used in pericardial fluid diagnosis are,
 Adenosine deaminase
Significantly high in tubercular effusion
136
10.9 Peritoneal fluid
Accumulation of peritoneal fluid in between the peritoneal membranes is

referred as ascites. This fluid is also known as ascitic fluid.
10.9.1 Abdominal lavage
 Lavage is collected by introducing normal saline into the peritoneal

cavity
 Used for the detection of abdominal injuries and intra-abdominal

bleeding in trauma.
 Analysis of peritoneal lavage is a very sensitive test
 RBC count obtained from the peritoneal lavage can be used to determine
the need of a surgery for the patient in case of blunt trauma injuries.
(Count >100000/µl)
10.9.2 Differentiation of peritoneal exudate and transudate
Earlier, fluid: serum LD and protein levels were used to differentiate

peritoneal transudates and exudates. However, due to the difficulty in
assessing of peritoneal fluid from those parameters, serum-ascites albumin
gradient is recommended now.
Serum albumin – Fluid albumin = Gradient (Difference)
Gradient ≥ 1.1 Gradient < 1.1

Transudate of hepatic origin Exudate effusion
Eg: Cirrhosis Eg: Peritonitis, Malignancy
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10.9.2 Laboratory analysis of peritoneal fluid – Appearance
Clear, pale yellow - Normal
Turbid - Bacterial / Fungal infection
Greenish/ dark brown- Gallbladder or pancreatic disorders. Colour is

due to presence of bile
Bloody - Trauma, tuberculosis, intestinal disorders,

malignancy
Milky - Lymphatic trauma or blockage of lymphatic

vessels
10.9.3 Differential count of peritoneal fluid
Normal WBC level of peritoneal fluid is <350cells/µl. If count increases

more than 500 cells/ µl, it indicates the presence of bacterial peritonitis and
cirrhosis. Neutrophil count can be used to differentiate the above two
conditions. Neutrophil count >250 cells/ µl or >50% of the total WBC count
indicates that the condition is bacterial infection. In tuberculosis,
lymphocytes are predominantly found. Other cells that can be seen in fluid
are mesothelial cells, macrophages, lipophages, microorganisms and
malignant cells.
10.9.4 Chemical analysis of ascitic fluid
 Glucose
Decreased in cases of malignancy and tubercular and bacterial peritonitis
 Amylase
Increased in pancreatitis and gastrointestinal perforations
 Alkaline phosphatase
Increased in gastrointestinal perforations
 Blood urea nitrogen/ creatinine
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Increased in ruptured or punctured bladder
 Adenosine deaminase
Increased in tubercular peritonitis
Summary
Other than blood and urine, there are many fluids in the body cavity. They
are CSF, synovial fluid, amniotic fluid, pleural fluid, peritoneal fluid,
pericardial fluid, sweat and saliva. These fluids are essential for laboratory
diagnosis of certain disease conditions and also to determine the patient
management and prognosis. Blood should be collected with care to avoid
haemolysis because it affects the analysis of many chemical constituents in
blood. Further, correct blood collection tube must be used for relevant test.
Transudates are fluid formed due to systemic disorders as a result of an
imbalance between fluid filtration and reabsorption process whereas
exudates are fluid produces as a result of disruption of membranes around
the cavities. Both controllable and uncontrollable factors are affecting the
fluid composition. Synovial fluid collected form synovial joints are analysed
to diagnose conditions such as osteoarthritis, rheumatoid arthritis and
pseudo-gut, pericardial, peritoneal and pleural fluid are three types of serous
fluid found in the body.
Review Questions
1. Briefly describe the factors that influence the composition of blood?

2. Compare and contrast the transudate fluid and exudate fluid?
3. Briefly describe the biochemical analysis of pleural fluid?
4. Briefly describe the biochemical analysis of synovial fluid?
5. State the importance of peritoneal lavage?
6. Describe how you do the analysis of pericardial fluid?
139
Learning outcome
 Describe the blood collection procedures
 Describe the influence of blood collection
 Describe the composition of blood
 State the anticoagulants used in blood collection
 Explain the differences between transudate and exudate
 Explain the factors affecting body fluid composition
 Describe the specimen collection procedure of synovial fluid
 Explain the laboratory diagnosis of synovial fluid
 State the types of serous fluid and sample collection
 Describe the laboratory diagnosis of peritoneal fluid
References

140
Session [11]: Analysis of stool
Session 11
Analysis of stool
Content
Introduction, p141
11.1 Formation of stool, p142
11.2 Clinical conditions associated with alimentary canal, p143
11.3 Specimen collection, p144
11.4 Constituents of normal feces, p145
11.5 Laboratory analysis of feces, p145
11.6 Macroscopic examination and Osmolality analysis of stool, p147
11.7 Microscopic examination of feces, p148
11.8 Chemical analysis of feces, p151
References, p158
Introduction
A stools’ analysis is a series of tests done on a stool (or fecal) sample to help
diagnose certain conditions affecting the digestive tract. These conditions
can include infection (parasitic, viral or bacterial), poor nutrient absorption,
or cancer.
Analysis of stool samples may be required to
1. Help identify diseases of the digestive tract (e.g. trypsin and elastase
levels to evaluate pancreatic function)
141
2. Help find the cause of symptoms affecting the digestive tract (prolonged
diarrhea, blood and mucous diarrhea, flatulence, abdominal pain etc.)
3. Screen for cancers of the digestive tract by checking for occult blood
(e.g. colon cancer).
4. Help identify parasitic infections of the digestive tract (pinworms,
giardiasis etc.) and other infections (bacterial, viral).
5. Help identify whether there is any problem of absorption of nutrients by
the digestive tract, especially in suspected fat malabsorption.
The clinical biochemistry laboratory too may be called upon to perform

some of these tests. Some of these tests performed in the biochemistry lab
include testing feces for occult blood, testing feces for lactase deficiency
and examining feces for excess fat.
11.1 Formation of stool
Once food is ingested, proteins, carbohydrates and fats get digested

throughout the alimentary canal. Digestion of food in the small intestine is
carried out by digestive enzymes such as trypsin, chymotrypsin, amino
peptidase, lipase secreted by pancreas. Bile salts facilitate fat digestion.
However, oligosaccharides are digested only in lower intestine by bacterial
metabolism producing large amount of strong odour intestinal gas referred
to as flatus.
Daily there will be an entry of approximately 9000 ml of saliva, ingested

fluid, gastric, liver and pancreas secretions to alimentary canal, but only
500-1500 ml reach the intestine. Finally 150 ml will be excreted with feces.
142
11.2 Clinical conditions associated with alimentary

canal
Diarrhoea and steatorrhea are the common clinical conditions associated

with alimentary canal.
11.2.1 Diarrhoea
It is the condition where daily stool output increases over 200g with increase
in frequency of passing, more than 3 times per day. Amount of fluid passing
through feces is high in diarrhoea. Diarrhoea may be due to secretory
mechanism, osmotic or altered motility which will be differentiated by fecal
electrolytes, osmolality and pH.
Secretary diarrhoea
Water and electrolyte secretion will be greater than the reabsorption

capacity by large intestine. Usually caused by bacterial/ viral/ protozoan
infections, laxatives, inflammatory bowel disease, endocrine disorders and
cancers. Osmotic gap will be <50 mOsm/Kg.
Laboratory diagnosis: Stool culture, Ova and parasite examination,

Leukocytes, Rotavirus immunoassay.
Osmotic diarrhoea
Excessive watery stool will be passed due to increased water retention at

large intestine. This happens due to incomplete breakdown of food or
reabsorption of food which will result in increased fecal matter at the large
intestine. Usually caused by malabsorption, maldigestion, disaccharide
deficiency, poorly absorbed sugars, magnesium containing antacids,
amoebiasis and certain antibiotics. Negligible electrolyte passing. Osmotic
gap is >50mOsm/Kg. Malabsorption of sugars will be indicated by pH<5.6.
143
Laboratory diagnosis: Fecal fat, Muscle fibre analysis, Trypsin analysis,

clinitest, D-xylose and Lactose tolerance tests, Electrolytes, Fecal pH and
osmolality.
Altered motility
Refer to increased motility or decreased motility or constipation. In

constipation more water will be reabsorbed producing small volume of hard
stool. Usually due to irritable bowel syndrome and rapid gastric emptying.
11.2.2 Steatorrhea
Passing of increased amount of fatty stool due to failure in digesting dietary

triglycerides due to the absence of bile salts for pancreatic lipase action.
Usually seen in pancreatic diseases and small bowel disorders.
Laboratory diagnosis: Low D-xylose level in D-xylose tolerance test.
11.3 Specimen collection

 Should be collected to a clean, dry container such as bedpan or
disposable container and transfer to the plastic laboratory container with
screw capped lid.
 Should not contaminate with urine, toilet water or any disinfectants.
 Do not contaminate the outside of the container.
 The sample collection vessel should not be fully filled and the container
should be loosely capped. This is to allow the gases that build up within
the sample to freely escape.
 For quantitative tests like fecal fat and occult blood, sample is collected
for 3 consecutive days due to varying bowel habitat and transit time to
pass stool.
144
 The sample should be sent to the laboratory immediately on collection

and as warm stools are the best for parasitological analysis, it should not
be refrigerated if stools for ova and parasites are to be checked
 If infections are suspected, stools analysis should be performed before

starting antibiotic therapy.
 In selecting a sample to be analyzed in the laboratory, only a small

amount of stools should be selected and if mucous and blood are present,
the selected sample should include them as part of the sample to be
examined
11.4 Constituents of normal feces
1. Indigestible material in food.

2. Bile pigments and bile salts.
3. Epithelial cells shed from the intestine.
4. Secretions which originate in the intestine.
5. White blood cells that have come in from the blood stream.
6. Bacteria that colonize the intestine.
7. Calcium, phosphate and other inorganic material.
8. Small quantity of digested food.
In disease states involving the gastro-intestinal tract the composition of

feces may change with the quantity of the normal constituents increasing or
decreasing and abnormal constituents being found in a fecal sample.
11.5 Laboratory analysis of feces
In stools analysis macroscopic, microscopic and chemical examinations are

performed.
145
The parameters assessed as macroscopic and microscopic analysis and the

normal characteristics are listed below:
1. Colour – Brown, darkens on standing

2. Odour - varies with pH of stools and the bacterial action
3. Consistency –Varies with the diet of the person.
4. Size and shape – Formed
5. Gross blood – Should be absent
6. Mucous – Absent
7. Pus – Absent
8. Parasites – absent
9. Fat – Small amount of neutral fats may be present.
10. Undigested particles – Very small amounts only
11. Amoeba, ova and cysts – Absent
12. Yeasts – Absent
13. White blood cells – Absent
Thus a simple inspection alone of a stool sample may give indicators for the
diagnosis of diseases like parasitic infection, obstructive jaundice (pale
stools, decreased urobilinogen), diarrhoea, malabsorption etc.
The parameters assessed in the chemical analysis of stools samples and the
normal values are listed below:
1. pH – Neutral to weakly alkaline (this varies depending on age and the

food intake e.g. whether breast fed or not)
2. Osmolality – 290 mOsm/Kg
3. Occult blood – Negative
4. Urobilinogens – 50-300 mg/24 hours
5. Bile - Negative (may be positive in children)
6. Others – Porphyrins, Nitrogen, trypsin, lactose.
146
Activity 11.1
1. State the advices that should be given to a patient during specimen collection?
11.6 Macroscopic examination and Osmolality analysis

of stool
Colour and appearance are reported under macroscopic examination of

stool.
11.6.1 Colour
 Red - Lower gastrointestinal bleeding, Beetroot and food

colouring, Rifampin.
 Black - Upper gastrointestinal bleeding, Iron therapy,

charcoal. Bismuth antacids.
 Green - Biliverdin production due to oral antibiotics, green

vegetables and food colouring.
 Pale yellow - Bile duct obstruction, Barium sulphate containing

diagnostic procedures.
11.6.2 Appearance
 Bulky, frothy stool with foul smell, greasy and float like
Bile duct obstruction, pancreatic disorders
 Ribbon like/ slender
Intestinal constriction
 Mucus stool
Intestinal inflammation or irritation, Pathologic colitis, constipation
147
 Blood streaked mucus stool
Bacterial/ amoebic dysentery intestinal wall damage, malignancy
11.6.3 Fecal osmolality
Using fecal sodium and potassium concentration osmotic gap is calculated

to differentiate diarrhoeal mechanisms
Total fecal osmolality = ≈ serum osmolality 290mOsm/Kg
Osmotic gap = 290 – [2 (fecal sodium + fecal potassium)]
11.7 Microscopic examination of feces

Fecal leukocytes, faecal muscle fibres and faecal fat are reported under
microscopic examination of feces.
11.7.1 Fecal leukocytes
 Most common leukocyte is neutrophils
 Indicates the presence of ulcerative colitis, bacterial dysentery, bacterial

diarrhoea and infections in intestinal mucosa
 Viral and parasitic diarrhoea does not show leukocytes
 Over 3 neutrophils / HPF will indicate an invasive condition
 Wet smears stained with methylene blue or dry smears stained with gram
stain./ Wright’s stain can be used.
148
 Although methylene blue technique is rapid, analysis is not easy.

However, Wright’s stain gives permanent analysis of slide and gram stain
helps to identify gram positive/ negative bacteria
 Leukocytes in refrigerated or frozen specimens can be analyzed by

Lactoferrin latex agglutination test.
11.7.2 Fecal muscle fibres
 Striated fibres are seen in conditions associated with pancreatic

insufficiency, cystic fibrosis, biliary obstruction and gastro-colic fistulas.
Procedure:
 Analyse the specimen within 24 hours of collection. It is better to advice

patient to eat red meat in the diet prior to collection to enhance the fibre
visualization.
 Slide preparation is done by emulsifying small stool volume with 10%

alcoholic eosin stain.
 Observe for at least 5 minutes.
 Count the undigested, red stained fibres with preserved striations
 If count is >10, condition is pathological.
 Digested fibres do not have striations. However, undigested fibres have

both vertical and horizontal striations while partially digested fibres have
striations only in one direction.
Activity 11.2
1. Briefly discuss the importance of assessment of physical properties of faecal

specimens.
149
11.7.3 Fecal fat
 Analysed in the condition steatorrhea
 Observed for neutral fats (triglycerides), fatty acid salts (soaps), fatty
acids and cholesterol
 Slides are stained with Sudan III (routinely used), Sudan IV and Oil red
O.
Neutral fat stain
Neutral fats on microscopic examination are highly retractile, colorless, and

variable in size and shape with an oily look. Large orange-red oil droplets
can be seen near cover slip edge with a concentrated alcoholic solution of
Sudan III or a saturated solution of Oil red O in isopropanol. If a drop of
ethanol or diethyl ether is added, the fat globules will dissolve. If >60
droplets/HPF, it indicates the presence of steatorrhea.
Fatty acid crystals

Could be identified and differentiated from neutral fats by their needle like
colorless appearance and by not being stained by Sudan III or oil red 0.
They melt easily if gentle heat is applied to the preparation and dissolve in
ethanol and diethyl ether.
Soaps and fatty acids
Soaps in feces also form masses of needle-like colorless crystals. They can
be differentiated form fatty acid crystals because they do not melt with heat
and they do not dissolve in ethanol or diethyl ether unless first treated with
acetic acid.
Cannot be visualized directly with Sudan III stain. Before the slide is
prepared, sample should be treated with acetic acid followed by heating.
This procedure is called as split fat stain. Both free fatty acids and fatty
acids produced by hydrolysis of soaps and neutral fats can be examined and
150
both number and size of fat droplets should be considered. Normal feces
have at least 100 droplets with 4µm size. If 100 droplets with size 1-8µm is
counted, considered as slightly increased and 6-75µm as highly increased.
The presence of these three types of fats in feces may indicate different
disorders concerning the digestive tract.
e.g. - Presence of excess neutral fat globules in a person taking a normal

diet indicates lipase deficiency due to pancreatic disease.
- The presence of excess fatty acids and soapy fats in feces indicate
malabsorption.
Cholesterol
Specimens are heated first and followed by Sudan III stain and allowed to
cool before analysis.
Activity 11.3
1. Discuss the importance of assessment of macroscopic and microscopic

examination of stool samples.
11.8 Chemical analysis of feces
Chemical analysis for feces include occult blood, fat, fetal haemoglobin,
fecal enzymes and carbohydrates.
11.8.1 Fecal occult blood
Diagnosis: Iron deficiency anemia

Bleeding lesions of the gastrointestinal tract
Colorectal cancer
151
Normal stool can have blood up to 2.5ml. Bleeding more than 2.5ml / 150g
of stool is pathological. However, in some cases bleeding may not be visible.
In such cases, occult blood test is done.
Principle:
Reaction is based on pseudo peroxidase activity on hemoglobin. Along with
H2O2 and chromogenic compound (Guaiac), colorless compound is oxidized
to colored compound. Colour of oxidized compound may vary on
chromogen used and sensitivity of chromogens which is indicted in
increasing order gum guaiac> ortho-tolidine> benzidine.
pseudo peroxidase
Hemoglobin H2O2 Guaiac
Oxidize
Oxidized Guaiac + H2O

(Colored)
False positives : Haemoglobin and myoglobin in ingested red meat, fish,

aspirin and anti-inflammatory medications, horseradish, raw broccoli,
cauliflower, radish, turnip, melons, menstrual and haemorrhoid
contaminations
False negatives: Vitamin C>250mg/d and Iron supplements containing

vitamin C
 At least 2 samples from stool samples collected on 3 different days

should be tested before reporting as negative.
152
Patient preparation:
 Avoid taking red meat, horseradish, raw broccoli, cauliflower, radish,

turnip, melons, vitamin C and iron supplements containing vitamin C for
3 days prior to collection
 Avoid taking NSAIDs and aspirin for 7 days prior to collection
Other available techniques:
1. Commercial kit – Has guaiac impregnated filter paper. Stool sample and
H2O2 is added prior to testing. Specimen is obtained from center of sample
to avoid external contaminations. Blue colour is given if the test is positive.
Specimen applied on paper should be dried before analysis. Dried papers
can be kept for about 6 days.
2. Hemoquant fluorometric test for haemoglobin and porphyrin is more

sensitive and specific.
3. Hemoccult immunochemical test is more sensitive to lower GI tract

bleeding. Avoid many false positive contaminations.
11.8.2 Fecal fat
 Usually done to detect steatorrhea.
 Needs a 3 day specimen collected into a paint can if possible to make it

homogenous prior to analysis.
 Refrigerating the specimen prevents bacterial degradation.
 Patient should maintain a regulated fat intake of 100g/dl before and

during the collection period.
153
1) Van de Kamer titration
Principle:
Fecal lipids are converted to fatty acids by saponification. Liberated

fatty acids are titrated with NaOH.
Reporting:
Two reporting ways:
1. As grams of fat. Normal diet of 100g/dl intake will have 1-6g/dl fecal fat
2. Coefficient of fat retention per 24 hours. Normal is 95%.
Coefficient of fat = (dietary fat – fecal fat) × 100

Dietary fat
2) Steatocrit and acid steatocrit
 Rapid detection of fat excretion which is a gravimetric assay using a

microhematocrit technique.
 Useful in determining patient’s response to therapy and fecal fat in

paediatric patients.
 Required only a 72-hours sample collection.
3) Near infrared reflectance spectroscopy
 Rapid technique which requires only a small volume of stool collected

for 48-72 hours.
 Able to determine water and nitrogen content in faeces other than fat in
grams/ 24 hours.
154
11.8.3 APT Test for fetal haemoglobin
 Used to determine whether the blood in stool is either due to fetal blood
or maternal blood swallowed during delivery.
 Sample is centrifuged with water to release Hb from cells and treated

with NaOH. Alkali-resistant fetal Hb will gives a pink colour in the
supernatant after 2 minutes whereas maternal blood will get denatured
giving a yellow-brown colour.
11.8.4 Fecal enzymes
1) Trypsin
Gelatin test – When X-ray paper is inserted into the stool sample
emulsified in water, trypsin will digest the gelatin on paper leaving a
clear area. However, due to many false positives and bacterial
proteolytic actions the test is insensitive.
2) Chymotrypsin
Spectrophotometric analysis is done. More stable and sensitive than

trypsin. Indicate pancreatic insufficiency. Stable for 10 days at room
temperature.
3) Fecal Elastase I
ELISA technique is used for quantification. Enzyme is produced

specifically in pancreas. Therefore used to determine exocrine
pancreatic insufficiency and steatorrhea. Enzyme is not affected by
degradations or any motility disorders.
155
4) Lactase
Lactose is the main sugar found in milk and the enzyme lactase converts
ingested lactose into glucose and galactose. In persons with deficiency of
lactase, lactose is not broken down to its constituents i.e. digested and it is
not absorbed.
Lactose in the intestine ferments to lactic acid with the production of gas.
This causes abdominal pain and diarrhea which may be persistent and
severe.
Lactase deficiency may occur as a congenital defect in children (primary

lactase deficiency) or may be acquired in adults and children (secondary
lactase deficiency). When there is lactase deficiency, fecal specimens
usually contain lactose which on fermentation to lactic acid lower the pH.
The easiest method of detecting lactose in feces is by performing the

Benedict’s test. Compared to the method of performing the Benedict’s test
in urine samples, 8 drops of freshly passed fecal fluid specimen are used
instead of urine.
As mentioned above, lactase deficiency gives rise to an acidic pH

due to the presence of lactic acid. Thus the pH of the fecal sample could be
assessed by using a pH meter or narrow range pH papers in the diagnosis of
lactase deficiency.
Lactase deficiency is indicated if the stool sample has a pH less than

6.0 and a sugar level of ++ or more.
11.8.5 Carbohydrate analysis
156
 Done to determine osmotic diarrhea or celiac disease as there’s a high

carbohydrate content in feces
 Copper reduction test using clinitest tablets is commonly carried out to

detect fecal carbohydrates.
 Differentiate normal diarrhea and excretion of carbohydrates due to

action of parasites and viruses.
 Most important test in determining fecal carbohydrates in infants along

with pH determination is to detect lactase deficiency. Normal pH of stool
is 7-8. However, if pH is less than 5.5 it indicates presence of lactic acid
by bacterial fermentation oflactose.
 Sucrose is not detectable as it is not a reducing sugar.
 Stool chromatography is also available to detect unabsorbed

carbohydrates in stool, but not routinely used.
Summary
Stool analysis is important in finding the disorders associated with
alimentary canal, nutritional deficiencies and parasitic infections in the
patient. The common clinical conditions associated with alimentary canal
are diarrhea and steatorrhea. Stool is collected to wide mouthed, clean and
dry container with screw capped lid. Although many constituents are present
in the stool sample, only few are analyzed mainly in the biochemical
laboratory. Macroscopic analysis includes colour and appearance of stool. In
addition, fecal osmolality is tested. Microscopic analysis includes fecal
leukocytes, muscle fibres and fat. Chemical analysis includes fecal occult
blood, fat, enzymes, fetal hemoglobin and carbohydrates.
157
Review Questions
1. List the laboratory tests that one should perform to diagnose diarrhea?
2. What is the importance of fecal fat analysis?
3. How do you analyze the presence of fecal muscle fibers?
Learning outcome
 State the pathophysiology behind stool formation
 Describe the pathological conditions related to stool examination
 State the constituents of normal stool
 State the laboratory tests available in stool examination
 Describe the macroscopic examination of stool
 Describe the microscopic examination of stool
 Explain the principles, procedures and importance of chemical tests

available in stool examination
References

158
Session [12]: Disorders of reproduction in male and laboratory diagnosis
Session 12
Disorders of reproduction in males
and laboratory diagnosis
Contents
Introduction, p159
12.1 Formation of semen, p160
12.2 Specimen collection, p160
12.3 Constituents of normal serum, p162
12.4 Sperm morphology, p163
12.5 Seminal fluid analysis guidelines, p166
12.6 Laboratory tests, p168
12.7 Sperm separation methods, p173
References, p176
Introduction
When married couples presenting to infertility clinics are investigated, at
least 50% of couples are found to have a contributing male factor. Male
factor infertility can represent a variety of defects, which gives rise to
abnormalities in sperm number, morphology or function. Thus in the
investigation of male fertility, semen analysis is the most important
diagnostic tool in the initial evaluation. A freshly ejaculated semen sample
is necessary for semen analysis.
Although seminal fluid analysis provides an indication of male fertility, by
itself it does not give an indication on the ability to conceive. The reason for
159
Unit [5]: Disorders of Reproduction and Laboratory Diagnosis
this is that it does not assess important aspects of sperm function which is
the ability of the sperm to locate and penetrate an ovum. It is also important
to note that finding of semen of poor quality points to a reduced chance of
pregnancy and there is a possibility that natural conception may still occur
in some cases although the chances decrease as the severity of semen
defects increase.
Other than routine biochemical tests on semen, andrology laboratories
perform special tests like in-vitro fertilization, post-vasectomy semen
analysis and forensic analysis.
12.1 Formation of semen
Semen is fluid composed of four different compositions released from

seminal vesicles, epididymis, prostate glands and bulbourethral glands; 60-
70% of semen is from seminal vesicles, 20- 30% from prostate glands, 5%
from bulbourethral glands and 5% is spermatozoa.
Seminiferous tubule in the testes is composed of germ cells which produce
spermatozoa. With the nutrients released from Sertoli cells, spermatozoa
undergo spermatogenesis and immature sperms are produced. Later,
matured sperms with flagella at epididymis utilize fructose released from
seminal fluid as an energy source for sperm motility in the female
reproductive tract. Acid phosphatase, citric acid and proteolytic enzymes
helps coagulation and liquefaction of semen after ejaculation and mucus
released from bulbourethral glands reduces the vaginal and prostatic acidity.
12.2 Specimen collection
The highest sperm number with the best motility is observed in the first part
of the ejaculate. The proper assessment of semen quality is essential in the
diagnosis of several treatable disorders of male fertility.
160
 According to the WHO guidelines the semen sample should be collected

after a period of sexual abstinence (minimum of two days but not more
than 5 days of). In patients where more than one sample has to be
analyzed the number of days of sexual abstinence should be kept constant
for each analysis. Reason is that more days of sexual abstinence will
increase the volume and reduce the sperm motility.
 At least 2-3 samples should be analyzed in 2 weeks’ time for fertility

determination.
 The man should be given clear written and spoken instructions regarding
how to collect the sample and it should be emphasized that loss of any
fraction of the sample in the process of collection should be informed.
 The laboratory should have a private room nearby for the person to
collect the sample. Specimen is collected by masturbation. If not, non-
lubricant polyurethane condoms should be used.
 Should be collected to a wide mouthed, warm sterile glass/plastic

container and keep at room temperature. Should be transported to
laboratory for analysis within 1 hour of collection at room temperature. If
delayed, store at 370C.
 All details regarding the person and the collection of the sample should
be recorded and it should include the man’s name, birth date, reference
number, period of sexual abstinence, date and time of collection of
sample, the completeness of the sample, any difficulties in producing the
sample, and the interval between collection and the start of the semen
analysis.
The above precautions and procedures are necessary as the results of

laboratory measurements of semen quality depend on:
 Whether a complete sample is collected - During ejaculation the first

semen that is voided through the penis contain mainly sperm rich
prostatic fluids. Later fractions contain seminal vesicular fluid. Thus if a
161
man loses the first sperm rich portion of the ejaculate the sperm counts
may be lower than what they really are.
 The time since the last sexual activity - This is of importance as if there is
no ejaculation, spermatozoa accumulate in the epididymis and then
overflow into the urethra and are expelled in urine.
 The penultimate abstinence period - As the epididymis are not

completely emptied by a single ejaculation some spermatozoa may
remain within the epididymis form the last ejaculation and this will have
an influence on the quality of the spermatozoa as there will be a mixture
of aged and new spermatozoa in the next ejaculate.
 The size of the testes - It has been found that the size of the testes has an
influence on the number of spermatozoa per ejaculate and the size also
reflects the level of sperm producing activity, which affects sperm
morphology.
 Should treat the specimen as a highly infectious sample since it may be

from an individual with sexually transmitted diseases.
12.3 Constituents of normal semen
1. Volume : 2-5ml
2. Viscosity : Pours in droplets
3. pH : 7.2-8.0
4. Sperm concentration : >20 million/ml
5. Sperm count : >40 million/ ejaculate
6. Motility : >50% within 1hour
7. Quality : >2.0 – Slow forward progression and
Noticeable lateral movement
8. Morphology : >30% normal forms in routine analysis
9. Round cells : >1 million/ml
162
Secretions of seminal vesicles
 Fructose, which is present in seminal plasma, is the main energy source

of sperm cells.
 Prostaglandins involved in suppressing an immune response by the

female against the semen
 Others: Amino acids, citrate, enzymes, flavins, proteins and vitamin C
Prostatic secretions
 Zinc helps to stabilize the DNA-containing chromatin in the sperm cells.

Thus in zinc deficiency the sperms may become fragile and this could be
a reason for male infertility.
 Prostate specific antigen
 Acid phosphatase, citric acid, fibrinolysis, proteolytic enzymes
Bulbourethral glands
 Mucus creates a less viscous channel for the sperms to migrate in the
vagina and the cervix thus increasing the mobility of the sperm.
 Galactose
12.4 Sperm morphology
Normal and abnormal spermatozoa characteristics are stated below.
12.4.1 Normal spermatozoa characteristics
 Total length between 50-70µm.
163
 Head
Oval shaped head in 3-6 x 2-3µm in size. Head should be smooth and of
regular contour with a well-defined acrosomal region comprising 40-70% of
the head area. The acrosomal region should not have large vacuoles and if
small vacuoles are there, their number should be less than three and should
not occupy more than 20% of the sperm head. Post acrosomal region should
not have any vacuoles.
 A short middle piece
It is slender, regular and about the same length as the sperm head. The major
axis of the middle piece and the head should be aligned. Should not contain
excess residual cytoplasm (i.e. not more than 30%).
 A principle piece (tail)
This should have a uniform calibre along its length. Should be thinner than
the middle piece. Length about 10 times the head length (i.e. a principle
piece length of about 45 µm). May loop back on itself without a sharp angle
(a sharp angle usually indicates a flagella breakdown which impairs
mobility).
A normal sperm in seminal plasma shows rapidly progressive movement. In

a normal seminal fluid sample, abnormal spermatozoa should not exceed
20%.
12.4.2 Abnormal sperm characteristics
 Head defects
Increased or decreased size of head
Abnormalities in shape (tapered, pyriform, round)
Two heads
164
Unevenly distributed chromatin in the head
Presence of vacuoles in head (more than two vacuoles or >20% of the head
area occupied by unstained vacuolar areas)
Vacuoles in the post-acrosomal region
Small or large acrosomal areas (<40% or >70% of the head area)
 Middle piece or neck defects

Absence of middle piece
Increased size
Bifurcated middle piece
Asymmetrical insertion of the middle piece into the head
Thick or irregular
Sharply bent
Abnormally thin
 Principle piece defects (Tail)

Absence of tail
Marked reduction in the length
Double/multiple tail
Broken tail
Smooth hairpin bends
Sharply angulated bends
Irregular width
Coiled middle piece
 Presence of excess residual cytoplasm
The morphology of abnormal spermatozoa can be seen in figure 12.1
165
Figure 12.1: Spermatozoa showing abnormalities in morphology
(Source: https://images.app.goo.gl/nePgwxnCwVSoMrip7)
12.5 Seminal fluid analysis guidelines
The steps in the seminal fluid analysis and the time frames in which they
should ideally be done according to the WHO guidelines are listed below:
a) During the first 5 minutes:

Placing the specimen container on the bench or in an incubator (37 °C) for
liquefaction.
b) Between 30 and 60 minutes:

 Assessing liquefaction and appearance of the semen.
Semen is a thick gel at the time of ejaculation and normally becomes liquid
within 30-60 minutes after ejaculation. Semen should liquefy before
analysis. Liquefaction time is a measure of the time it takes for the semen to
166
liquefy. If semen has not liquefied within 2 hours, alpha-chymotrypsin

proteolytic enzyme is added before analysis. This liquefaction delayed time
indicates that there is a prostatic enzyme deficiency.
Appearance of normal semen is typically translucent with white, grey or

even yellowish tint. Blood in the semen can cause a pink or reddish color
and may indicate an underlying problem which needs medical evaluation.
Turbid nature is due to the presence of WBCs during an infection. Yellow
coloration indicates the urine contamination, but the toxicity of urine affects
sperm motility.
 Measuring semen volume
 The average volume of semen produced at ejaculation is 2-5 ml.

Prolonged abstinence results in higher volumes and low volumes indicate
infertility.
 Measuring semen pH
 Measuring semen viscosity
Normal semen when released from pipette, forms a thin string. If this string
is >2cm, semen has high viscosity. Then, liquefaction is incomplete.
 Preparing a wet preparation for assessing microscopic appearance, sperm

motility and the dilution required for assessing sperm number.
 Assessing sperm vitality (if the percentage of motile cells is low).
 Making semen smears for assessing sperm morphology.
 Making semen dilutions for assessing sperm concentration.
 Assessing sperm number.
 Performing the mixed antiglobulin reaction (MAR) test (if required).
 Assessing peroxidase-positive cells (if round cells are present).
 Preparing spermatozoa for the immunobead test (if required).
167
 Centrifuging semen (if biochemical markers are to be assayed).
c) Within 3 hours:
Sending samples to the microbiology laboratory if microbiological
assessment is required.
d) After 4 hours:
Fixing, staining and assessing smears for sperm morphology.
e) Later on the same day (or on a subsequent day if samples are frozen)
 Assaying accessory gland markers (if required).
 Performing the indirect immunobead test (if required).
Activity 12.1
1. Briefly describe the information which can be gathered from assessment of pH and
viscosity of seminal fluid.
12.6 Laboratory tests
Routine analysis - volume, viscosity, pH, fructose, measurement of sperm

concentration, count, motility, viability, and morphology
Special tests - sperm autoantibodies, cervical mucus penetration, the

acrosomal reaction test, and computer assisted sperm analysis (CASA) etc.
1. For WBCs
Leukocyte-esterase reagent strip is used as screening test
168
2. Sperm concentration/ count
Total sperm count for ejaculate = sperm concentration × specimen volume
Total sperm count > 40 million/ ejaculate is considered as normal.
Sperm concentration is done using a Neubauer chamber. Semen dilution is

1:20 with sodium bicarbonate and formalin to immobilize and preserve the
sperms. Sperms are counted in the four corner squares and 5 small squares
in the large centre square just as in RBC counting. Usually average of two
counts is taken.
Sperm concentration/ml = Number of sperms in 5 squares in large centre

square × 1000000
Sperms are counted using phase or bright field microscope. Addition of

crystal violet stain to diluting fluid will help clear visualization of sperms
under bright field microscopy.
Count only mature sperms. Immature sperms (round cells) and leukocytes
are not counted along with mature sperms. If immature sperm count is >
1million/ml, indicates incomplete spermatogenesis either due to viral
infection, genetic abnormality or toxicity. If WBC count is >1million/ ml,
inflammation or infection is there affecting the fertility.
3. Sperm motility
Procedure:
 Within 1 hour collection, place a 10µl of sample on a clean glass slide,
cover with a coverslip and observe under phase contrast microscope at
x200 (or x400) magnification.
 Observe 20 HPF to calculated sperm percentage with actual forward

movement. Motility is determined by WHO grading system which
includes both speed and motility direction ( 0 – 4 system)
169
 Computer assisted semen analysis is a modified technique which replace

the manual technique providing speed and direction of motility,
concentration and morphology.
Types of sperm motility observed in a motility assay
 Progressively motile (PR)
Spermatozoa moving actively, either linearly or in a Large circle (swimming

in small circles not counter under PR), regardless of speed.
 Non-progressive motility (NP)
Patterns of movement which does not show any progression, e.g. swimming
in small circles, the flagella force hardly displacing the head, or when only a
flagella beat can be observed.
 Immotile (IM)
No sperm motility at all.
4. Sperm morphology
Procedure:
 Prepare a thin smear of semen
 Stain with Wright’s, Giemsa or Papanicolaou stain
 Air dry the smear
 Observe under oil immersion within 24 hours.
 Observe for approximately 200 sperms and get the percentage of normal
and abnormal sperm count.
 In a normal male, normal sperm count should be >30%
From this procedure, number of leukocytes or spermatids (round cells) too

can be calculated.
170
Leukocyte / Spermatid concentration / ml = (N × S) / 100
Where, N = Leukocyte or spermatid count / 100 mature sperm
S = Sperm concentration (106/ml)
5. Sperm viability
This parameter is checked sperm concentration is normal, but motility is

markedly low.
Procedure:
 Mix the semen specimen with eosin-nigrosin stain
 Prepare a thin smear
 Living cells are bluish-white colour, dead cells are red in a purple
background
 Calculate the number of dead cells in 100 sperms
 Normal viability should be 75%
6. Fructose concentration
Usually tested when there is a low sperm count as a result of decreased
seminal vesicle support medium.
Procedure:
 Sample should be tested within 2 hours. If delayed, should be frozen to

prevent fructolysis.
 Mix 1ml of semen with 9ml of resornicol-conc. HCl solution.
 Boil the solution
 Orange colouration will appear in the presence of fructose.
 Quantitative determination could be done by spectrophotometry
 Normal fructose concentration should be 13µmol/ ejaculation
171
7. Antisperm antibodies
Usually tested in semen, serum and cervical mucosa in infertility cases.

Generally tested for males.
In male routine serum analysis, if sperm clumping is observed it indicates

the presence of antisperm antibodies in male semen. This results in
decreased sperm motility.
If infertility is observed even with normal semen analysis, presence

antisperm antibodies in females are tested. Semen is mixed with female
cervical mucosa or serum and clumping is observed.
Available rapid tests are: Mixed agglutination reaction – Detect IgG
Immunobead tests – Detect IgG, IgM and IgA
Activity 12.2
1. Briefly describe how antisperm antibodies affect the fertility.
8. Other biochemical tests
Table 12.1 Other biochemical tests done on semen
Test Abnormality Normal range
Neutral α-glucosidase Disorder in epididymis ≥20 mU/ ejaculate
Zinc ≥2.4 µmol/ ejaculate
Citric acid ≥52 µmol/ ejaculate

Lack of prostatic fluid
Prostatic Acid
≥200 Units/ ejaculate
Phosphatase
172
9. Post vasectomy semen analysis
Analysed for viable (Motile) sperms using wet preparation observed under
phase microscope
10. Sperm functional tests
Tested for functional ability of sperms using special techniques such as:
Hamster egg preparation, cervical mucus penetration, Hypo-osmotic
swelling and in-vitro acrosome reaction.
12.7 Sperm separation methods
With the advent of assisted reproductive techniques, the need to separate,

select and enrich motile and functionally competent spermatozoa from the
ejaculate has become an important and routine part of work in the
reproductive laboratory.
Some of the more common methods of sperm separation are: Classical swim
up, migration sedimentation, density gradient centrifugation and glass wool
filtration.
12.7.1 Classical swim-up
Swim-up technique is the standard technique of sperm separation for

patients with normal sperm counts and motility (normozoospermia) and
female infertility. Excellent fertilization rates have been observed when
sperm preparations using the swim-up technique were used to inseminate
human oocytes in vitro.
In this method specific medium is gently layered over semen in a sterile
conical-based centrifuge tube. The tube is inclined at an angle of 45º and
incubated for 1 hour at 37 °C. The tube is then gently returned to the upright
position and the upper layer containing the motile cells removed. This upper
173
layer is then diluted with a large volume of medium, centrifuged at 500g for
5 minutes, and finally suspended in 0.5 ml of culture medium. This is then
used in the artificial reproduction techniques.
12.7.2 Migration-sedimentation
The migration sedimentation technique of sperm separation is a swim-up

technique combined with a sedimentation step. In this technique, special
glass or plastic tubes with an inner cone are used and the spermatozoa swim
up directly from liquefied semen into the supernatant medium and
subsequently sediment in that inner cone within an hour's time.
12.7.3 Density gradient centrifugation
In density gradient centrifugation, the ejaculate is placed on top of a density

media with higher density and is then centrifuged for 15–30 minutes. At the
end of centrifugation all semen will sediment. Then the highly motile
spermatozoa move actively in the direction of the sedimentation gradient
and can therefore penetrate the boundary quicker than poorly motile or
immotile cells. These highly motile sperm cells are concentrated in the soft
pellet at the bottom.
12.7.4 Glass wool filtration
In glass wool filtration, motile spermatozoa are separated from immotile

sperm cells by means of densely packed glass wool fibres. The motile
sperms separate themselves from the immotile sperms and cell debris by the
self-propelled movement of the spermatozoa and the filtration effect of the
glass wool
174
Summary
Semen is the collection of fluid from seminal vesicles, epididymis, prostate
glands and bulbourethral glands. The highest sperm number with the best
motility is observed in the first part of the ejaculate. The proper assessment
of semen quality is essential in the diagnosis of several treatable disorders of
male fertility. Semen is collected to a wide mouthed, warm sterile
glass/plastic container and keep at room temperature. Spermatozoa has 3
main sections; head, short middle piece and tail. Abnormal morphology in
these parts will form inactive forms in varying degrees. The steps in the
seminal fluid analysis and the time frames are described under WHO
guidelines. Routine analysis of semen include volume, viscosity, pH, and
fructose, measurement of sperm concentration, count, motility, viability, and
morphology. Special tests for semen include sperm autoantibodies, cervical
mucus penetration, the acrosome reaction test, and computer assisted sperm
analysis (CASA). Other common tests for semen are WBC, sperm count,
sperm motility, sperm morphology, sperm viability, fructose concentration,
antisperm antibodies, post vasectomy semen analysis and sperm functional
tests. Additional biochemical tests include zinc, citric acid, prostatic acid
phosphatase and neutral alpha glucosidase. Sperm separation can be done
by classical swim up, migration sedimentation, density gradient
centrifugation and glass wool filtration methods. Progressive motility, non-
progressive motility and immobility is checked under sperm motility.
Review Questions
1. Describe the semen collection procedure?

2. Describe the normal morphology of sperm?
3. List the sperm concentration techniques?
4. State the WHO guidelines on semen analysis?
5. List the biochemical analysis parameters in semen and the expected diagnosis?
175
Learning outcome
At the end of the session student should be able to
 State the pathophysiology behind semen formation
 Describe the structure and function of sperm
 State the constituents of normal semen
 Describe the abnormal morphology of sperm
 Describe the specimen collection procedure of semen
 State the WHO guidelines on semen analysis
 Describe the laboratory test, their procedures and expected diagnosis
 State the sperm separation methods
References

176
Session [13]: Disorders of Reproduction in Female and Laboratory Diagnosis
Session 13
Disorders of Reproduction in Female
and Laboratory Diagnosis
Content
Introduction, p177
3.1 Disorders of reproduction in female, p178
3.2 Factors causing female infertility, p178
3.3 Laboratory Diagnosis of Disorders in Reproduction in Females, p180
3.4 Analytical Methods, p182
Summary, p184
References, p185
Introduction
The female reproductive system consists of vagina, uterus, fallopian tubes

and ovaries. The ovaries are responsible for the production of ova and
secretion of female sex hormones namely, estrogens and progesterone.
These hormones are steroid derivatives. Steroids that feminize are classified
as estrogens. In adult females, menstruation is regulated by a tightly
controlled feedback system exists between the hypothalamus, anterior
pituitary and ovaries. The gonadotrophic releasing hormone secreted by
hypothalamus, stimulates the release of follicular stimulating (FSH) and
luteinizing hormone (LH) from the anterior pituitary. The FSH stimulates
follicular growth whereas LH stimulates ovulation and progesterone
secretion from the developing corpus luteum.
177
3.1 Disorders of reproduction in female
Infertility is defined as the inability to conceive after 1 year of unprotected,

regular sexual intercourse. It can be due to either male infertility factors or
female infertility factors. Infertility problems commonly arise as a result of
hormonal dysfunction of the hypothalamic-pituitary-gonadal axis. Therefore
measurement of serum levels of both peptide and steroid hormones related
to reproduction are essential in the evaluation of infertility. Apart from those
psychosocial and immunological factors also common. Anti-sperm
antibodies may be present in female serum, in cervical mucus or in seminal
fluid.
3.2 Factors causing female infertility

Ovulatory disorders are the commonest causes for infertility in females.
However, most of ovulatory problems can be treated successfully with
medications. Additionally, uterine, fallopian tube defects and cervical
problems also responsible for reproduction problems associated with
females (Table 13.1).
Table 3.1 Factors causing female infertility

(Source: Burtis, C. A., Ashwood, E. R., Bruns, D.E. 2006. Teitz’s textbook of
clinical chemistry and molecular diagnostics)
Ovarian or hormonal factors
Disorders of hormonal metabolism
 Thyroid
 Liver
 Obesity
 Androgen excess
 Polycystic ovarian syndrome (PCOS)
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Hypergonadotropic hypogonadism
 Menopause
 Luteal phase deficiency
 Gonadal dysgenesis
 Premature ovarian failure (autoimmune, cytotoxic chemotherapy,
tumor)
 Resistant ovary syndrome
Hypogonadotropic hypogonadism
 Hyperprolactinemia (tumor, drugs)

 Hypothalamic insufficiency (Kallmann syndrome)
 Pituitary insufficiency (tumor, necrosis, thrombosis, stress,
exercise, anorexia)
Uterine factors
 Leiomyomata
 Benign polyps/tumors
 Adhesions
 Endometriosis
 Uterine abnormalities/congenital abnormalities of the uterus
Cervical factors
 Stenosis
 Inflammations/infections
 Abnormalities in mucus viscosity
Tubal factors
 Occlusion or scarring
 Salpingitis isthmica nodosa
 Infectious salpingitis
Psychosocial factors
 Decreased libido
 Anorgasmia
Immunological reactions (Producing anti-sperm antibodies)
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3.3 Laboratory Diagnosis of Disorders in Reproduction

in Females
Endocrine parameters and ovulation parameters are evaluated.
3.3.1 Evaluation of ovulation
Progesterone measurement
The measurement of serum progesterone concentration is regarded as the

primary assay of ovulation. At the beginning of ovulation, serum
progesterone concentration rises, and peak within 5-9 days during the
midluteal phase (usually between days 21 to 23, if the woman has a 28-day
cycle). If ovulation does not happen, expected rise in progesterone
concentration is subnormal as corpus luteum fails to form. When the
pregnancy occurs, corpus luteum will exists and progesterone production
will be increased. However, it is important to note that sometimes, increase
in progesterone level may not confirm that the egg was actually release (but
can confirm that the corpus luteum has been formed).
A progesterone level (mid-luteal) >10 ng/mL is evidence of ovulation,

whereas concentrations <10 ng/mL suggests, anovulation, inadequate luteal
phase production or inappropriate timing of sample collection.
Basal Body Temperature
Although the basal body temperature charts have been practiced as a simple
and cost-effective indicators of ovulation, it is also suggested that basal
body temperature measurements is cumbersome and does not predict the
exact time of ovulation. However, when the ovulation occurs, there is a
rapid rise in body temperature, by 0.5 °F, which then persists through the
luteal phase. The increased progesterone level causes the body temperature
to be increased.
180
Measurement of the Luteinizing hormone Surge
The Luteinizing hormone appears in the urine just after the serum LH surge
and also 24-36 hours before the ovulation. Although the measurement of LH
unable to confirm the presence of ovulation or cause of anovulation, it can
provide a better guide with which to time intercourse.
Most of the ovulation kits consisted of monoclonal technology which are

ready to use (dipsticks) even at home with effective prediction of ovulation
in 70 % women.
3.3.2 Evaluation of Endocrine Parameters

Hypergonadotropic hypogonadism
Hypergonadotropic hypogonadism is characterized by hypogonadism due to

impaired response of gonads to gonadotropins (FSH and LH). As a result
there will be lack of sex steroid hormone production and elevated
gonadotropins later as a body compensator mechanism.
A disorder of the ovary, premature ovarian failure (POF) is indicated by

repeatedly elevated basal FSH concentrations (>30 IU/L) or single elevation
of FSH >40 IU/L. The measurement of basal serum concentration of FSH
also performed a as an indicator of relative ovarian age. When the serum
FSH concentration increases, the rate of successful pregnancies decreases.
Hypogonadotropic hypogonadism
Hypogonadotropic hypogonadism is a form of hypogonadism that is due to

a problem with the pituitary gland or hypothalamus. When the
hypothalamus is affected, there will be abnormalities of gonadotropin
releasing hormones, while pituitary gland problems will cause deficiencies
of gonadotropins from the anterior pituitary. In this condition, there will be
decreased serum estradiol (<40 pg/mL), LH (<10 IU/L) and FSH (<10 IU/L)
levels. Hyperprolactinemia also leads to hypogonadotropic hypogonadism.
181
Activity 13.1
1. Briefly describe how measurement of reproductive hormones of female can useful

in diagnosis of reproductive disorders of females
3.4 Analytical Methods
There are various methods available for measurement of female

reproductive and related hormones.
3.4.1 Estrogen
Specimen collection and storage

 Specimen – Serum/plasma (EDTA or heparin can be used as anti-
coagulants)
 Storage – samples should be centrifuged and separated within 24 hours

of collection. Samples can be stored for 24 hours at 4 °C or frozen for 1
year.
 Precautions - Administration of steroids, adrenocorticotropic hormone

(ACTH), gonadotropin or estradiol medications should be avoided within
48 hours of sample collection.
Methodology
a) Chromatographic methods
Gas chromatography–mass spectrometry (GC-MS) methods provide the

most accurate and reliable results in estradiol.
b) Immunoassay methods
Most of the laboratories have substituted with immunoassay methods which

are easier and faster than chromatographic methods. Both indirect and direct
immunoassay methods are available. Direct immunoassays do not need
182
extraction whereas, indirect immune assays required an extraction. The

commonest antigen used to prepare estradiol antibodies is estradiol-6-oxime,
which is conjugated to bovine serum albumin.
3.4.2 Progesterone
Specimen collection and storage

 Specimen – Serum/plasma (EDTA or heparin can be used as anti-
coagulants).
 Storage – Serum should be separated within 24 hours. Samples can be

refrigerated for up to 3 days at 4 °C to 8 °C or up to 1 years at 20 °C.
 Precautions – Patients should not be on corticosteroids, ACTH, estrogen

or gonadotropin medications for at least 48 hours of sample collection.
Methodology
Gas chromatography–mass spectrometry (GC-MS), double isotope
derivative methods, competitive protein binding assays, immunoassay
methods are used for the estimation of serum progesterone levels. Among
these, double isotope derivative method and competitive protein binding
assays requires extensive purification of the progesterone and also labor
intensive. GC-MS procedures also time consuming and requires extraction
of progesterone, chromatography initially. However, GC-MS is regarded as
a reference method for progesterone estimation. Majority of progesterone
assays in clinical laboratories nowadays have been replaced by
immunoassays using steroid specific antibodies.
3.4.3 Gonadotropins (Follicle-Stimulating hormone, Luteinizing

Hormone)
Gonadotropins are measured by immunoassay methods which are developed

to assay FSH and LH in blood and urine. Commercial kits are available for
either for manual and automated testing. However, most of the
183
immunoassay methods to measure FSH and LH show cross-reactivity with

Thyroid stimulating hormone or human chorionic gonadotropin.
Summary
Infertility can be due to male or female factors. Variety of female factors are
responsible for female infertility, such as ovarian/hormonal factors, uterine
factors, cervical factors, tubal factors, psychosocial factors and
immunological factors. Selection of laboratory investigations related to
reproduction depends on the factors leading to infertility. Basic laboratory
investigations include evaluation of ovulation and evaluation of endocrine
hormones.
Learning Outcomes
At the end of the session the student should be able to,
 Explain the female factors of disorders in reproduction.
 List and explain the laboratory diagnosis related to disorders of

reproduction in females.
 Explain the analytical methods of steroid and peptide reproductive

hormones.
Review Questions
1. Briefly explain the female factors of disorders in reproduction.

2. List the analytical methods available for the estimation of steroid hormones
associated with reproduction.
184
References
Burtis, C. A., Ashwood, E. R., Bruns, D.E. 2006. Teitz’s textbook of clinical
chemistry and molecular diagnostics. 6th Edition. Missouri. Saunders.
185
186
MDU4303 Block 1 Contributors: Session

Authors and Content Editors
Authors
Ms. N.D. Withanage - Session 13

Dr. D.I. Uluwaduge - Session 2, 3, 4
Dr. A.M.B. Priyadarshani - Session 5, 6, 7, 8
Ms. B. Yasassri Alvitigala - Session 9, 10, 11, 12
Ms. R.G.L. Rathnayake - Session 1
Content Editor
Professor. R. Shivakaneshan - Session 1 - 13
187
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FACULTY OF HEALTH SCIENCES

Course Team Chair Educational Technologist

Authors Desktop Publisher

Ms. N.D. Withanage Ms. B. Y. Alvitigala

The Open University of Sri Lanka

© 2019 The Open University of Sri Lanka

The course team members of MDU4303 – Clinical Biochemistry II is

Introduction to the Course

Welcome to MDU4303 Clinical Biochemistry. This level 4 course is one of

Structure of the Course

Day Schools 1 − Session 01− 04

Your overall performance during the course will be determined by your

Continuous Assessment - 40%

Introduction to the Course iv

Introduction to the Units 1

Unit 1: Disorders of carbohydrate metabolism and laboratory diagnosis

Session 1 Disorders of carbohydrate metabolism 5

Session 2 Diabetes Mellitus 23

Session 3 Blood Glucose determination and diagnosis of Diabetes 33

Session 4 Complications of Diabetes and laboratory diagnosis 43

Unit 2: Disorders of Lipid metabolism and laboratory diagnosis

Session 5 Disorders of Lipid and Lipoprotein metabolism 51

Unit 3: Serum / Plasma Protein

Session 7 Changes of protein levels in disorders 69

Unit 4: Other body fluids

Session 9 Analysis of Cerebrospinal fluid (CSF) 91

Session 10 Analysis body cavity fluids 113

Session 11 Stool analysis 141

Unit 5: Disorders of reproduction and laboratory diagnosis

Session 12 Disorders of reproduction in male and laboratory diagnosis 159

Session 13 Disorders of reproduction in female and laboratory diagnosis 177

Introduction to the Units

Disorders of carbohydrate metabolism and laboratory diagnosis will be discussed

 State the clinical significance of disorders of lipid and lipoprotein

Unit III is focused on Serum / Plasma Protein. Changes of protein levels in

 Draw and label the normal serum electrophoretic and densitometry

states, acute phase protein response pattern, hyperlipoproteinemia, chronic

Laboratory procedures including specimen collection and investigation methods of

 Describe the laboratory diagnosis of pleural fluid

Disorders of reproduction and laboratory diagnosis will be discussed in unit V. The

Disorders of Carbohydrate Metabolism

Disorders of carbohydrate metabolism occur in many forms. The

Inborn Errors of Metabolism (IEM) comprise a group of disorders in which

1.1 Disorders of galactose metabolism

Galactosaemia is a disorder in which the body cannot catabolize galactose

There are three separate disorders of galactose metabolism of clinical

2. Galactose-1-phosphate uridyl transferase deficiency

3. UDP galactose-4-epimerase deficiency

1.1.1 Galactokinase deficiency

This deficiency causes the elevation of galactose in blood and subsequent

1.1.2 Galactose-1-phosphate uridyl transferase deficiency

Classical galactosaemia is caused by this deficiency and it is the most

Here the trapping of inorganic phosphate as galactose 1 P leads to a

Figure 1.1 Galactose metabolism

1.1.3 UDP galactose-4-epimerase deficiency

Deficiency of GALE is the rarest condition among above discussed 3

 Assays for galactitol and galactose-1-phosphate

 Assessment of enzyme activities of GALT, GALK and GALE

1.2 Disorders of fructose metabolism