Essentials

of
Clinical Pathology

G
R
dV
te
ni
-U

ir
s.
ns
9
ir9

ia
rs
h
ta

e
p .p
vi

tahir99 - UnitedVRG
vip.persianss.ir
 G
R
dV
te
ni
-U

ir
s.
ns
9
ir9

ia
rs
h
ta

e
p .p
vi

tahir99 - UnitedVRG
vip.persianss.ir
 Essentials
of
Clinical Pathology

G
R
dV
te
Shirish M Kawthalkar
Associate Professor
ni
Department of Pathology
Government Medical College
-U

ir
Nagpur, Maharashtra, India

s.
ns
9
ir9

ia
rs
h
ta

e
p .p
vi

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD

Nagpur • St Louis (USA) • Panama City (Panama) • London (UK) • New Delhi • Ahmedabad
Bengaluru • Chennai • Hyderabad • Kochi • Kolkata • Lucknow • Mumbai

tahir99 - UnitedVRG
vip.persianss.ir
 G
Published by
Jitendar P Vij

R
Jaypee Brothers Medical Publishers (P) Ltd
Corporate Office

dV
4838/24 Ansari Road, Daryaganj, New Delhi - 110002, India,
Phone: +91-11-43574357, Fax: +91-11-43574314
Registered Office
B-3 EMCA House, 23/23B Ansari Road, Daryaganj, New Delhi - 110 002, India
Phones: +91-11-23272143, +91-11-23272703, +91-11-23282021 te
ni
+91-11-23245672, Rel: +91-11-32558559, Fax: +91-11-23276490, +91-11-23245683
e-mail: jaypee@jaypeebrothers.com, Website: www.jaypeebrothers.com
-U

ir
Offices in India
•
•
Ahmedabad, Phone: Rel: +91-79-32988717, e-mail: ahmedabad@jaypeebrothers.com
Bengaluru, Phone: Rel: +91-80-32714073, e-mail: bangalore@jaypeebrothers.com
s.
ns
• Chennai, Phone: Rel: +91-44-32972089, e-mail: chennai@jaypeebrothers.com
9

• Hyderabad, Phone: Rel:+91-40-32940929, e-mail: hyderabad@jaypeebrothers.com

ir9

• Kochi, Phone: +91-484-2395740, e-mail: kochi@jaypeebrothers.com

ia

• Kolkata, Phone: +91-33-22276415, e-mail: kolkata@jaypeebrothers.com

• Lucknow, Phone: +91-522-3040554, e-mail: lucknow@jaypeebrothers.com
rs
h

• Mumbai, Phone: Rel: +91-22-32926896, e-mail: mumbai@jaypeebrothers.com

• Nagpur, Phone: Rel: +91-712-3245220, e-mail: nagpur@jaypeebrothers.com
ta

Overseas Offices
.p

• North America Office, USA, Ph: 001-636-6279734, e-mail: jaypee@jaypeebrothers.com, anjulav@jaypeebrothers.com

• Central America Office, Panama City, Panama, Ph: 001-507-317-0160, e-mail: cservice@jphmedical.com, Website: www.jphmedical.com
p

• Europe Office, UK, Ph: +44 (0) 2031708910, e-mail: info@jpmedpub.com

vi

Essentials of Clinical Pathology

© 2010, Shirish M Kawthalkar

All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means:
electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the author and the publisher.

This book has been published in good faith that the material provided by author is original. Every effort is made to ensure accuracy of material,
but the publisher, printer and author will not be held responsible for any inadvertent error (s). In case of any dispute, all legal matters are to be
settled under Delhi jurisdiction only.

First Edition: 2010

ISBN 978-93-80704-19-7
Typeset at JPBMP typesetting unit
Printed at

tahir99 - UnitedVRG
vip.persianss.ir
 Preface

The major aims of this book are discussion of (i) use of laboratory tests in the investigation and management of
common diseases, and (ii) basic biochemical and pathological principles underlying the application of laboratory
tests. The book has been written keeping in mind mainly the curricula of undergraduate students of pathology. It
should also prove to be appropriate for postgraduate residents and students of medical laboratory technology. The
laboratory tests that are demonstrated to and performed by medical students in pathology practical class and during

G
university examination are given in more detail. To keep pace with new knowledge and advances, principles of
currently performed techniques in clinical laboratory practice have also been outlined. Most of the chapters are

R
followed by reference ranges and critical values for ready access. Critical values or action values are those laboratory
results that require immediate attention of the treating clinician. While interpreting results of laboratory tests, it is

dV
necessary to follow two fundamental rules of laboratory medicine: (i) diagnosis should never be made from a single
abnormal test result (since it is affected by a number of preanalytical and analytical factors), and (ii) try to arrive at
a single diagnosis (rather than multiple diagnoses) from all the abnormal test results obtained.

te
Clinical pathology is the second major subdivision of the discipline of pathology after anatomic pathology. It is
concerned with laboratory investigations for screening, diagnosis, and overall management of diseases by analysis
ni
of blood, urine, body fluids, and other specimens. The specialties included under the discipline of clinical pathology
are clinical chemistry, hematology, blood banking, medical microbiology, cytogenetics, and molecular genetics.
-U

ir
However, scope of this book does not allow microbiology and genetics to be included in this book.

s.
I must appreciate and recognize the unstinting support of my parents, my beloved wife Dr Anjali, and my two
children, Ameya and Ashish during preparation of this book. I am thankful to Dr HT Kanade, Dean, Government
ns
Medical College, Akola, Dr Smt Deepti Dongaonkar, Dean, Government Medical College, Nagpur, Dr BB Sonawane,
9

Professor and Head, Department of Pathology, Government Medical College, Akola, and Dr WK Raut, Professor
ir9

and Head, Department of Pathology, Government Medical College, Nagpur, for encouraging me in undertaking
ia

this project for the benefit of medical students.

I express my thanks to Mr JP Vij and his outstanding team of M/s Jaypee Brothers Medical Publishers for
rs
h

undertaking to publish this book, being patient with me during the preparation of the manuscript, and bringing it
ta

out in an easy-to-read and reader-friendly format.

e

Although I have made every effort to avoid any mistakes and errors, some may persist and feedback in this
.p

regard will be highly appreciated.

p

Shirish M Kawthalkar
vi

tahir99 - UnitedVRG
vip.persianss.ir
 G
R
dV
te
ni
-U

ir
s.
ns
9
ir9

ia
rs
h
ta

e
p .p
vi

tahir99 - UnitedVRG
vip.persianss.ir
 Contents

Section 1
Chemical Pathology and Related Studies

1. Examination of Urine ................................................................................................................................................. 3

2. Renal Function Tests ................................................................................................................................................ 30
3. Diabetes Mellitus ..................................................................................................................................................... 39
4. Liver Function Tests ................................................................................................................................................. 52
5. Disorders of Lipids and Biochemical Cardiac Markers .................................................................................... 69
6. Examination of Cerebrospinal Fluid .................................................................................................................... 80
7. Examination of Pleural and Peritoneal Fluids .................................................................................................... 91
8. Examination of Sputum........................................................................................................................................... 99
9. Examination of Feces ............................................................................................................................................. 104
10. Gastric Analysis ...................................................................................................................................................... 121
11. Tests for Malabsorption and Pancreatic Function ........................................................................................... 127
12. Thyroid Function Tests ......................................................................................................................................... 137
13. Pregnancy Tests ...................................................................................................................................................... 146
14. Infertility .................................................................................................................................................................. 150
15. Semen Analysis ....................................................................................................................................................... 159

Section 2
Laboratory Hematology
16. Hematopoiesis ......................................................................................................................................................... 169
17. Collection of Blood ................................................................................................................................................. 179
18. Estimation of Hemoglobin ................................................................................................................................... 183
19. Packed Cell Volume ............................................................................................................................................... 188
20. Total Leukocyte Count .......................................................................................................................................... 192
21. Reticulocyte Count ................................................................................................................................................. 196
22. Blood Smear ............................................................................................................................................................. 200
23. Red Cell Indices ...................................................................................................................................................... 213
24. Erythrocyte Sedimentation Rate .......................................................................................................................... 215
25. Examination of Bone Marrow .............................................................................................................................. 220
26. Diagnosis of Malaria and Other Parasites in Blood ........................................................................................ 229
27. Laboratory Tests in Anemia ................................................................................................................................. 244
28. Laboratory Tests in Hematological Malignancies ........................................................................................... 273
29. Laboratory Tests in Bleeding Disorders ............................................................................................................ 288
30. Laboratory Tests in Thrombophilia .................................................................................................................... 311
31. Laboratory Tests in Porphyrias ............................................................................................................................ 314
32. Automation in Hematology .................................................................................................................................. 319

tahir99 - UnitedVRG
vip.persianss.ir
viii Essentials of Clinical Pathology

Section 3
Practical Blood Transfusion

33. Blood Group Systems ............................................................................................................................................ 329

34. Blood Grouping ...................................................................................................................................................... 336
35. Collection of Donor Blood, Processing and Storage ........................................................................................ 341
36. Screening Tests for Infections Transmissible by Transfusion ...................................................................... 347
37. Compatibility Test (Cross-match) ....................................................................................................................... 352
38. Adverse Effects of Transfusion ............................................................................................................................ 354
39. Blood Components ................................................................................................................................................. 359

General References ...................................................................................................................................................... 365

Index ........................................................................................................................................................................... 367

tahir99 - UnitedVRG
vip.persianss.ir
Chemical Pathology and
Related Studies

tahir99 - UnitedVRG
vip.persianss.ir
tahir99 - UnitedVRG
vip.persianss.ir
 1

Examination of Urine

COMPOSITION OF NORMAL URINE COLLECTION OF URINE

Urinalysis is one of the most commonly performed There are various methods for collection of urine. Method
laboratory tests in clinical practice. Composition of of collection to be used depends on the nature of
normal urine is shown in Table 1.1. investigation (Boxes 1.1 and 1.2).

INDICATIONS FOR URINALYSIS
1. Suspected renal diseases like glomerulonephritis
nephrotic syndrome, pyelonephritis, and renal failure
2. Detection of urinary tract infection
3. Detection and management of metabolic disorders
like diabetes mellitus
4. Differential diagnosis of jaundice
5. Detection and management of plasma cell dyscrasias
6. Diagnosis of pregnancy.
Time of Collection
1. A single specimen: This may be a first morning
voiding, a random specimen, or a post-prandial
specimen.
The first voided specimen in the morning is the
most concentrated and has acidic pH in which formed
elements (cells and casts) are well preserved. This
specimen is used for routine examination, fasting
glucose, proteins, nitrite, microscopic analysis for
cellular elements, pregnancy test, orthostatic
proteinuria, and bacteriological analysis.

Table 1.1: Composition of normal urine (24 hour) in adults

Parameters Values

1. Volume 600-2000 ml
2. Specific gravity 1.003-1.030
3. Osmolality 300-900 mOsm/kg
4. pH 4.6-8.0
5. Glucose <0.5 gm
6. Proteins <150 mg
7. Urobilinogen 0.5-4.0 mg
8. Porphobilinogen 0-2 mg
9. Creatinine 14-26 mg/kg (men), 11-20 mg/kg (women)
10. Urea nitrogen 12-20 gm
11. Uric acid 250-750 mg
12. Sodium 40-220 mEq
13. Potassium 25-125 mEq
14. Chloride 110-250 mEq
15. Calcium (low calcium diet) 50-150 mg
16. Formiminoglutamic acid (FIGlu) < 3 mg
17. Red cells, epithelial cells, and white blood cells <1-2/high power field
 4 Essentials of Clinical Pathology
Box 1.1: Collection of urine sample
• First morning, midstream: Preferred for routine urine
examination.
• Random, midstream: Routine urine examination.
• First morning, midstream, clean catch: Bacteriological
examination.
• Postprandial: Estimation of glucose, urobilinogen
• 24-hour: Quantitative estimation of proteins or hormones.
• Catheterised: Bacteriological examination in infants,
bedridden patients, and in obstruction of urinary tract.
• Plastic bag (e.g. colostomy bag) tied around genitals:
Infants; incontinent adults.
The random specimen is a single specimen collected
at any time of day. It is used for routine urine exami-
nation.
Post-prandial specimen (collected 2 hours after a
meal in the afternoon) is sometimes requested for
estimation of glucose (to monitor insulin therapy in
diabetes mellitus) or of urobilinogen.
2. 24-hour specimen: After getting up in the morning,
the first urine is discarded. All the urine voided
subsequently during the rest of the day and the night
is collected in a large bottle (clean bottle of 2 liter
capacity with a cap). The first urine after getting up
in the morning on the next day is also collected. The
urine should be preserved at 4-6°C during the period
of collection. The container is then immediately
transported to the laboratory. The urine is thoroughly
mixed and an aliquot is used for testing. This method
is used for quantitative estimation of proteins and
hormones.
Collection Methods
1. Midstream specimen: This is used for all types of
examinations. After voiding initial half of urine into
the toilet, a part of urine is collected in the bottle. First
half of stream serves to flush out contaminating cells
and microbes from urethra and perineum. Subse-
quent stream is collected which is from the urinary
bladder.
2. Clean-catch specimen: This is recommended for
bacteriologic culture. In men, glans penis is suffi-
ciently exposed and cleaned with soap and water. In
women urethral opening should be exposed, washed
with soapy cotton balls, rinsed with water-saturated
cotton, and holding the labia apart, the initial urine
is allowed to pass into the toilet and the remaining is
voided into the bottle (amount 20-100 ml). This
method avoids contamination of urine with the
vaginal fluids.
Box 1.2: Collection of urine for routine and culture
examination
Collection for routine urinalysis
For routine examination of urine, a wide-mouthed glass bottle
of 20-30 ml capacity, which is dry, chemically clean, leak-
proof, and with a tight fitting stopper is used. About 15 ml
of midstream sample is cleanly collected.
Collection for bacterial culture
• Use sterile container
• Collect midstream, clean catch sample
• Must be plated within 2 hours of collection
• If refrigerated, must be plated within 24 hours of
collection
• No preservative should be added.
3. Catheter specimen: This is used for bacteriological
study or culture in bedridden, ill patients or in
patients with obstruction of urinary tract. It is usually
avoided in ambulatory patients since it carries the
risk of introduction of infection.
4. Infants: In infants, a clean plastic bag can be attached
around the baby’s genitalia and left in place for some
time. For bacteriologic examination, urine is aspirated
from bladder by passing a needle just above
symphysis pubis.
Changes which Occur in Standing Urine at
Room Temperature
If urine is left standing at room temperature for long after
collection, following changes occur:
• Increase in pH due to production of ammonia from
urea by urease-producing bacteria.
• Formation of crystals due to precipitation of phos-
phates and calcium (making the urine turbid)
• Loss of ketone bodies, since they are volatile.
• Decrease in glucose due to glycolysis and utilization
of glucose by cells and bacteria.
• Oxidation of bilirubin to biliverdin causing false-
negative test for bilirubin
• Oxidation of urobilinogen to urobilin causing false-
negative test for urobilinogen
• Bacterial proliferation
• Disintegration of cellular elements, especially in
alkaline and hypotonic urine.
Urine sample must be tested in the laboratory within 2
hours of collection to get the correct results.
Preservation of Urine Sample
The urine sample should ideally be examined within 1-2
hours of voiding. If delay in examination is expected,
 Examination of Urine 5

then to slow down the above changes, sample can be
kept in the refrigerator for a maximum of 8 hours.
Refrigeration (4-6°C) is the best general method of
preservation up to 8 hours. Before analysis, refrigerated
samples should be warmed to room temperature. For
routine urinalysis, preservatives should be avoided, as
they interfere with reagent strip techniques and
chemical test for protein. Following chemical preser-
vatives can be added to the 24-hour urine sample:
• Hydrochloric acid: It is used for preservation of a 24-
hour urine sample for adrenaline, noradrenaline,
vanillylmandelic acid, and steroids.
• Toluene: It forms a thin layer over the surface and
acts as a physical barrier for bacteria and air. It is used
for measurement of chemicals.
• Boric acid: A general preservative.
• Thymol: It inhibits bacteria and fungi.
• Formalin: It is an excellent chemical for preservation
of formed elements.
PHYSICAL EXAMINATION
The parameters to be examined on physical examination
of urine are shown in Box 1.3.
Volume
Volume of only the 24-hr specimen of urine needs to be
measured and reported. The average 24-hr urinary
output in adults is 600-2000 ml. The volume varies
according to fluid intake, diet, and climate. Abnormalities
of urinary volume are as follows:
• Polyuria means urinary volume > 2000 ml/24 hours.
This is seen in diabetes mellitus (osmotic diuresis),
Box 1.3: Physical examination
• Volume • Odor
• Color • Specific gravity
• Appearance • pH
diabetes insipidus (failure of secretion of antidiuretic
hormone), chronic renal failure (loss of concentrating
ability of kidneys) or diuretic therapy.
• Oliguria means urinary volume < 400 ml/24 hours.
Causes include febrile states, acute glomerulo-
nephritis (decreased glomerular filtration), congestive
cardiac failure or dehydration (decreased renal blood
flow).
• Anuria means urinary output < 100 ml/24 hours or
complete cessation of urine output. It occurs in acute
tubular necrosis (e.g. in shock, hemolytic transfusion
reaction), acute glomerulonephritis, and complete
urinary tract obstruction.
Color
Normal urine color in a fresh state is pale yellow or amber
and is due to the presence of various pigments
collectively called urochrome. Depending on the state
of hydration urine may normally be colorless (over
hydration) or dark yellow (dehydration). Some of the
abnormal colors with associated conditions are listed in
Table 1.2.

Table 1.2: Different colors of urine

Colors Conditions

Colorless Dilute urine (diabetes mellitus, diabetes insipidus, overhydration)

Red Hematuria, Hemoglobinuria, Porphyria, Myoglobinuria
Dark brown or black Alkaptonuria, Melanoma
Brown Hemoglobinuria
Yellow Concentrated urine
Yellow-green or Biliverdin
green
Deep yellow with Bilirubin
yellow foam
Orange or orange- Urobilinogen
brown Porphobilinogen
Milky-white Chyluria
Red or orange Porphyria
fluorescence with
UV light

Note: Many drugs cause changes in urine color; drug history should be obtained if there is abnormal coloration of urine
 6 Essentials of Clinical Pathology

Appearance Causes of decrease in SG of urine are diabetes insipidus

Normal, freshly voided urine is clear in appearance.
Causes of cloudy or turbid urine are listed in Table 1.3.
Foamy urine occurs in the presence of excess proteins or
bilirubin.
Odor
Freshly voided urine has a typical aromatic odor due to
volatile organic acids. After standing, urine develops
ammoniacal odor (formation of ammonia occurs when
urea is decomposed by bacteria). Some abnormal odors
with associated conditions are:
• Fruity: Ketoacidosis, starvation
• Mousy or musty: Phenylketonuria
• Fishy: Urinary tract infection with Proteus, tyrosinae-
mia.
• Ammoniacal: Urinary tract infection with Escherichia
coli, old standing urine.
• Foul: Urinary tract infection
• Sulfurous: Cystinuria.
(SG consistently between 1.002-1.003), chronic renal
failure (low and fixed SG at 1.010 due to loss of
concentrating ability of tubules) and compulsive water
drinking.
Methods for measuring SG are urinometer method,
refractometer method, and reagent strip method.
1. Urinometer method: This method is based on the
principle of buoyancy (i.e. the ability of a fluid to exert
an upward thrust on a body placed in it). Urinometer
(a hydrometer) is placed in a container filled with
urine (Fig. 1.1A). When solute concentration is high,
upthrust of solution increases and urinometer is
pushed up (high SG). If solute concentration is low,
urinometer sinks further into the urine (low SG).
Accuracy of a urinometer needs to be checked with
distilled water. In distilled water, urinometer should

Specific Gravity (SG)

This is also called as relative mass density. It depends on
amount of solutes in solution. It is basically a comparison
of density of urine against the density of distilled water
at a particular temperature. Specific gravity of distilled
water is 1.000. Normal SG of urine is 1.003 to 1.030 and
depends on the state of hydration. SG of normal urine is
mainly related to urea and sodium. SG increases as solute
concentration increases and decreases when temperature
rises (since volume expands with rise in temperature).
SG of urine is a measure of concentrating ability of
kidneys and is determined to get information about
this tubular function. SG, however, is affected by
proteinuria and glycosuria.
Causes of increase in SG of urine are diabetes mellitus
(glycosuria), nephrotic syndrome (proteinuria), fever, Fig. 1.1: (A) Urinometer method and (B) Reagent strip
and dehydration. method for measuring specific gravity of urine

Table 1.3: Causes of cloudy or turbid urine

Cause Appearance Diagnosis

1. Amorphous phosphates White and cloudy on standing in Disappear on addition of a drop of

alkaline urine dilute acetic acid
2. Amorphous urates Pink and cloudy in acid urine Dissolve on warming
3. Pus cells Varying grades of turbidity Microscopy
4. Bacteria Uniformly cloudy; do not settle at the bottom Microscopy, Nitrite test
following centrifugation
 Examination of Urine 7

show SG of 1.000 at the temperature of calibration. If not, 7.0). On standing, urine becomes alkaline because of loss
then the difference needs to be adjusted in test readings of carbon dioxide and production of ammonia from urea.
taken subsequently. Therefore, for correct estimation of pH, fresh urine
The method is as follows: should be examined.
1. Fill a measuring cylinder with 50 ml of urine. There are various methods for determination of
2. Lower urinometer gently into the urine and let it float reaction of urine: litmus paper, pH indicator paper, pH
freely.
meter, and reagent strip tests.
3. Let urinometer settle; it should not touch the sides or
bottom of the cylinder. 1. Litmus paper test: A small strip of litmus paper is
4. Take the reading of SG on the scale (lowest point of dipped in urine and any color change is noted. If blue
meniscus) at the surface of the urine. litmus paper turns red, it indicates acid urine. If red
5. Take out the urinometer and immediately note the paper turns blue, it indicates alkaline urine (Fig. 1.2A).
temperature of urine with a thermometer. 2. pH indicator paper: Reagent area (which is impreg-
Correction for temperature: Density of urine increases at nated with bromothymol blue and methyl red) of
low temperature and decreases at higher temperature. indicator paper strip is dipped in urine sample and
This causes false reading of SG. Therefore, SG is corrected the color change is compared with the color guide
for difference between urine temperature and calibration provided. Approximate pH is obtained.
temperature. Check the temperature of calibration of the
3. pH meter: An electrode of pH meter is dipped in urine
urinometer To get the corrected SG, add 0.001 to the
reading for every 3°C that the urine temperature is above sample and pH is read off directly from the digital
the temperature of calibration. Similarly subtract 0.001 display. It is used if exact pH is required.
from the reading for every 3°C below the calibration 4. Reagent strip test: The test area (Fig. 1.2B) contains
temperature. polyionic polymer bound to H+; on reaction with
Correction for dilution: If quantity of urine is not sufficient cations in urine, H+ is released causing change in color
for measurement of SG, urine can be appropriately of the pH-sensitive dye.
diluted and the last two figures of SG are multiplied by Normal pH range is 4.6 to 8.0 (average 6.0 or slightly
the dilution factor. acidic). Urine pH depends on diet, acid base balance,
Correction for abnormal solute concentration: High SG in the water balance, and renal tubular function.
presence of glycosuria or proteinuria will not reflect true Acidic urine is found in ketosis (diabetes mellitus,
kidney function (concentrating ability). Therefore it is starvation, fever), urinary tract infection by Escherichia
necessary to nullify the effect of glucose or proteins. For coli, and high protein diet. Alkaline urine may result from
this, 0.003 is subtracted from temperature-corrected SG
for each 1 gm of protein/dl urine and 0.004 for every 1
gm of glucose/dl urine.
2. Refractometer method: SG can be precisely deter-
mined by a refractometer, which measures the
refractive index of the total soluble solids. Higher the
concentration of total dissolved solids, higher the
refractive index. Extent of refraction of a beam of light
passed through urine is a measure of solute concen-
tration, and thus of SG. The method is simple and
requires only 1-2 drops of urine. Result is read from
a scale or from digital display.
3. Reagent strip method: Reagent strip (Fig. 1.1B)
measures the concentration of ions in urine, which
correlates with SG. Depending on the ionic strength
of urine, a polyelectrolyte will ionize in proportion.
This causes a change in color of pH indicator
(bromothymol blue).

Reaction and pH
The pH is the scale for measuring acidity or alkalinity Fig. 1.2: Testing pH of urine with litmus paper (A) and
(acid if pH is < 7.0; alkaline if pH is > 7.0; neutral if pH is with reagent strip test (B)
 8 Essentials of Clinical Pathology

urinary tract infection by bacteria that split urea to
ammonia (Proteus or Pseudomonas), severe vomiting,
vegetarian diet, old ammoniacal urine sample and
chronic renal failure.
Determining pH of urine helps in identifying various
crystals in urine. Altering pH of urine may be useful in
treatment of renal calculi (i.e. some stones form only in
acid urine e.g. uric acid calculi; in such cases urine is
kept alkaline); urinary tract infection (urine should be
kept acid); and treatment with certain drugs (e.g.
streptomycin is effective in urinary tract infection if urine
is kept alkaline). In unexplained metabolic acidosis,
measurement of urine pH is helpful in diagnosing renal
tubular acidosis; in renal tubular acidosis, urine pH is
consistently alkaline despite metabolic acidosis.
CHEMICAL EXAMINATION
The chemical examination is carried out for substances
listed in Box 1.4.
Box 1.4: Chemical examination of urine
• Proteins • Urobilinogen
• Glucose • Blood
• Ketones • Hemoglobin
• Bilirubin • Myoglobin
• Bile salts • Nitrite or leukocyte esterase
Box 1.5: Causes of proteinuria
• Glomerular proteinuria
• Tubular proteinuria
• Overflow proteinuria
• Hemodynamic (functional) proteinuria
• Post-renal proteinuria
disease, there is increased excretion of lower molecular
weight proteins like albumin and transferrin. When
glomeruli can retain larger molecular weight proteins
but allow passage of comparatively lower molecular
weight proteins, the proteinuria is called as selective.
With further glomerular damage, this selectivity is lost
and larger molecular weight proteins (γ globulins) are
also excreted along with albumin; this is called as
nonselective proteinuria.
Selective and nonselective proteinuria can be distin-
guished by urine protein electrophoresis. In selective
proteinuria, albumin and transferrin bands are seen,
while in nonselective type, the pattern resembles that of
serum (Fig. 1.3).
Causes of glomerular proteinuria are glomerular
diseases that cause increased permeability of glomerular
basement membrane. The degree of glomerular proteinu-

Proteins
Normally, kidneys excrete scant amount of protein in
urine (up to 150 mg/24 hours). These proteins include
proteins from plasma (albumin) and proteins derived
from urinary tract (Tamm-Horsfall protein, secretory
IgA, and proteins from tubular epithelial cells, leucocytes,
and other desquamated cells); this amount of proteinuria
cannot be detected by routine tests. (Tamm-Horsfall
protein is a normal mucoprotein secreted by ascending
limb of the loop of Henle).
Proteinuria refers to protein excretion in urine
greater than 150 mg/24 hours in adults.

Causes of Proteinuria
Causes of proteinuria can be grouped as shown in Box
1.5.
1. Glomerular proteinuria: Proteinuria due to increased
Fig. 1.3: Glomerular and tubular proteinuria. Upper figure shows
permeability of glomerular capillary wall is called as normal serum protein electrophoresis pattern. Lower part shows
glomerular proteinuria. comparison of serum and urine electrophoresis in (1) selective
There are two types of glomerular proteinuria: proteinuria, (2) non-selective proteinuria, and (3) tubular
selective and nonselective. In early stages of glomerular proteinuria

Box 1.6: Nephrotic syndrome
• Massive proteinuria (>3.5 gm/24 hr)
• Hypoalbuminemia (<3.0 gm/dl)
• Generalised edema
• Hyperlipidemia (serum cholesterol >350 mg/dl)
• Lipiduria
ria correlates with severity of disease and prognosis.
Serial estimations of urinary protein are also helpful in
monitoring response to treatment. Most severe degree
of proteinuria occurs in nephrotic syndrome (Box 1.6).
2. Tubular proteinuria: Normally, glomerular mem-
brane, although impermeable to high molecular
weight proteins, allows ready passage to low
molecular weight proteins like β2-microglobulin,
retinol-binding protein, lysozyme, α1-microglobulin,
and free immunoglobulin light chains. These low
molecular weight proteins are actively reabsorbed by
proximal renal tubules. In diseases involving mainly
tubules, these proteins are excreted in urine while
albumin excretion is minimal.
Urine electrophoresis shows prominent α- and β-
bands (where low molecular weight proteins migrate)
and a faint albumin band (Fig. 1.3).
Tubular type of proteinuria is commonly seen in
acute and chronic pyelonephritis, heavy metal
poisoning, tuberculosis of kidney, interstitial
nephritis, cystinosis, Fanconi syndrome and rejection
of kidney transplant.
Purely tubular proteinuria cannot be detected by
reagent strip test (which is sensitive to albumin), but
heat and acetic acid test and sulphosalicylic acid test
are positive.
3. Overflow proteinuria: When concentration of a low
molecular weight protein rises in plasma, it “over-
flows” from plasma into the urine. Such proteins are
immunoglobulin light chains or Bence Jones proteins
(plasma cell dyscrasias), hemoglobin (intravascular
hemolysis), myoglobin (skeletal muscle trauma), and
lysozyme (acute myeloid leukemia type M4 or M5).
4. Hemodynamic proteinuria: Alteration of blood flow
through the glomeruli causes increased filtration of
proteins. Protein excretion, however, is transient. It
is seen in high fever, hypertension, heavy exercise,
congestive cardiac failure, seizures, and exposure to
cold.
Postural (orthostatic) proteinuria occurs when the
subject is standing or ambulatory, but is absent in
recumbent position. It is common in adolescents (3-5%)
Examination of Urine 9
and is probably due to lordotic posture that causes
inferior venacaval compression between the liver and
vertebral column. The condition disappears in adulthood.
Amount of proteinuria is <1000 mg/day. First-morning
urine after rising is negative for proteins, while another
urine sample collected after patient performs normal
activities is positive for proteins. In such patients, periodic
testing for proteinuria should be done to rule out renal
disease.
5. Post-renal proteinuria: This is caused by inflamma-
tory or neoplastic conditions in renal pelvis, ureter,
bladder, prostate, or urethra.
Tests for Detection of Proteinuria
1. Heat and acetic acid test (Boiling test): This test is
based on the principle that proteins get precipitated
when boiled in an acidic solution.
Method: Urine should be clear; if not, filter or use
supernatant from a centrifuged sample.
Urine should be just acidic (check with litmus paper);
if not, add 10% acetic acid drop by drop until blue litmus
paper turns red.
A test tube is filled 2/3rds with urine. The tube is
inclined at an angle and the upper portion is boiled over
the flame. (Only the upper portion is heated so that
convection currents generated by heat do not disturb the
precipitate and the upper portion can be compared with
the lower clear portion). Compare the heated part with
the lower part. Cloudiness or turbidity indicates presence
of either phosphates or proteins (Fig. 1.4). A few drops
of 10% acetic acid are added and the upper portion is
boiled again. Turbidity due to phosphates disappears
while that due to proteins does not.
Fig. 1.4: Principle of heat test for proteins
 10 Essentials of Clinical Pathology

False-positive test occurs with tolbutamide and large

doses of penicillins.
2. Reagent strip test: The reagent area of the strip is
coated with an indicator and buffered to an acid pH
which changes color in the presence of proteins
(Figs 1.5 and 1.6). The principle is known as “protein
error of indicators”.
The reagent area is impregnated with bromo-
phenol blue indicator buffered to pH 3.0 with citrate.
When the dye gets adsorbed to protein, there is
change in ionization (and hence pH) of the indicator
that leads to change in color of the indicator. The
intensity of the color produced is proportional to the
concentration of protein. The test is semi-quantitative.
Reagent strip test is mainly reactive to albumin.
Fig. 1.6: Grading of proteinuria with reagent strip test
It is false-negative in the presence of Bence Jones (above) and sulphosalicylic acid test (below)
proteins, myoglobin, and hemoglobin. Overload
(Bence Jones) proteinuria and tubular proteinuria
denatured by organic acids and precipitate out of
may be missed entirely if only reagent strip method
solution).
is used. This test should be followed by sulpho-
Take 2 ml of clear urine in a test tube. If reaction of
salicylic acid test, which is a confirmatory test. Highly
urine is neutral or alkaline, a drop of glacial acetic acid is
alkaline urine, gross hematuria, and contamination
added. Add 2-3 drops of sulphosalicylic acid (3 to 5%),
with vaginal secretions can give false-positive
and examine for turbidity against a dark background
reactions. (Fig. 1.6).
3. Sulphosalicylic acid test: Addition of sulphosalicylic This test is more sensitive and reliable than boiling
acid to the urine causes formation of a white test.
precipitate if proteins are present (Proteins are False-positive test may occur due to gross hematuria,
highly concentrated urine, radiographic contrast media,
excess uric acid, tolbutamide, sulphonamides, salicylates,
and penicillins.
False-negative test can occur with very dilute urine.
The test can detect albumin, hemoglobin, myoglobin,
and Bence Jones proteins.
Comparison of reagent strip test and sulphosalicylic
acid test is shown in Table 1.4.
Fig. 1.5: Principle of reagent strip test for proteins. The principle
Quantitative Estimation of Proteins
is called as ‘protein error of indicators’ meaning that one color
appears if protein is present and another color if protein is Indications for quantitative estimation of proteins in
absent. Sensitivity is 5-10 mg/dl. The test does not detect Bence urine are:
Jones proteins, hemoglobin, and myoglobin • Diagnosis of nephrotic syndrome

Table 1.4: Comparison of two tests for proteinuria

Parameter Reagent strip test Sulphosalicylic acid test
1. Principle Colorimetric Acid precipitation
2. Proteins detected Albumin All (albumin, Bence Jones proteins,
hemoglobin, myoglobin)
3. Sensitivity 5-10 mg/dl 20 mg/dl
4. Indicator Color change Turbidity
5. Type of test Screening Confirmatory

Table 1.5: Grading of albuminuria
Condition mg/24 hr mg/L mg/g creatinine
Normal < 30 < 20 < 20
Microalbuminuria 30-300 20-200 20-300
Overt albuminuria >300 >200 >300
• Detection of microalbuminuria or early diabetic
nephropathy
• To follow response to therapy in renal disease
Proteinuria >1500 mg/ 24 hours indicates glomerular
disease; proteinuria >3500 mg/24 hours is called as
nephrotic range proteinuria; in tubular, hemodynamic
and post renal diseases, proteinuria is usually < 1500 mg/
24 hours.
Grading of albuminuria is shown in Table 1.5.
There are two methods for quantitation of proteins:
(1) Estimation of proteins in a 24-hour urine sample, and
(2) Estimation of protein/creatinine ratio in a random
urine sample.
1. Quantitative estimation of proteins in a 24-hour
urine sample: Collection of a 24-hour sample is given
earlier. Adequacy of sample is confirmed by
calculating expected 24-hour urine creatinine
excretion. Daily urinary creatinine excretion depends
on muscle mass and remains relatively constant in
an individual patient. In adult males creatinine
excretion is 14-26 mg/kg/24 hours, while in women
it is 11-20 mg/kg/24 hours. Various methods are
available for quantitative estimation of proteins:
Esbach’s albuminometer method, turbidimetric
methods, biuret reaction, and immunologic methods.
2. Estimation of protein/creatinine ratio in a random
urine sample: Because of the problem of incomplete
collection of a 24-hour urine sample, many labora-
tories measure protein/creatinine ratio in a random
urine sample. Normal protein/creatinine ratio is
< 0.2. In low-grade proteinuria it is 0.2-1.0; in
moderate, it is 1.0-3.5; and in nephrotic- range
proteinuria it is > 3.5.
Microalbuminuria
This is defined as urinary excretion of 30 to 300 mg/24
hours (or 2-20 mg/dl) of albumin in urine.
Significance of microalbuminuria
1. Microalbuminuria is considered as the earliest sign
of renal damage in diabetes mellitus (diabetic
nephropathy). It indicates increase in capillary
Examination of Urine 11
μg/min μg/mg creatinine g/mol creatinine
< 20 < 30 < 2.5
20-200 30-300 2.5-25
>200 >300 >25
permeability to albumin and denotes microvascular
disease. Microalbuminuria precedes the development
of diabetic nephropathy by a few years. If blood
glucose level and hypertension are tightly controlled
at this stage by aggressive treatment then progression
to irreversible renal disease and subsequent renal
failure can be delayed or prevented.
2. Microalbuminuria is an independent risk factor for
cardiovascular disease in diabetes mellitus.
Detection of microalbuminuria: Microalbuminuria cannot
be detected by routine tests for proteinuria. Methods for
detection include:
• Measurement of albumin-creatinine ratio in a random
urine sample
• Measurement of albumin in an early morning or
random urine sample
• Measurement of albumin in a 24 hr sample
Test strips that screen for microalbuminuria are
available commercially. Exact quantitation can be done
by immunologic assays like radioimmunoassay or
enzyme linked immunosorbent assay.
Bence Jones Proteinuria
Bence Jones proteins are monoclonal immunoglobulin
light chains (either κ or λ) that are synthesized by
neoplastic plasma cells. Excess production of these light
chains occurs in plasma cell dyscrasias like multiple
myeloma and primary amyloidosis. Because of their low
molecular weight and high concentration they are
excreted in urine (overflow proteinuria).
Bence Jones proteins have a characteristic thermal
behaviour. When heated, Bence Jones proteins precipi-
tate at temperatures between 40°C to 60°C (other proteins
precipitate between 60-70°C), and precipitate disappears
on further heating at 85-100°C (while precipitate of other
proteins does not). When cooled (60-85°C), there is
reappearance of precipitate of Bence Jones proteins. This
test, however, is not specific for Bence Jones proteins and
both false-positive and -negative results can occur. This
test has been replaced by protein electrophoresis of
concentrated urine sample (Fig. 1.7).
 12 Essentials of Clinical Pathology
Fig. 1.7: Urine protein electrophoresis showing heavy Bence
Jones proteinuria (red arrow) along with loss of albumin and
other low molecular weight proteins in urine
Further evaluation of persistent overt proteinuria is
shown in Figure 1.8.
Glucose
The main indication for testing for glucose in urine is
detection of unsuspected diabetes mellitus or follow-up
of known diabetic patients.
Practically all of the glucose filtered by the glomeruli
is reabsorbed by the proximal renal tubules and returned
to circulation. Normally a very small amount of glucose
is excreted in urine (< 500 mg/24 hours or <15 mg/dl)
that cannot be detected by the routine tests. Presence of
detectable amounts of glucose in urine is called as
Note: Quantitation of proteins and creatinine clearance are done in all patients with persistent proteinuria
Fig. 1.8: Evaluation of proteinuria
glucosuria or glycosuria (Box 1.7). Glycosuria results if
the filtered glucose load exceeds the capacity of renal
tubular reabsorption. Most common cause is hyper-
glycemia from diabetes mellitus.
Causes of Glycosuria
1. Glycosuria with hyperglycemia:
• Endocrine diseases: diabetes mellitus, acromegaly,
Cushing’s syndrome, hyperthyroidism, pancrea-
tic disease
• Non-endocrine diseases: central nervous system
diseases, liver disorders
• Drugs: adrenocorticotrophic hormone, cortico-
steroids, thiazides
• Alimentary glycosuria (Lag-storage glycosuria):
After a meal, there is rapid intestinal absorption
of glucose leading to transient elevation of blood
glucose above renal threshold. This can occur in
persons with gastrectomy or gastrojejunostomy
and in hyperthyroidism. Glucose tolerance test
reveals a peak at 1 hour above renal threshold
(which causes glycosuria); the fasting and 2-hour
glucose values are normal.
2. Glycosuria without hyperglycemia
• Renal glycosuria: This accounts for 5% of cases of
glycosuria in general population. Renal threshold
 Examination of Urine 13

Box 1.7: Urine glucose

• Urine should be tested for glucose within 2 hours of collection (due to lowering of glucose by glycolysis and by contaminating
bacteria which degrade glucose rapidly)
• Reagent strip test is a rapid, inexpensive, and semi-quantitative test
• In the past this test was used for home-monitoring of glucose; the test is replaced by glucometers.
• Urine glucose cannot be used to monitor control of diabetes since renal threshold is variable amongst individuals, no
information about level of blood glucose below renal threshold is obtained, and urinary glucose value is affected by
concentration of urine.

is the highest glucose level in blood at which
glucose appears in urine and which is detectable
by routine laboratory tests. The normal renal
threshold for glucose is 180 mg/dl. Threshold
substances need a carrier to transport them from
tubular lumen to blood. When the carrier is
saturated, the threshold is reached and the
substance is excreted. Up to this level glucose
filtered by the glomeruli is efficiently reabsorbed
by tubules. Renal glycosuria is a benign condition
in which renal threshold is set below 180 mgs/dl
but glucose tolerance is normal; the disorder is
transmitted as autosomal dominant. Other
conditions in which glycosuria can occur with
blood glucose level remaining below 180 mgs/dl
are renal tubular diseases in which there is
decreased glucose reabsorption like Fanconi’s
syndrome, and toxic renal tubular damage. During
pregnancy, renal threshold for glucose is
decreased. Therefore it is necessary to estimate
blood glucose when glucose is first detected in
urine.
Tests for Detection of Glucose in Urine
1. Copper reduction methods
A. Benedict’s qualitative test: When urine is boiled in
Benedict’s qualitative solution, blue alkaline copper
sulphate is reduced to red-brown cuprous oxide if a
reducing agent is present (Fig. 1.9). The extent of
reduction depends on the concentration of the reducing
substance. This test, however, is not specific for glucose.
Other carbohydrates (like lactose, fructose, galactose,
pentoses), certain metabolites (glucuronic acid, homo-
gentisic acid, uric acid, creatinine), and drugs (ascorbic
acid, salicylates, cephalosporins, penicillins, strepto-
mycin, isoniazid, para-aminosalicylic acid, nalidixic acid,
etc.) also reduce alkaline copper sulphate solution.
Method
1. Take 5 ml of Benedict’s qualitative reagent in a test
tube (composition of Benedict’s qualitative reagent:
copper sulphate 17.3 gram, sodium carbonate 100
gram, sodium citrate 173 gram, distilled water 1000
ml).
2. Add 0.5 ml (or 8 drops) of urine. Mix well.
3. Boil over a flame for 2 minutes.
4. Allow to cool at room temperature.
5. Note the color change, if any.
Sensitivity of the test is about 200 mg reducing
substance per dl of urine. Since Benedict’s test gives
positive reaction with carbohydrates other than glucose,
it is also used as a screening test (for detection of
galactose, lactose, fructose, maltose, and pentoses in
urine) for inborn errors of carbohydrate metabolism in
infants and children. For testing urine only for glucose,
reagent strips are preferred (see below).
The result is reported in grades as follows (Fig. 1.10):
Nil: no change from blue color
Trace: Green without precipitate
1+ (approx. 0.5 grams/dl): Green with precipitate
2+ (approx. 1.0 grams/dl): Brown precipitate
3+ (approx. 1.5 grams/dl: Yellow-orange precipitate
4+ (> 2.0 grams/dl): Brick- red precipitate.

Fig. 1.9: Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl
 14 Essentials of Clinical Pathology
Fig. 1.10: Grading of Benedict’s test (above) and reagent
strip test (below) for glucose
B. Clinitest tablet method (Copper reduction tablet test): This
is a modified form of Benedict’s test in which the reagents
are present in a tablet form (copper sulphate, citric acid,
sodium carbonate, and anhydrous sodium hydroxide).
Sensitivity is 200 mgs/dl of glucose.
2. Reagent strip method This test is specific for glucose
and is therefore preferred over Benedict’s and Clinitest
methods. It is based on glucose oxidase-peroxidase
reaction. Reagent area of the strips is impregnated with
two enzymes (glucose oxidase and peroxidase) and a
chromogen. Glucose is oxidized by glucose oxidase with
the resultant formation of hydrogen peroxide and
gluconic acid. Oxidation of chromogen occurs in the
presence of hydrogen peroxide and the enzyme peroxi-
dase with resultant color change (Fig. 1.11). Nature of
chromogen and buffer system differ in different strips.
The strip is dipped into the urine sample and color is
observed after a specified time and compared with the
color chart provided (Fig. 1.10).
This test is more sensitive than Benedict’s qualitative
test and specific only for glucose. Other reducing agents
give negative reaction.
Fig. 1.11: Principle of reagent strip test for glucose in urine. Each mole of glucose produces one mole of peroxide,
and each mole of peroxide reduces one mole of oxygen. Sensitivity is 100 mg glucose/100 ml
Sensitivity of the test is about 100 mg glucose/dl of
urine.
False positive test occurs in the presence of oxidizing
agent (bleach or hypochlorite used to clean urine
containers), which oxidizes the chromogen directly.
False-negative test occurs in the presence of large
amounts of ketones, salicylates, ascorbic acid, and severe
Escherichia coli infection (catalase produced by organisms
in urine inactivates hydrogen peroxide).
Ketones
Excretion of ketone bodies (acetoacetic acid, β-hydroxy-
butyric acid, and acetone) in urine is called as ketonuria.
Ketones are breakdown products of fatty acids and their
presence in urine is indicative of excessive fatty acid
metabolism to provide energy.
Causes of Ketonuria
Normally ketone bodies are not detectable in the urine
of healthy persons. If energy requirements cannot be met
by metabolism of glucose (due to defective carbohydrate
metabolism, low carbohydrate intake, or increased
metabolic needs), then energy is derived from break-
down of fats. This leads to the formation of ketone bodies
(Fig. 1.12).
1. Decreased utilization of carbohydrates
a. Uncontrolled diabetes mellitus with ketoacidosis: In
diabetes, because of poor glucose utilization, there is
compensatory increased lipolysis. This causes
increase in the level of free fatty acids in plasma.
Degradation of free fatty acids in the liver leads to
the formation of acetoacetyl CoA which then forms
ketone bodies. Ketone bodies are strong acids and
produce H+ ions, which are neutralized by bicar-
bonate ions; fall in bicarbonate (i.e. alkali) level
produces ketoacidosis. Ketone bodies also increase
the plasma osmolality and cause cellular dehydration.
Children and young adults with type 1 diabetes are

Fig. 1.12: Formation of ketone bodies. A small part of
acetoacetate is spontaneously and irreversibly converted to
acetone. Most is converted reversibly to β-hydroxybutyrate
especially prone to ketoacidosis during acute illness
and stress. If glycosuria is present, then test for ketone
bodies must be done. If both glucose and ketone
bodies are present in urine, then it indicates presence
of diabetes mellitus with ketoacidosis (Box 1.8).
In some cases of diabetes, ketone bodies are increased
in blood but do not appear in urine.
Presence of ketone bodies in urine may be a warning
of impending ketoacidotic coma.
b. Glycogen storage disease (von Gierke’s disease)
2. Decreased availability of carbohydrates in the diet:
a. Starvation
b. Persistent vomiting in children
c. Weight reduction program (severe carbohydrate
restriction with normal fat intake)
3. Increased metabolic needs:
a. Fever in children
b. Severe thyrotoxicosis
c. Pregnancy
d. Protein calorie malnutrition
Tests for Detection of Ketones in Urine
The proportion of ketone bodies in urine in ketosis is
variable: β-hydroxybutyric acid 78%, acetoacetic acid
20%, and acetone 2%.
Box 1.8: Urine ketones in diabetes
Indications for testing
• At diagnosis of diabetes mellitus
• At regular intervals in all known cases of diabetes,
and in gestational diabetes
• In known diabetic patients during acute illness, persistent
hyperglycemia (>300 mg/dl), pregnancy, clinical evidence
of diabetic acidosis (nausea, vomiting, abdominal pain)
Examination of Urine 15
No method for detection of ketonuria reacts with all
the three ketone bodies. Rothera’s nitroprusside method
and methods based on it detect acetoacetic acid and
acetone (the test is 10-20 times more sensitive to
acetoacetic acid than acetone). Ferric chloride test detects
acetoacetic acid only. β-hydroxybutyric acid is not
detected by any of the screening tests.
Methods for detection of ketone bodies in urine are
Rothera’s test, Acetest tablet method, ferric chloride test,
and reagent strip test.
1. Rothera’s’ test (Classic nitroprusside reaction) Acetoacetic
acid or acetone reacts with nitroprusside in alkaline
solution to form a purple-colored complex (Fig. 1.13).
Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate
and to 10-25 mg/dl of acetone.
Method
1. Take 5 ml of urine in a test tube and saturate it with
ammonium sulphate.
2. Add a small crystal of sodium nitroprusside. Mix
well.
3. Slowly run along the side of the test tube liquor
ammonia to form a layer.
4. Immediate formation of a purple permanganate
colored ring at the junction of the two fluids indicates
a positive test (Fig. 1.14).
False-positive test can occur in the presence of L-dopa
in urine and in phenylketonuria.
2. Acetest tablet test This is Rothera’s test in the form of a
tablet. The Acetest tablet consists of sodium nitro-
prusside, glycine, and an alkaline buffer. A purple-
lavender discoloration of the tablet indicates the presence
of acetoacetate or acetone (≥ 5 mg/dl). A rough estimate
of the amount of ketone bodies can be obtained by
comparison with the color chart provided by the
manufacturer.The test is more sensitive than reagent strip
test for ketones.
Fig. 1.13: Principles of Rothera’s test and reagent strip test
for ketone bodies in urine. Ketones are detected as acetoacetic
acid and acetone but not β-hydroxybutyric acid
 16 Essentials of Clinical Pathology
Fig. 1.14: Rothera’s tube test and reagent strip test for
ketone bodies in urine
3. Ferric chloride test (Gerhardt’s): Addition of 10% ferric
chloride solution to urine causes solution to become
reddish or purplish if acetoacetic acid is present. The test
is not specific since certain drugs (salicylate and L-dopa)
give similar reaction. Sensitivity of the test is 25-50 mg/
dl.
4. Reagent strip test: Reagent strips tests are modifications
of nitroprusside test (Figs 1.13 and 1.14). Their sensitivity
is 5-10 mg/dl of acetoacetate. If exposed to moisture,
reagent strips often give false-negative result. Ketone pad
on the strip test is especially vulnerable to improper
storage and easily gets damaged.
Bile Pigment (Bilirubin)
Bilirubin (a breakdown product of hemoglobin) is
undetectable in the urine of normal persons. Presence of
bilirubin in urine is called as bilirubinuria.
There are two forms of bilirubin: conjugated and
unconjugated. After its formation from hemoglobin in
reticuloendothelial system, bilirubin circulates in blood
bound to albumin. This is called as unconjugated
bilirubin. Unconjugated bilirubin is not water-soluble,
is bound to albumin, and cannot pass through the
glomeruli; therefore it does not appear in urine. The liver
takes up unconjugated bilirubin where it combines with
glucuronic acid to form bilirubin diglucuronide
(conjugated bilirubiun). Conjugated bilirubin is water-
soluble, is filtered by the glomeruli, and therefore appears
in urine.
Detection of bilirubin in urine (along with urobili-
nogen) is helpful in the differential diagnosis of
jaundice (Table 1.6).
Table 1.6: Urine bilirubin and urobilinogen in jaundice
Urine test Hemolytic Hepatocellular Obstructive
jaundice jaundice jaundice
1. Bilirubin Absent Present Present
2. Urobilinogen Increased Increased Absent
In acute viral hepatitis, bilirubin appears in urine
even before jaundice is clinically apparent. In a fever
of unknown origin bilirubinuria suggests hepatitis.
Presence of bilirubin in urine indicates conjugated
hyperbilirubinemia (obstructive or hepatocellular
jaundice). This is because only conjugated bilirubin is
water-soluble. Bilirubin in urine is absent in hemolytic
jaundice; this is because unconjugated bilirubin is
water-insoluble.
Tests for Detection of Bilirubin in Urine
Bilirubin is converted to non-reactive biliverdin on
exposure to light (daylight or fluorescent light) and on
standing at room temperature. Biliverdin cannot be
detected by tests that detect bilirubin. Therefore fresh
sample that is kept protected from light is required.
Findings associated with bilirubinuria are shown in
Box 1.9.
Methods for detection of bilirubin in urine are foam
test, Gmelin’s test, Lugol iodine test, Fouchet’s test,
Ictotest tablet test, and reagent strip test.
1. Foam test: About 5 ml of urine in a test tube is shaken
and observed for development of yellowish foam.
Similar result is also obtained with proteins and
highly concentrated urine. In normal urine, foam is
white.
2. Gmelin’s test: Take 3 ml of concentrated nitric acid
in a test tube and slowly place equal quantity of urine
over it. The tube is shaken gently; play of colors
(yellow, red, violet, blue, and green) indicates positive
test (Fig. 1.15).
3. Lugol iodine test: Take 4 ml of Lugol iodine solution
(Iodine 1 gm, potassium iodide 2 gm, and distilled
water to make 100 ml) in a test tube and add 4 drops
of urine. Mix by shaking. Development of green color
indicates positive test.
Box 1.9: Clinical and laboratory findings in bilirubinuria
• Jaundice
• Urine color: Dark yellow with yellow foam
• Elevated serum conjugated bilirubin

Fig. 1.15: Positive Gmelin’s test for bilirubin showing
play of colors
4. Fouchet’s test: This is a simple and sensitive test.
i. Take 5 ml of fresh urine in a test tube, add 2.5
ml of 10% of barium chloride, and mix well. A
precipitate of sulphates appears to which bilirubin
is bound (barium sulphate-bilirubin complex).
ii. Filter to obtain the precipitate on a filter paper.
iii. To the precipitate on the filter paper, add 1drop
of Fouchet’s reagent. (Fouchet’s reagent consists
of 25 grams of trichloroacetic acid, 10 ml of 10%
ferric chloride, and distilled water 100 ml).
iv. Immediate development of blue-green color
around the drop indicates presence of bilirubin
(Fig. 1.16).
5. Reagent strips or tablets impregnated with diazo
reagent: These tests are based on reaction of bilirubin
with diazo reagent; color change is proportional to
the concentration of bilirubin. Tablets (Ictotest) detect
0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests
are less sensitive (0.5 mg/dl).
Bile Salts
Bile salts are salts of four different types of bile acids:
cholic, deoxycholic, chenodeoxycholic, and lithocholic.
These bile acids combine with glycine or taurine to form
complex salts or acids. Bile salts enter the small intestine
through the bile and act as detergents to emulsify fat and
reduce the surface tension on fat droplets so that enzymes
(lipases) can breakdown the fat. In the terminal ileum,
bile salts are absorbed and enter in the blood stream from
where they are taken up by the liver and re-excreted in
bile (enterohepatic circulation).
Bile salts along with bilirubin can be detected in urine
in cases of obstructive jaundice. In obstructive jaundice,
Examination of Urine 17
Fig. 1.16: Positive Fouchet’s test for bilirubin in urine
bile salts and conjugated bilirubin regurgitate into blood
from biliary canaliculi (due to increased intrabiliary
pressure) and are excreted in urine. The test used for their
detection is Hay’s surface tension test. The property of
bile salts to lower the surface tension is utilized in this
test.
Take some fresh urine in a conical glass tube. Urine
should be at the room temperature. Sprinkle on the
surface particles of sulphur. If bile salts are present,
sulphur particles sink to the bottom because of lowering
of surface tension by bile salts. If sulphur particles remain
on the surface of urine, bile salts are absent.
Thymol (used as a preservative) gives false positive
test.
Urobilinogen
Conjugated bilirubin excreted into the duodenum
through bile is converted by bacterial action to urobilino-
gen in the intestine. Major part is eliminated in the feces.
A portion of urobilinogen is absorbed in blood, which
undergoes recycling (enterohepatic circulation); a small
amount, which is not taken up by the liver, is excreted in
urine. Urobilinogen is colorless; upon oxidation it is
converted to urobilin, which is orange-yellow in color.
Normally about 0.5-4 mg of urobilinogen is excreted in
urine in 24 hours. Therefore, a small amount of urobili-
nogen is normally detectable in urine.
Urinary excretion of urobilinogen shows diurnal
variation with highest levels in afternoon. Therefore, a
2-hour post-meal sample is preferred.
Causes of Increased Urobilinogen in Urine
1. Hemolysis: Excessive destruction of red cells leads
to hyperbilirubinemia and therefore increased
formation of urobilinogen in the gut. Bilirubin, being
of unconjugated type, does not appear in urine.
Increased urobilinogen in urine without bilirubin is
 18 Essentials of Clinical Pathology

typical of hemolytic anemia. This also occurs in

megaloblastic anemia due to premature destruction
of erythroid precursors in bone marrow (ineffective
erythropoiesis).
2. Hemorrhage in tissues: There is increased formation
of bilirubin from destruction of red cells.

Causes of Reduced Urobilinogen in Urine

1. Obstructive jaundice: In biliary tract obstruction,
delivery of bilirubin to the intestine is restricted and
very little or no urobilinogen is formed. This causes
stools to become clay-colored.
2. Reduction of intestinal bacterial flora: This prevents
conversion of bilirubin to urobilinogen in the
intestine. It is observed in neonates and following
antibiotic treatment.
Testing of urine for both bilirubin and urobilinogen
can provide helpful information in a case of jaundice
(Table 1.6). Fig. 1.17: Ehrlich’s aldehyde test for urobilinogen

Tests for Detection of Urobilinogen in Urine

Fresh urine sample should be used because on standing
urobilinogen is converted to urobilin, which cannot be
detected by routine tests. A timed (2-hour postprandial)
sample can also be used for testing urobilinogen.
Methods for detection of increased amounts of urobili-
nogen in urine are Ehrlich’s aldehyde test and reagent
strip test.
1. Ehrlich’s aldehyde test: Ehrlich’s reagent (p-
dimethylaminobenzaldehyde) reacts with urobili-
nogen in urine to produce a pink color. Intensity of
color developed depends on the amount of urobili-
nogen present. Presence of bilirubin interferes with
the reaction, and therefore if present, should be
removed. For this, equal volumes of urine and 10%
barium chloride are mixed and then filtered. Test for
urobilinogen is carried out on the filtrate. However,
similar reaction is produced by porphobilinogen (a
substance excreted in urine in patients of porphyria).
Method: Take 5 ml of fresh urine in a test tube. Add 0.5
ml of Ehrlich’s aldehyde reagent (which consists of
hydrochloric acid 20 ml, distilled water 80 ml, and para-
dimethylaminobenzaldehyde 2 gm). Allow to stand at
room temperature for 5 minutes. Development of pink
color indicates normal amount of urobilinogen. Dark
red color means increased amount of urobilinogen (Fig.
1.17).
Since both urobilinogen and porphobilinogen
produce similar reaction, further testing is required to
distinguish between the two. For this, Watson-Schwartz
test is used. Add 1-2 ml of chloroform, shake for 2
minutes and allow to stand. Pink color in the chloroform
layer indicates presence of urobilinogen, while pink
coloration of aqueous portion indicates presence of
porphobilinogen. Pink layer is then decanted and shaken
with butanol. A pink color in the aqueous layer indicates
porphobilinogen (Fig. 1.18).
False-negative reaction can occur in the presence of
(i) urinary tract infection (nitrites oxidize urobilinogen
to urobilin), and (ii) antibiotic therapy (gut bacteria which
produce urobilinogen are destroyed).
2. Reagent strip method: This method is specific for
urobilinogen. Test area is impregnated with either
p-dimethylaminobenzaldehyde or 4-methoxy-
benzene diazonium tetrafluoroborate.
Blood
The presence of abnormal number of intact red blood
cells in urine is called as hematuria. It implies presence
of a bleeding lesion in the urinary tract. Bleeding in urine
may be noted macroscopically or with naked eye (gross
hematuria). If bleeding is noted only by microscopic
examination or by chemical tests, then it is called as
occult, microscopic or hidden hematuria.
Causes of Hematuria
1. Diseases of urinary tract
• Glomerular diseases: Glomerulonephritis, Berger’s
disease, lupus nephritis, Henoch-Schonlein
purpura

Fig. 1.18: Interpretation of Watson-Schwartz test
• Nonglomerular diseases: Calculus, tumor, infec-
tion, tuberculosis, pyelonephritis, hydronephrosis,
polycystic kidney disease, trauma, after strenuous
physical exercise, diseases of prostate (benign
hyperplasia of prostate, carcinoma of prostate).
2. Hematological conditions: Coagulation disorders, sickle
cell disease
Presence of red cell casts and proteinuria along with
hematuria suggests glomerular cause of hematuria.
Tests for Detection of Blood in Urine
1. Microscopic examination of urinary sediment:
Definition of microscopic hematuria is presence of 3
or more number of red blood cells per high power
field on microscopic examination of urinary sediment
in two out of three properly collected samples. A
small number of red blood cells in urine of low specific
gravity may undergo lysis, and therefore hematuria
may be missed if only microscopic examination is
done. Therefore, microscopic examination of urine
should be combined with a chemical test.
2. Chemical tests: These detect both intracellular and
extracellular hemoglobin (i.e. intact and lysed red
Fig. 1.19: Principle of chemical test for red cells, hemoglobin, or myoglobin in urine
Examination of Urine 19
cells) as well as myoglobin. Heme proteins in
hemoglobin act as peroxidase, which reduces
hydrogen peroxide to water. This process needs a
hydrogen donor (benzidine, orthotoluidine, or
guaiac). Oxidation of hydrogen donor leads to
development of a color (Fig. 1.19). Intensity of color
produced is proportional to the amount of hemo-
globin present.
Chemical tests are positive in hematuria, hemo-
globinuria, and myoglobinuria.
• Benzidine test: Make saturated solution of benzidine
in glacial acetic acid. Mix 1 ml of this solution with 1
ml of hydrogen peroxide in a test tube. Add 2 ml of
urine. If green or blue color develops within 5
minutes, the test is positive.
• Orthotoluidine test: In this test, instead of benzidine,
orthotoluidine is used. It is more sensitive than
benzidine test.
• Reagent strip test: Various reagent strips are
commercially available which use different
chromogens (o-toluidine, tetramethylbenzidine).
 20 Essentials of Clinical Pathology
Fig. 1.20: Evaluation of positive chemical test for blood in urine
Causes of false-positive tests:
• Contamination of urine by menstrual blood in
females
• Contamination of urine by oxidizing agent (e.g.
hypochlorite or bleach used to clean urine containers),
or microbial peroxidase in urinary tract infection.
Causes of false-negative tests:
• Presence of a reducing agent like ascorbic acid in high
concentration: Microscopic examination for red cells
is positive but chemical test is negative.
• Use of formalin as a preservative for urine
Evaluation of positive chemical test for blood is
shown in Figure 1.20.
Hemoglobin
Presence of free hemoglobin in urine is called as
hemoglobinuria.
Causes of Hemoglobinuria
1. Hematuria with subsequent lysis of red blood cells
in urine of low specific gravity.
2. Intravascular hemolysis: Hemoglobin will appear in
urine when haptoglobin (to which hemoglobin binds
in plasma) is completely saturated with hemoglobin.
Intravascular hemolysis occurs in infections (severe
falciparum malaria, clostridial infection, E. coli
septicemia), trauma to red cells (march hemo-
globinuria, extensive burns, prosthetic heart valves),
glucose-6-phosphate dehydrogenase deficiency
following exposure to oxidant drugs, immune
hemolysis (mismatched blood transfusion, paroxy-
smal cold hemoglobinuria), paroxysmal nocturnal
hemoglobinuria, hemolytic uremic syndrome, and
disseminated intravascular coagulation.
Tests for Detection of Hemoglobinuria
Tests for detection of hemoglobinuria are benzidine test,
orthotoluidine test, and reagent strip test.
Hemosiderin
Hemosiderin in urine (hemosiderinuria) indicates
presence of free hemoglobin in plasma. Hemosiderin
appears as blue granules when urine sediment is stained
with Prussian blue stain (Fig. 1.21). Granules are located
inside tubular epithelial cells or may be free if cells have
disintegrated. Hemosiderinuria is seen in intravascular
hemolysis.
Myoglobin
Myoglobin is a protein present in striated muscle (skeletal
and cardiac) which binds oxygen. Causes of myoglo-
binuria include injury to skeletal or cardiac muscle, e.g.
crush injury, myocardial infarction, dermatomyositis,
severe electric shock, and thermal burns.
 Examination of Urine 21

reagent strip format that can detect significant

Fig. 1.21: Staining of urine sediment with Prussian blue
stain to demonstrate hemosiderin granules (blue)
Chemical tests used for detection of blood or
hemoglobin also give positive reaction with myoglobin
(as both hemoglobin and myoglobin have peroxidase
activity). Ammonium sulfate solubility test is used as a
screening test for myoglobinuria (Myoglobin is soluble
in 80% saturated solution of ammonium sulfate, while
hemoglobin is insoluble and is precipitated. A positive
chemical test for blood done on supernatant indicates
myoglobinuria).
Distinction between hematuria, hemoglobinuria, and
myoglobinuria is shown in Table 1.7.
Chemical Tests for Significant Bacteriuria
(Indirect Tests for Urinary Tract Infection)
In addition to direct microscopic examination of urine
sample, chemical tests are commercially available in a
bacteriuria: nitrite test and leucocyte esterase test. These
tests are helpful at places where urine microscopy is not
available. If these tests are positive, urine culture is
indicated.
1. Nitrite test: Nitrites are not present in normal urine;
ingested nitrites are converted to nitrate and excreted
in urine. If gram-negative bacteria (e.g. E.coli,
Salmonella, Proteus, Klebsiella, etc.) are present in urine,
they will reduce the nitrates to nitrites through the
action of bacterial enzyme nitrate reductase. Nitrites
are then detected in urine by reagent strip tests. As E.
coli is the commonest organism causing urinary tract
infection, this test is helpful as a screening test for
urinary tract infection.
Some organisms like Staphylococci or Pseudomonas do
not reduce nitrate to nitrite and therefore in such
infections nitrite test is negative. Also, urine must be
retained in the bladder for minimum of 4 hours for
conversion of nitrate to nitrite to occur; therefore, fresh
early morning specimen is preferred. Sufficient dietary
intake of nitrate is necessary. Therefore a negative nitrite
test does not necessarily indicate absence of urinary
tract infection.
The test detects about 70% cases of urinary tract
infections.
2. Leucocyte esterase test: It detects esterase enzyme
released in urine from granules of leucocytes. Thus
the test is positive in pyuria. If this test is positive,
urine culture should be done. The test is not sensitive
to leucocytes < 5/HPF.
MICROSCOPIC EXAMINATION
Microscopic examination of urine is also called as the
“liquid biopsy of the urinary tract”.
Urine consists of various microscopic, insoluble, solid
elements in suspension. These elements are classified as

Table 1.7: Differentiation between hematuria, hemoglobinuria, and myoglobinuria

Parameter Hematuria Hemoglobinuria Myoglobinuria

1. Urine color Normal, smoky, red, Pink, red, or Red or brown

or brown brown
2. Plasma color Normal Pink Normal
3. Urine test based on Positive Positive Positive
peroxidase activity
4. Urine microscopy Many red cells Occasional red cell Occasional red cell
5. Serum haptoglobin Normal Low Normal
6. Serum creatine kinase Normal Normal Markedly increased
 22 Essentials of Clinical Pathology

organized or unorganized. Organized substances
include red blood cells, white blood cells, epithelial cells,
casts, bacteria, and parasites. The unorganized sub-
stances are crystalline and amorphous material. These
elements are suspended in urine and on standing they
settle down and sediment at the bottom of the container;
therefore they are known as urinary deposits or urinary
sediments. Examination of urinary deposit is helpful in
diagnosis of urinary tract diseases as shown in Table 1.8.
Different types of urinary sediments are shown in
Figure 1.22. The major aim of microscopic examination
of urine is to identify different types of cellular elements
and casts. Most crystals have little clinical significance.
Specimen: The cellular elements are best preserved in
acid, hypertonic urine; they deteriorate rapidly in
alkaline, hypotonic solution. A mid-stream, freshly
voided, first morning specimen is preferred since it is
the most concentrated. The specimen should be
examined within 2 hours of voiding because cells and
casts degenerate upon standing at room temperature. If
preservative is required, then 1 crystal of thymol or 1
drop of formalin (40%) is added to about 10 ml of urine.
Method: A well-mixed sample of urine (12 ml) is
centrifuged in a centrifuge tube for 5 minutes at 1500
rpm and supernatant is poured off. The tube is tapped
at the bottom to resuspend the sediment (in 0.5 ml of
urine). A drop of this is placed on a glass slide and
covered with a cover slip (Fig. 1.23). The slide is examined
immediately under the microscope using first the low
power and then the high power objective. The condenser
should be lowered to better visualize the elements by
reducing the illumination.
Cells
Cellular elements in urine are shown in Figure 1.24.

Table 1.8: Urinary findings in renal diseases

Condition Albumin RBCs/HPF WBCs/HPF Casts/LPF Others

1. Normal 0-trace 0-2 0-2 Occasional –

(Hyaline)
2. Acute 1-2+ Numerous; 0-few Red cell, Smoky urine or
glomerulonephritis dysmorphic granular hematuria
3. Nephrotic syndrome >4+ 0-few 0-few Fatty, hyaline, Oval fat bodies,
Waxy, epithelial lipiduria
4. Acute pyelonephritis 0-1+ 0-few Numerous WBC, granular WBC clumps,
bacteria, nitrite test

HPF: High power field; LPF: Low power field; RBCs: Red blood cells; WBCs: White blood cells.

Fig. 1.22: Different types of urinary sediment

tahir99 - UnitedVRG
vip.persianss.ir
 Examination of Urine 23

Red Blood Cells

Normally there are no or an occasional red blood cell in

Fig. 1.23: Preparation of urine sediment for
microscopic examination
urine. In a fresh urine sample, red cells appear as small,
smooth, yellowish, anucleate biconcave disks about 7 μ
in diameter (called as isomorphic red cells). However,
red cells may appear swollen (thin discs of greater
diameter, 9-10 μ) in dilute or hypotonic urine, or may
appear crenated (smaller diameter with spikey surface)
in hypertonic urine. In glomerulonephritis, red cells are
typically described as being dysmorphic (i.e. markedly
variable in size and shape). They result from passage of
red cells through the damaged glomeruli. Presence of
> 80% of dysmorphic red cells is strongly suggestive of
glomerular pathology.
The quantity of red cells can be reported as number
of red cells per high power field.
Causes of hematuria have been listed earlier.

Fig. 1.24: Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red cells, (4) Dysmorphic red
cells, (5) White blood cells (pus cells), (6) Squamous epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial
cells, (9) Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa

tahir99 - UnitedVRG
vip.persianss.ir
 24 Essentials of Clinical Pathology
White Blood Cells (Pus Cells)
White blood cells are spherical, 10-15 μ in size, granular
in appearance in which nuclei may be visible. Degene-
rated white cells are distorted, smaller, and have fewer
granules. Clumps of numerous white cells are seen in
infections. Presence of many white cells in urine is called
as pyuria. In hypotonic urine white cells are swollen and
the granules are highly refractile and show Brownian
movement; such cells are called as glitter cells; large
numbers are indicative of injury to urinary tract.
Normally 0-2 white cells may be seen per high power
field. Pus cells greater than 10/HPF or presence of
clumps is suggestive of urinary tract infection.
Increased numbers of white cells occur in fever,
pyelonephritis, lower urinary tract infection, tubulo-
interstitial nephritis, and renal transplant rejection.
In urinary tract infection, following are usually seen
in combination:
• Clumps of pus cells or pus cells >10/HPF
• Bacteria
• Albuminuria
• Positive nitrite test
Simultaneous presence of white cells and white cell
casts indicates presence of renal infection (pyelo-
nephritis).
Eosinophils (>1% of urinary leucocytes) are a
characteristic feature of acute interstitial nephritis due to
drug reaction (better appreciated with a Wright’s stain).
Renal Tubular Epithelial Cells
Presence of renal tubular epithelial cells is a significant
finding. Increased numbers are found in conditions
causing tubular damage like acute tubular necrosis,
pyelonephritis, viral infection of kidney, allograft
rejection, and salicylate or heavy metal poisoning.
These cells are small (about the same size or slightly
larger than white blood cell), polyhedral, columnar, or
oval, and have granular cytoplasm. A single, large,
refractile, eccentric nucleus is often seen.
Renal tubular epithelial cells are difficult to distin-
guish from pus cells in unstained preparations.
Squamous Epithelial Cells
Squamous epithelial cells line the lower urethra and
vagina. They are best seen under low power objective
(×10). Presence of large numbers of squamous cells in
urine indicates contamination of urine with vaginal fluid.
These are large cells, rectangular in shape, flat with
abundant cytoplasm and a small, central nucleus.
Transitional Epithelial Cells
Transitional cells line renal pelvis, ureters, urinary
bladder, and upper urethra. These cells are large, and
diamond- or pear-shaped (caudate cells). Large numbers
or sheets of these cells in urine occur after catheterization
and in transitional cell carcinoma.
Oval Fat Bodies
These are degenerated renal tubular epithelial cells filled
with highly refractile lipid (cholesterol) droplets. Under
polarized light, they show a characteristic “Maltese
cross” pattern. They can be stained with a fat stain such
as Sudan III or Oil Red O. They are seen in nephrotic
syndrome in which there is lipiduria.
Spermatozoa
They may sometimes be seen in urine of men.
Telescoped urinary sediment: This refers to urinary
sediment consisting of red blood cells, white blood cells,
oval fat bodies, and all types of casts in roughly equal
proportion. It occurs in lupus nephritis, malignant
hypertension, rapidly proliferative glomerulonephritis,
and diabetic glomerulosclerosis.
Organisms
Organisms detectable in urine are shown in Figure 1.25.
Bacteria
Bacteria in urine can be detected by microscopic
examination, reagent strip tests for significant bacteriuria
(nitrite test, leucocyte esterase test), and culture.
Method of collection for bacteriologic examination
is given earlier in Box 1.2.
Significant bacteriuria exists when there are >105
bacterial colony forming units/ml of urine in a clean-
catch midstream sample, >104 colony forming units/ml
of urine in catheterized sample, and >10 3 colony-
forming units/ml of urine in a suprapubic aspiration
sample.
Fig. 1.25: Organisms in urine: (A) Bacteria, (B) Yeasts,
(C) Trichomonas, and (D) Egg of Schistosoma haematobium
tahir99 - UnitedVRG
vip.persianss.ir

1. Microscopic examination: In a wet preparation,
presence of bacteria should be reported only when
urine is fresh. Bacteria occur in combination with pus
cells. Gram’s-stained smear of uncentrifuged urine
showing 1 or more bacteria per oil-immersion field
suggests presence of > 105 bacterial colony forming
units/ml of urine. If many squamous cells are present,
then urine is probably contaminated with vaginal
flora. Also, presence of only bacteria without pus cells
indicates contamination with vaginal or skin flora.
2. Chemical or reagent strip tests for significant
bacteriuria: These are given earlier.
3. Culture: On culture, a colony count of >105/ml is
strongly suggestive of urinary tract infection, even
in asymptomatic females. Positive culture is followed
by sensitivity test. Most infections are due to Gram-
negative enteric bacteria, particularly Escherichia coli.
If three or more species of bacteria are identified on
culture, it almost always indicates contamination by
vaginal flora.
Negative culture in the presence of pyuria (‘sterile’
pyuria) occurs with prior antibiotic therapy, renal
tuberculosis, prostatitis, renal calculi, catheterization,
fever in children (irrespective of cause), female genital
tract infection, and non-specific urethritis in males.
Yeast Cells (Candida)
These are round or oval structures of approximately the
same size as red blood cells. In contrast to red cells, they
show budding, are oval and more refractile, and are not
soluble in 2% acetic acid.
Presence of Candida in urine may suggest immuno-
compromised state, vaginal candidiasis, or diabetes
Fig. 1.26: Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts
Examination of Urine 25
mellitus. Usually pyuria is present if there is infection
by Candida. Candida may also be a contaminant in the
sample and therefore urine sample must be examined in
a fresh state.
Trichomonas vaginalis
These are motile organisms with pear shape, undulating
membrane on one side, and four flagellae. They cause
vaginitis in females and are thus contaminants in urine.
They are easily detected in fresh urine due to their
motility.
Eggs of Schistosoma haematobium
Infection by this organism is prevalent in Egypt.
Microfilariae
They may be seen in urine in chyluria due to rupture of
a urogenital lymphatic vessel.
Casts
Urinary casts are cylindrical, cigar-shaped microscopic
structures that form in distal renal tubules and collecting
ducts. They take the shape and diameter of the lumina
(molds or ‘casts’) of the renal tubules. They have parallel
sides and rounded ends. Their length and width may be
variable. Casts are basically composed of a precipitate of
a protein that is secreted by tubules (Tamm-Horsfall
protein). Since casts form only in renal tubules their
presence is indicative of disease of the renal parenchyma.
Although there are several types of casts, all urine casts
are basically hyaline; various types of casts are formed
when different elements get deposited on the hyaline
material (Fig. 1.26). Casts are best seen under low power
tahir99 - UnitedVRG
vip.persianss.ir
 26 Essentials of Clinical Pathology
objective (×10) with condenser lowered down to reduce
the illumination.
Casts are the only elements in the urinary sediment
that are specifically of renal origin.
Casts (Fig. 1.27) are of two main types:
• Noncellular: Hyaline, granular, waxy, fatty
• Cellular: Red blood cell, white blood cell, renal
tubular epithelial cell.
Hyaline and granular casts may appear in normal or
diseased states. All other casts are found in kidney
diseases.
Non-cellular Casts
Hyaline casts: These are the most common type of casts
in urine and are homogenous, colorless, transparent, and
refractile. They are cylindrical with parallel sides and
blunt, rounded ends and low refractive index. Presence
of occasional hyaline cast is considered as normal. Their
presence in increased numbers (“cylinduria”) is
abnormal. They are composed primarily of Tamm-
Horsfall protein. They occur transiently after strenuous
Fig. 1.27: Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast, (E) Red cell cast,
(F) White cell cast, and (G) Epithelial cast
muscle exercise in healthy persons and during fever.
Increased numbers are found in conditions causing
glomerular proteinuria.
Granular casts: Presence of degenerated cellular debris in
a cast makes it granular in appearance. These are
cylindrical structures with coarse or fine granules (which
represent degenerated renal tubular epithelial cells)
embedded in Tamm-Horsfall protein matrix. They are
seen after strenuous muscle exercise and in fever, acute
glomerulonephritis, and pyelonephritis.
Waxy cast: These are the most easily recognized of all
casts. They form when hyaline casts remain in renal
tubules for long time (prolonged stasis). They have
homogenous, smooth glassy appearance, cracked or
serrated margins and irregular broken-off ends. The ends
are straight and sharp and not rounded as in other casts.
They are light yellow in color. They are most commonly
seen in end-stage renal failure.
Fatty casts: These are cylindrical structures filled with
highly refractile fat globules (triglycerides and cholesterol
tahir99 - UnitedVRG
vip.persianss.ir

esters) in Tamm-Horsfall protein matrix. They are seen
in nephrotic syndrome.
Broad casts: Broad casts form in dilated distal tubules and
are seen in chronic renal failure and severe renal tubular
obstruction. Both waxy and broad casts are associated
with poor prognosis.
Cellular Casts
To be called as cellular, casts should contain at least three
cells in the matrix. Cellular casts are named according to
the type of cells entrapped in the matrix.
Red cell casts: These are cylindrical structures with red
cells in Tamm-Horsfall protein matrix. They may appear
brown in color due to hemoglobin pigmentation. These
have greater diagnostic importance than any other cast.
If present, they help to differentiate hematuria due to
glomerular disease from hematuria due to other causes.
RBC casts usually denote glomerular pathology e.g. acute
glomerulonephritis.
White cell casts: These are cylindrical structures with white
blood cells embedded in Tamm-Horsfall protein matrix.
Leucocytes usually enter into tubules from the inter-
stitium and therefore presence of leucocyte casts indicates
tubulointerstitial disease like pyelonephritis.
Renal tubular epithelial cell casts: These are composed of
renal tubular epithelial cells that have been sloughed off.
They are seen in acute tubular necrosis, viral renal
disease, heavy metal poisoning, and acute allograft
rejection. Even an occasional renal tubular cast is a
significant finding.
Crystals
Crystals are refractile structures with a definite geometric
shape due to orderly 3-dimensional arrangement of its
atoms and molecules. Amorphous material (or deposit)
has no definite shape and is commonly seen in the form
of granular aggregates or clumps.
Crystals in urine (Fig. 1.28) can be divided into two
main types: (1) Normal (seen in normal urinary
sediment), and (2) Abnormal (seen in diseased states).
However, crystals found in normal urine can also be seen
in some diseases in increased numbers.
Most crystals have no clinical importance
(particularly phosphates, urates, and oxalates). Crystals
can be identified in urine by their morphology. However,
before reporting presence of any abnormal crystals, it is
necessary to confirm them by chemical tests.
Examination of Urine 27
Normal Crystals
Crystals present in acid urine
a. Uric acid crystals: These are variable in shape
(diamond, rosette, plates), and yellow or red-brown
in color (due to urinary pigment). They are soluble in
alkali, and insoluble in acid. Increased numbers are
found in gout and leukemia. Flat hexagonal uric acid
crystals may be mistaken for cysteine crystals that also
form in acid urine.
b. Calcium oxalate crystals: These are colorless, refractile,
and envelope-shaped. Sometimes dumbbell-shaped
or peanut-like forms are seen. They are soluble in
dilute hydrochloric acid. Ingestion of certain foods
like tomatoes, spinach, cabbage, asparagus, and
rhubarb causes increase in their numbers. Their
increased number in fresh urine (oxaluria) may also
suggest oxalate stones. A large number are seen in
ethylene glycol poisoning.
c. Amorphous urates: These are urate salts of potassium,
magnesium, or calcium in acid urine. They are usually
yellow, fine granules in compact masses. They are
soluble in alkali or saline at 60°C.
Crystals present in alkaline urine:
a. Calcium carbonate crystals: These are small, colorless,
and grouped in pairs. They are soluble in acetic acid
and give off bubbles of gas when they dissolve.
b. Phosphates: Phosphates may occur as crystals (triple
phosphates, calcium hydrogen phosphate), or as
amorphous deposits.
• Phosphate crystals
 Triple phosphates (ammonium magnesium
phosphate): They are colorless, shiny, 3-6 sided
prisms with oblique surfaces at the ends (“coffin-
lids”), or may have a feathery fern-like appearance.
 Calcium hydrogen phosphate (stellar phosphate):
These are colorless, and of variable shape (star-
shaped, plates or prisms).
• Amorphous phosphates: These occur as colorless
small granules, often dispersed.
All phosphates are soluble in dilute acetic acid.
c. Ammonium urate crystals: These occur as cactus-like
(covered with spines) and called as ‘thornapple’
crystals. They are yellow-brown and soluble in acetic
acid at 60°C.
Abnormal Crystals
They are rare, but result from a pathological process.
These occur in acid pH, often in large amounts. Abnormal
tahir99 - UnitedVRG
vip.persianss.ir
 28 Essentials of Clinical Pathology

Fig. 1.28: Crystals in urine. (A) Normal crystals: (1) Calcium oxalate, (2) Triple phosphates, (3) Uric acid, (4) Amorphous
phosphates, (5) Amorphous urates, (6) Ammonium urate. (B) Abnormal crystals: (1) Cysteine, (2) Cholesterol, (3) Bilirubin,
(4) Tyrosine, (5) Sulfonamide, and (6) Leucine

crystals should not be reported on microscopy alone;
additional chemical tests are done for confirmation.
1. Cysteine crystals: These are colorless, clear, hexagonal
(having 6 sides), very refractile plates in acid urine.
They often occur in layers. They are soluble in 30%
3. Bilirubin crystals: These are small (5 μ), brown crystals
hydrochloric acid. They are seen in cysteinuria, an
inborn error of metabolism. Cysteine crystals are often
associated with formation of cysteine stones.
2. Cholesterol crystals: These are colorless, refractile, flat
rectangular plates with notched (missing) corners,
and appear stacked in a stair-step arrangement. They
are soluble in ether, chloroform, or alcohol. They are
seen in lipiduria e.g. nephrotic syndrome and hyper-
cholesterolemia. They can be positively identified by
polarizing microscope.
of variable shape (square, bead-like, or fine needles).
Their presence can be confirmed by doing reagent
strip or chemical test for bilirubin. These crystals are
soluble in strong acid or alkali. They are seen in severe
obstructive liver disease.

tahir99 - UnitedVRG
vip.persianss.ir

4. Leucine crystals: These are refractile, yellow or brown,
spheres with radial or concentric striations. They are
soluble in alkali. They are usually found in urine along
with tyrosine in severe liver disease (cirrhosis).
5. Tyrosine crystals: They appear as clusters of fine,
delicate, colorless or yellow needles and are seen in
liver disease and tyrosinemia (an inborn error of
metabolism). They dissolve in alkali.
6. Sulfonamide crystals: They are variably shaped
crystals, but usually appear as sheaves of needles.
They occur following sulfonamide therapy. They are
soluble in acetone.
REFERENCE RANGES
Volume in 24 hours: Adults: 600-2000 ml
Color: Pale yellow to colorless
Appearance: Clear
Odor: Aromatic
Specific gravity: 1.003-1.030
Osmolality: 300-900 mOsm/kg of water
pH: 4.6-8.0 (Average: 6.0)
Proteins: Qualitative test: Negative
Quantitative test: < 150 mg/24 hours
Albumin: < 30 mg/24 hours
Glucose: Qualitative test: Negative
Quantitative test: < 500 mg/24 hours (< 15 mg/dl)
Ketones: Qualitative test: Negative
Bilirubin: Negative
Bile salts: Negative
Occult blood: Negative
Urobilinogen: 0.5-4.0 mg/24 hours
Myoglobin (Ammonium sulphate solubility test):
Negative
Microscopy: 1-2 red cells, pus cells, or epithelial cells/
HPF; occasional hyaline cast/LPF; Phosphate, oxalate,
or urate crystals depending on urine pH.
Examination of Urine 29
CRITICAL FINDINGS
• Strongly positive test for glucose and ketone bodies
• Positive test for reducing sugar in an infant
• Hemoglobinuria
• Red cell casts or >50% dysmorphic red cells on
microscopic examination
• Abnormal crystals like cysteine, leucine, or tyrosine.
BIBLIOGRAPHY
1. Burtis CA, Ashwood ER (Eds). Tietz fundamentals of
clinical chemistry (5th Ed). Philadelphia; WB Saunders
Company, 2001.
2. Carroll MF, Temte JL. Proteinuria in adults: A diagnostic
approach. Am Fam Physician 2000;62:1333-40.
3. Cheesbrough M. District laboratory practice in tropical
countries. Part 1 and Part 2. Cambridge; Cambridge
University Press, 1998.
4. Grossfeld GD, Wolf JS, Litwin MS, et al. Asymptomatic
microscopic hematuria in adults: Summary of the AUA
best policy recommendations. Am Fam Physician 2001;
63:1145-54.
5. Henry JB (Ed): Clinical diagnosis and management by
laboratory methods. (20th Ed). Philadelphia; WB Saunders
Company, 2001.
6. King M. A medical laboratory for developing countries.
London. Oxford University Press, 1973.
7. Mathieson PW. The cellular basis of albuminuria. Clinical
Science 2004;107:533-8.
8. Simerville JA, Maxted WC, Pahira JJ. Urinalysis: A
comprehensive review. Am Fam Physician 2005;71:
1153-62.
9. Wallach J. Interpretation of diagnostic tests. (7th Ed).
Philadelphia. Lippincott Williams and Wilkins, 2000.
10. World Health Organization. Manual of basic techniques
for a health laboratory (2nd Ed). Geneva; World Health
Organization, 2003.
tahir99 - UnitedVRG
vip.persianss.ir
 2
Renal Function Tests
Kidney is a highly specialized organ that performs
following functions:
• Maintenance of extracellular fluid volume and
composition: Kidney regulates water and electrolyte
balance, acid-base balance, and fluid osmotic
pressure.
• Excretion of metabolic waste products (blood urea,
creatinine, uric acid) and drugs, but retention of
essential substances (like glucose and amino acids).
• Regulation of blood pressure by renin-angiotensin
mechanism
• Synthesis of erythropoietin, a hormone which
stimulates erythropoiesis
• Production of vit. D3 (active form of vit. D) from vit.
D2, which stimulates absorption of calcium from
gastrointestinal tract.
FACTORS AFFECTING RENAL FUNCTION
Kidney function is affected by following factors:
• Diffuse renal disease.
• Pre-renal conditions—Decreased renal blood flow as
in dehydration, congestive cardiac failure and shock.
• Post-renal conditions—Obstruction to urinary
outflow.
INDICATIONS FOR RENAL FUNCTION TESTS
1. Early identification of impairment of renal function
in patients with increased risk of chronic renal
disease: Early detection and treatment of renal
impairment in chronic renal disease prevent compli-
cations of chronic renal failure and is associated with
improved prognosis. Laboratory tests can be applied
in individuals who are at increased risk of developing
chronic renal disease (Box 2.1) to detect renal
functional impairment at an early stage and to detect
degree of kidney damage.
2. Diagnosis of renal disease
3. Follow the course of renal disease and assess
response to treatment.
Box 2.1: Conditions with increased risk of
chronic renal disease
• Diabetes mellitus
• Hypertension
• Autoimmune diseases like systemic lupus erythematosus
• Older age (GFR declines with age)
• Family history of renal disease
• Systemic infection
• Urinary tract infection
• Lower urinary tract obstruction
4. Plan renal replacement therapy (dialysis or renal
transplantation) in advanced renal disease.
5. Adjust dosage of certain drugs (e.g. chemotherapy)
according to renal function.
CLASSIFICATION OF
RENAL FUNCTION TESTS
Renal function tests can be classified as shown in Table
2.1.
In practice, the commonly performed renal function
tests are routine urinalysis, estimation of serum
creatinine, blood urea nitrogen (BUN), BUN/Serum
creatinine ratio, creatinine clearance test (or estimation
of GFR from serum creatinine value by a prediction
equation), and estimation of urine concentrating ability
(water deprivation test). Urine examination is the first
test performed in patients suspected of having renal
disease. It is the simplest and the least expensive renal
function test. In urine examination parameters that can
assess renal function are urine volume in 24 hours,
specific gravity, osmolality, proteinuria, and microscopic
examination of urinary sediment.
Tests to Evaluate Glomerular Function
The best test to assess overall kidney function is
estimation of glomerular filtration rate or GFR (Box 2.2).
GFR varies according to age, sex, and body surface area.
tahir99 - UnitedVRG
vip.persianss.ir

Table 2.1: Classification of renal function tests
Tests to evaluate glomerular function
1. Clearance tests to measure glomerular
filtration rate: Inulin clearance, 125I-iothalamate
clearance, 51Cr-EDTA clearance, Cystatin C
clearance, Creatinine clearance, and Urea
clearance
2. Calculation of creatinine clearance from
prediction equations
3. Blood biochemistry: Serum creatinine,
Blood urea nitrogen (BUN), and BUN/serum creatinine ratio
4. Microalbuminuria and albuminuria
Box 2.2: Glomerular filtration rate (GFR)
• Best test for assessment of excretory renal function
• Varies according to age, sex, and body weight of an
individual; a normal GFR also depends on normal
renal blood flow and pressure.
• Normal GFR in young adults is 120-130 ml/min per
1.73 m2.
• Creatinine clearance is commonly used as a measure
of GFR. Equations can be used to estimate GFR
from serum creatinine value.
• GFR declines with age (due to glomerular arteriolo-
sclerosis)
• GFR <60 ml/min per 1.73 m2 indicates loss of ≥50%
of kidney function.
• Fall in GFR leads to accumulation of waste products
of metabolism in blood. GFR <15 ml/min per 1.73 m2
is associated with uremia.
Normal GFR in young adults is 120-130 ml/min per 1.73
m2 of body surface area. GFR declines progressively with
age (due to arteriolosclerosis of glomeruli). After 40 years
of age, there is a steady and progressive fall in the GFR
at the rate of 1 ml/minute/year because of reduction in
the number of glomeruli due to arteriolosclerosis.
GFR is measured to (i) detect suspected incipient
kidney disease (i.e. early detection), (ii) monitor course
of established kidney disease, (iii) plan renal replacement
therapy in advanced renal disease, and (iv) adjust dosage
of certain drugs which are nephrotoxic.
Based on GFR, chronic kidney disease is divided into
following stages (US National Kidney Foundation
Kidney Disease Quality Outcomes Initiative Classifi-
cation of Chronic Kidney Disease, 2002):
• Stage 1: Kidney damage with normal or increased
GFR (GFR ≥ 90 ml/min/1.73 m2)
• Stage 2: Kidney damage with mildly reduced GFR
(GFR 60-89 ml/min/1.73 m2)
Renal Function Tests 31
Tests to evaluate tubular function
1. Tests to assess proximal tubular
function:
• Glycosuria, phosphaturia, uricosuria
• Generalized aminoaciduria
• Tubular proteinuria
• Fractional sodium excretion
2. Tests to assess distal tubular function:
• Specific gravity and osmolality of urine
• Water-deprivation test and water-loading test
• Ammonium chloride loading test
• Stage 3: Moderately reduced GFR (GFR 30-59 ml/
min/1.73 m2)
• Stage 4: Severely reduced GFR (GFR 15-29 ml/min/
1.73 m2)
• Stage 5: Kidney failure (GFR < 15 ml/min/1.73 m2)
Kidney damage refers to presence of pathological
abnormalities or markers of damage like abnormalities
in blood or urine tests or imaging studies. Symptoms
usually develop at or after stage 3. GFR <60 ml/min per
1.73 m2 indicates loss of ≥ 50% of kidney function. GFR
<15 ml/min per 1.73 m2 is associated with kidney failure
and uremia. Following methods are used to measure
GFR: (1) Clearance tests and (2) Prediction equations.
Clearance Tests to Measure Glomerular
Filtration Rate (GFR)
Glomerular filtration rate refers to the rate in ml/min at
which a substance is cleared from the circulation by the
glomeruli. The ability of the glomeruli to filter a substance
from the blood is assessed by clearance studies. If a
substance is not bound to protein in plasma, is completely
filtered by the glomeruli, and is neither secreted nor
reabsorbed by the tubules, then its clearance rate is equal
to the glomerular filtration rate (GFR). Clearance of a
substance refers to the volume of plasma, which is
completely cleared of that substance per minute; it is
calculated from the following formula:
UV
Clearance = ——
P
where, U = concentration of a substance in urine in
mg/dl; V = volume of urine excreted in ml/min; and P
= concentration of the substance in plasma in mg/dl.
Since U and P are in the same units, they cancel each
other and the clearance value is expressed in the same
unit as V i.e. ml/min. All clearance values are adjusted
to a standard body surface area i.e. 1.73 m2.
tahir99 - UnitedVRG
vip.persianss.ir
 32 Essentials of Clinical Pathology
The agents used for measurement of GFR are:
• Exogenous: Inulin, Radiolabelled ethylenediamine
tetraacetic acid (51Cr- EDTA), 125I-iothalamate
• Endogenous: Creatinine, Urea, Cystatin C
The agent used for measurement of GFR should have
following properties: (1) It should be physiologically inert
and preferably endogenous, (2) It should be freely filtered
by glomeruli and should be neither reabsorbed nor
secreted by renal tubules, (3) It should not bind to plasma
proteins and should not be metabolized by kidneys, and
(4) It should be excreted only by the kidneys. However,
there is no such ideal endogenous agent.
Clearance tests are cumbersome to perform,
expensive, and not readily available. One major
problem with clearance studies is incomplete urine
collection.
Abnormal clearance occurs in: (i) pre-renal factors:
reduced blood flow due to shock, dehydration, and
congestive cardiac failure; (ii) renal diseases; and
(iii) obstruction to urinary outflow.
Inulin Clearance
Inulin, an inert plant polysaccharide (a fructose polymer),
is filtered by the glomeruli and is neither reabsorbed nor
secreted by the tubules; therefore it is an ideal agent for
measuring GFR. A bolus dose of inulin (25 ml of 10%
solution IV) is administered followed by constant
intravenous infusion (500 ml of 1.5% solution at the rate
of 4 ml/min). Timed urine samples are collected and
blood samples are obtained at the midpoint of timed
urine collection. This test is considered as the ‘gold
standard’ (or reference method) for estimation of GFR.
However, this test is rarely used because it is time
consuming, expensive, constant intravenous infusion of
inulin is needed to maintain steady plasma level, and
difficulties in laboratory analysis. Average inulin
clearance for males is 125 ml/min/1.73 m2 and for
females is 110 ml/min/1.73 m2. In children less than 2
years and in older adults, clearance is low. This test is
largely limited to clinical research.
Clearance of Radiolabeled Agents
Urinary clearance of radiolabeled iothalamate (125I-
iothalamate) correlates closely with inulin clearance.
However, this method is expensive with risk of exposure
to radioactive substances. Other radiolabelled substances
used are 51Cr-EDTA and 99Tc-DTPA.
Cystatin C Clearance
This is a cysteine protease inhibitor of MW 13,000, which
is produced at a constant rate by all the nucleated cells.
It is not bound to protein, is freely filtered by glomeruli
and is not returned to circulation after filtration. It is a
more sensitive and specific marker of impaired renal
function than plasma creatinine. Its level is not affected
by sex, diet, or muscle mass. It is thought that cystatin C
is a superior marker for estimation of GFR than creatinine
clearance. It is measured by immunoassay.
Creatinine Clearance
This is the most commonly used test for measuring GFR.
Creatinine is being produced constantly from creatine
in muscle. It is completely filtered by glomeruli and is
not reabsorbed by tubules; however, a small amount is
secreted by tubules.
A 24-hour urine sample is preferred to overcome the
problem of diurnal variation of creatinine excretion and
to reduce the inaccuracy in urine collection.
After getting up in the morning, the first voided urine
is discarded. Subsequently all the urine passed is
collected in the container provided. After getting up in
the next morning, the first voided urine is also collected
and the container is sent to the laboratory. A blood
sample for estimation of plasma creatinine is obtained
at midpoint of urine collection. Creatinine clearance is
calculated from (1) concentration of creatinine in urine
in mg/ml (U), (2) volume of urine excreted in ml/min
(V) (this is calculated by the formula: volume of urine
collected/collection time in minutes e.g. volume of urine
collected in 24 hours ÷ 1440), and (3) concentration of
creatinine in plasma in mg/dl (P). Creatinine clearance
in ml/min per 1.73 m2 is then derived from the formula
UV/P.
Because of secretion of creatinine by renal tubules,
the above formula overestimates GFR by about 10%. In
advanced renal failure, secretion of creatinine by tubules
is increased and thus overestimation of GFR is even more.
Jaffe’s reaction (see later under serum creatinine) used
for estimation of creatinine measures creatinine as well
as some other substances (non-creatinine chromogens)
in blood and thus gives slightly higher result. Thus effect
of tubular secretion of creatinine is somewhat balanced
by slight overestimation of serum creatinine by Jaffe’s
reaction.
To provide values closer to the actual GFR, cimetidine
(which blocks secretion by renal tubules) can be
administered before commencing urine collection
(cimetidine-enhanced creatinine clearance).
Creatinine clearance is not an ideal test for estimation
of GFR because of following reasons:
1. A small amount of creatinine is secreted by renal
tubules that increase even further in advanced renal
failure.
2. Collection of urine is often incomplete.
tahir99 - UnitedVRG
vip.persianss.ir

3. Creatinine level is affected by intake of meat and
muscle mass.
4. Creatinine level is affected by certain drugs like
cimetidine, probenecid, and trimethoprim (which
block tubular secretion of creatinine).
Urea Clearance
Urea is filtered by the glomeruli, but about 40% of the
filtered amount is reabsorbed by the tubules. The
reabsorption depends on the rate of urine flow. Thus it
underestimates GFR, depends on the urine flow rate, and
is not a sensitive indicator of GFR.
BUN and serum creatinine, by themselves, are not
sensitive indicators of early renal impairment since
values may be normal e.g. if baseline values of serum
creatinine is 0.5 mg/dl, then 50% reduction in kidney
function would increase it to 1.0 mg/dl. Thus clearance
tests are more helpful in early cases. If biochemical tests
are normal and renal function impairment is suspected,
then creatinine clearance test should be carried out. If
biochemical tests are abnormal, then clearance tests need
not be done.
Estimation of Creatinine Clearance from Serum
Creatinine by Prediction Equations
One can estimate GFR from age, sex, body weight, and
serum creatinine value of a person from the following
formula (Cockcroft and Gault):
Creatinine clearance
(140 - Age in years) × (Body weight in kg)
in ml/min =
(72 × Serum creatinine in mg/dl)
In females, the value obtained from above equation
is multiplied by 0.85 to get the result.
Fig. 2.1: Formation of urea from protein breakdown
Renal Function Tests 33
It is recommended by National Kidney Foundation
(USA) to calculate creatinine clearance by Cockcroft and
Gault or other equation from serum creatinine value
rather than estimating creatinine clearance from a 24-
hour urine sample. This is because the latter test is
inconvenient, time-consuming, and often inaccurate.
Blood Biochemistry
Two biochemical parameters are commonly used to
assess renal function: blood urea nitrogen (BUN) and
serum creatinine. Although convenient, they are
insensitive markers of glomerular function.
Blood Urea Nitrogen (BUN)
Urea is produced in the liver from amino acids (ingested
or tissue-derived). Amino acids are utilized to produce
energy, synthesize proteins, and are catabolized to
ammonia. Urea is produced in the liver from ammonia
in the Krebs urea cycle. Ammonia is toxic and hence is
converted to urea, which is then excreted in urine
(Fig. 2.1).
The concentration of blood urea is usually expressed
as blood urea nitrogen. This is because older methods
estimated only the nitrogen in urea. Molecular weight
of urea is 60, and 28 grams of nitrogen are present in a
gm mole of urea. As the relationship between urea and
BUN is 60/28, BUN can be converted to urea by
multiplying BUN by 2.14, i.e. the real concentration of
urea is BUN × (60/28).
Urea is completely filtered by the glomeruli, and
about 30-40% of the filtered amount is reabsorbed in the
renal tubules depending on the person’s state of
hydration
Blood level of urea is affected by a number of non-
renal factors (e.g. high protein diet, upper gastrointestinal
 34 Essentials of Clinical Pathology

hemorrhage, liver function, etc.) and therefore utility of
BUN as an indicator of renal function is limited. Also
considerable destruction of renal parenchyma is required
before elevation of blood urea can occur.
The term azotemia refers to the increase in the blood
level of urea; uremia is the clinical syndrome resulting
from this increase. If renal function is absent, BUN rises
by 10-20 mg/dl/day.
Causes of increased BUN:
1. Pre-renal azotemia: shock, congestive heart failure,
salt and water depletion
2. Renal azotemia: impairment of renal function
3. Post-renal azotemia: obstruction of urinary tract
4. Increased rate of production of urea:
• High protein diet
• Increased protein catabolism (trauma, burns,
fever)
• Absorption of amino acids and peptides from a
large gastrointestinal hemorrhage or tissue
hematoma
• It is not reabsorbed, and very little is secreted by
tubules.
With muscle mass remaining constant, increased
creatinine level reflects reduction of glomerular filtration
rate. However, because of significant kidney reserve,
increase of serum creatinine level (from 1.0 mg/dl to 2.0
mg/dl) in blood does not occur until about 50% of kidney
function is lost. Therefore, serum creatinine is not a
sensitive indicator of early renal impairment. Also,
laboratory report showing serum creatinine “within
normal range” does not necessarily mean that the level
is normal; the level should be correlated with body
weight, age, and sex of the individual. If renal function
is absent, serum creatinine rises by 1.0 to 1.5 mg/dl/day
(Fig. 2.2).

Methods for estimation of BUN:

Two methods are commonly used.
1. Diacetyl monoxime urea method: This is a direct
method. Urea reacts with diacetyl monoxime at high
temperature in the presence of a strong acid and an
oxidizing agent. Reaction of urea and diacetyl
monoxime produces a yellow diazine derivative. The
intensity of color is measured in a colorimeter or
spectrophotometer.
2. Urease- Berthelot reaction: This is an indirect method.
Enzyme urease splits off ammonia from the urea
molecule at 37°C. Ammonia generated is then reacted Fig. 2.2: Relationship between glomerular filtration rate and
serum creatinine. Significant increase of serum creatinine does
with alkaline hypochlorite and phenol with a catalyst
not occur till a considerable fall in GFR
to produce a stable color (indophenol). Intensity of
color produced is then measured in a spectro-
Causes of Increased Serum Creatinine Level
photometer at 570 nm.
Reference range for BUN in adults is 7-18 mg/dl. In 1. Pre-renal, renal, and post-renal azotemia
adults > 60 years, level is 8-21 mg/dl. 2. Large amount of dietary meat
3. Active acromegaly and gigantism
Serum Creatinine Causes of Decreased Serum Creatinine Level
Creatinine is a nitrogenous waste product formed in 1. Pregnancy
muscle from creatine phosphate. Endogenous production 2. Increasing age (reduction in muscle mass)
of creatinine is proportional to muscle mass and body
weight. Exogenous creatinine (from ingestion of meat) Methods for Estimation of Serum Creatinine
has little effect on daily creatinine excretion. The test for serum creatinine is cheap, readily available,
Serum creatinine is a more specific and more sensitive and simple to perform. There are two methods that are
indicator of renal function as compared to BUN because: commonly used:
• It is produced from muscles at a constant rate and its 1. Jaffe’s reaction (Alkaline picrate reaction):
level in blood is not affected by diet, protein This is the most widely used method. Creatinine
catabolism, or other exogenous factors; reacts with picrate in an alkaline solution to produce

a yellow-red color. The color is measured in a
spectrophotometer at 485 nm. Certain substances in
plasma (such as glucose, protein, fructose, ascorbic
acid, acetoacetate, acetone, and cephalosporins) react
with picrate in a similar manner; these are called as
non-creatinine chromogens (and can cause false
elevation of serum creatinine level). Thus ‘true’
creatinine is less by 0.2 to 0.4 mg/dl when estimated
by Jaffe’s reaction.
2. Enzymatic methods: These methods use enzymes
that cleave creatinine; hydrogen peroxide produced
then reacts with phenol and a dye to produce a
colored product, which is measured in a spectro-
photometer.
Reference range:
Adult males: 0.7-1.3 mg/dl.
Adult females: 0.6-1.1 mg/dl.
Serum creatinine alone should not be used to assess
renal function. This is because serum creatinine
concentration depends on age, sex, muscle mass,
glomerular filtration and amount of tubular secretion.
Thus, normal serum creatinine range is wide. Serum
creatinine begins to rise when GFR falls below 50% of
normal. Minor rise of serum creatinine is associated with
significant reduction of GFR (Fig 2.2). Therefore early
stage of chronic renal impairment cannot be detected by
measurement of serum creatinine alone.
BUN/Serum Creatinine Ratio
Clinicians commonly calculate BUN/creatinine ratio to
discriminate pre-renal and post-renal azotemia from
renal azotemia. Normal ratio is 12:1 to 20:1.
Causes of Increased BUN/Creatinine Ratio (>20:1):
1. Increased BUN with normal serum creatinine:
• Pre-renal azotemia (reduced renal perfusion)
• High protein diet
• Increased protein catabolism
• Gastrointestinal hemorrhage
2. Increase of both BUN and serum creatinine with
disproportionately greater increase of BUN:
• Post-renal azotemia (Obstruction to the outflow
of urine)
Obstruction to the urine outflow causes diffusion
of urinary urea back into the blood from tubules
because of backpressure.
Renal Function Tests 35
Causes of Decreased BUN/Creatinine Ratio (<10:1)
• Acute tubular necrosis
• Low protein diet, starvation
• Severe liver disease
Microalbuminuria and Albuminuria
Normally, a very small amount of albumin is excreted
in urine. The earliest evidence of glomerular damage in
diabetes mellitus is occurrence of microalbuminuria
(albuminuria in the range of 30 to 300 mg/24 hours). An
albuminuria > 300-mg/24 hour is termed clinical or overt
and indicates significant glomerular damage. (See
“Proteinuria” under Chapter 1 “Examination of Urine”).
Tests to Evaluate Tubular Function
Tests to Assess Proximal Tubular Function
Renal tubules efficiently reabsorb 99% of the glomerular
filtrate to conserve the essential substances like glucose,
amino acids, and water.
1. Glycosuria: In renal glycosuria, glucose is excreted in
urine, while blood glucose level is normal. This is
because of a specific tubular lesion which leads to
impairment of glucose reabsorption. Renal glycosuria
is a benign condition. Glycosuria can also occur in
Fanconi syndrome.
2. Generalized aminoaciduria: In proximal renal tubular
dysfunction, many amino acids are excreted in urine
due to defective tubular reabsorption.
3. Tubular proteinuria (Low molecular weight proteinuria):
Normally, low molecular weight proteins (β 2 –
microglobulin, retinol-binding protein, lysozyme, and
α1-microglobulin) are freely filtered by glomeruli and
are completely reabsorbed by proximal renal tubules.
With tubular damage, these low molecular weight
proteins are excreted in urine and can be detected by
urine protein electrophoresis. Increased amounts of
these proteins in urine are indicative of renal tubular
damage.
4. Urinary concentration of sodium: If both BUN and serum
creatinine are acutely increased, it is necessary to
distinguish between prerenal azotemia (renal
underperfusion) and acute tubular necrosis. In
prerenal azotemia, renal tubules are functioning
normally and reabsorb sodium, while in acute tubular
necrosis, tubular function is impaired and sodium
absorption is decreased. Therefore, in prerenal
azotemia, urinay sodium concentration is < 20 mEq/
L while in acute tubular necrosis, it is > 20 mEq/L.
 36 Essentials of Clinical Pathology
5. Fractional excretion of sodium (FENa): Measurement of
urinary sodium concentration is affected by urine
volume and can produce misleading results. There-
fore, to avoid this, fractional excretion of sodium is
calculated. This refers to the percentage of filtered
sodium that has been absorbed and percentage that
has been excreted. Measurement of fractional sodium
excretion is a better indicator of tubular absorption
of sodium than quantitation of urine sodium alone.
This test is indicated in acute renal failure. In oliguric
patients, this is the most reliable means of early
distinction between pre-renal failure and renal failure
due to acute tubular necrosis. It is calculated from the
following formula:
(Urine sodium × Plasma creatinine)
× 100%
(Plasma sodium × Urine creatinine)
In pre-renal failure this ratio is less than 1%, and in
acute tubular necrosis it is more than 1%. In pre-renal
failure (due to reduced renal perfusion), aldosterone
secretion is stimulated which causes maximal sodium
conservation by the tubules and the ratio is less than 1%.
In acute tubular necrosis, maximum sodium reabsorption
is not possible due to tubular cell injury and consequently
the ratio will be more than 1%. Values above 3% are
strongly suggestive of acute tubular necrosis.
Tests to Assess Distal Tubular Function
1. Urine specific gravity: Normal specific gravity is 1.003
to 1.030. It depends on state of hydration and fluid intake.
i. Causes of increased specific gravity:
a. Reduced renal perfusion (with preservation of
concentrating ability of tubules),
b. Proteinuria,
c. Glycosuria,
d. Glomerulonephritis.
e. Urinary tract obstruction.
ii. Causes of reduced specific gravity:
a. Diabetes insipidus
b. Chronic renal failure
c. Impaired concentrating ability due to diseases of
tubules.
As a test of renal function, it gives information about
the ability of renal tubules to concentrate the glomerular
filtrate. This concentrating ability is lost in diseases of
renal tubules.
Fixed specific gravity of 1.010, which cannot be
lowered or increased by increasing or decreasing the fluid
intake respectively, is an indication of chronic renal
failure.
2. Urine osmolality: The most commonly employed test
to evaluate tubular function is measurement of urine/
plasma osmolality. This is the most sensitive method for
determination of ability of concentration. Osmolality
measures number of dissolved particles in a solution.
Specific gravity, on the other hand, is the ratio of mass of
a solution to the mass of water i.e. it measures total mass
of solute. Specific gravity depends on both the number
and the nature of dissolved particles while osmolality is
exact number of solute particles in a solution. Specific
gravity measurement can be affected by the presence of
solutes of large molecular weight like proteins and
glucose, while osmolality is not. Therefore measurement
of osmolality is preferred.
When solutes are dissolved in a solvent, certain
changes take place like lowering of freezing point,
increase in boiling point, decrease in vapor pressure, or
increase of osmotic pressure of the solvent. These
properties are made use of in measuring osmolality by
an instrument called as osmometer.
Osmolality is expressed as milliOsmol/kg of water.
Urine/plasma osmolality ratio is helpful in distin-
guishing pre-renal azotemia (in which ratio is higher)
from acute renal failure due to acute tubular necrosis (in
which ratio is lower). If urine and plasma osmolality are
almost similar, then there is defective tubular reabsorp-
tion of water.
3. Water deprivation test If the value of baseline osmolality
of urine is inconclusive, then water deprivation test is
performed. In this test, water intake is restricted for a
specified period of time followed by measurement of
specific gravity or osmolality. Normally, urine osmolality
should rise in response to water deprivation. If it fails to
rise, then desmopressin is administered to differentiate
between central diabetes insipidus and nephrogenic
diabetes insipidus. Urinary concentration ability is
corrected after administration of desmopressin in central
diabetes insipidus, but not in nephrogenic diabetes
insipidus.
If urine osmolality is > 800 mOsm/kg of water or
specific gravity is ≥1.025 following dehydration,
concentrating ability of renal tubules is normal. However,
normal result does not rule out presence of renal disease.
False result will be obtained if the patient is on low-salt,
low-protein diet or is suffering from major electrolyte
and water disturbance.
4. Water loading antidiuretic hormone suppression test This
test assesses the capacity of the kidney to make urine
dilute after water loading.
After overnight fast, patient empties the bladder and
drinks 20 ml/kg of water in 15-30 minutes. The urine is
 Renal Function Tests 37

collected at hourly intervals for the next 4 hours for Indications for Renal Biopsy
measurements of urine volume, specific gravity, and
1. Nephrotic syndrome in adults (most common
osmolality. Plasma levels of antidiuretic hormone and
indication)
serum osmolality should be measured at hourly intervals.
2. Nephrotic syndrome not responding to cortico-
Normally, more than 90% of water should be excreted
steroids in children.
in 4 hours. The specific gravity should fall to 1.003 and
3. Acute nephritic syndrome for differential diagnosis
osmolality should fall to < 100 mOsm/kg. Plasma level
4. Unexplained renal insufficiency with near-normal
of antidiuretic hormone should be appropriate for serum
kidney dimensions on ultrasonography
osmolality. In renal function impairment, urine volume
5. Asymptomatic hematuria, when other diagnostic
is reduced (<80% of fluid intake is excreted) and specific
tests fail to identify the source of bleeding
gravity and osmolality fail to decrease. The test is also
6. Isolated non-nephrotic range proteinuria (1-3 gm/
impaired in adrenocortical insufficiency, malabsorption,
24 hours) with renal impairment
obesity, ascites, congestive heart failure, cirrhosis, and
7. Impaired function of renal graft
dehydration.
8. Involvement of kidney in systemic disease like
This test is not advisable in patients with cardiac
systemic lupus erythematosus or amyloidosis
failure or kidney disease. If there is failure to excrete
water load, fatal hyponatremia can occur.
Contraindications
5. Ammonium chloride loading test (Acid load test): Diagnosis
1. Uncontrolled severe hypertension
of renal tubular acidosis is usually considered after
2. Hemorrhagic diathesis
excluding other causes of metabolic acidosis. This test is
3. Solitary kidney
considered as a ‘gold standard’ for the diagnosis of distal
4. Renal neoplasm (to avoid spread of malignant cells
or type 1 renal tubular acidosis. Urine pH and plasma
along the needle track)
bicarbonate are measured after overnight fasting. If pH
5. Large and multiple renal cysts
is less than 5.4, acidifying ability of renal tubules is
6. Small, shrunken kidneys
normal. If pH is greater than 5.4 and plasma bicarbonate
7. Acute urinary tract infection like pyelonephritis
is low, diagnosis of renal tubular acidosis is confirmed.
8. Urinary tract obstruction
In both the above cases, further testing need not be
performed. In all other cases in which neither of above
Complications
results is obtained, further testing is carried out. Patient
is given ammonium chloride orally (0.1 gm/kg) over 1 1. Hemorrhage: As renal cortex is highly vascular, major
hour after overnight fast and urine samples are collected risk is bleeding in the form of hematuria or peri-
hourly for next 6-8 hours. Ammonium ion dissociates nephric hematoma. Severe bleeding may occasionally
into H+ and NH3. Ammonium chloride makes blood necessitate blood transfusion and rarely removal of
acidic. If pH is less than 5.4 in any one of the samples, kidney.
acidifying ability of the distal tubules is normal. 2. Arteriovenous fistula
3. Infection
RENAL BIOPSY 4. Accidental biopsy of another organ or perforation of
viscus (liver, spleen, pancreas, adrenals, intestine, or
Renal biopsy refers to obtaining a small piece of kidney gallbladder)
tissue for microscopic examination. Percutaneous renal 5. Death (rare).
biopsy was first performed by Alwall in 1944. In renal
disease, renal biopsy is helpful to: Procedure
• Establish the diagnosis 1. Patient’s informed consent is obtained.
• Assess severity and activity of disease 2. Ultrasound/CT scan is done to document the
• Assess prognosis by noting the amount of scarring location and size of kidneys.
• To plan treatment and monitor response to therapy 3. Blood pressure should be less than 160/90 mm of
Renal biopsy is associated with the risk of procedure- Hg. Bleeding time, platelet count, prothrombin
related morbidity and rarely mortality. Therefore, before time, and activated partial thromboplastin time
performing renal biopsy, risks of the procedure and should be normal. Blood sample should be drawn
benefits of histologic examination should be evaluated for blood grouping and cross matching, as blood
in each patient. transfusion may be needed.
 38 Essentials of Clinical Pathology

4. Patient is sedated before the procedure. REFERENCE RANGES

5. Patient lies in prone position and kidney is
identified with ultrasound. Inulin clearance:
6. The skin over the selected site is disinfected and a Men: 90-139 ml/min/1.73 m2
local anesthetic is infiltrated. Women: 80-125 ml/min/1.73 m2
7. A small skin incision is given with a scalpel (to Creatinine clearance:
insert the biopsy needle). Localization of kidney Men: 110-150 ml/min/1.73 m2
is done with a fine bore 21 G lumbar puncture Women: 105-132 ml/min/1.73 m2
needle. A local anesthetic is infiltrated down to Blood urea nitrogen (BUN):
the renal capsule. Adults: 7-18 mg/dl
8. A tru-cut biopsy needle or spring loaded biopsy >60 years: 8-21 mg/dl
gun is inserted under ultrasound guidance and
advanced down to the lower pole. Biopsy is Serum creatinine:
usually obtained from lateral border of lower pole. Adult males: 0.7-1.3 mg/dl
Patient should hold his/her breath in full Adult females: 0.6-1.1 mg/dl
inspiration during biopsy. After obtaining the Children (<20 years):
biopsy and removal of needle, patient is allowed Boys: 0.35 + Age in years/40
to breath normally. Girls: 0.35 + Age in years/55
9. The biopsy should be placed in a drop of saline BUN/Serum creatinine ratio: 12: 1 to 20:1
and examined under a dissecting microscope for
adequacy.
CRITICAL VALUES
10. Patient is turned to supine position. Vital signs
and appearance of urine should be monitored at Serum creatinine: > 5 mg/dl
regular intervals. Patient is usually kept in the BUN: > 80 mg/dl
hospital for 24 hours.
Kidney biopsy can be divided into three parts for light
microscopy, immunofluorescence, and electron micro- BIBLIOGRAPHY
scopy. For light microscopy, renal biopsy is routinely 1. Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart
fixed in neutral buffered formaldehyde. Sections are MJ, Shepherd J. Clinical Biochemistry: An Illustrated
stained by: Colour Text (3rd Ed). Edinburgh: Churchill Livingstone
• Hematoxylin and eosin (for general architecture of 2004.
kidney and cellularity) 2. Johnson CA, Levey AS, Coresh J, Levin A, Lau J, Eknoyan
• Periodic acid Schiff: To highlight basement mem- G. Clinical practice guidelines for chronic kidney disease
brane and connective tissue matrix. in adults: Part II. Glomerular filtration rate, proteinuria,
• Congo red: For amyloid. and other markers Am Fam Physician 2004;70:1091-7.
For electron microscopy, tissue is fixed in glutaraldeyde. 3. Stevens LA, Coresh J, Green T, Levey AS. Assessing
In immunohistochemistry, tissue deposits of IgG, IgA, kidney function-measured and estimated glomerular
IgM, C3, fibrin, and κ and λ light chains can be detected filtration rate. N Engl J Med 2006;354:2473-83.
by using appropriate antibodies. Many kidney diseases 4. Stevens LA, Levey AS. Measurement of kidney function.
are immune-complex mediated. Med Clin N Am 2005;89:457-73.
 3

Diabetes Mellitus

Diabetes mellitus (DM) is a metabolic group of disorders

characterized by persistent hyperglycemia due to
deficiency and/or diminished effectiveness of insulin.
There are derangements of carbohydrate, protein, and
fat metabolism due to failure of insulin action on target
cells. Typical features of DM are as follows:
• Fasting hyperglycemia
• Glycosuria
• Symptoms due to marked hyperglycemia: polyuria,
polydipsia, weight loss, weakness, polyphagia, and
blurred vision
• Long-term complications like atherosclerosis (leading
to ischemic heart disease, cerebrovascular disease,
and peripheral vascular disease) and microangio-
pathy (which can cause nephropathy with risk of
renal failure; retinopathy with potential loss of vision;
and peripheral neuropathy with risk of foot ulcers,
amputations, or Charcot joints).
• Acute metabolic complications (hyperosmolar
hyperglycemic state, diabetic ketoacidosis).
• Susceptibility to infections especially of skin,
respiratory tract, and urinary tract.

METABOLIC ACTIONS OF INSULIN
Insulin is the major hormone regulating blood glucose
level. Insulin is synthesized by β cells of pancreas as
preproinsulin, which is rapidly converted to proinsulin.
Proinsulin is a single chain polypeptide. In the Golgi
apparatus, proinsulin is broken down into 2 units- insulin
(51 amino acids) and C (connecting)-polypeptide (31
amino acids) (Fig. 3.1). Both insulin and C peptide are
stored in membrane-bound granules in the cytoplasm
of β cells. Upon stimulation (mainly by blood glucose),
both insulin and C peptide are released in circulation.
C peptide is often measured as a marker of activity of β
cells. C peptide has no known function.
Insulin acts on various cells (especially those of liver,
muscle, and adipose tissue) through receptors.
Important actions of insulin are shown in Box 3.1.
Fig. 3.1: Proinsulin, insulin, and C-peptide. The biochemical
cleavage of proinsulin to insulin and C-peptide occurs in Golgi
apparatus of β cell. Secretion of insulin is stimulated by glucose,
mannose, amino acids, and sulfonylureas
“Stress hormones” like glucagons, glucocorticoids,
growth hormone, and adrenaline oppose action of
insulin.
CLASSIFICATION OF DIABETES MELLITUS
According to American Diabetes Association (1997), DM
is classified into following types:
• Type 1 (Absolute deficiency of insulin due to
destruction of β cells of pancreas)
– Immune mediated
– Idiopathic
 40 Essentials of Clinical Pathology

Box 3.1: Major actions of insulin

• Increases:
– Uptake of glucose in skeletal muscle and adipose
tissue
– Glycogenesis in liver and muscle
– Fatty acid and triglycerides in liver and adipose
tissue
– Protein synthesis in liver and muscle
• Decreases:
– Gluconeogenesis in liver
– Glycogenolysis in liver and muscle
– Lipolysis in adipose tissue
– Ketogenesis in liver

• Type 2 (Insulin resistance along with relative

deficiency of insulin secretion)
• Other specific types
• Gestational DM (onset or first recognition of glucose
intolerance during pregnancy).
Fig. 3.2: Pathogenesis of type I diabetes mellitus
Type 1 Diabetes Mellitus
It accounts for 5-10% of all cases of DM. This was
infiltration by cytotoxic CD8+ T lymphocytes in and
previously called as insulin-dependent DM or IDDM
around islets. It is thought that many cases follow a viral
(because insulin therapy is essential to prevent ketosis),
juvenile-onset DM (because it commonly presents during infection that has damaged the islet cells of pancreas (Fig.
childhood or adolescence), brittle DM, or ketosis-prone 3.2). Markers of immune destruction of β cells, which
DM. It is characterized by absolute deficiency of insulin can be detected in peripheral blood, are islet cell
secretion. antibodies, autoantibodies to insulin, autoantibodies to
Cell-mediated autoimmune destruction of β cells glutamic acid decarboxylase (GAD65), and autoanti-
of pancreas is responsible for majority of cases of type bodies to tyrosine phosphatases (IA-2 and IA-2b). The
1 DM (immune-mediated type 1 DM), leading to disease has strong association with HLA DR3 and HLA
inability of pancreas to synthesize insulin. There is DR4 haplotypes (Fig. 3.3). This type occurs mainly in

Fig. 3.3: HLA-linked genetic predisposition to type 1 diabetes mellitus


children and adolescents, but can occur at any age. These
patients are also at risk of other autoimmune disorders
like Graves’ disease, Hashimoto’s thyroiditis, vitiligo,
Addison’s disease, pernicious anemia, etc.
Some cases of type 1 DM do not have any known
etiologies or evidence of autoimmunity. These indivi-
duals are of Asian or African origin and their disease is
strongly inherited. This form of type 1 DM is called as
idiopathic DM.
Type 2 Diabetes Mellitus
This is the most common form of DM comprising about
90-95% of all patients of DM. This was previously called
as non-insulin-dependent DM (NIDDM), maturity-onset
DM (because onset usually occurs during adult life),
stable DM, or ketosis-resistant DM. It is characterized
by insulin resistance along with relative deficiency of
insulin secretion (i.e. inadequate insulin secretory
response to overcome peripheral insulin resistance).
(Fig. 3.4). Type 2 DM is not HLA-linked and there is no
role of autoimmunity in its pathogenesis. It has a strong
genetic predisposition. Type 2 DM occurs more
frequently in individuals with positive family history
(parents or siblings with DM), obesity (≥ 20% over ideal
body weight or body mass index ≥ 25 kg/m2), hyper-
tension (>140/90 mm Hg in adults), dyslipidemia, lack
of physical activity, pre-diabetes (impaired fasting
glucose or impaired glucose tolerance), and prior
gestational DM.
Type 2 diabetes is more common in certain racial
groups like South Asians and Africans. Rising trend of
type 2 DM is due to increasing tendency towards obesity
in urban populations coupled with high-calorie diet.
Differences between type 1 and type 2 DM are listed
in Table 3.1.
Fig. 3.4: Pathogenesis of type 2 diabetes mellitus
Diabetes Mellitus 41
Other Specific Types
There are several forms of DM associated with under-
lying conditions:
• Genetic defects of β cell function: In these disorders,
insulin secretion from β cells is impaired. These are
called as maturity onset diabetes of the young
(MODY). They are inherited in an autosomal
dominant manner and they are caused by mutations
in genetic loci such as hepatic nuclear factor,
glucokinase, etc.
• Genetic defects in insulin action: These result from
mutations in insulin receptor gene.
• Diseases of exocrine pancreas: Diseases causing
generalized pancreatic damage can result in DM.
These include cystic fibrosis, hemochromatosis,
chronic pancreatitis, trauma, pancreatectomy, and
pancreatic cancer.
• Endocrine disorders: Several hormones inhibit the
action of insulin. Excessive secretion of these
hormones will cause DM. Hyperglycemia is corrected
following resolution of the primary endocrinopathy.
Endocrine disorders associated with hyperglycemia
are:
– Acromegaly: Excess growth hormone.
– Cushing’s syndrome: Excess cortisol.
– Glucagonoma: Excess glucagon
– Pheochromocytoma: Excess epinephrine.
– Hyperthyroidism: Excess thyroxine
• Drug- or chemical-induced DM: Drugs or chemicals can
impair insulin secretion or insulin action. Destruction
of β cells and formation of islet cell antibodies have
also been reported with some drugs. Examples
include thiazide diuretics, α-interferon, and gluco-
corticoids.
• Infections: Certain viral infections (such as Coxsackie
virus B, congenital rubella, cytomegalovirus) can
cause destruction of β cells.
• Other genetic syndromes sometimes associated with DM:
Many genetic syndromes (e.g. Down’s syndrome,
Klinefelter’s syndrome, Turner’s syndrome) are
associated with increased risk of developing DM.
Gestational Diabetes Mellitus (GDM)
This refers to the onset or first recognition of glucose
intolerance during pregnancy. GDM occurs in 2-3% of
all pregnant women. It is associated with increased risk
of high birth weight of the newborn, cardiac defects,
polyhydramnios, intrauterine fetal loss (due to
placental insufficiency), premature birth, hypertension
during pregnancy, pre-eclampsia, and alteration in
duration of pregnancy. Early diagnosis and treatment
 42 Essentials of Clinical Pathology

Table 3.1: Differences between type 1 and type 2 diabetes mellitus

Parameter Type 1 DM Type 2 DM

1. Previous names Type I, IDDM, Juvenile-onset, Type II, NIDDM, maturity-onset,

Ketosis-prone, Brittle Ketosis-resistant, Stable
2. % of all DM cases 5-10% 90-95%
3. Age of onset < 35 years > 35 years
4. Obesity No (weight loss common) Common
5. Presentation Acute; symptoms (classical) Insidious; symptoms since few
since few weeks months or years
6. Genetic predisposition Low Strong
7. Glucose intolerance Marked Mild
8. Ketoacidosis Common Uncommon
9. Hyperosmolar hyperglycemic state Uncommon Common
10. Serum insulin Undetectable Normal or high
11. Autoimmunity (Islet cell antibodies) Majority of cases No
12. HLA-linkage Yes (DR3, DR4) No
13. Pathogenesis Absolute insulin deficiency Insulin resistance with relative
insulin deficiency
14. Serum C-peptide level Very low to absent Normal or high
15. Predisposing factors Viral infections, toxins Obesity, lack of physical activity
16. Insulin requirement for treatment Always In some situations

of GDM are essential to prevent perinatal morbidity and
mortality. After delivery, GDM can have following
course:
1. Return to normal glucose tolerance (however, many
of these patients are likely to develop DM during
subsequent years), or
2. Persistence of DM or impaired glucose tolerance.
Patient should be reassessed 6 weeks or later
following delivery.
Risk factors for GDM are shown in Box 3.2.
PREDIABETES
Prediabetes is a state in which plasma glucose level is
higher than normal but not high enough for diagnosis of
DM. It is also referred to as impaired fasting glucose (IFG)
or impaired glucose tolerance (IGT), depending on which
test is used for its detection. Studies have shown that
majority of individuals with prediabetes develop type 2
DM within 10 years. Prediabetic persons do not develop
Box 3.2: Risk factors for gestational DM
• Past history of GDM
• Previous high-birth-weight baby
• Obesity
• Family history of diabetes mellitus
• High-risk ethnic group: South Asian or African.
acute complications of DM and microangiopathy, but are
at increased risk of cardiovascular disease (1.5 times
normal individuals). Development of DM can be delayed
or prevented through modest amount of weight loss, diet
modification, and regular moderate exercise in patients
with prediabetes.
METABOLIC SYNDROME (INSULIN
RESISTANCE SYNDROME, REAVEN’S
SYNDROME, SYNDROME X)
The metabolic syndrome refers to a constellation of lipid
and non-lipid risk factors that are of metabolic origin and
associated with risk of cardiovascular disease. The Adult
Treatment Panel III (ATP III) 1 of the National Cholesterol
Education Program in 2001 proposed criteria for
diagnosing the metabolic syndrome. The metabolic
syndrome is diagnosed when three or more out of
following five criteria are present.
• Abdominal obesity
– Men: Waist circumference > 40 inches (102 cm)
– Women: Waist circumference > 35 inches (88 cm)
• Fasting glucose ≥ 110 to < 126 mg/dl (As these criteria
were proposed in 2001, the fasting plasma glucose
value should be reduced to 100 mg/dl according to
revised criteria proposed by American Diabetes
Association in 2004).
• Blood pressure ≥ 130/> 85 mm Hg
• Triglycerides ≥ 150 mg/dl (> 1.7 mmol/L)

• Plasma high density lipoprotein (HDL)-cholesterol
– Men: < 40 mg/dl (< 1 mmol/L)
– Women: < 50 mg/dl (< 1.3 mmol/L)
Metabolic syndrome is common in Asian Indians in
Britain and in Africa. It is said that insulin resistance is
common to all above risk factors and plays an etiological
role. These persons have increased risk of developing
Type 2 DM.
METABOLIC ALTERATIONS IN
DIABETES MELLITUS
In DM, there are abnormalities of carbohydrate
metabolism (hyperglycemia, glycosuria, impaired
glucose tolerance), protein metabolism (increased protein
catabolism with muscle wasting, gluconeogenesis), and
fat metabolism (increased fatty acid synthesis, ketosis)
(Fig. 3.5). Metabolic alterations in type 2 DM are less
severe than in type 1 DM.
• Hyperglycemia: Hyperglycemia is due to deficient
uptake of glucose by muscle and fat cells (due to
Fig. 3.5: Metabolic alterations in diabetes mellitus
Diabetes Mellitus 43
insulin deficiency). In reaction to this, there are
compensatory glycogenolysis (breakdown of glyco-
gen to glucose) and gluconeogenesis (formation of
glucose from non-carbohydrates like proteins), which
contribute to hyperglycemia. Typical clinical features
of hyperglycemia are polyuria, polydipsia, poly-
phagia, weakness, weight loss, and blurring of vision.
• Glycosuria: Glycosuria results when blood glucose
level exceeds renal threshold (180 mg/dl or 10 mmol/
L in most individuals). Excess glucose increases
osmolality of glomerular filtrate with resultant
osmotic diuresis and polyuria. This causes depletion
of water and electrolytes, cellular dehydration, and
intense thirst (polydipsia). Insulin deficiency leads to
catabolism of proteins, and released amino acids are
used for formation of glucose (gluconeogenesis).
Breakdown of lipids also occurs and coupled with
proteolysis, lead to negative energy balance and
weight loss. This in turn induces polyphagia
(increased appetite).
 44 Essentials of Clinical Pathology
• Ketosis: Insulin deficiency leads to increased
degradation of lipids (lipolysis), resulting in increased
levels of free fatty acids in circulation. These are
converted to acetoacetyl CoA in the liver. Acetoacetyl
CoA, in turn, is converted to ketone bodies. If muscles
or other tissues do not utilize ketone bodies equal to
the rate of their formation, they accumulate in blood.
With the rise in blood levels of ketone bodies
(ketonemia), they are excreted in urine (ketonuria).
Ketone bodies are strong acids, which dissociate and
release H+ ions. Bicarbonate removes these H+ ions
in plasma and the level of bicarbonate falls. This
produces metabolic acidosis. Symptoms of metabolic
acidosis are nausea, vomiting, and hyperpnea (air
hunger). Diabetic ketoacidosis (DKA) is a typical
feature of type 1 DM.
• Hyperosmolar hyperglycemic state (HHS): This
occurs in type 2 patients due to combination of severe
dehydration (secondary to sustained osmotic diuresis
coupled with inadequate fluid intake) and hyper-
glycemia. It occurs usually in elderly with unrecog-
nized DM or DM of recent onset.
LONG-TERM COMPLICATIONS OF
DIABETES MELLITUS
In long standing DM of both types, a wide variety of
lesions develop in many organs, which are important
causes of morbidity and mortality.
Macroangiopathy (Macrovascular disease): In DM,
atherosclerosis of aorta and of medium size arteries (like
coronary, cerebral, and peripheral) occurs earlier in life
and is more extensive than in non-diabetic patients. It
can cause myocardial infarction, cerebrovascular
accident, and gangrene of lower extremities. Patho-
genesis of atherosclerosis in DM is related to hyper-
insulinemia with peripheral insulin resistance and
dyslipidemia (raised triglycerides, low high density
lipoprotein or HDL, and raised low density lipoprotein
or LDL).
Microangiopathy (Microvascular disease): Micro-
angiopathy is due to poor diabetes control (Box 3.3).
Microangiopathy (thickening of walls of small blood
vessels with narrowing of lumina) is common in kidneys
(leading to renal insufficiency), retina (visual impair-
ment), and peripheral nerves (sensory, motor or
autonomic neuropathy).
Infections: Diabetic patients are susceptible to infections
of skin, respiratory tract (pneumonia, tuberculosis), and
kidneys (pyelonephritis).
Box 3.3: Microangiopathy in diabetes mellitus
• Risk is directly related to presence of high glucose
level for long duration
• Improved glycemic control significantly reduces the
risk
• Consists of:
– Retinopathy: Visual loss can occur due to
vitreous hemorrhage (from proliferating retinal
vessels) and maculopathy
– Nephropathy: Early stage is characterized by
increased glomerular filtration rate and micro-
albuminuria; with progressive renal damage, overt
proteinuria and renal failure develop.
– Neuropathy: Postural hypotension, impotence,
sensory and motor neuropathy, foot ulcer.
Average life expectancy of DM patients is reduced.
Usual causes of death in DM include myocardial
infarction, stroke, renal failure, infections, and keto-
acidotic or hyperosmolar hyperglycemic coma.
ROLE OF LABORATORY TESTS IN
DIABETES MELLITUS
In DM, applications of laboratory tests are as follows:
• Diagnosis of DM
• Screening of DM
• Assessment of glycemic control
• Assessment of associated long-term risks
• Management of acute metabolic complications.
Laboratory Tests for Diagnosis of
Diabetes Mellitus
Diagnosis of DM is based exclusively on demonstration
of raised blood glucose level (hyperglycemia).
The current criteria (American Diabetes Association,
2004) for diagnosis of DM are as follows:
Typical symptoms of DM (polyuria, polydipsia, weight
loss) and random plasma glucose ≥ 200 mg/dl (≥ 11.1
mmol/L)
Or
Fasting plasma glucose ≥ 126 mg/dl (≥ 7.0 mmol/L)
Or
2-hour post glucose load (75 g) value during oral
glucose tolerance test ≥ 200 mg/dl (≥ 11.1 mmol/L).
If any one of the above three criteria is present,
confirmation by repeat testing on a subsequent day is
necessary for establishing the diagnosis of DM. However,
such confirmation by repeat testing is not required if

patient presents with (a) hyperglycemia and ketoacidosis,
or (b) hyperosmolar hyperglycemia.
The tests used for laboratory diagnosis of DM are (1)
estimation of blood glucose and (2) oral glucose tolerance
test.
Estimation of Blood Glucose
Measurement of blood glucose level is a simple test to
assess carbohydrate metabolism in DM (Fig. 3.6). Since
glucose is rapidly metabolized in the body, measurement
of blood glucose is indicative of current state of
carbohydrate metabolism.
Glucose concentration can be estimated in whole
blood (capillary or venous blood), plasma or serum.
However, concentration of blood glucose differs
according to nature of the blood specimen. Plasma
glucose is about 15% higher than whole blood glucose
(the figure is variable with hematocrit). During fasting
state, glucose levels in both capillary and venous blood
are about the same. However, postprandial or post
glucose load values are higher by 20-70 mg/dl in
capillary blood than venous blood. This is because venous
blood is on a return trip after delivering blood to the
tissues.
When whole blood is left at room temperature after
collection, glycolysis reduces glucose level at the rate of
about 7 mg/dl/hour. Glycolysis is further increased in
the presence of bacterial contamination or leucocytosis.
Addition of sodium fluoride (2.5 mg/ml of blood)
maintains stable glucose level by inhibiting glycolysis.
Sodium fluoride is commonly used along with an
anticoagulant such as potassium oxalate or EDTA.
Fig. 3.6: Blood glucose values in normal individuals,
prediabetes, and diabetes mellitus
Diabetes Mellitus 45
Addition of sodium fluoride is not necessary if plasma
is separated from whole blood within 1 hour of blood
collection.
Plasma is preferred for estimation of glucose since
whole blood glucose is affected also by concentration of
proteins (especially hemoglobin).
There are various methods for estimation of blood
glucose:
• Chemical methods:
– Orthotoluidine method
– Blood glucose reduction methods using
neocuproine, ferricyanide, or copper.
Chemical methods are less specific but are cheaper as
compared to enzymatic methods.
• Enzymatic methods: These are specific for glucose.
– Glucose oxidase-peroxidase
– Hexokinase
– Glucose dehydrogenase
Chemical methods have now been replaced by
enzymatic methods.
Terms used for blood glucose specimens: Depending
on the time of collection, different terms are used for
blood glucose specimens.
• Fasting blood glucose: Sample for blood glucose is
withdrawn after an overnight fast (no caloric intake
for at least 8 hours).
• Post meal or postprandial blood glucose: Blood
sample for glucose estimation is collected 2 hours after
the subject has taken a normal meal.
• Random blood glucose: Blood sample is collected at
any time of the day, without attention to the time of
last food intake.
Oral Glucose Tolerance Test (OGTT)
Glucose tolerance refers to the ability of the body to
metabolize glucose. In DM, this ability is impaired or
lost and glucose intolerance represents the fundamental
pathophysiological defect in DM. OGTT is a provocative
test to assess response to glucose challenge in an
individual (Fig. 3.7).
American Diabetes Association does not
recommend OGTT for routine diagnosis of type 1 or
type 2 DM. This is because fasting plasma glucose cut-
off value of 126 mg/dl identifies the same prevalence of
abnormal glucose metabolism in the population as
OGTT. World Health Organization (WHO) recommends
OGTT in those cases in which fasting plasma glucose is
in the range of impaired fasting glucose (i.e. 100-125 mg/
dl). Both ADA and WHO recommend OGTT for
diagnosis of gestational diabetes mellitus.
 46 Essentials of Clinical Pathology
Fig. 3.7: Oral glucose tolerance curve
Preparation of the Patient
• Patient should be put on a carbohydrate-rich,
unrestricted diet for 3 days. This is because carbo-
hydrate-restricted diet reduces glucose tolerance.
• Patient should be ambulatory with normal physical
activity. Absolute bed rest for a few days impairs
glucose tolerance.
• Medications should be discontinued on the day of
testing.
• Exercise, smoking, and tea or coffee are not allowed
during the test period. Patient should remain seated.
• OGTT is carried out in the morning after patient has
fasted overnight for 8-14 hours.
Test
1. A fasting venous blood sample is collected in the
morning.
2. Patient ingests 75 g of anhydrous glucose in 250-300
ml of water over 5 minutes. (For children, the dose is
1.75 g of glucose per kg of body weight up to
maximum 75 g of glucose). Time of starting glucose
drink is taken as 0 hour.
Table 3.2: Interpretation of oral glucose tolerance test
Parameter Normal Impaired
fasting glucose
1. Fasting (8 hr) < 100 100-125
2. 2 hr OGTT < 140 <140
3. A single venous blood sample is collected 2 hours
after the glucose load. (Previously, blood samples
were collected at ½, 1, 1½, and 2 hours, which is no
longer recommended).
4. Plasma glucose is estimated in fasting and 2-hour
venous blood samples.
Interpretation of blood glucose levels is given in Table
3.2.
OGTT in gestational diabetes mellitus: Impairment of
glucose tolerance develops normally during pregnancy,
particularly in 2nd and 3rd trimesters. Following are the
recent guidelines of ADA for laboratory diagnosis of
GDM:
• Low-risk pregnant women need not be tested if all of
the following criteria are met, i.e. age below 25 years,
normal body weight (before pregnancy), absence of
diabetes in first-degree relatives, member of an ethnic
group with low prevalence of DM, no history of poor
obstetric outcome, and no history of abnormal glucose
tolerance.
• Average-risk pregnant women (i.e. who are in
between low and high risk) should be tested at 24-28
weeks of gestation.
• High-risk pregnant women i.e. those who meet any
one of the following criteria should be tested
immediately: marked obesity, strong family history
of DM, glycosuria, or personal history of GDM.
Initially, fasting plasma glucose or random plasma
glucose should be obtained. If fasting plasma glucose is
≥ 126 mg/dl or random plasma glucose is ≥ 200 mg/dl,
repeat testing should be carried out on a subsequent day
for confirmation of DM. If both the tests are normal, then
OGTT is indicated in average-risk and high-risk pregnant
women.
There are two approaches for laboratory diagnosis
of GDM
• One step approach
• Two step approach
In one step approach, 100 gm of glucose is adminis-
tered to the patient and a 3-hour OGTT is performed.
This test may be cost-effective in high-risk pregnant
women.
Impaired glucose Diabetes mellitus
tolerance
— ≥ 126
140-199 ≥ 200

In two-step approach, an initial screening test is done
in which patient drinks a 50 g glucose drink irrespective
of time of last meal and a venous blood sample is
collected 1 hour later (O’Sullivan’s test). GDM is excluded
if glucose level in venous plasma sample is below 140
mg/dl. If level exceeds 140 mg/dl, then the complete
100 g, 3-hour OGTT is carried out.
In the 3-hour OGTT, blood samples are collected in
the morning (after 8-10 hours of overnight fasting), and
after drinking 100 g glucose, at 1, 2, and 3 hours. For
diagnosis of GDM, glucose concentration should be
above the following cut-off values in 2 or more of the
venous plasma samples:
• Fasting: 95 mg/dl
• 1 hour: 180 mg/dl
• 2 hour: 155 mg/dl
• 3 hour: 140 mg/dl
Laboratory Tests for Screening of
Diabetes Mellitus
Aim of screening is to identify asymptomatic individuals
who are likely to have DM. Since early detection and
prompt institution of treatment can reduce subsequent
complications of DM, screening may be an appropriate
step in some situations.
Screening for type 2 DM: Type 2 DM is the most common
type of DM and is usually asymptomatic in its initial
stages. Its onset occurs about 5-7 years before clinical
diagnosis. Evidence indicates that complications of type
2 DM begin many years before clinical diagnosis.
American Diabetes Association recommends screening
for type 2 DM in all asymptomatic individuals ≥ 45 years
of age using fasting plasma glucose. If fasting plasma
glucose is normal (i.e. < 100 mg/dl), screening test should
be repeated every three years.
Another approach is selective screening i.e. screening
individuals at high risk of developing type 2 DM i.e. if
one or more of the following risk factors are present-
obesity (body mass index ≥ 25.0 kg/m2), family history
of DM (first degree relative with DM), high-risk ethnic
group, hypertension, dyslipidemia, impaired fasting
glucose, impaired glucose tolerance, or history of GDM.
In such cases, screening is performed at an earlier age
(30 years) and repeated more frequently.
Recommended screening test for type 2 DM is fasting
plasma glucose. If ≥126 mg/dl, it should be repeated on
a subsequent day for confirmation of diagnosis. If <126
mg/dl, OGTT is indicated if clinical suspicion is strong.
A 2-hour post-glucose load value in OGTT ≥200 mg/dl
is indicative of DM and should be repeated on a different
day for confirmation.
Diabetes Mellitus 47
Screening for type 1 DM: Type 1 DM is detected early after
its onset since it has an acute presentation with
characteristic clinical features. Therefore, it is not
necessary to screen for type 1 DM by estimation of blood
glucose. Detection of immunologic markers (mentioned
earlier) has not been recommended to identify persons
at risk.
Screening for GDM: Given earlier under OGTT in
gestational diabetes mellitus.
Laboratory Tests to Assess Glycemic Control
There is a direct correlation between the degree of blood
glucose control in DM (both type 1 and type 2) and the
development of microangiopathic complications i.e.
nephropathy, retinopathy, and neuropathy. Maintenance
of blood glucose level as close to normal as possible
(referred to as tight glycemic control) reduces the risk of
microvascular complications. There is also association
between persistently high blood glucose values in DM
with increased cardiovascular mortality.
Following methods can monitor degree of glycemic
control:
• Periodic measurement of glycated hemoglobin (to
assess long-term control).
• Daily self-assessment of blood glucose (to assess day-
to-day or immediate control).
Glycated Hemoglobin
(Glycosylated Hemoglobin, HbA1C)
Glycated hemoglobin refers to hemoglobin to which
glucose is attached nonenzymatically and irreversibly;
its amount depends upon blood glucose level and
lifespan of red cells.
Hemoglobin + Glucose ↔ Aldimine → Glycated hemoglobin
Plasma glucose readily moves across the red cell
membranes and is being continuously combined with
hemoglobin during the lifespan of the red cells (120 days).
Therefore, some hemoglobin in red cells is present
normally in glycated form. Amount of glycated
hemoglobin in blood depends on blood glucose
concentration and lifespan of red cells. If blood glucose
concentration is high, more hemoglobin is glycated. Once
formed, glycated hemoglobin is irreversible. Level of
glycated hemoglobin is proportional to the average
glucose level over preceding 6-8 weeks (about 2 months).
Glycated hemoglobin is expressed as a percentage of total
hemoglobin. Normally, less than 5% of hemoglobin is
glycated.
 48 Essentials of Clinical Pathology
Numerous prospective studies have demonstrated
that a good control of blood glucose reduces the
development and progression of microvascular
complications (retinopathy, nephropathy, and
peripheral neuropathy) of diabetes mellitus. Mean
glycated hemoglobin level correlates with the risk of
these complications.
The terms glycated hemoglobin, glycosylated
hemoglobin, glycohemoglobin, HbA1, and HbA1c are
often used interchangeably in practice. Although these
terms refer to hemoglobins that contain nonenzy-
matically added glucose residues, hemoglobins thus
modified differ. Most of the studies have been carried
out with HbA1c.
Glycated hemoglobin should be routinely measured
in all diabetic patients (both type 1 and type 2) at regular
intervals to assess degree of long-term glycemic control.
Apart from mean glycemia (over preceding 120 days),
glycated hemoglobin level also correlates with the risk
of the development of chronic complications of DM.
In DM, it is recommended to maintain glycated
hemoglobin level to less than 7%.
Spurious results of glycated hemoglobin are seen in
reduced red cell survival (hemolysis), blood loss, and
hemoglobinopathies.
In DM, if glycated hemoglobin is less than 7%, it
should be measured every 6 months. If >8%, then more
frequent measurements (every 3 months) along with
change in treatment are advocated.
There are various methods for measurement of
glycated hemoglobin such as chromatography, immuno-
assay, and agar gel electrophoresis.
Role of glycated hemoglobin in management of DM
is highlighted in Box 3.4.
Box 3.4: Glycated hemoglobin
• Hemoglobin A1C of 6% corresponds to mean serum glucose
level of 135 mg/dl. With every rise of 1%, serum glucose
increases by 35 mg/dl. Approximations are as follows:
– Hb A1C 7%: 170 mg/dl
– Hb A1C 8%: 205 mg/dl
– Hb A1C 9%: 240 mg/dl
– Hb A1C 10%: 275 mg/dl
– Hb A1C 11%: 310 mg/dl
– Hb A1C 12%: 345 mg/dl
• Assesses long-term control of DM (thus indirectly
confirming plasma glucose results or self-testing results).
• Assesses whether treatment plan is working
• Measurement of glycated hemoglobin does not replace
measurement of day-to-day control by glucometer devices.
Self-Monitoring of Blood Glucose (SMBG)
Diabetic patients are taught how to regularly monitor
their own blood glucose levels. Regular use of SMBG
devices (portable glucose meters) by diabetic patients has
improved the management of DM. With SMBG devices,
blood glucose level can be monitored on day-to-day basis
and kept as close to normal as possible by adjusting
insulin dosage. SMBG devices measure capillary whole
blood glucose obtained by fingerprick and use test strips
that incorporate glucose oxidase or hexokinase. In some
strips, a layer is incorporated to exclude blood cells so
that glucose in plasma is measured. Aim of achieving
tight glycemic control introduces the risk of severe
hypoglycemia. Daily use of SMBG devices can avoid
major hypoglycemic episodes.
SMBG devices yield unreliable results at very high
and very low glucose levels. It is necessary to periodically
check the performance of the glucometer by measuring
parallel venous plasma glucose in the laboratory.
Portable glucose meters are used by patients for day-
to-day self-monitoring, by physicians in their OPD
clinics, and by health care workers for monitoring
admitted patients at the bedside. These devices should
not be used for diagnosis and population screening of
DM as they lack precision and there is variability of
results between different meters.
Goal of tight glycemic control in type 1 DM patients
on insulin can be achieved through self-monitoring of
blood glucose by portable blood glucose meters.
Glycosuria
Semiquantitative urine glucose testing for monitoring
of diabetes mellitus in home setting is not recommen-
ded. This is because (1) even if glucose is absent in
urine, no information about blood glucose concen-
tration below the renal threshold (which itself is
variable) is obtained (Normally, renal threshold is
around 180 mg/dl; it tends to be lower in pregnancy (140
mg/dl) and higher in old age and in long-standing
diabetics; in some normal persons it is low), (2) urinary
glucose testing cannot detect hypoglycemia, and (3)
concentration of glucose in urine is affected by urinary
concentration. Semiquantitative urine glucose testing
for monitoring has now been replaced by self-testing
by portable glucose meters.
 Diabetes Mellitus 49

Laboratory Tests to Assess Long-term Risks
Urinary Albumin Excretion
Diabetes mellitus is one of the leading causes of renal
failure. Diabetic nephropathy develops in around 20-30%
of patients with type 1 or type 2 DM. Diabetic nephro-
pathy progresses through different stages as shown in
Figure 3.8. Hypertension also develops along the course
of nephropathy with increasing albumin excretion.
Evidence indicates that if diabetic nephropathy is
detected early and specific treatment is instituted, further
progression of nephropathy can be significantly
ameliorated. Early detection of diabetic nephropathy is
based on estimation of urinary albumin excretion. In all
adult patients with DM, usual reagent strip test for
proteinuria should be carried out periodically. Positive
test means presence of overt proteinuria or clinical
proteinuria and may be indicative of overt nephropathy.
In all such patients quantitation of albuminuria should
be carried out to plan appropriate therapy. If the routine
dipstick test for proteinuria is negative, test for
microalbuminuria should be carried out.
The term ‘microalbuminuria’ refers to the urinary
excretion of albumin below the level of detection by
routine dipstick testing but above normal (30-300 mg/
24 hrs, 20-200 μg/min, or 30-300 μg/mg of creatinine).
Albumin excretion rate is intermediate between normal
(normal albumin excretion in urine is < 30 mg/24 hours)
and overt albuminuria (> 300 mg/24 hours). Significance
of microalbuminuria in DM is as follows:
• It is the earliest marker of diabetic nephropathy.
Early diabetic nephropathy is reversible.
• It is a risk factor for cardiovascular disease in both
type 1 and type 2 patients.
• It is associated with higher blood pressure and poor
glycemic control.
Specific therapeutic interventions such as tight
glycemic control, administration of ACE (angiotensin-
converting enzyme) inhibitors, and aggressive treatment
of hypertension significantly slow down the progression
of diabetic nephropathy.
In type 2 DM, screening for microalbuminuria
should begin at the time of diagnosis, whereas in type
1 DM, it should begin 5 years after diagnosis. At this
time, a routine reagent strip test for proteinuria is carried
out; if negative, testing for microalbuminuria is done.
Thereafter, in all patients who test negative, screening
for microalbuminuria should be repeated every year.
Screening tests for microalbuminuria include:
• Albumin to creatinine ratio in a random urine sample
• Urinary albumin excretion in a 24-hour urine sample.
Reagent strip tests to detect microalbuminuria are
available. Positive results should be confirmed by more

Fig. 3.8: Evolution of diabetic nephropathy. In 80% of patients with type 1 DM, microalbuminuria progresses in 10-15 years
to overt nephropathy that is then followed in majority of cases by progressive fall in GFR and ultimately end-stage renal
disease. Amongst patients with type 2 DM and microalbuminuria, 20-40% of patients progress to overt nephropathy, and
about 20% of patients with overt nephropathy develop end-stage renal disease. Abbreviation: GFR: Glomerular filtration rate
 50 Essentials of Clinical Pathology

specific quantitative tests like radioimmunoassay and
enzyme immunoassay. For diagnosis of microalbumi-
nuria, tests should be positive in at least two out of three
different samples over a 3 to 6 month period.
Lipid Profile
Abnormalities of lipids are associated with increased risk
of coronary artery disease (CAD) in patients with DM.
This risk can be reduced by intensive treatment of lipid
abnormalities. Lipid parameters which should be
measured include:
• Total cholesterol
• Triglycerides
• Low-density lipoprotein (LDL) cholesterol
• High-density lipoprotein (HDL) cholesterol
The usual pattern of lipid abnormalities in type 2
DM is elevated triglycerides, decreased HDL choleste-
rol and higher proportion of small, dense LDL particles.
Patients with DM are categorized into high, intermediate
and low-risk categories depending on lipid levels in
blood (Table 3.3).
Annual lipid profile is indicated in all adult patients
with DM.
Table 3.3: Categorization of cardiovascular risk in diabetes mellitus
according to lipid levels (American Diabetes Association)
Category Low density lipoproteins High density lipoproteins
Laboratory Tests in the Management of Acute
Metabolic Complications of Diabetes Mellitus
The three most serious acute metabolic complications of
DM are:
• Diabetic ketoacidosis (DKA)
• Hyperosmolar hyperglycemic state (HHS)
• Hypoglycemia
The typical features of DKA are hyperglycemia,
ketosis, and acidosis. The common causes of DKA are
infection, noncompliance with insulin therapy, alcohol
abuse and myocardial infarction. Patients with DKA
present with rapid onset of polyuria, polydipsia,
polyphagia, weakness, vomiting, and sometimes
abdominal pain. Signs include Kussmaul’s respiration,
odour of acetone on breath (fruity), mental clouding, and
dehydration. Classically, DKA occurs in type 1, while
HHS is more typical of type 2 DM. However, both
complications can occur in either types. If untreated, both
events can lead to coma and death.
Hyperosmolar hyperglycemic state is characterized by
very high blood glucose level (> 600 mg/dl), hyper-
osmolality (>320 mOsmol/kg of water), dehydration, lack
of ketoacidosis, and altered mental status. It usually occurs
in elderly type 2 diabetics. Insulin secretion is adequate to
prevent ketosis but not hyperglycemia. Causes of HHS are
illness, dehydration, surgery, and glucocorticoid therapy.
Differences between DKA and HHS are presented in
Table 3.4.
Triglycerides

High-risk ≥130 < 35 (men) ≥ 400

< 45 (women)
Intermediate risk 100-129 35-45 200-399
Low-risk < 100 > 45 (men) < 200
> 55 (women)

Table 3.4: Comparison of diabetic ketoacidosis and hyperosmolar hyperglycemic state

Parameter Diabetic ketoacidosis Hyperosmolar hyperglycemic state

1. Type of DM in which more common Type 1 Type 2

2. Age Younger age Older age
3. Prodromal clinical features < 24 hrs Several days
4. Abdominal pain, Kussmaul’s respiration Yes No
5. Acidosis Moderate/Severe Absent
6. Plasma glucose > 250 mg/dl Very high (>600 mg/dl)
7. Serum bicarbonate <15 mEq/L >15 mEq/L
8. Blood/urine ketones ++++ ±
9. β-hydroxybutyrate High Normal or raised
10. Arterial blood pH Low (<7.30) Normal (>7.30)
11. Effective serum osmolality* Variable Increased (>320)
12. Anion gap** >12 Variable
*Osmolality: Number of dissolved (solute) particles in solution; normal: 275-295 mOsmol/kg
** Anion gap: Difference between sodium and sum of chloride and bicarbonate in plasma; normal average value is 12

Laboratory evaluation consists of following investiga-
tions:
• Blood and urine glucose
• Blood and urine ketone
• Arterial pH, Blood gases
• Serum electrolytes (sodium, potassium, chloride,
bicarbonate)
• Blood osmolality
• Serum creatinine and blood urea.
Testing for ketone bodies: Ketone bodies are formed
from metabolism of free fatty acids and include
acetoacetic acid, acetone and β-hydroxybutyric acid.
Indications for testing for ketone bodies in DM include:
• At diagnosis of diabetes mellitus
• At regular intervals in all known cases of diabetes,
during pregnancy with pre-existing diabetes, and in
gestational diabetes
• In known diabetic patients: during acute illness,
persistent hyperglycemia (> 300 mgs/dl), pregnancy,
and clinical evidence of diabetic acidosis (nausea,
vomiting, abdominal pain).
An increased amount of ketone bodies in patients
with DM indicate impending or established diabetic
ketoacidosis and is a medical emergency. Method based
on colorimetric reaction between ketone bodies and
nitroprusside (by dipstick or tablet) is used for detection
of both blood and urine ketones.
Test for urine ketones alone should not be used for
diagnosis and monitoring of diabetic ketoacidosis. It is
recommended to measure β-hydroxybutyric acid (which
accounts for 75% of all ketones in ketoacidosis) for
diagnosis and monitoring DKA.
REFERENCE RANGES
• Venous plasma glucose:
Fasting: 60-100 mg/dl
At 2 hours in OGTT (75 gm glucose): <140 mg/dl
• Glycated hemoglobin: 4-6% of total hemoglobin
• Lipid profile:
– Serum cholesterol: Desirable level: <200 mg/dl
– Serum triglycerides: Desirable level: <150 mg/dl
– HDL cholesterol: ≥60 mg/dl
– LDL cholesterol: <130 mg/dl
– LDL/HDL ratio: 0.5-3.0
• C-peptide: 0.78-1.89 ng/ml
• Arterial pH: 7.35-7.45
• Serum or plasma osmolality: 275-295 mOsm/kg of
water.
Diabetes Mellitus 51
Serum Osmolality can also be calculated by the
following formula recommended by American Diabetes
Association:
Effective serum osmolality (mOsm/kg) =
(2 × sodium mEq/L) + Plasma glucose (mg/dl)
18
• Anion gap:
– Na+ – (Cl– + HCO3–): 8-16 mmol/L (Average 12)
– (Na + + K + ) – (Cl – + HCO3 – ): 10-20 mmol/L
(Average 16)
• Serum sodium: 135-145 mEq/L
• Serum potassium: 3.5-5.0 mEq/L
• Serum chloride: 100-108 mEq/L
• Serum bicarbonate: 24-30 mEq/L
CRITICAL VALUES
• Venous plasma glucose: > 450 mg/dl
• Strongly positive test for glucose and ketones in
urine
• Arterial pH: < 7.2 or > 7.6
• Serum sodium: < 120 mEq/L or > 160 mEq/L
• Serum potassium: < 2.8 mEq/L or > 6.2 mEq/L
• Serum bicarbonate: < 10 mEq/L or > 40 mEq/L
• Serum chloride: < 80 mEq/L or > 115 mEq/L
BIBLIOGRAPHY
1. American Diabetes Association. Diagnosis and
classification of diabetes mellitus. Diabetes Care 2004;
24:S5-S10.
2. American Diabetes Association. Gestational diabetes
mellitus. Diabetes Care 2004;27:S88-S90.
3. American Diabetes Association. Hyperglycemic crises
in diabetes. Diabetes Care 2004;27:S94-S102.
4. American Diabetes Association. Screening for type 2
diabetes. Diabetes Care 2004;27:S11-S14.
5. American Diabetes Association. Tests of glycemia in
diabetes. Diabetes Care 2004;27:S91-S93.
6. Lernmark A. Type 1 diabetes. Clin Chem 1999;45:
1331-38.
7. Lebovitz HE. Type 2 diabetes: An overview. Clin Chem
1999;45:1339-45.
8. Reaven GM. The metabolic syndrome: Requiescat in
pace. Clin Chem 2005;51:931-8.
9. Reinauer H, Home PD, Kanagasabapathy AS, Heuck
C. Laboratory diagnosis and monitoring of diabetes
mellitus. Geneva. World Health Organization, 2002.
10. Sacks DB, Bruns DE, Goldstein DE, Maclaren NK,
McDonald JM, Parrott M. Guidelines and recommen-
dations for laboratory analysis in the diagnosis and
management of diabetes mellitus. Clin Chem 2002;
48:436-72.
11. Trachtenbarg DE. Diabetic ketoacidosis. Am Fam
Physician 2005;71:1705-14.
 52 Essentials of Clinical Pathology
4

Liver Function Tests

FUNCTIONS OF LIVER
1. Excretory function: Liver cells metabolize and excrete
endogenous as well as exogenous substances. Liver
regulates bilirubin metabolism by secretion and
excretion of bilirubin.
2. Synthetic function: Synthesis of proteins like
albumin, α- and β-globulins, transport proteins and
many coagulation proteins occurs in the liver. Liver
also produces triglycerides, cholesterol, lipoproteins,
and primary bile acids. Albumin maintains osmotic
pressure of plasma, transports various compounds,
and acts as a protein reserve. Liver does not
synthesize immunoglobulins and complement.
3. General metabolic functions: Liver regulates carbo-
hydrate, lipid, and protein metabolism.
Glycogen, derived from monosaccharides, is
stored in the liver. When carbohydrate intake is
reduced, blood glucose level is maintained by
breakdown of stored glycogen (glycogenolysis). If
needed, amino acids and fat are converted to glucose
by the liver (gluconeogenesis).
Synthesis of triglycerides, phospholipids, choles-
terol, and lipoproteins occurs in the liver. Liver also
esterifies cholesterol, and forms bile acids from
cholesterol (Fig. 4.1). Bile acids are essential for fat Fig. 4.1: Formation of bile acids and bile salts
absorption from the intestine. Lipoproteins help in
transport of fats.
Besides synthesis of various proteins and NORMAL BILIRUBIN METABOLISM
enzymes, liver is the site for deamination and
transamination of amino acids. Ammonia is Bilirubin is mostly (85%) produced from breakdown
converted to urea in the urea cycle and detoxified in of hemoglobin of old red cells in reticuloendothelial
the liver. cells (macrophages), mainly in spleen. A smaller
4. Liver is the storage site for iron, glycogen, and amount is derived from premature destruction of red cell
vitamins. precursors in bone marrow, and from myoglobin,
5. During fetal life, hematopoiesis occurs in the liver. It
cytochromes, and peroxidases. Steps in metabolism of
is also a site for destruction of damaged red cells
(immune hemolysis). bilirubin are outlined below (Fig. 4.2).
6. Liver is the major organ for catabolism of steroid 1. Hemoglobin is degraded within macrophages to form
hormones. heme and globin; globin consists of amino acids

Fig. 4.2: Normal bilirubin metabolism
which are recycled. Heme (iron + protoporphyrin)
releases iron, which is stored as ferritin.
Protoporphyrin is first converted to biliverdin, which
is then reduced to bilirubin.
2. Bilirubin is released from macrophages into the
circulation where it binds with albumin. This is called
as unconjugated bilirubin. It is soluble in lipid but
insoluble in water.
3. Bilirubin-albumin complex reaches the liver where
it is taken up by the hepatocytes. Bilirubin is set free
in the cytoplasm, while albumin is released back into
the circulation.
4. Bilirubin is conjugated with glucuronic acid to form
bilirubin monoglucuronide and diglucuronide
(conjugated bilirubin); this process is mediated by the
enzyme glucuronyl transferase. Conjugated bilirubin
is more soluble in water.
5. Conjugated bilirubin is secreted from the hepatocyte
into the biliary canaliculi, from where it passes into
the bile duct and gallbladder along with bile (bilirubin
monoglucuronide 25% and bilirubin diglucuronide
75%). Bilirubin reaches the small intestine via the
common bile duct. Bile also contains bile salts, which
Liver Function Tests 53
are necessary for digestion and absorption of fat from
the small intestine.
6. When bilirubin reaches the large intestine, it is
converted by bacterial action to a group of
compounds known as urobilinogen by the action of
bacterial enzymes.
7. Most of the urobilinogen is excreted in feces as
urobilin and is responsible for brown coloration of
feces. A part of urobilinogen is absorbed into the
circulation from where it reaches the liver, is taken
by the hepatocytes, and is again re-excreted in bile
(enterohepatic circulation). A small amount of
urobilinogen in circulation escapes clearance by the
liver and is excreted in urine.
INDICATIONS AND LIMITATIONS OF
LIVER FUNCTION TESTS
Liver function tests (LFT) are the various laboratory tests
that are used to:
• Screen for liver disease;
• Identify the nature of liver disease (hepatocellular,
cholestatic, or infiltrative);
 54 Essentials of Clinical Pathology
• Assess severity and prognosis of liver disease; and
• Follow up the course of liver disease
Box 4.1: Limitations of liver function tests
• Do not necessarily assess liver function
• Lack sensitivity (i.e. may be normal in some liver
diseases like cirrhosis)
• Lack specificity (i.e. may be abnormal in non-liver
disorders e.g. serum albumin is low in nephrotic
syndrome and in cirrhosis)
The term ‘liver function tests’ is a misnomer since
most of these tests are used for identification of liver
disease, its severity, and its type and do not necessarily
assess liver function. Generally these tests, which are
performed in combination, are abnormal in liver disease,
and the pattern of abnormality is indicative of the nature
of liver disease. Since liver has a large amount of
anatomical and functional reserve and capacity for rapid
regeneration, functional deficiency becomes apparent if
there is an extensive liver damage. Also, these tests are
not specific for liver disease (except serum bile acids) and
are abnormal in various non-hepatic conditions (Boxes
4.1 and 4.2). Therefore, laboratory tests should be
selected and interpreted in the context of clinical
features and other investigations. An isolated
abnormality of a single liver function test usually means
a non-hepatic cause. If several liver function tests are
simultaneously abnormal, then hepatic etiology is likely.
Box 4.2: Non-hepatic causes of abnormal liver
function tests
• Increased serum bilirubin:
– Hemolysis
– Ineffective erythropoiesis
– Resorption of a large hematoma
• Increased aminotransferases:
– Muscle injury
– Alcohol abuse
– Myocardial infarction
• Increased serum alkaline phosphatase:
– Pregnancy
– Bone disease
• Low serum albumin:
– Poor nutritional status
– Proteinuria
– Malabsorption
– Severe illness causing protein catabolism
Table 4.1: Commonly performed liver function tests
Test Hepatic cause of abnormality
1. Serum alanine Hepatocellular injury
aminotransferase
2. Serum aspartate Hepatocellular injury
aminotransferase
3. Serum alkaline Cholestasis
phosphatase
4. Serum bilirubin Defective conjugation or excretion
5. Serum albumin Decreased synthesis
CLASSIFICATION OF
LIVER FUNCTION TESTS
Liver function tests can be classified as follows:
1. Tests that assess excretory function of the liver:
Bilirubin in serum and urine, and urobilinogen in
urine and feces.
2. Tests that assess synthetic and metabolic functions
of the liver: Serum proteins, serum albumin, serum
albumin/globulin (A/G) ratio, prothrombin time
(PT), and blood ammonia level.
3. Tests that assess hepatic injury (liver enzyme
studies): Serum alanine aminotransferase (ALT),
serum aspartate aminotransferase (AST), serum
alkaline phosphatase, serum γ-glutamyl transferase
(GGT), and 5’-nucleotidase (5’-NT).
4. Tests that assess clearance of exogenous substances
by the liver: Bromosulphthalein excretion test.
Commonly performed liver function tests are listed
in Table 4.1.
Tests that Assess Excretory
Function of the Liver
Jaundice
Jaundice (from French jaune, meaning yellow) or icterus
refers to yellow discoloration of skin, sclera, and mucous
membranes due to increased level of serum bilirubin.
Jaundice becomes clinically evident when serum
bilirubin level exceeds 2.0 mg/dl.
There are various methods for classification of
jaundice as follows:
1. According to the main type of bilirubin increased
in plasma:
• Predominantly unconjugated hyperbilirubinemia:
Indirect or unconjugated bilirubin is > 85% of total;
causes are hemolysis, resorption of a large
hematoma, ineffective erythropoiesis, Gilbert’s
syndrome, physiologic jaundice of newborn, and
Crigler-Najjar syndrome.
 Liver Function Tests 55

• Predominantly conjugated hyperbilirubinemia:
Direct or conjugated bilirubin is >50% of total;
causes are hepatitis, cirrhosis, cholestasis, drugs
(anabolic steroids, oral contraceptives), toxins,
Dubin-Johnson syndrome, and Rotor syndrome.
• Mixed (conjugated + unconjugated) hyperbili-
rubinemia: Conjugated bilirubin is 20-50% of total;
it results from viral or alcoholic hepatitis.
2. According to etiology:
• Hemolytic: The increased rate of red cell destruc-
tion causes increased haemoglobin breakdown to
bilirubin in reticuloendothelial cells; this exceeds
the capacity of conjugation in liver.
• Hepatocellular: Inability of hepatocytes to conju-
gate and/or excrete bilirubin.
• Obstructive: Failure of excretion of conjugated
bilirubin into the intestine, causing its
regurgitation in circulation.
3. According to site of disease:
• Prehepatic
• Hepatic
• Posthepatic
A simple classification is division of jaundice into
three main types: prehepatic, hepatic, and
posthepatic. This classification is the basis for
identifying the cause of jaundice (Table 4.2).
1. Prehepatic jaundice: There is excessive formation of
bilirubin exceeding the capacity of the liver to
conjugate it for excretion. The type of bilirubin
increased in serum is of unconjugated type. Bilirubin
is absent in urine since unconjugated bilirubin is
water-insoluble. Urobilinogen is increased in urine
and feces. Jaundice is usually mild (serum bilirubin
<5.0 mg/dl; conjugated bilirubin is <15% of total).
The cause can usually be identified by examination
of a stained blood smear and hematological
investigations. In jaundiced newborns, rapidly rising
unconjugated bilirubin needs careful management to
prevent kernicterus.
2. Hepatic jaundice: In hepatic disease, unconjugated,
conjugated, or both are increased.
a. Unconjugated hyperbilirubinemia:
1. Defective uptake of bilirubin by liver cells from
blood: Gilbert’s syndrome is the most common
cause of unconjugated hyperbilirubinemia,
affecting 5% of general population. It is a
familial, benign disease with autosomal domi-
nant mode of inheritance. Raised bilirubin is
usually noticed during routine laboratory
examination. There is defective uptake by
hepatocytes and mild deficiency of glucuronyl
transferase. Jaundice is mild and fluctuating,
and may go unnoticed for years. Jaundice
becomes noticeable during illness or after
fasting. Mildly elevated serum bilirubin is the
sole abnormality; other LFTs are normal.
2. Defective conjugation of bilirubin:
Crigler-Najjar syndrome: This is of two types.
Type I is characterized by autosomal recessive
mode of inheritance, complete absence of
glucuronyl transferase, severe unconjugated
hyperbilirubinemia, and kernicterus (deposition
of bilirubin in basal ganglia of brain). Type II is a

Table 4.2: Classification of jaundice according to the site of disease

Prehepatic jaundice
• Hemolytic anemia
• Ineffective erythropoiesis (megaloblastic anemia, thalassemias)
• Resorption of a large hematoma
Hepatic jaundice
• Predominantly unconjugated hyperbilirubinemia
– Gilbert’s syndrome
– Crigler-Najjar syndrome
– Physiologic jaundice of newborn
• Predominantly conjugated hyperbilirubinemia
– Hepatocellular diseases: viral hepatitis, toxic hepatitis, alcoholic hepatitis, active cirrhosis
– Intrahepatic cholestasis: Dubin-Johnson syndrome, drugs, primary biliary cirrhosis, primary sclerosing
cholangitis, biliary atresia
Posthepatic jaundice
• Carcinoma of head of pancreas
• Carcinoma of ampulla of Vater
• Secondaries in porta hepatis
• Gallstones in or stricture of common bile duct
56 Essentials of Clinical Pathology

less severe disease in which some amount of
enzyme activity is present.
Physiologic jaundice of newborn: This is a tran-
sient increase of unconjugated bilirubin which is
observed in almost all newborns. It usually
develops during the 2nd to 4th day after birth
with return to normal bilirubin level by 7th to
10th day. It is because of deficiency of glucuronyl
transferase leading to impaired conjugation
during the first few days of life.
b. Conjugated hyperbilirubinemia:
1. Hepatocellular disease: Liver enzymes (aspar-
tate aminotransferase and alanine amino-
transferase) are markedly elevated, and serum
bilirubin is usually in the range of 4.0 to 8.0
mg/dl. Conjugated bilirubin is 20-50% of total
bilirubin. In hepatocellular injury, both conju-
gated and unconjugated bilirubins are increa-
sed. Unconjugated bilirubin is increased due
to reduced ability of liver cells to conjugate
bilirubin. Conjugated bilirubin is raised from
cholestasis due to hepatocyte swelling.
2. Intrahepatic cholestasis: In intrahepatic choles-
tasis, there may be (1) impairment of secretion
of bilirubin from hepatocytes into the biliary
canaliculi; (2) obstruction of bile flow in
canaliculi by swollen hepatocytes;or (3)
damage to intrahepatic canaliculi.
In Dubin-Johnson syndrome, there is a
defect in the excretion of bilirubin by hepato-
cytes, and liver is darkly pigmented due to
accumulation of polymerized epinephrine
metabolites. Bromosulphthalein excretion test
is abnormal. In Rotor syndrome, there is impai-
red excretion of bilirubin but without pigmen-
tation of liver; bromosulphthalein test is
normal. Alkaline phosphatase is normal in both
conditions.
Drugs commonly associated with choles-
tatic injury are oral contraceptives, anabolic
steroids, oral anti-diabetics, phenothiazines,
and erythromycin.
In primary biliary cirrhosis, there is auto-
immune destruction of intrahepatic bile ducts.
It predominantly occurs in middle-aged
females and is characterized by chronic eleva-
tion of alkaline phosphatase and positive anti-
mitochondrial antibody in serum. There is an
association with other autoimmune disorders.
Primary sclerosing cholangitis is an auto-
immune disorder occurring in young to
middle-aged men in whom there is inflam-
mation and destruction of both intrahepatic
and extra-hepatic bile ducts. Associated
inflammatory bowel disease is often present.
Serum alkaline phosphatase is elevated and
many patients have circulating perinuclear
antineutrophil cytoplasmic antibodies.
3. Posthepatic jaundice: This is also called as
obstructive jaundice, surgical jaundice, or
extrahepatic cholestasis.
Different sites of cholestasis and causative
disorders are shown in Figure 4.3.
Obstruction of extrahepatic biliary tract
prevents flow of bile into the duodenum. This
causes “regurgitation” of conjugated bilirubin
into the circulation. (Biliary canaliculi distend
and rupture due to backpressure of bile and
conjugated bilirubin escapes into the
sinusoids). Conjugated bilirubin is usually
>50% of total in posthepatic jaundice. Urinary
and fecal urobilinogen are decreased, faeces are
clay-colored, and bilirubin (being conjugated
and water-soluble) appears in urine.
Differences between three main types of jaundice are
given in Table 4.3.
Investigation of a case of jaundice is shown in Figure 4.4.
The tests employed to assess excretory function of
liver are serum/urine bilirubin and urine/fecal
urobilinogen.
Serum Bilirubin
Normal serum bilirubin is less than 1 mg/dl. There are two
types of bilirubin in plasma: unconjugated (indirect-
reacting) and conjugated (direct-reacting) (Box 4.3).
Normally, conjugated bilirubin is 10% or less, while
Fig. 4.3: Sites of cholestasis
 Liver Function Tests 57

Table 4.3: Differential diagnosis of three main types of jaundice

Parameter Prehepatic jaundice Hepatocellular Posthepatic

jaundice jaundice

1. Basic mechanism of Hemolysis leading to excess Deficient uptake, Deficient excretion due to
raised bilirubin production conjugation, or obstruction of biliary tract
excretion by hepato-
cytes
2. Type of serum bilirubin Mainly unconjugated Unconjugated + Mainly conjugated (>50%)
increased Conjugated
3. Urine bilirubin Absent Present Present
4. Urine urobilinogen Increased Variable Decreased
5. Prototype Hemolytic anemia Viral hepatitis Common duct stone
6. Prothrombin time Normal Abnormal that is not Abnormal that is corrected
corrected with with vit K
vitamin K
7. Additional features Features of haemolysis on Marked rise of serum Marked rise of serum ALP
blood smear (reticulocytosis, ALT and AST (>3 times normal)
low haptoglobin, low hemoglobin)

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase

Fig. 4.4: Investigation of jaundice. ALT: alanine aminotransferase; AST: aspartate aminotransferase;
ALP: alkaline phosphatase

unconjugated bilirubin is 90% or more. This is because
conjugated bilirubin is rapidly secreted into the bile after
its formation and removed through the gut. Conjugated
bilirubin is composed of blirubin glucuronide, bilirubin
diglucuronide, and delta (δ) bilirubin. Delta bilirubin
represents bilirubin covalently bound to albumin in
circulation. Normally, δ bilirubin is absent or present in
very small amount. In cholestasis, proportion of δ-bili-
rubin increases. Owing to its longer half-life, it is cleared
slowly from circulation. Conjugated bilirubin is weakly
bound to albumin, is water-soluble, and can be excreted
in urine. Unconjugated bilirubin is tightly bound to
albumin and is water-insoluble. As bilirubin is altered by
exposure to light, sample should be kept in the dark.
 58 Essentials of Clinical Pathology
Fig. 4.5: Estimation of serum bilirubin by diazo method
There are two methods for estimation of serum
bilirubin: diazo method and spectrophotometry.
In diazo method, total and direct-reacting bilirubin
are measured, and indirect bilirubin is obtained by
subtracting direct from total bilirubin (Fig. 4.5).
Estimation of both types of bilirubin is helpful in the
differential diagnosis of jaundice. In post-hepatic type
of jaundice, direct bilirubin is the predominant form (>
50% of the total). In hepatocellular jaundice, direct
bilirubin is usually between 20-50% of total. Indirect
bilirubin predominates in hemolysis, Gilbert’s syndrome,
and Crigler-Najjar syndrome (direct bilirubin is < 15%
of total).
Box 4.3: Serum bilirubin
• Direct bilirubin (Conjugated bilirubin): It reacts
directly with diazo reagent. It consists of mono-
conjugated bilirubin, diconjugated bilirubin, and bilirubin
tightly bound to albumin (delta bilirubin).
• Indirect bilirubin (Unconjugated bilirubin): It reacts
with diazo reagent in the presence of alcohol. It consists
of bilirubin bound to albumin. It is calculated as ‘total
bilirubin minus direct bilirubin’.
Direct spectroscopic estimation is used for measurement
of total serum bilirubin in newborns and infants (<3
months of age). Concentration of serum bilirubin is
directly proportional to its absorbance in a
spectrophotometer at 454 nm. This method cannot be
used in older children and adults because their sera may
also contain carotene and other pigments, which absorb
light at the same wavelength. In newborns, other
pigments are absent.
Urine Bilirubin and Urobilinogen
See Chapter 1 “Examination of Urine”.
Tests which Assess Synthetic and Metabolic
Functions of Liver
Markers of hepatic synthetic function are serum albumin
and prothrombin time. Hypoalbuminemia and a pro-
longed prothrombin time indicate severe functional
impairment of liver.
Proteins
Liver is the sole site for the synthesis of most of the plasma
proteins, except gamma globulins which are synthesized
by plasma cells. Concentration of total serum proteins
in adults is about 5.5 to 8.0 gm/dl, while that of serum
albumin is 3.5 to 5.0 gm/dl. Serum albumin comprises
about 60% of total serum proteins.
Tests for proteins in liver disease include total
serum proteins, serum albumin, calculation of serum
albumin/globulin ratio (normal ratio is >1.5), and serum
protein electrophoresis.
1. Total serum proteins: These can be estimated by
refractometer method or by biuret method.
In refractometer method, refractive index of the
solution (which depends on solute concentration) is
measured. Refractive index varies mainly with concen-
tration of proteins and is affected very little by electrolytes

and other molecules. Protein concentration is read
directly from the scale on the refractometer. In biuret
method, copper ions react with peptide bonds of proteins
and form a violet-colored compound. Intensity of this
color (measured colorimetrically) is proportional to the
concentration of proteins.
Total serum protein level is affected by both albumin
and gamma globulins. In cirrhosis, decrease in albumin
level is often compensated by increase in the level of
gamma globulins; therefore, estimation of total serum
proteins is of limited value in cirrhosis. Estimation of
serum albumin and serum protein electrophoresis are
more helpful.
2. Serum albumin: Albumin is synthesized exclusively
in liver and constitutes about 60% of total proteins in
serum; therefore its estimation is an important investi-
gation in liver disease. Half-life of albumin is about 20
days and therefore fall in its level in response to decreased
synthesis is not immediately apparent. Therefore, in
acute liver disease (e.g. viral hepatitis), there is little
change in albumin level. Serum albumin level is low in
chronic liver disease (cirrhosis) and correlates with
synthetic capacity of hepatocytes; therefore, it is helpful
in following progression of cirrhosis. Also, fall in serum
albumin level correlates with severity of ascites. In
cirrhosis and in chronic active hepatitis, serum gamma
globulins are increased due to inflammation. Low
albumin and raised gamma globulins in serum cause
reversal of albumin/globulin ratio.
Serum albumin is estimated by bromocresol green
method. Bromocresol green is an indicator dye, which
when added to serum, binds selectively and tightly to
albumin and becomes blue in color. Absorbance (in a
spectrophotometer at 632 nm) is directly proportional to
concentration of albumin.
Causes of decreased serum albumin:
• Decreased intake: malnutrition.
• Decreased absorption: malabsorption syndromes.
• Decreased synthesis: liver disease, chronic infections.
• Increased catabolism: thyrotoxicosis, fever, malig-
nancy, infections.
• Increased loss: nephrotic syndrome, severe burns,
protein-losing enteropathies, ascites
• Increased blood volume: pregnancy, congestive
cardiac failure.
As low serum albumin occurs in diseases other than
those of liver, serum albumin is a sensitive but non-
specific test for liver disease.
3. Serum protein electrophoresis: Details of serum
protein electrophoresis are given in Chapter 28
“Laboratory Tests in Hematological Malignancies”.
In liver disease, following changes may be seen on
protein electrophoresis (Fig. 4.6):
Liver Function Tests 59
1. In cirrhosis, albumin may be reduced and there may
be polyclonal increase of IgG and IgA, with β-γ
bridging. (IgA migrates between β and γ regions which
obscures the demarcation between β and γ peaks).
2. In primary biliary cirrhosis, there is polyclonal
increase of IgM.
3. In α1-antitrypsin deficiency (associated with cirrhosis)
α1- globulin band is reduced.
4. In chronic active hepatitis, IgG is elevated.
Prothrombin Time (PT)
Most of the coagulation proteins are synthesized in the
liver. Vitamin K is required for the synthesis of factors
II, VII, IX, and X by the hepatocytes; therefore these
factors are called as vitamin K-dependent factors.
Synthesis of these factors is deficient in hepatocellular
disease. In obstructive jaundice, vitamin K (a fat-soluble
vitamin) cannot be absorbed due to the absence of bile
in the intestine.
PT measures three out of four vitamin K-dependent
factors (II, VII, and X) and is prolonged in hepatocellular
disease and in obstructive jaundice. Intramuscular
injection of vitamin K corrects prolonged PT in
obstructive jaundice but not in hepatocellular jaundice.
In acute fulminant liver failure, marked prolongation
of PT is an unfavourable prognostic sign.
To distinguish between a prolonged PT due to hepato-
cellular disease from that due to cholestasis with fat
malabsorption, PT is repeated after administration of
vitamin K. Reduction of prolonged PT occurs in
cholestatic liver disease, but not in hepatocellular
disease.
Blood Ammonia
Blood ammonia is mainly derived from gastrointestinal
tract. In the intestine, bacterial enzymes act on nitrogen-
containing foods to produce ammonia, which is carried
to the liver via portal vein. In the liver, ammonia is
converted to non-toxic urea in the urea cycle.
Increased blood ammonia levels are seen in:
• Fulminant hepatic failure
• Cirrhosis
• Reye’s syndrome
• “Shunting” of portal blood to systemic circulation
• Gastrointestinal hemorrhage (there is increased
production of ammonia from blood proteins by
bacterial enzymes). In hepatic disease, gastro-
intestinal hemorrhage is associated with increased
risk of hepatic encephalopathy.
• Inherited deficiencies of urea cycle enzymes.
If raised, estimation of blood ammonia is likely to be
helpful in patients with coma of unknown origin, since
it is indicative of hepatic encephalopathy.
 60 Essentials of Clinical Pathology

Fig. 4.6: Serum protein electrophoresis patterns and densitometer scans in normal individuals and in
cirrhosis of liver and α1-antitrypsin deficiency

Tests which Assess Hepatic Injury • 5’-nucleotidase (5’-NT)

(Liver Enzyme Studies)
Serum enzyme changes in liver disease result from
hepatocyte damage and do not indicate hepatic
functional capacity. In the investigation of liver disease,
following serum enzymes are measured:
• Serum aspartate aminotransferase or AST (formerly
called serum glutamic-oxaloacetic transaminase or
SGOT)
• Serum alanine aminotransferase or ALT (formerly
called serum glutamic-pyruvic transaminase or
SGPT)
• Serum alkaline phosphatase or ALP
• γ-Glutamyl transferase or GGT (also called as
γ-glutamyl transpeptidase)
Locations of enzymes in liver cell are shown in Figure
4.7. Degree of elevation of a particular enzyme depends
on pattern of hepatic damage.
Serum Aminotransferases
Serum aminotransferases are the sensitive markers of
acute hepatocellular injury. ALT is a cytosolic enzyme
while AST is both cytosolic and mitochondrial.
Normally, aminotransferases are present in serum at
a low level. When necrosis or death of cells containing
these enzymes occurs, aminotransferases are released
into the blood and their concentration in blood increases.
This level correlates with extent of tissue damage.
 Liver Function Tests 61

Most marked elevations of ALT and AST (>15 times
normal) are seen in acute viral hepatitis, toxin-induced
hepatocellular damage (e.g. carbon tetrachloride), and
centrilobular necrosis due to ischemia (congestive
cardiac failure).
Moderate elevations (5-15 times) occur in chronic
hepatitis, autoimmune hepatitis, alcoholic hepatitis, acute
biliary tract obstruction, and drug-induced hepatitis.
Mild elevations (1-3 times) are seen in cirrhosis, non-
alcoholic steatosis, and cholestasis.
Determinations of these enzymes are helpful in the
differential diagnosis of hepatocellular from cholestatic
jaundice. Increase of AST and ALT is much more in
hepatocellular jaundice (>500 units/ml) than in choles-
tatic jaundice (<200 units/ml) (Fig. 4.8).
Although determination of any one aminotransferase
is adequate, measurement of both can be helpful in the
calculation of AST/ALT ratio. Normal ratio is 0.7 to 1.4.
Increased ratio (>2.0) is highly suggestive of alcoholic
hepatitis, while ratio <1.0 is seen in acute viral hepatitis.
ALT and AST are elevated in acute viral hepatitis
even before the appearance of jaundice. Persistence of
elevated ALT and AST beyond 6 months in a case of
hepatitis indicates development of chronic hepatitis.
In massive liver necrosis, aminotransferase levels
gradually decrease. Therefore, falling levels do not
necessarily indicate recovery from acute hepatitis.
Serum Alkaline Phosphatase (ALP)
Alkaline phosphatase is distributed widely in various
tissues like liver, bones, intestine, kidney, and placenta.
In the liver, ALP, GGT, and 5’-NT are located normally
on canalicular surface of hepatocytes (See Fig. 4.7). In
cholestasis, accumulated bile acids dissolve canalicular
side of hepatocyte membrane and enzymes are released
in blood. Therefore, diseases that affect mainly
hepatocyte secretion have elevated levels of ALP.
Measurement of ALP is helpful in differentiation of
hepatocellular jaundice from cholestatic jaundice
(See Fig. 4.8).
Causes of increased ALP
1. Hepatobiliary disease: ALP is increased in most cases
of cholestatic type of jaundice. While hepatocellular
injury is characterized by marked elevation of ALT
and AST, cholestasis is characterized by marked
increase (more than 3 times normal) of ALP. Since
there are many other sources of ALP apart from liver,
simultaneous measurement of serum GGT and
serum 5’-NT may be used to ascertain whether
increase of ALP is of hepatic origin.
Hepatobiliary causes of increased alkaline
phosphatase are:
• Bile duct obstruction (cancer of head of pancreas,
stone in common bile duct, stricture of bile duct,
biliary atresia)

Fig. 4.7: Locations of enzymes in liver cell. The enzymes are found in specific locations as follows: Alanine aminotransferase
(ALT): cytosol; Aspartate aminotransferase (AST): mitochondria and cytosol; Lactate dehydrogenase (LDH): cytosol; Alkaline
phosphatase (ALP): canalicular surface; Gamma glutamyl transferase (GGT): canalicular surface and microsomes; 5’ NT:
canalicular surface. The pattern of hepatocyte injury determines the enzymes elevated: cytoplasmic damage: elevated AST,
ALT, and LDH; mitochondrial damage: elevated AST; cholestatic damage: elevated ALP and GGT
 62 Essentials of Clinical Pathology
Fig. 4.8: (A) Comparative serum alanine transferase (ALT) levels in liver diseases. Red horizontal line represents upper limit
of normal. (B) Comparison of percentage levels of serum alkaline phosphatase (ALP) in hepatocellular disease and cholestasis.
Red horizontal line represents discriminatory level between hepatocellular disease and cholestasis (i.e. 3 times normal), while
black horizontal line represents upper limit of normal
• Primary biliary cirrhosis
• Primary sclerosing cholangitis
• Infiltrative diseases of liver (granulomatous
diseases like tuberculosis or sarcoidosis, amyloi-
dosis, cysts, primary or secondary cancer)
Further work-up is necessary to diagnose the
cause of cholestasis depending on history and
physical examination findings. This usually consists
of abdominal ultrasonography or CT scan (for
detection of dilated bile ducts which indicate
extrahepatic obstruction, or for detection of focal or
diffuse parenchymal lesion of liver); endoscopic
retrograde cholangiopancreatography (ERCP) or
percutaneous transhepatic cholangiography (PTC) to
diagnose cause of biliary obstruction; and liver
biopsy.
2. Diseases of bones: ALP is present within osteoblasts.
Due to high osteoblastic activity during active bone
growth, serum ALP is higher in children than in
adults. Elevation of ALP occurs in conditions with
increased osteoblastic activity like osteomalacia,
rickets, hyperparathyroidism, Paget’s disease,
osteosarcoma, and osteoblastic type of metastatic
carcinoma.
3. Pregnancy: Serum ALP is increased during
pregnancy due to secretion from placenta.
Serum γ-glutamyl Transferase (GGT)
This is also called as γ-glutamyl transpeptidase. Relatively
high levels of this enzyme are present in liver, pancreas,
kidney, and prostate. Estimation of this enzyme is
particularly useful in following liver diseases:
1. Alcoholism: Increased enzyme activity is present in
alcoholism, and is a helpful clue in suspected cases
of occult alcoholism (even in the absence of alcoholic
liver disease). It is also helpful in follow up of patients
with chronic alcoholism. Marked elevation of GGT
occurs in acute alcoholic hepatitis.
2. Cholestasis: Elevation of GGT generally parallels that
of ALP and 5’-NT in liver disease. Elevation of ALP
is not specific for liver disease. Elevation of both ALP
and GGT points towards liver disease.
3. Recovery in acute hepatitis: Serum GGT is the last
enzyme to return to normal following acute hepatitis
and its normalization is indicative of a favourable
outcome.
5’-nucleotidase (5’-NT)
5’-NT is present in liver as well as in various other tissues.
It is located mainly along the cell membrane, similar to
ALP and GGT. Estimation of 5’-NT is helpful in deciding
tahir99 - UnitedVRG
vip.persianss.ir

whether increased ALP is due to liver disease or due to
increased osteoblastic activity in growing children.
Tests that Assess Clearance of Exogenous
Substances from the Liver
Some synthetic dyes, when introduced into the body, are
rapidly removed from the blood stream by the liver and
excreted into the bile. Disappearance of these dyes from
the blood is dependent on adequate hepatic circulation,
normal hepatocyte function, and uninterrupted bile flow.
Dye excretion tests are sensitive tests of liver function
and are usually indicated in those patients in whom liver
disease is suspected but have little or no jaundice. Three
synthetic dyes are commonly employed to test liver
function: bromosulphthalein (BSP), indocyanine green
(used by surgeons before resecting liver for hepato-
cellular cancer) , and Rose Bengal. However, because of
the risk of adverse reactions and advent of more sensitive
tests for detection of liver disease, dye excretion tests are
nowadays rarely used.
Bromosulphthalein (BSP) excretion test: After adminis-
tration, BSP is taken up by the hepatocytes, binds to
ligandin and Z protein, conjugated to glutathione, and
then excreted into the bile. In this test, BSP is injected
intravenously, a blood specimen is obtained after a
specified time, and the percent of dye retained in blood
is calculated. If the amount of dye retained in blood is
more than 50% at 45 minutes, abnormal liver function is
present.
BSP excretion test will yield falsely abnormal result
if hepatic circulation is impaired. Also, there is a risk of
adverse reactions including tissue necrosis and anaphy-
laxis in a small number of patients.
BSP test remains useful in the diagnosis of Dubin-
Johnson syndrome and its differentiation from Rotor
syndrome. A higher level of BSP in blood at 2 hours with
normal value at 45 minutes is seen in Dubin-Johnson
syndrome. In Rotor syndrome, value of BSP is higher at
45 minutes and lower at 2 hours.
INTERPRETATION OF
LIVER FUNCTION TESTS
A stepwise approach consists of laboratory tests for:
• Presence of liver disease: History, physical findings,
and liver function tests
• Nature of liver disease: whether hepatocellular (cell
injury) or cholestatic
• Specific cause of liver disease
• Assessment of severity of liver disease
Liver diseases can be broadly classified into two types:
hepatocellular (primary abnormality is liver cell
necrosis), and cholestatic (primary abnormality is
Liver Function Tests 63
impairment of bile flow that may be intrahepatic or
extrahepatic). History and physical examination findings
should be correlated with liver function tests.
Box 4.4: Typical LFT profile in hepatocellular disease
• Marked elevation of AST and ALT (usually >500 IU)
• Mild increase of ALP (<3 times normal)
• Hyperbilirubinemia, if present, is of both conjugated and
unconjugated type
Hepatocellular injury may be acute (e.g. acute viral
hepatitis, toxic hepatitis, ischemic hepatitis, alcoholic
hepatitis) or chronic (cirrhosis, chronic active hepatitis,
autoimmune hepatitis). Acute hepatocellular injury is
usually associated with marked elevation of ALT and
AST and mild elevation of alkaline phosphatase (Box 4.4).
In chronic hepatocellular injury, aminotransferases are
moderately elevated, and serum albumin is reduced.
Cholestasis is characterized by mild elevation of ALT
and AST and marked increase of ALP and GGT (Box
4.5). With complete obstruction of bile ducts, both serum
ALP and serum bilirubin are increased, stools are pale,
and urine contains no urobilinogen. The pattern of
elevated ALP but normal serum bilirubin is seen in
infiltrative diseases. Laboratory abnormalities often
overlap in cholestatic and infiltrative diseases; imaging
studies (such as ultrasound, CT scan, magnetic resonance
imaging, and cholangiography) and liver biopsy are
needed for distinction between them.
Box 4.5: Typical LFT profile in cholestatic jaundice
• Marked elevation of ALP (>3 times normal)
• Elevation of GGT and 5’-NT
• Mild or no increase of ALT and AST (usually <200 IU)
• Elevation of conjugated bilirubin
For specific or etiologic diagnosis of liver disease,
further testing is required:
• Hepatocellular disease: In patients with hepato-
cellular pattern of injury, common investigations to
detect underlying cause include viral serology (viral
hepatitis: IgM anti-hepatitis A antibody, hepatitis
surface B antigen (HBsAg), antihepatitis C antibody),
search for injurious drugs, toxins or alcohol,
autoantibodies like antinuclear antibodies and anti-
smooth muscle antibodies (autoimmune hepatitis),
and serum ceruloplasmin (Wilson disease). If cause
is not detected in the presence of persistent elevation
of aminotransferases, liver biopsy is performed (that
may reveal chronic viral hepatitis, autoimmune
disorders, Wilson disease, haemochromatosis, and
infiltrative diseases) (Table 4.4 and Figure 4.9).
tahir99 - UnitedVRG
vip.persianss.ir
 64 Essentials of Clinical Pathology

Table 4.4: Differentiation of causes of raised aminotransferases

Cause Clinical features Diagnosis

1. Viral hepatitis A H/o exposure in endemic areas IgM anti-hepatitis A antibodies

2. Viral hepatitis B
3. Viral hepatitis C
4. Viral hepatitis E
5. Alcoholic liver disease
6. Nonalcoholic steatohepatitis
7. Autoimmune hepatitis
8. Hemochromatosis
9. Wilson’s disease
H/o nonsterile injections, IV drug
abuse, blood transfusion, multiple
sexual partners, homosexuals
As for hepatitis B
H/o exposure in endemic area
H/o alcohol abuse
Type 2 DM, obesity, hyperlipidemia
Young female
Autosomal recessive, diabetes mellitus,
skin pigmentation, chronic liver disease,
multiorgan dysfunction
Young age, autosomal recessive,
Kayser-Fleischer ring, hemolytic
anemia
IgM anti-hepatitis B core antigen
antibodies, hepatitis B surface antigen
Hepatitis C-RNA, antihepatitis C
antibodies
Anti-HEV
AST/ALT ratio > 2.0, ↑ GGT
Liver biopsy
Raised immunoglobulins, low albumin,
antinuclear antibody+, anti-smooth
muscle antibody+, liver kidney
microsomal antibody+
Transferrin saturation >45%, genetic
testing
Low ALP, high serum bilirubin, low serum
ceruloplasmin

Fig. 4.9: Evaluation of acute hepatocellular injury

• In patients with cholestatic or infiltrative pattern of LIVER BIOPSY

injury, imaging studies should be done for diagnosis
of obstruction. If there is no evidence of obstruction,
liver biopsy is usually done (Table 4.5 and Fig. 4.10).
Severity of liver disease is assessed by serum
bilirubin, serum albumin, and prothrombin time.
Child-Turcotte-Pugh classification is commonly used to
assess severity of cirrhosis and is based on both clinical
and laboratory parameters (Table 4.6).
Liver biopsy is a procedure in which a small piece of
liver tissue is removed and examined microscopically to
determine the cause and severity of liver disease. Paul
Ehrlich performed the first percutaneous liver biopsy in
1883 in Germany. Since then the technique has been
modified and now a variety of approaches can be used
to obtain a liver biopsy sample. Before proceeding with

tahir99 - UnitedVRG
vip.persianss.ir
 Liver Function Tests 65

Table 4.5: Differentiation of causes of raised serum alkaline phosphatase in jaundice

Cause Clinical features Investigations

1. Primary biliary cirrhosis Middle-aged female; pruritus; Antimitochondrial antibody; raised

xanthelasma
2. Primary sclerosing cholangitis Adult male; associated inflammatory
bowel disease
3. Extrahepatic biliary obstruction Dark urine; clay-colored stools;
palpable gallbladder ±
serum cholesterol
Antinuclear cytoplasmic antibody,
endoscopic retrograde
cholangiopancreatography
Ultrasonography for dilated bile ducts;
endoscopic retrograde
cholangiopancreatography

Fig. 4.10: Evaluation of raised serum alkaline phosphatase in cholestatic injury

the biopsy, potential risks of the procedure and benefits TYPES OF LIVER BIOPSY
of histological examination should be assessed and the Currently, several methods are available for obtaining
procedure should be performed only when benefits out liver tissue specimen. Choice of the method depends on
weigh the risks. its availability, clinical situation, and preference of the

tahir99 - UnitedVRG
vip.persianss.ir
 66 Essentials of Clinical Pathology
Table 4.6: Child-Turcotte-Pugh grading system to assess severity of cirrhosis
Parameter Score 1
Serum bilirubin (mg/dl) <2
Serum albumin (g/dl) ≥ 3.5
Prothrombin time (difference in seconds) <4
Ascites None
Encephalopathy (grade) 0 1-2
Grade A (excellent prognosis): Score 5 or 6; Grade B (intermediate prognosis): Score 7 to 9; Grade C (very poor prognosis):
Score ≥ 10.
operator. An experienced clinician, gastroenterologist,
or hepatologist performs the liver biopsy. A radiologist
performs transjugular biopsy.
Methods of obtaining liver biopsy are:
• Percutaneous liver biopsy
– Percutaneous ‘blind’ liver biopsy
– Percutaneous guided liver biopsy
– Percutaneous plugged liver biopsy
• Transvenous (Transjugular) liver biopsy
• Laparoscopic liver biopsy
Percutaneous ‘Blind’ Liver Biopsy
In this technique, liver biopsy is carried out without any
real time imaging guidance. Borders of the liver are
defined by percussion and the biopsy needle is inserted
either through the intercostal or subcostal route.
Subcostal route is employed if liver is enlarged below
the costal margin.
Liver biopsy needles are of three main types: suction
needles (Menghini, Jamshidi), cutting needles (Tru-Cut,
Vim-Silverman) (Fig. 4.11), and spring-loaded cutting
needles. Cutting needles require longer time for biopsy
as compared to the suction needles and remain in liver
for a longer period, thus increasing the risk of bleeding.
Suction needles often cause fragmentation of cirrhotic
liver tissue.
Indications:
1. Acute hepatitis of unknown cause: (Liver biopsy
is not required in typical acute viral hepatitis).
Fig. 4.11: Vim-Silverman liver biopsy needle
Score 2 Score 3
2-3 >3
2.8-3.5 <2.8
4-6 >6
Slight or controlled by diuretics Moderate despite diuretics
3-4
2. Chronic liver disease:
• To define cause
• Grading of inflammatory activity
• Staging (prognosis) of chronic hepatitis C and
chronic hepatitis B
• Monitor response to therapy
3. Diagnosis of metabolic liver disorders like
haemochromatosis and Wilson’s disease and
quantitation of accumulated iron and copper
levels respectively.
4. Diagnosis and grading of alcoholic liver disease
5. Accurate staging of advanced cases of primary
biliary cirrhosis and primary sclerosing cholan-
gitis.
6. Investigation of persistently abnormal and
unexplained LFTs.
7. Investigation of pyrexia of unknown origin.
8. Diagnosis of infections like tuberculosis.
9. Diagnosis of focal hepatic lesions
10. Histologic monitoring following liver trans-
plantation.
Contraindications:
• Uncooperative patient: If patient is not cooperative,
movement during the procedure can lead to lacera-
tion of liver and bleeding.
• Extrahepatic biliary obstruction: Injury to dilated bile
ducts can lead to biliary peritonitis.
• Bleeding diathesis or abnormal coagulation profile:
Prolongation of prothrombin time >3-5 seconds above
control value, platelet count < 50,000/ml, or bleeding
time ≥10 minutes are considered as contraindications
to liver biopsy. If liver biopsy is essential, then biopsy
can be performed after administration of vitamin K,
fresh frozen plasma, or platelet concentrate. Alter-
natively, transjugular approach can be used.
In patients with hemophilia who are infected
with hepatitis C or B virus through blood transfusion,
liver biopsy can be carried out to assess liver damage
after correcting coagulation deficiency.
tahir99 - UnitedVRG
vip.persianss.ir
 Liver Function Tests 67

• Tense ascites: Tense ascites is associated with failure real time imaging of the liver (ultrasonography,
to obtain liver tissue (due to increased distance computed tomography, or magnetic resonance imaging).
between abdominal wall and liver) and risk of This approach is suitable for biopsy of focal lesions.
bleeding in ascites. Biopsy can be performed after
removal of ascitic fluid or through transjugular Percutaneous ‘Plugged’ Liver Biopsy
approach.
In this method, after taking the biopsy sample, the outer
• Hydatid cyst of liver: Puncture of hydatid cyst will
sheath of the needle is left in place and the obturator
lead to spread of cysts throughout the abdomen and
holding the liver biopsy tissue is removed. A cannula is
sometimes, anaphylactic reaction.
then introduced through the outer sheath and gel or gel
• Suspected hemangioma or other highly vascular
foam is injected to seal or ‘plug’ the needle track in the
tumor
liver. This method is said to reduce the risk of bleeding
• Amyloidosis is associated with increased risk of
in individuals with impaired coagulation.
bleeding.
Patients with encephalopathy, hepatic failure with
Transvenous (Transjugular) Liver Biopsy
severe jaundice, severe congestive cardiac failure, and
advanced age are at increased risk of complications Liver biopsy is obtained from within the vascular system
following liver biopsy. of liver. A small catheter is inserted through the jugular
vein in the neck and radiologically guided, via right
Pre-requisites:
atrium and inferior vena cava, into the hepatic vein. The
• Prior imaging of liver should be carried out to identify
biopsy needle is then inserted through the catheter,
any abnormality, to define borders, and to determine
advanced into the liver, and biopsy is taken.
relative positions of gallbladder, lungs, and kidneys.
This method is suitable in severe coagulation
• Routine hemogram
disorders and in massive ascites. Due to the risk of cardiac
• Coagulation profile
arrhythmias (as the catheter passes through the atrium),
• Informed consent
close cardiac monitoring is required during this
• Blood grouping and cross matching
procedure.
Method:
1. Patient lies in supine position. Laparoscopic Liver Biopsy
2. After selecting the site for liver biopsy, a local
A laparoscope is introduced through an incision in the
anesthetic is injected.
abdominal wall, and liver biopsy is obtained under direct
3. A small incision is made and the biopsy needle is
visualization. Usually, such a biopsy is taken when a focal
passed into the liver. Patient is asked to hold his/her
lesion is incidentally detected on diagnostic laparoscopy
breath in expiration. Method of obtaining liver tissue
of abdomen.
depends on the type of needle used.
4. After needle is removed, patient lies supine or on the
right side and is closely monitored especially during
REFERENCE RANGES
first 6 hours for early detection of complications. Serum alanine aminotransferase (ALT, SGPT): 5-42 U/L
Complications: Serum aspartate aminotransferase (AST, SGOT): 5-40 U/L
1. Pain Serum alkaline phosphatase (ALP):
2. Intraperitoneal hemorrhage: Patients at particular • Children: 25-350 U/L
risk of bleeding are those with cirrhosis and malig- • Adult males: 25-120 U/L
nancy in liver. These patients should not be biopsied • Adult females: 25-90 U/L
on outpatient basis. AST/ALT ratio: 0.7-1.4
3. Biliary peritonitis due to puncture of gallbladder. Serum bilirubin:
4. Puncture of other organs like kidney, lung, and colon. • Total: 0.3-1.0 mg/dl
Overall mortality following liver biopsy is reported • Direct (Conjugated): 0-0.2 mg/dl
to be 0.1%. Serum proteins, total: 5.5-8.0 gm/dl
Serum albumin: 3.5-5.0 gm/dl
Percutaneous Guided Liver Biopsy
Serum globulins: 1.8-3.5 gm/dl
In percutaneous guided liver biopsy, needle is inserted
into the liver through the abdomen or lower chest during Albumin/Globulin (A/G) ratio: >1.5

tahir99 - UnitedVRG
vip.persianss.ir
 68 Essentials of Clinical Pathology

Serum protein electrophoresis: 6. Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff
Albumin 52-65% LB. Diagnosis and monitoring of hepatic injury. II.
α1 globulin: 2.5-5% Recommendations for use of laboratory tests in
α2 globulin: 7-13% screening, diagnosis, and monitoring. Clin Chem 2000;
β globulin: 8-14% 46:2050-68.
γ globulin: 12-22% 7. Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff
LB. Diagnosis and monitoring of hepatic injury. I.
Prothrombin time: 11-15 seconds Performance characteristics of laboratory tests. Clin
Chem 2000;46:2027-49.
Plasma ammonia: 9-33 μmol/L
8. Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart
Serum gammaglutamyl transferase: MJ, Shepherd J. Clinical Biochemistry. An Illustrated
• Males: Up to 40 U/L Colour Text, 3rd Ed. Edinburgh. Churchill Livingstone.
• Females: Up to 25 U/L 2004.
9. Grant A, Neuberger J. Guidelines on the use of liver
CRITICAL VALUES biopsy in clinical practice. Gut 1999; 45 (Suppl IV):
IV1-IV11.
Plasma ammonia: >40 μmol/L 10. Johnston DE. Special considerations in interpreting liver
function tests. Am Fam Physician 1999;59:2223-30.
Serum bilirubin: >15 mg/dl in newborns 11. Limdi JK, Hyde GM. Evaluation of abnormal liver
function tests. Postgrad Med J 2003;79;307-12.
BIBLIOGRAPHY 12. Lucey MR, Brown KA, Everson GT, et al. Minimal
criteria for placement of adults on the liver transplant
1. American Gastroenterological Association Clinical
waiting list; a report of a national conference organized
Practice Committee: AGA technical review on liver
by the American Society of Transplant Physicians and
chemistry tests. Gastroenterology 2002;123:1367-84.
2. American Gastroenterological Association Medical the American Association for the Study of Liver
Position Statement: Evaluation of liver chemistry tests. Disease. Liver Transpl Surg 1997;3:628-37.
Gastroenterology 2002;123:1364-6 13. Pugh RN, Murray-Lyon IM, Dawson JL, et al.
3. Beckingham IJ, Ryder SD. Investigation of liver and Transection of the oesophagus for bleeding oesopha-
biliary disease. BMJ 2001;322:33-6. geal varices. Br J Surg 1973;60:646-9.
4. Black ER. Diagnostic strategies and test algorithms in 14. Roche SP, Kobos R. Jaundice in the adult patient. Am
liver disease. Clin Chem 1997;43:1555-60. Fam Physician 2003;69:299-304.
5. Burke MD. Liver function: test selection and interpreta- 15. Thapa BR, Walia A. Liver function tests and their
tion of results. Clin Lab Med 2002;22:377-90. interpretation. Indian J Pediatr 2007;74:663-71.

tahir99 - UnitedVRG
vip.persianss.ir
 5
Disorders of Lipids and
Biochemical Cardiac Markers
DISORDERS OF LIPIDS
The major lipids present in blood are cholesterol, fatty
acids, and triglycerides. Lipid disorders are common in
clinical practice, and some of them are associated with
an increased risk of atherosclerotic cardiovascular
disease. Cardiovascular disease is a major cause of
mortality in persons under the age of 60, and proper
management of lipid abnormalities significantly reduces
this risk.
Lipids are insoluble in plasma and are therefore
transported in circulation in association with proteins.
These complexes of lipids and proteins are known as
lipoproteins. Dyslipidemias are disorders of lipoprotein
metabolism.
PHYSIOLOGY
Cholesterol and Triglycerides
The two major lipids in blood are cholesterol and
triglycerides. Since they are insoluble in water, they are
carried by lipoproteins.
Cholesterol is a lipid found in all cell membranes and
in blood plasma. Cholesterol is an essential component
of the cell membranes, and is necessary for synthesis of
steroid hormones, and for the formation of bile acids.
Cholesterol is synthesized by liver and many other
organs, and is also ingested in the diet.
Triglycerides are lipids in which three long-chain fatty
acids are attached to glycerol. Triglycerides serve as a
source of energy. They are present in dietary fat and also
synthesized by liver and adipose tissue.
Lipoproteins
Cholesterol and triglycerides are not soluble in water and
are transported in blood incorporated into lipoproteins.
Lipoproteins are spherical macromolecular complexes
consisting of (i) a central core of lipids (cholesterol ester,
triglycerides, fat-soluble vitamins) that is surrounded by
(ii) a surface monolayer composed of phospholipids, free
cholesterol, and apoproteins (Fig. 5.1). The surface of
lipoproteins is water-soluble or polar while core is
hydrophobic or non-polar.
There are five major lipoprotein classes: chylo-
microns, very low-density lipoproteins (VLDL), inter-
mediate density lipoproteins (IDL), low-density lipo-
proteins (LDL), and high-density lipoproteins (HDL)
(Fig. 5.2 and Table 5.1). Triglycerides are carried mainly
by chylomicrons, VLDL, and LDL, while major lipo-
proteins for cholesterol transport are LDL and HDL.
Chylomicrons: These are the largest and the least dense
of the lipoproteins, and transport exogenous lipids to
various cells. Dietary fat is incorporated into chylo-
microns by intestinal epithelial cells. The lipid core of
Fig. 5.1: Basic structure of a lipoprotein molecule. Lipoproteins
are spherical aggregates of lipids and apolipoproteins. They
consist of a core of triglycerides and cholesterol esters
surrounded by a shell of phospholipids and cholesterol.
Apolipoproteins are embedded in the shell. The larger the lipid
core, the lower is its density. Lipoproteins are classified into
5 types: chylomicrons, very low density lipoprotein (VLDL),
intermediate density lipoprotein (IDL), low density lipoprotein
(LDL), and high density lipoprotein (HDL)
tahir99 - UnitedVRG
vip.persianss.ir
 70 Essentials of Clinical Pathology

Fig. 5.2: Lipoproteins and their composition

Table 5.1: Summary of major lipoproteins

Lipoprotein Major lipid Formation Function

carried

1. Chylomicron Triglyceride Secreted by intestinal Transport of exogenous triglycerides, cholesterol,

epithelial cells and fat-soluble vitamins absorbed from food to
the peripheral tissues

2. Very low density Triglycerides, Secreted by liver Transport of endogenous triglycerides to adipose
lipoprotein Cholesterol tissue and muscle from liver
3. Low density Cholesterol Formed from modification Transport of cholesterol from liver to peripheral
lipoprotein of VLDL by lipoprotein tissues
lipase in peripheral
tissues
4. High density Cholesterol Secreted from liver “Reverse cholesterol transport”, i.e. from peripheral
lipoprotein tissues to liver

chylomicrons consists mainly of triglycerides, some
cholesterol and fat-soluble vitamins. Intestinal cells
secrete chylomicrons into the lymphatics, which then
enter the bloodstream via the thoracic duct. In circulation,
chylomicrons are acted upon by lipoprotein lipase to
release triglycerides; further hydrolysis of triglycerides
by lipoprotein lipase causes release of free fatty acids that
are then taken up by adipose tissue and muscle. Liver
takes up the cholesterol-rich chylomicron remnant
particle and cholesterol enters the metabolic pathway.
Very low-density lipoproteins (VLDL): VLDL particle
is synthesized by the liver. It transports triglycerides and
cholesterol. It carries most of the endogenous
triglyceride from liver to adipose tissue and muscle.
Triglyceride is removed by the action of lipoprotein lipase
in the circulation and VLDL particle becomes smaller,
when it is called as intermediate density lipoprotein
(IDL). Further processing of IDL leads to the formation
of low-density lipoprotein (LDL), which is the major
carrier for cholesterol.
Intermediate density lipoprotein (IDL): This is the
remnant of VLDL formed when triglycerides are
removed from VLDL by lipoprotein lipase in circulation.

tahir99 - UnitedVRG
vip.persianss.ir
 Disorders of Lipids and Biochemical Cardiac Markers
About half of IDL is cleared from blood by the liver and
the remaining half is further processed to form LDL.
Normally, IDL is not detected in plasma as it is formed
transiently.
Low-density lipoprotein (LDL): LDL is the major carrier
lipoprotein for cholesterol from liver to the peripheral
tissues. It is formed from VLDL. LDL plays a major role
in the genesis of atherosclerosis.
LDL is taken up by the cells through the LDL receptor,
a glycoprotein. The LDL receptor is present on the surface
of all cells and recognizes apolipoprotein B on the surface
of LDL particle. After internalization of LDL particle, the
lipoprotein is catabolized and the receptor is recycled
back to the cell surface. Intracellularly, LDL is degraded
to free cholesterol that is needed for cellular needs. The
level of LDL in circulation is determined by number and
function of LDL receptors. Joseph Goldstein and Michael
Brown were awarded the Nobel Prize for Physiology or
Medicine in 1985 for characterizing the LDL receptor.
Genetic absence of LDL receptors leads to familial hyper-
cholesterolemia.
High-density lipoprotein (HDL): HDL binds to
peripheral tissues that have apolipoprotein A receptors
and takes up cholesterol. HDL cholesterol is either taken
by the liver or is incorporated into IDL to form LDL.
Lipoprotein (a) or Lp (a): Attachment of apolipoprotein
(a) molecule to apolipoprotein B molecule on the surface
of LDL particle leads to the formation of a new particle
called as lipoprotein (a). Excess Lp (a) is associated with
risk of atherosclerosis.
Lipoprotein Metabolism
There are two pathways of lipoprotein metabolism:
exogenous and endogenous.
Exogenous pathway: Small intestinal cells absorb fatty
acids and cholesterol, esterify them into triglycerides and
Fig. 5.3: Exogenous lipid pathway showing route of metabolism of lipid absorbed from the intestine
71
cholesterol esters and incorporate them into the core of
the chylomicrons. Chylomicrons are secreted into the
lymphatics from where they reach the bloodstream via
thoracic duct. In the circulation, endothelial-bound
lipoprotein lipase hydrolyzes triglycerides in the
chylomicron core and releases free fatty acids that are
then taken up by the adipose tissue and muscle; in these
the free fatty acids are converted to triglycerides and
stored. The remaining smaller chylomicron is taken by
the liver, degraded, and cholesterol contained therein is
then used for the formation of bile acids, incorporated
into cell membranes, secreted in blood as lipoprotein
cholesterol, or excreted in bile (Fig. 5.3).
Endogenous pathway: This pathway is divided into:
• Apo B-100 lipoprotein system
• Apo A-I lipoprotein system
Apo B-100 lipoprotein system: In the liver, triglycerides and
cholesterol are assembled with apo B-100 and
phospholipids to produce VLDL. VLDL represents the
major export pathway for cholesterol from liver. After
its secretion from the liver, lipoprotein lipase on capillary
endothelium hydrolyzes triglyceride in the core of the
VLDL particles resulting in the formation of (cholesterol
ester-rich) intermediate density lipoproteins (IDL).
Further degradation results in the formation of LDL
particles that are rich in cholesterol ester. LDL particles
are taken up by all nucleated cells through LDL receptors
(receptor- mediated endocytosis) or by other scavenger
routes (e.g. monocytes or foam cell in atheromatous
plaques).
Apo A-I lipoprotein system: High-density lipoprotein
(HDL) particles are synthesized by liver and intestine
and participate in reverse cholesterol transport. HDL, rich
in apo A-I, acquires free cholesterol from peripheral
tissues, esterifies it, and either transfers it directly to the
liver or to other lipoproteins (IDL and LDL), which then
transport it to liver (Fig. 5.4).
tahir99 - UnitedVRG
vip.persianss.ir
 72 Essentials of Clinical Pathology

Fig. 5.4: Endogenous lipid pathway showing route of metabolism of endogenously synthesized lipid Abbreviations: VLDL:
very low density lipoprotein; IDL: intermediate density lipoprotein; LDL: Low density lipoprotein; HDL: high density lipoprotein;
LCAT: lecithin-cholesterol acyltransferase

Apolipoproteins commonly classified according to the Fredrickson or

These are proteins located on the surface of lipoproteins.
They aid in lipid transport and delivery in three ways:
(i) stabilizing structure of lipoproteins, (ii) serving as
regulatory cofactors for enzymes that act on lipoproteins,
and (iii) acting as ligands for binding to lipoprotein
receptors. Important apolipoproteins are:
• A-I: Activates lecithin-cholesterol acyl transferase
(LCAT)
• B-100: Ligand for LDL receptors
• C-II: Activates lipoprotein lipase
• E: Ligand for chylomicron remnant receptor in liver
CLASSIFICATION OF LIPOPROTEIN
DISORDERS
In clinical practice, lipoprotein disorders are classified
as being primary (inherited disorders) or secondary
(acquired disorders). Primary lipoprotein disorders are
World Health Organization classification (Table 5.2). This
classification is based on the particular pattern of lipid
and lipoprotein abnormality. On electrophorsis, four
principal bands are observed from cathode (–) to anode
(+): chylomicrons, β (LDL), pre β (VLDL), and α (HDL)
(Fig. 5.5). Fredrickson classification of hyperlipidemia is
as follows:
• Type I: Increased chylomicrons
• Type IIA: Increased β lipoproteins (LDL)
• Type IIB: Increased β and pre-β lipoproteins (LDL,
VLDL)
• Type III: Broad β lipoproteins (IDL)
• Type IV: Increased pre-β lipoproteins (VLDL)
• Type V: Increased chylomicrons and pre-β lipo-
proteins (chylomicrons, VLDL)
Secondary lipoprotein diseases arise from an
underlying cause such as diabetes mellitus, alcohol abuse,
hypothyroidism, nephrotic syndrome, renal failure, and
biliary cirrhosis of liver.

Table 5.2: Frederickson’s classification of primary lipoprotein disorders

Types Prevalence Appearance of plasma Increased particles Blood lipids

(fasting)

I Rarest Creamy layer at the top Chylomicron Marked elevation of triglycerides

IIA Common; 1:500 Clear (orange-yellow tint) Low density lipoprotein Increased total cholesterol
IIB Common; 1:300 Clear to slightly turbid Low density lipoprotein, Increased triglycerides and total
Very low density lipoprotein cholesterol
III Uncommon Thin creamy layer at the top; Intermediate density Increased triglycerides
turbid to opaque plasma lipoprotein and total cholesterol
IV Common; 1:500 Turbid to opaque Very low density lipoprotein Increased triglycerides
V Uncommon Creamy layer at the top; Chylomicron, Very low Marked elevation of triglycerides
plasma turbid to opaque density lipoprotein

In routine clinical practice lipid abnormalities are identified by standard lipid assays.

tahir99 - UnitedVRG
vip.persianss.ir
 Disorders of Lipids and Biochemical Cardiac Markers 73

Fig. 5.5: Classification of lipoproteins by agarose gel

electrophoresis

The main risk factors for coronary artery disease are

age (men > 45 years, women > 55 years), dyslipidemia,
family history of premature coronary heart disease,
hypertension, and cigarette smoking. Lipid abnor-
malities associated with atherosclerosis and increased
risks of coronary artery disease are (i) elevated LDL
cholesterol, (ii) low HDL-cholesterol, and (iii) elevated
triglycerides.

LABORATORY TESTS FOR

LIPOPROTEIN DISORDERS
Collection of sample: Sample for lipid analysis should
be collected in dry EDTA anticoagulant (1 mg/ml). For
estimation of triglycerides and lipoproteins, a 12-hour
fasting sample is necessary. Triglycerides and LDL are
affected by recent ingestion of food. Patient should be
on the usual diet for 2-3 weeks before analysis. Lipid
analysis should not be performed during acute illness
and should be deferred for 3 months after major illness.
Drugs affecting lipid levels should be avoided like
steroids, oral contraceptives, etc.
Approach to the evaluation of lipid disorder is shown
in Figure 5.6.
Identification of a lipid disorder: Following investi-
gations on a fasting blood sample are usually adequate
for identifying lipid abnormalities in majority of cases:
• Total cholesterol
• Triglycerides
• HDL-cholesterol
• LDL-cholesterol
Fig. 5.6: Approach to the evaluation of a lipid disorder
It is recommended by National Cholesterol Education
Program that a fasting lipid profile (consisting of
triglycerides, total cholesterol, HDL, and LDL) be carried
out every 5 years beginning at the age of 20 years.
Desirable and high risk levels of serum lipids are shown
in Table 5.3.
(1) Total cholesterol: Causes of elevated serum
cholesterol are listed in Table 5.4.
(2) Serum triglycerides: Hypertriglyceridemia is a risk
factor for coronary heart disease, but less significant than
total cholesterol and lipoproteins. Patients with serum
triglycerides >200 mg/dl have risk of atherosclerosis, and
>1000 mg/dl are at increased risk of acute pancreatitis.
Increase in triglyceride is often associated with low HDL.
Causes of hypertriglyceridemia are listed in Table 5.5.

Table 5.3: Guidelines of National Cholesterol Education Program Adult treatment Panel III (All values in mg/dl)

• Total cholesterol: Desirable: <200; Borderline high: 200-239; High: ≥ 240

• Triglycerides: Normal: <150; Borderline high:150-199; High:200-499; Very high: ≥ 500
• LDL: Optimal: <100; Near optimal: 100-129; Borderline high: 130-159; High: 160-189; Very high: ≥ 190
• HDL: Low: <40; High: ≥ 60.
 74 Essentials of Clinical Pathology
Table 5.4: Causes of hypercholesterolemia
Primary
1. Type IIA and type IIB hyperlipidemia (Familial hypercholesterolemia)
2. Common or polygenic hypercholesterolemia
3. Familial apolipoprotein B100 defect
4. Familial alpha lipoproteinemia
Table 5.5: Causes of hypertriglyceridemia
Primary
1. Type I hyperlipidemia
2. Type V hyperlipidemia
3. Deficiency of lipoprotein lipase
4. Deficiency of apolipoprotein C-II
VLDL level can be derived from serum triglyceride
level by the formula:
Triglycerides
VLDL =
5 and
(3) HDL-cholesterol: HDL contains 20-30% of total
serum cholesterol. Low HDL-cholesterol is a significant
risk factor for coronary artery disease even if total
cholesterol level is normal. HDL is involved in “reverse
cholesterol transport” (i.e. from peripheral tissues to the
liver where it is excreted in bile) and thus decreases
cholesterol accumulated in blood vessel walls. Therefore,
it is called as cardioprotective cholesterol. Concentration
of HDL-cholesterol is inversely related with the risk of
atherosclerotic coronary artery disease. HDL-cholesterol
<40 mg/dl is an important risk factor for coronary artery
disease (positive risk factor), while level >60 mg/dl is
cardioprotective (negative risk factor).
(4) LDL-cholesterol: LDL contains about 60% of total
serum cholesterol. High LDL-cholesterol is a strong risk
factor for atherosclerotic heart disease, and is the major
atherogenic lipoprotein. It is the primary lipoprotein that
mediates atherosclerotic heart disease, and is the primary
target of lipid-lowering therapy. High LDL levels are
associated with obesity, high carbohydrate intake,
diabetes mellitus, lack of exercise, smoking, and some
drugs. Intensive therapy to lower LDL cholesterol slows
the progression of atherosclerosis, reduces coronary
events, and decreases mortality. LDL-cholesterol can be
Secondary
1. Hypothyroidism
2. Nephrotic syndrome
3. Cholestasis
4. Drugs: Protease inhibitors
Secondary
1. Obesity
2. Poorly controlled diabetes mellitus
3. Alcoholism
4. Renal failure
5. Drugs: Estrogen, glucocorticoids
measured directly or can be derived from Friedewald’s
equation as follows:
LDL cholesterol = Total cholesterol – (HDL cholesterol – VLDL)
Triglycerides
VLDL =
5
Identification of cause of dyslipidemia: After the
presence of dyslipidemia is established, it is necessary
to determine whether it is primary or secondary on the
basis of clinical features, family history, and laboratory
studies. Initially, the secondary causes should be ruled
out. Laboratory studies for identification of the secondary
nature of the disorder are blood glucose (diabetes
mellitus), liver function tests (biliary cirrhosis), thyroid
function tests, and plasma and urine albumin (nephrotic
syndrome).
BIOCHEMICAL CARDIAC MARKERS
Acute Coronary Syndrome
The term acute coronary syndrome comprises of condi-
tions characterized by acute myocardial ischemia and
includes (i) unstable angina, (ii) non-ST segment
elevation myocardial infarction (NSTEMI) and (iii) ST
segment elevation myocardial infarction (STEMI)
(Table 5.6). The basic pathogenetic mechanism is
atherosclerosis of a coronary artery; acute coronary
syndrome results from rupture or erosion of an athero-
matous plaque with subsequent superimposition of
thrombosis (Fig. 5.7). Clinical presentation is decided by
 Disorders of Lipids and Biochemical Cardiac Markers 75

Table 5.6: Characteristics of acute coronary syndromes

Parameter Unstable angina (UA) Non-ST segment elevation ST segment elevation myocardial
myocardial infarction infarction (STEMI)
(NSTEMI)

1. Clinical features Three main presentations:
1. New angina of severe onset,
2. Angina at rest, or
3. Recent increase in frequency,
duration and severity of angina
2. ECG ST depression and/or T wave
inversion OR Normal
3. Biomarkers of Not raised
myocardial
injury in blood
Similar to UA or STEMI Pain similar to angina but more severe
and persistent; not completely relieved
by rest or nitroglycerin; nausea, sense
of apprehension, and sweating;
asymptomatic in 25%
ST depression and/or ST segment elevation followed by
T wave inversion OR appearance of Q wave and T
Normal wave inversion
Raised Raised

Fig. 5.7: Pathogenesis and classification of acute coronary syndrome. Acute coronary syndrome
results from acute reduction of myocardial blood supply due to disruption of atheromatous plaque

the degree of ischemia, amount of collateral circulation,
myocardial oxygen demand, and certain other patient-
specific determinants. As the condition is life-threatening,
early recognition of acute coronary syndrome is essential
so that proper and timely treatment can be instituted.
The essential parameters for diagnosis are clinical
features, electrocardiographic changes, and detection of
biochemical markers released in blood from myocardial
damage.
The term acute coronary syndrome is coined to
distinguish between chronic stable angina from unstable
angina and acute myocardial infarction.
Majority of patients with acute coronary syndrome
have prior history of effort angina or coronary artery
disease. The usual clinical manifestation of myocardial
ischemia is chest pain. The pain is typically located in
centre or left side of chest, and variously described as
pressure, squeezing, constriction, crushing, tightness or
 76 Essentials of Clinical Pathology

shown in Figure 5.8) but is more severe and persistent

Fig. 5.8: Myocardial infarction about 1-2 days old showing
disappearance of nuclei of myocardial fibers, contraction bands,
and beginning of acute inflammation. Rise in CK-MB enzyme
occurs at this time
heaviness. The pain radiates to left arm, shoulder, neck,
and jaw. It develops after exertion, meals, or emotional
stress. In angina pectoris, pain is relieved by rest and
nitroglycerin. Pain is similar in acute myocardial
infarction (necrosis of myocardium due to ischemia as
(>30 minutes), and not readily relieved by rest or
nitroglycerin. In myocardial infarction pain is often
accompanied by other features such as sweating, nausea,
vomiting, breathlessness, and palpitations. Acute
myocardial infarction may be clinically silent (in 25%
cases, especially those with diabetes or hypertension).
Unstable angina can present with (i) new angina of
severe onset, (ii) angina at rest, or (iii) recent increase in
frequency or pattern of angina.
Previously, diagnosis of acute myocardial infarction
required any two of the following criteria (World Health
Organization, 1974):
• Symptoms of myocardial ischemia, i.e. severe and
prolonged chest pain,
• Unequivocal changes consistent with acute
myocardial infarction on electrocardiogram
(development of Q wave)
• Elevation of cardiac enzymes in blood
A 12-lead electrocardiogram (ECG) is an important
test for diagnosis and should be obtained as soon as
possible after presentation (Fig. 5.9). This is because the

Fig. 5.9: (1) Normal electrocardiogram pattern; (2) to (6): Sequential electrocardiographic changes after acute myocardial
infarction. Initially T wave becomes tall, peaked, and pointed (first few minutes) and there is ST segment elevation. T wave
inversion occurs after a few hours and there is development of an abnormal Q wave. After some duration, ST segment returns
to normal and T wave becomes normal. Q wave changes, however, persist.
 Disorders of Lipids and Biochemical Cardiac Markers 77

test is noninvasive, inexpensive and rapid. The test is

useful for diagnosis, prognosis, and monitoring of
myocardial infarction. General ECG changes of
myocardial ischemia are ST elevation/depression and
deep symmetric T wave inversion. Persistent elevation
of ST segment differentiates STEMI from unstable angina
and NSTEMI. Normal appearing ECG does not exclude
cardiac ischemia or myocardial infarction.
ECG changes like ST segment elevation or depression
and T wave inversion are not sufficient on their own for
diagnosis of myocardial infarction. Revised criteria for
diagnosis of myocardial infarction (acute, evolving, or
recent) have been proposed by European Society of
Cardiology (ESC) and American Society of Cardiology
(ACC) (2000) (Box 5.1).

Box 5.1: Revised criteria for diagnosis of myocardial

infarction (ESC/ACC, 2000)
Any one of the following is satisfactory for diagnosis of Fig. 5.10: Cardiac muscle cell. Appearance of biochemical
acute/evolving/recent myocardial infarction: markers and their kinetics in bloodstream depend on their (i)
1. Typical myocardial necrosis-associated rise and fall intracellular location (whether free or bound to other organelles),
of troponin or CK-MB PLUS atleast one of the (ii) molecular weight, and (iii) rate of elimination from blood
following:
• Symptoms of ischemia
• Pathologic Q wave on ECG Table 5.7: Biochemical markers of myocardial injury
• ST segment elevation or depression on ECG
(indicative of ischemia) • Creatine kinase (CK)
• Coronary artery intervention (e.g. coronary – Total CK
angioplasty) – Isoenzymes
2. Pathologic features of an acute myocardial infarction – CK-MB (activity)
– CK-MB (mass)*
• Aspartate aminotransferase (AST) activity
Necrosis of myocardial cells leads to the release of
• Lactate dehydrogenase (LDH)
intracellular macromolecules from the cells into the – LDH activity
blood- stream. Detection of significant amounts of these – Isoenzymes
biochemical markers in blood under appropriate clinical • Cardiac troponins (cTn)*
setting can allow diagnosis of myocardial infarction to – cTnT
be made. Measurement of these markers can also – cTnI
distinguish between unstable angina and acute • Myoglobin*
myocardial infarction.
*Currently recommended markers
Biochemical Cardiac Marker Studies
troponin (a definitive marker) are the recommended
Myocardial cell necrosis leads to membrane damage and markers for diagnosis of MI; if cardiac troponin is not
leakage of cell contents into the bloodstream. This forms available, CK-MB (mass assay) is the next best alterna-
the basis for measurement of biochemical markers of tive.
myocardial injury in blood (Fig. 5.10). Various Usefulness of cardiac markers depends on time of
biochemical markers for myocardial injury are listed in specimen collection after myocardial infarction. Use of
Table 5.7. combination of markers and serial changes are more
Previously, total creatine kinase, LD, and AST were helpful (Table 5.8).
used for diagnosis of MI. However, due to their low An ideal cardiac marker should be highly sensitive,
specificity and due to the availability of more specific highly specific, should help to improve patient outcome,
markers, these enzymes are now rarely measured. and be cost-effective. Such a marker is currently
Currently, myoglobin (an early marker) and cardiac unavailable.
 78 Essentials of Clinical Pathology
Table 5.8: Timeline of cardiac markers after acute
myocardial infarction
Markers Time for Peak Return to
detection normal
Myoglobin 1-3 hr 6-9 hr 1 day
CK-MB 3-6 hr 12-24 hr 2-3 days
Troponin 4-8 hr 12-24 hr 5-10 days
Role of biochemical cardiac markers in acute coronary
syndrome:
• To confirm or exclude the diagnosis of acute
myocardial infarction in cases with sudden onset of
chest pain: In the presence of typical history and ECG
findings consistent with acute MI, measurement of
biochemical markers does not provide additional
information for initial management. These patients
need urgent treatment in the form of thrombolytic
therapy or angioplasty or both. If diagnosis is
uncertain from clinical features and ECG in an
emergency setting, biochemical markers are helpful
in ruling out myocardial infarction.
Serial determinations (at admission, 6-9 hours, and
12-24 hours) are recommended to rule in or rule out
diagnosis of acute myocardial infarction. Use of CK-MB
(mass) and cardiac troponin is recommended for
diagnosis.
• Detection of old (by some days) myocardial infarction
by cardiac troponin
• Diagnosis of reinfarction (CK-MB mass assay)
• To assess effectiveness of immediate reperfusion
therapy (thrombolysis or percutaneous coronary
intervention) in STEMI.
• Risk stratification to determine the likelihood of acute
coronary syndrome
Creatine kinase: Highest activity of CK is present in
striated muscle, brain, and heart.
Causes of increased CK are as follows:
• Disorders of skeletal muscle: Trauma, intramuscular
injection, vigorous exercise, dermatomyositis, mus-
cular dystrophy
• Disorders of heart: Myocardial infarction, myocarditis
• Disorders of central nervous sytem: Cerebrovascular
accident, head injury, generalized convulsions
• Disorders of thyroid: Hypothyroidism
CK-BB, CK-MB, and CK-MM are the isoenzymes of
CK. CK-BB predominates in brain, CK-MB in cardiac
muscle, while CK-MM in skeletal muscle and heart. CK-
MB is the most cardiac-specific CK isoenzyme, and it is
recommended to measure CK-MB mass.
Total serum CK and CK-MB are always elevated
following MI. However, serum CK and CK-MB can arise
from tissues other than heart. Following MI, CK-MB rises
within 3-6 hours after the onset of symptoms, peaks
within 12-24 hours, and returns to normal level by 48-72
hours.
Relative index or RI ([CK-MB/total CK × 100) is used
to distinguish cardiac from skeletal muscle damage; RI
above 5% is highly suggestive of acute myocardial
infarction.
It is recommended to obtain sequential samples (one
at presentation and subsequently at 8-hour intervals for
24 hours).
Myoglobin: Myoglobin is the oxygen-binding low
molecular weight protein of cardiac and skeletal muscle
cells. Myoglobin rises early after MI (1-3 hours) and is
currently the earliest marker. Myoglobin of cardiac
muscle cannot be distinguished from that of skeletal
muscle. Myoglobin levels are raised following MI, open
heart surgery, muscle injury, muscle dystrophy, renal
failure, shock, and trauma. Thus, although myoglobin
rises early following MI, it is not cardiac-specific.
However, non-elevation of myoglobin (in two sequential
samples 2-4 hours apart) is helpful for exclusion of early
MI in patients presenting with chest pain at emergency
department.
Troponins (Tn): Cardiac troponin T (cTnT) and cardiac
troponin I (cTnI) are the most sensitive and specific of
the available markers of myocardial necrosis and are
considered ideal markers for definitive diagnosis (either
cTnT or cTnI). Troponins regulate the interaction of actin
and myosin filaments during myocardial contraction.
Following MI, troponins appear in blood at about the
same time as CK-MB. If troponins are elevated atleast 12
or more hours following onset of chest pain, their
diagnostic sensitivity is 100%. TnI is more cardio-specific
as it is found only in heart muscle. It is not elevated
following skeletal muscle injury. Following myocardial
damage, TnI rises 4-8 hours the onset of chest pain, peaks
within 12-24 hours, and remains elevated for 7-10 days.
Development of assays for TnI and TnT represent a major
advance in the diagnosis of MI. As troponins remain
elevated for 7-10 days, they are useful in cases presenting
late. If onset of chest pain is 9-12 hours before admission,
only troponin needs to be measured.
 Disorders of Lipids and Biochemical Cardiac Markers 79

REFERENCE RANGES 2. Bock JL. Test strategies for the detection of myocardial
damage. Clin Lab Med 2002;22:357-75.
• Lipid profile: 3. Crook MA. Clinical chemistry and metabolic medicine
– Serum cholesterol: Desirable level: <200 mg/dl 7th Ed. London. Edward Arnold (Publishers) Ltd. 2006.
– Serum triglycerides: Desirable level: <150 mg/ 4. Eaton CB. Hyperlipidemia. Prim Care Clin Office Pract
2005;32:1027-55.
dl
5. Executive Summary of The Third Report of The
– HDL cholesterol: >60 mg/dl
National Cholesterol Education Program (NCEP).
– LDL cholesterol: <130 mg/dl Expert Panel on Detection, Evaluation, and Treatment
– LDL/HDL ratio: 0.5-3.0 of High Blood Cholesterol in Adults (Adult Treatment
• Biochemical cardiac markers: Panel III). JAMA 2001;285:2486-97.
– CK-MB: <5% or <10 μg/L 6. Koolman J, Roehm KH. Color Atlas of Biochemistry,
– Cardiac Troponin T: <0.1 μg/L 2nd Ed. Stuttgart. Thieme 2005.
– Myoglobin: < 90 μg/L 7. McDermott MT. Endocrine secrets. 4th Ed.
Philadelphia: Mosby, 2005.
8. Senger AK, Jaffe AS. The use of biomarkers for the
CRITICAL VALUES evaluation and treatment of patients with acute
• CK-MB: >5% or >10 μg/L coronary syndromes. Med Clin N Am 2007;91:657-81.
9. The Joint European Society of Cardiology/American
• Cardiac Troponin T: >0.1 μg/L
College of Cardiology Committee: Myocardial infarc-
• Myoglobin: >110 μg/L tion redefined-A consensus document of The Joint
European Society of Cardiology/American College of
BIBLIOGRAPHY Cardiology Committee for the redefinition of myo-
1. Achar SA, Kundu S, NorcrossWA. Diagnosis of acute cardial infarction. Eur Heart J 2000;21:1502-13.
coronary syndrome. Am Fam Physician 2005;72: 10. Tiyyagura SR, Smith DA. Standard lipid profile. Clin
119-26. Lab Med 2006;26:707-32.
 80 Essentials of Clinical Pathology
6

Examination of
Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a clear, colorless fluid formed
in the ventricles of the brain mainly by choroid plexus
(meshwork of tiny small blood vessels in lateral third
and fourth ventricles). It is mainly an ultrafiltrate of
plasma. CSF is contained within the cerebral ventricles,
the spinal canal and the subarachnoid space (space
between arachnoid externally and pia mater internally)
surrounding the brain and spinal cord (Fig. 6.1). CSF is
reabsorbed into the blood through the arachnoid villi of
dural venous sinuses.
COMPOSITION OF NORMAL
CEREBROSPINAL FLUID IN ADULTS
• Total volume: 100-150 ml (10-60 ml in the newborn)
• Opening pressure: 60-180 mm of water (10-100 mm
in infants and young children)
• Appearance: Clear and colorless with no clots
(viscosity similar to water)
• Cells:
– Adults: 0-5 cells/cmm
– Infants: 0-30 cells/cmm
– 1-4 years: 0-20 cells/cmm
– 5-18 years: 0-10 cells/cmm
• Glucose: 45-80 mg/dl. (Normally CSF glucose is 60%
or 2/3rds of blood glucose)
• Proteins: 15-45 mg/dl. (Normally CSF proteins are
1% of plasma proteins).
• Oligoclonal bands: Negative
• Chloride: 120-130 mEq/L (20 mEq/L more than
serum level)
• Bilirubin: Absent.
FUNCTIONS OF CEREBROSPINAL FLUID
1. Protection of brain and spinal cord from injury by
acting as a shock absorber.
2. To serve as a medium between blood and brain for
supply of nutrients to and removal of waste products
from brain.
COLLECTION OF CEREBROSPINAL FLUID
Some diseases produce characteristic alterations in
composition of CSF, thus providing the basis for
examination of CSF. Lumbar puncture or LP (first
performed by Quincke in 1891) is the procedure whereby
a sample of CSF is obtained. Spinal or LP needle is passed
between 3rd and 4th or between 4th and 5th lumbar
vertebra (L3-L4 or L4-L5) and CSF is obtained from the
subarachnoid space (Fig. 6.2). LP is carried out at these

Fig. 6.2: Site of lumbar puncture. Structures through which

needle passes are skin/superficial fascia, ligaments, epidural
space, dura mater, subdural space, arachnoid mater and
Fig. 6.1: Cranial meninges subarachnoid space
 Examination of Cerebrospinal Fluid 81

Fig. 6.3: Lumbar puncture needle

levels to prevent injury to the spinal cord (spinal cord

ends at about T12, below which are nerve roots or cauda
equina).
Patient is in a side-lying (lateral recumbent) position
with his back absolutely vertical and at the edge of the
bed, with the knees drawn up and the head flexed onto
his chest. This position increases the space between the
lumbar vertebrae. Alternately, patient may be in a sitting
position.
The selected site is thoroughly disinfected with
povidone-iodine or chorhexidine-containing solution,
covered with sterile drapes, and after injecting a local
anesthetic, a sterile lumbar puncture needle, preferably
22 gauge (Fig. 6.3), is inserted slowly. There is an
increased resistance as the needle passes through the
spinal ligaments and dura mater. As the needle enters
the subarachnoid space, a sudden 'give' (or loss of
resistance) is felt. The stylet is withdrawn slowly. When
CSF drops appear, a pre-assembled manometer is
attached to the needle and the opening pressure is
recorded. To measure opening pressure, patient should
be in the lateral recumbent position.
With the three-way stopcock in appropriate position,
CSF is collected in sterile plain tubes as follows (Fig. 6.4):
• Tube 1: Chemistry (glucose and protein) and
serology
• Tube 2: Microbiology (Gram's staining, bacterial
culture, and sensitivity)
• Tube 3: Hematology (Total cell count and
differential count)
• Tube 4: Cytology, special studies.
Typically, 3-5 ml of CSF is collected. After CSF
collection, the closing pressure is recorded (if the opening
pressure was high).
The needle is withdrawn after replacing the stylet,
and a sterile dressing is applied. Before or immediately
after LP, venous blood should be obtained for concurrent
determination of blood glucose.
INDICATIONS FOR LUMBAR PUNCTURE
1. Examination of CSF for diagnosis in suspected cases
of:
• Central nervous system infections, especially
meningitis (inflammation of leptomeninges) and
encephalitis
Fig. 6.4: Laboratory tests for evaluation of CSF in
different tubes
• Meningeal involvement by leukemia or
malignancy
• Subarachnoid hemorrhage (if CT scan is not
available)
• Inflammatory diseases, e.g. multiple sclerosis (for
diagnostic gammaglobulin findings), Guillain-
Barré syndrome
• Neoplasms of central nervous system (CNS).
Indications for emergency lumbar puncture are
shown in Box 6.1.
2. To reduce CSF pressure in benign intracranial
hypertension (pseudotumor cerebri)
3. Administration of medications:
• Anesthetic agents
• Antibiotics (e.g. amphotericin B in fungal menin-
gitis)
• Anticancer drugs (e.g. methotrexate in acute
lymphoblastic leukemia)
4. Introduction of radiographic contrast media for
myelography
5. For Queckenstedt's test: If opening CSF pressure is
normal and clinically subarachnoid block or a tumor
or a sinus thrombosis is suspected, then
Queckenstedt's test may be preformed. However, the
test is contra-indicated if CSF pressure is raised since
 82 Essentials of Clinical Pathology
it can precipitate herniation of brain. There is a direct
correlation between CSF pressure and pressure in
jugular vein (because of continuity with dural venous
sinuses in which arachnoid villi project). In
Queckenstedt's test, both jugular veins are
compressed and then released, and subsequent
changes in CSF pressure are observed. Normally,
upon compression of jugular veins, CSF pressure
rapidly rises and with the release of pressure on
jugular veins, CSF pressure rapidly falls. In the
presence of a spinal block, there is no rise of CSF
pressure with jugular vein compression (or a pressure
rise is low or delayed). This test is positive in about
80% of patients with cord compression. With the
advent of myelography, Queckenstedt's test is rarely
performed.
Box 6.1: Indications for emergency lumbar puncture
• Suspected meningitis
• Suspected subarachnoid hemorrhage
• Suspected meningeal involvement by leukemia
COMPLICATIONS OF LUMBAR PUNCTURE
1. Post-puncture headache: This is the most common
side effect and results from leakage of CSF from
puncture site at a rate faster than the rate of CSF
production. With the use of a large bore needle,
greater CSF leak occurs. Use of a smaller bore needle
(22 G) for LP and keeping the patient flat after the
procedure for 2-3 hours reduces the risk of headache.
2. Introduction of infection in spinal canal if aseptic
precautions are not observed, if septicemia is present,
or if infection is present at the site of LP.
3. Subdural hematoma with resultant neurologic deficit
in patients with bleeding diathesis.
4. Failure to obtain CSF (dry tap) which may be due to
the incorrect positioning of the patient or spinal block.
5. Herniation of brain through tentorium (uncus of
temporal lobe) or foramen magnum (cerebellar
tonsils), if intracranial pressure is high. This can
damage the brainstem. This risk has led to the
reduced use of lumbar puncture over the last few
years.
6. Subarachnoidal epidermal cyst (due to traumatic
implantation of a skin plug in subarachnoid space)
may develop after a few years if LP is preformed
without a stylet.
CONTRAINDICATIONS TO LUMBAR PUNCTURE
1. Raised intracranial pressure due to a space-
occupying lesion (e.g. brain abscess, posterior fossa
tumor, subdural hematoma, epidural abscess): These
patients usually have headache, altered pupillary
response, absent Doll's eye reflex, abnormal
respiratory pattern, papilledema, bradycardia,
hypertension, and decerebrate or decorticate
posturing. Lumbar puncture in such cases can lead
to herniation of brain. If a mass lesion is clinically
suspected, cranial computerized tomography (CT) or
magnetic resonance imaging (MRI) should be
obtained first.
2. Cardiorespiratory compromise
3. Bleeding diathesis that has not been corrected
4. Local infection at the site of lumbar puncture
LABORATORY EXAMINATION OF
CEREBROSPINAL FLUID
After collection, specimen of CSF should be transported
immediately to the laboratory and examined without
delay. This is because (i) cells disintegrate rapidly, and
(ii) reduction of glucose level occurs due to glycolysis.
At the latest, CSF should be examined within 1 hour of
collection, and CSF cell counts are always done within
30-60 minutes of collection. Glass tubes should not be
used for collection since cell adherence to glass reduces
the cell count. Specimen for bacterial culture should not
be refrigerated as fastidious organisms (Hemophilus
influenzae, Neisseria meningitidis) do not survive in the cold
temperature.
CSF chemical examination results should always be
compared with those in plasma since any change in
plasma is reflected in CSF.
Examination of CSF includes:
1. Opening pressure
2. Appearance
3. Total and differential cell counts
4. Chemical examination
5. Microbiological examination
6. Special investigations
1. Opening Pressure
After attaching the manometer to the hub of the spinal
needle, patient's legs should be gently extended and neck
returned to neutral position. The CSF pressure is then
measured. CSF pressure is directly related to jugular and
vertebral venous pressures. Patient should be relaxed
since tension, straining, or breath-holding will increase
the CSF pressure, while hyperventilation will lower the
opening pressure.
 Examination of Cerebrospinal Fluid 83

Normal opening pressure of CSF is:

• 60-180 mm of water in adults in lateral recumbent
position
• 10-100 mm of water in children less than 8 years
Causes of increased CSF pressure:
• Tense and anxious patient
• Intracranial mass lesion (e.g. neoplasm, abscess,
hemorrhage)
• Meningitis
• Cerebral edema
• Subarachnoid hemorrhage
• Congestive cardiac failure
• Benign intracranial hypertension (pseudotumor
cerebri).
Causes of decreased CSF pressure: Fig. 6.5: Appearance of CSF. (1) Normal CSF is clear and
• Leakage of spinal fluid following trauma or previous transparent. (2) Bloody CSF (either due to traumatic LP or
lumbar puncture subarachnoid haemorrhage). (3) After centrifugation, bloody
• Complete spinal block (obstruction of spinal sub- CSF due to traumatic LP shows clear supernatant, while that
arachnoid space due to tumor, abscess, adhesions, due to subarachnoid haemorrhage shows, (4) yellowish
herniated intervertebral disk). supernatant (xanthochromia) (5) Turbid CSF
A large difference between opening and closing
pressures usually indicates presence of a partial or – Leukocytes >200 cells/cmm
complete spinal block. – Red cells >400 cells/cmm
If opening CSF pressure is >200 mm, no more than 1- – Microorganisms like bacteria, fungi, or amebae
2 ml of CSF should be removed. – Radiographic contrast media
– Aspiration of epidural fat during LP
2. Gross Appearance of Cerebrospinal Fluid – Raised proteins.
Normal CSF is clear and colorless like distilled water, • Blood-mixed CSF: Blood-stained CSF may result
and does not clot. Abnormal CSF may appear turbid, from traumatic tap (due to injury to venous plexus in
blood-mixed, xanthochromic, or viscous (Fig. 6.5). Clot spinal wall) or subarachnoid hemorrhage. Distinction
formation in CSF is abnormal and indicates increased of traumatic tap from subarachnoid hemorrhage is
proteins. vitally important. Differences between the two are
• Turbid CSF may be due to the presence of: given in Table 6.1.

Table 6.1: Differences between traumatic lumbar puncture and subarachnoid hemorrhage

Cerebrospinal fluid finding Traumatic lumbar puncture Subarachnoid hemorrhage

1. Gross appearance Blood more in initial tubes as Blood uniform in all tubes; Blood does not
compared to later tubes; clot on standing
Blood clots on standing.
2. Supernatant after centrifugation Clear Pink or yellow (xanthochromia); yellow
within 1 hour of collection xanthochromia develops 12 hours after
hemorrhage
3. Microscopy Progressive decrease of red cell Red cell counts uniform in all tubes;
counts in later tubes Hemosiderin-laden macrophages
present
4. Latex agglutination test for Negative Positive
D-dimer*
5. Cerebrospinal fluid pressure Normal Increased
6. Cerebrospinal fluid protein Normal Increased

*D-dimer: A cross-linked fibrin degradation product

 84 Essentials of Clinical Pathology
• Xanthochromia: This refers to yellow discoloration
of CSF. For its detection, CSF is centrifuged and the
supernatant is compared with another tube of same
size filled with distilled water. Causes of
xanthochromia are:
– Subarachnoid hemorrhage (12 hours after
bleeding episode): In subarachnoid hemorrhage,
bleeding occurs in subarachnoid space usually
due to rupture of a cerebral aneurysm. Patient
presents with severe bursting headache of sudden
onset in occipital region that may be followed by
loss of consciousness. Red blood cells in CSF are
hemolyzed with release of oxyhemoglobin. About
a week after bleeding, macrophages and other
cells of leptomeninges convert oxyhemoglobin to
bilirubin. This produces yellowish discoloration
of CSF supernatant (See Fig. 6.5).
The investigation of choice, if available, for
diagnosis of subarachnoid hemorrhage is CT scan
to detect blood in basal cisterns. When CT scan is
negative or equivocal and xanthochromia cannot
be appreciated visually, spectroscopic
examination of CSF is helpful for diagnosis of
subarachnoid hemorrhage. It will show
absorption peaks of oxyhemoglobin and bilirubin.
– Jaundice, when serum bilirubin is >6.0 mg/dl
– CSF protein >150 mg/dl.
Froin's syndrome is a combination of xanthochromia,
excess proteins in CSF, and spontaneous formation of a
coagulum in CSF on standing. It results from complete
block of subarachnoid space.
• Other abnormal colors of CSF: These are:
– Pink: Red cell lysis and hemoglobin breakdown
– Brownish: Meningeal metastatic melanoma
– Orange: High carotene ingestion
• Clot formation: Pellicle (thin membrane or scum on
the surface of CSF) or clot formation (after 10 minutes
of collection) indicates increased proteins (>150 mg/
dl). It occurs in tuberculous meningitis (fine cobweb-
like clot after 12-24 hours), purulent meningitis
(pellicle forms early followed by a large clot), spinal
block (complete clotting of CSF), and traumatic LP.
Clot formation does not occur in subarachnoid
hemorrhage.
• Thick viscous CSF: This is seen cryptococcal
meningitis, meningeal metastatic mucinous adeno-
carcinoma, severe meningitis, and release of nucleus
pulposus fluid in CSF due to needle injury to the
intervertebral disk.
3. Cell Counts in Cerebrospinal Fluid
(1) Total leukocyte count: Cell count on CSF is done
manually on undiluted sample in a counting chamber.
Total leukocyte count increases in various disorders
and along with differential count provides important
diagnostic information. An increase in cell count in CSF
is called as pleocytosis.
It is essential to do microscopic examination of all
CSF samples since white blood cell (WBC) count upto
200/cmm and red cell count upto 400/cmm are associated
with clear appearance of CSF.
For correct results:
• Cell count should be done as soon as possible after
collection of CSF since cellular disintegration occurs
rapidly. Cells also adhere to the walls of the glass
tubes.
• CSF specimen collected in tube 3 should be used.
• No dilution of CSF is usually required. A diluent
should be used only if CSF is cloudy and likely to
contain increased leukocytes.
Method:
i. CSF sample should be properly mixed. If CSF is
clear, it is not diluted. If CSF appears cloudy or
turbid, 1:20 dilution is made using 0.05 ml of CSF
and 0.95 ml of Turk solution. (Composition of Turk
solution: Glacial acetic acid 4 ml, methylene blue
solution 10 drops, and distilled water to make 200
ml).
ii. The counting chamber is covered with the coverslip
provided.
iii. The counting chamber is filled with the fluid and
allowed to stand for 2 minutes for cells to settle.
iv. For counting cells in CSF, Fuchs-Rosenthal counting
chamber is preferred because its depth is twice that
of improved Neubauer chamber. In this, cells are
counted in 5 large squares (4 corner squares and
one central square).
v. If undiluted CSF is used, total number of cells
counted in 5 squares represents total count per cmm
of CSF. If CSF is diluted, the number of cells counted
is multiplied by the dilution factor (i.e. 20).
Causes of increased cell count in CSF:
• Meningitis and other infections of CNS
• Intracranial hemorrhage
• Meningeal infiltration by malignancy
• Repeated lumbar punctures
• Injection of foreign substances (e.g. radiographic
contrast media, drugs) in subarachnoid space.
• Multiple sclerosis
Presence of blood in CSF due to traumatic tap or
subarachnoid hemorrhage artefactually raises the
 Examination of Cerebrospinal Fluid 85

If CSF contains only a few cells, then it is centrifuged

Fig. 6.6: Cells in CSF: (1) Many neutrophils in bacterial
meningitis, (2) lymphocytes seen in normal CSF in adults, (3)
Many red cells and a small lymphocyte (traumatic tap), (4)
Increased monocytes in CSF, (5) Neutrophils, lymphocytes,
and red cells in CSF, (6) Lining cells of pia and arachnoid
in CSF, (7) Blast cells in CSF, (8) a malignant cell in CSF
at high speed (3000 g) for 10 minutes and a smear is made
from the sediment. If CSF contains many cells, then a
smear is made directly from the uncentrifuged sample.
After staining with a Romanowsky stain, smear is
examined under the microscope (Fig. 6.6).
Simple centrifugation of CSF often causes cell
breakage and distortion. Cytospin preparation with
cytocentrifuge (high speed centrifugation to concentrate
cells on a slide in a uniform monolayer) has been
recommended as it improves the cell yield and preserves
the cell morphology well.
In normal adults, differential count shows 70%
lymphocytes and 30% monocytes. In young children, a
higher proportion of monocytes (up to 70%) are present.
Table 6.2 shows causes of increase in different types of
leukocytes in CSF.
(3) Other cells: Apart from mature blood cells, CSF may
contain immature hematopoietic cells, tissue cells
(ependymal cells, pia arachnoid mesothelial cells) and
malignant cells. CSF examination is commonly carried
out in acute lymphoblastic leukemia to detect
involvement of CNS. Increased WBC count (>5/μl) with
lymphoblasts is evidence of CNS involvement.

leucocyte count by 1 WBC per 1000 red cells. This
correction factor should be used if patient's hemogram
is normal. If significant anemia or leukocytosis is present,
then leukocyte count in CSF should be corrected as
follows:
Corrected WBC count in CSF =
WBC count in blood ×
Red cell count in CSF
WBC count in CSF (cells/cmm) –
Red cell count in blood
(2) Differential leukocyte count: This provides informa-
tion about relative proportion of various leukocytes.
4. Chemical Examination of Cerebrospinal Fluid
Routine chemical examination of CSF consists of estima-
tion of proteins and glucose. CSF from tube 1 is used for
chemical examination.
(1) Estimation of proteins in CSF: Normal CSF protein
level in adults is 15-45 mg/dl. An increase in CSF protein
is a sensitive but non-specific indicator of CNS disease.
CSF proteins may be normal during early stages of
meningitis. Significant elevation (>150 mg/dl) occurs in
bacterial meningitis.

Table 6.2: Differential cell count in CSF

Predominant neutrophils Predominant Mixed cell pattern Predominant

lymphocytes (neutrophils, lympho- eosinophils
cytes, monocytes)

1. Meningitis: bacterial,
early viral, fungal,
early tuberculous
2. Subarachnoid hemorrhage
3. Repeated lumbar punctures
4. Introduction of anticancer
drugs or contrast media in
subarachnoid space
5. Meningeal metastasis
1. Meningitis: viral, tuberculous 1. Tuberculous meningitis 1. Parasitic and fungal
2. Incompletely treated bacterial 2. Fungal meningitis infections
meningitis 3. Chronic bacterial 2. Reaction to foreign
3. Cysticercosis, toxoplasmosis meningitis material (e.g. shunts)
4. Multiple sclerosis
5. Subacute sclerosing
panencephalitis
 86 Essentials of Clinical Pathology

There are various methods for estimation of CSF

proteins. Turbidimetric method using trichloroacetic acid
for precipitation of proteins is commonly used. In
principle, trichloroacetic acid, when added to CSF, causes
precipitation of proteins and a turbid solution is obtained.
Amount of turbidity is compared with the turbidity of a
known (standard) concentration of protein in a photo-
electric colorimeter.
If a sample is contaminated with blood while doing
the lumbar puncture (traumatic tap), false elevation of
proteins will occur. This can be corrected by deducting
1 mg/dl of protein for every 1000 red cells per cmm. For
this correction to be accurate, red cell count and proteins
should be estimated in the sample from the same tube of
CSF. Fig. 6.7: Pandy's test for globulins
If facilities for estimation of CSF proteins are not
vs. increased intrathecal synthesis of immunoglobulin
available in the laboratory, then Pandy's test for globulins
G) can be made from parameters shown in Table 6.3.
may be performed. In this test, CSF is added to saturated
Albumin is neither synthesized nor metabolized in CNS.
solution of phenol. If cloudiness develops immediately,
Therefore, increased CSF albumin/serum albumin ratio
it indicates presence of increased globulins and the test
indicates increased permeability of blood-brain barrier.
is reported as positive. If no cloudiness develops, the test
Immunoglobulin G (IgG) can be synthesized in CNS.
is reported as negative (Fig. 6.7).
Therefore, increased CSF IgG/serum IgG ratio indicates
Along with CSF proteins, it is necessary to simulta-
either increased permeability of blood-brain barrier or
neously measure serum proteins for proper increased intrathecal synthesis of IgG. Increased CSF
interpretation of results. IgG/albumin index indicates local IgG production.
CSF proteins are elevated in following conditions:
• Increased capillary permeability of blood-brain
barrier: Meningitis
• Mechanical obstruction to circulation of CSF
(causing increased fluid reabsorption due to stasis):
Spinal cord tumor
• Increased local (intrathecal) immunoglobulin (IgG)
production: Multiple sclerosis, neurosyphilis, sub-
acute sclerosing panencephalitis
• Both increased capillary permeability and increased
local immunoglobulin (IgG) production: Guillain-
Barré syndrome
• Hemorrhage in CSF: Traumatic tap, subarachnoid (2) Estimation of glucose in CSF: Normal CSF glucose
hemorrhage. is 2/3rds of blood glucose (CSF to blood glucose ratio is
Marked elevation (>500 mg/dl) is noted in complete 0.6). A sample for blood glucose should be drawn 1 hour
spinal block by a tumor, bacterial meningitis, and bloody before LP for comparison with CSF glucose. After
CSF. collection, CSF sample should be immediately processed
Differential diagnosis of elevated proteins in CSF for glucose estimation because falsely low result due to
(increased capillary permeability of blood-brain barrier glycolysis may occur.

Table 6.3: Differentiation of causes of elevated proteins in cerebrospinal fluid

CSF/Serum albumin ratio CSF/Serum IgG ratio CSF IgG/Albumin index Causes

1. Increased Increased Normal Increased permeability of blood-brain barrier

2. Normal Increased Increased Increased intrathecal synthesis of proteins

CSF glucose is measured by glucose oxidase method.
Normal range is 45-80 mg/dl. CSF glucose <40 mg/dl is
abnormal.
CSF glucose is decreased due to utilization by bacteria
(pyogenic or tuberculous), leucocytes, or cancer cells in
CSF.
Decreased CSF glucose occurs in following
conditions:
• Acute bacterial meningitis
• Tuberculous meningitis
• Fungal meningitis
• Meningeal involvement by malignant tumor
(meningeal carcinomatosis)
• Hypoglycemia
CSF glucose is normal in viral meningitis.
5. Microbiological Examination
Microbiological tests which can be carried out on CSF
sample are—
• Direct wet mount of CSF: in suspected cases of
cryptococcosis, amebic meningoencephalitis,
Candida infection, and trypanosomiasis
• Gram's smear: should be done if CSF is turbid and
neutrophils are increased.
• Ziehl-Neelsen smear: if tuberculous meningitis is
suspected.
• Latex agglutination tests: for detection of bacterial
and cryptococcal antigens.
• Serologic tests for syphilis
• Culture for bacteria and Mycobacterium tuberculosis
• Polymerase chain reaction for Mycobacterium
tuberculosis and viruses.
(a) Direct wet mount of CSF: One drop of CSF deposit
(obtained after centrifugation) is placed on a glass slide,
covered with a cover glass, and examined under the
microscope with reduced illumination. Observe for
motile trypanosomes or sluggishly moving amebae
(Naegleria fowleri). N. fowleri is a free-living ameba in water
which enters through the nose and reaches central
Fig. 6.9: Gram stained smears of CSF showing (1) Neisseria meningitidis,
(2) Streptococcus pneumoniae, and (3) Haemophilus influenzae
Examination of Cerebrospinal Fluid 87
Fig. 6.8: India ink preparation of CSF showing single and
budding yeast cells of Cryptococcus neoformans
nervous system. It causes a fatal type of hemorrhagic
meningoencephalitis. Candida albicans may be seen in
unstained wet mount; it appears as oval budding forms
and as pseudohyphae.
When cryptococcal meningitis is clinically suspected,
the wet mount is examined by dark-field microscopy.
Alternatively, a drop of India ink is added to the drop of
sediment on a glass slide, a coverslip is placed, and
examined under the microscope using ×40 objective.
Cryptococcus neoformans appears as spherical, budding
yeast forms, 2-10 μ in diameter and surrounded by a large
unstained capsule (Fig. 6.8). Cryptococci can be
demonstrated by India ink preparation in 50% cases of
cryptococcal meningitis.
(b) Gram's smear: This must be done if CSF is purulent
and neutrophils are increased. Gram's smear is positive
in 80% untreated cases and 60% partially treated cases
of bacterial meningitis. Therefore, absence of bacteria on
Gram's smear does not rule out bacterial infection.
CSF is centrifuged and a smear of the deposit is made
on a glass slide. (If CSF is purulent, smear is made directly
without centrifugation). Smear is air-dried, stained with
Gram's method, and examined under the oil-immersion
lens for bacteria (Fig. 6.9). Bacteria which commonly
cause meningitis are:
• Meningococci: Gram-negative diplococci located
inside neutrophils.
 88 Essentials of Clinical Pathology
• Pneumococci: Gram-positive diplococci surrounded
by an unstained capsule.
• Hemophilus influenzae: Gram-negative coccobacilli.
• Escherichia coli: Gram-negative rods.
In all the above types of meninigitis, the
accompanying cells are polymorphonuclear neutrophils.
Detection of typical bacteria on Gram-staining
suggests the etiologic agent of meningitis. However,
definitive diagnosis requires culture of CSF.
There is an association between age of the patient and
the causative organism of meningitis (Box 6.2).
(c) Ziehl-Neelsen staining for Mycobacterium tuber-
culosis: In tuberculous meningitis, number of tubercle
bacilli is usually low in CSF. Ziehl-Neelsen or AFB
staining is not a very sensitive method for detection of
M. tuberculosis in CSF. AFB smears are negative in about
70% of cases of tuberculous meningitis. Fluorescent
auramine stains have better sensitivity than Ziehl-
Neelsen stain.
Box 6.2: Association between age and organisms
causing meningitis
• 0 to 6 months: Group B streptococci, Escherichia
coli, Listeria monocytogenes
• 6 months to 6 years: Streptococcus pneumoniae,
Neisseria meningitidis, Hemophilus influenzae type
B, Enteroviruses
• 6 to 60 years: Neisseria meningitides, Streptococcus
pneumoniae, Enteroviruses, herpes simplex virus
• >60 years: Streptococcus pneumoniae, gram-
negative bacilli, Listeria monocytogenes
(d) Latex agglutination tests: Latex agglutination tests
for bacterial antigens are available commercially and are
sensitive, rapid, and simple to perform. Currently
available tests can detect N. meningitidis (groups A, B, C,
Y, and W135), H. influenzae (capsular type B), S.
pneumoniae and S. agalactiae. These tests are expensive
and their sensitivity is similar to that of Gram's smear.
Owing to the occurrence of false-positive results, these
tests are not recommended for routine diagnosis of
meningitis. These tests are particularly useful in patients
who have been partially treated and in whom Gram's
stain and culture are negative.
Latex agglutination tests for cryptococcal antigens
are also available and have sensitivity of 90%. These tests
have largely replaced India ink preparation for diagnosis
of cryptococcal meningitis.
(e) Limulus lysate assay for endotoxin produced by
gram-negative bacteria: Limulus amebocyte lysate assay
is a rapid, sensitive, and specific test for the presence of
endotoxin. Endotoxin is produced by gram-negative
bacteria like N. meningitides, H. influenzae type b, E. coli,
and Pseudomonas. This test is particularly useful as a rapid
test in newborns in whom these infections are common.
(f) Serologic tests for syphilis: If fluorescent treponemal
antibody with absorption (FTA-ABS) test is positive in
serum, Venereal Disease Research Laboratory (VDRL)
test should be done on CSF if neurosyphilis is suspected.
VDRL test is highly specific but lacks sensitivity.
Therefore, a positive test rules in but does not rule out
the diagnosis of neurosyphilis. Other serological tests for
syphilis are not suitable for diagnosis of neurosyphilis
in CSF.
Combination of positive FTA-ABS test in serum and
reactive VDRL test in CSF is diagnostic of active
neurosyphilis
(g) CSF culture: Culture of CSF is indicated if bacteria
are seen on Gram-stained smear, or leukocytes or
proteins are increased. Culture remains the gold standard
for diagnois of bacterial meningitis. For culture, CSF
collected in tube 2 is used. Sensitivity of culture for
identification of bacteria is about 90%. In incompletely
treated cases, sensitivity is less. Usually CSF sample is
inoculated on chocolate (heated blood) agar and blood
agar. In newborn infants, sample is also inoculated on
McConkey's agar.
In tuberculous meningitis, culture for M. tuberculosis
is positive in about 56% of cases. If larger volume of CSF
is used (i.e. 10 ml) for inoculation, sensitivity for detection
increases.
In cryptococcal meningitis, culture is positive in 95%
of cases.
(h) Polymerase chain reaction (PCR): PCR is a highly
specific and sensitive tool for diagnosis of infections of
CNS. It uses probes to detect genes specific for the
infecting organism. The test is rapid and requires only a
small amount of CSF. High cost and availability only in
a few specialist laboratories are major limitations.
CSF PCR is mainly useful for diagnosis of viral
infections of CNS (e.g. herpes simplex, enteroviruses,
herpes zoster, etc.) and of tuberculous meningitis.
CSF findings in different types of meningitis are
shown in Table 6.4.
6. Special Investigations
(a) CSF protein electrophoresis: Protein electrophoresis
of normal CSF differs from normal serum in (i) presence
of a prominent transthyretin band and (ii) an extra
transferrin band (called β2-transferrin or tau protein).
Protein electrophoresis of CSF is used:
 Examination of Cerebrospinal Fluid 89

Table 6.4: Cerebrospinal fluid findings in different types of meningitis

Condition Appearance Leukocytes Proteins in Glucose in Additional

mg/dl mg/dl investigations

1. Normal Clear, <5/μl (mostly lymphocytes) 15-45 45-80 –

colorless
2. Acute pyogenic Turbid or Increased (>1000/μl); Increased; Decreased; Gram's stain;
meningitis purulent mostly neutrophils 50-1500 <40 culture; Latex
agglutination test
3. Tuberculous Clear or cloudy Increased (100-600/μl); Increased; Decreased; AFB stain; culture;
meningitis mostly lymphocytes or both 45-300 10-45 polymerase chain
lymphocytes and neutrophils reaction
4. Viral meningitis Clear or cloudy Increased (6-300/μl); Increased Normal Polymerase chain
lymphocytes reaction

Fig. 6.11: An example of positive tau protein: Lane 1: Normal

CSF; Lane 2: Serum; Lane 3: Nasal fluid. Lane 3 (nasal fluid)
shows two bands in the same position as normal CSF
confirming CSF leakage from nose

identification of such fluid as CSF is essential.

Fig. 6.10: Oligoclonal bands in CSF in gamma region
(arrows) in a case of multiple sclerosis
• For identification of oligoclonal bands, and
• To determine whether fluid submitted for
examination is CSF.
1. Oligoclonal bands: Agarose gel electrophoresis of
concentrated CSF is used for detection of oligoclonal
bands. These are two or more discrete bands in the
gamma region. Presence of oligoclonal bands on CSF
protein electrophoresis that are absent on
concurrently run serum protein electrophoresis is
indicative of intrathecal syntheis of immunoglobulins
(Fig. 6.10). Oligoclonal bands have been identified in
majority of patients (90%) with multiple sclerosis;
however, they are not specific for this disorder. They
are also seen in subacute sclerosing panencephalitis,
viral CNS infections, neurosyphilis, and Guillain-
Barré syndrome.
2. CSF leakage: Occasionally, clear fluid leaking through
the nose or ear after trauma or surgery is submitted
for examination to determine whether it is CSF. Due
to the risk of recurrent meningitis, accurate
Recommended test for this purpose is protein
electrophoresis with immunofixation for transferrin.
Protein electrophoresis of CSF shows an extra
transferrin band (called as 'tau' protein), which is
absent in other body fluids and secretions. Tau protein
is an enzymatically modified transferrin that moves
just behind the unaltered transferrin in β region.
Identification of two isoform bands of transferrin on
protein electrophoresis is a highly sensitive and
specific test for identification of fluid as CSF (Fig.
6.11). Other body fluids or secretions do not show
the second isoform band.
(b) Measurement of albumin and immunoglobulin G
(IgG): Comparison of IgG and albumin CSF/plasma ratio
can be helpful for diagnosis of multiple sclerosis (high
ratio due to intrathecal synthesis of IgG).
REFERENCE RANGES
Reference ranges are given at the beginning of this
chapter under "Composition of normal cerebrospinal
fluid in adults".
 90 Essentials of Clinical Pathology

CRITICAL VALUES • Detection of blast cells or malignant cells

• Positive polymerase chain reaction for herpes simplex
• Cells: >10/cmm
• Glucose: <45 mg/dl BIBLIOGRAPHY
• Proteins: >45 mg/dl
• Detection of pathogens on Gram stain, latex aggluti- 1. Ellenby MS et al. Lumbar puncture. N Engl J Med 2006;
355:e12. Downloaded from www.nejm.org on January 14,
nation tests, Ziehl-Neelsen stain, or India ink
2007.
preparation 3. Seehusen DA, Reeves MM, Fomin DA. Cerebrospinal
• Positive culture fluid analysis. Am Fam Physician 2003;68:1103-8.
2. Riordan FAI, Cant AJ. When to do a lumbar puncture.
Arch Dis Child 2002;87:235-7.
 7
Examination of Pleural and
Peritoneal Fluids
PLEURAL FLUID
Pleural cavity is a potential space between chest wall and
lung lined by a layer of flattened epithelium called as
mesothelium. It contains a small amount of fluid (<10
ml in each pleural cavity) which is an ultra-filtrate of
plasma and which lubricates the two opposing parietal
and visceral pleural layers. Pleural fluid is being formed
continuously depending on hydrostatic pressure, plasma
colloid oncotic pressure, and permeability of capillaries
in parietal pleura. Resorption of pleural fluid occurs
through lymphatics and venules in visceral pleura.
Composition of pleural fluid is similar to that of plasma,
except that it has lower protein level (< 1.5 gm/dl).
Normal pleural fluid is clear and straw or pale yellow in
color.
Accumulation of excess fluid in pleural cavity is called
as pleural effusion (Fig. 7.1). Presence of pleural effusion
is usually confirmed by lateral decubitus chest X-ray or
ultrasonography. Radiography can also provide clues
about the cause of effusion e.g. bilateral effusion with
enlarged heart suggests congestive cardiac failure,
radiological signs of pneumonia suggest parapneumonic
effusion.
Fig. 7.1: Accumulation of fluid in pleural cavity on
X-ray chest
Causes of Pleural Effusion
There are various causes of pleural effusion (Table 7.1).
Common causes include congestive cardiac failure,
bacterial pneumonia, tuberculosis, cirrhosis with ascites,
and cancer. Cause of pleural effusion may be evident
clinically such as congestive cardiac failure (raised
jugular venous pressure, edema of feet), cirrhosis of liver
(ascites, other signs of portal hypertension), lymphoma
or carcinoma (enlargement of lymph nodes, liver or
spleen), and pulmonary embolism (deep vein thrombosis
of lower limbs, hemoptysis, pleuritic type of chest pain,
and dyspnea out of proportion to amount of fluid
accumulated). Accumulated pleural fluid may be either
a transudate or an exudate. Transudates result from non-
inflammatory conditions and are due to increase in
capillary hydrostatic pressure (e.g. congestive cardiac
failure, portal hypertension) or low oncotic pressure of
plasma (e.g. hypoproteinemia). Exudates result from
inflammatory conditions and are due to increased
capillary permeability (e.g. infections) or obstruction of
pleural lymphatics (e.g. malignancy). Further testing of
transudates is not required, while it is necessary to
analyze exudates further. Comparative features of
transudates and exudates are presented in Table 7.2.
Collection of Pleural Fluid
Procedure for collection of pleural fluid by inserting a
needle in the chest wall is called as thoracentesis. It may
be either diagnostic or therapeutic. Diagnostic thora-
centesis is carried out to determine the cause of pleural
effusion. Conditions that can be definitely diagnosed are
empyema, malignancy, tuberculous effusion, fungal
effusion, esophageal rupture, chylothorax, hemothorax,
rheumatoid pleurisy, and lupus pleurisy. It is
recommended to perform diagnostic thoracentesis when
pleural effusion is clinically significant (i.e. >10 mm thick
on lateral decubitus chest X-ray or ultrasonography) and
 92 Essentials of Clinical Pathology

Table 7.1: Causes of pleural effusion

Transudates Exudates

1. Congestive cardiac failure 1. Infections

2. Cirrhosis of liver • Parapneumonic effusion in bacterial pneumonia
3. Hypoproteinemia • Tuberculosis
4. Pulmonary embolism 2. Pulmonary infarction
3. Collagen disorders
• Rheumatoid arthritis
• Systemic lupus erythematosus
4. Acute pancreatitis
5. Trauma to chest wall
6. Malignancy
• Secondary deposits from bronchogenic carcinoma, carcinoma of
breast, etc.
• Lymphoma
• Mesothelioma

Table 7.2: Differences between transudate and exudate in pleural effusion

Parameters Transudates Exudates

1. Cause Non-inflammatory Inflammatory

2. Unilateral or bilateral Usually bilateral Usually unilateral
3. Mechanism Raised hydrostatic pressure or Increased capillary permeability or
reduced oncotic pressure in lymphatic obstruction
capillaries
4. Appearance Clear or straw-colored, does Turbid or cloudy, often clots
not clot
5. Specific gravity <1.016 >1.016
6. Proteins <3 gm/dl >3 gm/dl
7. Pleral fluid protein/serum protein ratio <0.5 >0.5
8. Lactate dehydrogenase (LDH) <200 >200
9. Pleural fluid LDH/serum LDH ratio <0.6 >0.6
10. Glucose Equivalent to plasma May be low
11. Cells Few lymphocytes and Many inflammatory cells
mesothelial cells
12. Organisms Nil May be present
13. Pleural fluid pH 7.45-7.55 <7.30

Note: No single criterion is adequate for differentiation. Test combinations are more sensitive and accurate

if cause is not obvious. Therapeutic thoracentesis is include introduction of infection, pneumothorax,

performed either to relieve respiratory insufficiency due
to massive pleural effusion or to introduce antineoplastic
drugs in pleural space following withdrawal of pleural
fluid.
A qualified and experienced physician performs
thoracentesis. It is contraindicated in non-cooperative
patients, coagulation disorders, respiratory insufficiency,
infection at the site of chest wall puncture, mechanical
ventilation, a small volume of fluid, unstable angina, and
cardiac arrhythmias. Complications of the procedure
hemothorax, air embolism, injury to the spleen or liver,
and pulmonary edema if large amount of pleural fluid
is rapidly removed.
For diagnostic studies, pleural fluid is collected in
heparinised tubes to prevent clotting. For cell counts,
sample is collected in EDTA anticoagulant. For micro-
biologic studies, sterile sample tubes are necessary; for
culture, aliquots of sample are best inoculated in blood
culture bottles at the bedside. In suspected malignancy,
maximum amount of fluid should be submitted to
enhance the cellular yield.

Examination of Pleural Fluid
This consists of:
• Appearance
• Chemical examination
• Cell counts
• Microbiological examination
• Cytological examination.
1. Appearance: Transudates are clear, pale yellow, or
straw-colored and do not clot on standing. Exudates are
opaque or turbid due to increased proteins, leukocytes,
or presence of malignant cells, and often clot on standing
due to high fibrinogen content. Frank pus with putrid
odor indicates empyema.
Straw-colored transudative fluid occurs in congestive
cardiac failure, pulmonary embolism, and cirrhosis of
liver.
Thick exudative fluid occurs in pneumonia and
cancer.
Bloody or hemorrhagic fluid indicates traumatic tap,
pulmonary infarction, or malignancy. Hemorrhage in
pleural space is referred to as hemothorax. A traumatic
tap progressively becomes less bloodstained during
removal of pleural fluid.
Accumulation of a milky or chylous pleural fluid is
referred to as chylothorax. True chylous effusion results
from obstruction of lymphatic duct (due to injury,
carcinoma, or lymphoma). A characteristic feature of
chylous effusion is triglyceride level >110 mg/dl.
Microscopy of such fluid shows lymphocytosis and fat
droplets that stain with Sudan III. Following
centrifugation of a chylous fluid, a layer of creamy
chylomicrons forms at the top. A pseudochylous effusion
results from long-standing chronic pleural effusion
through breakdown of cellular lipids (tuberculous or
rheumatoid effusion). It has a bright, shiny appearance,
and microscopic examination shows mixed cellularity
and cholesterol crystals. Triglyceride level is ≤50 mg/dl
and chylomicrons are absent.
A foul smelling pleural fluid may indicate a possible
anerobic infection, while urinous (ammoniacal) odor
indicates possible urinothorax.
In mesothelioma, pleural fluid is viscous.
2. Chemical examination: If old criteria are used for
differentiation of exudates and transudates (like protein
level, specific gravity, cell count, and clots in sample),
significant cases are misclassified. Therefore, in pleural
fluid, differentiation of exudates from transudates is
based on following criteria (known as Light’s criteria):
• Pleural fluid protein/serum protein ratio > 0.5
• Pleural fluid LDH/serum LDH ratio > 0.6
Examination of Pleural and Peritoneal Fluids 93
• Pleural fluid LDH above 2/3rds of upper limit of
normal for serum LDH.
Exudates have at least one of the above criteria.
Presence of all the three criteria best differentiates an
exudate from a transudate. Transudates have none of
these criteria. If the fluid is an exudate, further
investigations are indicated like cell counts, estimation
of glucose, cytological examination, test for tuberculosis,
Gram smear, and culture. In case of transudates,
diagnostic considerations include congestive cardiac
failure, cirrhosis, and pulmonary embolism (Fig. 7.2).
Low glucose levels are found in empyema,
rheumatoid pleurisy, tuberculous pleurisy, and
malignant effusion.
There is a direct correlation between lactate dehydro-
genase levels in pleural fluid with degree of pleural
inflammation.
3. Cell counts: Total leukocyte count should routinely
be obtained on all fluids. In transudates, leukocytes are
<1000/ml and are mostly small, mature lymphocytes.
Predominance of neutrophils (>50%) indicates an
acute process (e.g. parapneumonic effusion, pulmonary
infarct), while predominance of lymphocytes indicates
a chronic process (e.g. tuberculosis, rheumatoid pleurisy,
malignancy).
Very high leukocyte count (>50,000/ml) with
predominance of neutrophils suggests parapneumonic
effusion (usually empyema). Presence of many small,
mature lymphocytes with only a few or no mesothelial
cells is suggestive of tuberculosis.
Blood-tinged pleural fluid is not diagnostic of any
condition and can occur in a transudate or an exudate.
Grossly hemorrhagic pleural fluids have red cells above
100,000/ml and are seen in pulmonary infarction,
malignancy, or traumatic tap. Traumatic bloody effusion
is suggested by gradual clearing with aspiration, clotting
of fluid after some time, and lack of hemosiderin-laden
macrophages.
4. Microbiological examination: Gram’s smear and
culture should be carried out on exudates, especially on
purulent, bloodstained, or cloudy samples. The
likelihood of isolation of organisms is increased if pleural
fluid is inoculated in blood culture bottles at the bedside.
Ziehl-Neelsen stained smear is positive in <20% of
tuberculous pleural effusions and culture in <40% of
cases. If tuberculosis is suspected and culture is negative,
polymerase chain reaction for mycobacterial DNA or
increased level of adenosine deaminase (released in
pleural fluid from activated lymphocytes) can establish
the diagnosis.
5. Cytological examination: For cytological examination,
pleural fluid is centrifuged, smears are prepared from
 94 Essentials of Clinical Pathology
Fig. 7.2: Evaluation of pleural effusion
the sediment, and are stained with Papanicolaou stain.
In older age, metastatic malignancy is responsible for
majority of pleural effusions. The common malignancies
are bronchogenic carcinoma, carcinoma of breast, and
lymphoma. Pleural effusion results from lymphatic
obstruction by tumor cells. Typically, effusion is blood
tinged or hemorrhagic. Examination of three separately
obtained pleural fluid samples increases sensitivity of
detection of cancer cells to 80%.
6. Other tests: For diagnosis of pulmonary embolism,
D-dimer test (a breakdown product of fibrin) should be
carried out on peripheral blood sample. Flow cytometric
analysis of pleural fluid sample should be done if
lymphoma is suspected. In parapneumonic effusion, if
pleural fluid pH is <7.20, drainage is indicated; if pH is
>7.30, complete resolution will occur with medical
treatment. In malignant pleural effusion, low pH
indicates poor prognosis and poor response to
tetracycline pleurodesis. A pleural fluid rheumatoid
factor ≥ 1:320 is suggestive of rheumatoid pleurisy.
Presence of LE cells in pleural fluid makes diagnosis of
lupus nephritis virtually certain. Elevated pleural fluid
amylase occur in pancreatitis and esophageal rupture.
Pleural fluid triglyceride ≥ 110 mg/dl indicates chylous
effusion.
Box 7.1: Special studies on pleural fluid to determine the
cause of pleural effusion
• Tuberculous pleural effusion: Adenosine deaminase,
gamma interferon, polymerase chain reaction
• Rheumatoid effusion: Rheumatoid factor
• Lupus pleuritis: Antinuclear antibodies
• Pancreatic disease, esophageal disease: Amylase
• Malignancy: Carcinoembryonic antigen
• Chylothorax: Triglycerides
Pleural fluid findings in main causes of pleural
effusion are shown in Table 7.3. Evaluation of pleural
effusion is shown in Figure 7.2. Special studies on pleural
fluid are listed in Box 7.1.
PLEURAL BIOPSY
Percutaneous needle biopsy of the parietal pleura is
indicated in patients with exudative pleural effusion if
examination of pleural fluid does not yield a specific
 Examination of Pleural and Peritoneal Fluids 95

Table 7.3: Pleural fluid findings in main causes of pleural effusion

Causes Appearance Type of fluid Cells Special studies

1. Tuberculosis Straw-colored Exudate 5000-10000/μl; mostly Ziehl-Neelsen smear;

lymphocytes; <5% culture; polymerase
mesothelial cells chain reaction
2. Parapneumonic effusion* Turbid Exudate 5000-40000/μl; mostly Gram stain; culture
neutrophils
3. Empyema** Purulent Exudate >25000/cmm; mostly Gram stain; culture; low
neutrophils pH; low glucose
4. Malignancy*** Bloody Exudate Lymphocytes, Cytology, pleural biopsy
malignant cells
5. Congestive cardiac failure Clear Transudate Few mesothelial cells –

*Parapneumonic effusion: an exudative pleural effusion that occurs in bacterial pneumonia due to inflammation of pleura
adjacent to the affected lobe; **Empyema: Presence of purulent exudate in pleural cavity; it can result from parapneumonic
effusion, rupture of lung abscess in pleural space, injury, or rupture of subdiaphragmatic or hepatic abscess in pleural space;
*** Common causes of malignant pleural effusion are carcinoma of lung, carcinoma of breast, and lymphoma

diagnosis. It is done especially if tuberculosis or
malignancy is suspected. For better results, thoracoscopy
(direct visualization of pleural surface by introducing a
thoracoscope through the chest wall into the pleural
cavity previously filled with air) can be helpful in
localizing the site of biopsy. For diagnosis of tuberculosis,
pleural biopsy is more sensitive than pleural fluid
examination and culture. In malignancy, improved
techniques in pleural fluid cytology have increased the
diagnostic yield to 80%; combination of pleural fluid
cytology and pleural biopsy can increase the detection
rate further.
For pleural biopsy, special needles like Abrams, Cope
and tru-cut are available. Needle is inserted with its
cutting chamber closed into the pleural effusion. It is
recommended to obtain at least three biopsy samples
(from 3, 6, and 9 ‘O’ clock positions of cutting chamber)
for histology and culture.
Risks of hemothorax and pneumothorax are higher
as compared to thoracentesis. Spread of malignant cells
can occur along the needle track.
Thoracoscopy: In thoracoscopy, an endoscope is
introduced into the pleural cavity for direct visualization
of pleural surfaces. It is indicated if pleural fluid analysis
is non-diagnostic in a case of pleural effusion and to
localize the site of biopsy.
Adequate pleural space is created by removing
pleural fluid and replacing it with an equal amount of
atmospheric air. Thoracoscope is inserted through a skin
incision via a trocar into the pleural cavity.
It is especially helpful for diagnosis of metastatic
cancer and mesothelioma. Appearance of pleura is also
diagnostic in tuberculosis and rheumatoid pleurisy. The
procedure can be combined with aspiration of pleural
fluid, pleural biopsy, and pleurodesis. Thorascopy is
better than blind pleural biopsy for diagnosis of
malignancy.
Thoracoscopy is contraindicated if pleural space is
obliterated and in the presence of coagulopathy and
severe cardiac disease.
Complications include subcutaneous emphysema,
empyema, spread of malignant cells along the access
track, and hemorrhage. Reported mortality rate is <0.1%.
PERITONEAL FLUID
Peritoneal cavity is a potential space in the abdomen lined
by mesothelial cells and normally containing about 30-
50ml of serous fluid. The fluid is an ultrafiltrate of plasma
and its formation is dependent upon hydrostatic pressure,
plasma oncotic pressure, and capillary permeability.
Pathological accumulation of fluid in peritoneal cavity is
called as ascites, and the accumulated fluid is called as
ascitic fluid.
Causes of Ascites
Causes of ascites are listed in Table 7.4. Causes are
classified based on whether the fluid is a transudate or
an exudate. Unlike pleural fluid, there are no well-
defined criteria for distinction between transudates and
exudates. Majority of patients with ascites have cirrhosis
of liver; presence of ascites in a patient with cirrhosis is a
poor prognostic sign.
Indications for Abdominal Paracentesis
Abdominal paracentesis refers to removal of ascitic fluid
through puncture of the peritoneal cavity. Indications
for abdominal paracentesis are given in Table 7.5.
 96 Essentials of Clinical Pathology
Table 7.4: Causes of ascites
Transudate (increased hydrostatic pressure or plasma
oncotic pressure)
1. Cirrhosis of liver
2. Congestive cardiac failure
3. Hypoproteinemia
Table 7.5: Indications for abdominal paracentesis
1. All patients with new-onset ascites
2. At admission in all patients with ascites for detection of
asymptomatic infection
3. All patients with ascites who develop clinical features
of bacterial infection, hepatic encephalopathy,
gastrointestinal hemorrhage, or impairment of renal
function.
4. Symptomatic ascites (therapeutic paracentesis)
Collection of Sample
Presence of ascites can usually be detected by clinical
examination; if clinical examination is not definitive,
ultrasound can be helpful. Ultrasonography can also be
useful for determining the cause of ascites.
A hollow needle is inserted through the abdominal
wall (usually left lower quadrant of abdomen below the
border of shifting dullness) into the peritoneal cavity
(Fig. 7.3) and fluid (20-50 ml) is removed under aseptic
precautions. For cytology, to maximize the yield of
malignant cells, 100 ml should be submitted. For cell
count sample is collected in EDTA-containing tube. For
microbiologic culture, sample is inoculated in blood
culture bottles at bedside.
Complications of the procedure include hemorrhage,
perforation of viscus, and introduction of infection.
Evidence of fibrinolysis or of disseminated intra-
vascular coagulation in liver disease is a contraindication
for paracentesis.
Examination of Ascitic Fluid
Laboratory analysis of ascitic fluid helps in the
differential diagnosis of ascites. A variety of tests can be
carried out; however, the tests should be decided in an
individual patient according to the clinical presentation.
The commonly performed tests include estimation of
total proteins and albumin, cell count, cytological
examination, and bacterial culture (Box 7.2).
Exudate (increased capillary permeability or
lymphatic obstruction)
1. Bacterial peritonitis (primary or secondary)
2. Tuberculosis
3. Malignancy (lymphoma, hepatoma, metastatic
carcinoma)
4. Abdominal injury
5. Biliary peritonitis (rupture of gallbladder)
6. Pancreatitis
7. Chylous ascites (obstruction of or injury to thoracic
duct)
Fig. 7.3: Sites for paracentesis (blue filled circles)
Box 7.2: Tests commonly done on ascitic fluid
• White cell count: This is the most important test as it
rapidly provides information about possible bacterial
infection. Neutrophil count >250/cmm is a strong
indication of bacterial infection, whereas lymphocytosis
indicates peritoneal tuberculosis or carcinomatosis.
• Albumin: Estimation of serum and ascitic fluid albumin
allows calculation of serum-ascites albumin gradient
(SAAG) that allows categorization of ascites into low and
high SAAG.
• Microbiological tests: Gram stain, Ziehl-Neelsen stain,
culture
• Cytological examination: For detection of malignant
cells when peritoneum is involved by cancer.
1. Appearance: Transudates are pale yellow or straw-
colored and clear, whereas exudates are opaque or turbid.
Turbid fluid results from leucocytes, malignant cells, or
proteins. Bloody or hemorrhagic fluid indicates
traumatic tap, recent surgery, abdominal trauma, or
malignancy. A traumatic tap shows gradual clearing of
fluid during aspiration. Milky or chylous fluid results
 Examination of Pleural and Peritoneal Fluids 97

Fig. 7.4: Classification of ascites into high and low albumin gradient. This system has replaced test for total protein concentration
in ascitic fluid for classification of ascites into transudate and exudate. Patients with high albumin gradient respond to sodium
restriction and diuretics, while patients with low albumin gradient require specific treatment

from obstruction of lymphatic duct due to inflammation
or malignancy (lymphoma, carcinomatosis), or from
abdominal injury.
2. Chemical examination:
i. Proteins: Traditionally, fluid is called as a transudate
if protein content is low, and an exudate if its protein
content is high. However, this criterion alone is not
always sufficient. In ascitic fluid, distinction
between transudates and exudates cannot be
reliably made by estimation of proteins. A better
indicator is albumin gradient (calculated as serum
iii. Amylase: Normally, amylase in ascitic fluid is
albumin minus ascitic fluid albumin done on the
same day) (Fig 7.4).
Total protein concentration in ascitic fluid can be
helpful in differentiating spontaneous (total protein
<1.0 gm/dl) from secondary bacterial peritonitis
(total protein > 1.0 gm/dl).
ii. Lactate dehydrogenase: Lactate dehydrogenase in
ascitic fluid is elevated in spontaneous bacterial
peritonitis (i.e. there is no obvious source of
infection), secondary bacterial peritonitis (i.e.
identifiable source of infection is present), and in
peritoneal carcinomatosis.
Ascitic fluid findings in various diseases are shown
in Table 7.6. Distinction between spontaneous and
secondary bacterial peritonitis is presented in Table
7.7 and Figure 7.5.
similar to serum amylase. If ascitic fluid amylase is
three times greater than serum amylase, ascites is
most likely to be due to pancreatic disease such as
acute pancreatitis.

Table 7.6: Ascitic fluid findings in various diseases

Cause Appearance Type of fluid Cells Special studies

1. Spontaneous bacterial peritonitis Cloudy or turbid Exudate Neutrophils Single organism isolated
≥250/μl on culture; Proteins
<1.0 gm/dl; Glucose normal
2. Secondary bacterial peritonitis Cloudy or turbid Exudate Neutrophils Multiple organisms isolated
≥1000/μl on culture
3. Cirrhosis of liver Clear, straw- Transudate Lymphocytes Albumin gradient ≥1.1 gm/dl
colored <500/μl
4. Tuberculous peritonitis Serosanguineous Exudate Lymphocytes Ziehl-Neelsen stain; culture;
>500/μl Low albumin gradient;
Total proteins ≥2.5 gm/dl
5. Malignancy Bloody Exudate Lymphocytes, Cytology
malignant cells
 98 Essentials of Clinical Pathology

Table 7.7: Distinction between spontaneous and secondary bacterial peritonitis

Parameter Spontaneous bacterial peritonitis Secondary bacterial peritonitis

1. Obvious source of infection Absent Present, e.g. perforation of viscus, abscess

2. Total ascitic fluid proteins <1.0 gm/dl ≥1.0 gm/dl
3. Severity Less severe More severe
4. Culture Single organism Multiple organisms
5. Treatment Rapid response to antibiotics Requires surgical treatment

Fig 7.5: Differentiation of spontaneous from secondary bacterial peritonitis

iv. Bilirubin: Ascitic fluid bilirubin greater than 6.0 mg/
dl and ascitic fluid bilirubin/serum bilirubin ratio
greater than 1.0 indicate perforation of biliary tract
(biliary peritonitis). Ascitic fluid is bile-stained.
3. Cell count: Cell count is usually done to distinguish
cirrhotic ascites from spontaneous bacterial
peritonitis. In ascitic fluid, total leukocyte count >
500/ml and absolute neutrophil count >250/ml
constitute the presumptive evidence of spontaneous
bacterial peritonitis.
4. Microbiological examination: Gram smear is positive
in 25% cases of spontaneous bacterial peritonitis. If
ascitic fluid is inoculated in blood-culture bottles
at bedside, sensitivity of isolation rises to 85% (as
compared to conventional method of inoculation in
broth and agar plates in laboratory). In spontaneous
bacterial peritonitis, a single organism is isolated,
while secondary bacterial peritonitis is polymicrobial.
In case of tuberculosis, Ziehl-Neelsen stain has
sensitivity of 25-30%, while culture is positive in about
50% of cases. Laparoscopic biopsy is more helpful in
diagnosis of tuberculous peritonitis.
5. Cytological examination: Cytological examination of
peritoneal fluid can detect 40-65% cases of malignant
ascites.
BIBLIOGRAPHY
1. Light RW. Pleural effusion. N Engl J Med 2002;346:
1971-77.
2. Runyon BA. Care of patients with ascites. N Engl J Med
1994;330:337-42.
3. Tarn AC and Lapworth R. Biochemical analysis of
pleural fluid; What should we measure? Ann Clin
Biochem 2001;38:311-22.
4. Thomsen TW, DeLaPena J, Setnik GS. Thoracentesis.
N Engl J Med 2006;355:e16. Downloaded from
www.nejm. org on January 14, 2007.
5. Thomsen TW, Shaffer RW, White B, Setnik GS.
Paracentesis. N Engl J Med 2006;355: e21. Downloaded
from www.nejm.org on January 14, 2007.
 8
Examination of Sputum
Sputum examination refers to the examination of the
material coughed out from the lungs, bronchi, trachea,
and larynx. Normally, sputum is composed predomi-
nantly of mucus and also certain cellular and non-cellular
components of host origin. During expectoration, sputum
gets contaminated with cells and normal bacterial flora
from the mouth and pharynx.
Examination of sputum is mainly carried out for:
• Identification of the causative organism in a
suspected infection of the lower respiratory tract, e.g.
– Pneumonia especially if severe or in an immuno-
compromised host.
– Suspected tuberculosis
– Suspected fungal infection
– Pneumocystic carinii pneumonia in HIV-positive
patients
– Infective exacerbation of a chronic disease like
bronchiectasis
• Cytologic examination for malignant cells, investi-
gation of asbestosis, investigation of viral infections
(viral inclusions in cytomegalovirus and herpes
simplex infections) and fungal infections.
COLLECTION OF SPUTUM
1. Sputum sample is preferably collected in the morning
(since secretions accumulate overnight), soon after
awakening and before taking any mouthwash or
food.
2. Sample is collected in a clean, dry, wide-mouthed
container with a securely fitting screw cap. The
container should be leak proof and break-resistant to
prevent aerosol formation and desiccation, and
should have the capacity of about 25 ml.
3. Patient takes a deep breath 2-3 times filling his/her
lungs, coughs deeply, and spits into the container.
About 2-5 ml of sputum is collected. Sample
consisting only of saliva (foamy, clear, and watery
appearance) is not acceptable; in such a case, another
sample should be obtained. The container is capped
securely and labeled.
Induction of Sputum
If the patient is unable to bring out the sputum
spontaneously, inhaling aerosol of 15% sodium chloride
and 20% propylene glycol for 20 minutes can induce
expectoration. Sputum can also be induced by inhaling
nebulized hypertonic saline or distilled water in
association with chest physiotherapy.
For microbiologic studies, sample should be sent to
the laboratory immediately. If sputum is allowed to
stand, rapid proliferation of contaminating bacterial flora
from oral cavity and throat will occur leading to incorrect
results. In addition, pathogenic organisms, especially
Haemophilus influenzae, do not survive in collected
samples for long. Sample for bacterial culture should not
be refrigerated.
If sample is to be transported to a distant laboratory
for mycobacterial culture, sputum should be collected
in 25 ml of following solution:
• N-acetylpyridinium chloride 5 gm
• Sodium chloride 10 gm
• Distilled water upto 1000 ml
APPEARANCE OF SPUTUM
Appearance of sputum is often suggestive of the
underlying pathologic process as follows:
• Bloody: Hemoptysis (pulmonary tuberculosis, lung
abscess, bronchiectasis, bronchogenic carcinoma,
mitral stenosis, pulmonary infarction)
• Rusty: Pneumococcal lobar pneumonia
• Bloody and gelatinous (red current jelly): Klebsiella
pneumonia
• Green: Pseudomonas infection
• Purulent and separating into 3 layers on standing:
Bronchiectasis, lung abscess
• Pink, frothy (air bubbles): Pulmonary edema
• Copious amounts of purulent sputum: Lung abscess,
bronchiectasis, bronchopleural fistula
100 Essentials of Clinical Pathology
MICROBIOLOGICAL EXAMINATION
Sputum sample is often contaminated with normal flora
of the oral cavity and pharynx (Box 8.1).
Box 8.1: Normal flora of the oral cavity and pharynx
Gram-positive: Staphylococci (S. aureus, S. epidermidis),
streptococci (S. viridans, S. pneumoniae), Diptheroids,
enterococci, micrococci, lactobacilli, Yeasts (Candida spp.).
Gram-negative: Neisseria spp; Haemophilus spp; fuso-
bacteria, coliforms, Moraxella catarrhalis.
Gram Staining
Pathogenic organisms found in sputum include—
1. Gram-positive: Streptococcus pneumoniae, Streptococcus
pyogenes, Staphylococcus aureus.
2. Gram-negative: Haemophilus influenzae, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Yersinia pestis,
Moraxella catarrhalis.
For bacteriologic examination, sputum sample should
be processed in the laboratory within 1 hour of collection.
The sample is transferred to a sterile Petri dish and its
appearance is noted. A thin smear is made on a glass
slide from the purulent portion of the sputum with a
clean stick, air-dried, fixed, and stained with Gram’s
stain. Purely mucoid, watery, frothy, or white samples
usually show squamous epithelial cells covered with
masses of bacteria; this indicates that the sample consists
mainly of secretions from the mouth and the throat. Such
samples are not suitable for bacteriological examination
(Fig. 8.1). If polymorphonuclear neutrophils are less than
10 per epithelial cell, culture is not carried out.
Fig. 8.1: Unacceptable sputum sample: Sputum sample
shows many squamous cells covered with masses of bacteria
Gram stained smear of sputum should be interpreted
carefully because of the presence of various contami-
nating gram-positive and gram-negative organisms
originating from mouth and throat (normal bacterial
flora).
Morphological appearance on Gram stained smear
is suggestive of a particular organism as follows:
• Gram-positive diplococci with surrounding clear
space (capsule): S. pneumoniae (Fig. 8.2).
• Gram-positive cocci in grape-like clusters: S. aureus.
• Gram-positive yeast cells with budding and pseudo-
hyphae: Candida.
• Gram-negative diplococci, both intra- and extra-
cellular: Moraxella catarrhalis.
• Gram-negative coccobacilli: H. influenzae.
• Large granules with center gram-negative and
periphery gram-positive: Actinomyces.
Bacteriological Culture
A floccule of purulent portion of sputum is inoculated
on culture media for definitive identification of
organisms. Sputum sample is considered as unsatis-
factory for culture if it contains >25 squamous cells/low
power field. Ideal sputum sample for culture contains
alveolar macrophages, numerous neutrophils (>5/high
power field), bronchial epithelial cells, and few squamous
cells (<10/high power field). To reduce the amount of
contaminating normal bacterial flora in the inoculum,
saliva is washed away from sputum with sterile normal
saline. The washed sputum is inoculated on (i) blood agar
plate and (ii) chocolate agar (heated blood agar). The
blood agar plate is incubated aerobically and chocolate
agar plate is incubated in an atmosphere of extra carbon
dioxide. Inoculated plates are inspected for growth after
Fig. 8.2: Gram stained smear of sputum showing
gram-positive diplococci (Streptococcus pneumoniae)

incubation for 18 hours; if growth is not satisfactory,
incubation for further 24 hours is indicated. Antibiotic
sensitivity test is carried out only if amount of growth is
significant.
EXAMINATION OF SPUTUM FOR
MYCOBACTERIUM TUBERCULOSIS
Tuberculosis is one of the major public health problems
in India. Early diagnosis of pulmonary tuberculosis will
lead to early institution of therapy enabling cure, and
also prevention of spread of disease to others. Number
of cases of tuberculosis has increased in recent times, with
World Health Organization calling it a global emergency.
Multidrug-resistant tuberculosis is also emerging on a
large scale.
Mycobacterium tuberculosis complex comprises of M.
tuberculosis, M. bovis, and M. africanum. These tubercle
bacilli are the aetiologic agents of human tuberculosis.
Other mycobacteria are called as non-tuberculous.
There are two main approaches for diagnosis of
tuberculosis:
• Direct tests: This consists of detection of M.
tuberculosis or its components
• Indirect tests: This involves detection of humoral or
cellular immune response to tuberculous infection.
These tests have not been considered in this chapter.
Direct tests for diagnosis of tuberculosis on sputum
sample are as follows:
• Examination of sputum smear
1. Ziehl-Neelsen technique
2. Fluorescence microscopy
• Culture on conventional media
• Commercial automated culture systems
• Molecular methods.
Examination of Sputum Smear
For detection of M. tuberculosis, at least three sputum
samples collected on three separate occasions (including
at least one early morning specimen) need to be
examined. A thin smear is prepared on a clean glass slide
from blood-tinged, opaque, grayish, or yellowish portion.
Children often swallow sputum and may not be able to
cough it up; in them a sample of fasting gastric juice can
be aspirated and examined like sputum.
The smear is stained with Ziehl-Neelsen stain and
examined under the ordinary light microscope. If
fluorescent microscope is available, smear can be
examined after staining it with a fluorochrome
(auramine-rhodamine or auramine O).
Examination of Sputum 101
Ziehl-Neelsen stain of sputum smear: This simple,
inexpensive, and rapid technique is mainly useful for:
• Diagnosis of infectious cases of pulmonary tubercu-
losis. (Sputum smear-positive cases are a major source
of spread of infection).
• Assessment of response to anti-tuberculous treat-
ment.
• Determining cure or treatment failure.
Ziehl-Neelsen-stained sputum smear is positive if at
least 5000-10000 tubercle bacilli/ml are present in the
sputum. Sensitivity of the technique is reported to be 60-
80%. Chances of detection of tubercle bacilli are increased
if multiple sputum samples are examined or if bleach
concentration technique is used. In bleach concentration
technique, a solution of bleach (concentrated sodium
hypochlorite) is added to the sputum sample, which
leads to the liquefaction of mucus and killing of
mycobacteria. After centrifugation (or overnight
sedimentation), smears are prepared from the sediment,
stained, and examined.
With Ziehl-Neelsen staining, mycobacteria appear as
bright red straight or slightly curved beaded rods (2-4 μ
in length and 0.2-0.5 μ in width) against a blue or green
background (Fig. 8.3). Mycobacteria are both acid- and
alcohol-fast and are termed acid-fast bacilli (AFB). If acid-
fast bacilli are seen, their number should be reported. At
least 100 fields are examined before reporting the smear
as negative.
A negative smear does not rule out the diagnosis of
tuberculosis since organisms may be few in number,
sputum sample may not have been collected satisfac-
torily, or smear may be of poor quality.
Fig. 8.3: Sputum smear stained with Ziehl-Neelsen stain
showing acid-fast bacilli
102 Essentials of Clinical Pathology
Fig. 8.4: Demonstration of mycobacteria in sputum by
fluorescence microscopy. Bacilli appear as yellow fluorescing
rods with auramine O against dark background
Fluorescence microscopy: M. tuberculosis can also be stained
with a fluorochrome (auramine-rhodamine or auramine
O) and the preparation is examined under fluorescence
microscope. Mycobacteria fluoresce bright yellow against
a dark background (Fig. 8.4). This technique is rapid
(since the smear is examined under low power) and is
especially helpful if organisms are few in number. It is
necessary to confirm a positive smear by Ziehl-Neelsen
stain since false-positive rate is high.
Culture (Conventional)
The definitive diagnosis of tuberculosis is based on
isolation of M. tuberculosis from culture of sputum
sample. Culture is usually carried out for:
• Drug susceptibility testing
• Precise species identification, if organism other than
M. tuberculosis is suspected (for epidemiologic
purpose).
• Diagnosis in patients who have typical clinical and
radiological features of tuberculosis but are sputum
smear-negative.
Culture is more sensitive than sputum smear exami-
nation as it can detect 10-100 microorganisms per ml of
sputum sample. Its sensitivity for diagnosis of tuber-
culosis is 80-85% and specificity is 98%. However culture
is expensive, around 6 weeks are required for result and
even longer for drug susceptibility testing, and prior
decontamination of sputum is required to kill normal
bacterial flora.
Sputum samples are contaminated to a varying
degree with normal bacterial flora. These bacteria are
rapidly growing and digest the culture medium before
tubercle bacilli begin to grow. Therefore, before
inoculation, digestion and decontamination of sputum
sample is carried out to liquefy the organic debris so that
the decontaminating agent can reach the bacteria, and
kill the contaminating organisms. The usual agent
employed for this purpose is 4% sodium hydroxide.
Traditional culture media for isolation of M.
tuberculosis are:
• Solid media: Egg-based (Lowenstein-Jensen medium)
or agar-based (Middlebrook 7H10 or 7H11).
• Liquid media: Middlebrook 7H9, Middlebrook 7H12.
The most commonly used solid medium for culture
is Lowenstein-Jensen medium. Visible mycobacterial
growth may require up to 6 weeks. Further biochemical
tests are required for determination of species.
Commercial Automated Culture Systems
New rapid automated culture systems have been
introduced commercially which can give result within 2
weeks (instead of 6 weeks with conventional media).
These methods, however, are expensive. One such
system is BACTEC 460TB system (Becton-Dickinson
Diagnostic Instruments Systems, Maryland, USA). This
is a well-established, sensitive, and rapid method for
detection of M. tuberculosis in clinical samples. This
method uses a broth in which radiolabeled 14C-palmitate
has been incorporated. Mycobacteria metabolize 14C-
palmitate to radiolabelled 14CO2, which is then detected
by the instrument.
Molecular Methods
There are two approaches for molecular diagnosis of
tuberculosis in sputum samples:
• Direct detection of Mycobacterium tuberculosis in
sputum sample
• Detection of Mycobacterium tuberculosis in isolates
from culture by nucleic acid probes.
M. tuberculosis can be rapidly identified directly in
sputum samples by detecting DNA sequences specific
to it. A commonly used DNA target for this purpose is
IS 6110 that is observed only in M. tuberculosis complex.
Multiple copies of this sequence are present in the
genome of M. tuberculosis. This method can detect 10-
1000 organisms per ml of sputum. Other DNA and RNA
sequences specific for M. tuberculosis complex can also
be targeted.
Laboratory cross-contamination (due to aerosolized
PCR product) is responsible for significant number of
false-positive results. This test is also expensive. PCR
amplifies DNA sequences of both live and dead bacilli.
Therefore the test cannot be used to assess response to
therapy.
PCR-based assays should be interpreted in the light
of clinical features, prevalence of tuberculosis in the
population, and findings on Ziehl-Neelsen smear.

EXAMINATION FOR OTHER ORGANISMS
Additional investigations given below are indicated
when infection by following organisms is suspected:
• Pneumocystis carinii: Bronchoalveolar lavage fluid
stained with silver stain (Fig. 8.5) and Giemsa stain
• Yersinia pestis (pneumonic plague): Giemsa smear
• Yeast-like organisms on Gram’s smear: Sabouraud
dextrose agar
• Histoplasmosis: Giemsa smear
• Aspergillus: Potassium hydroxide wet mount of
sputum
• Paragonimus: Saline wet mount of sputum for eggs.
CYTOLOGICAL EXAMINATION OF SPUTUM
Cytological examination of sputum is usually carried out
for investigation of bronchogenic carcinoma. Sometimes,
it may also be helpful in the identification of viral
inclusions (like those of cytomegalovirus and Herpes
simplex virus), fungi, protozoa, and asbestos bodies.
For cytological examination, fresh early morning
sputum sample is preferred. For detection of lung cancer,
it is recommended to collect sputum sample daily for
five consecutive days. This is because multiple sputum
samples increase the chances of detection of malignant
cells (Fig. 8.6).
Sputum sample may be either spontaneously
produced or artificially induced. If patient is unable to
produce sputum spontaneously by deep coughing, he is
made to inhale a heated, aerosolized solution of 15%
sodium chloride and 20% propylene glycol for 20
minutes. This usually results in induction of a satisfactory
sputum sample.
Ideally, freshly collected sputum should be sent
immediately to the laboratory without addition of any
Fig. 8.5: Pneumocystis carinii cysts in bronchoalveolar
lavage fluid (silver methenamine stain)
Examination of Sputum 103
Fig. 8.6: Sputum examination showing malignant cells
of squamous type
fixative. If delay is anticipated, prefixation of sputum
with Saccomano’s fixative is recommended. This consists
of collection of sputum in a mixture of 50% ethyl alcohol
and 2% carbowax.
In the laboratory, smears are made from blood-tinged
portion or from tissue fragments in sputum and stained
with Papanicolaou technique. Sputum sample is
considered as adequate for cytological evaluation if
alveolar macrophages or bronchial epithelial cells are
seen in the smear.
The average sensitivity of sputum examination for
detection of malignant cells is about 65%. Sensitivity
increases if:
• Increased numbers of sputum samples are examined
• Lesion is centrally located rather than at the periphery
of the lung
• Size of tumor is large
• Histologic type of carcinoma is of squamous nature
rather than adenocarcinoma or small cell carcinoma.
BIBLIOGRAPHY
1. American Thoracic Society. Diagnostic standards and
classification of tuberculosis in adults and children. Am
J Respir Crit Care Med 2000;161:1376-95.
2. Indian Council of Medical Research. What is new in
the diagnosis of tuberculosis? Part I: Techniques for
diagnosis of tuberculosis. ICMR Bulletin 2002;32;No 8
3. Laszlo A. Tuberculosis: Laboratory aspects of diagnosis.
CMAJ 1999;160:1275-9.
4. Soini H, Musser JM. Molecular diagnosis of myco-
bacteria. Clin Chem 2001;47:809-14.
5. Thunissen FBJM. Sputum examination for early
detection of lung cancer. J Clin Path 2003;56:805-10.
6. Watterson SA, Drobniewski FA. Modern laboratory
diagnosis of mycobacterial infections. J Clin Path 2000;
53:727-32.
tahir99 - UnitedVRG
vip.persianss.ir
 9

Examination of Feces

Waste products excreted from the digestive tract are
composed of water (up to 75%), indigestible residue,
undigested food, food which is digested but not
absorbed, bile, epithelial cells, secretions from digestive
tract, inorganic material, and bacteria. Normal amount
of feces in an adult is 100-200 grams per day.
Examination of feces is helpful in the investigation
of diseases of the gastrointestinal tract as follows:
• Detection of parasites: Stool examination is done for
detection of worms (adult worms, segments of
tapeworms, larvae, ova), and protozoa (trophozoites
or cysts).
• Evaluation of chronic diarrhea: Chronic diarrhea
refers to passage of 3 or more loose or liquid stools
per day lasting for more than 4 weeks. Acute diarrhea
is defined as passage of 3 or more loose or liquid stools
per day for less than 4 weeks. In diarrhea, stool
examination is an important part of laboratory work-
up. Causes of chronic and acute diarrhea are listed in
Table 9.1 and Figure 9.1 respectively. Depending on
the nature of information sought, either a random
stool sample or 48- or 72-hour sample is collected.
Random diarrheal sample can be tested for occult
blood, fat, pH, white blood cells, culture, or micro-
scopy. A 48- or 72-hour sample is examined for
weight, fat content, carbohydrate, osmolality, or
chymotrypsin activity.
• Evaluation of dysentery: Identification of causative
organism is definitive in differentiating amebic from
bacillary dysentery.
• Bacteriologic examination: Infection by bacteria such
as Salmonella, Shigella, Vibrio, Yersinia, or Clostridium
difficile can be identified by stool culture. Bacterial
toxins (such as those released by Clostridium botulinum
or Clostridium difficile) can also be identified.
• Chemical examination: Chemical tests can be applied
on feces to detect occult blood (in ulcerated lesions of
gastrointestinal tract, especially occult carcinoma of
colon), excess fat excretion (malabsorption syn-
drome), and presence or absence of urobilinogen
(obstructive jaundice).

Table 9.1: Classification and causes of chronic diarrhea

1. Watery diarrhea
i. Osmotic
• Carbohydrate malabsorption
• Osmotic laxatives
ii. Secretory
• Bacterial toxins
• Bile acid malabsorption
• Laxative abuse
• Hormonal disorders: VIPoma, carcinoid syndrome, gastrinoma, hyperthyroidism
2. Inflammatory diarrhea
i. Invasive bacterial and parasitic infections
ii. Inflammatory bowel disease
iii. Pseudomembranous colitis
iv. Infectious diseases
v. Neoplasia
3. Fatty diarrhea
• Malabsorption syndromes

tahir99 - UnitedVRG
vip.persianss.ir
 Examination of Feces 105

Fig. 9.1: Classification of causes of acute diarrhea

Fig. 9.2: Preliminary evaluation of acute diarrhea. Examination of feces for white blood cells is helpful in
narrowing differential diagnosis in intestinal infections in acute diarrhea

• Differentiating infection by invasive bacteria (like rescence, or enzyme-linked immunosorbent assay

Salmonella or Shigella) from that due to toxin-
producing bacteria (like Escherichia coli or Vibrio
cholerae): Increased numbers of polymorphonuclear
neutrophils (identified by methylene blue stain) are
seen in the former (Fig. 9.2).
• Identification of Rotavirus: Rotavirus is a common
cause of diarrhea in infants and young children. It
can be identified by examination of stool by electron
microscopy, latex agglutination, immunofluo-
(ELISA).
MICROSCOPIC EXAMINATION
Microscopic examinations done on fecal sample are
shown in Figure 9.3.
Collection of Specimen for Parasites
A random specimen of stool (at least 4 ml or 4 cm3) is
collected in a clean, dry, container with a tightly fitting
lid (a tin box, plastic box, glass jar, or waxed cardboard

tahir99 - UnitedVRG
vip.persianss.ir
106 Essentials of Clinical Pathology
Fig. 9.3: Microscopic examinations carried out on fecal sample
box) and transported immediately to the laboratory (this
is because trophozoites of Entameba histolytica rapidly
degenerate and alter in morphology). About 20-40 grams
of formed stool or 5-6 tablespoons of watery stool should
be collected. Stool should not be contaminated with urine,
water, soil, or menstrual blood. Urine and water destroy
trophozoites; soil will introduce extraneous organisms
and also hinder proper examination. Parasites are best
detected in warm, freshly passed stools and therefore
stools should be examined as early as possible after
receipt in the laboratory (preferably within 1 hour of
collection). If delay in examination is anticipated, sample
may be refrigerated. A fixative containing 10% formalin
(for preservation of eggs, larvae, and cysts) or polyvinyl
alcohol (for preservation of trophozoites and cysts, and
for permanent staining) may be used if specimen is to be
transported to a distant laboratory.
One negative report for ova and parasites does not
exclude the possibility of infection. Three separate
samples, collected at 3-day intervals, have been
recommended to detect all parasite infections.
Patient should not be receiving oily laxatives, anti-
diarrheal medications, bismuth, antibiotics like tetra-
cycline, or antacids for 7 days before stool examination.
Patient should not have undergone a barium swallow
examination.
In the laboratory, macroscopic examination is done
for consistency (watery, loose, soft or formed) (Fig. 9.4),
color, odor, and presence of blood, mucus, adult worms
or segments of tapeworms.
Trophozoites are most likely to be found in loose
or watery stools or in stools containing blood and
mucus, while cysts are likely to be found in formed
stools. Trophozoites die soon after being passed and
therefore such stools should be examined within 1 hour
of passing. Examination of formed stools can be delayed
but should be completed on the same day.
Fig. 9.4: Consistency of feces
Color/Appearance of Fecal Specimens
• Brown: Normal
• Black: Bleeding in upper gastrointestinal tract
(proximal to cecum), Drugs (iron salts, bismuth salts,
charcoal)
• Red: Bleeeding in large intestine, undigested tomatoes
or beets
• Clay-colored (gray-white): Biliary obstruction
• Silvery: Carcinoma of ampulla of Vater
• Watery: Certain strains of Escherichia coli, Rotavirus
enteritis, cryptosporidiosis
• Rice water: Cholera
• Unformed with blood and mucus: Amebiasis,
inflammatory bowel disease
• Unformed with blood, mucus, and pus: Bacillary
dysentery
• Unformed, frothy, foul smelling, which float on
water: Steatorrhea.
Preparation of Slides
After receipt in the laboratory, saline and iodine wet
mounts of the sample are prepared (Fig. 9.5).
A drop of normal saline is placed near one end of a
glass slide and a drop of Lugol iodine solution is placed
tahir99 - UnitedVRG
vip.persianss.ir

Fig. 9.5: Saline and iodine wet mounts of fecal sample
near the other end. A small amount of feces (about the
size of a match-head) is mixed with a drop each of saline
and iodine using a wire loop, and a cover slip is placed
over each preparation separately. If the specimen
contains blood or mucus, that portion should be included
for examination (trophozoites are more readily found in
mucus). If the stools are liquid, select the portion from
the surface for examination.
Saline wet mount is used for demonstration of eggs
and larvae of helminths, and trophozoites and cysts of
protozoa. It can also detect red cells and white cells.
Iodine stains glycogen and nuclei of the cysts. The iodine
wet mount is useful for identification of protozoal cysts.
Trophozoites become non-motile in iodine mounts. A
liquid, diarrheal stool can be examined directly without
adding saline.
Concentration Procedure
Concentration of fecal specimen is useful if very small
numbers of parasites are present. However, in concen-
trated specimens, amebic trophozoites can no longer be
detected since they are destroyed. If wet mount
examination is negative and there is clinical suspicion of
parasitic infection, fecal concentration is indicated. It is
used for detection of ova, cysts, and larvae of parasites.
Various concentration methods are available; the
choice depends on the nature of parasites to be identified
and the equipment/reagent available in a particular
laboratory. Concentration techniques are of two main
types:
• Sedimentation techniques: Ova and cysts settle at the
bottom. However, excessive fecal debris may make
the detection of parasites difficult. Example: Formol-
ethyl acetate sedimentation procedure.
Examination of Feces 107
Fig. 9.6: Formol-ethyl acetate concentration technique
• Floatation techniques: Ova and cysts float on surface.
However, some ova and cysts do not float at the top
in this procedure. Examples: Saturated salt floatation
technique and zinc sulphate concentration technique.
The most commonly used sedimentation method is
formol-ethyl acetate concentration method since: (i) it can
detect eggs and larvae of almost all helminths, and cysts
of protozoa, (ii) it preserves their morphology well, (iii) it
is rapid, and (iv) risk of infection to the laboratory worker
is minimal because pathogens are killed by formalin.
In this method, fecal suspension is prepared in 10%
formalin (10 ml formalin + 1 gram feces). This suspension
is then passed through a gauze filter till 7 ml of filtered
material is obtained. To this, ethyl acetate (3 ml) is added
and the mixture is centrifuged for 1 minute. Eggs, larvae,
and cysts sediment at the bottom of the centrifuge tube
(Fig. 9.6). Above this deposit, there are layers of formalin,
fecal debris, and ether. Fecal debris is loosened with an
applicator stick and the supernatant is poured off. One
drop of sediment is placed on one end of a glass slide
and one drop is placed at the other end. One of the drops
is stained with iodine, cover slips are placed, and the
preparation is examined under the microscope.
Classification of
Intestinal Parasites of Humans
Intestinal parasites of humans are classified into two
main kingdoms: protozoa and metazoa (helminths)
(Fig. 9.7).
tahir99 - UnitedVRG
vip.persianss.ir
108 Essentials of Clinical Pathology
Fig. 9.7: Classification of intestinal parasites of humans
PROTOZOA
Entamoeba histolytica
E. histolytica is worldwide in distribution, and endemic
in tropical and subtropical countries. It is transmitted by
fecal-oral route (ingestion of food or water contaminated
by cysts of E. histolytica).
Infection by E. histolytica may be asymptomatic or
may cause amebic dysentery or amebic liver abscess.
Amebic trophozoites invade the large intestinal mucosa,
multiply in submucosa, spread laterally and produce
flask-shaped ulcers. Symptoms include low-grade fever,
diarrhea with blood and mucus, weight loss, and
cramping abdominal pain. The cecum, ascending colon,
and rectosigmoid are commonly affected. Excessive
granulation tissue may form in the intestine at the site of
lesion (ameboma) to produce constriction, which may
be mistaken clinically for a neoplasm.
In some cases, amebae penetrate the portal vessels
and are transported to the liver where they form liver
abscess. Amebic abscesses can also form in lungs or brain.
Life cycle: Infection is acquired by ingestion of food or
water contaminated with infective (quadrinucleate) cysts
of E. histolytica. Dissolution of cyst wall in the small
intestine occurs with formation of trophozoites. Actively
motile trophozoites invade large intestinal mucosa, lodge
in submucosa, multiply, and cause disease (colitis).
Extraintestinal spread to liver and other sites can occur.
Under unfavorable conditions, encystation of tropho-
zoites leads to the formation of cysts in the intestinal
lumen. Cysts are discharged in feces and can survive in
moist environmental conditions for weeks to months and
propagate the life cycle by further fecal-oral spread.
Laboratory Diagnosis
1. Identification of trophozoites and cysts on stool
examination: Demonstration of trophozoites of E.
histolytica in fecal specimens is required for diagnosis
of amebic dysentery. For diagnosis, at least three fresh
stool samples should be examined to increase
sensitivity. Trophozoites vary from 15 to 40 μ in
diameter. In saline wet mounts, trophozoites show
motility in one direction via pseudopodia, which form
rapidly. Cytoplasm shows outer transparent ecto-
plasm and inner finely granular endoplasm. Diag-
nostic feature of E. histolytica trophozoites is the
presence of ingested red cells. Nucleus is visible in
the iodine preparation (Fig. 9.8). Fine granules of
peripheral nuclear chromatin are evenly distributed
along the nuclear membrane. Karyosome is small and
centrally placed (Motility is lost in iodine mount).
tahir99 - UnitedVRG
vip.persianss.ir
 Examination of Feces 109

Fig. 9.8: Trophozoite (left) and uninucleate cyst (right) of E. histolytica. Nuclear chromatin is finely beaded and
evenly coats regular nuclear membrane. Karyosome is central. Chromatoid body is long with rounded ends

Cysts of E. histolytica are spherical and measure 10-

15 μ in diameter. Nuclei are 1, 2, 3, or 4 and are similar in
morphology to trophozoite nucleus (mature cyst contains
4 nuclei). Nuclear membrane is regular and thin with
finely granular peripheral chromatin. Karyosome is small
and central. Immature cysts may show chromatoid
bodies (aggregates of ribosomes) that are oblong
structures with rounded ends, and glycogen clumps
(Fig. 9.9).
Entameba dispar is a morphologically identical
organism; however, it is non-pathogenic. If red blood
cells are identified within trophozoites, then the species
is E. histolytica. However, there is no morphologic feature
to distinguish between the cysts of these two organisms.
Therefore, if only cysts are identified on stool exami-
nation, they are reported as “Cysts of E. histolytica/dispar”.
Asymptomatic infections with E. histolytica reported in
the past are now known to be due to E. dispar. Monoclonal
antibodies can distinguish between these two organisms.
Fig. 9.9: Uninucleate, binucleate, trinucleate, and
quadrinucleate cysts of Entamoeba histolytica
Entamoeba histolytica can be definitively identified in
saline wet mount if it shows definite directional motility
and contains ingested red cells.

It is also necessary to distinguish E. histolytica from

other non-pathogenic amebae found in stools like
Entamoeba coli, Entamoeba hartmanii, Endolimax nana, and
Iodameba butschlii.
Wet mount examination of stool has low sensitivity
(25-60%) and also false-positivity due to E. dispar.
For identification of trophozoites, stool smears can
be prepared and stained with trichrome stain (Fig. 9.10).
2. Other findings on stool examination: Plenty of red Fig. 9.10: Trophozoite of Entamoeba histolytica showing
cells and very few white cells are helpful in ingested red cells (Trichrome stain)

tahir99 - UnitedVRG
vip.persianss.ir
110 Essentials of Clinical Pathology
Table 9.2: Differences between amebic and bacillary dysentery
Parameter Amebic dysentery
1. Cause Entamoeba histolytica
2. Onset Gradual
3. Fever/vomiting Not significant
4. Appearance of fecal sample Unformed with blood and mucus
5. Microscopic examination of stool
• Red cells Clumps
• Pus cells Nil or few
• Macrophages Not seen
• Charcot-Leyden crystals May be present
• Trophozoites of E. histolytica Present
• Bacteria Many, motile
6. Antigen test for E. histolytica Positive
7. Stool culture Negative
differentiating amebic from bacillary dysentery.
Charcot-Leyden crystals may be seen. Differences
between amebic and bacillary dysentery are listed in
Table 9.2.
3. Detection of antigen of E. histolytica in stools: Direct
detection of antigen specific to E. histolytica is possible
by commercially available tests based on enzyme
immunoassay. These tests are specific and sensitive
(90%) and can differentiate E. histolytica from E. dispar.
These tests are indicated if direct stool examination
is negative for organisms and intestinal amebiasis is
suspected clinically.
4. Detection of DNA specific to E. histolytica is possible
by polymerase chain reaction-based assays.
5. Serologic tests: Serologic tests, which detect anti-
bodies to E. histolytica, are performed to support the
diagnosis of invasive amebiasis (i.e. colitis, liver
abscess, or ameboma) when organisms cannot be
demonstrated on stool examination. Various tests are
available like latex agglutination test, indirect
hemagglutination test, enzyme immunoassay, and
counter immunoelectrophoresis. Enzyme immuno-
assay is the method of choice since it is most sensitive
and specific. Serologic tests, however, remain positive
for many years after infection and thus cannot
distinguish between recent and past infections.
6. Endoscopic biopsy of ulcer in the intestine: This can
demonstrate trophozoites of E. histolytica in 50% of
cases. Staining with periodic acid Schiff stain
facilitates identification of parasites.
Bacillary dysentery
Shigella (most common)
Acute
Significant
Unformed with blood, mucus, and pus
Discrete
Numerous
Many, some with ingested red cells
Not seen
Not seen
Few, nonmotile
Negative
Positive for Shigella
Giardia intestinalis (lamblia)
G. intestinalis (lamblia), a pathogenic intestinal protozoan,
has a worldwide distribution. It is transmitted by fecal-
oral route, and is usually water-borne. Giardia is resistant
to chlorine levels in tap water and is commonly found in
cold mountain streams. Giardiasis frequently occurs in
persons who spend time camping outdoors or in woods
and is also called as “Backpacker’s diarrhea” or “beaver
fever”. It can cause asymptomatic infection, mild
diarrhea, or a severe disease with diarrhea, mal-
absorption, weight loss and steatorrhea.
There are two stages in the life cycle: cyst and
trophozoite. After ingestion of cysts, excystation occurs
due to action of gastric acid, and trophozoites are released
which migrate to the duodenum and proximal jejunum
where they attach to the mucosa and replicate.
Laboratory Diagnosis
1. Demonstration of trophozoites or cysts: G. lamblia
trophozoites are found in fresh liquid stools,
particularly in flakes of mucus. They often occur in
clusters. Cysts are more likely to be found in formed
or loose stools.
Duodenal aspirates may be obtained if repeated
fecal examinations are negative for G. lamblia and
there is a strong clinical suspicion of giardiasis.
However, the test is invasive and is usually not
necessary for diagnosis.
Trophozoites of G. lamblia are pear-shaped,
12-15 μ in diameter, have 4 pairs of flagellae, 2 large
and oval nuclei, 2 axonemes (axial filaments of
flagella), and 1 or 2 curved median bodies. Motility
is likened to that of “falling leaf”.
tahir99 - UnitedVRG
vip.persianss.ir

Fig. 9.11: Cyst and trophozoite of Giardia lamblia
Cysts of G. lamblia are 8-12 μ in diameter, oval,
and contain 4 nuclei, axonemes, median bodies, and
remains of flagella (Fig. 9.11).
2. Detection of antigen of G. lamblia in stool sample:
Antigen of G. lamblia can be demonstrated by enzyme
immunoassay technique with high sensitivity (90-
99%) and specificity (95-100%). This test can be used
as the initial test for diagnosis of giardiasis; however,
stool examination is still important for detection of
other concomitant parasite infections.
3. Direct fluorescent antibody assay: This test is
available commercially in a kit form and is highly
sensitive and specific. Cysts are labeled with
immunofluorescent antibodies and are detected
under fluorescence microscope.
Coccidia
Isospora belli, Cryptosporidium parvum, and Cyclospora
cayetanensis are human intestinal coccidia. They have a
worldwide distribution. Transmission is by fecal-oral
route (ingestion of infective oocysts). These protozoan
organisms cause self-limited, mild diarrheal illness;
however, in immunocompromised patients (such as
patients with acquired immunodeficiency syndrome)
they can induce severe and protracted diarrhea, which
may sometimes be life-threatening.
Life cycle: Ingestion of infective oocysts by humans leads
to infection. Release of sporozoites from oocysts occurs
which infect intestinal epithelial cells. Sporozoites
multiply within epithelial cells with formation of
merozoites (asexual reproduction by fission or schizo-
gony), which infect other epithelial cells. Some mero-
Examination of Feces 111
zoites develop into male and female gametes. Fertili-
zation of male and female gametes produces a zygote.
Oocyst is formed by encystation around the conjugating
gametes. Sporozoites form within oocysts by sexual
reproduction or sporogony. Oocysts are excreted in feces
and contaminate food or water.
Laboratory Diagnosis
1. Examination of stools for demonstration of oocysts
of I. belli, C. parvum, or C. cayetanensis:
Isospora belli: Oocysts of I. belli can usually be found
in direct wet mounts of feces (unstained). Formalin-
ether concentration technique may sometimes be
necessary. Oocysts of I. belli are oval and about 32 ×
16 μ in size. Immature oocysts contain a granular
zygote. Mature oocysts contain two sporocysts, each
with four sporozoites. With modified Ziehl-Neelsen
stain (on a fecal smear prepared from the sediment
after formalin-ether concentration), oocysts stain
uniform red-pink. Under ultraviolet light, oocysts
show autofluorescence.
Cryptosporidium parvum: Oocysts of C. parvum are
difficult to demonstrate in direct fecal wet mounts.
They are demonstrated either by modified Ziehl-
Neelsen staining of concentrated fecal smear or by
immunofluorescence technique. They are 4-6 μ in size,
round to oval, and stain pink-red.
Cyclospora cayetanensis: In direct wet mounts of feces,
oocysts measure 8-10 μ in diameter, and contain a
cluster of refractile globules (morula-like appearance).
With modified Ziehl-Neelsen stain, they appear
similar to C. parvum, but are larger. Under ultraviolet
light (365 nm), oocysts show intense blue auto-
fluorescence.
2. Detection of antigen in stool samples: Enzyme
immunoassay for detection of specific antigen of C.
parvum is available. It is more sensitive and specific
than Ziehl-Neelsen staining.
3. Direct fluorescent antibody assay: This assay is
available for Cryptosporidium parvum and is highly
sensitive and specific. Oocysts of Cryptosporidium
labeled with fluorescent antibody are readily detected
under fluorescence microscope.
Microsporidia
Microsporidia are obligate intracellular protozoa, which
cause opportunistic infection in immunocompromised
patients leading to persistent diarrhea and weight loss.
Common species causing infection in humans are
Enterocytozoon bieneusi, Encephalitozoon intestinalis, and
Encephalitozoon hellem.
tahir99 - UnitedVRG
vip.persianss.ir
112 Essentials of Clinical Pathology

Transmission of infection is by ingestion of spores. and intestinal perforation. Sometimes worms can
The organisms develop and multiply in intestinal cells invade pancreatic duct or common bile duct and
and form infective spores; rupture of host cells releases cause obstruction; this can lead to pancreatitis or
spores some of which infect newer cells while others are obstructive jaundice respectively. From the bile duct,
excreted in feces. adult worms can reach liver and cause abscess. Adult
Some microsporidia can cause keratoconjunctivitis, worms can migrate to the appendix to cause
hepatitis, peritonitis, respiratory infection, and kidney appendicitis.
disease. Microsporidia cannot be demonstrated on wet • Malabsorption
mounts because of their very small size.
Laboratory Diagnosis
Laboratory Diagnosis
1. Demonstration of eggs of A. lumbricoides: Diagnosis
1. Modified trichrome stain of stool sample: Spores are
of A. lumbricoides infection is made by demonstration
very small (1-5 μ), stain red, and may show a
of eggs on stool examination. Eggs can be demons-
transverse band.
trated in direct wet mount of feces in moderate to
2. Small intestinal biopsy for demonstration of spores
heavy infections. The recommended procedure is
within intestinal cells.
formol-ethyl acetate sedimentation technique for
concentration of eggs. In feces, four types of eggs are
HELMINTHS found: fertilized (double-shelled or decorticated) and
Ascaris lumbricoides (Roundworm) unfertilised (double-shelled or decorticated).
Fertilized eggs: These are oval, yellow-brown, and
This is the most common helminthic infection of humans.
about 70 μ × 50 μ in size. They have outer and inner
Children are more commonly affected than adults. Mode
shells. Outer shell is uneven, brown (due to staining
of transmission is fecal-oral route (ingestion of infective
by bile), and rough (mamillated), while the inner shell
eggs). Adult worms live in the small intestine (duodenum
is thick, smooth , and colorless. The egg contains a
and jejunum) of the host. Eggs are laid by adult female
single central granular mass (fertilized ovum).
worms (about 200,000 per day), which are excreted in
feces. Eggs can remain viable in soil for many years. Unfertilized eggs: Single female worms discharge these
Contamination can occur when untreated human feces eggs. They are slightly larger and more elongated
are used as a fertilizer or by soiling of hands of playing than the fertilized eggs (90 μ in length). Outer shell is
children. Adult worms can live in the intestine for 1-2 dark brown and more irregular, while the inner shell
years. is thinner. This egg is filled with a mass of large
refractile granules (Fig. 9.12).
Life cycle: Infection is acquired by ingestion of infective
Decorticated eggs do not have the outer uneven shell
eggs via contaminated food or hands. Eggs hatch to
and resemble the hookworm eggs.
release larvae in intestine. Larvae penetrate the mucosa
and enter the bloodstream. Larvae circulate, reach lungs
and penetrate alveolar walls to enter the respiratory tree.
Larvae migrate up the trachea to the epiglottis from
where they are swallowed. Maturation of larvae to adult
worms occurs in the small intestine. Female worms lay
down eggs, which are excreted in feces. Eggs become
embryonated (infective) in 4-6 weeks in favorable
environment.

Clinical Features
• Asymptomatic if infection is light.
• Loeffler’s syndrome: Migration of larvae through the
lungs can induce cough, wheezing, eosinophilia, and
bilateral, irregular pulmonary densities.
• Local effects: These include abdominal pain, diarrhea, Fig. 9.12: Fertilized and unfertilized eggs of
intestinal obstruction due to a large mass of worms, Ascaris lumbricoides

tahir99 - UnitedVRG
vip.persianss.ir

2. Identification of adult worms: Occasionally adult
worms are passed spontaneously in the feces and
brought to the laboratory for identification. Adult
ascaris worms are cylindrical or round, pinkish, and
measure about 15 cm (male) or 30 cm (female) in
length. Diameter is about 0.5 cm and tail is curved
(male) or straight (female). There are three lips at the
anterior end (mouth).
Hookworms
The hookworms are Ancylostoma duodenale (old world
hookworm) and Necator americanus (new world hook-
worm).
Life cycle: Infection occurs when there is penetration of
skin of foot by filariform larvae present in soil. Larvae
enter the circulation, and through veins are carried to
the heart and then reach lungs, migrate up the respiratory
tree and are swallowed. Maturation of larvae to the adult
worms occurs in small intestine. Adult worms attach to
the mucosa and suck blood. Adult female worms produce
eggs, which are excreted in feces. Rhabditiform larvae
are released from eggs into the soil and mature into
infective filariform larvae. Both A. duodenale and N.
americanus infection are acquired when infective
filariform larvae penetrate the skin. A. duodenale infection
is also acquired by ingestion of infective larvae.
Clinical Features
Hookworm infection can cause:
• “Ground itch”: This is inflammation and marked
itching on the skin at the site of larval penetration.
• Loeffler’s syndrome: This is due to migration of larvae
through the lungs.
• Iron deficiency anemia due to chronic blood loss: This
is a well-known and most common complication of
hookworm infection. Adult worms attach themselves
to the small intestinal mucosa by teeth-like structures
or cutting plates, and suck blood. They then change
their sites of attachment (every 4-8 hours) while blood
continues to ooze from the previous site. One adult
A. duodenale causes loss of 0.15 ml of blood while one
N. americanus causes loss of 0.03 ml per day.
• Abdominal pain and diarrhea if worm load is high.
Laboratory Diagnosis
1. Demonstration of hookworm eggs: Diagnosis is based
on identification of hookworm eggs on stool
examination. Technique of formol-ethyl acetate
sedimentation is preferred; if not available, direct wet
Examination of Feces 113
Fig. 9.13: Hookworm egg in fresh stools. Egg in oval shape
with a thin shell, has a clear space between wall and developing
cleavage, and contains granular cells
mount of fecal sample can demonstrate eggs in
moderate to heavy infections. Eggs of A. duodenale
and N. americanus are morphologically similar. They
are 50-75 μ in length and 40 μ in width, oval,
colourless, and have a thin shell. In fresh stools, eggs
show 4-8 gray, granular cells (Fig. 9.13). If stool is
more than 12 hours old, eggs will show a rhabditiform
larva folded upon itself. Such egg is called as
embryonated. If feces are more than 24 hours old, then
free rhabditiform larvae will be seen. This should be
differentiated from larvae of Strongyloides stercoralis
(buccal cavity of hookworm larva is longer).
2. Other laboratory features: Blood examination often
shows eosinophilia. Microcytic hypochromic anemia
develops due to chronic blood loss. Test for occult
blood in stools is positive.
Trichuris trichiura
Infection is acquired by ingestion of infective eggs.
Larvae, which are released, develop into adult worms
and attach to the mucosa. Released eggs are excreted in
feces, and mature in soil to infective stage under suitable
conditions.
Heavy infection can cause diarrhea with blood and
mucus in stools, iron deficiency anaemia, or rectal
prolapse.
Diagnosis depends on identification of typical eggs
on stool examination. Eggs measure 50 × 25 μ in size, are
yellow-brown and barrel-shaped. A rounded, trans-
parent plug is present at both poles (Fig. 9.14). Eggs
tahir99 - UnitedVRG
vip.persianss.ir
114 Essentials of Clinical Pathology
Fig. 9.14: Egg of Trichuris trichiura in iodine wet mount
contain central, uniformly granular mass. Eggs are often
quantitated to assess the severity of infection.
Strongyloides stercoralis
Life cycle: Life cycle is more complex than that of other
nematodes. Penetration of skin by infective filariform
larvae in soil causes infection. Larvae enter the circulation
and migrate to the lungs, penetrate the alveolar spaces,
move up the trachea, and are swallowed. Maturation to
adult worms occurs in the small intestine. Female worms
lay down eggs (parthenogenesis). Eggs hatch to release
rhabditiform larvae in small intestine, which are excreted
along with feces (Sometimes, rhabditiform larvae can
mature into filariform larvae, which penetrate mucosa,
or perianal skin and enter the circulation; this is called
as autoinfection). Maturation of rhabditiform larvae to
infective filariform larvae occurs in soil, which can
penetrate the skin and cause infection. If this does not
happen, larvae develop into adult male and female
worms which mate and lay down eggs, from which
rhabditiform larvae are released which further mature
into infective filariform larvae.
Clinical Features
1. Redness and itching at the site of penetration of
filariform larva.
2. Loeffler’s syndrome due to migration of larva through
the lungs.
3. Heavy infection can cause abdominal pain, diarrhea,
and malabsorption.
4. Chronic infection causes abdominal pain, diarrhea,
and urticaria.
5. In immunocompromised persons, potentially fatal
hyper-infection (secondary to auto-infection) can
occur causing severe pneumonia, neurologic compli-
cations, abdominal pain, shock, and septicemia.
Laboratory Diagnosis
Identification of larvae of S. stercoralis: Diagnosis of S.
stercoralis infection depends on demonstration of
rhabditiform larvae in fresh stool specimens. Eggs of S.
stercoralis are rarely seen in stool samples because they
hatch and release rhabditiform larvae in the intestine.
Rhabditiform larvae are 200-300 μ in length and 15 μ in
width and are actively motile. They have two esopha-
geal swellings, a prominent genital primordium and a
short buccal cavity. Sometimes, in old fecal samples,
rhabditiform larvae of hookworms are seen which
resemble those of S. stercoralis; the differentiating feature
is the longer buccal cavity of the former.
Excretion of larvae is often irregular and their number
may be few. Therefore, fecal examination may yield
negative result. In suspected cases, concentration
technique (formol-ethyl acetate) is helpful.
Duodenal fluid can be aspirated for detection of
larvae or Entero-test (String test) can be performed.
(Entero-test: A commercially available gelatin capsule
consists of a textured string. One end of the string is
attached or taped to the cheek and the capsule is then
swallowed. The end of the string reaches and is exposed
in the duodenum after several hours. After removal, the
terminal part of the string should be bile-stained. The
mucus from the end of the string is wiped onto a glass
slide and examined for larvae). In disseminated infection,
larvae may be detected in sputum.
Enzyme immunoassay test that detects IgG antibodies
to S. stercoralis is available and is indicated if organism is
not detected in feces, duodenal aspirate, or string test
and clinical suspicion is strong. It cannot differentiate
between recent and past infection.
Enterobius vermicularis
E. vermicularis is also called as pinworm, seatworm, or
oxyuris. It is distributed worldwide. Infection is common
in children. Mode of transmission is ingestion of food or
water contaminated with infective eggs through fingers.
Life cycle: After ingestion, eggs hatch to release larvae in
the intestine. Larvae mature to the adult worms in the
cecum or appendix. Female worms migrate at night to
perianal skin to deposit up to 15,000 eggs. Marked

irritation at the site leads to contamination of fingers
through scratching which causes self-infection by
transferring eggs to the mouth. Enterobiasis can also be
acquired by handling contaminated clothes, linen, etc.
Clinical Features
Intense nocturnal perineal or perianal itching is usual.
Acute appendicitis can occur due to the presence of worm
in the appendix. Sometimes, worm can invade female
genital tract leading to vulvovaginitis, and formation of
pelvic and peritoneal granulomas.
Special technique for collection and demonstration of pinworm
eggs: Adult female pinworm migrates at night from the
intestine (cecum) to the perianal skin and deposits eggs.
Pinworm eggs are, therefore, detected in perianal skin
folds and are often not found on routine stool exami-
nation.
Pinworm eggs can be collected either by a transparent
adhesive tape (“cellophane tape test”) or by anal swab.
Specimen should preferably be collected late into the
night or early morning before patient passes urine, feces,
or takes a bath.
A transparent adhesive tape is folded over the end
of a glass slide, spoon handle or a wooden tongue
depressor (sticky surface outwards). Patient’s buttocks
are separated and the slide or spoon handle covered with
tape is pressed over perianal skin at many sites. The tape
is then spread over a glass slide with adhesive side down
and pressed flat onto the slide surface. Slide covered with
tape is then examined under the microscope.
In another method, a cotton swab is rubbed around
the anus and then rinsed in a test tube containing 0.5 ml
saline. The fluid is drawn in a pipette; a drop is placed
on a glass slide, and observed under the microscope with
reduced illumination.
Fig. 9.15: (A) Enterobius eggs, (B) Enterobius eggs collected by transparent tape method
Examination of Feces 115
Laboratory Diagnosis
Diagnosis depends on demonstration of eggs in samples
collected from perianal skin (transparent adhesive tape
method) or demonstration of adult worms. Eggs of E.
vermicularis measure 60 μ × 30 μ in size, are oval, and
flattened on one side. They are colorless, transparent with
a double-lined smooth shell, and contain a small granular
mass (Fig. 9.15) or a larva. Adult pinworms may be
recovered from perianal skin folds (by adhesive tape) or
may be found in children’s feces. They are white, motile,
and small in size (male: 0.5 cm; female: 1 cm).
Taenia solium
T. solium occurs mainly in India, Pakistan, China, S.
Africa, and Latin America. It is transmitted by ingestion
of raw or undercooked pork containing infective
cysticercus larvae (cysticercus cellulosae).
Life cycle: Infection is acquired by ingestion of raw or
undercooked pork containing infective encysted
cysticercus larva by man who is a definitive host. Scolex
of cysticercus is freed and attaches to the wall of the
intestine by its suckers and hooks. Development of
cysticercus into an adult tapeworm occurs by addition
of multiple segments or proglottids to the scolex. Length
of the adult tapeworm is about 2-7 meters. Each
proglottid is a functional hermaphroditic reproductive
unit, which produces numerous eggs. Egg-filled segment
at the end of the worm detaches itself from the worm
and releases eggs in feces; segment is also excreted. Eggs
contaminate the soil and are ingested by the pigs
(intermediate hosts) while feeding. Embryo (released
from the egg) penetrates intestinal wall of the pig and is
carried through the bloodstream to the muscles where it
develops into infective cysticercus larvae.
116 Essentials of Clinical Pathology

Man can become an intermediate host if he eats food

contaminated with eggs or if there is self-contamination
(through contaminated fingers). In such an event, embryo
enters the bloodstream and cysticercus develops at any
body site.

Clinical Features
1. Intestinal infection: Clinical features are usually
insignificant. The patient may notice passage of a flat
segment of the worm in feces.
2. Cysticercosis: This occurs if man accidentally ingests
food contaminated with T. solium eggs (or if there is
auto infection). Nodules containing cysticercus
develop in skeletal muscle, subcutaneous tissue,
heart, liver, brain, etc. Cysticercus (or bladder worm)
is a small cyst (< 1.5 cm) containing clear fluid and
inverted scolex.
Involvement of brain (neurocysticercosis) can cause
seizures (neurocysticercosis is a common cause of
epilepsy in endemic areas in children), raised intracranial
tension (due to obstruction to cerebrospinal fluid flow
by intraventricular cysts), psychiatric disturbances, and
localizing signs; sometimes sudden death can occur.
Laboratory Diagnosis
1. Examination of feces
i. Identification of eggs: Morphologically, eggs of T.
solium and T. saginata are identical. Distinction
between the two species requires examination of
proglottids or scolices. Egg measures 30-40 μ in
diameter, is round to oval, and has a thick, brown
wall with transverse lines. The egg contains an
embryo, which is a round granular mass contai-
ning 3 pairs of hooklets and surrounded by a fine
membrane (Fig. 9.16). Occasionally, the egg is
enclosed in a clear sac. Eggs are discharged
intermittently by the tapeworm and therefore may
not be detected easily. Repeated stool exami-
nations and formol-ether concentration technique
are often required for their demonstration.
ii. Identification of gravid segments or proglottids: This
allows identification of species. The segment is
flattened between two glass slides and examined
under a magnifying glass. Gravid segment is 13
mm × 8 mm in size, translucent, and pale blue. It
has a central uterine stem with 8-13 lateral
branches. (Uterine branches are >13 in T. saginata).
iii. Identification of scolex (head): Scolex of a tapeworm
is very small (pinhead size) and is rarely seen.
When examined with a magnifying glass, scolex
Fig. 9.16: Egg of Taenia solium/saginata
of T. solium shows 4 suckers and a crown of
hooklets.
2. Diagnosis of cysticercosis: Cysticercosis can be
diagnosed by serologic tests, radiologic studies, and
biopsy of the lesion.
Indirect hemagglutination assay has sensitivity of
about 80%. A titer of ≥ 1:64 indicates cysticercosis. Newer
glycoprotein immunoblot assay is more sensitive and
specific for diagnosis of neurocysticercosis. .
X-ray is helpful in detecting calcified cysts. Computed
tomography scans are helpful in diagnosing neuro-
cysticercosis.
Taenia saginata
T. saginata (beef tapeworm) has worldwide distribution
with particularly high prevalence in the Middle East,
Africa, Asia, and Latin America. Mode of transmission
is eating raw or undercooked beef (containing infective
cysticercus bovis larvae).
Life cycle is similar to that of T. solium except: (i)
animal host is cattle, (ii) eggs are not infectious to humans
and therefore human cysticercosis does not occur after
ingestion of eggs, and (iii) segments of T. saginata also
migrate to the perianal skin and deposit eggs.
Clinical Features
These are usually insignificant. Sometimes abdominal
pain and diarrhea can occur.
Laboratory Diagnosis
1. Identification of eggs: Eggs of T. saginata can be
identified in feces and perianal skin. They are
morphologically similar to those of T. solium.

2. Identification of gravid segments: Segments measure 20
mm × 6 mm in size, and are ivory white. They contain
a central uterine stem with more than 13 side branches
(i.e. 15-20 branches).
3. Identification of head or scolex: Scolex of T. saginata has
4 suckers but no hooklets. It measures 2 mm in width.
Various laboratory tests for diagnosis of parasitic
infection of intestine are summarized in Table 9.3.
CHEMICAL EXAMINATION
Chemical examination of feces is usually carried out for
the following tests (Fig. 9.17):
• Occult blood
• Excess fat excretion (malabsorption)
• Urobilinogen
• Reducing sugars
Fig. 9.17: Chemical examinations done on fecal sample
Table 9.3: Summary of laboratory tests for diagnosis of intestinal parasites
Test
1. Direct saline mount
2. Direct iodine mount
3. Faecal concentration
4. Cellophane tape technique
5. Trichrome stain of fecal smear
6. AFB stain of fecal smear
7. Detection of antigen in random stool sample
8. Molecular methods based on polymerase
chain reaction on stool sample
9. Serologic methods
Examination of Feces 117
• Fecal osmotic gap
• Fecal pH
Test for Occult Blood in Stools
Presence of blood in feces which is not apparent on gross
inspection and which can be detected only by chemical
tests is called as occult blood. Causes of occult blood in
stools are:
i. Intestinal diseases: hookworms, amebiasis,
typhoid fever, ulcerative colitis, intussusception,
adenoma, cancer of colon or rectum.
ii. Gastric and esophageal diseases: peptic ulcer,
gastritis, esophageal varices, hiatus hernia.
iii. Systemic disorders: bleeding diathesis, uremia.
iv. Long distance runners.
Occult blood test is recommended as a screening
procedure for detection of asymptomatic colorectal
Use
Trophozoites, cysts, ova, and larvae
Protozoal cysts
Recovery of protozoal cysts, helminth ova and larvae
Ova of Enterobius vermicularis
Protozoal cysts and trophozoites
Oocysts of Cryptosporidium, Cyclospora, and Isospora
E. histolytica, G. lamblia, Cryptosporidium
E. histolytica, G. lamblia, Cryptosporidium, Microsporidia,
Cyclospora
E. histolytica, S. stercoralis, Cysticercus
118 Essentials of Clinical Pathology
cancer. Yearly examinations should be carried out after
the age of 50 years. If the test is positive, endoscopy and
barium enema are indicated.
Tests for detection of occult blood in feces: Many tests are
available which differ in their specificity and sensitivity.
These tests include tests based on peroxidase-like activity
of hemoglobin (benzidine, orthotolidine, aminophena-
zone, guaiac), immunochemical tests, and radioisotope
tests.
Tests Based on Peroxidase-like
Activity of Hemoglobin
Principle: Hemoglobin has peroxidase-like activity and
releases oxygen from hydrogen peroxide. Oxygen
molecule then oxidizes the chemical reagent (benzidine,
orthotolidine, aminophenazone, or guaiac) to produce a
colored reaction product.
Benzidine and orthotolidine are carcinogenic and are
no longer used. Benzidine test is also highly sensitive
and false-positive reactions are common.
Since bleeding from the lesion may be intermittent,
repeated testing may be required.
Causes of False-positive Tests
1. Ingestion of peroxidase-containing foods like red
meat, fish, poultry, turnips, horseradish, cauliflower,
spinach, or cucumber. Diet should be free from
peroxidase-containing foods for at least 3 days prior
to testing.
2. Drugs like aspirin and other anti-inflammatory drugs,
which increase blood loss from gastrointestinal tract
in normal persons.
Causes of False-negative Tests
1. Foods containing large amounts of vitamin C.
2. Conversion of all hemoglobin to acid hematin (which
has no peroxidase-like activity) during passage
through the gastrointestinal tract.
Immunochemical Tests
These tests specifically detect human hemoglobin.
Therefore there is no interference from animal hemo-
globin or myoglobin (e.g. meat) or peroxidase-containing
vegetables in the diet.
The test consists of mixing the sample with latex
particles coated with anti-human haemoglobin antibody,
and if agglutination occurs, test is positive. This test can
detect 0.6 ml of blood per 100 grams of feces.
51
Radioisotope Test Using Cr
In this test, 10 ml of patient’s blood is withdrawn, labeled
with 51Cr, and re-infused intravenously. Radioactivity
is measured in fecal sample and in simultaneously
collected blood specimen. Radioactivity in feces indicates
gastrointestinal bleeding. Amount of blood loss can be
calculated. Although the test is sensitive, it is not suitable
for routine screening.
Apt test: This test is done to decide whether blood in the
vomitus or in the feces of a neonate represents swallowed
maternal blood or is the result of bleeding in the
gastrointestinal tract. The test was devised by Dr. Apt
and hence the name. The baby swallows blood during
delivery or during breastfeeding if nipples are cracked.
Apt test is based on the principle that if blood is of
neonatal origin it will contain high proportion of
hemoglobin F (Hb F) that is resistant to alkali denatu-
ration. On the other hand, maternal blood mostly
contains adult hemoglobin or Hb A that is less resistant.
Test for Malabsorption of Fat
Dietary fat is absorbed in the small intestine with the
help of bile salts and pancreatic lipase. Fecal fat mainly
consists of neutral fats (unsplit fats), fatty acids, and soaps
(fatty acid salts). Normally very little fat is excreted in
feces (<7 grams/day in adults). Excess excretion of fecal
fat indicates malabsorption and is known as steatorrhea.
It manifests as bulky, frothy, and foul-smelling stools,
which float on the surface of water.
Causes of Malabsorption of Fat
i. Deficiency of pancreatic lipase (insufficient
lipolysis): chronic pancreatitis, cystic fibrosis.
ii. Deficiency of bile salts (insufficient emulsification
of fat): biliary obstruction, severe liver disease, bile
salt deconjugation due to bacterial overgrowth in
the small intestine.
iii. Diseases of small intestine: tropical sprue, celiac
disease, Whipple’s disease.
Tests for fecal fat are qualitative (i.e. direct micro-
scopic examination after fat staining), and quantitative
(i.e. estimation of fat by gravimetric or titrimetric
analysis).
1. Microscopic stool examination after staining for fat:
A random specimen of stool is collected after putting
the patient on a diet of >80 gm fat per day. Stool
sample is stained with a fat stain (oil red O, Sudan
III, or Sudan IV) and observed under the microscope
for fat globules (Fig. 9.18). Presence of ≥60 fat
droplets/HPF indicates steatorrhea. Ingestion of
mineral or castor oil and use of rectal suppositories
can cause problems in interpretation.
2. Quantitative estimation of fecal fat: The definitive
test for diagnosis of fat malabsorption is quantitation

Fig. 9.18: Sudan stain on fecal sample: (A) Negative; (B) Positive
of fecal fat. Patient should be on a diet of 70-100 gm
of fat per day for 6 days before the test. Feces are
collected over 72 hours and stored in a refrigerator
during the collection period. Specimen should not be
contaminated with urine. Fat quantitation can be
done by gravimetric or titrimetric method. In
gravimetric method, an accurately weighed sample
of feces is emulsified, acidified, and fat is extracted
in a solvent; after evaporation of solvent, fat is
weighed as a pure compound. Titrimetric analysis is
the most widely used method. An accurately weighed
stool sample is treated with alcoholic potassium
hydroxide to convert fat into soaps. Soaps are then
converted to fatty acids by the addition of hydro-
chloric acid. Fatty acids are extracted in a solvent and
the solvent is evaporated. The solution of fat made in
neutral alcohol is then titrated against sodium
hydroxide. Fatty acids comprise about 80% of fecal
fat. Values >7 grams/day are usually abnormal.
Values >14 grams/day are specific for diseases
causing fat malabsorption.
Test for Urobilinogen in Feces
Fecal urobilinogen is determined by Ehrlich’s aldehyde
test (see Chapter 1 “Examination of Urine”). Specimen
should be fresh and kept protected from light. Normal
amount of urobilinogen excreted in feces is 50-300 mg
per day. Increased fecal excretion of urobilinogen is seen
in hemolytic anemia. Urobilinogen is deceased in biliary
Examination of Feces 119
tract obstruction, severe liver disease, oral antibiotic
therapy (disturbance of intestinal bacterial flora), and
aplastic anemia (low hemoglobin turnover). Stools
become pale or clay-colored if urobilinogen is reduced
or absent.
Test for Reducing Sugars
Deficiency of intestinal enzyme lactase is a common
cause of malabsorption. Lactase converts lactose (in milk)
to glucose and galactose. If lactase is deficient, lactose is
converted to lactic acid with production of gas. In infants
this leads to diarrhea, vomiting, and failure to thrive.
Benedict’s test or ClinitestTM tablet test for reducing
sugars is used to test freshly collected stool sample for
lactose. In addition, oral lactose tolerance test is abnormal
(after oral lactose, blood glucose fails to rise above 20
mg/dl of basal value) in lactase deficiency. Rise in blood
glucose indicates that lactose has been hydrolysed and
absorbed by the mucosa. Lactose tolerance test is now
replaced by lactose breath hydrogen testing. In lactase
deficiency, accumulated lactose in the colon is rapidly
fermented to organic acids and gases like hydrogen.
Hydrogen is absorbed and then excreted through the
lungs into the breath. Amount of hydrogen is then
measured in breath; breath hydrogen more than 20 ppm
above baseline within 4 hours indicates positive test.
Fecal Osmotic Gap
Fecal osmotic gap is calculated from concentration of
electrolytes in stool water by formula 290-2([Na+] + [K+]).
(290 is the assumed plasma osmolality). In osmotic
diarrheas, osmotic gap is >150 mOsm/kg, while in
secretory diarrhea, it is typically below 50 mOsm/kg.
Evaluation of chronic diarrhea is shown in Figure 9.19.
Fecal pH
Stool pH below 5.6 is characteristic of carbohydrate
malabsorption.
REFERENCE RANGES
Bulk: 100-200 grams/day
Color: Brown
Water: Up to 75%
pH: 7.0-7.5
Red blood cells: Absent
White blood cells: Few
Epithelial cells: Present
Crystals: Calcium oxalate, triple phosphate
120 Essentials of Clinical Pathology

Fig. 9.19: Evaluation of chronic diarrhea

Fat (Adults): <7 grams/day (gravimetric method), <6 BIBLIOGRAPHY

grams/day (titrimetric method) 1. American Gastroenterological Association. AGA
Fat droplets: Average 2.5 per high power field in random technical review on the evaluation and management
sample of chronic diarrhea. Gastroenterology 1999;116:
1464-86.
Urobilinogen: 50-300 mg/24 hours 2. American Gastroenterological Association Medical
Parasites: Nil Position Statement: Guidelines for the evaluation and
management of chronic diarrhoea. Gastroenterology
Ova, cysts, trophozoites: Nil 1999;116:1461-3.
3. Haque R, Huston CD, Hughes M, Houpt E, Petri, WA
Critical Values Jr. Amebiasis. New Engl J Med 2003;348:1565:73.
Stool culture positive for Salmonella, Shigella, Campylo- 4. Kucik CJ, Martin GL, Sortor BV. Common intestinal
bacter, Vibrio, or Yersinia. parasites. Am Fam Physician 2004;69:1161-8.
 10
Gastric Analysis
Gastric analysis consists of quantitation of gastric acid
(basal and pentagastrin-stimulated) produced by the
stomach. Gastric juice is collected by a nasogastric tube
and gastric acid is quantitated by its titration with sodium
hydroxide solution.
NORMAL GASTRIC ANATOMY AND
PHYSIOLOGY
Anatomically, stomach is divided into four parts: cardia,
fundus, body, and pyloric part. Cardia is the upper part
surrounding the entrance of the esophagus and is lined
by the mucus-secreting epithelium. The epithelium of
the fundus and the body of the stomach is composed of
different cell types including: (i) mucus-secreting cells
which protect gastric mucosa from self-digestion by
forming an overlying thick layer of mucus, (ii) parietal
cells which secrete hydrochloric acid and intrinsic factor,
and (iii) peptic cells or chief cells which secrete the
proteolytic enzyme pepsinogen. Pyloric part is divided
Fig. 10.1: Parts of stomach and their lining cells
into pyloric antrum and pyloric canal. It is lined by
mucus-secreting cells and gastrin-secreting neuro-
endocrine cells (G cells) (Fig. 10.1).
In the stomach, ingested food is mechanically and
chemically broken down to form semi-digested liquid
called chyme. Following relaxation of pyloric sphincter,
chyme passes into the duodenum.
There are three phases of gastric acid secretion:
cephalic, gastric, and intestinal.
• Cephalic or neurogenic phase: This phase is initiated
by the sight, smell, taste, or thought of food that causes
stimulation of vagal nuclei in the brain. Vagus nerve
directly stimulates parietal cells to secrete acid; in
addition, it also stimulates antral G cells to secrete
gastrin in blood (which is also a potent stimulus for
gastric acid secretion) (Fig. 10.2). Cephalic phase is
abolished by vagotomy.
• Gastric phase: Entry of swallowed food into the
stomach causes gastric distension and induces gastric
phase. Distension of antrum and increase in pH due
122 Essentials of Clinical Pathology

Fig. 10.2: Stimulation of gastric acid secretion. Three receptors on parietal cells stimulate acid secretion: histamine (H2) receptor,
acetylcholine or cholinergic receptor, and gastrin/CCK-B receptor. Histamine is released by enterochromaffin-like cells in lamina
propria. Acetylcholine is released from nerve endings. Gastrin is released from G cells in antrum (in response to amino acids
in food, antral distention, and gastrin-releasing peptide). After binding to receptors, H+ is secreted in exchange for K+ by
proton pump

to neutralization of acid by food stimulate antral G
cells to secrete gastrin into the circulation. Gastrin, in
turn, causes release of hydrochloric acid from parietal
cells.
• Intestinal phase: Entry of digested proteins into the
duodenum causes an increase in acid output from
the stomach. It is thought that certain hormones and
absorbed amino acids stimulate parietal cells to
secrete acid.
The secretion from the stomach is called as gastric juice.
The chief constituents of the gastric juice are:
• Hydrochloric acid (HCl): This is secreted by the parietal
cells of the fundus and the body of the stomach. HCl
provides the high acidic pH necessary for activation
of pepsinogen to pepsin. Gastric acid secretion is
stimulated by histamine, acetylcholine, and gastrin
(Fig. 10.2). HCl kills most microorganisms entering
the stomach and also denatures proteins (breaks
hydrogen bonds making polypeptide chains to
unfold). Its secretion is inhibited by somatostatin
(secreted by D cells in pancreas and by mucosa of
intestine), gastric inhibitory peptide (secreted by K
cells in duodenum and jejunum), prostaglandin, and
secretin (secreted by S cells in duodenum).
• Pepsin: Pepsin is secreted by chief cells in stomach.
Pepsin causes partial digestion of proteins leading to
the formation of large polypeptide molecules (optimal
function at pH 1.0 to 3.0). Its secretion is enhanced by
vagal stimulation.
• Mucus
• Intrinsic factor (IF): IF is necessary for absorption of
vitamin B12 in the terminal ileum. It is secreted by
parietal cells of stomach.
INDICATIONS FOR GASTRIC ANALYSIS
In gastric analysis, amount of acid secreted by the
stomach is determined on aspirated gastric juice sample.
Gastric acid output is estimated before and after
stimulation of parietal cells (i.e. basal and peak acid
output). This test was introduced in the past mainly for
the evaluation of peptic ulcer disease (to assess the need
for operative intervention). However, diminishing
frequency of peptic ulcer disease and availability of safe
and effective medical treatment have markedly reduced
the role of surgery.
1. To determine the cause of recurrent peptic ulcer
disease:
• To detect Zollinger-Ellison (ZE) syndrome: ZE
syndrome is a rare disorder in which multiple
mucosal ulcers develop in the stomach, duode-
num, and upper jejunum due to gross hyper-
secretion of acid in the stomach. The cause of

excess secretion of acid is a gastrin-producing
tumor of pancreas. Gastric analysis is done to
detect markedly increased basal and pentagastrin-
stimulated gastric acid output for diagnosis of ZE
syndrome (and also to determine response to acid-
suppressant therapy). However, a more sensitive
and specific test for diagnosis of ZE syndrome is
measurement of serum gastrin (fasting and
secretin-stimulated).
• To decide about completeness of vagotomy
following surgery for peptic ulcer disease: See
Hollander’s test, later.
2. To determine the cause of raised fasting serum
gastrin level: Hypergastrinemia can occur in
achlorhydria, Zollinger-Ellison syndrome, and antral
G cell hyperplasia.
3. To support the diagnosis of pernicious anemia (PA):
Pernicious anemia is caused by defective absorption
of vitamin B12 due to failure of synthesis of intrinsic
factor secondary to gastric mucosal atrophy. There is
also absence of hydrochloric acid in the gastric juice
(achlorhydria). Gastric analysis is done for demons-
tration of achlorhydria if facilities for vitamin assays
and Schilling’s test are not available (Achlorhydria
by itself is insufficient for diagnosis of PA).
4. To distinguish between benign and malignant
ulcer: Hypersecretion of acid is a feature of duodenal
peptic ulcer, while failure of acid secretion
(achlorhydria) occurs in gastric carcinoma. However,
anacidity occurs only in a small proportion of cases
with advanced gastric cancer. Also, not all patients
with duodenal ulcer show increased acid output.
5. To measure the amount of acid secreted in a patient
with symptoms of peptic ulcer dyspepsia but
normal X-ray findings: Excess acid secretion in such
cases is indicative of duodenal ulcer. However,
hypersecretion of acid does not always occur in
duodenal ulcer.
6. To decide the type of surgery to be performed in a
patient with peptic ulcer: Raised basal as well as peak
acid outputs indicate increased parietal cell mass and
need for gastrectomy. Raised basal acid output with
normal peak output is an indication for vagotomy.
CONTRAINDICATIONS TO GASTRIC
ANALYSIS
• Gastric intubation for gastric analysis is contra-
indicated in esophageal stricture or varices, active
nasopharyngeal disease, diverticula, malignancy,
recent history of severe gastric hemorrhage, hyper-
tension, aortic aneurysm, cardiac arrhythmias,
Gastric Analysis 123
congestive cardiac failure, or non-cooperative patient.
• Pyloric stenosis: Obstruction of gastric outlet can
elevate gastric acid output due to raised gastrin
(following antral distension).
• Pentagastrin stimulation is contraindicated in cases
with allergy to pentagastrin, and recent severe gastric
hemorrhge due to peptic ulcer disease.
Gastric analysis is not a commonly performed proce-
dure because of following reasons:
• It is an invasive and cumbersome technique that is
traumatic and unpleasant for the patient.
• Information obtained is not diagnostic in itself.
• Availability of better tests for diagnosis such as
endoscopy and radiology (for suspected peptic ulcer
or malignancy); serum gastrin estimation (for ZE
syndrome); vitamin assays, Schilling test, and anti-
parietal cell antibodies (for pernicious anemia); and
tests for Helicobacter pylori infection (in duodenal or
gastric ulcer).
• Availability of better medical line of treatment that
obviates need for surgery in many patients.
Method of Gastric Analysis
To assess gastric acid secretion, acid output from the
stomach is measured in a fasting state and after injection
of a drug which stimulates gastric acid secretion.
Basal acid output (BAO) is the amount of hydro-
chloric acid (HCl) secreted in the absence of any external
stimuli (visual, olfactory, or auditory).
Maximum acid output (MAO) is the amount of
hydrochloric acid secreted by the stomach following
stimulation by pentagastrin. MAO is calculated from the
first four 15-minute samples after stimulation.
Peak acid output (PAO) is calculated from the two
highest consecutive 15-minute samples. It indicates
greatest possible acid secretory capacity and is preferred
over MAO as it is more reproducible.
Acidity is estimated by titration (see later).
Collection of Sample
All drugs that affect gastric acid secretion (e.g. antacids,
anticholinergics, cholinergics, H2-receptor antagonists,
antihistamines, tranquilizers, antidepressants, and
carbonic anhydrase inhibitors) should be withheld for
24 hours before the test. Proton pump inhibitors should
be discontinued 5 days prior to the test. Patient should
be relaxed and free from all sources of sensory stimula-
tion.
Patient should drink or eat nothing after midnight.
124 Essentials of Clinical Pathology
Gastric juice can be aspirated through an oral or
nasogastric tube (polyvinyl chloride, silicone, or
polyurethane) or during endoscopy.
Oral or nasogastric tube (Fig. 10.3) is commonly used.
It is a flexible tube having a small diameter and a bulbous
end that is made heavy by a small weight of lead. The
end is perforated with small holes to allow entry of gastric
juice into the tube. As the end is radiopaque, the tube
can be positioned in the most dependent part of the
stomach under fluoroscopic or X-ray guidance. The tube
is lubricated and can be introduced either through the
mouth or the nose. The patient is either sitting or reclining
on left side. The tube has three or four markings on its
outer surface that correspond with distance of the tip of
the tube from the teeth, i.e. 40 cm (tip to cardio-
esophageal junction), 50 cm (body of stomach), 57 cm
(pyloric antrum), and 65 cm (duodenum). The position
of the tube can be verified either by fluoroscope or by
‘water recovery test’. In the latter test, 50 ml of water is
introduced through the tube and aspirated again;
recovery of > 90% of water is indicative of proper
placement. The tube is usually positioned in the antrum.
A syringe is attached to the outer end of the tube for the
aspiration of gastric juice.
For estimation of BAO, sample is collected in the
morning after 12-hour overnight fast. Gastric secretion
Fig. 10.3: Oral or nasogastric Ryle’s tube. The tube is marked
at 40, 50, 57, and 65 cm with radiopaque lines for accurate
placement. The tip is bulbous and contains a small weight
of lead to assist the passage during intubation and to know
the position under fluoroscopy or X-ray guidance. There are
four perforations or eyes to aspirate contents from the stomach
through a syringe attached to the base
that has accumulated overnight is aspirated and
discarded. This is followed by aspiration of gastric
secretions at 15-minute intervals for 1 hour (i.e. total 4
consecutive samples are collected). All the samples are
centrifuged to remove any particulate matter. Each 15-
minute sample is analyzed for volume, pH, and acidity.
The acid output in the four samples is totaled and the
result is expressed as concentration of acid in milliequi-
valents per hour or in mmol per hour.
After the collection of gastric juice for determination
of BAO, patient is given a subcutaneous or intramuscular
injection of pentagastrin (6 μg/kg of body weight), and
immediately afterwards, gastric secretions are aspirated
at 15-minute intervals for 1 hour (for estimation of MAO
or PAO). MAO is calculated from the first four 15-minute
samples after stimulation. PAO is calculated from two
consecutive 15-minute samples showing highest acidity.
Titration
Gastric acidity is estimated by titration, with the end
point being determined either by noting the change in
color of the indicator solution or till the desired pH is
reached.
In titration, a solution of alkali (0.1 N sodium
hydroxide) is added from a graduated vessel (burette)
to a known volume of acid (i.e. gastric juice) till the end
point or equivalence point of reaction is reached. The
concentration of acid is then determined from the
concentration and volume of alkali required to neutralize
the particular volume of gastric juice. Concentration of
acid is expressed in terms of milliequivalents per liter or
mmol per liter.
Free acidity refers to the concentration of HCl present
in a free, uncombined form in a solution. The volume of
alkali added to the gastric juice till the Topfer’s reagent
(an indicator added earlier to the gastric juice) changes
color or when the pH (as measured by the pH meter)
reaches 3.5 is a measure of free acidity. A screening test
can be carried out for the presence of free HCl in the
gastric juice. If red color develops after addition of a drop
of Topfer’s reagent to an aliquot of gastric juice, free HCl
is present and the diagnosis of pernicious anaemia
(achlorhydria) can be excluded.
Combined acidity refers to the amount of HCl
combined with proteins and mucin and also includes
small amount of weak acids present in gastric juice.
Total acidity is the sum of free and combined acidity.
The amount of alkali added to the gastric juice till
phenolphthalein indicator (added earlier to the gastric
juice) changes color is a measure of total acidity
(Box 10.1).

Box 10.1: Determination of basal acid output, maximum
acid output, and peak acid output
• Basal acid output (BAO)= Total acid content in all four
15-minute basal samples in mEq/L
• Maximum acid output (MAO) = Total acid content in all
four 15-minute post-pentagastrin samples in mEq/L
• Peak acid output (PAO) = Sum of two consecutive 15-
minute post-pentagastrin samples showing highest acidity
×2 (mEq/L)
Interpretation of Results
1. Volume: Normal total volume is 20-100 ml (usually
< 50 ml). Causes of increased volume of gastric juice
are—
• Delayed emptying of stomach: pyloric stenosis
• Increased gastric secretion: duodenal ulcer,
Zollinger-Ellison syndrome.
2. Color: Normal gastric secretion is colorless, with a
faintly pungent odor. Fresh blood (due to trauma, or
recent bleeding from ulcer or cancer) is red in color.
Old hemorrhage produces a brown, coffee-ground
like appearance (due to formation of acid hematin).
Bile regurgitation produces a yellow or green color.
3. pH: Normal pH is 1.5 to 3.5. In pernicious anemia,
pH is greater than 7.0 due to absence of HCl.
4. Basal acid output:
• Normal: Up to 5 mEq/hour.
• Duodenal ulcer: 5-15 mEq/hour.
• Zollinger-Ellison syndrome: >20 mEq/hour.
Normal BAO is seen in gastric ulcer and in some
patients with duodenal ulcer.
Table 10.1: Causes of alterations in gastric acid output
Increased gastric acid output
• Duodenal ulcer
• Zollinger-Ellison syndrome
• Hyperplasia of antral G cells
• Systemic mastocytosis
• Basophilic leukemia
Gastric Analysis 125
5. Peak acid output:
• Normal: 1-20 mEq/hour.
• Duodenal ulcer: 20-60 mEq/hour.
• Zollinger-Ellison syndrome: > 60 mEq/hour.
• Achlorhydria: 0 mEq/hour.
Normal PAO is seen in gastric ulcer and gastric
carcinoma. Values up to 60 mEq/hour can occur in some
normal individuals and in some patients with Zollinger-
Ellison syndrome.
In pernicious anemia, there is no acid output due to
gastric mucosal atrophy. Achlorhydria should be
diagnosed only if there is no free HCl even after
maximum stimulation.
6. Ratio of basal acid output to peak acid output (BAO/
PAO):
• Normal: < 0.20 (or < 20%).
• Gastric or duodenal ulcer: 0.20-0.40 (20-40%).
• Duodenal ulcer: 0.40-0.60 (40-60%).
• Zollinger-Ellison syndrome: > 0.60 (> 60%).
Normal values occur in gastric ulcer or gastric
carcinoma.
Conditions associated with change in gastric acid
output are listed in Table 10.1.
It is to be noted that values of acid output are not
diagnostic by themselves and should be correlated with
clinical, radiological, and endoscopic features.
OTHER TESTS FOR GASTRIC ANALYSIS
1. Hollander’s test (Insulin hypoglycemia test): In the
past, this test was used for confirmation of complete-
ness of vagotomy (done for duodenal ulcer).
Hypoglycemia is a potent stimulus for gastric acid
secretion and is mediated by vagus nerve. This
response is abolished by vagotomy.
Decreased gastric acid output
• Chronic atrophic gastritis
1. Pernicious anemia
2. Rheumatoid arthritis
3. Thyrotoxicosis
• Gastric ulcer
• Gastric carcinoma
• Chronic renal failure
• Post-vagotomy
• Post-antrectomy
126 Essentials of Clinical Pathology
In this test, after determining BAO, insulin is
administered intravenously (0.15-0.2 units/kg) and
acid output is estimated every 15 minutes for 2 hours
(8 post-stimulation samples). Vagotomy is considered
as complete if, after insulin-induced hypoglycemia
(blood glucose < 45 mg/dl), no acid output is
observed within 45 minutres.
The test gives reliable results only if blood glucose
level falls below 50 mg/dl at some time following
insulin injection. It is best carried out after 3-6 months
of vagotomy.
The test is no longer recommended because of the
risk associated with hypoglycemia. Myocardial
infarction, shock, and death have also been reported.
2. Fractional test meal: In the past, test meals (e.g. oat
meal gruel, alcohol) were administered orally to
stimulate gastric secretion and determine MAO or
PAO. Currently, parenteral pentagastrin is the gastric
stimulant of choice.
3. Tubeless gastric analysis: This is an indirect and
rapid method for determining output of free
hydrochloric acid in gastric juice. In this test, a cation-
exchange resin tagged to a dye (azure A) is orally
administered. In the stomach, the dye is displaced
from the resin by the free hydrogen ions of the
hydrochloric acid. The displaced azure A is absorbed
in the small intestine, enters the bloodstream, and is
excreted in urine. Urinary concentration of the dye is
measured photometrically or by visual comparison
with known color standards. The quantity of the dye
excreted is proportional to the gastric acid output.
However, if kidney or liver function is impaired, false
results may be obtained. The test is no longer in use.
4. Spot check of gastric pH: According to some
investigators, spot determination of pH of fasting
gastric juice (obtained by nasogastric intubation) can
detect the presence of hypochlorhydria (if pH>5.0 in
men or >7.0 in women).
5. Congo red test during esophagogastroduodeno-
scopy: This test is done to determine the completeness
of vagotomy. Congo red dye is sprayed into the
stomach during esophagogastroduodenoscopy; if it
turns red, it indicates presence of functional parietal
cells in stomach with capacity of producing acid.
REFERENCE RANGES
• Volume of gastric juice: 20-100 ml
• Appearance: Clear
• pH: 1.5 to 3.5
• Basal acid output: Up to 5 mEq/hour
• Peak acid output: 1 to 20 mEq/hour
• Ratio of basal acid output to peak acid output: <0.20
or < 20%
BIBLIOGRAPHY
1. Burtis CA, Ashwood ER (Eds). Tietz Fundamentals of
Clinical Chemistry, 4th ed. Philadelphia: WB Saunders
Co, 1996.
2. Drossman DA, Shaheen NJ, Grimm IS (Eds). Handbook
of Gastroenterologic Procedures (4th Ed). Philadelphia:
Lippincott Williams and Wilkins, 2005.
3. Rosenfeld L. Gastric tubes, meals, acid, and analysis-
rise and decline. Clin Chem 1997;43:837-42.
4. Wallach J. Interpretation of Diagnostic tests (7th Ed).
Philadelphia. Lippincott: Williams and Wilkins, 2000.
5. Wolfe MM, Soll AH. The physiology of gastric acid
secretion. N Engl J Med 1988;319:1707-14.
 11

Tests for Malabsorption and

Pancreatic Function

MALABSORPTION SYNDROMES gastrointestinal tract. Causes of malabsorption are listed

Normal Absorption of Carbohydrates,
Fats, and Proteins
Normal digestive processes of carbohydrates, fats, and
proteins are presented in Figures 11.1 to 11.3.
Causes of Malabsorption
Malabsorption is a term that includes disorders that cause
deficient digestion or absorption of nutrients in the
in Table 11.1.
Evaluation of Malabsorption Syndromes
Clinical Features
The manifestations of malabsorption include diarrhea
(of osmotic type as non-absorption of solutes increases
intraluminal osmotic pressure and outpouring of water),
steatorrhea (passage of frothy, bulky, and malodorous
stools that are difficult to flush, with oil droplets floating

Fig. 11.1: Carbohydrate digestion and absorption. Carbohydrate digestion and absorption depend mainly on normal
pancreatic function (amylase) and normal intestinal mucosal cells (disaccharidases)
128 Essentials of Clinical Pathology
Fig. 11.2: Digestion and absorption of fat. This requires presence of normal functioning and
adequate absorptive area of intestinal mucosa, bile salts, and pancreatic enzymes
Fig. 11.3: Protein digestion and absorption. Proteolytic enzymes
of pancreas are trypsin, chymotrypsin, elastase, and carboxy-
peptidase. Protein absorption mainly depends on pancreatic
proteolytic enzymes and small intestinal cells (enterocytes)
Table 11.1: Causes of malabsorption
Impaired digestion
1. Diseases of pancreas: Chronic pancreatitis, cystic fibrosis,
resection of pancreas
2. Deficiency of bile salts: Impaired excretion of bile
(cholestasis), blind loop syndrome (bile salt deconjugation),
intestinal resection (impaired enterohepatic circulation)
Impaired absorption
1. Disorders of mucosa: Tropical sprue, giardiasis, celiac
disease, Crohn’s disease, Whipple’s disease, radiation
enteritis, amyloidosis, disaccharidase deficiency (e.g.
lactase), abetalipoproteinemia
2. Intestinal resection
3. Lymphatic obstruction: Lymphoma
on toilet water; results from malabsorption of fat), or
deficiency of nutrients (e.g. iron, vitamin B12, folate,
vitamin K, vitamin D, calcium and electrolytes leading
to anemia, bleeding, bone pain, neurological manifes-
tations, etc.). Other manifestations include peripheral
edema, abdominal distention, weight loss and fatigue.
Laboratory Evaluation
A variety of laboratory tests are available for assessment
of a patient presenting with clinical manifestations of

malabsorption. In the beginning, complete blood count
(for detecting anemia); iron studies, vitamin B12, and
folate levels; serum albumin, and serum calcium;
erythrocyte sedimentation rate (raised in Crohn’s disease
and Whipple’s disease), and prothrombin time (for
malabsorption of vitamin K) should be obtained.
Radiological investigations (such as small bowel barium
enema, CT scan of abdomen, plain X-ray film of
abdomen, etc.) can be helpful in certain cases.
Laboratory tests for malabsorption can be classified
as follows:
• Tests for malabsorption of fat
• Tests for malabsorption of carbohydrates
• Tests for pancreatic function
• Tests for malabsorption of vitamin B12 (See ‘Schilling
test’ in Chapter on ‘Approach to Diagnosis of
Anemias’)
• Tests for bacterial overgrowth in small intestine
• Tests for laxative abuse.
Tests for malabsorption of fat These tests include:
• Microscopic examination of feces for fat
• Butter fat test
• Estimation of fecal fat
• 14CO2 breath tests, e.g. 14C-triolein breath test.
1. Microscopic examination for fecal fat: This test (that
detects fat globules in feces after staining with a fat
Fig. 11.4: Evaluation of steatorrhea or malabsorption of fat
Tests for Malabsorption and Pancreatic Function 129
stain) is poorly validated and often misses mild forms
of steatorrhoea (See Chapter 9 “Examination of
Feces”).
2. Butter fat test: In this test, a standard amount of fat
is ingested and if chylomicrons are observed in blood
after the fatty meal, fat digestion and absorption are
considered to be adequate.
3. Estimation of fecal fat: Patient should take 70 g per
day fat diet for at least 6 days. This is followed by
collection of feces for atleast 3 days and measurement
of fecal mass or volume and fat. Fecal fat excretion >7%
is considered as abnormal. The test is unpleasant to
perform and misleading results are often obtained due
to incompleteness of collection and poor analytical
performance (See Chapter 9 “Examination of feces”).
4. 14CO2-breath tests: Labeled triglycerides (e.g. 14C-
triolein) are administered orally; after hydrolysis by
pancreatic lipase to monoglycerides and free fatty
acids, micelles are formed with the aid of bile acids
that are then absorbed. Ultimately metabolism leads
to their excretion as CO2. Measurement of breath 14CO2
should show peak radioactivity at 5 to 7 hours. Result
depends both on fat digestion and absorption. To
distinguish between the two, the test is repeated after
administration of a radiolabeled free fatty acid. This
test is indirect and qualitative rather than quantitative.
Evaluation of steatorrhea is shown in Figure 11.4.
130 Essentials of Clinical Pathology
Tests for malabsorption of carbohydrates
These tests include:
• Lactose tolerance test
• Breath hydrogen test
• D-xylose absorption test.
1. Lactose tolerance test: Lactose is a carbohydrate
consisting of one molecule of glucose and one
molecule of galactose. It is also called as milk sugar
as it comprises of 2-8% of milk. A 50 g oral dose of
lactose in 400 ml of water is given and blood glucose
response is measured as in oral glucose tolerance test
(at 0, 30, 60, and 120 minutes) and patient is also
observed for development of symptoms. Failure of
expected rise of blood glucose (i.e. peak rise of blood
glucose <20 mg/dl) and provocation of symptoms
3. D-xylose absorption test: This test assesses the
are indicative of lactose intolerance (Fig. 11.5). To
improve specificity, test is repeated following
administration of glucose (25 g) and galactose (25 g);
a normal rise in blood glucose indicates lactose
intolerance. This test is less reliable than breath
hydrogen test.
2. Breath hydrogen test: An oral dose of lactose is
administered and breath hydrogen is measured. In
lactase deficiency, lactose is not absorbed, and reaches
Fig. 11.5: Diagnosis of lactose intolerance
the large intestine, where hydrogen is produced by
bacterial fermentation (Note: Hydrogen cannot be
produced by mammalian cells and bacterial fermen-
tation of unabsorbed carbohydrate is responsible for
its presence in the expired air). About 20% of such
hydrogen is absorbed by mucosa and excreted
through respiration. In lactase deficiency, breath
hydrogen is increased following lactose load.
Clinically such patients also develop symptoms of
lactose intolerance such as abdominal distention,
discomfort, flatulence and diarrhea.
Lactase deficiency may be congenital or acquired.
Transient deficiency occurs following damage to gut
mucosa, e.g. gastroenteritis, celiac disease, and
inflammatory bowel disease.
absorptive capacity of small intestinal mucosa. D-
xylose, a plant sugar, is completely absorbed in the
small intestine (without prior digestion) after
ingestion (5 g dose) and excreted largely unchanged
in urine. If excretion is less than 20% of ingested dose,
absorption is considered as impaired (small intestinal
disease). Misleading results are obtained in renal
failure, delayed gastric emptying and incomplete
urine collection.
 Tests for Malabsorption and Pancreatic Function 131

Tests for bacterial overgrowth in small intestine Bacterial urine or of faecal water, while magnesium abuse can be
overgrowth in the small intestine occurs if there is stasis identified by measuring fecal osmotic gap and
of small bowel contents (e.g. due to stricture or magnesium concentration in fecal water.
diverticulosis). Bacteria cause deconjugation of bile salts
Other tests for malabsorption Some intestinal disorders
leading to failure of mixed micelle formation and fat
have characteristic radiological abnormalities like
malabsorption. Various tests for detection of small bowel
Crohn’s disease, diverticula, enterocolic fistula, etc. CT
bacterial overgrowth include:
scan can detect changes associated with chronic
• Aspiration and culture of duodenal/jejunal contents
pancreatitis.
• Breath hydrogen tests (e.g. with lactulose)
A definitive diagnosis can be established in certain
• Breath CO2 tests (e.g. with 14C-glycocholic acid or 14C-
malabsorption syndromes by endoscopically-directed
xylose)
biopsy of small intestinal mucosa.
• Measurement of urinary indican
1. Aspiration and culture of duodenal/jejunal contents:
PANCREATIC FUNCTION TESTS
Duodenal or jejunal contents are aspirated at
endoscopy and sent for bacterial culture. This is the The pancreas is a major gland having both exocrine and
most reliable diagnostic test for bacterial overgrowth; endocrine functions. The exocrine component secretes
however, it is invasive and occasionally false-negative its fluid into the pancreatic duct that opens into the
result is obtained. duodenum and is concerned with digestion of food. The
2. Breath hydrogen tests: Lactulose, a non-absorbable endocrine component secretes its hormones directly into
carbohydrate, is administered orally (10 g). In the bloodstream.
bacterial overgrowth, fermentation of unabsorbed
carbohydrate occurs in the small as well as large Anatomy and Physiology of Pancreas
intestine with the production of hydrogen. Breath 1. Exocrine component: Exocrine pancreas is the major
hydrogen is increased early (40 minutes) following component of the gland. It is made up of closely
the oral dose of lactulose. packed secretory acini that drain their secretions via
3. Breath CO 2 tests: In 14C-xylose breath test, 1 g small, highly branched ducts into the main pancreatic
radiolabeled xylose is given orally. In bacterial duct. The main pancreatic duct joins the common bile
overgrowth a large amount of xylose is metabolized duct and opens into the duodenum via the ampulla
by bacteria to 14CO2 that is absorbed and excreted of Vater. A small accessory pancreatic duct is also
through breath. This isotopically labeled CO 2 is present in most individuals and opens into the
measured in expired breath. duodenum separately (Fig. 11.6).
This test is better than 14C-glycocholic acid test. In
the later test, 14C-glycocholic acid (a radiolabeled
conjugated bile acid) is given orally. Normally this
bile acid is absorbed intact. Bacterial overgrowth
causes deconjugation of glycocholic acid with the
release of 14C-glycine which is absorbed in the small
intestine, and metabolized in the liver releasing
labeled carbon dioxide. Excretion of labeled CO2 is
measured in expired breath.
4. Test for urinary indican: Indican is a bacterial
metabolic product of dietary tryptophan.
Tests for laxative abuse Diarrhea due to malabsorption
should be distinguished from that due to laxative abuse.
Laxatives or their metabolites can be detected in urine
or loose fecal sample by thin layer chromatography.
Phenolphthalein can also be detected by alkalization of Fig. 11.6: Gross anatomy of pancreas
132 Essentials of Clinical Pathology
Each acinus is lined by pyramidal secretory cells,
apices of which are packed with zymogen secretory
granules (Figs 11.7 and 11.8). These granules contain
inactive forms of enzymes synthesized by acinar cells.
Zymogen granules are released into the acinar lumina
by the process of exocytosis. Secretions in acinar lumina
initially drain into intercalated ducts, lining cells of which
secrete water and bicarbonate ions. High concentration
of bicarbonate ions is responsible for alkaline pH of
pancreatic juice, which serves to neutralize the acidic pH
of the chyme entering into the duodenum.
The pancreatic enzymes entering into the duodenum
breakdown proteins, carbohydrates, lipids, and nucleic
acids, making them suitable for digestion and absorption.
Pancreatic secretions also neutralize gastric acid entering
into the small intestine. Gastric acid entering the
Fig. 11.7: Major components of exocrine pancreas
Fig. 11.8: Diagrammatic representation of pancreatic
acini and terminal ductule
duodenum stimulates secretion of secretin that in turn
stimulates release of bicarbonate from the pancreas into
the small intestine.
Proteolytic enzymes are secreted in an inactive form
(trypsinogen and chymotrypsinogen) into the pancreatic
juice to prevent the autodigestion of pancreas. Entero-
kinase, secreted by duodenal mucosa, activates the
inactive trypsinogen to trypsin in duodenal lumen.
Trypsin converts chymotrypsinogen to the active enzyme
chymotrypsin. Trypsin and chymotrypsin degrade large
polypeptide molecules to smaller ones. Peptidase, an
intestinal enzyme bound to enterocyte cell membrane,
further degrades smaller polypeptides to amino acids.
Other proteolytic enzymes elaborated by the pancreas
include elastase, carboxypeptidases, ribonuclease, and
collagenase (Box 11.1).
Carbohydrates are broken down by amylase in the
pancreatic juice and by intestinal cell membrane-bound
disaccharidases to monosaccharides.
Lipids are emulsified in the duodenum by the action
of bile; they are then broken down by the pancreatic
lipase into free fatty acids, monoglycerides, and glycerol.
Exocrine pancreatic function is regulated by neural
factors and by certain hormones of the gastrointestinal
tract like cholecystokinin-pancreozymin (CCK-PZ),
gastrin, and secretin.
CCK-PZ stimulates contraction of gallbladder and
also secretion of enzyme-rich pancreatic juice. It is a
polypeptide hormone synthesized by duodenal endo-
crine cells in response to contact with partially digested
proteins and gastric hydrochloric acid.
Gastrin is produced mainly by G cells of the antral
mucosa of the stomach and has actions on the pancreas
similar to that of CCK-PZ. Gastrin secretion is promoted
Box 11.1: Composition of pancreatic juice
• Digestive enzymes (secreted by exocrine acinar cells
of pancreas)
– Proteases: Digest proteins. Pancreatic proteases are
chymotrypsin and trypsin, which digest proteins to smaller
peptides. Other proteases are elastase, carboxypeptidase,
ribonuclease, and collagenase.
– Lipase: Digestion of dietary triglycerides to mono-
glycerides and free fatty acids
– Amylase: Hydrolysis of starch to maltose (a glucose-
glucose disaccharide)
• Bicarbonate and water (secreted by lining cells of
small pancreatic ducts): Neutralization of gastric acid
entering duodenum

by antral distention and the presence of partially digested
proteins.
Secretin is a polypeptide synthesized by duodenal
endocrine S cells in response to contact with gastric
hydrochloric acid. It enhances secretion of bicarbonate-
rich pancreatic fluid into the duodenum.
Normal pancreatic juice is colorless, has alkaline pH,
and its 24-hour volume is 1000-2500 ml. It consists of
water, sodium, potassium, chloride, bicarbonate, and
pancreatic enzymes.
2. Endocrine component: Isolated clusters of endocrine
cells are scattered throughout the exocrine pancreatic
tissue and are known as islets of Langerhans.
Endocrine cells are also present singly in between the
pancreatic acini. Hormones secreted by the endocrine
pancreas include insulin, glucagon, somatostatin,
vasoactive intestinal polypeptide, and pancreatic
polypeptide.
Tests of Pancreatic Function
Tests for assessing pancreatic function measure the
physiologic function of exocrine component. These tests
either determine the functional activity of certain
pancreatic enzymes, or directly quantitate the products
of pancreatic secretions.
Pancreatic function tests are listed in Table 11.2.
Nowadays, the direct tests have largely been replaced
by the indirect (tubeless) tests.
Pancreatic insufficiency can be readily diagnosed in
advanced cases showing steatorrhea, pancreatic
calcification, and diabetes mellitus. Clinical features of
pancreatic insufficiency do not appear until about 90%
of the gland is destroyed. If pancreatic insufficiency is
detected early, appropriate treatment can be given and
improved outlook can be expected. Ideally, exocrine
pancreatic function tests should be simple to perform,
specific, sensitive, and should be able to detect early or
Table 11.2: Pancreatic function tests
Direct tests (Invasive)
• Secretin-CCK-PZ test (or secretin-cerulin test)
• Lundh test
Indirect tests (Non-invasive)
• NBT-PABA (Bentiromide) test
• Pancreolauryl test
• Breath tests
• Estimation of faecal enzymes
• Estimation of faecal fat
Tests for Malabsorption and Pancreatic Function 133
mild pancreatic insufficiency (i.e. when imaging studies
are normal). However, no such non-invasive pancreatic
function test is currently available. Also, the results of
these tests in pancreatic insufficiency often overlap with
normal values.
Direct (Invasive or Tube) Tests
In direct tests, duodenum is intubated, a pancreatic
stimulant (such as secretin-CCK-PZ, secretin-cerulin, or
Lundh meal) is administered, and pancreatic secretions
entering into the duodenum are aspirated. Quantity of
bicarbonate and enzyme secretions is measured, which
correlates with functional mass of pancreas.
1. Secretin-CCK-PZ or Secretin-Cerulin test: This test
is the ‘gold standard’ for detection of exocrine pancreatic
insufficiency.
This test is based on the principle that output of
pancreatic juice, bicarbonate, and enzymes from pancreas
(after maximum stimulation by an exogenously adminis-
tered hormone) is dependent on the amount of functional
pancreatic tissue. The method, in short, is as follows:
i. In a fasting patient, duodenum is intubated with a
radioopaque tube under fluoroscopic guidance and
contents are aspirated until the contents are clear.
ii. Secretin and cholecystokinin-pancreozymin (or
secretin-caerulin) are administered intravenously.
Secretin stimulates secretion of watery, bicarbonate-
rich fluid, while CCK-PZ or cerulin stimulate
secretion of enzymes from the pancreas.
iii. Three samples of duodenal secretions are aspirated:
basal, following intravenous secretin, and following
intravenous CCK-PZ. Total volume of fluid, pH and
concentrations of bicarbonate and enzymes (amylase,
trypsin) are measured, and compared with the
normal values.
In exocrine pancreatic insufficiency, bicarbonate
secretion is lost earlier than enzyme secretions. Also,
enzyme activity and bicarbonate content are reduced
before there is any reduction in volume of pancreatic
juice.
The specificity and sensitivity of this test is high
(around 90%). However, the test is invasive, laborious,
time-consuming, and expensive. It also requires special
skills for performance and interpretation.
2. Lundh meal: Lundh meal is a physiologic test meal
consisting of protein (15 gm), fat (18 gm), carbohydrates
(40 gm glucose), and water (300 ml). It induces pancreatic
secretion by stimulating release of endogenous secretin
and CCK-PZ. Following administration of the meal,
duodenal contents are aspirated and activity of one or
134 Essentials of Clinical Pathology
more enzymes (usually trypsin) and the volume of the
pancreatic juice are measured. The results are usually
abnormal in pancreatic insufficiency. As compared to the
direct hormonal stimulation, this test is more physio-
logical and cheaper.
Disadvantages of this test are:
i. Lower specificity and sensitivity (70-80%) as
compared to the direct secretin-pancreozymin test.
ii. Abnormal result is also obtained in small bowel
mucosal disease.
Indirect (Non-invasive or Tubeless) Tests
As direct tests are invasive, labor-intensive, and need a
specialized set-up, various indirect (tubeless) function
tests have been developed. Deficient activity of pancreatic
enzymes is indirectly demonstrated by reduced digestion
or increased faecal excretion of fat, low concentration of
pancreatic enzymes in feces or in blood, or decreased
hydrolysis of ingested synthetic compounds causing their
reduced urinary excretion. Although simpler, indirect
tests have low sensitivity and specificity and cannot
detect mild pancreatic insufficiency.
1. NBT-PABA test (Bentiromide test): When a synthetic
compound N-benzoyl-l tyrosyl-p-aminobenzoic acid
(NBT-PABA, bentiromide) is ingested orally, it is
hydrolyzed by pancreatic chymotrypsin in duodenum
to release p-aminobenzoic acid (PABA). PABA is
absorbed in the small intestine and metabolized in the
liver. The urinary excretion of metabolites of PABA is
measured for 6 hours; if excretion is less than 50% of
ingested dose, exocrine pancreatic insufficiency is
present. Thus, amount of PABA excreted in urine
indirectly reflects activity of pancreatic chymotrypsin and
pancreatic function.
False results are obtained in small bowel disease
(decreased absorption), liver disease (defective conju-
gation), and renal disease (decreased excretion). To guard
against false results, urinary excretion of PABA after
administration of bentiromide is compared against
urinary excretion of free radiolabeled PABA given at the
same time.
2. Pancreolauryl test (Test for pancreatic arylesterase):
This test is similar in principle to NBT-PABA test.
Fluorescein dilaurate (Pancreolauryl), a synthetic ester,
is ingested along with a standard breakfast. Fluorescein
dilaurate is hydrolysed by pancreas-specific cholesterol
ester hydrolase in the presence of bile acids to release
fluorescein, which is then absorbed in the small intestine,
partially conjugated by the liver, and excreted in urine.
Amount of fluorescein excreted in urine for 10 hours is
measured (Alternatively, serum concentrations can also
be measured). Test is repeated a day later, but following
ingestion of free fluorescein to correct for individual
variation in intestinal absorption, conjugation in liver,
and urinary excretion. Ratio of excretion of fluorescein
dilaurate to excretion of free fluorescein is determined.
If it is less than 0.20, pancreatic insufficiency is present.
Sensitivity and specificity are similar to NBT-PABA test.
3. Breath tests: Breath tests have been developed which
indirectly assess pancreatic function by measuring 13CO2
or 14CO2 in breath following ingestion of a substrate such
as 14C-triglyceride, 14C-triolein, mixed triglyceride, 13C-
D-hiolein, or cholesteryl octanoate.
Principle of one such test using 14C-triglyceride is as
follows. When 14C-triglyceride is ingested, it is hydro-
lysed by pancreatic lipase to free fatty acids and
monoglycerides. After absorption, metabolism of free
fatty acids ultimately leads to the formation of 14CO2 that
is excreted in the breath. The amount of 14CO2 formed is
proportional to the rate of absorption of fatty acids in
the intestine. Normally there is a peak radioactivity in
breath after about 6 hours. Low radioactivity in breath
can be due to deficient fat digestion or deficient fat
absorption. To distinguish between the two, the test is
repeated after 2 weeks using radiolabeled free fatty acids
(instead of radiolabeled triglycerides). If the result is
normal, pancreatic insufficiency is present.
Disadvantages of the test are:
• Time consuming.
• Exposure to radioisotopes.
• Inability to detect mild pancreatic insufficiency.
• False-positive test in respiratory or metabolic disease
in which excretion of CO2 is affected.
4. Estimation of fecal enzymes: Concentration of
enzymes secreted by the pancreatic acinar cells into the
gut is lowered in pancreatic insufficiency and obstruction
of pancreatic duct. Output of elastase-1 or chymotrypsin
(both of which remain intact during their passage
through the gut) is measured in a fecal sample, which
correlates well with their secretion from the pancreas into
the duodenum.

Activity of elastase-1 is measured in a random fecal
sample by immunoassay. Its concentration is reduced in
pancreatic insufficiency. The test is sensitive and specific.
Although the test is simple and noninvasive, it is
expensive.
Activity of chymotrypsin is measured by spectro-
photometric assay of 4-nitroaniline in a fecal sample (A
synthetic pentapeptide is mixed with fecal extract that is
hydrolyzed by chymotrypsin in feces to release 4-
nitroaniline). Although the test is cheaper, it has low
sensitivity. It is commonly used as a screening test for
detection of pancreatic insufficiency in cystic fibrosis.
5. Estimation of fecal fat: Quantitation of fecal fat is a
definitive test for diagnosis of steatorrhea but not for
identification of its cause. The standard indirect test for
pancreatic insufficiency is estimation of 72-hour faecal
fat. (See Chapter 9: Examination of Feces).
Normal fat excretion of fat is <7% of total amount of
fat ingested. In pancreatic insufficiency, fat excretion is
often >20%.
Aside from pancreatic insufficiency, excess fecal fat
excretion also occurs in conditions such as biliary
obstruction, small bowel mucosal disease, lymphatic
obstruction, liver disease, abetalipoproteinemia, and
small bowel bacterial overgrowth. Apart from its
nonspecificity, the test is inconvenient and unpleasant.
It cannot differentiate between malabsorption and
deficient digestion, and the result is normal in mild
pancreatic insufficiency.
Pancreatic Enzymes Used as Markers of
Active Pancreatic Disease
Two enzymes, serum or urinary amylase and serum
lipase, are often measured to determine the presence of
pancreatic disease, especially acute pancreatitis.
1. Amylase: Serum amylase is mainly derived from
pancreas and salivary glands. Amylase is a small
molecule and is filtered by the glomeruli and excreted in
urine.
Fig. 11.9: Evaluation of raised serum amylase
Tests for Malabsorption and Pancreatic Function 135
Elevation of serum amylase occurs in:
• Pancreatic diseases: acute and chronic pancreatitis,
pseudocyst
• Parotitis
• Intestinal diseases: perforation, ischemia, obstruction.
• Biliary tract disease
• Ectopic pregnancy
• Malignant tumors of lung or ovary
• Macroamylasemia.
Binding of normal serum amylase with high mole-
cular weight plasma proteins (immunoglobulins) leads
to the formation of macroamylase. Because of large size,
they cannot be excreted in urine. Macroamylasemia is
not associated with any clinical features, but it must be
distinguished from other causes of elevated serum
amylase. In renal failure, and in macroamylasemia,
urinary amylase is low or absent.
2. Serum lipase: Serum lipase is elevated in pancreatic
diseases, intestinal diseases, acute cholecystitis, and renal
failure. Values are normal in parotitis and in macro-
amylasemia.
In acute pancreatitis, serum amylase begins to rise
within 3-6 hours, peaks at 24 hours, and returns to normal
levels by 2-3 days. Urinary amylase is also high in acute
pancreatitis and remains elevated for 7-10 days. Serum
lipase starts to increase within 3-6 hours, reaches
maximum at 24 hours, and remains elevated for 8-14
days.
Very high levels of both serum amylase and serum
lipase (i.e. >5 times the upper limit of normal) are
observed in acute pancreatitis; in other intra-abdominal
disorders, elevations are moderate or slight. It has been
recommended to measure both serum amylase and
serum lipase if diagnosis of acute pancreatitis is suspected
(Fig. 11.9).
136 Essentials of Clinical Pathology
REFERENCE RANGES
• Fecal fat (24 hour): < 7 g/24 hours
• Fecal fat globules: Average 2.5 per high power field
in random sample
• 14CO breath test: Peak radioactivity at 5-7 hours
2 •
• D-xylose absorption test: >1.2 g/5 hour (5 g dose);
> 4.0 g/5 hours (25.0 g dose)
• Lactulose breath hydrogen test: 30-40 minutes
• Lactose tolerance test: Blood glucose >30 mg/dl above
fasting value.
Urinary excretion of
PABA
• NBT-PABA (Bentiromide) test:——————————> 0.75
Urinary excretion of
free PABA
Urinary excretion of
fluorescein dilaurate
• Pancreolauryl test: ——————————————>0.30
Urinary excretion of
free dilaurate
• Fecal elastase: >190 μg/g feces
Fecal chymotrypsin: 120-1265 μg/g (Mean: 290 μg/g)
• Serum amylase: 35-115 U/L
• Serum lipase: 56-239 U/L
BIBLIOGRAPHY
1. Dimagno EP. A perspective on the use of tubeless
pancreatic function tests. Gut 1998;43:2-3.
2. Provan D and Krentz A. Oxford Handbook of Clinical
and Laboratory Investigation. Oxford University Press.
Oxford, 2002.
 12

Thyroid Function Tests

ANATOMY AND PHYSIOLOGY

The thyroid gland is a butterfly-shaped endocrine gland
composed of two lateral lobes connected by a thin band
of tissue called as isthmus. It is located in the neck in
front of the upper part of trachea (Fig. 12.1). Normal
weight is 15 to 25 grams.
The thyroid follicle is the functional unit of the thyroid
gland. It consists of a single layer of cuboidal epithelial
cells that rest on a basement membrane and enclose
amorphous material called as colloid (Fig. 12.2). The
follicles synthesize and secrete iodine-containing
hormones tri-iodothyronine (T3) and tetra-iodothyronine
or thyroxine (T4). The colloid consists mainly of
thyroglobulin (Tg) that stores the thyroid hormones
before secretion. Fig. 12.1: Anatomy of thyroid gland
The thyroid gland also consists of a second type of
cell called as parafollicular or C (clear) cells that are
scattered amongst the follicular cells. These cells have a
completely distinct embryological origin, and synthesize
and secrete the hormone calcitonin, which regulates
blood calcium levels along with parathyroid hormone.
The thyroid gland plays a major role in the meta-
bolism and growth and development of tissues.
Biosynthesis of thyroid hormones: Iodine is essential for
synthesis of thyroid hormones. Iodine is obtained from
the diet; dietary content of iodine is dependent on
availability of iodine in land and water in a particular
region. Various steps in the synthesis of thyroid
hormones are as follows (Fig. 12.3):
• Trapping of inorganic iodide circulating in blood by
follicular cells by means of an iodide pump (sodium
iodide symporter)
• Diffusion of iodide towards the apical surface of Fig. 12.2: Normal histology of thyroid showing follicles
follicular cell lined by follicular cells and containing colloid
138 Essentials of Clinical Pathology

Fig. 12.3: Synthesis of thyroid hormones

• Oxidation of iodide to iodine by thyroid peroxidase

• Iodination of tyrosine residues of thyroglobulin
(organification) to form monoiodotyrosine (MIT) and
diiodotyrosine (DIT). This process occurs at the
interface of follicular cell and colloid. The rate of
synthesis of DIT is twice that of MIT.
Fig. 12.4: Regulation of thyroid hormone production.
• Coupling of MIT and DIT to form T3 (MIT+DIT) +: Stimulation; –: Inhibition
and T4 (DIT + DIT).
• Endocytosis of colloid droplets by follicular cell and
proteolysis of Tg-hormone complex leads to the
formation of a cytoplasmic vacuole within the cell.
Fusion of lysosome with the vacuole is followed by
action of hydrolytic enzymes and release of the
hormone from the thyroglobulin. MIT and DIT are
deiodinated for recycling of iodine.
• T3 and T4 are released into the circulation. Traces
of thyroglobulin are also released.
Thyroid stimulating hormone (TSH) from anterior
lobe of the pituitary gland regulates the synthesis and
secretion of thyroid hormones by a feedback mechanism.
Thyrotropin releasing hormone (TRH) produced by
hypothalamus acts on pituitary to stimulate synthesis
and release of TSH. An increase in the level of thyroid
hormone inhibits the release of TRH from hypothalamus
and TSH from pituitary, while fall in the level of thyroid
hormone leads to an increase in the level of TRH and
TSH (Fig. 12.4).

Metabolism of Thyroid Hormones

Thyroid gland primarily secretes free or unbound T4 in Fig. 12.5: Metabolism of thyroid hormones.
circulation. Only a small amount of T3 is secreted directly rT3: Reverse T3

Fig. 12.6: In conditions with altered TBG concentration, total T4 is altered. Therefore measurement of free
T4 is more reliable as a test of thyroid function. TBG: Thyroid-binding globulin
by the thyroid gland (10%). Majority of T3 in circulation
(90%) is derived from removal of one iodothyronine unit
from T4 (Fig. 12.5). T4 may also be converted in liver to
reverse T3 (rT3) which is an inactive hormone.
In circulation, T4 and T3 are bound reversibly to
carrier proteins like thyroxine-binding globulin (TBG),
thyroxine-binding prealbumin (TBPA), and albumin.
Very small amounts of T3 (0.5%) and T4 (0.05%) are
unbound and free. Only the free forms of the hormone
(T3 and T4) are biologically active. T3 is more potent
than T4, although levels of T4 are 50-times more than
those of T3. Variations in the levels of carrier proteins
affect the levels of total T4 (Fig. 12.6) and T3, even in
normal individuals e.g. increased TBG concentration
occurs during pregnancy due to stimulation of its
synthesis in liver by high oestrogen levels. Total T4 and
T3 levels are dependent on thyroid function and level of
TBG, and therefore total levels will be abnormal if TBG
is abnormal Therefore, free levels of T4 and T3 are better
indicators of thyroid status than total levels.
DISORDERS OF THYROID
Among the endocrine disorders, disorders of thyroid
are common and are only next in frequency to diabetes
mellitus. They are more common in women than in men.
Functional thyroid disorders can be divided into two
types depending on activity of the thyroid gland:
hypothyroidism (low thyroid hormones), and hyper-
thyroidism (excess thyroid hormones). Any enlargement
of thyroid gland is called as a goiter. Terminology related
to thyroid disorders is shown in Box 12.1.
Hyperthyroidism
Hyperthyroidism is a condition caused by excessive
secretion of thyroid hormone. Causes of hyperthyroidism
are listed in Table 12.1.
Thyroid Function Tests 139
Box 12.1: Terminology in thyroid disorders
• Primary hyper-/hypothyroidism: Increased or decreased
function of thyroid gland due to disease of thyroid itself
and not due to increased or decreased levels of TRH
or TSH.
• Secondary hyper-/hypothyroidism: Increased or
decreased function of thyroid gland due to increased or
decreased levels of TSH.
• Tertiary hypothyroidism: Decreased function of thyroid
gland due to decreased function of hypothalamus.
• Subclinical thyroid disease: A condition with abnormality
of thyroid hormone levels in blood but without specific
clinical manifestations of thyroid disease and without any
history of thyroid dysfunction or therapy.
• Subclinical hyperthyroidism: A condition with normal
thyroid hormone levels but with low or undetectable TSH
level.
• Subclinical hypothyroidism: A condition with normal
thyroxine and triiodothyronine level along with mildly
elevated TSH level.
Table 12.1: Causes of hyperthyroidism
1. Graves’ disease (Diffuse toxic goiter)
2. Toxicity in multinodular goiter
3. Toxicity in adenoma
4. Subacute thyroiditis
5. TSH-secreting pituitary adenoma (secondary hyperthy-
roidism)
6. Trophoblastic tumours that secrete TSH-like hormone:
choriocarcinoma, hydatidiform mole
7. Factitious hyperthyroidism
Clinical Characteristics
Clinical characteristics of hyperthyroidism are nervous-
ness, anxiety, irritability, insomnia, fine tremors; weight
loss despite normal or increased appetite; heat intole-
rance; increased sweating; dyspnea on exertion;
amenorrhea and infertility; palpitations, tachycardia,
140 Essentials of Clinical Pathology

cardiac arrhythmias, heart failure (especially in elderly); Table 12.2: Differences between primary and
and muscle weakness, proximal myopathy, and secondary hyperthyroidism
osteoporosis (especially in elderly). Parameter Primary Secondary
The triad of Graves’ disease consists of hyper- hyperthyroidism hyperthyroidism
thyroidism, ophthalmopathy (exophthalmos, lid
1. Serum TSH Low Normal or high
retraction, lid lag, corneal ulceration, impaired eye
2. Serum free High High
muscle function), and dermopathy (pretibial myxo- thyroxine
edema). 3. TSH receptor May be Negative
antibodies positive
Laboratory Features 4. Causes Graves’ disease,
In most patients, free serum T3 and T4 are elevated. In toxic multinodular
goiter, toxic Pituitary
T3 thyrotoxicosis (5% cases of thyrotoxicosis), serum T4
adenoma adenoma
levels are normal while T3 is elevated. Serum TSH is low
or undetectable (< 0.1 mU/L) (Box 12.2).
Undetectable or low serum TSH along with normal Hypothyroidism
levels of T3 and T4 is called as subclinical hyper- Hypothyroidism is a condition caused by deficiency of
thyroidism; subtle signs and symptoms of thyrotoxicosis thyroid hormones. Causes of hypothyroidism are listed
may or may not be present. Subclinical hyperthyroidism in Table 12.3. Primary hypothyroidism results from
is associated with risk of atrial fibrillation, osteoporosis, deficient thyroid hormone biosynthesis that is not due
and progression to overt thyroid disease. to disorders of hypothalamus or pituitary. Secondary
Features of primary and secondary hyperthyroidism hypothyroidism results from deficient secretion of TSH
are compared in Table 12.2. from pituitary. Deficient or loss of secretion of thyro-
Evaluation of hyperthyroidism is presented in Figure
12.7.
Table 12.3: Causes of hypothyroidism

Box 12.2: Thyroid function tests in hyperthyroidism 1. Primary hypothyroidism (Increased TSH)
• Iodine deficiency
• Thyrotoxicosis: • Hashimoto’s thyroiditis
– Serum TSH low or undetectable • Exogenous goitrogens
– Raised total T4 and free T4. • Iatrogenic: surgery, drugs, radiation
• T3 toxicosis: 2. Secondary hypothyroidism (Low TSH): Diseases of
– Serum TSH undetectable pituitary
– Normal total T4 and free T4 3. Tertiary hypothyroidism (Low TSH, Low TRH):
– Raised T3 Diseases of hypothalamus

Fig. 12.7: Evaluation of hyperthyroidism. TSH: thyroid stimulating hormone; FT4: free T4; FT3: free T3; TRAb: TSH
receptor antibody; TRH: Thyrotropin releasing hormone
 Thyroid Function Tests 141

Table 12.4: Differences between primary and secondary hypothyroidism

Parameter Primary Secondary

hypothyroidism hypothyroidism

1. Cause Hashimoto’s thyroiditis Pituitary disease

2. Serum TSH High Low
3. Thyrotropin releasing hormone Exaggerated response No response
stimulation test
4. Antimicrosomal antibodies Present Absent

tropin releasing hormone from hypothalamus results in

tertiary hypothyroidism. Secondary and tertiary
hypothyroidism are much less common than primary.
Plasma TSH is high in primary and low in secondary
and tertiary hypothyroidism. Differences between
primary and secondary hypothyroidism are shown in
Table 12.4.
Clinical features of primary hypothyroidism are:
lethargy, mild depression, disturbances in menstruation,
weight gain, cold intolerance, dry skin, myopathy,
constipation, and firm and lobulated thyroid gland (in
Hashimoto’s thyroiditis).
In severe cases, myxoedema coma (an advanced stage
with stupor, hypoventilation, and hypothermia) can
occur.
Laboratory Features
Laboratory features in hypothyroidism are shown in Box
12.3.
Normal serum thyroxine (T4 and FT4) coupled with
a moderately raised TSH (>10 mU/L) is referred to as
subclinical hypothyroidism. It is associated with bad
Box 12.3: Thyroid function tests in hypothyroidism
• Primary hypothyroidism
– Serum TSH: Increased (proportional to degree of
hypofunction)
– Free T4: Decreased
– TRH stimulation test: Exaggerated response
• Secondary hypothyroidism
– Serum TSH: Decreased
– Free T4: Decreased
– TRH stimulation test: Absent response
• Tertiary hypothyroidism
– Serum TSH: Decreased
– FT4: Decreased
– TRH stimulation test: Delayed response
obstetrical outcome, poor cognitive development in
children, and high risk of hypercholesterolemia and
progression to overt hypothyroidism.
Evaluation of hypothyroidism is presented in Figure 12.8.

Fig. 12.8: Evaluation of hypothyroidism. TSH: thyroid stimulating hormone;

FT4: free T4; TRH: Thyrotropin releasing hormone
142 Essentials of Clinical Pathology
THYROID FUNCTION TESTS
Biochemical tests for diagnosis of a thyroid disorder are
called as thyroid function tests. The first-line tests are
serum TSH, total T4 or free T4, and total T3 and free T3.
A variety of non-thyroidal diseases can alter the
results of thyroid function tests in patients with normal
thyroid status. These disorders include infections, liver
disease, malignancy, trauma, surgery, renal failure, and
cardiac failure. To avoid misinterpretation, thyroid
function tests should not be performed during an acute
non-thyroidal illness.
Thyroid Stimulating Hormone (TSH)
Currently, the most important single test to assess
thyroid function and to monitor thyroid hormone
replacement therapy is a sensitive TSH assay. This is
because most cases of thyroid dysfunction result from
primary thyroid disease. TSH is a hormone secreted by
anterior pituitary gland. A normal TSH level excludes
thyroid disease. TSH is low in primary hyperthyroidism
and high in primary hypothyroidism. The standard
method for measurement of TSH is immunoassay. Newer
sensitive methods can reliably distinguish between
extremely low or undetectable levels seen in hyper-
thyroidism and low normal or below-normal levels seen
in some euthyroid patients.
Normal reference range in adults is 0.5 –5.0 mU/L
and in newborns < 20 mU/L. In adults, borderline
increase is 5-10 mU/L, while values >10 mU/L are
considered as high. Values less than 0.1 mU/L are low.
Third and fourth generations TSH assays have detection
limits of 0.01 to 0.02 mU/L and 0.001 to 0.002 mU/L,
respectively.
TSH levels are affected by non-thyroidal illness and
certain drugs; therefore for confirmation of thyroid
dysfunction, estimation of free T4 and total T4 should
also be done. With the availability of sensitive TSH
assays, TRH stimulation test is usually not required (see
later).
Causes of low and increased TSH are shown in Table
12.5.
Uses of TSH Measurement
1. Screening for euthyroidism: The availability of
sensitive TSH assay has made the TSH measurement
the first-line test for assessment of thyroid function.
In most patients a normal TSH test indicates normal
function, and no further testing is required
Table 12.5: Causes of low and increased TSH
Low TSH Increased TSH
Primary hyperthyroidism Primary hypothyroidism
T3 toxicosis Secondary hyperthyroidism
(pituitary adenoma secreting
TSH)
Secondary and tertiary
hypothyroidism
2. Screening for hypothyroidism in newborn
3. Diagnosis of primary and secondary hypothyroidism,
and subclinical hypothyroidism
4. Diagnosis of clinical and subclinical hyperthyroidism
5. Follow-up of T3 and T4 replacement therapy in
hypothyroidism.
The best single test for initial assessment of thyroid
function is a sensitive (3rd or 4th generations) TSH
assay, which is sufficiently sensitive and specific for
early detection of primary hyper- and hypothyroidism.
• In primary hyperfunctioning of thyroid, TSH is low.
• In primary hypofunction of thyroid, TSH is high.
• Serum free T4 should be measured in all patients
with elevated TSH.
• Serum free T4 and total T3 or free T3 should be
measured in all patients with low TSH.
TOTAL THYROXINE (T4)
Total serum thyroxine includes both free and protein-
bound thyroxine and is usually measured by competitive
immunoassay. Normal level in adults is 5.0-12.0 μg/dl.
Test for total thyroxine or free thyroxine is usually
combined with TSH measurement and together they
give the best assessment of thyroid function.
Causes of Increased Total T4
1. Hyperthyroidism: Elevation of both T4 and T3 values
along with decrease of TSH are indicative of primary
hyperthyroidism.
2. Increased thyroxine-binding globulin: If concentration
of TBG increases, free hormone level falls, release of
TSH from pituitary is stimulated, and free hormone
concentration is restored to normal. Reverse occurs
if concentration of binding proteins falls. In either
case, level of free hormones remains normal, while

concentration of total hormone is altered. Therefore,
estimation of only total T4 concentration can cause
misinterpretation of results in situations that alter
concentration of TBG.
3. Factitious hyperthyroidism
4. Pituitary TSH-secreting tumor.
Causes of Decreased Total T4
1. Primary hypothyroidism: The combination of
decreased T4 and elevated TSH are indicative of
primary hypothyroidism.
2. Secondary or pituitary hypothyroidism
3. Tertiary or hypothalamic hypothyroidism
4. Hypoproteinaemia, e.g. nephrotic syndrome
5. Drugs: oestrogen, danazol
6. Severe non-thyroidal illness.
Free Thyroxine (FT4)
FT4 comprises of only a small fraction of total T4, is
unbound to proteins, and is the metabolically active form
of the hormone. It constitutes about 0.05% of total T4.
Normal range is 0.7 to 1.9 ng/dl. Free hormone
concentrations (FT4 and FT3) correlate better with
metabolic state than total hormone levels (since they are
not affected by changes in TBG concentrations). .
Measurement of FT4 is helpful in those situations in
which total T4 level is likely to be altered due to alteration
in TBG level (e.g. pregnancy, oral contraceptives,
nephrotic syndrome).
Total and Free Triiodothyronine (T3)
Uses
1. Diagnosis of T3 thyrotoxicosis: Hyperthyroidism with
low TSH and elevated T3, and normal T4/FT4 is
termed T3 thyrotoxicosis.
2. Early diagnosis of hyperthyroidism: In early stage of
hyperthyroidism, total T4 and free T4 levels are
normal, but T3 is elevated.
A low T3 level is not useful for diagnosis of
hypothyroidism since it is observed in about 25% of
normal individuals.
For routine assessment of thyroid function, TSH and
T4 are measured. T3 is not routinely estimated because
normal plasma levels are very low.
Normal T3 level is 80-180 ng/dl.
Thyroid Function Tests 143
Free T3: Measurement of free T3 gives true values in
patients with altered serum protein levels (like preg-
nancy, intake of estrogens or oral contraceptives, and
nephrotic syndrome). It represents 0.5% of total T3.
Thyrotropin Releasing Hormone (TRH)
Stimulation Test
Uses
1. Confirmation of diagnosis of secondary hypo-
thyroidism
2. Evaluation of suspected hypothalamic disease
3. Suspected hyperthyroidism
This test is not much used nowadays due to the
availability of sensitive TSH assays.
Procedure
• A baseline blood sample is collected for estimation
of basal serum TSH level.
• TRH is injected intravenously (200 or 500 μg) followed
by measurement of serum TSH at 20 and 60 minutes.
Interpretation
1. Normal response: A rise of TSH > 2 mU/L at 20
minutes, and a small decline at 60 minutes.
2. Exaggerated response: A further significant rise in
already elevated TSH level at 20 minutes followed
by a slight decrease at 60 minutes; occurs in primary
hypothyroidism.
3. Flat response: There is no response; occurs in
secondary (pituitary) hypothyroidism.
4. Delayed response: TSH is higher at 60 minutes as
compared to its level at 20 minutes; seen in tertiary
(hypothalamic) hypothyroidism.
Antithyroid Antibodies
Various autoantibodies (TSH receptor, antimicrosomal,
and antithyroglobulin) are detected in thyroid disorders
like Hashimoto’s thyroiditis and Graves’ disease.
Antimicrosomal (also called as thyroid peroxidase) and
anti-thyroglobulin antibodies are observed in almost all
patients with Hashimoto’s disease. TSH receptor
antibodies (TRAb) are mainly tested in Graves’ disease
to predict the outcome after treatment (Box 12.4).
144 Essentials of Clinical Pathology

– Toxic multinodular goiter: Multiple discrete areas

Box 12.4: Thyroid autoantibodies
of increased uptake
• Useful for diagnosis and monitoring of autoimmune thyroid – Adenoma: Single area of increased uptake
diseases. • Evaluation of a solitary thyroid nodule:
• Antimicrosomal or antithyroid peroxidase antibodies: – ‘Hot’ nodule: Hyperfunctioning
Hashimoto’s thyroiditis – ‘Cold’ nodule: Non-functioning; about 20% cases
• Anti-TSH receptor antibodies: Graves’ disease are malignant.
Interpretation of thyroid function tests is shown in
Table 12.6.
Radioactive Iodine Uptake (RAIU) Test
This is a direct test that assesses the trapping of iodide Table 12.6: Interpretation of thyroid function tests
by thyroid gland (through the iodine symporters or Test results Interpretations
pumps in follicular cells) for thyroid hormone synthesis.
Patient is administered a tracer dose of radioactive iodine 1. TSH Normal, FT4 Normal Euthyroid
2. Low TSH, Low FT4 Secondary hypothyroidism
(131I or 123I) orally. This is followed by measurement of
3. High TSH, Normal FT4 Subclinical hypothyroidism
amount of radioactivity over the thyroid gland at 2 to 6
4. High TSH, Low FT4 Primary hypothyroidism
hours and again at 24 hours. RAIU correlates directly
5. Low TSH, Normal FT4, Subclinical
with the functional activity of the thyroid gland. Normal Normal FT3 hyperthyroidism
RAIU is about 10-30% of administered dose at 24 hours, 6. Low TSH, Normal FT4, T3 toxicosis
but varies according to the geographic location due to High FT3
differences in dietary intake. 7. Low TSH, High FT4 Primary hyperthyroidism

Causes of Increased Uptake Neonatal Screening for Hypothyroidism

• Hyperthyroidism due to Graves’ disease, toxic
multinodular goiter, toxic adenoma, TSH-secreting
tumor.
Causes of Decreased Uptake
• Hyperthyroidism due to administration of thyroid
hormone, factitious hyperthyroidism, subacute
thyroiditis.
Thyroid hormone deficiency during neonatal period can
cause severe mental retardation (cretinism) that can be
prevented by early detection and treatment. Estimation
of TSH is done on dry blood spots on filter paper or cord
serum between 3rd to 5th days of life. Elevated TSH is
diagnostic of hypothyroidism. In infants with confirmed
hypothyroidism, RAIU (123I) scan should be done to
distinguish between thyroid agenesis and dyshormono-
genesis.

Uses REFERENCE RANGES

RAIU is most helpful in differential diagnosis of
hyperthyroidism by separating causes into those due to
increased uptake and due to decreased uptake.
Thyroid Scintiscanning
An isotope (99mTc-pertechnetate) is administered and a
gamma counter assesses its distribution within the
thyroid gland.
Interpretation
• Differential diagnosis of high RAIU thyrotoxicosis:
– Graves’ disease: Uniform or diffuse increase in
uptake
1. Total serum thyroxine (T4): 5.0-12.0 μg/dl
2. Free thyroxine (FT4): 0.7-1.9 ng/dl
3. Free thyroxine index (FTI): 4-11
4. Total tri-iodothyronine (T3): 80-180 ng/dl
5. Free tri-iodothyronine (FT3): 2.3-4.2 pg/ml
5. Radioactive iodine uptake: 10-30%
6. Thyroid stimulating hormone: 0.5-5.0 mU/L
7. Serum reverse T3 (rT3): 10-40 ng/dl
BIBLIOGRAPHY
1. Demers LM. Thyroid disease: pathophysiology and
diagnosis. Clin Lab Med 2004;24:19-28.
 Thyroid Function Tests 145

2. Heuck CC, Kallner A, Kanagasabapathy AS, Riesen W.
Diagnosis and monitoring of diseases of thyroid. World
Health Organization. 2000 WHO/DIL/0.004.
3. Kaplan MM. Clinical perspectives in the diagnosis of
thyroid disease. Clin Chem 1999;45:1377-83.
4. Lazarus JH, Obuobie K. Thyroid disorders—an update.
Postgrad Med J 2000;76:529-36.
5. McDermott MT. Endocrine Secrets (4th Ed).
Philadelphia. Mosby, 2005.
6. US Preventive Services Task Force: Screening for
thyroid disease: Recommended statement. Ann Intern
Med 2004;140:125-7.
7. Woeber KA. The year in review: the thyroid. Ann Intern
Med 1999;131:959-62.
 13

Pregnancy Tests

Pregnancy tests detect human chorionic gonadotropin CLINICAL APPLICATIONS OF TESTS FOR
(hCG) in serum or urine. Although pregnancy is the most HUMAN CHORIONIC GONADOTROPIN
common reason for ordering the test for hCG, measure-
1. Early diagnosis of pregnancy: Qualitative serum hCG
ment of hCG is also indicated in other conditions as
test becomes positive 3 weeks after last menstrual
shown in Box 13.1.
period (LMP), while urine hCG test becomes positive
Human chorionic gonadotropin is a glycoprotein
5 weeks after LMP.
hormone produced by placenta that circulates in
2. Exclusion of pregnancy before prescribing certain
maternal blood and excreted intact by the kidneys. It
medications (like oral contraceptives, steroids, some
consists of two polypeptide subunits: α (92 amino acids)
antibiotics), and before ordering radiological studies,
and β (145 amino acids) which are non-covalently bound
radiotherapy, or chemotherapy. This is necessary to
to each other. Structurally, hCG is closely related to three
prevent any teratogenic effect on the fetus.
other glycoprotein hormones, namely, luteinizing
3. Early diagnosis of ectopic pregnancy: Trans-vaginal
hormone (LH), follicle-stimulating hormone (FSH), and
ultrasonography (USG) and quantitative estimation
thyroid-stimulating hormone (TSH). The α subunits of
of hCG are helpful in early diagnosis of ectopic
hCG, LH, FSH, and TSH are similar, while β subunits
pregnancy (before rupture).
differ and confer specific biologic and immunologic
4. Evaluation of threatened abortion: Serial quantitative
properties. Immunological tests use antibodies directed
estimation of hCG is helpful in following the course
against β-subunit of hCG to avoid cross-reactivity against
of threatened abortion.
LH, FSH, and TSH.
5. Diagnosis and follow-up of gestational trophoblastic
Syncytiotrophoblastic cells of conceptus and later of
disease (GTD).
placenta synthesize hCG. Human chorionic gonado-
6. Maternal triple test screen: This consists of measure-
tropin supports the corpus luteum of ovary during early
ment of hCG, α-fetoprotein, and unconjugated estriol
pregnancy. Progesterone, produced by corpus luteum,
in maternal serum at 14-19 weeks of gestation. The
prevents ovulation and thus maintains pregnancy. After
maternal triple screen identifies pregnant women
7-10 weeks of gestation, sufficient amounts of proges-
with increased risk of Down syndrome and major
terone are synthesized by placenta, and hCG is no longer
congenital anomalies like neural tube defects.
needed and its level declines.
7. Follow-up of ovarian or testicular germ cell tumors,
which produce hCG.
Box 13.1: Indications for measurement of β human
chorionic gonadotropin Normal Pregnancy
• Early diagnosis of pregnancy In women with normal menstrual cycle, conception
• Diagnosis and management of gestational trophoblastic (fertilization of ovum to form a zygote) occurs on day 14
disease in the fallopian tube. Zygote travels down the fallopian
• As a part of maternal triple test screen
tube into the uterus. Division of zygote produces a
• Follow-up of malignant tumors that produce β human
chorionic gonadotropin. morula. At 50-60-cell stage, morula develops a primitive
yolk sac and is then called as a blastocyst. About 5 days

after fertilization, implantation of blastocyst occurs in the
uterine wall. Trophoblastic cells (on the outer surface of
the blastocyst) penetrate the endometrium and develop
into chorionic villi. There are two main forms of
trophoblasts—syncytiotrophoblast and cytotrophoblast.
Placental development occurs from chorionic villi. After
formation of placenta, the conceptus is called as an
embryo. When embryo develops most major organs, it
is called as fetus (after 10 weeks of gestation).
Human chorionic gonadotropin is synthesized by
syncytiotrophoblasts (of placenta) and detectable
amounts (~5 mIU/ml) appear in maternal serum about
8 days after conception (3 weeks after LMP). In the first
trimester (first 12 weeks, calculated from day 1 of LMP)
of pregnancy, hCG levels rapidly rise with a doubling
time of about 2 days. Highest or peak level is reached at
8-10 weeks (about 100,000 mIU/ml). This is followed by
a gradual fall, and from 15-16 weeks onwards, a steady
level of 10,000-20,000 mIU/ml is maintained for the rest
of the pregnancy (Fig. 13.1). After delivery, hCG becomes
non-detectable by about 2 weeks.
Box 13.2 shows minimum time required for the
earliest diagnosis of pregnancy by hCG test and
ultrasonography (USG).
Two types of pregnancy tests are available:
• Qualitative tests: These are positive/negative result
types that are done on urine sample.
• Quantitative tests: These give numerical result and
are done on serum or urine. They are also used for
Fig. 13.1: Level of human chorionic gonadotropin
during pregnancy
Pregnancy Tests 147
Box 13.2: Diagnosis of early pregnancy
• Positive serum hCG test: 8 days after conception or 3
weeks after last menstrual period (LMP)
• Positive urine hCG test: 21 days after conception or 5
weeks after LMP
• Ultrasonography for visualization of gestational sac:
– Transvaginal: 21 days after conception or 5 weeks
after LMP
– Transabdominal: 28 days after conception or 6 weeks
after LMP
evaluation of ectopic pregnancy, failing pregnancy,
and for follow-up of gestational trophoblastic disease.
Ectopic Pregnancy
Ectopic pregnancy refers to the implantation of blastocyst
at a site other than the cavity of uterus. The most common
of such sites (>95% cases) is fallopian tube. Early diagnosis
and treatment of tubal ectopic pregnancy is essential since
it can lead to maternal mortality (from rupture and
hemorrhage) and future infertility. Ectopic pregnancy is
a leading cause of maternal death during first trimester.
Diagnosis of ectopic pregnancy can be readily made in
most cases by ultrasonography and estimation of β-
subunit of human chorionic gonadotropin.
Early diagnosis of unruptured tubal pregnancy can
be made by quantitative estimation of serum hCG and
ultrasonography. In normal intrauterine pregnancy, hCG
titer doubles every 2 days until first 40 days of gestation.
If hCG rise is abnormally slow, then an unviable
pregnancy (either ectopic or abnormal intrauterine
pregnancy) should be suspected.
Transabdominal USG can detect gestational sac in
intrauterine pregnancy 6 weeks after LMP. The level of
hCG in serum at this stage is >6500 mIU/ml. If
gestational sac is not visualized at this level of hCG, then
there is a possibility of ectopic pregnancy. Transvaginal
ultrasonography can detect ectopic pregnancy average
1 week earlier than abdominal ultrasonography; it can
detect gestational sac if β-hCG level is 1000-1500 mIU/
ml. Therefore, if gestational sac is not visualized in the
presence of >1500 mIU/ml of β-hCG level, an ectopic
pregnancy can be suspected.
Early diagnosis of ectopic pregnancy provides the
option of administration of intramuscular methotrexate
(rather than surgery), which causes dissolution of
conceptus. This improves the chances of patient’s future
fertility.
148 Essentials of Clinical Pathology
Serial measurements of hCG after surgical removal
of ectopic pregnancy can help in detecting persistence
of trophoblastic tissue.
Abortion
Termination of pregnancy before fetus becomes viable
(i.e. before 20 weeks) is called as abortion.
In threatened abortion, vaginal bleeding is present
but internal os is closed and process of abortion, though
started, is still reversible. It is possible that pregnancy
will continue.
Serial quantitative titers of hCG showing lack of
expected doubling of hCG level and USG are helpful in
diagnosis and management of abortion.
Gestational Trophoblastic Disease (GTD)
It is characterized by proliferation of pregnancy-
associated trophoblastic tissue. The two main forms of
GTD are hydatidiform (vesicular) mole (benign) and
choriocarcinoma (malignant). Clinical features of GTD
are as follows:
• Short history of amenorrhea followed by vaginal
bleeding.
• Size of uterus larger than gestational age; uterus is
soft and doughy on palpation with no fetal parts and
no fetal heart sounds.
• Excessive nausea and vomiting due to high hCG.
• Characteristic snowstorm appearance on pelvic USG.
Quantitative estimation of hCG is helpful in diagnosis
and management of GTD.
Trophoblastic cells of GTD produce more hCG as
compared to the trophoblasts of normal pregnancy for
the same gestational age. Concentration of hCG parallels
tumor load. Also, hCG continues to rise beyond 10 weeks
of gestation without reaching plateau (as expected at the
end of first trimester).
After evacuation of uterus, weekly estimation of hCG
is advised till subsequent three (weekly) results are
negative; following evacuation of vesicular mole, hCG
becomes undetectable (after 2-3 months) on follow-up
in 80% of cases. Plateau or rising hCG indicates persistent
GTD. In such cases, chemotherapy is indicated.
Negative results for hCG after therapy should be
regularly followed up every 3 months for 1-2 years.
LABORATORY TESTS FOR
HUMAN CHORIONIC GONADOTROPIN
These are classified into two main groups:
• Biological assays or bioassays
• Immunological assays
Bioassays
In bioassay, effect of hCG is tested on laboratory animals
under standardized conditions. There are several
limitations of bioassays like need for animal facilities,
need for standardization of animals, long time required
for the test results, low sensitivity, and high cost.
Therefore, bioassays have been replaced by immuno-
logical assays.
In Ascheim-Zondek test, urine from pregnant woman
is injected into immature female mice. Formation of
hemorrhagic corpora lutea in ovaries (after 4 days) is a
positive test. Friedman test is similar except that urine is
injected into female rabbit. In rapid rat test, injection of
urine containing hCG into female rats is followed by
hyperaemia and hemorrhage in ovaries. Yet another test
measures release of spermatozoa from male frog after
injection of urine containing hCG.
Immunological Assays
These are rapid and sensitive tests for detection and
quantitation of hCG. Variable results are obtained by
different immunological tests with the same serum
sample; this is due to differences in specificity of different
immunoassays to complete hCG, β-subunit, and β-core
fragment. A number of immunological tests are
commercially available based on different principles like
agglutination inhibition assay, enzyme immunoassay
including enzyme linked immunosorbent assay or
ELISA, radioimmunoassay (RIA), and immunoradio-
metric assay.
A commonly used qualitative urine test is aggluti-
nation inhibition assay. Early morning urine specimen
is preferred because it contains the highest concentration
of hCG. Causes of false-positive test include red cells,
leukocytes, bacteria, some drugs, proteins, and excess
luteinizing hormone (menopause, midcycle LH surge)
in urine. Some patients have anti-mouse antibodies (that
are used in the test), while others have hCG-like material
in circulation, producing false-positive test. Anti-mouse
antibodies also interfere with other antibody-based tests

Fig. 13.2: Principle of agglutination inhibition test for
diagnosis of pregnancy
and are known as ‘heterophil’ antibodies. Fetal death,
abortion, dilute urine, and low sensitivity of a particular
test are causes of false-negative test. Renal failure leads
to accumulation of interfering substances causing
incorrect results.
In latex particle agglutination inhibition test
(Fig. 13.2), anti-hCG antibodies are incubated with
patient’s urine. This is followed by addition of hCG-
coated latex particles. If hCG is present in urine, anti-
Pregnancy Tests 149
hCG serum is neutralized, and no agglutination of latex
particles occurs (positive test). If there is no hCG in urine,
there is agglutination of latex particles (negative test).
This is commonly used as a slide test and requires only a
few minutes.
Sensitivity of agglutination inhibition test is >200
units/liter of hCG.
Radioimmunoassay, enzyme immunoassay, and
radioimmunometric assay are more sensitive and reliable
than agglutination inhibition assay.
Quantitative tests are employed for detection of very
early pregnancy, estimation of gestational age, diagnosis
of ectopic pregnancy, evaluation of threatened abortion,
and management of GTD.
REFERENCE RANGES
• Serum human chorionic gonadotropin:
– Non-pregnant females: <5.0 mIU/ml
– Pregnancy: 4 weeks after LMP: 5-100 mIU/ml
– 5 weeks after LMP: 200-3000 mIU/ml
– 6 weeks after LMP: 10,000-80,000 mIU/ml
– 7-14 weeks: 90,000-500,000 mIU/ml
– 15-26 weeks: 5000-80000 mIU/ml
– 27-40 weks: 3000-15000 mIU/ml
BIBLIOGRAPHY
1. Burtis CA, Ashwood ER. Tietz fundamentals of clinical
chemistry (5th Ed). Philadelphia: WB Saunders
Company, 2001.
2. Davies S, Byrn F, Cole LA. Human chorionic gonado-
tropin testing for early pregnancy viability and
complications. Clin Lab Med 2003;23:257-64.
3. Tenore JL. Ectopic pregnancy. Am Fam Physician
2000;61:1080-8.
 14
Infertility
Infertility refers to failure of a couple to achieve concep-
tion after one year of unprotected regular sexual
intercourse.
In primary infertility, there is a failure of the couple
to conceive. In secondary infertility, there is a failure to
achieve conception after a previous pregnancy.
About 80% of couples achieve conception within their
first year of attempt, another 10% will achieve within 2
years, while remaining 10% fail to conceive after 2 years.
With time and therapeutic intervention, many subfertile
couples can achieve a pregnancy and deliver a child.
About 40% couples have primary, and 60% of couples
have secondary infertility.
Source of infertility can be solely in female (30%),
solely in male (30%), or in both partners (30%); the cause
remains unknown in the remaining 10% cases even after
extensive investigations.
Fig. 14.1: Hypothalamus-pituitary-testis axis. + indicates stimulation; – indicates negative feedback
MALE INFERTILITY
The male reproductive system consists of testes (paired
organs located in the scrotal sac that produce sperma-
tozoa and secrete testosterone), a paired system of ducts
comprising of epididymis, vasa deferentia, and ejacu-
latory ducts (collect, store, and conduct spermatozoa),
paired seminal vesicles and a single prostate gland
(produce nutritive and lubricating seminal fluid),
bulbourethral glands of Cowper (secrete lubricating
mucus), and penis (organ of copulation).
The hypothalamus secretes gonadotropin releasing
hormone (GnRH) that regulates the secretion of the two
gonadotropins from the anterior pituitary: luteinizing
hormone (LH) and follicle stimulating hormone (FSH)
(Fig. 14.1). Luteinizing hormone primarily stimulates the
production and secretion of testosterone from Leydig
 Infertility 151

cells located in the interstitial tissue of the testes.

Testosterone stimulates spermatogenesis, and plays a
role in the development of secondary sexual characters.
Testosterone needs to be converted to an important
steroidal metabolite, dihydrotestosterone within cells to
perform most of its androgenic functions. Testosterone
inhibits LH secretion by negative feedback. Follicle
stimulating hormone acts on Sertoli cells of seminiferous
tubules to regulate the normal maturation of the sperms.
Sertoli cells produce inhibin that controls FSH secretion
by negative feedback.
During sexual intercourse, semen is deposited into
the vagina. Liquefaction of semen occurs within 20-30
minutes due to proteolytic enzymes of prostatic fluid.
For fertilization to occur in vivo, the sperm must undergo
capacitation and acrosome reaction. Capacitation refers
to physiologic changes in sperms that occur during their
passage through the cervix of the female genital tract.
With capacitation, the sperm acquires (i) ability to
undergo acrosome reaction, (ii) ability to bind to zona
pellucida, and (iii) hypermotility. Sperm then travels
through the cervix and uterus up to the fallopian tube.
Binding of sperm to zona pellucida induces acrosomal
reaction (breakdown of outer plasma membrane by
enzymes of acrosome and its fusion with outer acrosomal
membrane, i.e. loss of acrosome). This is necessary for Fig. 14.2: Steps before and after fertilization of ovum
fusion of sperm and oocyte membranes. Acrosomal
reaction and binding of sperm and ovum surface proteins egg in the ampullary portion of the fallopian tube
is followed by penetration of zona pellucida of ovum by (Fig. 14.2).
the sperm. Following penetration by sperm, hardening
of zona pellucida occurs that inhibits penetration by Causes of Male Infertility
additional sperms. A sperm penetrates and fertilizes the Causes of male infertility are listed in Table 14.1.
Table 14.1: Causes of male infertility
1. Idiopathic
2. Hypothalamic-pituitary dysfunction (hypogonadotropic hypogonadism)
3. Testicular dysfunction:
• Radiation, cytotoxic drugs, antihypertensives, antidepressants
• General factors like stress, emotional factors, drugs like marijuana, anabolic steroids, and cocaine, alcoholism, heavy
smoking, undernutrition
• Mumps orchitis after puberty
• Varicocele (dilatation of pampiniform plexus of scrotal veins)
• Undescended testes (cryptorchidism)
• Endocrine disorders like diabetes mellitus, thyroid dysfunction
• Genetic disorders: Klinefelter’s syndrome, microdeletions in Y chromosome, autosomal Robertsonian translocation,
immotile cilia syndrome (Kartagener’s syndrome), cystic fibrosis, androgen receptor gene defect
4. Dysfunction of passages and accessory sex glands:
• Infections of epididymis: tuberculosis, gonorrhea, Chlamydia
• Congenital bilateral absence of vasa deferentia (cystic fibrosis), vasectomy
• Prostatitis
5. Dysfunction of sexual act:
• Impotence, erectile dysfunction
• Defects in ejaculation: retrograde (semen is pumped backwards in to the bladder), premature, or absent
• Hypospadias
152 Essentials of Clinical Pathology

Investigations of Male Infertility 4. Chromosomal analysis: This can reveal Klinefelter’s

syndrome (e.g. XXY karyotype) (Fig. 14.5), deletion
1. History: This includes type of lifestyle (heavy
in Y chromosome, and autosomal Robertsonian
smoking, alcoholism), sexual practice, erectile
translocation. It is necessary to screen for cystic
dysfunction, ejaculation, sexually transmitted
fibrosis carrier state if bilateral congenital absence of
diseases, surgery in genital area, drugs, and any vas deferens is present.
systemic illness. 5. Hormonal studies: This includes measurement of
2. Physical examination: Examination of reproductive FSH, LH, and testosterone to detect hormonal
system should includes testicular size, undescended abnormalities causing testicular failure (Table 14.2).
testes, hypospadias, scrotal abnormalities (like 6. Testicular biopsy: Testicular biopsy is indicated
varicocele), body hair, and facial hair. Varicocele can when differentiation between obstructive and non-
occur bilaterally and is the most common surgically
removable abnormality causing male infertility.
3. Semen analysis: See Chapter 15: Semen Analysis.
Evaluation of azoospermia is shown in Figure 14.3.
Evaluation of low semen volume is shown in Figure 14.4.

If all the values of semen analysis are normal

(according to WHO criteria), further testing is not
required. Further andrological tests are indicated only
if at least two semen examinations (done 1 month apart)
are abnormal. In patients with abnormal semen
analysis, the further basic laboratory tests done are
estimation of serum luteinizing hormone, follicle
stimulating hormone and testosterone.
Fig. 14.4: Evaluation of low semen volume

Fig. 14.3: Evaluation of azoospermia. FSH: Follicle stimulating hormone;

LH: Luteinizing hormone
 Infertility 153

Fig. 14.5: Karyotype in Klinefelter’s syndrome (47, XXY)

Table 14.2: Interpretation of hormonal studies in male infertility

Follicle Luteinizing Testosterone Interpretation

stimulating hormone
hormone

Low Low Low Hypogonadotropic hypogonadism

(Hypothalamic or pituitary disorder)

High High Low Hypergonadotropic hypogonadism

(Testicular disorder)

Normal Normal Normal Obstruction of passages,

dysfunction of accessory glands
154 Essentials of Clinical Pathology

Box 14.1: Common initial investigations for

diagnosis of infertility

Males
• Semen analysis
• Blood glucose
• Endocrine tests: Serum FSH, LH, testosterone

Females
• Hemogram, X-ray chest, Mantoux test, erythrocyte
sedimentation rate
• Thyroid hormones, serum prolactin
• Endometrial biopsy
• Laparoscopy with hysteroscopy and/or hysterosalpin-
gography
• Transvaginal ultrsonography
Fig. 14.6: The hypothalamus-pituitary-ovarian axis

obstructive azoospermia is not evident (i.e. normal
FSH and normal testicular volume).
Common initial investigations for diagnosis of cause
of infertility are shown in Box 14.1.
FEMALE INFERTILITY
The ovaries are the sites of production of female gametes
or ova by the process of oogenesis. The ova are released
by the process of ovulation in a cyclical manner at regular
intervals. Ovary contains numerous follicles that contain
ova in various stages of development. During each
menstrual cycle, up to 20 primordial follicles are activated
for maturation; however, only one follicle becomes fully
mature; this dominant follicle ruptures to release the
secondary oocyte from the ovary. Maturation of the
follicle is stimulated by follicle stimulating hormone
(FSH) secreted by anterior pituitary (Fig. 14.6). Maturing
follicle secretes estrogen that causes proliferation of
endometrium of the uterus (proliferative phase).
Follicular cells also secrete inhibin which regulates
release of FSH by the anterior pituitary. Fall in FSH level
is followed by secretion of luteinizing hormone (LH) by
the anterior pituitary (LH surge). This causes follicle to
rupture and the ovum is expelled into the peritoneal
cavity near the fimbrial end of the fallopian tube. The
fallopian tubes conduct ova from the ovaries to the
uterus. Fertilization of ovum by the sperm occurs in the
fallopian tube.
The ovum consists of the secondary oocyte, zona
pellucida and corona radiata. The ruptured follicle in the
ovary collapses and fills with blood clot (corpus luteum).
LH converts granulose cells in the follicle to lutein cells
which begin to secrete progesterone. Progesterone
stimulates secretion from the endometrial glands
(secretory phase) that were earlier under the influence
of estrogen. Rising progesterone levels inhibit LH
production from the anterior pituitary. Without LH, the
corpus luteum regresses and becomes functionless
corpus albicans. After regression of corpus luteum,
production of estrogen and progesterone stops and
endometrium collapses, causing onset of menstruation.
If the ovum is fertilized and implanted in the uterine
wall, human chorionic gonadotropin (hCG) is secreted
by the developing placenta into the maternal circulation.
Human chorionic gonadotropin maintains the corpus
luteum for secetion of estrogen and progesterone till 12th
week of pregnancy. After 12th week, corpus luteum
regresses to corpus albicans and the function of synthesis
of estrogen and progesterone is taken over by placenta
till parturition.
The average duration of the normal menstrual cycle
is 28 days. Ovulation occurs around 14th day of the cycle.
The time interval between ovulation and menstruation
is called as luteal phase and is fairly constant (14 days)
(Fig. 14.7).
Causes of Female Infertility
Causes of female infertility are shown in Table 14.3.
 Infertility 155

Fig. 14.7: Normal menstrual cycle

Table 14.3: Causes of female infertility

1. Hypothalamic-pituitary dysfunction: 3. Dysfunction in passages

• Hypothalamic causes • Fallopian tubes
– Excessive exercise – Infections: Tuberculosis, gonorrhea, Chlamydia
– Excess stress – Previous surgery (e.g. laparotomy)
– Low weight – Tubectomy
– Kallman’s syndrome – Congenital hypoplasia, non-canalization
– Idiopathic – Endometriosis
• Pituitary causes • Uterus
– Hyperprolactinemia – Uterine malformations
– Hypopituitarism (Sheehan’s syndrome, – Asherman’s syndrome
Simmond’s disease) – Tuberculous endometritis
– Craniopharyngioma – Fibroid
– Cerebral irradiation • Cervix: Sperm antibodies
2. Ovarian dysfunction • Vagina: Septum
• Polycystic ovarian disease 4. Dysfunction of sexual act: Dyspareunia
(Stein-Leventhal syndrome)
• Luteinized unruptured follicle
• Turner’s syndrome
• Radiation or chemotherapy
• Surgical removal of ovaries
• Idiopathic

Investigations Anovulatory cycles are clinically characterized by

Evaluation of female infertility is shown in Figure 14.8. amenorrhea, oligomenorrhea, or irregular menstrua-
tion. However, apparently regular cycles may be
associated with anovulation.
Tests for Ovulation
2. Endometrial biopsy: Endometrial biopsy is done during
Most common cause of female infertility is anovulation. premenstrual period (21st-23rd day of the cycle). The
1. Regular cycles, mastalgia, and laparoscopic direct secretory endometrium during the later half of the
visualization of corpus luteum indicate ovulatory cycles. cycle is an evidence of ovulation.
156 Essentials of Clinical Pathology

Fig. 14.8: Evaluation of female infertility. FSH: Follicle stimulating hormone; LH: Luteinizing hormone;
DHEA-S: Dihydroepiandrosterone; TSH: Thyroid stimulating hormone; ↑ : Increased; ↓ : Decreased

3. Ultrasonography (USG): Serial ultrasonography is done

from 10th day of the cycle and the size of the
dominant follicle is measured. Size >18 mm is
indicative of imminent ovulation. Collapse of the
follicle with presence of few ml of fluid in the pouch
of Douglas is suggestive of ovulation. USG also is
helpful for treatment (i.e. timing of coitus or of
intrauterine insemination) and diagnosis of luteinized
unruptured follicle (absence of collapse of dominant
follicle). Transvaginal USG is more sensitive than
abdominal USG.
4. Basal body temperature (BBT): Patient takes her oral
temperature at the same time every morning before
arising. BBT falls by about 0.5 ° F at the time of
ovulation. During the second (progestational) half of Fig. 14.9: Ferning of cervical mucosa
the cycle, temperature is slightly raised above the pre-
ovulatory level (rise of 0.5° to 1°F). This is due to the
• Spinnbarkeit test: Cervical mucus is elastic and
slight pyrogenic action of progesterone and is
withstands stretching upto a distance of over 10
therefore presumptive evidence of functional corpus
cm. This phenomenon is called Spinnbarkeit or
luteum. the thread test for the estrogen activity. During
5. Cervical mucus study: the secretory phase, viscosity of the cervical mucus
• Fern test: During estrogenic phase, a characteristic increases and it gets fractured when stretched.
pattern of fern formation is seen when cervical This change in cervical mucus is evidence of
mucus is spread on a glass slide (Fig. 14.9). This ovulation.
ferning disappears after the 21st day of the cycle. 6. Vaginal cytology: Karyopyknotic index (KI) is high
If previously observed, its disappearance is during estrogenic phase, while it becomes low in
presumptive evidence of corpus luteum activity. secretory phase. This refers to percentage of super-

ficial squamous cells with pyknotic nuclei to all
mature squamous cells in a lateral vaginal wall smear.
Usually minimum of 300 cells are evaluated. The peak
KI usually corresponds with time of ovulation and
may reach upto 50 to 85.
7. Estimation of progesterone in mid-luteal phase (day 21 or
7 days before expected menstruation): Progesterone
level > 10 nmol/L is a reliable evidence of ovulation
if cycles are regular (Fig. 14.10). A mistimed sample
is a common cause of abnormal result.
Tests to Determine the Cause of Anovulation
1. Measurement of LH, FSH, and estradiol during days 2 to
6: All values are low in hypogonadotropic hypo-
gonadism (hypothalamic or pituitary failure).
2. Measurement of TSH, prolactin, and testosterone if cycles
are irregular or absent:
Increased TSH: Hypothyroidism
Increased prolactin: Pituitary adenoma
Fig. 14.10: Serum progesterone during
normal menstrual cycle
Fig. 14.11: Hysterosalpingography
Infertility 157
Increased testosterone: Polycystic ovarian disease
(PCOD), congenital adrenal hyperplasia (To differen-
tiate PCOD from congenital adrenal hyperplasia,
ultrasound and estimation of dihydroepiandros-
terone or DHEA are done).
3. Transvaginal ultrasonography: This is done for detection
of PCOD.
Investigations to Assess Tubal and Uterine Status
1. Infectious disease: These tests include endometrial
biopsy for tuberculosis and test for chlamydial IgG
antibodies for tubal factor in infertility.
2. Hysterosalpingography (HSG): HSG is a radiological
contrast study for investigation of the shape of the
uterine cavity and for blockage of fallopian tubes
(Fig. 14.11). A catheter is introduced into the cervical
canal and a radiocontrast dye is injected into the
uterine cavity. A real time X-ray imaging is carried
out to observe the flow of the dye into the uterine
cavity, tubes, and spillage into the uterine cavity.
3. Hysterosalpingo-contrast sonography: A catheter is
introduced into the cervical canal and an echocontrast
fluid is introduced into the uterine cavity. Shape of
the uterine cavity, filling of fallopian tubes, and
spillage of contrast fluid are noted. In addition,
ultrasound scan of the pelvis provides information
about any fibroids or polycystic ovarian disease.
4. Laparoscopy and dye hydrotubation test with hysteroscopy:
In this test, a cannula is inserted into the cervix and
methylene blue dye is introduced into the uterine
cavity. If tubes are patent, spillage of the dye is
observed from the ends of both tubes. This technique
also allows visualization of pelvic organs, endo-
metriosis, and pelvic adhesions. If required, endo-
metriosis and tubal blockage can be treated during
the procedure.
158 Essentials of Clinical Pathology
Possible pregnancy and active pelvic or vaginal
infection are contraindications to tubal patency tests.
REFERENCE RANGES
• Serum follicle stimulating hormone:
– Adult males: 1.4-15.4 mIU/ml
– Adult females: Follicular phase: 1-10 mIU/ml;
Ovulatory peak: 6-17 mIU/ml; Luteal phase: 1-9
mIU/ml
• Serum luteinizing hormone:
– Adult males: 1.24-7.8 mU/ml
– Adult females: Follicular phase: 1.68-15 mIU/ml;
Ovulatory peak: 21.9-56.6 mIU/ml; Luteal phase:
0.61-16.3 mIU/ml
• Serum testosterone:
– Adult males: 280-1100 ng/dl
– Adult females: 15-70 ng/dl
• Serum prolactin:
– Adult males: 3.0-14.7 ng/ml
– Adult females: 3.8-23.2 ng/ml
• Serum dehydroepiandrosterone sulfate (DHEA-S):
– Adult males: 59-452 μg/ml
– Adult females: 12-379 μg/ml
BIBLIOGRAPHY
1. Brugh III, VM, Lipshultz LI. Male factor infertility: Evaluation
and management. Med Clin N Am 2004;88:367-85.
2. Burtis CA, Ashwood ER. Tietz fundamentals of clinical
chemistry. 5th Ed. Philadelphia: WB Saunders
Company, 2001.
3. Dohle GR, Jungwirth A, Colpi G, Papp G, Pomerol J,
Hargreave TB. Guidelines on male infertility. European
Association of Urology, 2006.
4. Hirsh A. Male subfertility. BMJ 2003;327:669-72.
5. Hamilton-Fairley D, Taylor A. Anovulation. BMJ
2003;327:546-9.
6. Jarrow JP. Diagnostic approach to the infertile male
patient. Endocrinol Metab Clin N Am 2007;36:297-311.
7. Khalaf Y. Tubal subfertility. BMJ 2003;327:610-3.
8. Kolettis PN. Evaluation of the subfertile man. Am Fam
Physician 2003;67:2165-73.
9. Williams C, Giannopoulos T, Sherriff EA. Investigation
of infertility with the emphasis on laboratory testing
and with reference to radiological imaging. J Clin Pathol
2003;56:261-7.
 15
Semen Analysis
Semen (or seminal fluid) is a fluid that is emitted from
the male genital tract and contains sperms that are
capable of fertilizing female ova. Structures involved in
production of semen are (Box 15.1):
• Testes: Male gametes or spermatozoa (sperms) are
produced by testes; constitute 2-5% of semen volume.
• Epididymis: After emerging from the testes, sperms
are stored in the epididymis where they mature;
potassium, sodium, and glycerylphosphorylcholine
(an energy source for sperms) are secreted by
epididymis.
• Vas deferens: Sperms travel through the vas deferens
to the ampulla which is another storage area. Ampulla
secretes ergothioneine (a yellowish fluid that reduces
chemicals) and fructose (source of nutrition for
sperms).
• Seminal vesicles: During ejaculation, nutritive and
lubricating fluids secreted by seminal vesicles and
prostate are added. Fluid secreted by seminal vesicles
consists of fructose (energy source for sperms), amino
acids, citric acid, phosphorous, potassium, and
prostaglandins. Seminal vesicles contribute 50% to
semen volume.
• Prostate: Prostatic secretions comprise about 40% of
semen volume and consist of citric acid, acid
phosphatase, calcium, sodium, zinc, potassium,
proteolytic enzymes, and fibrolysin.
• Bulbourethral glands of Cowper secrete mucus.
Box 15.1: Contributions to semen volume
• Testes and epididymis: 10%
• Seminal vesicles: 50%
• Prostate: 40%
• Cowper’s glands: Small volume
Normal values for semen analysis are shown in Tables
15.1 and 15.2.
INDICATIONS FOR SEMEN ANALYSIS
Availability of semen for examination allows direct
examination of male germ cells that is not possible with
female germ cells. Semen analysis requires skill and
should preferably be done in a specialized andrology
laboratory.
1. Investigation of infertility: Semen analysis is the first
step in the investigation of infertility. About 30% cases
of infertility are due to problem with males.
2. To check the effectiveness of vasectomy by confir-
ming absence of sperm.
3. To support or disprove a denial of paternity on the
grounds of sterility.
4. To examine vaginal secretions or clothing stains for
the presence of semen in medicolegal cases.
5. For selection of donors for artificial insemination.
6. For selection of assisted reproductive technology, e.g.
in vitro fertilization, gamete intrafallopian transfer
technique.
COLLECTION OF SEMEN FOR
INVESTIGATION OF INFERTILITY
Semen specimen is collected after about 3 days of sexual
abstinence. Longer period of abstinence reduces motility
of sperms. If the period of abstinence is shorter than 3
days, sperm count is lower. The sample is obtained by
masturbation, collected in a clean, dry, sterile, and leak-
proof wide-mouthed plastic container, and brought to
the laboratory within 1 hour of collection. The entire
ejaculate is collected, as the first portion is the most
concentrated and contains the highest number of sperms.
160 Essentials of Clinical Pathology

Table 15.1: Normal values of semen analysis (World Health Organization, 1999)

Test Result
1. Volume ≥2 ml
2. pH 7.2 to 8.0
3. Sperm concentration ≥20 million/ml
4. Total sperm count per ejaculate ≥40 million
5. Morphology ≥30% sperms with normal morphology
6. Vitality ≥75% live
7. White blood cells <1 million/ml
8. Motility within 1 hour of ejaculation
• Class A ≥25% rapidly progressive
• Class A and B ≥50% progressive
9. Mixed antiglobuiln reaction (MAR) test <50% motile sperms with adherent particles
10. Immunobead test <50% motile sperms with adherent particles

Table 15.2: Biochemical variables of semen analysis (World Helath Organization, 1992)

1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate

2. Total zinc (Prostate marker) ≥2.4 μmol/ejaculate
3. Total acid phosphatase (Prostate marker) ≥200U/ejaculate
4. Total citric acid (Prostate marker) ≥52 μmol/ejaculate
5. α-glucosidase (Epididymis marker) ≥20 mU/ejaculate
6. Carnitine (Epididymis marker) 0.8-2.9 μmol/ejaculate

During transport to the laboratory, the specimen should

be kept as close to body temperature as possible (i.e. by
carrying it in an inside pocket). Ideally, the specimen
should be obtained near the testing site in an adjoining
room. Condom collection is not recommended as it
contains spermicidal agent. Ejaculation after coitus
interruptus leads to the loss of the first portion of the
ejaculate that is most concentrated; therefore this method
should not be used for collection. Two semen specimens
should be examined that are collected 2-3 weeks apart;
if results are significantly different additional samples
are required.
EXAMINATION OF SEMINAL FLUID
Box 15.2: Tests done on seminal fluid
• Physical examination: Time to liquefaction, viscosity,
volume, pH, color
• Microscopic examination: Sperm count, vitality, motility,
morphology, and proportion of white cells
• Immunologic analysis: Antisperm antibodies (SpermMAR
test, Immunobead test)
• Bacteriologic analysis: Detection of infection
• Biochemical analysis: Fructose, zinc, acid phosphatase,
carnitine.
• Sperm function tests: Postcoital test, cervical mucus
penetration test, Hamster egg penetration assay, hypo-
osmotic swelling of flagella, and computer-assisted semen
analysis

The tests that can be done on seminal fluid are shown in Box 15.3: Semen analysis for initial
Box 15.2. Tests commonly done in infertility are shown investigation of infertility
in Box 15.3. The usual analysis consists of measurement • Volume
of semen volume, sperm count, sperm motility, and • pH
• Microscopic examination for (i) percentage of motile
sperm morphology.
spermatozoa, (ii) sperm count, and (iii) sperm morphology
Terminology in semen analysis is shown in Box 15.4.

Box 15.4: Terminology in semen analysis
• Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration <20 million/ml
(mild to moderate: 5-20 million/ml; severe: <5 million/ml)
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility; <50% of
sperms showing class (a) and class (b) type of motility
OR <25% sperms showing class (a) type of motility.
• Teratozoospermia: Spermatozoa with reduced proportion
of normal morphology (or increased proportion of abnormal
forms)
• Leukocytospermia: >1 million white blood cells/ml of
semen
• Oligoasthenoteratozoospermia: All sperm variables are
abnormal
• Necrozoospermia: All sperms are non-motile or non-viable
Physical Examination
Examination is carried out after liquefaction of semen
that occurs usually within 20-30 minutes of ejaculation.
1. Visual appearance: Normal semen is viscous and
opaque gray-white in appearance. After prolonged
abstinence, it appears slightly yellow.
2. Viscosity: Immediately following ejaculation, normal
semen is thick and viscous. It becomes liquefied
within 30 minutes by the action of proteolytic
enzymes secreted by prostate. If liquefaction does not
occur within 60 minutes, it is abnormal. The viscosity
of the sample is assessed by filling a pipette with
semen and allowing it to flow back into the container.
Normal semen will fall drop by drop. If droplets form
‘threads’ more than 2 cm long, then viscosity is
increased. Increased semen viscosity affects sperm
motility and leads to poor invasion of cervical mucus;
it results from infection of seminal vesicles or prostate.
3. Volume: Volume of ejaculated semen sample should
normally be > 2 ml. It is measured after the sample
has liquefied. Volume < 2.0 ml is abnormal, and is
associated with low sperm count.
4. pH: A drop of liquefied semen is spread on pH paper
(of pH range 6.4-8.0) and pH is recorded after 30
seconds. Normal pH is 7.2 to 8.0 after 1 hour of
ejaculation. The portion of semen contributed by
seminal vesicles is basic, while portion from prostate
is acidic. Low pH (< 7.0) with absence of sperms
Semen Analysis 161
(azoospermia) suggests obstruction of ejaculatory
ducts or absence of vas deferens. Low pH is usually
associated with low semen volume (as most of the
volume is supplied by seminal vesicles).
Microscopic Examination
The most important test in semen analysis for infertility
is microscopic examination of the semen.
Sperm Motility
The first laboratory assessment of sperm function in a
wet preparation is sperm motility (ability of the sperms
to move). Sperm motility is essential for penetration of
cervical mucus, traveling through the fallopian tube, and
penetrating the ovum. Only those sperms having rapidly
progressive motility are capable of penetrating ovum and
fertilizing it.
Principle: All motile and non-motile sperms are counted
in randomly chosen fields in a wet preparation under
40× objective. Result is expressed as a percentage of
motile spermatozoa observed.
Method: A drop of semen is placed on a glass slide,
covered with a coverslip that is then ringed with
petroleum jelly to prevent dehydration, and examined
under 40× objective. Atleast 200 spermatozoa are counted
in several different microscopic fields. Result is expressed
as a percentage of (a) rapidly progressive spermatozoa
(moving fast forward in a straight line), (b) slowly
progressive spermatozoa (slow linear or non-linear, i.e.
crooked or curved movement), (c) non-progressive
spermatozoa (movement of tails, but with no forward
progress), and (d) immotile spermatozoa (no movement
at all) (WHO critera). Sperms of grades (c) and (d) are
considered to be poorly motile (asthenospermia).
Normally, ≥ 25% of sperms show rapid progressive
motility, or ≥ 50% of sperms show rapid progressive and
slow progressive motility.
If the proportion of motile spermatozoa is < 50%, then
proportion of viable sperms should be determined by
examining an eosin preparation.
Sperm Viability or Vitality
Principle: A cell with intact cell membrane (a vital or
viable cell) will not take up the eosin Y and will not be
stained, while a non-viable or dead cell will have
damaged cell membrane, will take up the dye, and will
162 Essentials of Clinical Pathology
Fig. 15.1: Eosin-nigrosin stain. Dead sperms are stained
pink-red, while live sperms are stained white
be stained pink-red (Fig. 15.1). Another stain (e.g.
nigrosin) may be used to stain the background material.
The test is performed if motility is abnormal.
Method
1. Mix one drop of semen with 1 drop of eosin-nigrosin
solution and incubate for 30 seconds.
2. A smear is made from a drop placed on a glass slide.
3. The smear is air-dried and examined under oil-
immersion objective. White sperms are classified as
live or viable, and red sperms are classified as dead
or non-viable. At least 200 spermatozoa are examined.
4. The result is expressed as a proportion of viable
sperms against non-viable as an integer percentage.
Seventy-five percent or more of sperms are normally
live or viable.
Sperm Count
Principle: The sperm count is done after liquefaction in a
counting chamber following dilution and the total
number of spermatozoa is reported in millions/ml (106/
ml).
Method
1. Semen is diluted 1:20 with sodium bicarbonate-
formalin diluting fluid (Take 1 ml liquefied semen in
a graduated tube and fill with diluting fluid to 20 ml
mark. Mix well).
2. A coverslip is placed over the improved Neubauer
counting chamber and the counting chamber is filled
with the well-mixed diluted semen sample using a
Pasteur pipette. The chamber is then placed in a
humid box for 10-15 minutes for spermatozoa to
settle.
3. The chamber is placed on the microscope stage. Using
the 20× or 40× objective and iris diaphragm lowered
sufficiently to give sufficient contrast, number of
spermatozoa is counted in 4 large corner squares.
Spermatozoa whose heads are touching left and
upper lines of the square should be considered as
‘belonging’ to that square.
4. Sperm count per ml is calculated as follows:
Sperms counted × correction factor
for dilution
Sperm count = × 1000
Number of × Volume of
squares counted 1 square
Sperms counted × 20
= 1000
4 × 0.1
= Sperms counted × 50, 000
5. Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 ×
10 6/ml). Sperm count < 20 million/ml may be
associated with infertility in males.
Sperm Morphology
A smear is prepared by spreading a drop of seminal fluid
on a glass slide, stained, and percentages of normal and
abnormal forms of spermatozoa are counted. The
staining techniques used are Papanicolaou, eosin-
nigrosin, hematoxylin-eosin, and Rose Bengal-toluidine
blue stain. Atleast 200 spermatozoa should be counted
under oil immersion. Percentages of normal and
abnormal spermatozoa should be recorded.
Normal morphology: A spermatozoon consists of three
main components: head, neck, and tail. Tail is further
subdivided into midpiece, main (principle) piece, and
end piece (Fig. 15.2 and Box 15.5).
Head is pear-shaped. Most of the head is occupied
by the nucleus which has condensed chromatin and few
areas of dispersed chromatin (called nuclear vacuoles).
The anterior 2/3rds of the nucleus is surrounded by
acrosomal cap. Acrosomal cap is a flattened membrane-
bound vesicle containing glycoproteins and enzymes.
These enzymes are required for separation of cells of
corona radiata and dissolution of zona pellucida of ovum
during fertilization.
 Semen Analysis 163

Box 15.5: Normal sperm morphology

• Total length of sperm: About 60 μ

• Head:
– Length: 3-5 μ
– Width: 2-3 μ
– Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
– Length: 3-5 μ
– Width: 1.0 μ
• Principal piece:
– Length: 40-50 μ
– Width: 0.5 μ
• End piece: 4-6 μ

Abnormal morphology (Fig. 15.3): WHO morphological

classification of human spermatozoa (1999) is given
below:
1. Normal sperm
2. Defects in head:
• Large heads
• Small heads
• Tapered heads
Fig. 15.2: Morphology of spermatozoa • Pyriform heads
• Round heads
• Amorphous heads
Neck is a very short segment that connects the head
• Vacuolated heads (> 20% of the head area occu-
and the tail. Centriole in the neck gives rise to axoneme
pied by vacuoles)
of the flagellum. Axoneme consists of 20 microtubules
• Small acrosomes (occupying < 40% of head area)
(arranged as a central pair surrounded by 9 peripheral
• Double heads
doublets) and is surrounded by condensed fibrous rings.
3. Defects in neck:
Middle piece is the first part of the tail and consists
• Bent neck and tail forming an angle >90° to the
of central axoneme surrounded by coarse longitudinal
long axis of head
fibers. These are surrounded by elongated mitochondria
4. Defects in middle piece:
that provide energy for movement of tail.
• Asymmetric insertion of midpiece into head
Principle or main piece constitutes most of the tail
• Thick or irregular midpiece
and is composed of axoneme that is surrounded by 9 • Abnormally thin midpiece
coarse fibers. This central core is surrounded by many 5. Defects in tail:
circularly arranged fibrous ribs. • Bent tails
Endpiece is the short tapering part composed of only • Short tails
axoneme. • Coiled tails
Normally, > 30% of spermatozoa should show normal • Irregular tails
morphology (WHO, 1999). The defects in morphology • Multiple tails
that are associated with infertility in males include • Tails with irregular width
defective mid-piece (causes reduced motility), an 6. Pin heads: Not to be counted
incomplete or absent acrosome (causes inability to 7. Cytoplasmic droplets
penetrate the ovum), and giant head (defective DNA • > 1/3rd the size of the sperm head
condensation). 8. Precursor cells: Considered abnormal
164 Essentials of Clinical Pathology
Fig. 15.3: Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered head, (5)
Pyriform head, (6) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, (10) Double
head, (11) Pin head, (12) Round head without acrosome and thick midpiece, (13) Coiled tail, and (14) Double tail
Round Cells
Round cells on microscopic examination may be white
blood cells or immature sperm cells. Special stain
(peroxidase or Papanicolaou) is required to differentiate
between them. White blood cells >1 million/ml indicate
presence of infection. Presence of large number of
immature sperm cells indicates spermatogenesis
dysfunction at the testicular level.
Immunologic Analysis
Antisperm Antibodies
The role of antisperm antibodies in causation of male
infertility is controversial. The immunological tests done
on seminal fluid include mixed antiglobulin reaction
(MAR test) and immunobead test.
The antibodies against sperms immobilize or kill
them, thus preventing their passage through the cervix
to the ovum. The antibodies can be tested in the serum,
seminal fluid, or cervical mucus. If the antibodies are
present bound to the head of the sperm, they will prevent
the penetration of the egg by the sperm. If antibodies are
bound to the tail of the sperm, they will retard motility.
a. SpermMARTM test: This test can detect IgG and IgA
antibodies against sperm surface in semen sample.
In direct SpermMARTM IgG test, a drop each of semen
(fresh and unwashed), IgG-coated latex particles, and
anti-human immunoglobulin are mixed together on
a glass slide. At least 200 motile spermatozoa are
examined. If the spermatozoa have antibodies on
their surface, antihuman immunoglobulin will bind
IgG-coated latex particles to IgG on the surface of the
spermatozoa; this will cause attachment of latex
particles to spermatozoa, and motile, swimming
sperms with attached particles will be seen. If the
spermatozoa do not have antibodies on their surface,
they will be seen swimming without attached
particles; the latex particles will show clumping due
to binding of their IgG to antihuman immuno-
globulin.
In direct SpermMARTM IgA test, a drop each of fresh
unwashed semen and of IgA-coated latex particles,
are mixed on a glass slide. The latex particles will
bind to spermatozoa if spermatozoa are coated with
IgA antibodies.
In indirect SpermMAR TM tests, fluid without
spermatozoa (e.g. serum) is tested for the presence of

antisperm antibodies. First, antibodies are bound to
donor spermatozoa which are then mixed with the
fluid to be analyzed. These antibodies are then
detected as described above for direct tests.
Atleast 200 motile spermatozoa should be counted.
If >50% of spermatozoa show attached latex particles,
immunological problem is likely.
b. Immunobead test: Antibodies bound to the surface of
the spermatozoa can be detected by antibodies
attached to immunobeads (plastic particles with
attached anti-human immunoglobulin that may be
either IgG, IgA,or IgM). Percentage of motile
spermatozoa with attached two or more immuno-
beads are counted amongst 200 motile spermatozoa.
Finding of >50% spermatozoa with attached beads is
abnormal.
Biochemical Analysis of Semen
Biochemical markers (Table 15.2) can be measured in
semen to test the secretions of accessory structures. These
include fructose (seminal vesicles), zinc, citric acid or acid
phosphatase (prostate), and α-glucosidase or carnitine
(epididymis).
Test for Fructose
Resorcinol method is used for detection of fructose. In
this test, 5 ml of resorcinol reagent (50 mg resorcinol
dissolved in 33 ml concentrated hydrochloric acid; dilute
up to 100 ml with distilled water) is added to 0.5 ml of
seminal fluid. The mixture is heated and brought to boil.
If fructose is present, a red-colored precipitate is formed
within 30 seconds.
Absence of fructose indicates obstruction proximal
to seminal vesicles (obstructed or absent vas deferens)
or a lack of seminal vesicles. In a case of azoospermia, if
fructose is absent, it is due to the obstruction of
ejaculatory ducts or absence of vas deferens, and if
present, azoospermia is due to failure of testes to produce
sperm.
Sperm Function Tests or Functional Assays
These tests are available only in specialized andrology
laboratories. The tests are not standardized thus making
interpretation difficult. If used singly, a sperm function
test may not be helpful in fertility assessment. They are
more predictive if used in combination.
Semen Analysis 165
Postcoital (Sims-Huhner) Test
This is the examination of the cervical mucus after coitus
and assesses the ability of the sperm to penetrate the
cervical mucus. The quality of the cervical mucus varies
during the menstrual cycle, becoming more abundant
and fluid at the time of ovulation (due to effect of
estrogen); this facilitates penetration of the mucus by the
spermatozoa. Progesterone in the secretory phase
increases viscosity of the mucus. Therefore cervical
mucus testing is scheduled just before ovulation
(determined by basal body temperature records or
follicular sizing by ultrasonography). Postcoital test is
the traditional method to detect the cervical factor in
infertility. Cervical mucus is aspirated with a syringe
shortly before the expected time of ovulation and 2-12
hours after intercourse. Gross and microscopic examina-
tions are carried out to assess the quality of cervical
mucus (elasticity and drying pattern) and to evaluate the
number and motility of sperms (Box 15.6). If ≥ 10 motile
sperms are observed the test is considered as normal.
An abnormal test may result from: (a) poor quality of
cervical mucus due to wrong judgment of ovulation,
cervicitis or treatment with antioestrogens (e.g. Clomid),
and (b) absence of motile sperms due to ineffective
technique of coitus, lack of ejaculation, poor semen
quality, use of coital lubricants that damage the sperm,
or presence of antisperm antibodies. Antisperm
antibodies cause immotile sperms, or agglutination or
clumping of sperms; they may be present in either
partner. If cervical factor is present, intrauterine
insemination is the popular treatment. The value of the
postcoital test is disputed in the medical literature.
This test can be carried out if semen analysis is
normal, and the female partner is ovulating and fallopian
tubes are not blocked. It is also done if antisperm
Box 15.6: Interpretation of postcoital test
• Normal: Sperms are normal in amount and moving forward
in the mucus; mucus stretches atleast 2 inches (5 cm)
and dries in a fern-like manner.
• Abnormal: Absence of sperms or large number of sperms
are dead or sperms are clumped; cervical mucus cannot
stretch 2 inches (5 cm) or does not dry in a fern-like
manner.
166 Essentials of Clinical Pathology
antibodies are suspected and male partner refuses semen
analysis.
Cervical Mucus Penetration Test
In this test, greatest distance traveled by the sperm in
seminal fluid placed and incubated in a capillary tube
containing bovine mucus is measured. Majority of fertile
men show score >30 mm, while most infertile men show
scores <20 mm.
Hamster Egg Penetration Assay
Hamster oocytes are enzymatically treated to remove the
outer layers (that inhibit cross-species fertilization). They
are then incubated with sperms and observed for
penetration rate. It can be reported as (a) Number of eggs
penetrated (penetration rate <15% indicates low fertility),
or as (b) Number of sperm penetrations per egg (Normal
>5). This test detects sperm motility, binding to oocyte,
and penetration of oocyte. There is a high incidence of
false-negative results.
Hypo-osmotic Swelling of Flagella
This test assesses the functional integrity of the plasma
membrane of the sperm by observing curling of flagella
in hypo-osmotic conditions.
Computer-assisted Semen Analysis
Computer software measures various characteristics of
the spermatozoa; however, its role in predicting fertility
potential is not confirmed.
EXAMINATION FOR THE PRESENCE OF
SEMEN IN MEDICOLEGAL CASES
This includes examination of material obtained from
vagina, stains from clothing, skin, hair, or other body
parts for semen. This is carried out in cases of alleged
rape or sexual assault.
Collection of Sample
• Vagina: Direct aspiration or saline lavage
• Clothing: When scanned with ultraviolet light, semen
produces green white fluorescence. A small piece (1
m2) of clothing from stained portion is soaked in 1-2
ml of physiologic saline for 1 hour. A similar piece of
clothing distant from the stain is also soaked in saline
as a control.
Tests
1. Microscopic examination for sperms: Presence of motile
sperms in vaginal fluid indicates interval of
< 8 hours. Smears prepared from collected samples
are stained and examined for the presence of sperms.
2. Acid phosphatase: Acid phosphatase is determined on
vaginal or clothing samples. Due to the high level of
acid phosphatase in semen, its presence indicates
recent sexual intercourse. Level of ≥50 U/sample is
considered as positive evidence of semen.
3. Determination of blood group substances: When semen
is positively identified in vaginal fluid or other
sample, test can be carried out for the presence of
blood group substances in the same sample. The
‘secretor’ individuals (80% individuals are secretors)
will secrete the blood group substances in body fluids,
including semen.
4. Florence test: This test detects the presence of choline
found in high concentration in semen. To several
drops of sample, add equal volume of reagent (iodine
2.54 g, potassium iodide 1.65 g, distilled water 30 ml);
in positive test rhombic or needle-like crystals of
periodide of choline form. False-positive tests can
occur due to high choline content of some other body
fluids.
EXAMINATION OF SEMEN TO CHECK THE
EFFECTIVENESS OF VASECTOMY
The aim of post-vasectomy semen analysis is to detect
the presence or absence of spermatozoa. The routine
follow-up consists of semen analysis starting 12 weeks
(or 15 ejaculations) after surgery. If two successive semen
samples are negative for sperms, the semen is considered
as free of sperm. A follow-up semen examination at 6
months is advocated by some to rule out spontaneous
reconnection.
BIBLIOGRAPHY
1. Hirsh A. Male subfertility. BMJ 2003;327:669-72.
2. Phadke AM. Clinical Atlas of Sperm Morphology. New
Delhi: Jaypee Brothers Medical Publishers (P) Ltd. 2007.
3. World Health Organization. Laboratory Manual for the
Examination of Human Semen and Sperm-Cervical
Mucus Interactions, 3rd edition. Cambridge: Cambridge
University Press, 1992.
4. World Health Organization. WHO laboratory manual
for the examination of human semen and sperm-
cervical mucus interaction. 4th edition. Cambridge, UK:
New York, NY: Published on behalf of the World Health
Organization [by] Cambridge University Press; 1999.
Laboratory Hematology
 16

Hematopoiesis

The physiologic process of formation of blood cells is

called as hematopoiesis (Greek haima: blood; poiesis: to
make). During 3rd week of embryonic life, hematopoiesis
begins in the yolk sac, as aggregates of blood cells called
as blood islands. Around 3rd month of fetal life, some of
the hematopoietic stem cells migrate to the liver which
then becomes the main site of hematopoiesis. Some blood
cell formation also occurs in the spleen, lymph nodes,
and thymus. Bone marrow starts producing blood cells
around 4th month of fetal life, and becomes the sole site
of hematopoiesis after birth (Box 16.1 and Fig. 16.1). In
certain conditions, hematopoiesis can become re-
established in liver, spleen, and lymph nodes; this is
called as extramedullary hematopoiesis.
Bone marrow is the soft, gelatinous tissue supported
by a reticulin framework that fills the intertrabecular
spaces in the cavities of the bones. There are two types Fig. 16.1: Sites of hematopoiesis
of bone marrow: red marrow composed of hematopoietic
tissue (active marrow) and yellow marrow composed of term pleuripotent refers to the ability to produce many
fat cells (inactive marrow). Active sites of hematopoiesis cell types. Most of the PHSCs are located in the bone
are: pelvis, vertebra, skull, ribs, sternum, and proximal marrow, but they are able to enter and circulate freely in
ends of long bones. In children, 100% of the total marrow peripheral blood. Stem cells have the ability of self-
space is active, while in adults, 50% of total marrow space renewal (to maintain a constant pool of undifferentiated
is active. cells) and differentiation. Hematopoietic stem cells are
All the blood cells circulating in the peripheral blood rare in the bone marrow (1 stem cell per 10000 to 1000000
are derived from the primitive mesenchymal cells called bone marrow cells). Primitive hematopoietic cells express
as pleuripotent hematopoietic stem cells (PHSCs); the CD34 antigen. Stem cells do not have any distinguishing
morphological features, and resemble a lymphocyte.
Box 16.1: Stages of hematopoiesis Stem cells can differentiate along different pathways and
produce other tissue cells (stem cell plasticity). Stem cells,
• Mesoblastic stage: Begins at 3rd week of gestation; as part of their proliferation, become committed to a
occurs in yolk sac. lineage and are then called as progenitor cells. Progenitor
• Hepatic stage: Begins 4-8 weeks of gestation; occurs cells do not have capacity of self-renewal, and have
in liver, thymus, and spleen
restricted multilineage potential.
• Myeloid stage: Begins at 12 weeks of gestation;
Two multipotential cell lines originate from the
occurs in cancellous areas of bones
pleuripotent hematopoietic stem cell: myeloid and
170 Essentials of Clinical Pathology

lymphoid and all blood cells belong to either of these
two lineages. Myeloid cells include erythrocytes or red
cells, platelets, neutrophils, eosinophils, basophils, and
monocytes. Lymphoid cells are of two main types: B and
T lymphocytes. The myeloid stem cell is called as CFU-
GEMM (Colony forming unit granulocyte-erythrocyte-
monocyte-megakaryocyte). CFU refers to the develop-
ment of colonies of cells when marrow cells are injected
in a tissue culture medium. CFU-GEMM leads to the
formation of BFU-E (Burst Forming Unit-Erythroid),
CFU-Meg (Colony Forming Unit-Megakaryocyte), CFU-
GM (Colony Forming Unit-Granulocyte-Monocyte/
Macrophage), CFU-Eo (Colony Forming Unit-
Eosinophil), and CFU-Baso (Colony Forming Unit-
Basophil). CFU-GM further produces CFU-G (Colony
Forming Unit-Granulocyte) and CFU-M (Colony
Forming Unit-Monocyte/Macrophage) (Fig. 16.2). The
progenitor cells up to these stages are not identifiable
morphologically; they can however be identified by their
surface receptors.
Blood cells in the bone marrow can be considered to
be in three compartments or pools: stem cell pool
(comprises of pleuripotent and multipotent stem cells,
and commited CFUs), proliferating pool (morpholo-
gically identifiable precursors that are capable of DNA
synthesis and mitosis), and storage pool (maturing and
mature cells that are stored for later release in peripheral
blood).
Hematopoiesis is regulated by two main factors: (i)
hematopoietic growth factors (HGFs) or cytokines,
secreted by various cell types (Table 16.1), and (ii) stromal
cells in the microenvironment of the bone marrow. HGFs
are glycoproteins secreted by various cell types which
regulate proliferation and differentiation of progenitor
cells and function of mature blood cells. Stromal cells
secrete a variety of HGFs in the bone marrow micro-
environment; secondly hematopoietic progenitor cells
adhere to receptors on stromal cells and on the stromal
matrix that facilitates interaction of progenitor cells with
regulatory cytokines.

Fig. 16.2: Hierarchy of hematopoiesis. Abbreviations: SCF: Stem cell factor; IL: Interleukin; CFU-GEMM: Colony forming unit
granulocyte-erythrocyte-monocyte-megakaryocyte; CFU-GM: Colony forming unit granulocyte-macrophage; TPO: Thrombopoietin;
CFU- G: Colony forming unit granulocyte; CFU-M: Colony forming unit macrophage; CFU-Eo: Colony-forming unit eosinophil;
CFU-Baso: Colony forming unit basophil; CFU-Meg: Colony forming unit megakaryocyte; Epo: Erythropoietin; GM-CSF: granulocyte
macrophage colony stimulating factor; G-CSF: Granulocyte colony stimulating factor; M-CSF: Macrophage Colony stimulating
factor
 Hematopoiesis 171

Table 16.1: Hematopoietic growth factors

Growth factor Site of synthesis Site of action

1. Granulocyte macrophage T cells, fibroblasts, Granulocytes, monocytes,

colony stimulating factor endothelial cells platelets, red cells
2. Granulocyte colony Monocytes, fibroblasts Neutrophils
stimulating factor
3. Macrophage colony Monocytes, endothelial Monocytes
stimulating factor cells, fibroblasts
4. Erythropoietin Kidney Red cells
5. Thrombopoietin Liver Platelets
6. Stem cell factor — Pleuripotent stem cells
7. Interleukin 3 T cells Hematopoietic stem cells
8. Interleukin 5 T cells Eosinophils
9. Interleukin 6 T lymphocytes, B and T cells
monocytes, fibroblasts

Fig. 16.3: Stages of erythropoiesis

Table 16.2: Stages in maturation of red blood cells

Cell Size (μ) Nucleus Cytoplasm

1. Proerythroblast 15-20 Large with immature chromatin, Blue

usually a single nucleolus
2. Early erythroblast 12-16 Fine chromatin clumps, nucleolus Deep blue due to high RNA
(Basophilic) barely visible
3. Intermediate erythroblast 12-15 Smaller size, chromatin clumps Polychromatic due to
(Polychromatic) coarse beginning of
hemoglobinization
4. Late erythroblast 8-12 Small, dense, pyknotic, and Polychromatic
(Orthochromatic) eccentric
5. Reticulocyte (Polychromatic 8-10 No nucleus Polychromatic, remnants of
red cell) RNA visible as a network on
supravital staining
6. Erythrocyte 7-8 No nucleus Pink

RED BLOOD CELLS identifiable erythroid precursor. It proliferates to

Erythropoiesis
Stages of erythropoiesis are shown in Figure 16.3 and
Table 16.2. Proerythroblast is the earliest morphologically
generate sequentially basophilic, polychromatic, and
orthochromatic erythroblast; these cells are named after
staining characteristics of cytoplasm. Reticulocytes still
retain some cytoplasmic organelles (a fine reticulim of
172 Essentials of Clinical Pathology
ribosomal RNA). With each stage of development, cell
size and nuclear size become smaller, chromatin
clumping increases, and ultimately nucleus is extruded.
Color of cytoplasm gradually changes from basophilic
to pink due to hemoglobinization. A mature red cell or
erythrocyte is a biconcave, non-nucleated disk of 7-8 μ
size.
Hemoglobin
Hemoglobin (average molecular weight 64,000) consists
of heme (iron and protoporphyrin) and globin (two
polypeptide chains). A heme group is attached to each
polypeptide chain. Normally, different types of hemo-
globins are present during embryonic life, fetal life,
infancy, and adulthood (Table 16.3).
Hemoglobin (Hb) Gower I, Hb Gower II, and Hb
Portland predominate during embryonic life, while Hb
F predominates during fetal life. HbF starts to decline
after 36 weeks of gestation, and constitutes <1% of total
hemoglobin in adults. HbA becomes the main hemo-
globin after 3-4 months of age.
Steps in the synthesis of β globin chain of hemo-
globin A are shown in Figure 16.4.
Biosynthesis of heme is outlined in Chapter 31:
Laboratory Tests in Porphyrias.
The function of red cells is transport of oxygen and
carbon dioxide. Oxygen binds covalently and reversibly
with hemoglobin, with maximum 4 oxygen molecules
binding to four iron molecules (i.e. one molecule of
oxygen binds to each heme group). Oxygen affinity of
hemoglobin is affected by pH, temperature, and oxygen
pressure in capillaries.
Table 16.3: Types of hemoglobin during different periods of life
Type of hemoglobin
Hemoglobin Gower I
Hemoglobin Gower II
Hemoglobin Portland
Hemoglobin F (Fetal hemoglobin)
Hemoglobin A (Adult hemoglobin)
Hemoglobin A2
Red Cell Enzymes
Energy for various metabolic processes in the red cell
(transport of oxygen and carbon dioxide, membrane
pump, prevention of oxidant damage to hemoglobin, and
to convert methemoglobin to oxyhemoglobin) is
provided by glycolysis or Embden-Meyerhof pathway
and hexose monophosphate (HMP) shunt. A metabolic
shunt in glycolytic pathway is Rapoport-Luebering shunt
for generation of 2,3-diphosphoglycerate (2,3-DPG), an
important determinant for oxygen affinity of hemo-
globin. In HMP pathway, NADPH is produced that
reduces oxidized glutathione; reduced glutathione along
with glutathione peroxidase detoxifies hydrogen
peroxide and prevents oxidant damage to hemoglobin.
The enzyme glucose 6 phosphate dehydrogenase (G6PD)
is necessary for generation of NADPH (Fig. 16.5).
Red Cell Membrane
Red cell cytoskeletal proteins lie beneath the lipid bilayer
and consist of horizontally oriented spectrin hetero-
dimers (α and β spectrin intertwined together) that link
with ankyrin, proteins 4.1 and 4.2, and band 3 (vertical
elements) (Fig. 16.6). Cytoskeletal proteins maintain
shape and allow flexibility permitting red cells to traverse
through the capillaries of small diameter.
The lifespan of red cells is about 120 days, after which
they are ingested and degraded by mononuclear
phagocytes. Iron and amino acids are recycled for reuse,
while the remainder of the heme molecule is metabolized
to bilirubin and excreted by liver in bile.
Globin chains Period of life
when predominant
ζ 2ε2 Embryonic
α 2ε2 Embryonic
ζ 2γ2 Embryonic
α 2γ2 Fetal
α 2β2 Adult
α 2δ2 Adult
 Hematopoiesis 173

Fig. 16.4: (A) β globin gene cluster on chromosome 11; (B) Steps in the synthesis of β globin
polypeptide chain from β globin gene

Fig. 16.5: Role of glucose-6-phosphate dehydrogenase enzyme in detoxification of toxic peroxide. Abbreviations: HK: Hexokinase;
GSSG: Oxidized glutathione; GSH: Reduced glutathione; H2O2: Hydrogen peroxide; 6PGL: 6 Phosphogluconolaconase; 6PGD:
6 Phosphogluconate dehydrogenase; Ru5PI: Ribulose 5 phosphate isomerase
174 Essentials of Clinical Pathology

Fig. 16.6: Structure of red cell membrane

WHITE BLOOD CELLS Basophils

Neutrophils
Morphologically identifiable stages in maturation of
neutrophils are shown in Table 16.4 and Figure 16.7.
Neutrophils play an important role in acute infla-
mmation.
Eosinophils
Various stages in maturation of eosinophils are similar
to neutrophils with primary or specific granules first
appearing in myelocyte stage. The mature eosinophil (15-
16 μ) is slightly larger than a neutrophil, usually bilobed,
and contains numerous bright orange-red granules. The
eosinophil granules contain major basic protein that is
toxic to many parasites.
Stages in the maturation of basophils are similar to
neutrophils. Basophils are 9-12 μ in size, and their
cytoplasm is filled with coarse deep purple-black
granules that obscure the nucleus. Nucleus is segmented
into 2-3 lobes. The basophil granules contain histamine
and heparin. Basophils play a role in allergic and
anaphylactic reactions.
Monocytes
The maturation sequence is monoblast, promonocyte,
and monocyte (Fig. 16.8). Monocytes are the largest white
blood cells in peripheral blood (15-20 μ) with irregular
shape, oval or kidney-shaped nucleus, and fine reticular
chromatin. Cytoplasm is abundant, blue-gray with

Table 16.4: Stages in maturation of neutrophils

Cell Size (μ) Nucleus Cytoplasm

1. Myeloblast 15-20 Large, immature, fine dispersed chromatin, 2-5 nucleoli Scanty, light blue
2. Promyelocyte 16-20 Similar to myeloblast, slightly eccentric Azurophil granules
3. Myelocyte 14-18 Chromatin condensed, no nucleoli Specific granules
predominate
4. Metamyelocyte 14-18 Indented or kidney-shaped; peripheral clumping Faint pink; numerous
of chromatin specific granules
5. Band form 14-16 U-shaped or band-like nucleus with heavily Pink; numerous
clumped chromatin specific granules
6. Neutrophil 14-15 2-5 lobes joined by chromatin strands Pink; numerous
specific granules

Fig. 16.7: Stages of granulopoiesis
Fig. 16.8: Formation of monocytes
ground glass appearance and often contains fine
azurophil granules and vacuoles. After migration to
tissues, they are called as macrophages. Monocytes
function as phagocytic cells, antigen presenting cells, and
also produce a variety of cytokines.
Lymphocytes
There are three main types: B lymphocytes, T lympho-
cytes, and natural killer (NK) cells.
B lymphocytes comprise about 10-20% of lympho-
cytes in peripheral blood. In lymphoid organs they are
located in superficial cortex, germinal centers, and mantle
zone of lymph nodes, follicles in spleen, and mucosa-
associated lymphoid tissue (MALT) in gastrointestinal
and respiratory tracts. On antigen stimulation, B
lymphocytes differentiate into plasma cells that produce
immunoglobulins (antibodies).
T lymphocytes comprise 60-70% of circulating
lymphocytes. In lymphoid organs, they are located in
thymus, paracortex of lymph nodes, and periarteriolar
lymphoid sheaths in spleen. T lymphocytes are respon-
Hematopoiesis 175
sible for cell-mediated immunity; they also regulate
humoral response. The T lymphocyte precursor cell can
differentiate into (i) T helper inducer cells which
differentiate into TH1 and TH2 cells, (ii) T suppressor
cells, and (iii) cytotoxic T cells.
Natural killer cells comprise about 10-15% of
lymphocytes in peripheral blood. NK cells can attack and
kill virus-infected cells and some neoplastic cells without
previous sensitization.
Features of B and T lymphocytes are compared in
Table 16.5.
B cell development (B cell ontogeny): There are two stages
of B cell development: antigen-independent (occurring
in bone marrow) and antigen-dependent (occurring in
peripheral lymphoid organs). Heavy chain gene
rearrangement is followed by rearrangement of light
chain genes. Following expression of TdT and HLA-DR,
there is sequential appearance of antigens on the surface
of developing B cells: CD19, CD10, and CD20. Plasma
cells express CD38 antigen.
T cell development (T cell ontogeny): Pre-T cells are
transported from the bone marrow to the thymus, where
maturation occurs. Initially the immature thymocytes
express CD7, TdT, and cytoplasmic CD3. Subsequently,
both CD4 and CD8 antigens are acquired; with matu-
ration, either CD4 or CD8 is retained.
Normal mature white blood cells in peripheral blood
are shown in Figure 16.9.
Human Leukocyte Antigens
Human leukocyte antigens or HLA (so called because
they were first discovered on white blood cells) are any
Fig. 16.9: Normal mature white blood cells in
peripheral blood
176 Essentials of Clinical Pathology

Table 16.5: Comparison of T and B lymphocytes

Feature T lymphocytes B lymphocytes

1. Origin Stem cells in bone marrow Stem cells in bone marrow

2. Maturation
3. Name derivation
4. Lifespan
5. Differentiation
6. Function
7. Surface receptor
8. Fc and C3 receptor
9. Electron microscopy
10. Peripheral blood (%)
11. Location in lymph
nodes
12. Location in spleen
13. Immunological markers
Thymus Bone marrow, lymphoid organs
Thymus-derived, meaning Bone marrow-derived (however, the B
they mature in thymus stands for bursa of Fabricius, a lymphoid
organ of birds in which B cells were first
discovered)
Long-lived (2-4 years) Short-lived (weeks to a few months)
Helper, suppressor, and Plasma cells
cytotoxic cells
Cell-mediated immunity, Humoral immunity (Secretion of antibodies
regulation of function of B cells that circulate through humors or body fluids)
T cell receptor Immunoglobulin
Absent Present
Smooth surface Microvilli
60-70% 10-20%
Paracortex Germinal centers of follicles, medullary
cords
Periarteriolar lymphoid sheaths Germinal centers of follicles
CD1, CD2, CD3, CD4/CD8, CD5, CD19, CD20, CD21, CD22
CD6, CD7

of the numerous antigens that are encoded by a cluster

of genes on short arm of chromosome 6 (6p21) called as
major histocompatibility complex, that are extremely
polymorphic (Fig. 16.10). Each gene has an unusually
large number of alleles, so that it is very rare for two
individuals to have the same set of HLA molecules.
Alleles at the MHC loci are inherited in a codominant
Mendelian manner. HLA antigens are of three main
types: classes I, II, and III.
Class I HLA antigens: These include HLA-A, HLA-B, and
HLA-C with numerous alleles. They are associated non-
covalently with β2-microglobulin. They are expressed on
the surface of all nucleated cells. Their major function is
presentation of antigen to cytotoxic T cells. Class I
antigens are detected by lymphocytotoxicity test.
Class II antigens: These include HLA-DR, HLA-DQ, and
HLA-DP with numerous alleles. These consist of two
glycoprotein chains (α and β) bound non-covalently.
They are expressed primarily on antigen-presenting cells
like macrophages, B lymphocytes, and activated T Fig. 16.10: MHC complex (above) and HLA
lymphocytes. Their major function is antigen presen- antigens (below)

tation to T helper lymphocytes. Mixed lymphocyte
culture or mixed lymphocyte reaction is used for
detection of class II antigens. Primed lymphocyte typing
is used for detection of HLA-DP antigens.
Class III antigens: There are several class III genes that
encode certain complement components (C2, C4, and
Factor B) and cytokines (tumor necrosis factor).
Importance of HLA antigens
• Organ transplantation: The immune system relies on
HLA antigens to distinguish self from nonself. Kidney
and bone marrow transplantation depend heavily on
HLA antigen typing since these tissues express HLA
antigens to a great extent.
• Recognition of foreign antigens and immunity
• Association between certain HLA antigens and
diseases like ankylosing spondylitis, type 1 diabetes
mellitus, systemic lupus erythematosus, myasthenia
gravis, and Sjogren’s syndrome.
• Paternity testing.
Methods for HLA Antigen Typing
Serological Testing
1. Lympocytotoxicity test (Fig. 16.11): Lymphocytes are
separated from blood by density gradient centri-
fugation. Lymphocytes are then added to known
specific antisera (containing known specific anti-
bodies to HLA antigens). Complement is added to
Fig. 16.11: Principle of lymphocytotoxicity test
Hematopoiesis 177
lymphocyte-antiserum mixture. If corresponding
antigen is present on lymphocytes, cell damage and
death occurs due to antigen-antibody reaction
followed by complement activation. Dead and live
cells are differentiated by staining with a vital dye
(dead cells stain while live cells remain unstained). If
≥ 60% cells are stained, they are considered to be
positive for the particular HLA antigen.
2. Mixed lymphocyte culture or mixed lymphocyte reaction:
Lymphocytes from two different individuals are
obtained. Lymphocytes from one individual are
inactivated to suppress their division; these lympho-
cytes are called stimulator cells. The lymphocytes
from two individuals are cultured together. If
antigens on stimulator cells are foreign to the
responder lymphocytes (i.e. to lymphocytes from
other individual), responder cells show proliferation
and synthesis of DNA (blastogenic response). DNA
synthesis is quantitated by incorporation of 3thymi-
dine.If antigens on both responder and stimulator
cells are similar there is no blastogenic response.
3. Primed lymphocyte typing: This is a modification of
mixed lymphocyte culture. The culture of lympho-
cytes is extended during which stimulator cells die
and responder cells cease to proliferate. When
responder cells are re-exposed to the same antigen-
bearing cells as stimulator cells, rapid proliferation
of responder cells occurs.
Molecular Genetic Techniques
1. Restriction fragment length polymorphism: This method
relies on recognition of certain DNA nucleotide
sequences by restriction enzymes and cutting the
DNA at these points. For example, DNA for one HLA
antigen will have a different restriction site as
compared to DNA for another HLA antigen so that
fragments of different sizes are obtained with the
same enzyme which can be determined by electro-
phoresis.
2. Polymerase chain reaction (PCR): Various steps in this
technique are: (1) DNA is obtained from the nuclei of
white blood cells; (2) Double-stranded DNA is
denatured into single-stranded DNA; (3) An oligo-
nucleotide primer sequence (‘probe’) complementary
to a small segment of only one HLA allele is chosen,
that will attach to single stranded DNA; (3) After
attachment, that part of DNA becomes double
stranded (after addition of DNA polymerase and
deoxyrionucleotide triphosphates); (4) Thus two
copies of DNA of interest are obtained; the double
178 Essentials of Clinical Pathology

stranded DNA is again denatured and associated

with primers to yield four copies; the cycle is repeated
to obtain sufficient amount of DNA to be elctro-
phoresed and typed. (5) If any DNA is detected on
agarose gel electrophoresis, particular HLA is present.
A number of PCR-based methods are used like
sequence specific priming and sequence specific
oligonucleotide priming.

THROMBOPOIESIS
Platelets are produced by cytoplasmic fragmentation of
large cells in bone marrow called as megakaryocytes,
which in turn are derived from megakaryoblasts. The
morphologically identifiable stages in thrombopoiesis
are: megakaryoblast, promegakaryocyte, megakaryocyte,
and platelets (Fig. 16.12). Although nucleus of a mega-
karyocyte divides, cell division does not occur so that a
very large cell with multiple nuclear lobes (increased
ploidy) is formed (the process whereby nuclear DNA is
duplicated without cell division is called as endomitosis).
Megakaryocyte is the largest normally occurring hemato-
poietic cell in the bone marrow. Each megakaryocyte
produces about 4000 platelets during its lifespan. The
primary regulator of megakaryocyte differentiation is
thrombopoietin (secreted by liver and stromal cells of
bone marrow). A mature megakaryocyte extends
Fig. 16.12: Stages of platelet formation. The morphologically
recognizable stages are megakaryoblast (high nucleo-
cytoplasmic ratio with basophilic cytoplasm), promegakaryocyte
(nucleus typically horse-shoe shaped), and megakaryocyte
(cytoplasm granular, nucleus highly lobular). Megakaryocytes
undergo endomitosis and are the largest cells in the bone
marrow
cytoplasmic processes through the sinusoidal wall in
marrow and platelets are released directly in bloodstream
by fragmentation of cytoplasm. The lifespan of platelets
is about 7-10 days.
BIBLIOGRAPHY
1. Hoffbrand AV, Moss PAH, Pettit JE (Eds). Essential
Hematology (5th Ed). Blackwell Publishing Ltd, 2006.
2. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
 17

Collection of Blood

For reliable and accurate results of laboratory tests, it is

essential to follow a standard procedure for specimen
collection. For hematological investigations, blood
sample can be obtained from the skin puncture or
venepuncture.

SKIN PUNCTURE
This method is commonly used in infants and small
children and if the amount of blood required is small. It
is suitable for cell counts, estimation of hemoglobin,
determination of hematocrit by micro method, and
preparation of blood films.
Blood obtained by skin puncture is also called as
capillary blood. However, it is a mixture of blood from
capillaries, venules, and arterioles. It also contains some
tissue fluid.
In adults, blood is obtained from the side of a ring or
middle finger (distal digit) or ear lobe. In infants, it is
collected from the heel (lateral or medial aspect of plantar Fig. 17.1: (A) Blood lancet and sites of (B) finger
surface) or great toe (Fig. 17.1). puncture (cross) and (C) heel puncture (shaded areas)
The puncture site is cleansed with 70% ethanol or
other suitable disinfectant. After drying, a puncture,
VENOUS BLOOD COLLECTION
sufficiently deep to allow free flow of blood, is made with
a sterile, dry, disposable lancet. The first drop of blood is When multiple tests are to be done and larger quantity
wiped away with sterile, dry cotton as it contains tissue of blood is needed, anticoagulated venous blood should
fluid. Next few drops of blood are collected. Excessive be obtained.
squeezing should be avoided, as it will dilute the blood
Method
with tissue fluid. After collection a piece of sterile cotton
is pressed over the puncture site till bleeding ceases. 1. Due to the ease of access, blood is best obtained from
As compared to the venous blood, hemoglobin, the veins of the antecubital fossa (Fig. 17.2). A rubber
hematocrit, and red cell count are slightly higher in blood tourniquet (18 inches long × 3/4 or 1 inch in adults
from skin puncture. As platelets adhere to the puncture and 12 inches × 1/8 inch in children) is applied to
site, platelet count is lower. Because of small sample size, the upper arm. It should not be too tight and should
immediate repeat testing is not possible if the result is not remain in place for more than two minutes.
abnormal. Patient is asked to make a fist so that veins become
Blood should not be collected from cold, cyanosed more prominent and palpable.
skin since false elevation of values of hemoglobin and 2. Venepuncture site is cleansed with 70% ethanol and
red/white cell counts will be obtained. allowed to dry.
180 Essentials of Clinical Pathology
Fig. 17.2: Common sites of venepuncture in antecubital
fossa (red circles)
3. The selected vein is anchored by compressing and
pulling the soft tissues below the puncture site with
the left hand.
4. Sterile, disposable needles and syringes should be
used for venepuncture. Needle size should be 19- to
21-gauge in adults and 23-gauge in children.
Venepuncture is performed with the bevel of the
needle up and along the direction of the vein. Blood
is withdrawn slowly. Pulling the plunger quickly can
cause hemolysis and collapse of the vein.
Tourniquet should be released as soon as the blood
begins to flow into the syringe.
5. When the required amount of blood is withdrawn,
the patient is asked to open his/her fist. The needle
is withdrawn from the vein. A sterile cotton gauze is
pressed over the puncture site. Patient is asked to
press the gauze over the site till bleeding stops.
6. The needle is detached from the syringe and the
required amount of blood is carefully delivered into
the tube containing appropriate anticoagulant (see
later). If the blood is forced through the needle
without detaching it, hemolysis can occur. Containers
may be glass bottles or disposable plastic tubes with
caps and flat bottom.
7. Blood is mixed with the anticoagulant in the container
thoroughly by gently inverting the container several
times. The container should not be shaken vigorously
as it can cause frothing and hemolysis.
Check whether the patient is feeling faint and
bleeding has stopped. Cover the puncture site with an
adhesive bandage strip.
After use, disposable needles should be placed in a
puncture-proof container for proper disposal. Recapping
of needle by hand can cause needle-stick injury.
The container is labeled. Time of collection should be
noted on the label. Sample should be sent immediately
to the laboratory with accompanying properly filled
order form.
Precautions
1. Blood is never collected from an intravenous line or
from the arm being used for intravenous line (since
it will dilute the blood sample). Blood is not collected
from a sclerosed vein and from an area with hema-
toma.
2. Tourniquet should not be too tight and should not be
applied for more than 2 minutes as it will cause
hemoconcentration and alteration of test results.
3. Puncture site should be allowed to dry completely
after cleaning with alcohol (before performing the
venepuncture).
4. Tourniquet should be released before removing the
needle from the vein (to prevent hematoma
formation).
5. To avoid hemolysis, blood is withdrawn gradually, a
small-bore needle should not be used, and the needle
is detached from the syringe before dispensing blood
into the container.
6. All blood samples are considered as infectious and
proper precautions should be observed while
collecting blood either from a vein or a skin puncture.
Anticoagulated blood sample should be tested within
1-2 hours of collection. If this is not possible, sample can
be stored in a refrigerator at 4-6°C for maximum of 24
hours. After the sample is taken out of refrigerator, it
should be allowed to return to room temperature, mixed
properly, and then tested.
Complications
1. Failure to obtain blood: This happens if vein is missed,
or excessive pull is applied to the plunger causing
collapse of the vein.
2. Occurrence of hematoma, thrombosis, thrombo-
phlebitis, abscess, or bleeding.
3. Transmission of infections like hepatitis B or human
immunodeficiency virus if reusable needles and
syringes, which are not properly sterilised, are used.
 Collection of Blood 181

ANTICOAGULANTS
Anticoagulants used for hematological investigations are
ethylene diamine tetra-acetic acid (EDTA), heparin,
double oxalate, and trisodium citrate (Table 17.1).

Ethylene Diamine Tetra-acetic Acid (EDTA)

This is also called as Sequestrene or Versene. This is the
recommended anticoagulant for routine hematological
investigations. However, it cannot be used for coagu-
lation studies. Disodium and dipotassium salts of EDTA
are in common use. International Committee for
Standardization in Hematology recommends dipo-
tassium EDTA since it is more soluble. It is used in a
concentration of 1.5 mg/ml of blood. Dried form of
anticoagulant is used as it avoids dilution of sample. Its
mechanism of action is chelation of calcium.
Proportion of anticoagulant to blood should be
maintained. EDTA in excess of 2 mg/ml causes shrinkage
of and degenerative changes in red and white blood cells,
decrease in hematocrit, and increase in mean corpuscular
hemoglobin concentration. Excess EDTA also causes
swelling and fragmentation of platelets, which leads to
erroneously high platelet counts.
Prolonged storage of blood in EDTA anticoagulant
leads to alterations as shown in Figure 17.3 and Box 17.1.
EDTA is used for estimation of hemoglobin, hema-
tocrit, cell counts, making blood films, sickling test,
reticulocyte count, and hemoglobin electrophoresis.
Preparation
Dipotassium ethylene 20 gm
diamine tetra-acetic acid
Distilled water 200 ml
Fig. 17.3: Changes in blood cell morphology (crenation of red
cells, separation of nuclear lobes of neutrophil, vacuoles in
cytoplasm, and irregular lobulation of monocyte and lymphocyte
nuclei) due to storage of blood in EDTA anticoagulant for
prolonged time
Box 17.1: Changes occurring due to prolonged storage
of blood in EDTA
• Blood smear:
– Red cells: Crenation and spherocytic change
– Neutrophils: Separation of nuclear lobes, loss of
granules, vacuoles in cytoplasm
– Monocytes and lymphocytes: Irregular lobulation
of nucleus, vacuoles in cytoplasm
– Platelets: Disintegration
• Hematological tests:
– Packed cell volume and mean cell volume:
Increase due to red cell swelling
– Osmotic fragility: Increases
– Total leucocyte and platelet counts: Progressive
decrease

Table 17.1: Salient features of three main anticoagulants used in the hematology laboratory

Name Mode of action Concentration Main uses Disadvantages

1. EDTA Chelation of calcium 1.5 mg/ml Complete blood Cannot be used for
count, blood smear, coagulation tests; can cause
hemoglobin pseudothrombocytopenia on
electrophoresis, hematology analyzer
sickling test
2. Heparin Inhibition of thrombin 15 U/ml Osmotic fragility test, Causes blue background of
activity immunophenotyping blood smears; expensive
3. Sodium citrate Chelation of calcium 0.109 mg/ml Coagulation studies, Liquid anticoagulant: causes
estimation of ESR by dilution and cannot be used
Westergren method for complete blood counts
182 Essentials of Clinical Pathology
Mix to dissolve. Place 0.04 ml of this solution in a
bottle for 2.5 ml of blood. Anticoagulant should be dried
on a warm bench or in an incubator at 37°C before use.
For routine hematological investigations, 2-3 ml of
EDTA blood is required.
Heparin
Heparin prevents coagulation by enhancing the activity
of antithrombin III (AT III). AT III inhibits thrombin and
some other coagulation factors. It is used in the
proportion of 15-20 IU/ ml of blood. Sodium, lithium, or
ammonium salt of heparin is used.
Heparin should not be used for total leukocyte count
(since it causes leukocyte clumping) and for making of
blood films (since it imparts a blue background).
It is used for osmotic fragility test (since it does not
alter the size of cells) and for immunophenotyping.
Double Oxalate (Wintrobe Mixture)
This consists of ammonium oxalate and potassium
oxalate in 3:2 proportion. This combination is used to
balance the swelling of red cells caused by ammonium
oxalate and shrinkage caused by potassium oxalate.
Mechanism of anticoagulant action is removal of calcium.
It is used for routine hematological tests and for
estimation of erythrocyte sedimentation rate by Wintrobe
method.
As it causes crenation of red cells and morphologic
alteration in white blood cells, it cannot be used for
making of blood films.
Preparation
Ammonium oxalate 1.2 gm
Potassium oxalate 0.8 gm
Distilled water upto 100 ml
Place 0.5 ml of this solution in a bottle for 5 ml of
blood. Anticoagulant should be dried in an incubator at
37°C or on a warm bench before use.
Trisodium Citrate (3.2%)
This is the anticoagulant of choice for coagulation studies
and for estimation of erythrocyte sedimentation rate by
Westergren method.
Preparation
Trisodium citrate 3.2 gm
Distilled water upto 100 ml
Mix well to dissolve. Store in a refrigerator at 2-8°C.
Use 1:9 (anticoagulant: blood) proportion for coagu-
lation studies; for ESR, 1:4 proportion is recommended.
ESR should be measured within 4 hours of collection
of blood, while coagulation studies should be performed
within 2 hours.
Other Tubes for Collection of Blood
Plain tubes (i.e. without any anticoagulant) are used for
chemistry studies after separation of serum: liver function
tests (total proteins, albumin, aspartate aminotransferase,
alanine aminotransferase, bilirubin), renal function tests
(blood urea nitrogen, creatinine), calcium, lipid profile,
electrolytes, hormones, and serum osmolality.
Fluoride bulb is used for collection of whole blood
for estimation of blood glucose. Addition of sodium
fluoride (2.5 mg/ml of blood) maintains stable glucose
level by inhibiting glycolysis. Sodium fluoride is
commonly used along with an anticoagulant such as
potassium oxalate or EDTA.
SEQUENCE OF FILLING OF TUBES
Following order of filling of tubes should be followed
after withdrawal of blood from the patient if multiple
investigations are ordered:
1. First tube: Blood culture.
2. Second tube: Plain tube (serum).
3. Third tube: Tube containing anticoagulant (EDTA,
citrate, or heparin).
4. Fourth tube: Tube containing additional stabilizing
agent like fluoride.
USE OF PLASMA VS. SERUM
Plasma is the supernatant liquid obtained after centrifu-
gation of anticoagulated whole blood. Serum is the liquid
obtained after clotting of whole blood sample collected
in a plain tube. Some of the differences between the two
are as follows:
1. Plasma contains fibrinogen as well as all the other
proteins, while serum does not contain fibrinogen.
2. Plasma can be obtained immediately after sample
collection by centrifugation, while minimum of 30
minutes are required for separation of serum from
the clotted blood.
3. Amount of sample is greater with plasma than with
serum for a given amount of blood.
4. Use of anticoagulant may alter the concentration of
some constituents if they are to be measured like
sodium, potassium, lithium, etc.
BIBLIOGRAPHY
1. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
Hematology. 9th Ed. London: Churchill Livingstone,
2001.
2. World Health Organization. Manual of Basic Techni-
ques for a Health Laboratory. 2nd Ed. Geneva: World
Health Organization, 2003.
3. World Health Organization. Use of anticoagulants in
diagnostic laboratory investigations. 2002. WHO/DIL/
LAB/99.1 Rev.2
 18
Estimation of Hemoglobin
Hemoglobin carries oxygen from the lungs to the tissues
and carbon dioxide from tissues to the lungs. It is
composed of heme (iron + protoporphyrin) and globin
polypeptide chains. Hemoglobin is not homogeneous
and normally different variants and derivatives exist.
Normal hemoglobin variants are embryonic hemo-
globins (Gower I, Gower II, and Portland), fetal hemo-
globin (Hb F), adult hemoglobin (Hb A), and Hb A2.They
differ from each other in the type of polypeptide chains
(Box 18.1).
INDICATIONS FOR HEMOGLOBIN
ESTIMATION
1. To determine presence and severity of anemia:
Anemia refers to low hemoglobin concentration or
oxygen-carrying capacity of blood. Clinical signs of
anemia (pallor of skin, conjunctival vessels, or
mucous membranes) are unreliable for diagnosis of
anemia. Anemia is best assessed by estimation of
hemoglobin or packed cell volume (See Chapter 27:
Laboratory Tests in Anemia).
Box 18.1: Hemoglobin
• Normal physiological hemoglobins: Oxyhemoglobin,
Reduced hemoglobin (i.e. iron of heme not associated with
oxygen)
• Hemoglobin derivatives: Compounds formed from
physiological hemoglobins through the action of acids,
alkalis, oxidizing/reducing agents, etc. They are identified
by their characteristic absorption spectra in a spectro-
photometer.
• Hemoglobin variants: Different structural forms of
hemoglobin that differ in structure of polypeptide chains.
They are identified by hemoglobin electrophoresis.
– Normal: HbA (α2β2), HbF (α2γ2), HbA2 (α2δ2)
– Abnormal: HbS, HbC, HbD, HbE, etc.
Box 18.2: Polycythemia
• Primary: Increase in total red cell mass with low
erythropoietin level e.g. polycythemia vera
• Secondary: Increase in total red cell mass with
increased erythropoietin level, e.g. hypoxia
• Relative: Decrease in plasma volume with red cell
mass remaining normal, e.g. dehydration.
2. Screening for polycythemia: Polycythemia refers to
increased hemoglobin level above the normal range.
It may be primary, secondary, or relative (Box 18.2).
3. To assess response to specific therapy in anemia.
4. Estimation of red cell indices (along with packed cell
volume and red cell count) i.e. mean cell hemoglobin
and mean cell hemoglobin concentration.
5. Selection of blood donors.
METHODS FOR ESTIMATION OF
HEMOGLOBIN
There are various methods for estimation of hemoglobin.
These are:
1. Colorimetric methods: In these methods, color
comparison is made between the standard and the
test sample, either visually or by colorimetric
methods.
• Visual methods: Tallqvist chart, Sahli’s acid
hematin method, and WHO hemoglobin color
scale.
• Photoelectric methods: Cyanmethemoglobin (hemi-
globincyanide) method, oxyhemoglobin method,
and alkaline hematin method.
2. Gasometric method: Oxygen-carrying capacity of
blood is measured in a Van Slyke apparatus. The
amount of hemoglobin is then derived from the
formula that 1 gram of hemoglobin carries 1.34 ml of
oxygen. However, this method measures only physio-
logically active hemoglobin, which can carry oxygen.
184 Essentials of Clinical Pathology

It does not measure carboxyhemoglobin, sulfhemo-

globin, and methemoglobin. Also, this method is
time-consuming and expensive. The result is about
2% less than other methods.
3. Chemical method: Iron-content of hemoglobin is first
estimated. Value of hemoglobin is then derived
indirectly from the formula that 100 grams of
hemoglobin contain 374 mg of iron. This method is
tedious and time-consuming.
4. Specific gravity method: A rough estimate of
hemoglobin is obtained from the specific gravity of
blood as determined from copper sulphate technique.
This method is useful in mass screening like selection
of blood donors.

Tallqvist Hemoglobin Chart

Tallqvist hemoglobin chart consists of a series of
lithographed colors said to correspond to hemoglobin
values ranging from 10 to 100%. A drop of blood obtained
by finger puncture is placed on a piece of absorbent
paper. Color produced is matched against the color on
the chart and corresponding reading is taken. Although
this method is cheap and simple, margin of error is 20-
50%.
Sahli’s Acid Hematin Method
Principle: Blood is mixed with an acid solution so that
hemoglobin is converted to brown-colored acid hematin.
This is then diluted with water till the brown color
matches that of the brown glass standard. The
hemoglobin value is read directly from the scale.
Equipment (Fig 18.1)
1. Sahli’s hemoglobinometer: This consists of Sahli’s
graduated hemoglobin tube (marked in grams and
percent) and a comparator with a brown glass
standard.
2. Sahli’s pipette or hemoglobin pipette (marked at 20 μl
or 0.02 ml).
3. Small glass rod (stirrer).
4. Dropping pipette.
Reagents
1. N/10 hydrochloric acid
2. Distilled water
Specimen: EDTA-anticoagulated venous blood or blood
obtained by skin puncture.
Method
1. Place N/10 hydrochloric acid into Sahli’s graduated
hemoglobin tube up to the mark of 2 grams.
Fig. 18.1: Sahli’s hemoglobinometer
2. Take blood sample in Sahli’s pipette exactly up to 20
μl mark. Blood adhering to the exterior of the pipette
is wiped away using absorbent paper or gauze.
3. Add blood sample to the acid solution, mix with a
glass stirrer, and allow to stand for 10 minutes.
4. Add distilled water drop by drop till the color of the
solution matches that of the brown glass standard.
5. Take the reading of the lower meniscus from the
graduated tube in grams.
Note:
1. Although the graduated tube is marked in both grams
and percent figures, result should always be reported
in grams. This is because (a) no single hemoglobin
value can be considered as 100% since it varies with
the age and sex of the individual and altitude, and
(b) hemoglobinometers of different manufacturers
have different values as 100%, so that same sample
of blood will yield different results on different
instruments.
2. Disadvantages of Sahli’method:
• About 95% color of acid hematin is attained at the
end of 10 minutes. For maximum color develop-
ment, much longer time (1 hour) is required.
• Perfect matching with the brown glass standard
is not possible.
• Carboxyhemoglobin, methemoglobin, and sulf-
hemoglobin are not converted to acid hematin.
HbF is also not converted to acid hematin and
therefore this method is not suitable in small
infants.

• Development of color is slow and acid hematin
solution is not stable.
• Source of light (daylight or artificial) will influence
the visual comparison of colors.
• Personal error in matching brown glass standard
with test solution is 10%.
• Color of brown glass standard fades with time.
WHO Hemoglobin Color Scale
This method, devised by Stott and Lewis (1995), is similar
in principle to the now obsolete Tallqvist method. Certain
technical modifications have been made to improve the
accuracy and reliability. This method is rapid, simple,
inexpensive, and reliable within 1 gram/dl for diagnosis
of anemia. The hemoglobin color scale consists of a
printed set of colors corresponding to hemoglobin values
ranging from 4-14 grams/dl (Fig. 18.2). A drop of blood
is placed on a strip of chromatography paper and the
color developed is matched visually against the printed
color scale. Studies have shown that efficiency is greater
than 90% in detecting anemia and 86% in classifying its
grade. Hemoglobin color scale has been developed by
Fig. 18.2: Hemoglobin color scale
Estimation of Hemoglobin 185
World Health Organization after extensive field trials. It
is mainly intended for detection and management of
anemia in under-resourced countries. It is especially
useful for screening of blood donors, for screening
children and women in health programs, monitoring iron
therapy, decision- making regarding referral to a hospital,
and as a point of care device.
Cyanmethemoglobin (Hemiglobincyanide) Method
This is the method of choice for estimation of hemoglobin
and is recommended by International Committee for
Standardization in hematology. This is because (i) all
forms of hemoglobin are converted to cyanmethemo-
globin (except sulfhemoglobin), and (ii) a stable and
reliable standard is available.
Principle
Blood is mixed with a solution of potassium ferricyanide,
potassium cyanide and a non-ionic detergent (Drabkin’s
solution). Erythrocytes are lysed producing an evenly
distributed hemoglobin solution. Potassium ferricyanide
converts hemoglobin to methemoglobin, and methemo-
globin combines with potassium cyanide to form
cyanmethemoglobin (hemiglobincyanide). All forms of
hemoglobin present in blood are completely converted
to a single compound, cyanmethemoglobin. When the
reaction is completed, absorbance of the solution is
measured in a spectrophotometer at 540 nm. At this
wavelength, cyanmethemoglobin has a broad absorbance
peak. To obtain the amount of hemoglobin in the
unknown sample, its absorbance is compared with that
of the standard cyanmethemoglobin solution (the
hemoglobin concentration of which is known) by using
a formula or a previously prepared graph/table. The
reaction is linear up to 20 gm/dl.
Equipment
1. Photoelectric colorimeter or spectrophotometer
2. Sahli’s pipette marked at 20 μl (20 cmm).
3. Pipette 5 ml.
Reagents
1. Drabkin’s solution (pH 7.0-7.4):
Potassium ferricyanide 200 mg
Potassium cyanide 50 mg
Potassium dihydrogen phosphate 140 mg
Non-ionic detergent 1 ml
Distilled water to 1000 ml.
2. Cyanmethemoglobin standard solution with known
hemoglobin value
186 Essentials of Clinical Pathology

Specimen: EDTA-anticoagulated venous blood or blood

obtained from skin puncture.

Method
1. In a test tube, take 5 ml of Drabkin’s solution and to
it add 20 μl of blood (1:251 dilution). Stopper the tube,
mix by inverting several times, and allow to stand
for atleast 5 minutes. This time is adequate for
conversion of hemoglobin to cyanmethemoglobin.
2. Transfer the test sample to a cuvette. Read the
absorbance in a spectrophotometer at 540 nm or in a
photoelectric colorimeter using a yellow-green filter.
Also take the absorbance of the standard solution.
Absorbance should be read against reagent blank
(Drabkin’s solution).
3. Hemoglobin value is derived from the formula given
below or from the previously prepared graph or table.

Fig. 18.3: Standard curve for determining hemoglobin

Absorbance of test sample
Hemoglobin in gm/dl = × concentration by cyanmethemoglobin method
Absorbance of standard

Dilution factor
Concentration of standard ×
100
Preparation of graph and table: If a graph or a table is
prepared which correlates absorbance with hemoglobin
concentration, result can be obtained quickly. This is
particularly suitable when a large number of samples
are regularly processed on the same instrument.
Diluted cyanmethemoglobin standards are available
commercially for preparation of a calibration graph.
Alternatively, standard cyanmethemoglobin solution is
diluted serially with Drabkin’s solution. On a linear
graph paper, hemoglobin concentration (horizontal axis)
in each dilution is plotted against the absorbance (vertical
axis). A straight line joining the points and passing
through the origin is obtained. From this graph, a table
can be prepared relating absorbance to hemoglobin
concentration (Fig. 18.3).
Notes:
1. Lipemic blood (hypertriglyceridemia), high total
leukocyte count (> 25,000/μl), or abnormal plasma
proteins (e.g. in multiple myeloma, Waldenström’s
macroglobulinemia) can cause erroneous results.
2. Cyanmethemoglobin solution is stable so that delay
in taking the reading of absorbance does not affect
the result.
Oxyhemoglobin Method
In this method, blood sample is mixed with a weak
ammonia solution. Absorbance of this solution is
measured in a spectrophotometer at 540 nm or in a
photometer using a yellow-green filter. Absorbance of
the test sample is compared with that of the standard
solution.
This method is rapid and simple. However, no stable
standard solution is available, color of oxyhemoglobin
solution rapidly fades, and hemoglobin derivatives other
than oxyhemoglobin are not measured.
Specific Gravity Method
This is a rapid and simple method, which gives approxi-
mate hemoglobin value. It is commonly used for selection
of blood donors in blood banks.
A drop of blood is allowed to fall in copper sulphate
solution of specific gravity 1.053 from a height of 1 cm.
Specific gravity of 1.053 is equivalent to hemoglobin
concentration of 12.5 grams/dl. The drop of blood gets
covered with copper proteinate and remains discrete for
15-20 seconds. If the drop sinks within this time, its
specific gravity is higher than that of copper sulphate
solution (i.e. hemoglobin is >12.5 grams/dl) and
hemoglobin level is acceptable for donation. If it floats,
hemoglobin level is unacceptable. However, specific
gravity of whole blood is also affected by total leukocyte

count and concentration of plasma proteins. In the
presence of leukocytosis (e.g. as in chronic myeloid
leukemia) or hypergammaglobulinemia (e.g. multiple
myeloma), hemoglobin value will be misleadingly high.
GENERAL REMARKS
• The cyanmethemoglobin method is the most accurate
method for estimation of hemoglobin.
• If anemia is suspected, it is prudent to measure
packed cell volume and red cell count along with
hemoglobin concentration. This will be useful in
assessing the correctness of hemoglobin value and in
calculation of red cell indices (for morphological
classification of anemia). Packed cell volume is
roughly three-times the hemoglobin value. This rule,
however, does not apply to hypochromic anemia
since red cells contain lower hemoglobin for their size.
• Hemoglobin level is decreased in anemia, recumbent
position (by 5-6%), excess squeezing during finger
puncture, presence of clots in the sample, inadequate
mixing of blood with anticoagulant, and “spurious”
anemia. Causes of “spurious” or “pseudo” anemia
are increased plasma volume in third trimester of
pregnancy, pooling of red cells in splenomegaly, fluid
retention in congestive cardiac failure, and rise in
plasma proteins in paraproteinemias. Hemoglobin
level is increased following strenuous exercise, at high
altitude, in hemoconcentration (e.g. dehydration),
prolonged application of tourniquet during vene-
puncture, and in polycythemia.
• On most automated hematology analyzers, hemo-
globin is measured as cyanmethemoglobin.
Estimation of Hemoglobin 187
• Anemia is graded as mild (hemoglobin value from
lower limit of normal range to 10.0 grams/dl),
moderate (7.0-10.0 grams/dl), and severe (< 7.0
grams/dl).
REFERENCE RANGES (WORLD HEALTH
ORGANIZATION)
• Adult males: 13.0 - 17.0 gm/dl.
• Adult females (non-pregnant): 12.0 – 15.0 gm/dl.
• Adult females (pregnant): 11.0 - 14.0 gm/dl.
• Children, 6-12 years: 11.5 - 15.5 gm/dl.
• Children, 6 months to 6 years: 11.0 – 14.0 gm/dl.
• Children, 2 – 6 months: 9.5 – 14.0 gm/dl.
• At birth (full term): 13.6 – 19.6 gm/dl.
CRITICAL VALUES
• < 7 gm/dl (severe anemia)
• > 20 gm/dl (hyperviscosity)
BIBLIOGRAPHY
1. Cheesbrough M. District Laboratory Practice in Tropical
Countries. Part 2. Cambridge: Cambridge University
Press, 1998.
2. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
Hematology (9th Ed). London: Churchill Livingstone,
2001.
3. Wallach J. Interpretation of Diagnostic Tests (7th Ed).
Philadelphia: Lippincott Williams & Wilkins, 2000.
4. World Health Organization. Manual of Basic Techni-
ques for a Health Laboratory (2nd Ed). Geneva: World
Health Organization, 2003.
 19
Packed Cell Volume
Packed cell volume (PCV) is the volume occupied by the
red cells when a sample of anticoagulated blood is
centrifuged. It indicates relative proportion of red cells
to plasma. PCV is also called as hematocrit or erythrocyte
volume fraction. It is expressed either as a percentage of
original volume of blood or as a decimal fraction.
USES OF PCV
• Detection of presence or absence of anemia or
polycythemia
• Estimation of red cell indices (mean cell volume and
mean corpuscular hemoglobin concentration)
• Checking accuracy of hemoglobin value (Hemoglobin
in grams/dl × 3 = PCV).
There are two methods for estimation of PCV: macro
method (Wintrobe method) and micro method (micro-
hematocrit method). Micro method is preferred because
it is rapid, convenient, requires only a small amount of
blood, capillary blood from skin puncture can be used,
and a large number of samples can be tested at one time.
This method is also more accurate as plasma trapping in
red cell column is less.
MACRO METHOD (WINTROBE METHOD)
Principle
Anticoagulated whole blood is centrifuged in a Wintrobe
tube to completely pack the red cells. The volume of
packed red cells is read directly from the tube.
An advantage with this method is that before
performing PCV, test for erythrocyte sedimentation rate
can be set up.
Equipment
1. Wintrobe tube: This tube is about 110 mm in length
and has 100 markings, each at the interval of 1 mm.
Internal diameter is 3 mm. It can hold about 3 ml of
blood.
2. Pasteur pipette with a rubber bulb and a sufficient
length of capillary to reach the bottom of the Wintrobe
tube.
3. Centrifuge with a speed of 2300 g.
Specimen
Venous blood collected in EDTA (1.5 mg EDTA for 1 ml
of blood) or in double oxalate. Test should be performed
within 6 hours of collection.
Method
1. Mix the anticoagulated blood sample thoroughly.
2. Draw the blood sample in a Pasteur pipette and
introduce the pipette up to the bottom of the Wintrobe
tube. Fill the tube from the bottom exactly up to the
100 mark. During filling, tip of the pipette is raised,
but should remain under the rising meniscus to avoid
foaming.
3. Centrifuge the sample at 2300 g for 30 min (To
counterbalance a second Wintrobe tube filled with
blood from another patient or water should be placed
in the centrifuge).
4. Take the reading of the length of the column of red
cells. Hematocrit can be expressed either as a
percentage or as a fraction of the total volume of blood
sample.
Significance
In anemia, PCV is below the lower level of normal range.
PCV is raised in dehydration, shock, burns, and
polycythemia.
After centrifugation of anticoagulated whole blood,
three zones can be distinguished in the Wintrobe tube
from above downwards- plasma, buffy coat layer (a small
 Packed Cell Volume 189

Fig. 19.1: Anticoagulated blood-filled Wintrobe hematocrit tubes Fig. 19.2: Preparation of smear from buffy coat
after centrifugation, showing normal PCV, low PCV (anemia),
and thick buffy coat layer

greyish layer of white cells and platelets, about 1 mm

thick), and packed red cells (Fig. 19.1). Normal plasma is
straw-colored. It is colorless in iron deficiency anemia,
pink in the presence of hemolysis or hemoglobinemia,
and yellow if serum bilirubin is raised (jaundice). In
hypertriglyceridemia, plasma appears milky. Increased
thickness of buffy coat layer occurs if white cells or
platelets are increased in number (e.g. in leukocytosis,
thrombocytosis, or leukemia). Smears can be made from
the buffy coat layer (Fig. 19.2) for demonstration of lupus
erythematosus (LE) cells (Fig. 19.3), malaria parasites, or
immature cells.

MICRO METHOD Fig. 19.3: Lupus erythematosus or LE cell (red arrow) in buffy
coat smear. LE cell is a neutrophil with a homogeneous red
Principle purple inclusion distending the cytoplasm. It is seen in collagen
disorders, drug reactions, chronic hepatitis, and serum sickness
Anticoagulated whole blood is centrifuged in a capillary
tube of uniform bore to pack the red cells. Centrifugation
is done in a special microhematocrit centrifuge till
packing of red cells is as complete as possible. The
reading (length of packed red cells and total length of
the column) is taken using a microhematocrit reader, a
ruler, or arithmetic graph paper (Fig. 19.4).

Equipment
1. Microhematocrit centrifuge: It should provide relative
centrifugal force of 12000 g for 5 minutes.
2. Capillary hematocrit tubes: These are disposable glass Fig. 19.4: Microhematocrit capillary tubes following
tubes 75 mm in length and 1 mm in internal diameter. centrifugation in a microhematocrit centrifuge
190 Essentials of Clinical Pathology
They are of two types: plain (containing no anticoagu-
lant) and heparinised (coated with a dried film of 2
units of heparin). For plain tubes, anticoagulated
venous blood is needed. Heparinised tubes are used
for blood obtained from skin puncture.
3. Tube sealant like plastic sealant or modeling clay; if
not available, a spirit lamp for heat sealing.
4. Microhematocrit reader; if not available, a ruler or
arithmetic graph paper.
Specimen
Venous blood collected in EDTA (dipotassium salt) for
plain tubes or blood from skin puncture collected directly
in heparinised tubes. Venous blood should be collected
with minimal stasis to avoid hemoconcentration and
false rise in PCV.
Method
1. Fill the capillary tube by applying its tip to the blood
(either from skin puncture or anticoagulated venous
blood, depending on the type of tube used). About
2/3rds to 3/4ths length of the capillary tube should
be filled with blood.
2. Seal the other end of the capillary tube (which was
not in contact with blood) with a plastic sealant. If it
is not available, heat-seal the tube using a spirit lamp.
3. The filled tubes are placed in the radial grooves of
the centrifuge with the sealed ends toward the outer
rim gasket. Counterbalance by placing the tubes in
the grooves opposite to each other.
4. Centrifuge at relative centrifugal force 12000 g for 5
minutes to completely pack the red cells.
5. Immediately remove the tubes from the centrifuge
and stand them upright. The tube will show three
layers from top to bottom: column of plasma, thin
layer of buffy coat, and column of red cells.
6. With the microhematocrit reader, hematocrit is
directly read from the scale. If hematocrit reader is
not available, the tube is held against a ruler and the
hematocrit is obtained by the following formula:
Length of red cell column in mm
Length of total column in mm
To obtain PCV, the above result is multiplied by 100.
GENERAL NOTES
1. Prolonged application of tourniquet during
venepuncture causes hemoconcentration and rise
in hematocrit.
2. Excess squeezing of the finger during skin
puncture dilutes the sample with tissue fluid and
lowers the hematocrit.
3. Correct proportion of blood with anticoagulant
should be used. Excess EDTA causes shrinkage
of red cells and falsely lowers the hematocrit.
4. Inadequate mixing of blood with anticoagulant,
and inadequate mixing of blood before testing can
cause false results.
5. Low hematocrit can result if there are clots in the
sample.
6. Centrifugation at lower speed and for less time
falsely increases PCV.
7. A small amount of plasma is trapped in the lower
part of the red cell column which is usually
insignificant. Increased amount of plasma is
trapped in microcytosis, macrocytosis, sphero-
cytosis, and sickle cell anemia, which cause an
artifactual rise in hematocrit. Larger volume of
plasma is trapped in Wintrobe tube than in
capillary tube.
8. As PCV requires whole blood sample, it is affected
by plasma volume (e.g. PCV is higher in dehy-
dration, and lower in fluid overload).
9. Expression of PCV: Occasionally, PCV is
expressed as a percentage. In SI units, PCV is
expressed as a volume fraction. Conversion factor
from conventional to SI units is 0.1 and from SI to
conventional units is 100.
10. Rules of 3 and 9: These rules of thumb are
commonly used to check the accuracy of results
and are applicable only if red cells are of normal
size and shape.
Hemoglobin (gm/dl) × 3 = PCV
Red cell count (million/cmm) × 9 = PCV
11. Automated hematocrit: In automated hematology
analyzers, hematocrit is obtained by multiplying

red cell count (in millions/cmm) by mean cell
volume (in femtoliters).
REFERENCE RANGES
• Adult males: 40-50%
• Adult females (nonpregnant): 38-45%
• Adult females (pregnant): 36-42%
• Children 6 to 12 years: 37-46%
• Children 6 months to 6 years: 36-42%
• Infants 2 to 6 months: 32-42%
• Newborns: 44-60%
Packed Cell Volume 191
CRITICAL VALUES
• Packed cell volume: < 20% or > 60%
BIBLIOGRAPHY
1. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
Hematology (9th Ed). London: Churchill Livingstone,
2001.
2. Wallach J. Interpretation of Diagnostic tests (7th Ed).
Philadelphia: Lippincott Williams and Wilkins, 2000.
3. World Health Organization. Manual of Basic Techni-
ques for a Health Laboratory (2nd Ed). Geneva. World
Health Organization, 2003.
 20

Total Leukocyte Count

Total leukocyte count (TLC) refers to the number of white

blood cells in 1 μl of blood (or in 1 liter of blood if the
result is expressed in SI units). There are two methods
for estimation of TLC:
• Manual or microscopic method
• Automated method (See Chapter 32: Automation in
Hematology).
A differential leukocyte count should always be
performed along with TLC to obtain the absolute cell
counts.
The purpose of carrying out TLC is to detect increase
or decrease in the total number of white cells in blood,
i.e. leukocytosis or leukopenia respectively. TLC is carried
out in the investigation of infections, any fever,
hematologic disorders, malignancy, and for follow-up
of chemotherapy or radiotherapy.
Fig. 20.1: Neubauer counting chamber: (A) Surface view,
MANUAL METHOD (B) Side view

Principle
A sample of whole blood is mixed with a diluent, which
lyses red cells and stains nuclei of white blood cells. White
blood cells are counted in a hemocytometer counting
chamber under the microscope and the result is
expressed as total number of leukocytes per μl of blood
or per liter of blood.
Equipment
1. Hemocytometer or counting chamber with coverglass: The
recommended hemocytometer is one with improved
Neubauer rulings and metallized surface. There are
two ruled areas on the surface of the chamber
(Fig. 20.1). Each ruled area is 3 mm × 3 mm in size
and consists of 9 large squares with each large square
measuring 1 mm × 1 mm. When the special thick
coverglass is placed over the ruled area, the volume
occupied by the diluted blood in each large square is
0.1 ml. In the improved Neubauer chamber, the
central large square is divided into 25 squares, each
of which is further subdivided into 16 small squares.
A group of 16 small squares is separated by closely
ruled triple lines (Figs 20.2 and 20.3). Metallized
surface makes background rulings and cells easily
visible. The 4 large corner squares are used for
counting leukocytes, while the central large square
is used for counting platelets and red blood cells.
Only special coverglass, which is intended for
use with hemocytometer, should be used. It should
be thick and optically flat. When the special
coverglass is placed on the surface of the chamber, a
volumetric chamber with constant depth and volume
throughout its entire area is formed. Ordinary cover
slips should never be employed since they do not
provide constant depth to the underlying chamber
due to bowing.

Fig. 20.2: Neubauer counting chamber showing areas for
counting WBCs (W) and red blood cells and platelets (R)
Fig. 20.3: (A) Counting area for WBCs and (B) counting
area for RBCs and platelets in Neubauer counting chamber
When the special cover glass is placed over the
ruled area of the chamber and pressed, Newton’s
rings (colored refraction or rainbow colored rings)
appear between the two glass surfaces; their
formation indicates the correct placement of the cover
glass.
2. Pipette calibrated to deliver 20 μl (0.02 ml, 20 cmm): WBC
bulb pipettes (Fig. 20.4), which have a bulb for
dilution and mixing (Thoma pipettes) are no longer
recommended. This is because blood and diluting
fluid cannot be mixed adequately inside the bulb of
the pipette. Bulb pipettes are also difficult to calibrate,
costly, and charging of counting chamber is difficult.
Tips of pipettes often chip easily and unnecessarily
small volume of blood needs to be used.
Total Leukocyte Count 193
Fig. 20.4: (A) WBC pipette and (B) Sahli’s pipette
calibrated to deliver 20 μl
3. Graduated pipette, 1 ml.
4. Pasteur pipette
5. Test tube (75 × 12 mm).
Reagent
WBC diluting fluid (Turk’s fluid) consists of a weak acid
solution (which hemolyzes red cells) and gentian violet
(which stains leucocyte nuclei deep violet). Diluting fluid
also suspends and disperses the cells and facilitates
counting. Its composition is as follows:
• Acetic acid, glacial 2 ml
• Gentian violet, 1% aqueous 1 ml
• Distilled water to make 100 ml
Specimen
EDTA anticoagulated venous blood or blood obtained
by skin puncture is used. (Heparin should not be used
since it causes leukocyte clumping). While collecting
capillary blood from the finger, excess squeezing should
be avoided so as not to dilute blood with tissue fluid.
Method
1. Dilution of blood: Take 0.38 ml of diluting fluid in a
test tube. To this, add exactly 20 μl of blood and mix.
This produces 1:20 dilution. Alternatively, 0.1 ml of
blood can be added to 1.9 ml of diluting fluid to get
the same dilution.
2. Charging the counting chamber: Place a coverglass
over the hemocytometer. Draw some of the diluted
194 Essentials of Clinical Pathology
blood in a Pasteur pipette. Holding the Pasteur pipette
at an angle of 45° and placing its tip between the
coverglass and the chamber, fill one of the ruled areas
of the hemocytometer with the sample. The sample
should cover the entire ruled area, should not contain
air bubbles, and should not flow into the side
channels. Allow 2 minutes for settling of cells.
3. Counting the cells: Place the charged hemocytometer
on the microscope stage. With the illumination
reduced to give sufficient contrast, bring the rulings
and the white cells under the focus of the low power
objective (× 10). White cells appear as small black dots.
Count the number of white cells in four large corner
squares. (To reduce the error of distribution, counting
of cells in all the nine squares is preferable). To correct
for the random distribution of cells lying on the
margins of the square, cells which are touching the
left hand lines or upper lines of the square are
included in the count, while cells touching the lower
and right margins are excluded.
4. Calculation of TLC:
(1) TLC/μl = (Number of WBCs counted ×
Correction for dilution × Correction for volume)
———————————————————————
(Number of large squares counted)
Number of WBCs counted × 20 × 10
= —————————————————
4
= Number of WBCs counted × 50
(2) TLC/L = Number of WBCs counted × 50 × 106 (106 is
the correction factor to convert count in 1 μl to count in 1
liter).
Example: If 200 WBCs are counted in 4 large squares,
TLC/μl will be 10,000/μl and TLC/liter will be 10.0 ×
109/liter.
If TLC is more than 50,000/ml, then dilution of blood
should be increased to 1:40 to increase the accuracy of
the result.
If TLC is less than 2,000/ml then lesser dilution
should be used.
Expression of TLC: Conventionally, TLC is expressed as
cells/μl or cells/cmm or cells/mm3. In SI units, TLC is
expressed as cells × 109/liter. Conversion factors for
conventional to SI units is 0.001 and SI to conventional
units is 1000.
Correction of TLC for nucleated red cells: The diluting
fluid does not lyse nucleated red cells or erythroblasts.
Therefore, they are counted as leukocytes in hemo-
cytometer. If erythroblasts are markedly increased in the
blood sample, overestimation of TLC can occur. To avoid
this if erythroblasts are greater than 10 per 100 leukocytes
as seen on blood film, TLC should be corrected for
nucleated red cells by the following formula:
TLC/μl × 100
Corrected TLC/μl = ————————————
Nucleated red cells per
100 WBCs + 100
Sources of error in manual blood cell count: There are
two main errors: technical and inherent.
Technical Errors
Errors in blood collection:
• Prolonged, tight application of a tourniquet leads to
venous stasis and false elevation of cell count.
• Excessive squeezing of finger puncture results in
dilution of blood with tissue fluid.
• Inadquate mixing of blood with anticoagulant leads
to formation of clots in blood sample and falsely low
count.
• Non-mixing of anticoagulated venous blood imme-
diately before testing will cause falsely low count.
Errors in pipetting
• Use of wet, chipped, or dirty pipettes
• Use of improperly calibrated pipettes
• Not wiping blood adherent to outside of pipette
• Using bulb pipettes that are difficult to calibrate and
which break off easily.
Errors in filling of chamber
• Incorrect filling of chamber, spillage into moats
• Not allowing 2 minutes for cells to settle.
• Drying of edges of preparation
• Not using specified coverglass
• Chamber containing dirt or air bubble.
Inherent Error (Statistical Error)
In spite of the best technique, an error still occurs due to
random distribution of cells in the chamber. This results
 Total Leukocyte Count 195

in variation in cell number in different areas of the same CRITICAL VALUES

size. This distribution follows Poisson’s law. More Total leukocyte count< 2000/μl or > 50000/μl.
number of cells should be counted to reduce this error.
n
This variance is calculated from the formula  100% , BIBLIOGRAPHY
n
where n is the total number of cells counted. If 100 cells are
1. Cheesbrough M. District laboratory practice in tropical
counted, then variance will be 10%. In 95% of cases, the
countries. Part 1 and Part 2. Cambridge. Cambridge
range would be n ± 2σ i.e. 100 –2(10) to 100 + 2(10), i.e. 80 University Press, 1998.
to 120. If 200 cells are counted, the error is reduced to 2. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
about 7%. Haematology (9th Ed). London. Churchill Livingstone,
2001.
REFERENCE RANGES 3. The Expert Panel on Cytometry of the International
• Adults: 4000-11,000/μl Council for Standardization in Haematology: Recom-
mended methods for the visual determination of white
• At birth: 10,000-26000/μl
blood cell count and platelet count. World Health
• 1 year: 6,000-16,000/μl
Organization. WHO/DIL/00.3. 2000.
• 6-12 years: 5,000-13,000/μl 4. World Health Organization. Manual of Basic Techni-
• Pregnancy: up to 15,000/μl ques for a Health Laboratory (2nd Ed). Geneva: World
Health Organization, 2003.
 21
Reticulocyte Count
Reticulocytes are young or juvenile red cells released
from the bone marrow into the bloodstream and that
contain remnants of ribonucleic acid (RNA) and
ribosomes but no nucleus. After staining with a
supravital dye such as new methylene blue, RNA
appears as blue precipitating granules or filaments within
the red cells. Following supravital staining, any non-
nucleated red cell containing 2 or more granules of blue-
stained material is considered as a reticulocyte (The
College of American Pathology). Supravital staining
refers to staining of cells in a living state before they are
killed by fixation or drying or with passage of time.
Reticulocyte count is performed by manual method.
Recently methods based on flow cytometry have been
introduced which are rapid and more precise.
Reticulocyte count is performed to assess erythropoietic
activity of the bone marrow
USES
1. As one of the baseline studies in anemia with no
obvious cause
2. To diagnose anemia due to ineffective erythropoiesis
(premature destruction of red cell precursors in bone
marrow seen in megaloblastic anemia and thalasse-
mia) or due to decreased production of red cells: In
hypoplastic anemia or in ineffective erythropoiesis,
reticulocyte count is low as compared to the degree
of anemia. Increased erythropoiesis (e.g. in hemolytic
anemia, blood loss, or specific treatment of nutritional
anemia) is associated with increased reticulocyte
count. Thus reticulocyte count is used to differentiate
hypoproliferative anemia from hyperproliferative
anemia.
3. To assess response to specific therapy in iron
deficiency and megaloblastic anemias.
4. To assess response to erythropoietin therapy in
anemia of chronic renal failure.
5. To follow the course of bone marrow transplantation
for engraftment
6. To assess recovery from myelosuppressive therapy
7. To assess anemia in neonate
PRINCIPLE
A few drops of blood (collected in EDTA) are incubated
with new methylene blue solution which stains granules
of RNA in red cells. A thin smear is prepared on a glass
slide from the mixture and reticulocytes are counted
under the microscope. Number of reticulocytes is
expressed as a percentage of red cells.
REAGENT
New methylene blue solution is prepared as follows:
New methylene blue 1.0 gm
Sodium citrate 0.6 gm
Sodium chloride 0.7 gm
Distilled water 100 ml
Reagent should be kept stored in a refrigerator at
2-6°C and filtered before use.
Suitable alternatives to new methylene blue are
brilliant cresyl blue and azure B.
SPECIMEN
Capillary blood or EDTA-anticoagulated venous blood
can be used.
METHOD
1. Take 2-3 drops of filtered new methylene blue
solution in a 12 × 75 mm test tube.
2. Add equal amount of blood and mix well.
3. Keep the mixture at room temperature or at 37°C for
15 minutes.

Fig. 21.1: Reticulocytes stained with a supravital stain
4. After gentle mixing, place a small drop from the
mixture on a glass slide, prepare a thin smear, and
allow to dry in the air.
5. Examine under the microscope using oil-immersion
objective. Mature red cells stain pale green-blue.
Reticulocytes show deep blue precipitates of fine
granules and filaments in the form of a network
(reticulum) (Fig. 21.1). Most immature reticulocytes
show a large amount of precipitated material in the
form of a mass, while the most mature reticulocytes
show only a few granules or strands. Any non-
nucleated red cell is considered as a reticulocyte if it
contains 2 or more blue-stained particles of ribosomal
RNA.
6. Count 1000 red cells and note the number of red cells
that are reticulocytes. Counting error is minimized if
size of the microscopic field is reduced. This is
achieved by using a Miller ocular disk inserted in the
eyepiece (Fig. 21.2); it divides the field into two
squares (one nine times larger in size than the other).
Reticulocytes are counted in both the squares and the
red cells are counted in the smaller square.
REPORTING THE RESULT
1. Reticulocyte percentage: The most common method
of reporting is reticulocyte percentage which is
calculated from the following formula:
Reticulocyte
Number of reticulocytes counted
percentage = ———————————————— × 100
Number of red cells counted
Reference range is 0.5%-2.5% in adults and children.
Reticulocyte count is higher in newborns.
Reticulocyte Count 197
Fig. 21.2: Miller ocular disk to facilitate counting of reticulocytes.
The Miller ocular disk contains two squares: A and B. Square
A is 9 times larger than square B. Square B is used for counting
red cells, while reticulocytes are counted in square A. Only
the reticulocytes are counted in 20 large (i.e. A) squares, while
red cells are counted in 20 small (i.e. B) squares. Reticulocyte
count is obtained from the formula
Total reticulocytes counted in square A
——————————————————— × 100
Total red cells counted in square B × 9
2. Absolute reticulocyte count
= Reticulocyte percentage × Red cell count
Normal: 50,000 to 85,000/cmm
3. Corrected reticulocyte count (Reticulocyte index)
PCV of patient
= Reticulocyte percentage × ————————
Normal PCV
Corrected reticulocyte count > 2% indicates reticulo-
cyte release appropriate for the degree of anemia. If <
2%, reticulocyte release is inappropriate.
(4) Reticulocyte maturation production index
Corrected reticulocyte count
= ———————————————
Estimated maturation time in days
Notes:
1. Stages of maturation of reticulocytes (Heilmeyer and
Westhaeuser, 1932) (Fig. 21.3):
• Stage 0: Late or orthochromatic normoblast
(Nucleated red cell); strong staining for reticulum
(This cell type should not be included under
reticulocytes as it is not a stage of reticulocyte
maturation)
198 Essentials of Clinical Pathology
Fig. 21.3: Stages of reticulocyte maturation
• Stage I: Dense cohesive reticulum in non-nucleated
red cell (0.1% of reticulocyte count in normal
individuals)
• Stage II: Extensive network of loose reticulum
(0.7% of reticulocyte count in normal individuals)
• Stage III: Small reticulum along with scattered
granules (32% of reticulocyte count in normal
individuals)
• Stage IV: Scattered granules (61% of reticulocyte
count in normal individuals).
2. Time required for maturation of reticulocytes (i.e.
from extrusion of nucleus till no granules are seen in
the cytoplasm) is 4 to 4.5 days. Till the extrusion of
the nucleus, 97% of hemoglobin is synthesized;
reticulocytes synthesize remaining 3%. Reticulocytes
are produced in the bone marrow where they spend
about 3 to 3.5 days for maturation. In circulation, they
spend 1 more day before maturing into red cells. In
hemolytic anemia and following acute blood loss,
reticulocytes are released prematurely in circulation
where they require more time (about 2-3 days) for
maturation. Such prematurely released reticulocytes
are larger than reticulocytes released under basal
conditions. The maturation time is dependent upon
the hematocrit of the patient. Therefore, to avoid
overestimation of actual red cell production,
reticulocyte index is calculated. In a patient with
anemia, if reticulocyte index fails to rise 2-3 times
above the normal value, then decreased production
of red cells in the marrow should be suspected.
Maturation time according to the hematocrit is as
follows: hematocrit 45%: maturation time 1 day, 35%:
1.5 days, 25%: 2 days, and 15%: 2.5 days. There is
evidence that maturation of reticulocytes also occurs
in vitro following blood collection and therefore
specimen should be processed for reticulocyte
counting as soon as possible (within 4 hours of
collection).
3. An example illustrating importance of absolute
reticulocytes count and corrected reticulocyte count:
A 30-year-old male patient with anemia has PCV 15%,
red cell count 1.2 million count/cmm, and reticulo-
cyte count 3%. Reticulocyte count appears to be
increased (normal: 0.5-2.5%) indicating adequate
response to anemia. However, value of absolute
reticulocyte count (0.03 × 1200000) is 36,000 and
15
corrected reticulocyte count is 3 × or 1%; both of
45
which are low as compared to the degree of anemia.
Thus the patient has diminished production of red
cells in marrow (hypoproliferative anemia).
4. Causes of increased reticulocyte count (reticulo-
cytosis):
• Hemolytic anemias
• Blood loss
• Following specific therapy of nutritional anemia
(like iron in iron deficiency anemia, folate in folate
deficiency anemia, Vit B 12 in B 12 deficiency
anemia)
• Hemoglobinopathies, e.g. sickle cell anemia.
5. Causes of decreased reticulocyte count (reticulo-
cytopenia):
• Aplastic anemia and pure red cell aplasia
• Bone marrow infiltration (leukemia, lymphoma,
myelofibrosis, metastatic malignancy)
• Renal disease
• Anemia of chronic disease
• Alcoholism
• Myxedema
• Ineffective erythropoiesis: Megaloblastic anemia,
sideroblastic anemia, thalassemia, myelodysplasia
• Following blood transfusion.
6. Classification of anemia according to reticulocyte
count: Normocytic normochromic anemias are
classified into two types based on reticulocyte count:
• Normocytic normochromic anemia with increa-
sed reticulocyte count:
–Recent blood loss
–Hemolysis

• Normocytic normochromic anemia with low
reticulocyte count:
– Aplastic anemia
– Chronic renal failure
– Anemia of chronic disease
– Anemia due to ineffective erythropoiesis
7. Reticulocytes should be distinguished from (Fig. 21.4):
• Heinz bodies: These are precipitated globin chains
attached to the red cell membrane. They stain light
blue and are seen in glucose-6-phosphate dehy-
drogenase deficiency following exposure to
oxidant stress.
• Pappenheimer bodies: These are iron-containing
granules which appear as one or more small dots
in red cells. They give positive Prussian blue
reaction. They are confused with reticulocytes in
Romanowsky-counterstained preparations.
Fig. 21.4: Differentiation of reticulocytes from Howell-Jolly
bodies, Heinz bodies, and HbH inclusions
Reticulocyte Count 199
• HbH inclusions: These are round, golf ball like
inclusion bodies seen in α-thalassemias. They are
seen in most of the red cells while reticulocytes
are seen in only a few cells.
• Granules of new methylene blue superimposed on
red cells, if stain is not filtered before use.
• Howell-Jolly bodies: These are nuclear remnants in
red cells seen in certain anemias and following
splenectomy. As supravital dyes stain both DNA
and RNA, these structures are also stained.
8. The polychromatic red cells on Romanowsky-stained
blood smears represent reticulocytes. However, only
stages 1, 2, and 3 cause polychromasia, while stage 4
reticulocytes having small amount of RNA do not
cause polychromasia. Therefore, counting polychro-
matic cells cannot be used as a substitute for
reticulocyte count.
REFERENCE RANGES
• Reticulocyte percentage: 0.5-2.5%
• Absolute reticulocyte count: 50,000-85,000/cmm
BIBLIOGRAPHY
1. Pierre RV. Reticulocytes: Their usefulness and
measurement in peripheral blood. Clin Lab Med
2002;22:63-79.
2. The Expert Panel on Cytometry of the International
Council for Standardization in Hematology: ICSH
guidelines for reticulocyte counting by microscopy on
supravitally stained preparations. World Health
Organization. WHO/LBS/92.3 1992.
 22

Blood Smear

A blood smear or film is a specimen for microscopic blood is used, smear should be prepared and stained
examination prepared by spreading a drop of blood within 2 hours of blood collection. If venous blood
across a glass slide followed by staining with one of the collected in a syringe is used, the last drop of blood
Romanowsky's stains. in the needle after withdrawing (or first drop while
dispensing) should be used.
USES 2. A 'spreader' slide is placed at an angle of 30° in front
of the drop and then drawn back to touch the drop of
1. Blood smear is helpful in suggesting the cause of
blood. Blood spreads across the line of contact of two
anemia or thrombocytopenia, identifying and typing
slides.
of leukemia, and in diagnosing hemoparasitic
3. Smear is made by smooth, forward movement of the
infections (malaria, filaria, and trypanosomiasis). It
'spreader' along the slide. The whole drop should be
is also helpful in the management of these conditions.
used up 1 cm before the end of the slide. The length
2. To monitor the effect of chemotherapy and radio-
of the smear should be about 3 cm (Fig. 22.1). The
therapy on bone marrow.
'spreader' should not be raised above the slide surface
3. To provide direction for further investigations that
till whole drop of blood is spread out.
will help in arriving at the correct diagnosis (e.g. in
infections, drug toxicity, etc.).
Blood smear examination is therefore indicated in
clinically suspected cases of anemia, thrombocytopenia,
hematological malignancies (leukemia, lymphoma,
multiple myeloma), disseminated intravascular coagu-
lation, parasitic infections (like malaria or filaria),
infectious mononucleosis, and various inflammatory, or
malignant diseases.

PREPARATION OF BLOOD SMEAR

(WEDGE METHOD)
1. A small drop of blood (2-3 mm in diameter) is placed
in the center line about 1 cm away from one end of a
glass slide (typical size of slide is 75 × 25 mm;
thickness about 1mm) with a wooden stick or glass
capillary. Slide should be clean, dry, and grease-free.
Blood sample may be venous (anticoagulated with
EDTA) or capillary (finger prick). Better blood cell
morphology is obtained if smear is made directly
from a skin puncture. If EDTA-anticoagulated venous Fig. 22.1: Preparation of blood smear

4. Smear is rapidly dried by waving it in the air or
keeping it under an electric fan. Slow drying causes
shrinkage artifact of red cells.
5. Patient's name or laboratory number and date are
written (with a lead pencil, a permanent marker pen,
or a diamond pencil) on the thicker end of the smear.
6. The smear is fixed immediately with absolute methyl
alcohol (which should be moisture- and acetone-free)
for 2-3 minutes in a covered jar (Absolute ethyl
alcohol can also be used, but not methylated spirit as
it contains water). Aim of fixation is to prevent
washing off of the smear from the slide. Following
this, color of the smear becomes light brown. This
fixation is desirable even when Leishman stain is used
which contains methyl alcohol. This is because
Leishman stain may have absorbed moisture leading
to poor fixation. If methanol is contaminated with
water, sharpness of cell morphology is lost and there
is vacuolation of red cells. Methanol should be
acetone-free since acetone washes out nuclear stain.
(In many laboratories, slide is stained immediately
after air-drying without prior fixation, and the results
are satisfactory; however, if delay of >4 hours is
anticipated between air-drying and staining, the slide
should be fixed. If not, a background gray-blue
staining of plasma occurs).
Notes
1. Making a 'spreader' slide—a glass slide with
absolutely smooth edges should be selected and one
or both corners at one end of the slide should be
broken off. The 'spreader' slide should be narrower
(width of about 15 mm) so that edges of the smear
can be examined microscopically (Fig. 22.1). The
'spreader' slide should be discarded after use. If the
same is to be reused, its edge should be thoroughly
cleaned and dried (otherwise carryover of cells or
parasites can occur).
2. A well-spread blood smear (a) is tongue-shaped with
a smooth tail, (b) does not cover the entire area of the
slide, (c) has both thick and thin areas with gradual
transition, and (d) does not contain any lines or holes.
Smears unsatisfactory for examination are shown in
Figure 22.2.
3. By changing the angle of the 'spreader' and its speed,
thickness of the blood smear can be controlled. In
patients with anemia, a thicker smear can be obtained
by increasing the angle and the speed of spreading.
Blood Smear 201
Fig. 22.2: Blood smears that are unsatisfactory for
evaluation
In patients with polycythemia, a thinner smear is
obtained by decreasing the 'spreader' angle and the
speed of spreading.
4. Anticoagulant used may be EDTA (dipotassium salt)
or sodium citrate. Heparin should not be used as an
anticoagulant for making blood films since it causes
platelet clumping and imparts a blue background to
the film.
5. It is recommended to stain blood films in reagent
filled Coplin jars (rather than covering them with the
staining solution) to avoid formation of stain
precipitates due to evaporation.
6. A drawback of this method is uneven distribution of
leukocytes (i.e. monocytes, neutrophils, and abnormal
cells are pushed towards the extreme tail end of the
smear) and distortion of red cell morphology at the
edges.
7. Blood smear is covered with a coverslip and mounted
in a mounting medium (e.g. DPX) for protection
against mechanical damage and deterioration of
staining with time on exposure to air.
8. Cleaning of slides: (A) New slides: If new slides are
not clean and grease-free, they are left overnight in a
detergent solution, washed in running tap water,
rinsed in distilled water, and wiped dry with a clean
cloth. Before use, they are wiped with 95% methyl
alcohol, dried, and then kept covered to protect from
dust. (B) Used slides: The used slides are soaked in a
detergent solution at 60°C for 20 minutes, washed in
running tap water, rinsed in distilled water, and then
wiped dry. Before use, they are wiped with 95%
methyl alcohol, dried, and then kept covered to
protect from dust.
202 Essentials of Clinical Pathology
STAINING OF BLOOD SMEAR
Blood smears are routinely stained by one of the
Romanowsky stains (Box 22.1). Romanowsky stains
consist of a combination of acidic and basic dyes and
after staining various intermediate shades are obtained
between the two polar (red and blue) stains. Romanow-
sky stains include May-Grunwald-Giemsa, Jenner,
Wright's, Leishman's, and Field's stains. Staining
properties of the Romanowsky stains are dependent on
two synthetic dyes: methylene blue and eosin. Inter-
national Committee for Standardization in Haematology
has recommended a highly purified standardized stain,
which contains azure B and eosin Y; it, however, is very
expensive. Romanowsky stains are insoluble in water but
soluble in methyl alcohol. Methyl alcohol acts as a solvent
as well as a fixative. Staining reaction is pH-dependent.
These stains have a tendency towards precipitation and
should be filtered before use.
Methylene blue and azure B are basic (cationic) dyes
and have affinity for acidic components of the cells (like
nucleic acids or basophil granules) and impart purple-
violet color to the nuclear chromatin, dark blue-violet
color to the basophil granules, and deep blue color to the
cytoplasm of lymphocytes. Eosin is an acidic (anionic)
dye and has affinity for basic components like hemo-
globin (stained pink-red), and granules in eosinophils
(stained orange-red). Neutrophil granules are slightly
basic and stain violet-pink or lilac.
Romanowsky stains impart more colours than just
blue (from methylene blue or azure B) and red-orange
(from eosin Y). Usefulness of the Romanowsky stains lies
in their ability to differentially stain leucocyte granules.
Box 22.1: Romanowsky stains
Two main components of all Romanowsky stains are an acidic
dye (eosin Y) and a basic dye (oxidized methylene blue).
• Basic or cationic dye: It is positively charged and binds to
anionic sites and imparts blue-gray color to nucleic acids,
nucleoproteins, and granules of basophils. Examples:
methylene blue, azure B.
• Acidic or anionic dye: It is negatively charged and binds
to cationic sites and imparts orange-red color to
hemoglobin and eosinophil granules. Example: eosin Y.
Examples of Romanowsky stains: May-Grunwald-Giemsa,
Jenner’s, Leishman’s, Wright’s, Field’s.
A well-stained smear is pink in color in thinner
portion and purple-blue in thicker portion. Excess blue
coloration can be due to: (i) excessively thick smear, (ii)
low concentration of eosin, (iii) impure dyes, (iv) too long
staining time, (v) inadequate washing, or (vi) excessive
alkaline pH of stain, buffer, or water. Excess red
coloration can be due to: (i) impure dyes or incorrect
proportion of dyes, (ii) excessive acid pH of stain, buffer,
or water (as the red cells take up more acid dye i.e. eosin),
(iii) too short staining time, or (iv) excessive washing. If
there are granules of stain precipitate (masses of small
black dots) on smear, stain needs to be filtered.
Method of Leishman staining is given below:
Reagents
1. Leishman stain: William Boog Leishman, a British
pathologist, modified the original Romanowsky
method and devised a stain which is widely known
as Leishman's stain. This consists of methylene blue
and eosin dissolved in absolute methyl alcohol.
Commercially available Leishman stain powder (0.6
gram) is mixed with water-free absolute methyl
alcohol (400 ml). Prepared stain should be kept tightly
stoppered in a brown bottle and stored in a cool, dark
place at room temperature. Exposure to direct
sunlight causes deterioration of the stain. After
preparation, stain should be kept for 3-5 days before
using since it improves the quality of the stain.
2. Buffered water (pH 6.8).
Method
1. Air-dry the smear and fix with methanol for 2-3
minutes.
2. Cover the smear with Leishman stain for 2 minutes.
3. After 2 minutes, add twice the volume of buffered
water and leave for 5-7 minutes. A scum of metallic
sheen forms on the surface.
4. Wash the stain away in a stream of buffered water.
Tap water can also be used for washing if it is not
highly alkaline or highly acid.
5. Wipe the back of the slide clean and set it upright in
the draining rack to dry.
6. Mount the slide in a suitable mounting medium (e.g.
DPX) with a clean and dry 25 × 25 mm coverslip.
 Blood Smear 203

A well-stained smear shows following features

(Fig. 22.3):
• Red cells: pink-red or deep pink
• Polychromatic cells (Reticulocytes): Gray-blue
• Neutrophils: Pale pink cytoplasm; mauve-purple
granules
• Eosinophils: Pale-pink cytoplasm; orange-red
granules
• Basophils: Blue cytoplasm; dark blue-violet
granules
• Monocytes: Gray-blue cytoplasm; fine reddish
(azurophil) granules
• Small lymphocytes: Dark blue cytoplasm
• Platelets: Purple
• Nuclei of all cells: Purple-violet

EXAMINATION OF BLOOD SMEAR
A blood smear is examined for:
• Red cells: Morphology, immature forms, inclusion
bodies, arrangement of cells.
• White cells: Differential count, abnormal or immature
forms.
• Platelets: Adequacy, abnormal forms.
• Parasites: Malaria, filaria.
A peripheral blood smear has three parts: Head, body,
and tail (Fig. 22.4). A blood smear should be examined
in an orderly manner. Initially, blood smear should be
observed under low power objective (10×) to assess
whether the film is properly spread and stained, to assess
Fig. 22.4: Area for examination of blood cells and for
differential cell count in blood smear
cell distribution, and to select an area for examination of
blood cells. Best morphologic details are seen in the area
where red cells are just touching one another. Low power
view is also helpful for the identification of Rouleaux
formation, autoagglutination of red cells, and micro-
filaria. High power objective (45×) is suitable for
examination of red cell morphology and for differential
leukocyte count. A rough estimate of total leukocyte
count can be obtained which also serves to crosscheck
the total leukocyte count done by manual counting or
automated method. Oil-immersion objective (100×) is
used for more detailed examination of any abnormal
cells.

Fig. 22.3: Normal blood smear showing red cells (R), a
neutrophil (N), a lymphocyte (L), eosinophil (E), basophil (B),
monocyte (M), and platelets (P)
Red Cells
Red cells are best examined in an area where they are
just touching one another (towards the tail of the film).
Normal red cells are 7-8 µm in size, round with smooth
contours, and stain deep pink at the periphery and paler
in the center. Area of central pallor is about 1/3rd the
diameter of the red cell. Size of a normal red cell
corresponds roughly with the size of the nucleus of a
small lymphocyte. Normal red cells are described as
normocytic (of normal size) and normochromic (with
normal staining intensity i.e. hemoglobin content).
Morphologic abnormalities of red cells in peripheral
blood smear can be grouped as follows:
• Red cells with abnormal size (Fig. 22.5)
• Red cells with abnormal staining
• Red cells with abnormal shape (Fig. 22.5)
204 Essentials of Clinical Pathology

Fig. 22.5: Variations in size and shape of red cells: (A) Microcytic hypochromic red cells in iron deficiency anemia; (B) Oval
macrocytes and a hypersegmented neutrophil in megaloblastic anemia; (C) Sickle cells in sickle cell anemia; (D) Spherocytes
in hereditary spherocytosis; (E) Fragmented red cells or schistocytes in microangiopathic hemolytic anemia; (F) Target cells
in hemoglobinopathy; (G) Burr cells in chronic renal failure; (H) Tear drop red cells in myelofibrosis; (I) Bite cells and
(J) Blister cell in glucose-6-phosphate dehydrogenase deficiency

• Red cell inclusions (Fig. 22.6)

• Immature red cells (Fig. 22.7)
• Abnormal red cell arrangement (Fig. 22.8).
1. Red cells with abnormal size: Mild variation in red
cell size is normal. Increased variation in red cell size is
called as anisocytosis. This is a feature of most anemias
and is non-specific. Anisocytosis is due to the presence
of microcytes, macrocytes, or both in addition to red cells
of normal size.
Microcytes are red cells smaller in size than normal.
They are seen when hemoglobin synthesis is defective
i.e. in iron deficiency anemia, thalassemias, anemia of
chronic disease, and sideroblastic anemia.
Macrocytes are red cells larger in size than normal. Fig. 22.6: Red cell inclusions: (A) Basophilic stippling;
(B) Howell-Jolly bodies; (C) Pappenheimer bodies; (D) Cabot’s
Oval macrocytes (macro-ovalocytes) are seen in ring
megaloblastic anemia, myelodysplastic syndrome, and
in patients being treated with cancer chemotherapy. hemoglobin content. Red cells with increased area of
Round macrocytes are seen in liver disease, alcoholism, central pallor (i.e. containing less hemoglobin) are called
and hypothyroidism. as hypochromic. They are seen when hemoglobin
2. Red cells with abnormal staining (hemoglobin synthesis is defective, i.e. in iron deficiency, thalassemias,
content): Staining intensity of red cells depends on anaemia of chronic disease, and sideroblastic anemia.

Fig. 22.7: Immature red cells: (A) Polychromatic red cell;
(B) Nucleated red cell
Fig. 22.8: Abnormal red cell arrangement: (A) Rouleaux
formation; (B) Autoagglutination
In dimorphic anemia, there are two distinct popula-
tions of red cells in the same smear. An example is
presence of both normochromic and hypochromic red
cells seen in sideroblastic anemia, iron deficiency anemia
responding to treatment, and following blood transfusion
in a patient of hypochromic anemia. In myelodysplastic
syndrome, dimorphic picture results from admixture of
microcytic hypochromic cells and macrocytes.
3. Red cells with abnormal shape: Increased variation
in red cell shape is called as poikilocytosis and is a feature
of many anemias. A red cell that is abnormal in shape is
called as a poikilocyte.
Sickle cells are narrow and elongated red cells with
one or both ends pointed. Sickle form is assumed when
a red cell containing hemoglobin S is deprived of oxygen.
Sickle cells are seen in sickle cell disorders, particularly
sickle cell anemia. Sickle cells are not seen on blood smear
in neonates with sickle cell disease because high
percentage of fetal hemoglobin in red cells prevents
sickling.
Spherocytes are red cells, which are slightly smaller in
size than normal, round, stain intensely, and do not have
central area of pallor. The surface area of spherocytes is
less as compared to the volume. They are seen in
hereditary spherocytosis, autoimmune hemolytic anemia
(warm antibody type), and ABO hemolytic disease of
newborn.
Blood Smear 205
Schistocytes are fragmented red cells, which take
various forms like helmet, crescent, triangle, etc. and
usually have surface projections or spicules. They are
seen in microangiopathic hemolytic anaemia, cardiac
valve prosthesis, and severe burns.
Target cells are red cells with bull's eye appearance.
These red cells show a central stained area and a
peripheral stained rim with unstained cytoplasm in
between. They are seen in hemoglobinopathies (e.g.
thalassemias, hemoglobin C disease, sickle cell disease),
obstructive jaundice, and following splenectomy.
Burr cells or echinocytes are small red cells with
regularly placed small projections on surface. They are
seen in uremia.
Acanthocytes are red cells with irregularly spaced
sharp projections of variable length on surface. They are
seen in spur cell anemia of liver disease, McLeod
phenotype, and following splenectomy.
Teardrop cells or dacryocytes have a tapering drop-
like shape. Numerous teardrop red cells are seen in
myelofibrosis and myelophthisic anemia.
Blister cells or hemi ghost cells are irregularly
contracted cells in which hemoglobin is contracted and
condensed away from the cell membrane. This is seen in
glucose-6-phosphate dehydrogenase deficiency during
acute hemolytic episode.
Bite cells result from removal of Heinz bodies by the
pitting action of the spleen (i.e. a part of red cell is bitten
off by the splenic macrophages). They are seen in glucose-
6-phosphate dehydrogenase deficiency and unstable
hemoglobin disease.
4. Red cell inclusions: Those inclusions that can be
visualized on Romanowsky-stained smears are baso-
philic stippling, Howell-Jolly bodies, Pappenheimer
bodies, and Cabot's rings.
Basophilic stippling or punctate basophilia refers to the
presence of numerous, irregular basophilic (purple-blue)
granules which are uniformly distributed in the red cell.
These granules represent aggregates of ribosomes. Their
presence is indicative of impaired erythropoiesis and
they are seen in thalassemias, megaloblastic anemia,
heavy metal poisoning (e.g. lead), and liver disease.
Howell-Jolly bodies are small, round, purple-staining
nuclear remnants located peripherally in red cells. They
are seen in megaloblastic anemia, thalassemias,
hemolytic anemia, and following splenectomy.
206 Essentials of Clinical Pathology

Pappenheimer bodies are basophilic, small, iron- Box 22.2: Role of blood smear in anemias
containing granules in red cells. They give positive Perl's
Prussian blue reaction. Unlike basophilic stippling, • Macrocytic anemia: Differential diagnosis between
Pappenheimer bodies are few in number and are not megaloblastic anemia (oval macrocytosis and hyper-
distributed throughout the red cell. They are seen segmented neutrophils), liver disease (round macrocytosis
following splenectomy and in thalassemias and and target cells), hemolytic anemia (numerous poly-
chromatic cells), and myelodysplastic syndrome (dimor-
sideroblastic anemia.
phic red cells, pseudo-Pelger-Huet neutrophils, giant
Cabot's rings are fine, reddish-purple or red, ring-like platelets, occasional blast cell).
structures. They appear like loops or figure of eight • Microcytic anemias: Differential diagnosis between iron
structures. They indicate impaired erythropoiesis and are deficiency anemia (microcytic hypochromic red cells,
seen in megaloblastic anemia and lead poisoning. pencil cells), thalassemia minor (microcytic hypochromic
5. Immature red cells: red cells, basophilic stippling), and sideroblastic anemia
Polychromatic cells are young red cells containing (dimorphic anemia).
remnants of ribonucleic acid. These cells are slightly • Sickle cell disease: Differentaition between sickle cell trait
larger than normal red cells and have a diffuse bluish- (target cells with no sickle cells), sickle cell anemia (sickle
cells), and sickle cell β-thalassemia (microcytic hypo-
grey tint. (They represent reticulocytes when stained with
chromic red cells, sickle cells).
a supravital stain like new methylene blue). Poly-
• Hemolytic anemias: Spherocytosis (hereditary sphero-
chromasia is due to the uptake of acid stain by cytosis, autoimmune hemolytic anemia), fragmented cells
hemoglobin and basic stain by ribonucleic acid. Presence (microangiopathic haemolytic anemia), bite cells or blister
of polychromatic cells is indicative of active erythro- cells (Glucose-6-phosphate dehydrogenase deficiency),
poiesis and are increased in hemolytic anemia, acute and autoagglutination of red cells (cold hemagglutinin
blood loss, and following specific therapy for nutritional disease).
anemia.
Nucleated red cells are red cell precursors (erythro-
blasts), which are released prematurely in peripheral
blood from the bone marrow. They are a normal finding
in cord blood of newborns. Large number of nucleated
red cells in blood smear is seen in hemolytic disease of
newborn, hemolytic anemia, leukemias, myelophthisic
anemia, and myelofibrosis.
6. Abnormal red cell arrangement:
Rouleaux formation refers to alignment of red cells on top
of each other like a stack of coins. It occurs in multiple
myeloma, Waldenström's macroglobulinemia, hyper-
gammaglobulinemia, and hyperfibrinogenemia.
Autoagglutination refers to the clumping of red cells
in large, irregular groups on blood smear. It is seen in
cold agglutinin disease.
Role of blood smear in anemia is shown in Box 22.2
and Figures 22.9 to 22.11.

White Cells
Approximate idea about total leukocyte count can be
gained from the examination of the smear under high
power objective (40× or 45×). A differential leukocyte
count should be carried out. Abnormal appearing white
Fig. 22.9: Differential diagnosis of macrocytic anemia on blood
smear: (A) Megaloblastic anemia; (B) Hemolytic anemia;
(C) Liver disease; (D) Myelodysplastic syndrome

Fig. 22.10: Differential diagnosis of microcytic anemia on
blood smear: (A) Iron deficiency anemia; (B) Thalassemia
minor; (C) Thalassemia major; (D) Sideroblastic anemia
Fig. 22.11: Differential diagnosis of hemolytic anemia on
blood smear. (A) Microangiopathic hemolytic anemia showing
fragmented red cells, (B) Hereditary spherocytosis showing
spherocytes and a polychromatic red cell, and (C) Glucose-
6-phosphate dehydrogenase deficiency showing a blister cell
and a bite cell
cells are evaluated under oil-immersion objective.
1. Morphology of normal leukocytes (refer to Fig. 16.9
in Chapter 16: Hematopoiesis):
i. Polymorphonuclear neutrophil: Neutrophil measures
14-15 µm in size. Its cytoplasm is colorless or lightly
eosinophilic and contains multiple, small, fine,
mauve granules. Nucleus has 2-5 lobes that are
connected by fine chromatin strands. Nuclear
chromatin is condensed and stains deep purple in
color. A segmented neutrophil has at least 2 lobes
connected by a chromatin strand. A band neutro-
Blood Smear 207
phil shows non-segmented U-shaped nucleus of
even width. Normally band neutrophils comprise
less than 3% of all leukocytes. Majority of
neutrophils have 3 lobes, while less than 5% have
5 lobes.
In females, 2-3% of neutrophils show a small
projection (called drumstick) on the nuclear lobe.
It represents one inactivated X chromosome.
ii. Eosinophil: Eosinophils are slightly larger than
neutrophils (15-16 µm). The nucleus is often
bilobed and the cytoplasm is packed with
numerous, large, bright orange-red granules. On
blood smears, some of the eosinophils are often
ruptured.
iii. Basophils: Basophils are seen rarely on normal
smears. They are small (9-12 µm), round to oval
cells, which contain very large, coarse, deep purple
granules. It is difficult to make out the nucleus since
granules cover it.
iv. Monocytes: Monocyte is the largest of the leuko-
cytes (15-20 µm). It is irregular in shape, with oval
or clefted (kidney-shaped) nucleus and fine,
delicate chromatin. Cytoplasm is abundant, blue-
gray with ground glass appearance and often
contains fine azurophil granules and vacuoles.
After migration to the tissues from blood, they are
called as macrophages.
v. Lymphocytes: On peripheral blood smear, two types
of lymphocytes are distinguished: small and large.
The majority of lymphocytes are small (7-8 µm).
These cells have a high nuclear-cytoplasmic ratio
with a thin rim of deep blue cytoplasm. The
nucleus is round or slightly clefted with coarsely
clumped chromatin. Large lymphocytes (10-15
µm) have a more abundant, pale blue cytoplasm,
which may contain a few azurophil granules.
Nucleus is oval or round and often placed on one
side of the cell.
2. Morphology of abnormal leukocytes (Fig. 22.12):
i. Toxic granules: These are darkly staining, blue-
purple, coarse granules in the cytoplasm of
neutrophils. They are commonly seen in severe
bacterial infections.
ii. Döhle inclusion bodies: These are small, oval, pale
blue cytoplasmic inclusions in the periphery of
neutrophils. They represent remnants of riboso-
mes and rough endoplasmic reticulum. They are
208 Essentials of Clinical Pathology
Fig. 22.12: Morphological abnormalities of white blood cells:
(A) Toxic granules; (B) Döhle inclusion body; (C) Shift to left
in neutrophil series; (D) Hypersegmented neutrophil in
megaloblastic anemia; (E) Atypical lymphocyte in infectious
mononucleosis; (F) Blast cell in acute leukemia
often associated with toxic granules and are seen
in bacterial infections.
iii. Cytoplasmic vacuoles: Vacuoles in neutrophils are
indicative of phagocytosis and are seen in bacterial
infections.
iv. Shift to left of neutrophils: This refers to presence of
immature cells of neutrophil series (band forms
Fig. 22.13: Comparison of blood smears in (A) acute myeloid leukemia and (B) acute lymphoblastic leukemia
and metamyelocytes) in peripheral blood and
occurs in infections and inflammatory disorders.
v. Hypersegmented neutrophils: Hypersegmentation of
neutrophils is said to be present when >5% of
neutrophils have 5 or more lobes. They are large
in size and are also called as macropolycytes. They
are seen in folate or vitamin B12 deficiency and
represent one of the earliest signs.
vi. Pelger-Huet cells: In Pelger-Huet anomaly (a benign
autosomal dominant condition), there is failure
of nuclear segmentation of granulocytes so that
nuclei are rod-like, round, or have two segments.
Such granulocytes are also observed in myelo-
proliferative disorders (pseudo-Pelger-Huet cells).
vii. Atypical lymphocytes: These are seen in viral
infections, especially infectious mononucleosis.
Atypical lymphocytes are large, irregularly
shaped lymphocytes with abundant cytoplasm
and irregular nuclei. Cytoplasm shows deep
basophilia at the edges and scalloping of borders.
Nuclear chromatin is less dense and occasional
nucleolus may be present.
viii. Blast cells: These are most premature of the
leukocytes. They are large (15-25 µm), round to
oval cells, with high nuclear cytoplasmic ratio.
Nucleus shows one or more nucleoli and nuclear
chromatin is immature. These cells are seen in
severe infections, infiltrative disorders, and
leukemia. In leukemia and lymphoma, blood
smear suggests the diagnosis or differential
diagnosis and helps in ordering further tests
(Fig. 22.13 and Box 22.3).
3. Differential leukocyte count (DLC): DLC refers to
relative proportion of different leukocytes expressed
as a percentage.
 Blood Smear 209

Box 22.3: Role of blood smear in leukemia

• It suggests the diagnosis or differential diagnosis

• It suggests further investigations to be performed for
definitive diagnosis.

Principle: Leukocytes are counted on a blood smear and

percentage of each type of leukocyte is recorded.
Uses of DLC
1. To support the diagnosis of infectious, inflammatory,
or allergic disorders.
2. Diagnosis of malignant blood disorders.
Using a high power objective, leukocytes should be
classified and counted moving from one field to another
as shown in Figure 22.4. DLC should not be carried out
on the edges and in the tail of the film. In a badly spread
smear, polymorphonuclear neutrophils, monocytes, and
abnormal cells tend to accumulate in 'feather edge' (tail)
and lateral edges, while lymphocytes accumulate in the
body of the smear. Also, cells in the body of the smear
are shrunken and darkly stained. DLC on such a smear
will not be representative. If count is high and abnormal
cells are present, DLC should be done in the area just
before the tail of the film. This is because morphology is
better appreciated in this area.
4. Numerical abnormalities of leukocytes (Fig. 22.14):
For meaningful interpretation, absolute count of
leukocytes should be reported. These are obtained as
follows:
Fig. 22.14: Numerical abnormalities of white blood cells: (A)
Neutrophilia; (B) Eosinophilia; (C) Monocytosis; (D) Lympho-
cytosis
8. Malignant tumors
9. Physiologic causes: Exercise, labor, pregnancy,
emotional stress.
Leukemoid reaction: This refers to the presence of markedly
increased total leukocyte count (>50,000/cmm) with
immature cells in peripheral blood resembling leukaemia
but occurring in non-leukemic disorders (Fig. 22.15). Its
causes are:
• Severe bacterial infections, e.g. septicemia, pneumo-
nia
• Severe hemorrhage
• Severe acute hemolysis

Absolute leukocyte count = Percentage of leukocyte ×

Total leukocyte count/ml

Neutrophilia
An absolute neutrophil count greater than 7500/µl is
termed as neutrophilia or neutrophilic leukocytosis.
Causes
1. Acute bacterial infections: Abscess, pneumonia,
meningitis, septicemia, acute rheumatic fever, urinary
tract infection.
2. Tissue necrosis: Burns, injury, myocardial infarction.
3. Acute blood loss
4. Acute hemorrhage
5. Myeloproliferative disorders
6. Metabolic disorders: Uremia, acidosis, gout
7. Poisoning Fig. 22.15: Leukemoid reaction in blood smear
210 Essentials of Clinical Pathology

Table 22.1: Differences between leukemoid reaction and leukemia

Parameter Leukemoid reaction Leukemia

1. Clinical presentation Features of underlying Splenomegaly

2. Examination of blood
a. Total leukocyte count
b. Course of neutrophilia
c. Evidence of infection
d. Basophilia
e. Immature cells
3. Examination of marrow
4. Clonality
5. Karyotype
disease; fever common
< 50000/µl Variable, usually > 1 lac/µl
Disappears with resolution of Progressive increase
underlying disease
Toxic granules, Döhle Absent
inclusion bodies
Absent Present
Few; cells up to myelocyte stage Many; cells up to blasts
Myeloid hyperplasia Increased blasts and immature
cells of neutrophil series;
Suppression of other cell lines
Polyclonal Monoclonal
Normal Abnormal

• Poisoning II. Increased destruction in peripheral blood:

• Burns
• Carcinoma metastatic to bone marrow
Leukemoid reaction should be differentiated from
chronic myeloid leukemia (Table 22.1).
Neutropenia: Absolute neutrophil count less than 2000/
µl is neutropenia. It is graded as mild (2000-1000/µl),
moderate (1000-500/µl), and severe (< 500/µl).
Causes
I. Decreased or ineffective production in bone marrow:
1. Infections
a. Bacterial: typhoid, paratyphoid, miliary tuber-
culosis, septicemia
b. Viral: influenza, measles, rubella, infectious
mononucleosis, infective hepatitis.
c. Protozoal: malaria, kala azar
d. Overwhelming infection by any organism
2. Hematologic disorders: megaloblastic anemia,
aplastic anemia, aleukemic leukemia, myelo-
phthisis.
3. Drugs:
a. Idiosyncratic action: Analgesics, antibiotics,
sulfonamides, phenothiazines, antithyroid
drugs, anticonvulsants.
b. Dose-related: Anticancer drugs
4. Ionizing radiation
5. Congenital disorders: Kostman's syndrome, cyclic
neutropenia, reticular dysgenesis.
1. Neonatal isoimmune neutropaenia
2. Systemic lupus erythematosus
3. Felty's syndrome
III. Increased sequestration in spleen:
1. Hypersplenism.
Eosinophilia
This refers to absolute eosinophil count greater than 600/
µl.
Causes
1. Allergic diseases: Bronchial asthma, rhinitis, urticaria,
drugs.
2. Skin diseases: Eczema, pemphigus, dermatitis
herpetiformis.
3. Parasitic infection with tissue invasion: Filariasis,
trichinosis, echinococcosis.
4. Hematologic disorders: Chronic myeloproliferative
disorders, Hodgkin's disease, peripheral T cell
lymphoma.
5. Carcinoma with necrosis.
6. Radiation therapy.
7. Lung diseases: Loeffler's syndrome, tropical eosino-
philia
8. Hypereosinophilic syndrome.
Basophilia: Increased numbers of basophils in blood
(>100/μl) occurs in chronic myeloid leukemia, poly-
cythemia vera, idiopathic myelofibrosis, basophilic
leukemia, myxedema, and hypersensitivity to food or
drugs.

Monocytosis
This is an increase in the absolute monocyte count above
1000/µl.
Causes
1. Infections: Tuberculosis, subacute bacterial endocar-
ditis, malaria, kala azar.
2. Recovery from neutropenia.
3. Autoimmune disorders.
4. Hematologic diseases: Myeloproliferative disorders,
monocytic leukemia, Hodgkin's disease.
5. Others: Chronic ulcerative colitis, Crohn's disease,
sarcoidosis.
Lymphocytosis
This is an increase in absolute lymphocyte count above
upper limit of normal for age (4000/µl in adults, >7200/
µl in adolescents, >9000/µl in children and infants) (Box
22.4).
Causes
1. Infections:
• Viral: Acute infectious lymphocytosis, infective
hepatitis, cytomegalovirus, mumps, rubella,
varicella
• Bacterial: Pertussis, tuberculosis
• Protozoal: Toxoplasmosis
2. Hematological disorders: Acute lymphoblastic
leukemia, chronic lymphocytic leukemia, multiple
myeloma, lymphoma.
3. Other: Serum sickness, post-vaccination, drug
reactions.
Platelets
Platelets are small, 1-3 µm in diameter, purple structures
with tiny irregular projections on surface. In blood films
prepared from non-anticoagulated blood (i.e. direct
Fig. 22.16: Causes of false thrombocytopenia on automated hematology analyzer:
(A) Clumps of platelets; (B) Platelet satellitism
Blood Smear 211
Box 22.4: Differential diagnosis of lymphocytosis
• Mature lymphocytosis: Viral infections, whooping
cough, tuberculosis, infectious lymphocytosis, chronic
lymphocytic leukemia
• Atypical lymphocytosis: Infectious mononucleosis,
cytomegalovirus, toxoplasmosis, infectious hepatitis
• Lymphoblasts: Acute lymphoblastic leukemia
fingerstick), they occur in clumps. If platelet count is done
on automated blood cell counters using EDTA-anticoagu-
lated blood sample, about 1% of persons show falsely
low count due to the presence in them of EDTA-
dependent antiplatelet antibody.
Examination of a parallel blood film is useful in
avoiding the false diagnosis of thrombocytopenia in such
cases. Occasionally, platelets show rosetting around
neutrophils (platelet satellitism) (Fig. 22.16). This is seen
in patients with platelet antibodies and in apparently
normal persons.
Blood smear examination can be helpful in deter-
mining underlying cause of thrombocytopenia such as
leukemia, lymphoma, or microangiopathic hemolytic
anemia (Box 22.5).
Organisms
Common parasites seen in blood are malaria parasites
and microfilaria (See Chapter 26: Diagnosis of Malaria
and Other Parasites in Blood). Other parasites that can
Box 22.5: Role of blood smear in thrombocytopenia
It suggests probable cause, e.g. leukemia, microangio-
pathic process like thrombotic thrombocytopenic purpura
or disseminated intravascular coagulation, aplastic
anemia, May-Hegglin anomaly.
212 Essentials of Clinical Pathology

be seen in blood smear are Leishmania donovani, and • Eosinophils: 1-6%

trypanosomes. Sometimes bacteria such as meningococci • Basophils: 0-1%
and fungi (e.g. Histoplasma capsulatum) can be observed
within phagocytic leukocytes. Critical Values
• Blood smear showing sickle cells
REFERENCE RANGES • Blood smear showing blast cells
• Leukemoid reaction
Normal Blood Smear • Suspected aplastic anemia
• Malarial parasites
Red blood cells: Normocytic and normochromic
• Absolute neutrophil count <500/cmm.
White blood cells: Total and differential leucocyte
counts within normal limits BIBLIOGRAPHY
Platelets: Adequate 1. Bain BJ. Diagnosis from the blood smear. N Engl J Med
Parasites: Not seen. 2005;353:498-507.
2. Houwen B. Blood film preparation and staining
Differential Leukocyte Count procedures. Clin Lab Med 2002;22:1-14.
3. International Council for Standardization in Haema-
• Neutrophils: 40-75% tology (ICSH). ICSH reference method for staining of
• Lymphocytes: 20-40% (in children between 4 months blood and bone marrow by azure B and eosin Y
to 4 years of age, percentage of lymphocytes is more (Romanowsky stain). Br J Haematol 1984;57:707-10.
4. Kawthalkar SM. Essentials of Hematology. New Delhi.
than neutrophils; this is called as inverted differential Jaypee Brothers Medical Publishers (P) Ltd, 2006.
in children) 5. Woronzoff-Dashkoff KK. The Wright-Giemsa stain:
• Monocytes: 2-10% secrets revealed. Clin Lab Med 2002;22:15-23.
 23
Red Cell Indices
Red cell indices are mean cell volume (MCV), mean cell
hemoglobin (MCH), and mean cell hemoglobin concen-
tration (MCHC). They are also called as “absolute
values”. They are derived from the values of hemoglobin,
packed cell volume (PCV or hematocrit), and red cell
count. Red cell indices are accurately measured by
automated hematology analyzers. Recently, a new
parameter called red cell distribution width (RDW) has
been introduced.
USES OF RED CELL INDICES
1. Morphological classification of anemia: Based on
values of red cell indices, anemia is classified into
threee main types: normocytic normochromic,
microcytic hypochromic, and macrocytic normo-
chromic (See Chapter 27: Laboratory Tests in
Anemia). Calculation of red cell indices is especially
helpful in mild or moderate anemia when red cell
changes are subtle and often difficult to appreciate
on stained blood smear.
2. Differentiation of iron deficiency anemia from
thalassemia trait: In iron deficiency, MCV, MCH, and
MCHC are low, while in thalassemia trait, MCV and
MCH are low and MCHC is normal.
MEAN CELL VOLUME
MCV is a measure of average size of the red cells. It is
measured directly by automated instruments from the
measurement of volume of each red cell. With semi-
automated instruments and by manual method, it is
obtained by dividing PCV by red cell count.
PCV in%
MCV = —————————————— × 10
Red cell count in million/cmm
MCV is expressed in femtoliters or fl (10–15 of a liter).
It corresponds with red cell diameter on blood smear.
Normal MCV is 80-100 fl.
Causes of Increased MCV
• Megaloblastic anemia
• Non-megaloblastic macrocytosis: Chronic alcoholism,
liver disease, hypothyroidism, normal pregnancy,
reticulocytosis
• Newborns.
Causes of Low MCV
• Microcytic hypochromic anemia
MCV is normal in normocytic normochromic anemia
(acute blood loss, hemolysis, aplastic anemia).
In the presence of large number of abnormal red cells
like sickle cells, and in dimorphic anemia (e.g. mixed
normocytic and microcytic), MCV may be normal (since
it is an average value) and thus unreliable for morpho-
logical classification.
Mentzer index is derived by dividing MCV with red
cell count. Ratio of less than 13 is seen in thalassemia
while ratio is more than 13 in iron deficiency anemia.
MEAN CELL HEMOGLOBIN (MCH)
MCH is the average amount of hemoglobin in a single
red cell. It is obtained by dividing hemoglobin value by
red cell count.
Hemoglobin in grams/dl
MCH = ——————————————— × 10
Red cell count in millions/cmm
MCH is expressed in picograms or pg (10–12 gram).
Reference range is 27-32 pg.
214 Essentials of Clinical Pathology
MCH is decreased in microcytic hypochromic
anemia, and increased in macrocytic anemia and in
newborns.
MEAN CELL HEMOGLOBIN
CONCENTRATION (MCHC)
MCHC is obtained by dividing hemoglobin value by PCV
and expressed in grams/dl or grams/liter. It refers to
concentration of hemoglobin in 1 dl or 1 liter of packed
red cells.
Hemoglobin in grams/dl
MCHC = ——————————— × 100
PCV in %
Reference range is 30-35 grams/dl.
MCHC is raised in hereditary spherocytosis, and is
decreased in hypochromic anemia. MCHC corresponds
with degree of hemoglobinization of red cells on a blood
smear. If MCHC is normal, red cell is normochromic,
and if low, red cell is hypochromic.
Red Cell Distribution Width (RDW)
Some automated analyzers measure red cell distribution
width or RDW. It is a measure of degree of variation in
red cell size (anisocytosis) in a blood sample. It is helpful
in differential diagnosis of some anemias. Amongst
microcytic anemias, RDW is low in β thalassemia trait,
high in iron deficiency anemia, and normal in anemia of
chronic disease. Normal RDW is 9.0 to 14.5.
REFERENCE RANGES
• Mean cell volume: 80-100 fl
• Mean cell hemoglobin: 27-32 pg
• Mean cell hemoglobin concentration: 30-35 g/dl
• Red cell distribution width: 9.0-14.5
BIBLIOGRAPHY
1. Henry JB. Clinical diagnosis and management by
laboratory methods (20th Ed). Philadelphia: WB
Saunders Company, 2001.
2. Wallach J. Interpretation of Diagnostic Tests (7th Ed).
Philadelphia: Lippincott Williams and Wilkins, 2000.
tahir99 - UnitedVRG
vip.persianss.ir
 24
Erythrocyte
Sedimentation Rate

The erythrocyte sedimentation rate (ESR) measures the

rate of settling (sedimentation) of erythrocytes in
anticoagulated whole blood. Anticoagulated blood is
allowed to stand in a glass tube for 1 hour and the length
of column of plasma above the red cells is measured in
millimeters; this corresponds to ESR.
STAGES OF ERYTHROCYTE
SEDIMENTATION RATE
There are three stages of erythrocyte sedimentation:
• Stage 1: Formation of rouleaux or lag phase (10
minutes): Red cells stack together like a pack of coins.
The ESR depends mainly on this stage.
• Stage 2: Sinking of rouleaux or decantation phase (40
minutes): Rapid and constant sedimentation. The
longer the tube, the longer is this stage and higher
the value of ESR.
• Stage 3: Packing of rouleaux (10 minutes): Slow
sedimentation.
FACTORS AFFECTING ERYTHROCYTE
SEDIMENTATION RATE
Red cells, composition of plasma, and technical factors
affect ESR (Box 24.1).
1. Red cells: Alteration of ratio of red cells to plasma
affects ESR. Decreased red cell mass in anemia
increases ESR. Conversely, increased red cell mass
in polycythemia decreases ESR.
Macrocytes tend to sediment rapidly than microcytes.
Sickle cells and spherocytes are unable to form
rouleaux and therefore ESR is low in sickle cell disease
and hereditary spherocytosis. In these conditions,
ESR is not reliable as an indicator of illness.
2. Plasma: The most important factor affecting ESR is
the composition of plasma. Increase in fibrinogen,
Box 24.1: Factors affecting erythrocyte
sedimentation rate
• Factors increasing ESR
– Old age
– Pregnancy
– Anemia
– Elevated fibrinogen
– Macrocytosis
– Technical factors: High temperature, tilting of ESR tube
• Factors decreasing ESR
– Microcytosis
– Low fibrinogen
– Polycythemia
– Marked leukocytosis
– Technical factors: Vibration of tube during test
other acute phase proteins (C-reactive protein,
haptoglobin, ceruloplasmin, α1-antitrypsin, etc.) and
immunoglobulins increase ESR. Increased proteins
in plasma reduce negative charge on the surface of
red cells and reduce the zeta potential (the electrical
repulsion between red cells); this brings red cells
closer together and facilitates rouleaux formation.
Removal of fibrinogen by defibrination and increase
of albumin retard ESR.
3. Technical factors: ESR increases with room tempe-
rature. Tilting of the tube, and length and bore of the
tube affect ESR.
SIGNIFICANCE OF ERYTHROCYTE
SEDIMENTATION RATE
ESR is elevated in a wide range of organic diseases. ESR
is not a specific and diagnostic test for any disease.
However, it is helpful in differentiating functional from
organic disease. Raised ESR signifies presence of some

tahir99 - UnitedVRG
vip.persianss.ir
216 Essentials of Clinical Pathology

Table 24.1: Causes of increased erythrocyte
sedimentation rate
1. Infections • Acute rheumatic fever
• Osteomyelitis
• Bacterial endocarditis
• Pyogenic arthritis
• Pelvic inflammatory disease
• Tuberculosis
• Acute hepatitis
2. Inflammatory diseases • Rheumatoid arthritis
• Systemic lupus
erythematosus
• Temporal arteritis
• Polymyalgia rheumatica
3. Acute myocardial
infarction
4. Malignancy
5. Paraproteinemias • Multiple myeloma
• Waldenström’s
macroglobulinemia
• Cryoglobulinemia
6. Technical problems • Increased temperature
• Tilted ESR tube
7. Others • Ruptured ectopic pregnancy
• Anemia
• Renal disease with
azotemia
• Administration of dextran or
oral contraceptives
(≥100 mm at 1 hour) is seen in infections, parapro-
teinemias, metastatic malignancy, polymyalgia rheu-
matica, temporal arteritis, and rheumatoid arthritis.
ESR is decreased in polycythemia, congestive cardiac
failure, dehydration, sickle cell anemia, hereditary
spherocytosis, and hypofibrinogenemia.
In patients with chronic inflammatory conditions like
rheumatoid arthritis, ESR can be helpful in distinguishing
iron deficiency anemia from anemia of chronic disease.
In inflammatory conditions, ESR rises more than 24
hours after onset and returns to normal about 4 weeks
following resolution.
There is no role of ESR in the evaluation of asympto-
matic patients.
INDICATIONS FOR MEASUREMENT OF
ERYTHROCYTE SEDIMENTATION RATE
It is recommended to measure ESR (i) when infectious,
inflammatory, or a neoplastic disease is suspected in
symptomatic individuals in whom a specific diagnosis
has not been established, (ii) to monitor disease activity
in tuberculosis, inflammatory arthritis, rheumatic fever,
Hodgkin’s disease, giant cell arteritis, and polymyalgia
rheumatica, and (iii) as a diagnostic criterion for temporal
arteritis and polymyalgia rheumatica. ESR can also be
helpful in distinguishing iron deficiency anemia from
anemia of chronic disease in patients known to have
inflammatory disease.

METHODS FOR ESTIMATION OF

organic disease, which needs evaluation. Most of the
inflammatory and neoplastic diseases are associated with
an increase in ESR (Table 24.1).
ESR correlates with disease activity and therefore it
is helpful in monitoring disease activity and response to
therapy in acute rheumatic fever, bacterial endocarditis,
tuberculosis, rheumatoid arthritis, temporal arteritis,
polymyalgia rheumatica, and Hodgkin’s disease.
ESR is an important criterion in establishing the
diagnosis of temporal arteritis and polymyalgia
rheumatica.
ESR is not significantly raised in typhoid fever,
malaria, infectious mononucleosis, angina pectoris,
osteoarthritis, acute appendicitis, peptic ulcer, acute
allergy, and unruptured ectopic pregnancy. In emer-
gency situations, ESR may be helpful in distinguishing
angina pectoris from myocardial infarction, and acute
appendicitis from other causes of acute abdomen
particularly pelvic inflammatory disease or ruptured
ectopic pregnancy. Very high or extreme elevation of ESR
ERYTHROCYTE SEDIMENTATION RATE
These include:
• Westergren method
• Wintrobe method
• Zeta sedimentation ratio
• Micro-ESR
Westergren Method
International Council for Standardization in Hematology
recommends Westergren method for estimation of ESR.
Equipment and Reagent
1. Westergren ESR tube: This is a straight glass pipette
measuring 300 mm in length and calibrated in mm
from 0-200 (top to bottom). The markings are only
over the lower 2/3rds of the tube. The tube is open-
ended. Internal diameter should not be less than 2.55
mm (Fig. 24.1). The tube should be clean and dry.

tahir99 - UnitedVRG
vip.persianss.ir

Fig. 24.1: Westergren and Wintrobe tubes for measurement of ESR
Fig. 24.2: Westergren ESR stand with ESR tubes
2. Westergren stand: This holds the tube in a motionless,
vertical position (Fig. 24.2).
3. Anticoagulant-diluent solution: Trisodium citrate
dihydrate is the anticoagulant of choice. Its compo-
sition is as follows:
Trisodium citrate dihydrate 32.08 gm
Distilled water upto 1000 ml
It is filtered through a sterile membrane (0.22 μm)
into a sterile container and stored in a refrigerator at 4°C.
It keeps for several months, but if it becomes turbid (due
to the growth of moulds), it should be discarded.
Alternate anticoagulant is EDTA (1.5 mg/ml);
however, such anticoagulated blood sample should be
diluted with trisodium citrate just before the test
(1 volume of trisodium citrate to 4 volumes of blood).
Erythrocyte Sedimentation Rate 217
Specimen: Venous blood is collected in trisodium citrate
solution in 4:1 (blood: citrate) proportion. If specimen is
kept at room temperature, test should be carried out
within 4 hours of blood collection; if stored at 4°C, a delay
of up to 6 hours is permissible. Blood anticoagulated with
EDTA can be tested within 24 hours if stored at 4°C
(provided it is diluted with trisodium citrate before
testing).
Method
1. Mix anticoagulated blood sample thoroughly. The
Westergren tube is filled with the blood sample up
to the “0” mark. A rubber bulb or a mechanical device
should be used for filling. There should be no air
bubbles in the blood.
2. The tube is placed in a strictly vertical position in the
ESR stand and left undisturbed for 1 hour. It should
not be kept in direct sunlight and should not be
subjected to vibrations.
3. After exactly 1 hour, read the height of the column of
plasma above the red cell column in mm.
4. Express the result as:
Erythrocyte sedimentation rate = ——— mm in 1 hour.
Precautions
1. Use the correct proportion of blood and anticoagulant.
Mix blood and anticoagulant thoroughly. There
should be no clots and air bubbles in blood.
2. The reference range relates to test performed at room
temperature of 18-25°C. If temperature is higher, ESR
will increase and different reference range will have
to be derived.
tahir99 - UnitedVRG
vip.persianss.ir
218 Essentials of Clinical Pathology

3. ESR tube must be kept vertically. Even a slight tilting

will increase the ESR.

Other Methods

Wintrobe Method
Wintrobe tube (Fig. 24.1) can be used for estimation of
both ESR and packed cell volume (PCV). After obtaining
ESR in the first hour, the tube can be spun in a centrifuge
to get PCV. Wintrobe’s method is more reliable when
ESR is low, while Westergren’s method is more sensitive
for high ESR. EDTA or double oxalate is used as an
anticoagulant. Length of Wintrobe tube is shorter (110
mm) and internal diameter is about 3 mm.

Zeta Sedimentation Ratio (ZSR)

A special device called as zetafuge is required. ZSR is
not affected by anemia, unlike Westergren method.

Micro-ESR Fig. 24.3: Formation of acute phase proteins by

This method uses capillary blood that is collected in
anticoagulant-coated capillary tubes. It is more suitable
in small children.
ACUTE PHASE REACTANTS
The acute phase proteins are plasma proteins synthesized
in liver that undergo change in concentration in response
to infection, tissue injury, and inflammation (Table 24.2).
Acute phase proteins are synthesized by liver
following stimulation by cytokines like interleukin-1,
interleukin-6, and tumor necrosis factor-α synthesized
by monocytes and macrophages (Fig. 24.3). Acute phase
reactants play a role in the defensive process of
inflammation. They help in opsonization, neutralization
of proteolytic enzymes released from neutrophils
(thereby limiting tissue damage), clearance of cell debris,
and healing of wounds
Tests are available for estimation of C-reactive protein
(CRP) in serum. When it was first described in 1930, it
was found to bind to C-polysaccharide in the cell wall of
Streptococcus pneumonie; hence the name C-reactive
Table 24.2: Acute phase proteins
• C-reactive protein • Serum amyloid A protein
• Fibrinogen • Ceruloplasmin
• α1-antitrypsin • α1 acid glycoprotein
• Haptoglobin • Ferritin
liver in inflammation
protein. Test for CRP is helpful in determining the
presence and extent of inflammation and in monitoring
response to therapy. Among the acute phase reactants,
CRP is earliest to rise during inflammation and to return
rapidly to normal level following successful therapy.
Unlike other acute phase proteins, hormones or drugs
do not affect CRP levels and no deficiency state of CRP
has been reported. Also, CRP rises to a more significantly
higher level than other acute phase reactants. Therefore
among acute phase proteins, measurement of CRP is
preferred.
In acute disease, CRP level correlates well with ESR.
As compared to ESR, CRP is (i) earlier to rise in
inflammation and earlier to return to normal following
resolution, (ii) more sensitive, and (iii) more specific since
it is not affected by anemia, polycythemia, alteration of
red cell shape, and paraproteinemia (Table 24.3).
Normal level of CRP is less than 8 mg/L.
Uses of C-reactive Protein
1. Detection of infection: CRP level is elevated in bacterial
infections. It is especially helpful in neonates (bacterial
sepsis and meningitis). Sequential testing is helpful
in monitoring response to antimicrobial therapy. In
immunocompromised patients, due to absence of
fever and leukocytosis, elevation of CRP is helpful in
suggesting infection.

tahir99 - UnitedVRG
vip.persianss.ir
 Erythrocyte Sedimentation Rate 219

Table 24.3: Comparison of erythrocyte sedimentation rate and C-reactive protein

Erythrocyte sedimentation rate C-reactive protein

1. Affected by age, sex, pregnancy, temperature, 1. Not affected

level of plasma proteins, anemia and red cell morphology
2. Less sensitive and specific for acute phase response
3. Inexpensive and easy procedure
4. Specimen: Fresh whole anticoagulated blood
5. Indirect means of assessing acute phase response
2. More sensitive and more specific for acute phase
response
3. Expensive and requires sophisticated materials and
equipment
4. Specimen: Serum that may have been stored
5. Direct measurement of acute phase response

2. Assessment of severity of inflammatory disorders like Erythrocyte Sedimentation Rate by

rheumatoid arthritis and acute rheumatic fever.
3. Detection of postoperative infection: CRP level begins to
rise 4-6 hours after surgery, peaks in 2-3 days, and
returns to normal by 7th day. Failure of expected
normalization indicates postoperative infection.
4. Myocardial infarction: CRP rises over 24-48 hours,
peaks at 72 hours, and disappears by 7th day after
acute myocardial infarction. Failure to return to
normal indicates re-infarction.
5. Screening for presence or absence of organic disease.
REFERENCE RANGES
Erythrocyte Sedimentation Rate by
Westergren Method
• Males < 50 years: 0-15 mm in 1 hr
• Females<50 years: 0-20 mm in 1 hr
• Children: 0-10 mm in 1 hr
• Elderly (>50 years): Males: 0-20 mm in 1 hr and
females: 0-30 mm in 1 hr.
Wintrobe Method
• Males: 0-9 mm in 1 hour
• Females: 0-20 mm in 1 hour
• Children: 0-13 mm in 1 hour
C-reactive Protein
< 8 mg/L.
BIBLIOGRAPHY
1. Brigden ML. Clinical utility of the erythrocyte
sedimentation rate. Am Fam Physician 1999;60:
1443-50.
2. International Council for Standardization in Hema-
tology. ICSH recommendation for measurement of
erythrocyte sedimentation rate. J Clin Path 1993;46:
198-203.
3. Smellie WS, Forth JO, McNulty CAM, et al. Best practice
in primary care pathology: review 2. J Clin Pathol
2006;59:113-20.
4. Stuart J and Lewis SM. Recommendations, safety and
quality control of erythrocyte sedimentation rate. World
Health Organization. 1993. WHO/LBS/93.1.

tahir99 - UnitedVRG
vip.persianss.ir
 25
Examination of
Bone Marrow
NORMAL BONE MARROW
Bone marrow is the site of hematopoiesis in postnatal
life. Bone marrow is one of the largest organs in the body,
and although distributed widely in the skeleton,
functions as a single unit. It is located within the cavities
of the bones and mainly consists of hematopoietic cells,
vascular sinusoids, fibroblasts, fat cells, and macro-
phages. There are no lymphatic channels in the bone
marrow. There are two types of marrow: red and yellow.
Red marrow refers to the active hematopoietic tissue
while fatty tissue comprises the yellow (inactive)
marrow. Red coloration is due to presence of large
amounts of developing red cells. The average volume of
bone marrow (red and yellow) in an adult is 3000-4000
ml. Red or active marrow constitutes 1500 ml.
The hematopoietic cells are present as cords between
vascular sinusoids and are supported by a framework of
branching processes of fibroblasts and reticulin fibers.
Erythroid precursors are present as clusters (islands) and
are closely associated with centrally placed sinusoids.
An erythroid island consists of a centrally placed
macrophage around which erythroid precursors are
concentrically arranged. Early granulocyte precursors are
located close to the bony trabecule, while more mature
granulocytes are located more centrally, between
adjacent trabeculae. Megakaryocytes, the largest of the
hematopoietic cells, are closely apposed to the walls of
the sinusoids. Lymphocytes and plasma cells are mostly
present around capillaries and arterial vessels. In contrast
to other blood cells, lymphocytes also divide and mature
outside the bone marrow.
Hematopoiesis occurs extravascularly in between
interconnecting marrow sinusoids. The marrow sinu-
soids are lined by endothelial cells and are covered
partially by cytoplasmic processes of fibroblasts. After
development, mature blood cells leave the bone marrow
and enter the circulation by passing through or in
between the endothelial cells of the sinusoids.
Marrow fibroblasts (reticular cells) are highly
branched cells, which form reticulin fibers and form a
supporting framework for the hematopoietic cells.
Accumulation of lipid in reticular cells is said to produce
fat cells of the bone marrow.
Amount of fatty tissue depends on activity of
hematopoietic cells. The proportion of fat cells increases
with advancing age with corresponding decrease in
hematopoietic tissue. On demand, fatty tissue (yellow
marrow) can be rapidly replaced by hematopoietic tissue
(red marrow). In children and adolescents, fat cells
occupy about 20-30% of the bone marrow volume, in
adults about 50%, while in the elderly about 70%.
Macrophages in bone marrow serve various functions
like synthesis of hematopoietic growth factors and
inhibitory substances, phagocytosis of senescent red cells,
and transport of iron to the erythroblasts.
The stromal cells of the bone marrow (fibroblasts, fat
cells, macrophages, and endothelial cells) and vascular
sinusoids constitute the bone marrow microenvironment,
which is essential for normal hematopoiesis.
During infancy and early childhood, hematopoiesis
is occurring in almost all the bones of the body. By late
childhood, hematopoiesis becomes mainly restricted to
flat bones like sternum, ribs, iliac bones, and vertebre,
and proximal ends of long bones; other sites of red
marrow are transformed into yellow marrow. However,
when there is increased demand for blood cells, blood
cell production can resume in the yellow marrow. In
extremely severe cases (e.g. chronic hemolytic anemia),
resumption of hematopoiesis in other organs like liver
and spleen (extramedullary hematopoiesis) can occur.
tahir99 - UnitedVRG
vip.persianss.ir

Apart from its major function of hematopoiesis, bone
marrow also plays a role in the removal of senile and
defective red cells (via macrophages) and immunity
(processing of antigen by macrophages and synthesis of
antibodies by plasma cells).
There are two techniques for sampling of bone
marrow: aspiration and biopsy. In aspiration, bone
marrow fluid is obtained by a special needle and syringe.
Smears from this material are prepared on glass slides,
stained, and examined under the microscope. In bone
marrow trephine biopsy (core biopsy), a small tissue
piece of bone marrow is removed with a special needle,
processed to obtain histological sections, and examined.
INDICATIONS FOR BONE MARROW
EXAMINATION
Before performing bone marrow aspiration/biopsy, one
should assess clinical features, treatment received, and
relevant laboratory test results (especially basic blood
studies and peripheral blood smear). Based on above
data, if appropriate indication exists, then examination
of bone marrow is carried out and findings are correlated
to arrive at the final diagnosis.
INDICATIONS FOR BONE MARROW
ASPIRATION
1. Unexplained cytopenia: If the cause of cytopenia
(anemia, leukopenia, or thrombocytopenia) is not
apparent from blood investigations and clinical
details, bone marrow examination is indicated.
(A) To distinguish amongst causes of microcytic hypo-
chromic anemia (e.g. iron deficiency from chronic
disease), bone marrow examination can be done to
assess storage iron. Identification of ringed sidero-
blasts is helpful in diagnosis of sideroblastic anemia
or myelodysplastic syndrome. In macrocytic anemia,
in the absence of vitamin assays and typical features
of megaloblastic anemia on blood smear, marrow
aspiration is carried out to distinguish megaloblastic
macrocytosis from non-megaloblastic one. (B) In
leukopenia or thrombocytopenia, bone marrow
examination is helpful in distinguishing peripheral
destruction from deficient production. (C) In the
presence of isolated thrombocytopenia, if idiopathic
thrombocytopenic purpura (ITP) is suspected,
marrow examination is done for ruling out under-
lying hematologic disorder or deficient platelet
production.
Examination of Bone Marrow 221
2. Suspected acute leukemia: In majority of cases, acute
leukemia can be diagnosed from examination of a
blood smear and cell counts. Bone marrow exami-
nation is carried out in acute leukemia for:
• Detection of subleukemic or aleukemic leukemia
• Comparison of baseline marrow smear with
follow-up marrow aspiration smears during
treatment
• Diagnosis of acute myeloid leukemia with
trilineage dysplasia
• Cytogenetic analysis.
• Detection of remission
3. Suspected myelodysplastic syndrome.
4. Suspected myeloproliferative disorders including
chronic myeloid leukemia, polycythemia vera,
essential thrombocythemia, and myelofibrosis.
5. Suspected plasma cell dyscrasia: Bone marrow
aspiration is indicated if multiple myeloma is
suspected from clinical and radiologic features.
6. Suspected chronic lymphoid leukemias like chronic
lymphocytic leukemia, prolymphocytic leukemia,
hairy cell leukemia, etc.
7. Investigation of pyrexia of unknown origin:
Sometimes, bone marrow aspiration smears are
helpful in detecting Histoplasma capsulatum and
Leishmania donovani organisms in macrophages of
bone marrow. Aspirated material can also be cultured
to isolate above organisms or mycobacteria.
8. Suspected storage disorder like Gaucher’s disease or
Neimann-Pick disease.
9. Suspected infections like kala azar, miliary tuber-
culosis, or histoplasmosis.
INDICATIONS FOR BONE MARROW BIOPSY
1. Repeated failure of aspiration (dry tap) which may
be due to faulty technique, myelofibrosis or leuke-
mia.
2. Suspected aplastic anemia
3. Suspected myelofibrosis.
4. Suspected focal lesions like granuloma, metastatic
deposit, or infiltrate of lymphoma.
5. Suspected hairy cell leukemia.
6. Suspected bone disorder, e.g. osteopetrosis.
7. Staging of lymphoma.
CONTRAINDICATIONS
Bone marrow aspiration or biopsy is contraindicated in
hemophilia and other coagulation disorders; however,
tahir99 - UnitedVRG
vip.persianss.ir
222 Essentials of Clinical Pathology

in these patients it can be performed under cover of

appropriate replacement therapy. Thrombocytopenic
purpura is not a contraindication since applying firm
pressure for 5 minutes at the puncture site can prevent
excess bleeding.

SITES FOR BONE MARROW

ASPIRATION OR BIOPSY
• Iliac spines or crest: The most frequently used site
for both marrow aspiration and biopsy in children
(>1 year of age) as well as in adults is posterior iliac
crest (posterior superior iliac spine). This site has a
large reservoir of marrow, is located just beneath the
skin and therefore easily accessible. Also, there are
no large blood vessels or nerves close to this area,
and as the patient’s back is towards the physician,
patient’s apprehension is less.
In obese patients, anterior superior iliac spine,
Fig. 25.1: Sites of bone marrow aspiration
being more easily localized, can be used.
(shaded areas)
• Sternum: Previously, sternum was commonly used
for aspiration of bone marrow in adults (at the level
of second intercostal space in midline). However, it
is associated with the risk of perforation of posterior
sternal plate and puncturing of underlying large
blood vessels and right atrium with serious conse-
quences. It also causes greatest patient anxiety. This
site should not be used in children as the bone is thin
and marrow cavity is small.
• Spinous processes of lumbar vertebra: This is an
additional site for aspiration in adults.
• Tibia: In infants under 1 year of age, marrow can be
Fig. 25.2: Klima and Salah bone marrow aspiration needles.
aspirated from the medial aspect of upper end of tibia Adjustable guard prevents damage to deeper structures. Stylet
just beneath tibial tuberosity. In older children, the keeps needle patent during introduction
tibial cortical bone becomes hard and cellularity of
marrow decreases, and therefore this site is not used.
solution, clean and dry glass slides, spreader slide,
Sites for bone marrow aspiration are shown in Figure
gloves, drapes, gauze, and a skin antiseptic solution.
25.1.
All aseptic precautions should be observed during
the procedure. Various bone marrow aspiration
METHOD
needles are available. Salah and Klima needles are
Bone Marrow Aspiration commonly used (Fig. 25.2). Salah needle has a guard
with a side screw, while Klima needle has a guard
1. An informed consent should be obtained before the which screws along the length of the needle. Guards
procedure. Bone marrow aspiration or biopsy should on these needles are adjustable to control the depth
be performed by the physician. An assistant is of penetration. The guard may slip from the Salah
required for preparation of smears. needle during the procedure. For aspiration from iliac
2. A sterile tray should be prepared containing crest and tibia, if required, guard may be removed to
autoclaved bone marrow aspiration needle, sterile increase the length of the needle. Guard is essential
disposable syringes with needles, local anesthetic during sternal aspiration to prevent penetration

tahir99 - UnitedVRG
vip.persianss.ir

Fig. 25.3: Jamshidi bone marrow biopsy needle
deeper than necessary and avoid damage to heart or
large vessels. Jamshidi needle, which is longer, can
be used for both aspiration and biopsy from iliac crest
(Fig. 25.3).
3. For aspiration from posterior superior iliac spine,
patient should lie on one side with back towards the
physician, knees and hips flexed, and the knees
drawn towards the chest. The site for aspiration
should be selected and scrubbed with soap and water.
After wearing sterile gloves, antiseptic solution is
applied in a circular fashion, moving from center
towards periphery. A sterile drape is placed over the
area with its central opening over the aspiration site.
4. Skin and periosteum are infiltrated with a local
anesthetic. First inject beneath the skin surface and
advancing the needle further, a larger amount is
injected into the periosteal surface.
5. After waiting for 5 minutes for anesthesia to take
effect, bone marrow aspiration needle is inserted
along with the fitted stylet. (Stylet prevents blockage
of lumen of needle by tissues through which needle
passes). When the bone is reached, the needle is
rotated clockwise and anticlockwise and slowly
advanced into the bone, maintaining steady and firm
pressure. When the marrow is reached, a slight “give”
(decrease in resistance) will be noted. The needle is
advanced for 1-2 mm into the marrow and the stylet
is removed. If the needle is placed correctly, it will be
fixed by the surrounding bone and will remain rigid
and unmoving.
Examination of Bone Marrow 223
Fig. 25.4: (A) Bone marrow smear and (B) Bone marrow
biopsy sample
6. A 5 or 10 ml syringe is attached to the needle and a
small amount of marrow is aspirated (till the first drop
of blood appears i.e. 0.25-0.50 ml) by quickly pulling
the plunger of the syringe. Aspiration is associated
with sharp pain (suction pain). Aspiration of larger
amount of blood causes dilution of marrow sample
by peripheral blood with subsequent difficulties in
interpretation of smears. If no material is aspirated,
stylet is replaced, needle is redirected, and aspiration
attempted again.
7. The syringe should be handed over to the assistant
for preparation of smears on glass slides. The smears
should be made promptly, before clotting occurs, by
putting one drop of the aspirated material near one
end of a glass slide and spreading it similar to a blood
film (Fig. 25.4). Before making smears, any excess
blood on the slide should be sucked away by Pasteur
pipette, leaving behind marrow particles. If immuno-
phenotyping or cytogenetic analysis is to be carried
out, further marrow sample should be aspirated in a
second syringe and dispensed in a tube containing
heparin anticoagulant.
8. After completion of aspiration, the stylet should be
reinserted into the needle and the needle is removed.
Sterile gauze is placed over the site and light pressure
is applied till bleeding ceases. A larger dressing is
then applied.
Bone Marrow Trephine Biopsy
Preparation of the patient and local anesthesia are similar
to aspiration. A short acting intravenous sedative is
preferable in adults. In children, general anesthesia may
be necessary.
Percutaneous trephine biopsy of bone marrow is
commonly obtained from posterior superior iliac spine.
tahir99 - UnitedVRG
vip.persianss.ir
224 Essentials of Clinical Pathology
Jamshidi or Islam needles are commonly used. Often, bone
marrow aspiration and biopsy are combined together;
aspiration is carried out first followed by biopsy. If both
aspiration and biopsy are combined, then, after aspiration,
either (i) the needle is advanced a little further (1-3 cm)
into the bone, or (ii) the needle is withdrawn and reinserted
through the same skin incision but placed at a different
site in the bone (about 1 cm away). For biopsy, the needle
should be advanced through the bone rotating it clockwise
10 times. The needle should be removed by anticlockwise
rotation. The biopsy should be removed gently from the
hub end of the needle by inserting the stylet through the
point of the needle. For adequate assessment, biopsy
should measure at least 1.6 cm in length (Fig. 25.4). Biopsy
should be placed in a fixative solution (either 10% formalin
or preferably Helly’s fluid). Dressing should be applied to
the site similar to aspiration.
Along with bone marrow aspiration/biopsy, peri-
pheral blood smears should be prepared from finger
prick and venous blood should be collected in EDTA
anticoagulant for cell counts.
COMPLICATIONS OF BONE MARROW
ASPIRATION AND/OR BIOPSY
1. Local infection: This complication, which is more
likely to occur in neutropenic patients, can be
prevented if strict aseptic precautions are observed.
2. Hemorrhage: Serious hemorrhage can occur if (i)
marrow biopsy is done without adequate replace-
ment cover in coagulation disorders, and (ii) great
vessels or heart is injured during sternal aspiration.
3. Cardiac tamponade or mediastinitis: This is likely if
posterior sternal plate is penetrated during sternal
aspiration.
PROCESSING OF MARROW SPECIMENS
Bone Marrow Aspiration
A drop of aspirated marrow sample is put on each of the
several glass slides, excess blood is removed by sucking
with a Pasteur pipette, and smears are prepared with a
‘spreader’. The marrow particles are carried just behind
the spreader and cellular trails are produced while
spreading. The smears are allowed to dry in the air and
are labeled. They are stained with one of the
Romanowsky stains. The staining time for marrow films
is longer than that for blood films. On smears, marrow
particles are dragged towards the tail end of the smear
and after staining appear as dark blue-violet irregular
granules. Rest of the film stains even pink. Routinely,
one marrow smear (containing one or more marrow
particles) should also be stained with Perl’s stain for
assessment of bone marrow storage iron.
Bone marrow aspiration provides following information:
• Assessment of morphology of bone marrow cells.
• Assessment of nature of hematopoiesis (normal,
dyshematopoiesis)
• Cytogenetic analysis
• Immunophenotyping of abnormal cells in leukemias.
• Cytochemistry for typing of leukemia
• Iron stain for assessing iron stores and sideroblasts
• Microbial culture, e.g. for tuberculosis
With marrow aspiration, marrow architecture and
cellular relations cannot be studied.
Bone Marrow Trephine Biopsy
If prior marrow aspiration is not satisfactory, imprint
smears should be prepared by gently rolling the marrow
biopsy specimen on a glass slide. These smears are
stained with one of the Romanowsky stains for
cytological evaluation. Biopsy specimen is then fixed in
either 10% formalin or Helly’s fluid. (Helly’s fluid
consists of potassium dichromate 2.5 gm; mercuric
chloride 5 gm; 40% formalin 5 ml; and water 100 ml).
The biopsy is processed (by decalcification, dehydration,
clearing, and embedding) to obtain paraffin wax blocks.
Less than 4 μm thick sections are cut and stained with
hematoxylin and eosin stain and reticulin. It is
recommended to mount 5 stepwise serial sections on one
slide to increase the chance of detecting small focal
lesions. In addition, Giemsa stain is also recommended
for easier differentiation between blood and marrow cells
and mast cells. Iron stain is less informative on biopsy
since iron is usually lost during decalcification.
Bone marrow biopsy provides following information:
• Cellularity of bone marrow
• Bone marrow architecture
• Bone structure
• Marrow fibrosis
• Focal lesions (granulomas, metastatic deposits,
infiltration by lymphoma).
tahir99 - UnitedVRG
vip.persianss.ir
 Examination of Bone Marrow 225

Table 25.1: Comparison of bone marrow aspiration and biopsy

Parameter Bone marrow aspiration Bone marrow biopsy

1. Site Iliac spine, sternum, tibia, spinous Iliac spine (posterior superior)
process of vertebra
2. Information obtained Morphology, cytochemistry, iron stain, Cellularity, architecture, fibrosis,
immunophenotyping, culture focal lesions, bone structure
3. Main indications Unexplained cytopenia, suspected Repeated dry tap, aplastic anemia,
hematological malignancy myelofibrosis, focal lesions, hairy cell
leukemia, staging of lymphoma
4. Needle used Salah, Klima Jamshidi
commonly
5. Studies done Romanowsky stain, Iron stain, H and E stain, reticulin stain,
cytochemistry, cytogenetic or immunohistochemistry
molecular genetic analysis,
immunophenotyping, culture
6. Time for routine Same day Up to 7 days
examination report

With marrow biopsy, detailed morphologic assessment • Plasma cell series

of cells is often not satisfactory. Because of decalcification,
paraffin wax- embedded sections show cellular distortion
and shrinkage. Plastic embedding of marrow biopsy is
advocated to obtain superior cytologic details (as
decalcification step is not required).
Comparison of bone marrow aspiration and biopsy
is presented in Table 25.1.
EXAMINATION OF MARROW SPECIMENS
Bone Marrow Aspiration Smears
Peripheral blood smear in conjunction with routine
hemogram (hemoglobin and cell counts) should be
examined first before assessing bone marrow smears.
Findings should be interpreted in the light of clinical
features and relevant laboratory data.
Examination of marrow smear consists of assessment
of following features:
• Cellularity
• Differential count
• Myeloid:erythroid ratio
• Erythroid series: maturation sequence, type of
maturation (normoblastic, micronormoblastic,
megaloblastic), cytologic abnormalities.
• Myeloid series: maturation sequence, cytologic
abnormalities.
• Megakaryocyte series: number, abnormal forms.
• Lymphocyte series
• Abnormal cells: blasts, carcinoma cells, and necrotic
cells.
• Parasites: malaria parasites, microfilaria, Leishmania
donovani, and Histoplasma.
• Iron content of marrow (on iron stain).
Romanowsky-stained smears are first examined
under low power objective (×10) to assess:
• Cellularity of marrow particles
• Number of megakaryocytes
• Focal metastatic deposits
• Cell distribution and selection of suitable area for
detailed cytologic examination.
Assessment of cellularity is based on examination of
several marrow particles. Cellularity refers to the
proportion of hematopoietic cells as compared to the fat
cells in a marrow particle. Cellularity can also be assessed
by examining the density of hematopoietic cells in
cellular trails behind the marrow particles. Cellularity
can be expressed either in percentage or stating whether
marrow particles are normocellular, hypercellular, or
hypocellular for age.
Megakaryocytes are found in the tail of the smear or
near the marrow particles. About 1-3 megakaryocytes
are seen normally per low power field.
Cellular trails are examined under high power (×40)
and oil immersion objective (×100) for differential count
and assessing erythroid and myeloid maturation. For
differential count, at least 500 cells should be counted in
226 Essentials of Clinical Pathology
cellular trails of particles. Normal differential count in
bone marrow in adults is shown at the end of this chapter
under “Reference Ranges”.
Usually a 5-finger differential count is sufficient
which consists of counting relative percentages of
erythroid cells, myeloid cells, lymphoid cells, plasma
cells, and blast cells. In acute leukemia, myelodysplastic
syndrome, lymphoma infiltration, and plasma cell
dyscrasias, a more detailed differential count is essential.
Myeloid:erythroid (M:E) ratio is the ratio of all
granulocytes and their precursors to all erythroid
precursor cells. Normal M:E ratio ranges from 2:1 to 4:1.
On an average, there are 3 myeloid precursors for every
erythroid precursor. Since M:E ratio is relative it should
be interpreted carefully. Increased M:E ratio is observed
when myeloid series is hyperplastic as in infections and
myeloproliferative disorders (chronic or acute myeloid
leukemia) or when erythroid series is suppressed as in
aplastic crisis of hemolytic anemia. Reduced M:E ratio is
observed when myeloid series is depressed and in
erythroid hyperplasia (e.g. hemolytic anemia).
Maturation of erythroid and myeloid series should
be assessed. Nature of erythroid maturation may be
normoblastic, micronormoblastic, or megaloblastic.
Dyshematopoietic features may be suggestive of
myelodysplastic syndrome or congenital dyserythro-
poietic anemia.
Detailed cytological evaluation of abnormal cells like
blast cells, lymphoma cells, myeloma cells, storage cells,
Fig. 25.5: Iron stain on bone marrow aspiration smear.
Left part of the figure shows blue stained iron granules. Right part of the figure shows no stainable iron
necrotic cells, or metastatic cells should be carried out.
Parasites like Leishmania donovani and Histoplasma
capsulatum are detected in macrophages.
Routinely, Perl’s stain for iron is done on a marrow
smear (containing at least one marrow particle) to assess
storage iron and number and types of sideroblasts.
In acute leukemia, cytochemical stains like myelo-
peroxidase, nonspecific esterase, and periodic acid shiff
are done for typing of acute leukemia.
Iron Staining of Bone Marrow Aspiration Smears
Presence of iron in marrow aspiration smears can be
demonstrated by Perls’ Prussian blue reaction. In this
method, ionic iron reacts with acid ferrrocyanide solution
to form blue-colored ferric ferrocyanide. Iron appears as
bright blue granular aggregates. Perls’ stain does not
detect heme iron of hemoglobin.
Iron staining is a valuable test for detection of iron
deficiency and must be carried out as a routine on all
aspiration smears (Fig. 25.5). Bone marrow aspiration
smears containing at least one marrow particle are
required for demonstration of storage iron in macro-
phages. Bone marrow biopsy sections are not suitable
for evaluation of iron stores since some iron is lost during
processing (decalcification step) of tissue.
On marrow smears, stainable iron can be demons-
trated in siderocytes, sideroblasts, and macrophages of
reticuloendothelial system. Siderocytes are immature,
non-nucleated red cells, which contain 1-2 granules of

non-heme iron. On Romanowsky-stained smears, these
are called as Pappenheimer bodies. Erythroblasts
containing aggregates of iron are called as sideroblasts.
They are of three types—I, II, and III. In type I sideroblasts
(which are seen normally), iron granules are small and
few (1-4) in number. Normally, about 25-50% of
erythroblasts contain 1-4, small iron granules. Types II
and III sideroblasts are abnormal. In type II sideroblasts,
iron granules are large and numerous. Type II sidero-
blasts are seen in hemolytic anemia and iron overload.
In type III sideroblasts, iron granules are distributed in
the form of a ring around the nucleus. The ‘ringed’
sideroblasts are seen in sideroblastic anemias.
Iron granules in macrophages of reticuloendothelial
system represent storage form of iron. Normally, a few,
small granules are seen. In iron deficiency anemia, there
is complete lack of stainable iron in the erythroblasts and
macrophages. Lack of stainable iron in erythroblasts and
increased amount of iron in macrophages is a feature of
anemia of chronic disease. Marked increase of storage iron
in macrophages occurs in thalassemias and iron overload.
Bone Marrow Trephine Biopsy
Hematoxylin and eosin-stained sections of marrow
trephine biopsy are initially examined under low power
objective to assess cellularity, to identify focal lesions like
granuloma, lymphoma infiltrates and clumps of metastatic
malignancy, and to assess bone structure and number of
megakaryocytes. Because of variable cellularity between
particles and dilution with peripheral blood in marrow
aspiration smears, assessment of cellularity is more
reliably performed on biopsy sections.
Fig. 25.6: Cellularity of bone marrow on biopsy: (A) Normocellular; (B) Hypercellular; and (C) Hypocellular
Examination of Bone Marrow 227
Cellularity (Fig. 25.6) varies with the age of the patient.
There is a gradual reduction in red marrow (hematopoietic
tissue) and proportionate increase in fat cells as age
advances. A rough estimate of normal cellularity is
obtained by deducting 1 for each year of age from 100 and
expressing it as a percentage; expected normal cellularity
is ±10% of this value. For example, if age of the patient is
25 years, then expected normal cellularity is 65% to 85%.
The cellularity in the first decade of life is about 80% while
in adults it is about 50%. In older age (>70 years), cellularity
decreases to about 30%. Also, normally, the cellularity
immediately beneath the subcortical area is low as
compared to the deeper medullary areas.
Normally bone marrow biopsy shows bony trabecule
(lamellar bone) separated by interconnecting spaces
containing bone marrow. The bony trabecule are thin,
irregular, and show osteocytes within lacune. The
trabecule show lining of endosteum (osteoblasts and
osteocytes). In a disease called osteopetrosis, bony
trabecule are markedly thick with severe reduction in
marrow space; there are cartilaginous plates in trabecular
bone; and numerous osteoclasts are present along the
endosteal surface.
High power examination is carried out to assess relative
proportion of myeloid and erythroid cells, location of
hematopoietic precursors, pattern of infiltration of
lymphoma cells, morphology of abnormal cells, and
parasites.
Normally, erythroid precursors are located in clusters
in the center of marrow spaces, while granulocyte
228 Essentials of Clinical Pathology
precursors are present close to the trabecule. In
myelodysplastic syndrome, clusters of myeloblasts and
promyelocytes are located in central marrow cavity; this
is called as abnormal localization of immature precursors
or ALIP.
In infiltration of bone marrow by non-Hodgkin’s
lymphoma, pattern of infiltration may be paratrabecular,
focal non-paratrabecular, interstitial, and diffuse.
Similarly, distinct patterns of infiltration are found in
chronic lymphocytic leukemia like diffuse, interstitial,
nodular, or mixed (nodular and interstitial) which has
prognostic significance.
In idiopathic myelofibrosis, collagen deposition can
be made out readily on H and E stained sections. For
demonstration of reticulin fibers, a silver impregnation
technique is required. Reticulin fibers are increased in
acute leukemias, chronic myeloid leukemia, poly-
cythemia vera, myelofibrosis, hairy cell leukemia,
metastatic carcinoma, and certain inflammatory
conditions.
If necessary, special stains like Ziehl-Neelsen stain,
Giemsa stain, Congo red stain, stains for fungi, and
immunoperoxidase stains can be applied.
During decalcification of bone marrow biopsy
specimen for paraffin wax embedding, many cellular
enzymes are destroyed and therefore cytochemical stains
(for typing of leukemias), which depend on enzymatic
reactions within cells, cannot be applied. For plastic
embedding of tissues, decalcification is not required and
therefore cytochemistry is possible.
REFERENCE RANGES
Differential Count in Bone Marrow in Adults
• Myeloblasts: 0-3%
• Promelocytes: 2-5%
• Neutrophil myelocytes: 8-15%
• Metamyelocytes: 9-24%
• Neutrophils (including band forms): 14-26%
• Erythroblasts: 15-36%
• Lymphocytes: 5-20%
• Plasma cells: 0-3%
• Myeloid:erythroid (M:E) ratio: 2:1 to 4:1.
Iron Staining of Bone Marrow Smears
• Sideroblasts 30-50% of all erythroblasts
• No ringed sideroblasts
CRITICAL VALUES
• New diagnosis of a hematological malignancy or
aplastic anemia or severe thrombocytopenia
• Parasites.
BIBLIOGRAPHY
1. Bain BJ. Bone marrow aspiration. J Clin Pathol
2001;54:657-63.
2. Bain BJ. Bone marrow trephine biopsy. J Clin Pathol
2001;54:737-42.
3. Hoffbrand AV, Moss PAH, Pettit JE (Eds). Essential
Hematology. (5th Ed). Blackwell Publishing Ltd, 2006.
4. Hyun BH, Gulati GL, Ashton JK. Bone marrow exami-
nation: techniques and interpretation. Hematology/
Oncology Clinics of North America 1988;4:513-52.
5. Knowles S and Hoff brand AV. Bone marrow aspiration
and trephine biopsy. (1) BMJ 1980;281:204-5. (2) BMJ
1981;281:280-1.
 26
Diagnosis of Malaria and
Other Parasites in Blood
MALARIA
Malaria is endemic in tropical and subtropical develo-
ping countries where it is a major public health problem.
Every year there are about 300-500 million clinical cases
of malaria with 1-2 million deaths, most of them in Africa.
Inadequate administration and implementation of
malaria control measures, failure of insecticides, and
emergence of drug-resistance strains of the parasite are
responsible for resurgence of malaria.
There are four species of malaria parasites that infect
humans: Plasmodium vivax, P. falciparum, P. malariae, and
P. ovale. P. vivax is the most widely distributed species in
the world. In India, the prevalent forms of infections are:
P. vivax (70%), P. falciparum (25-30%), mixed P. vivax and
P. falciparum (4-8%), and P. malariae (< 1%). P. malariae is
localised to certain foci, especially in some areas of
Karnataka. P. ovale is mainly restricted to West Africa.
Fig. 26.1: Life cycle of malaria parasite
LIFE CYCLE OF MALARIA PARASITES
Life history of malaria parasite consists of two cycles of
development: asexual cycle or schizogony that occurs in
humans and sexual cycle or sporogony that occurs in
mosquitoes (Fig. 26.1).
1. Asexual cycle (human cycle, schizogony): This
occurs in the liver cells and red blood cells of infected
humans, and therefore humans are the intermediate
hosts of the malaria parasite (Schizogony refers to the
process of reproduction in protozoa in which there is
production of daughter cells by fission). The human
cycle begins when infected female Anopheles
mosquito bites a person and sporozoites are injected
into the circulation. There are four stages of human
cycle.
230 Essentials of Clinical Pathology
a. Pre-erythrocytic schizogony (Hepatic schizogony):
Inoculated sporozoites rapidly leave the circulation
to enter the liver cells where they develop into
hepatic (pre-erythrocytic) schizonts (Schizonts are
cells undergoing schizogony). One sporozoite
produces one tissue form. Hepatic schizonts
rupture to release numerous merozoites in
circulation (Merozoites are daughter cells
produced after schizogony). Up to 40,000 merozoi-
tes are produced in the hepatic schizont.
In P. falciparum infection, all of the hepatic
schizonts mature and rupture simultaneously;
dormant forms do not persist in hepatocytes. In
contrast, some of the sporozoites of P. vivax and
P. ovale remain dormant after entering liver cells
and develop into schizonts after some delay. Such
persistent forms are called as hypnozoites; they
develop into schizonts at a later date and are a
cause of relapse.
b. Erythrocytic schizogony: Merozoites released
from rupture of hepatic schizonts enter the red
blood cells via specific surface receptors. These
merozoites become trophozoites that utilize red
cell contents for their metabolism. A brown-black
granular pigment (malaria pigment or hemozoin)
is produced due to breakdown of hemoglobin by
malaria parasites. The fully formed trophozoite
develops into a schizont by multiple nuclear and
2. Sexual cycle (mosquito cycle, sporogony): The sexual
cytoplasmic divisions. Mature schizonts rupture
to release merozoites, red cell contents, malarial
toxins, and malarial pigment. (This pigment is
taken up by monocytes in peripheral blood and
by macrophages of reticulo-endothelial system. In
severe cases, organs which are rich in macro-
phages like spleen, liver, lymph nodes, and bone
marrow become slate-gray or black in color due
to hemozoin pigment). Rupture of red cell
schizonts corresponds with clinical attack of
malaria. Released merozoites infect new red cells
and enter another erythrocytic schizogony cycle.
This leads to rapid amplification of plasmodia in
the red cells of the human host. In P. falciparum,
P. vivax, and P. ovale infections, cycle of schizogony
lasts for 48 hours, while in P. malarie infection it
lasts for 72 hours. Merozoites of P. vivax and P.
ovale preferentially invade young red cells or
reticulocytes while those of P. falciparum infect red
cells of all ages. Senescent red cells are preferred
by P. malariae.
P. vivax, P. ovale, and P. malariae complete the
erythrocyte schizogony in general circulation.
Schizonts of P. falciparum induce membrane
changes in red cells, which causes them to adhere
to the capillary endothelial cells (cytoadherence).
Therefore, in P. falciparum infection, erythrocyte
schizogony is completed in capillaries of internal
organs and usually only ring forms are seen in
circulation.
c. Gametogony: After several cycles of erythrocytic
schizogony, some merozoites, instead of develo-
ping into trophozoites and schizonts, transform
into male and female gametocytes. These sexual
forms are infective to mosquito and the person
harboring them is called as a “carrier”. Gameto-
cytes are not pathogenic for humans.
d. Exoerythrocytic schizogony: In P. vivax and P.
ovale infections, some of the sporozoites in liver
cells persist and remain dormant. These dormant
forms in liver cells are called as hypnozoites. They
become active and develop into schizonts a few
days, months, or even years later. These schizonts
rupture, release merozoites, and cause relapse.
Exoerythrocytic schizogony is absent in P.
falciparum infection and therefore relapse does not
occur. Hence, P. vivax and P. ovale are called as
relapsing plasmodia while P. falciparum and
P. malariae are known as non-relapsing plasmodia.
cycle begins when a female Anopheles mosquito
ingests mature male and female gametocytes during
a blood meal. First, 4-8 microgametes are produced
from one male gametocyte (microgametocyte) in the
stomach of the mosquito; this is called as ex-
flagellation. The female gametocyte (macrogameto-
cyte) undergoes maturation to produce one macro-
gamete. By chemotaxis, microgametes are attracted
toward the macrogamete; one of the microgametes
fertilizes the macrogamete to produce a zygote. The
zygote becomes motile and is called as ookinete.
Ookinete penetrates the lining of the stomach and
comes to lie on the outer surface of the stomach where
it develops into an oocyst. On further growth and
maturation, multiple sporozoites are formed within
the oocyst. After complete maturation, oocyst
ruptures to release sporozoites into the body cavity
of the mosquito. Most of the sporozoites migrate to
the salivary glands. Infection is transmitted to the
humans by the bite of the mosquito through saliva
when it takes a blood meal.

CLINICAL FEATURES
Clinically malaria is characterized by paroxysms of high-
grade fever along with anemia and splenomegaly. There
are three stages of a typical malarial paroxysm: (i) cold
stage (lasts for 20 min to 1 hr): There is sudden onset of
fever, shivering, and extreme cold; (ii) hot stage (1-4 hr):
Fever rises to its maximum and there are associated
severe headache and body ache; (iii) sweating stage (2-3
hrs.): Patient perspires profusely and temperature falls.
The duration of each paroxysm is about 6-10 hours and
coincides with the rupture of erythrocytes and release of
showers of merozoites. Febrile paroxysms recur every
48 hours (in P. vivax, P. falciparum, and P. ovale infections)
that is called as tertian malaria or every 72 hours (in
P.malariae infection) called as quartan malaria.
Anemia usually results from destruction of para-
sitized red cells; other causes are suppression of
erythropoiesis in bone marrow and immune hemolysis.
Splenomegaly is an important feature and with
repeated paroxysms there is a significant enlargement
of spleen. Splenomegaly is secondary to activation and
hyperplasia of mononuclear phagocytic system.
Other clinical features include myalgia, joint pains,
thrombocytopenia, and hypoglycemia.
Relapse is a feature of malaria caused by P. vivax and
P. ovale and is due to persistence of hypnozoites in liver
cells. It may occur months or even years after the initial
attack. Relapse does not occur with P. falciparum infection.
Untreated P. falciparum infection can be severe and
fatal. This is because of high degree of parasitemia and
sequestration of parasitized red cells in capillaries of
internal organs. The red cells containing trophozoites and
schizonts of P. falciparum express, on their surface,
positively charged parasite proteins and become ‘sticky’.
These red cells adhere to each other and to endothelial
cells of capillaries of internal organs (cytoadhesion). This
leads to clogging of microcirculation by parasitized red
cells with resultant rupture of capillaries. Two serious
complications of falciparum malaria are cerebral malaria
and blackwater fever. Cerebral malaria clinically
manifests as hyperpyrexia, convulsions, coma, and
sometimes death. In blackwater fever, there is sudden
onset of massive intravascular hemolysis (which causes
hemoglobinemia, hemoglobinuria, and hyperbili-
rubinemia) and high-grade fever. Renal failure is
common. Parasites are not detectable in blood. Urine
Diagnosis of Malaria and Other Parasites in Blood 231
appears dark brown or black in color due to hemo-
globinuria.
Infection by P. malariae can cause nephrotic syndrome.
MODES OF TRANSMISSION
Malaria is transmitted by the bite of certain species of
infected female Anopheles mosquitoes. Other rare modes
of transmission are blood transfusion, use of conta-
minated syringes or needles, and congenital (from
mother to the fetus). Hepatic schizogony does not occur
when malaria is transmitted through transfusion,
contaminated syringes, or congenital route; therefore, in
case of P. vivax infection, relapse will not occur.
ROLE OF GENETIC FACTORS IN MALARIA
1. Sickle cell trait and β -thalassemia trait: Young
children with sickle cell trait (heterozygotes) develop
comparatively mild form of falciparum malaria.
However, persons with sickle cell anemia (homo-
zygous for sickle cell gene) are not protected. Theory
of balanced polymorphism proposes that selective
advantage gained by sickle heterozygotes is balanced
by disadvantage of homozygous state. β-thalassemia
trait is also protective against severe falciparum
malaria.
2. Glucose-6-phosphate dehydrogenase (G-6-PD)
deficiency: Protection against severe falciparum
malaria is afforded to G-6-PD-deficient female
heterozygotes.
3. Newborns: For the first few months of life, newborn
infant has high levels of hemoglobin F in red cells
that suppresses the growth of malaria parasite.
4. Duffy antigen: The Fy (a-b-) phenotype (i.e. Duffy
antigen-negative blood group) in blacks confers
resistance against P. vivax infection. This is because
P. vivax parasite enters the red cells at glycophorin
receptors present on Duffy antigen site. P. vivax
infection is very rare in West Africa due to the lack of
Duffy antigen on red cells of the population.
Methods of Diagnosis
Malaria can be diagnosed by following methods:
• Clinical diagnosis
• Microscopic examination of blood smears
• Rapid diagnostic tests (Detection of malaria parasite
antigen or enzyme)
232 Essentials of Clinical Pathology
• Fluorescence microscopy
• Serologic techniques
• Detection of nucleic acid of parasite by polymerase
chain reaction.
1. Clinical diagnosis: This is the most common method
for diagnosis of malaria in endemic countries.
However, this method is unreliable since symptoms
of malaria overlap with those of many other febrile
illnesses like viral fever, viral encephalitis, and
typhoid fever.
2. Microscopic examination of thick and thin smears:
The most commonly used, fairly rapid, and accepted
method for diagnosis of malaria is microscopic
examination of stained blood smears. It remains the
gold standard for diagnosis of malaria. Blood smear
should be obtained before the next expected febrile
paroxysm or at the onset of fever and chills. Collection
of blood immediately following a paroxysm of fever
is not likely to show intrerythrocytic parasites because
of lysis of parasitized red cells. Blood sample should
be taken before antimalarial drugs are given. After
administration of antimalarial drugs, parasites
become scarce in blood and alter in morphology.
Ideally, blood should be collected by skin puncture
and smears made immediately. Adhesion of blood to the
slide and subsequent staining are affected if antico-
agulated blood sample is used. If it is necessary to use
anticoagulated blood, blood should be collected in EDTA
and smear made as early as possible; delay causes
morphologic changes in parasites, which may make
diagnosis difficult.
One thick smear (for detection of parasites) and one
thin smear (for definitive identification of species) should
be prepared (Fig. 26.2).
Fig. 26.2: Thick and thin blood smears (unstained)
After complete drying, thick smear is stained with a
Giemsa stain or a Field’s stain. Red cells are lysed during
staining and parasites are better visualised against a clear
background. Thick smear (since it allows examination
of a relatively large amount of blood) is more sensitive
than thin smear for detection of malaria parasites,
particularly if they are few in number. For thick smear, a
large drop of blood is collected in the center of glass slide.
It is spread with the corner of a spreader slide or a stick
in such a manner that an evenly spread circular or
rectangular smear of size 15 × 15 mm is obtained. After
labeling, smears are allowed to dry in the air. For better
results, thick smear can be dried in an incubator at 37°C
for 15 min. Thick smear should not be fixed since it is to
be dehemoglobinised.
Thin smear is prepared as outlined in Chapter 22
(Blood Smear). It is air-dried, fixed with methanol, and
stained with a Giemsa stain or Leishman stain. Thin
smear is used for definitive identification of parasite
species, determination of parasite density (if parasitemia
is very high), and for detection of any associated
morphologic abnormalities of blood cells. It is fixed with
methanol for 1-2 minutes before staining.
Thick and thin smears can also be prepared on the
same slide.
It is recommended to examine at least 100 oil
immersion fields in thick smear and 200 oil immersion
fields in thin smear before reporting the smear as negative
for malaria parasite.
If blood smear is negative for malaria parasite despite
strong clinical suspicion, repeat smears should be taken.
A buffy coat preparation can also be helpful in such a
case. EDTA-anticoagulated blood is centrifuged and a
smear is prepared from buffy coat layer and from red
cells just below it. Parasitized red cells get concentrated
just beneath the buffy coat layer.
In P. vivax infection, almost all red cell stages of
parasite are represented in the blood smear. In P.
falciparum infection, usually only ring forms or gameto-
cytes are seen. Presence of mature trophozoites and
schizonts of P. falciparum in blood smear are indicative
of a serious infection. If only gametocytes of P. falciparum
are seen in untreated subjects, it denotes active,
suppressed infection; if seen in treated subjects, it carries
no significance.
Identification of malaria parasites: Different stages of
malaria parasites are found in peripheral blood. These
are trophozoites, schizonts, and gametocytes. The four
 Diagnosis of Malaria and Other Parasites in Blood 233

species of malaria parasites can be distinguished
morphologically by microscopic examination of thin
blood smear.
Microscopic examination of blood smears for
diagnosis of malaria is a sensitive test; it can detect about
50 parasites/μl in experienced hands.
Morphological features of malaria parasites are
summarized and compared in Table 26.1. Stages of P.
vivax and P.falciparum are shown in Figures 26.3 and 26.5.
Blood smears in P. vivax and P. falciparum are shown in
Figures 26.4 and 26.6 respectively.
As compared to the recently available rapid immuno-
chromatographic tests (see below), microscopic examina-
tion of the blood smear is relatively inexpensive.
However, it needs good quality control, is labor-
intensive, and takes more time than immunochromato-
graphic tests. Also, in some cases of falciparum malaria,
parasites get sequestered in the capillaries of internal
organs and are not detectable on blood smears (but can
be detected by immunochromatographic tests).
If P. falciparum is identified, it is necessary to estimate
parasite density. Since gametocytes are non-pathogenic,
they should be excluded while counting parasites. Two
methods for estimation of parasite density are given
below.
1. In a thick smear, number of parasites is counted till
200 white blood cells are observed. Number of
parasites present per μl of blood is calculated from
the following formula:
Number of parasites
Total leukocyte count/µl × ———————————
200

Table 26.1: Morphological comparison of Plasmodia

Plasmodium falciparum
Often only ring forms and gametocytes are seen; multiple rings in a single cell common; red cell size normal; high
parasite density.

Early trophozoite Late trophozoite Schizont Gametocyte

A delicate, small
uniformly fine cytoplasmic
ring with 1-2 small chromatin
dots; ring may be attached to
the red cell margin (accole form).
Compact blue ring with
1-2 red chromatin dots
Very rarely seen except Crescentic or banana-
in cerebral malaria; shaped; larger than a red
contains 18-32 cell
merozoites which fill
2/3rds of a red cell; a
single block of dark
brown pigment

Plasmodium vivax
Often all stages are seen; single parasite in a red cell; red cell enlarged and shows Schuffner’s dots (fine, pink
granules); medium parasite density.

Blue cytoplasmic ring Irregularly thick 12-24 merozoites Large and spherical
1/3rd the diameter of the cytoplasmic ring arranged rosette-like
red cell; one side of ring (ameboid); large granular yellow-brown
thicker; red chromatin at red chromatin dot pigment in center
thinner part of ring

Plasmodium ovale
Often all stages are seen; single parasite in a red cell; red cell slightly enlarged, oval, and with fimbriated margins;
prominent Schuffner’s dots.
Thick, dense blue ring Compact ring; slightly 8-12 irregularly placed Oval; prominent
with a large red amoeboid merozoites Schuffner’s dots
chromatin dot

Plasmodium malariae
Often all stages are seen; single parasite in a red cell; red cell normal in size; no Schuffner’s dots; low parasite
density.
Similar to P. vivax Compact band-like ring 6-12 merozoites in a Round or oval
across the red cell “daisy-head” pattern

234 Essentials of Clinical Pathology
Fig. 26.3: Stages of P. vivax
Fig. 26.4: P. vivax infection is associated with
simultaneous presence of multiple stages
Usually, total leukocyte count is taken as 8000/
μl and the number of parasites is multiplied by 40 to
get the result. However, if accurate leukocyte count
is known, then a better estimate of parasite density is
obtained.
To obtain percent parasitemia, figure of number
of parasites amongst 200 white blood cells is divided
by 1250.
Fig. 26.5: Stages of P. falciparum
•••••••••••••••••
2. In a thin smear, number of parasites amongst 1000
red cells is counted and reported as a percentage.
Number of parasites in 1 μl of blood can be calculated
if red cell count in millions/μl is known; if it is not
known, then it can be arbitrarily taken as 5 million/
μl.
Number of parasites in 1 μl of blood = Red cell count in
million/cmm × parasite percentage
Estimation of degree of parasitemia is of significance
in P. falciparum infection. Percentage parasitemia
exceeding 10% is an indication for exchange transfusion.
In patients taking antimalarial treatment, percent
parasitemia should be obtained daily till no more
parasites (excluding gametocytes) are detectable.
Reporting the result: In the presence of malaria parasite,
blood smear should be reported as follows:
• Blood smear is positive for malaria parasite
• Name of species
• Red cell stages seen
• Parasite density (in case of P. falciparum)
Comparison of morphology of P. vivax and
P. falciparum is shown in Figure 26.7.
3. Detection of malaria parasite antigen by rapid
immunochromatographic tests: In recent years,
commercial manufacturers have introduced simple
non-microscopic rapid diagnostic tests. These tests

Fig. 26.6: Blood smear in P. falciparum infection showing ring
forms.Usually a single stage is seen in P. falciparum infection
detect antigens of malaria parasites by immuno-
chromatographic method. The antigens against
which the commercial test kits are currently available
are:
• Histidine rich protein-2 (HRP-2) synthesized by
asexual blood stages and young gametocytes of
P. falciparum and expressed on red cell surface.
• Parasite lactate dehydrogenase (pLDH), an
enzyme of glycolytic pathway found in all four
human malaria species; there are different forms
of pLDH for each species. Level of pLDH
correlates with parasite density.
Fig. 26.7: Comparison of P. falciparum and P. vivax morphology
Diagnosis of Malaria and Other Parasites in Blood 235
Many commercial test kits are available with differing
test procedures. General principle of these tests is as
follows. Blood sample is collected by skin puncture. It is
then mixed with a buffer solution provided with the kit;
this solution causes lysis of red cells and also contains a
specific antibody labeled with a dye. If the antigen under
investigation is present in blood, formation of antigen-
antibody complex occurs. The labeled antigen-antibody
complex is then allowed to migrate along a nitrocellulose
test strip by capillary action (The manufacturer has
impregnated this test strip with a monoclonal antibody
against the antigen across the strip. The strip also contains
a line of positive control to check whether the test has
been performed correctly and whether the reagents are
functional). In the last step, a washing solution is added
to remove hemoglobin from lysed red cells and to
visualise the reaction. If the blood sample contains the
antigen under investigation, the labeled antigen-
antibody complex will form, it will migrate up the strip,
and will be captured by the pre-deposited monoclonal
antibody; this results in the appearance of a colored line
across the strip.
Tests that detect P. falciparum, P. vivax, and other
species are available. Tests which detect HRP-2 antigen
of P. falciparum have sensitivity and specificity of >90%
(if parasite density is greater than 100/μl).
Commercial test kits can be helpful in following
situations:
• Confirmation of P. falciparum infection if there is
uncertainty about the identity of malaria parasite on
blood smear.
• Confirmation of P. falciparum in mixed infections.
236 Essentials of Clinical Pathology
• Diagnosis of P. falciparum in endemic remote
peripheral areas where properly trained technicians
and facilities for microscopic diagnosis are not
available. Local health workers with little training can
perform rapid diagnostic tests.
• Rapid, self-diagnosis of malaria by tourists traveling
to endemic countries. Some manufacturers have
developed self-help diagnostic kits for travelers.
Positive rapid diagnostic tests (for HRP-2) persist for
several days following successful treatment of P.
falciparum infection (due to persistence of antigens in
blood). Therefore these tests are not useful in following
response to treatment. Also these tests are not able to
estimate parasite density. Distinction between non-
pathogenic gametocyte stage and other pathogenic stages
is not possible.
Comparison between blood smear and rapid diag-
nostic tests is given in Table 26.2.
4. Fluorescence microscopy: There are two fluorescence
microscopy techniques employed for diagnosis of
malaria: quantitative buffy coat (QBC) system (Becton
Dickinson), and Kawamoto technique.
a. Quantitative Buffy Coat (QBC) system (Becton
Dickinson): Blood is centrifuged in a special
capillary tube which contains a float and which is
coated with acridine orange (a fluorescent dye)
and an anticoagulant. Following centrifugation,
malaria parasites get concentrated in the upper
layer of red cells just below the buffy coat and are
stained with the fluorescent dye. When the
capillary tube is viewed using a special objective
(paralens) attached to the fluorescence micro-
scope, malaria parasites fluoresce green yellow
against a dark red-black background. Nuclei of
trophozoites fluoresce bright green.
Table 26.2: Comparison of blood smear and commercial rapid diagnostic tests for malaria diagnosis
Parameter
1. Sensitivity
2. Species identified
3. Parasite density
4. Performance
5. Time required
6. Detection of sequestered P. falciparum
7.Distinction between gametocytes and other stages
8. Cost
Acridine orange stains all the cells that contain
nucleic acids. Therefore, considerable experience
is required to distinguish fluorescing parasites
from other cells containing nucleic acids. This
method also needs expensive apparatus and
materials. Howell-Jolly bodies (nuclear remnants
in red cells in certain anemias) give a false positive
reaction. Schizonts and gametocytes get localized
in the buffy layer and are missed. Specific
identification of species is difficult and it is
necessary to use Romanowsky-stained smears for
this purpose. Despite these disadvantages, this
technique is a more rapid alternative to blood
smears.
b. Kawamoto technique: Blood smears are prepared,
stained with a fluorescent dye (acridine orange),
and examined by fluorescence microscopy. An
interference filter designed for acridine orange is
placed in the pathway of transmitted light beam
and a barrier filter is placed in the eyepiece. Nuclei
of malaria parasites fluoresce bright green and
cytoplasm red. Nuclei of white cells also fluoresce
bright green. Definitive species identification is
difficult; therefore Romanowsky-stained smears
are required for species diagnosis. Kawamoto
technique is less expensive than QBC system.
5. Detection of nucleic acid sequences of malaria
parasites: Malaria parasites can be detected by
identification of specific nucleic acid sequences in
their DNA. Methods based on polymerase chain
reaction (PCR) have been developed for identification
of DNA of malaria parasite. Species diagnosis is also
possible. PCR-based methods can detect very low
levels of parasites in blood (< 5 parasites/μl of blood)
with very high sensitivity and specificity.
Blood smear Rapid diagnostic tests
5-10 parasites/μl 40-100 parasites/μl
All Depends on test kit
Can be estimated Cannot be estimated
Laborious Easy
1 hour 15-20 minutes
No Yes
Yes No
Low High

Molecular methods can be useful in the diagnosis
of malaria, in following response to treatment, in
epidemiological surveys, and for screening of blood
donors. They can also be used as a standard to judge
other methods of malaria diagnosis. However, these
methods cannot be routinely applied because of the
high cost, need for special equipments and materials,
and lengthy procedure (24 hours). Presently they can
be used as a research tool in malaria control programs,
and to carry out quality control checks on microscopic
diagnosis.
6. Serologic methods: Indirect immunofluorescence or
hemagglutination tests are available which detect
antibodies against malaria parasites in patient’s
serum. These tests are helpful for: (i) retrospective
epidemiological surveys in endemic areas, (ii)
screening of blood donors for asymptomatic infec-
tion, and (iii) retrospective diagnosis. However, these
tests are not helpful for routine diagnosis of active
infection as they cannot distinguish between current
and past infection. Thus they cannot guide treatment.
LYMPHATIC FILARIASIS
Lymphatic filariasis is a parasitic infection caused by
nematode worms Wuchereria bancrofti, Brugia malayi, and
Brugia timori. Lymphatic filariasis affects more than 120
million people in the tropical and subtropical countries.
In India, lymphatic filariasis is caused by Wuchereria
bancrofti (majority of cases) and Brugia malayi (mainly in
southwest India). (Brugia timori is restricted to Indonesia).
According to World Health Organization, one-third of
the people affected by lymphatic filariasis are in India.
The prevalence of this disease appears to be on the rise
due mainly to proliferation of slums and expansion of
breeding sites for mosquitoes.
Life Cycle
Filarial worms pass their life cycle in two hosts: humans
(definitive host) and mosquitoes (intermediate host)
(Fig. 26.8).
The disease is transmitted to the humans by the bite
of infected female mosquitoes of the genera Culex,
Anopheles, or Aedes. The infective larvae are deposited
on the human skin when the mosquito takes a blood
meal. The larvae then enter the host through the puncture
wound. The larvae reach the lymphatic channels and
mature into adult male and female worms (5-18 months).
Diagnosis of Malaria and Other Parasites in Blood 237
Fig. 26.8: Life cycle of Wuchereria bancrofti
After fertilization, female worms produce numerous
microfilariae, which migrate into venous blood (via large
lymphatic ducts) and reach arterial circulation through
pulmonary capillaries. The adult worms inhabit the
lymphatics and live for many (5-10) years.
The microfilariae are ingested by the mosquitoe when
it takes a blood meal. Microfilariae cast off their sheaths
in the stomach of the mosquitoe, penetrate the gut wall,
and migrate into the thoracic muscles where they reside
and grow into infective larvae. After about 2 weeks of
development, the mature infective forms migrate to the
mouthparts of the mosquitoe and are transmitted to the
definitive host through the bite.
CLINICAL FEATURES
Clinical features are variable. Also, after acquisition,
infection may take years to manifest clinically. Many
infected individuals do not exhibit any clinical manifes-
tations even though they have numerous circulating
microfilariae and harbor adult worms in lymphatics.
In acute disease, repeated attacks of fever are
associated with lymphangitis (filarial fever). Infla-
mmation occurs in lymphatics of limbs, genitals
(epididymis, spermatic cord, and vulva), and breasts.
Lymphangitis is often associated with enlargement of
regional lymph nodes (inguinal or axillary). Repeated
episodes of lymphangitis cause fibrosis and obstruction
of lymphatics with accumulation of lymph fluid in
surrounding tissues (lymphedema).
The late manifestations of filariasis are hydrocele and
elephantiasis. In hydrocele, sac surrounding the testis is
filled with fluid. In elephantiasis, there is extreme
edematous enlargement of the affected part (limbs,
238 Essentials of Clinical Pathology
external genitals, or breasts) with coarse thickening and
fissuring of skin. Local bacterial and fungal infections
are common. Microfilariae are usually not found in blood
of patients with hydrocele and elephantiasis.
In some cases, there is passage of milky-white chyle
in urine (chyluria). This is due to rupture of distended
urogenital lymphatics, which are connected to the
intestinal lymphatic vessels carrying chyle. Microscopic
examination of such urine shows microfilariae.
LABORATORY DIAGNOSIS
Diagnostic methods in lymphatic filariasis include:
• Microscopic examination of blood for demonstration
of microfilaria
• Detection of filarial antigen in blood by rapid
immunochromatographic test
• Demonstration of microfilariae in hydrocele fluid or
chylous urine
• Demonstration of adult worms in lymph node biopsy
• Detection of parasite DNA by polymerase chain
reaction.
Microscopic Examination of Blood for
Demonstration of Microfilaria
Since some species exhibit periodicity (i.e. circulation of
microfilariae in increased numbers at certain times of the
day), blood should be collected at the correct time to
improve the chances of detection. For Wuchereria bancrofti
and Brugia malayi showing nocturnal periodicity, blood
should be collected at night between 10 p.m. to 4 a.m.
Microfilariae are present in greater numbers in capillary
blood than in venous blood; therefore, skin puncture is
preferred. Usually microfilariae are scanty in peripheral
blood so that concentration techniques may be necessary
for their demonstration.
Following microscopic methods can be used for
detection of microfilariae in peripheral blood:
• Thick blood smear
• Concentration techniques: membrane filtration,
microhematocrit centrifugation, lysed venous blood
technique, lysed capillary blood technique.
Thick Blood Smear
A thick blood smear is spread from 20 μl of capillary blood
on a glass slide, air-dried, and stained with a Romanowsky
stain. If microfilariae are not detected in thick smears
prepared from capillary blood collected at the appropriate
time, and if clinical suspicion is strong, concentration
techniques are employed. This is because circulating
microfilariae are often scanty and sensitivity of
microfilarial detection increases when volume of blood
sampled is increased.
Concentration techniques
Membrane filtration: This is a sensitive method but is
expensive for routine use in endemic areas. Anticoa-
gulated venous blood (10 ml) is passed through a
polycarbonate membrane filter of 3 μm or 5 μm pore size.
Following this 10 ml of methylene blue saline solution is
passed through the filter for staining the microfilariae.
Microfilariae are trapped and retained on the filter, which
is placed on a glass slide and examined under the
microscope.
Microhematocrit tube or capillary tube method: Two
heparinised capillary tubes are filled with blood from
skin punctures (or two plain capillary tubes are filled
with anticoagulated venous blood). After sealing the dry
ends with a suitable sealant, tubes are centrifuged in a
microhematocrit centrifuge for about 5 minutes. The
capillary tubes are placed on a glass slide and fixed with
adhesive tape. Plasma just above the buffy coat layer is
examined for motile microfilariae under the microscope
(Fig. 26.9).
Lysed venous blood method: 10 ml of venous blood is lysed
by saponin-saline solution. The hemolysate is centri-
fuged, supernatant is discarded, and the sediment is
placed on glass slide. After adding a drop of methylene
blue solution, a coverslip is placed, and the preparation
is examined under the microscope for microfilariae.
Lysed capillary blood method: 0.1 ml of blood obtained by
skin puncture is added to 1 ml of saponin-saline solution
to cause lysis of red cells. After centrifugation, super-
natant is discarded and sediment is placed on a glass
slide. A drop of methylene blue solution is added and a
coverslip is placed over it. The entire preparation is
examined under the microscope for motile microfilariae.
Morphology of Microfilariae on
Romanowsky-stained Blood Smears
Wuchereria bancrofti: Microfilariae measure about 300 μ in
length and 8 μ in breadth. They have a hyaline sheath,
which stains pink. There are distinct nuclei in the central

Fig. 26.9: Microhematocrit tube concentration technique for demonstration of microfilaria.
Fig. 26.10: Microfilaria of W. bancrofti in blood smear
axis of the body. Nuclei are not present in the tip of the
tail. Cephalic space (present at the anterior end) is as long
as it is broad. Tip of the tail is bent backwards and body
curves are few (Fig. 26.10).
Brugia malayi: These measure about 230 μ × 6 μ in size.
Sheath stains dark pink in color. The nuclei are crowded
in the body, are blurred in outline, and the tip of the tail
Diagnosis of Malaria and Other Parasites in Blood 239
shows two distinct nuclei. Cephalic space is twice as long
as it is broad. Instead of smooth curves to the body, there
are kinks.
Detection of Filarial Antigen in Blood by
Rapid Immunochromatographic Test
A highly sensitive and specific test for diagnosis of active
infection by Wuchereria bancrofti is ICT Filariasis Card Test
(ICT Diagnostics), which is available commercially. This
test detects circulating antigens of this organism in a
fingrprick blood sample of infected individuals. In
contrast to the microscopic tests, blood sample can be
collected at any time of the day. There is no cross-
reactivity with other filarial organisms like Brugia malayi.
Positive result is obtained even if microfilariae are not
circulating and live adult worms are present in the
lymphatics. The test remains positive for up to 18 months
following successful therapy of filariasis. Apart from
diagnosis of individual patients, this test can be applied
to assess the prevalence of filarial infection in a
population. Time required for the test is about 10-15
minutes.
Other tests have been developed for detection of
circulating filarial antigens like immunoradiometric
assay and enzyme-linked immunosorbent assay (ELISA).
However, these tests are expensive and more labor-
intensive for routine use. Also, they cannot be applied in
field conditions.
240 Essentials of Clinical Pathology
Detection of Filarial DNA by Polymerase
Chain Reaction
Filarial DNA can be detected in circulating blood by
polymerase chain reaction. Although this test is highly
sensitive and species-specific for diagnosis, it is laborious
and expensive for routine use in developing countries.
VISCERAL LEISHMANIASIS
Leishmaniases are a group of parasitic diseases caused
by obligate intracellular protozoa of the genus Leishmania
and transmitted by the bite of an infected female sandfly
of the genus Phlebotomus (Africa, Asia, and Europe) or
Lutzomyia (South and Central America).
Leishmaniases are endemic in tropical and sub-
tropical regions of 88 countries on five continents. There
are about 12 million cases of leishmaniases worldwide
and 1.5 to 2 million cases occur annually. Rising
prevalence of leishmaniases is attributed to massive rural
to urban migration, deforestation with development of
newer dwelling sites, and newer irrigation projects. In
addition, leishmania/human immunodeficiency virus
coinfection is rapidly emerging as a new, severe disease.
Leishmaniases occur in three different clinical forms:
visceral, cutaneous, and mucocutaneous. Only visceral
leishmaniasis is considered below.
In Asia, visceral leishmaniasis (VL) is also called as
kala azar or black sickness. This is the most severe form of
leishmaniases and is caused by Leishmania donovani
complex (which includes L. donovani, L. infantum, and L.
chagasi species). Majority of cases of VL occur in
Bangladesh, northeastern Brazil, northeast India, Nepal,
and Sudan.
In India, the causative organism is L. donovani and
infection is anthroponotic (i.e. transmitted from one
human to another through sandfly vector Phlebotomus). In
India, leishmaniasis is prevalent in Assam and Bengal
Fig. 26.11: Life cycle of Leishmania donovani
along Brahmaputra and Ganges, Bihar, Orissa, and
Andhra Pradesh.
L. infantum is endemic in the Mediterranean basin and
infection is zoonotic (i.e. transmitted to humans from
animals like dogs through sandfly vector).
L. chagasi occurs in Latin America with domestic dog
as the primary reservoir of infection.
Typical manifestations of VL include fever, enlarge-
ment of spleen, enlargement of liver, severe cachexia,
pancytopenia (anemia, leukopenia, and thrombo-
cytopenia), and hypergammaglobulinemia. Secondary
infections are frequent. The name “ Kala-azar” is derived
from the grayish coloration of skin that often develops
during the course of disease. If untreated, death often
follows.
Post kala azar dermal leishmaniasis (PKDL) is a
cutaneous form of leishmaniasis occurring after
resolution of visceral leishmaniasis. It is observed in India
and East Africa. It manifests as hypopigmented and
raised erythematous lesions most prominently on the
face. The lesions contain amastigote forms of L. donovani.
Since Leishmania are present in large numbers in
peripheral blood, they can be transmitted via sharing of
needles among intravenous drug abusers.
The typical clinical features of visceral leishmaniasis
are not always seen and atypical presentations make
diagnosis difficult. Response to treatment is poor and
relapses are common.
Life Cycle of Leishmania donovani
Leishmania donovani (LD) exists in two stages: promasti-
gote (flagellated form found in sandfly vector) and
amastigote (non-flagellated tissue-form found in
mammalian host) (Fig. 26.11). Infection is transmitted to
man when the infected female sandfly takes a blood meal
 Diagnosis of Malaria and Other Parasites in Blood 241

and promastigote forms are inoculated. These promasti-

gotes are taken up by the macrophages of the reticulo-
endothelial system and are transformed into amastigote
forms. The amastigote forms multiply by binary fission
inside the macrophages, are released in circulation when
the host cell ruptures, and are again taken up by new
macrophages. In VL, whole of the reticuloendothelial
system is progressively infected (including bone marrow,
spleen, liver, lymph nodes, and blood monocytes).
When the female sandfly takes a blood meal, it ingests
intracellular and free amastigote forms present in blood.
These then develop into promastigote forms in the
midgut of the sandfly. After multiplication, promastigote Fig. 26.12: Leishmania donovani bodies in bone marrow
forms fill the lumen of the midgut and spread forwards (within a macrophage as well as lying free)
to the pharynx and buccal cavity. These are transmitted
to man when the infected female sandfly takes a blood
(Fig. 26.12). In buffy coat smears, they are seen inside
meal.
monocytes or less commonly inside neutrophils.
LABORATORY DIAGNOSIS OF VISCERAL Amastigote forms should be distinguished from yeast
LEISHMANIASIS forms of Histoplasma capsulatum (which show budding and
do not show kinetoplast). Splenic aspirate has the highest
Laboratory studies for diagnosis of VL include:
sensitivity for detection of tissue forms of Leishmania
• Examination of splenic aspirate, bone marrow
donovani. Since splenic aspiration is associated with a
aspirate, or buffy coat preparation of peripheral blood
risk of fatal hemorrhage, platelet count and prothrombin
for amastigote forms
time should be obtained before the procedure. Splenic
• Detection of anti-Leishmania antibodies
aspirate is contra-indicated if platelet count is <40,000/μl
• Intradermal skin test
and prothrombin time exceeds the control value by >5
• Culture of parasite
seconds.
• Animal inoculation studies
• DNA studies Tests for Detection of Anti-Leishmania Antibodies
Tests are available for detection of raised non-specific
Examination of Splenic Aspirate, Bone Marrow
immunoglobulins and specific anti-Leishmania anti-
Aspirate, or Buffy Coat Preparation of Peripheral
bodies.
Blood for Amastigote Forms
a. Tests that detect raised non-specific immuno-
The most commonly used method for demonstration of globulins: In VL, increased amounts of non-
the parasite is the examination of Giemsa- or Leishman- specific polyclonal IgG and IgM antibodies are
stained smears from relevant tissues. Usually, smear is produced. Tests, which detect these, are formol
prepared from splenic aspirate (sensitivity 95-98%), bone gel (aldehyde) test and antimony test. However,
marrow aspirate (sensitivity 60-85%), or buffy coat these tests are nonspecific and therefore unreliable
(sensitivity 67-99%). After staining, smears are examined for diagnosis of VL.
under oil-immersion lens of the light microscope for Formol gel (aldehyde) test can be used to support
amastigote forms (also called as LD bodies). Amastigote the diagnosis of VL. In this test, 1-2 drops of 40%
forms are small (2-4 μ in diameter), round to oval, and formalin solution are added to 1 ml of patient’s
have nucleus and a rod-shaped kinetoplast. Cytoplasm serum in a test tube. The mixture is allowed to
is pale blue while nucleus and kinetoplast stain pinkish stand for 2 hr. In a positive test, serum becomes
red. Nucleus is relatively large and kinetoplast lies at right milky white and gels (usually within 2-20 min)
angles to it. In aspirate smears, amastigote forms are seen (Fig. 26.13). This test becomes positive only when
in groups inside macrophages or lying free between cells the disease is of greater than 3 months duration.
242 Essentials of Clinical Pathology
Fig. 26.13: Formol gel test for visceral leishmaniasis
This test is also positive in other conditions with
hypergammaglobulinemia like multiple myeloma
and chronic liver disease.
b. Tests that detect specific anti-Leishmania anti-
bodies in serum: Various tests have been developed
for detection of anti-Leishmania antibodies like
complement fixation test, indirect immuno-
fluorescence test, countercurrent immunoelectro-
phoresis, enzyme-linked immunosorbent assay,
direct agglutination test, and latex agglutination
test.
In direct agglutination test (DAT), the antigen
used is trypsin-treated promastigotes, which are
fixed in formalin and stained with Coomasie
brilliant blue. Patient’s serum is incubated with
this antigen and agglutination is noted. Due to
technical difficulties, the test is not widely
employed in field conditions.
A rapid and simple immunochromatographic
strip test has been developed for diagnosis of VL.
This test detects specific IgG anti-Leishmania
antibodies against rk39 antigen. This test has been
evaluated in field trials in India and is reported to
have high sensitivity and specificity.
Skin Test
The Montenegro (or Leishmanin) skin test is based on
delayed type of hypersensitivity reaction to Leishmaniasis.
In this test, 0.5 ml of killed promastigotes are injected
intradermally and the test is read after 72 hr. The test is
positive in persons who have recovered from kala azar. It
is negative in active infection.
Parasite Culture
Cultures are usually made on Novy-McNeal Nicolle
(NNN) medium. After inoculation with 1-2 drops of
splenic or bone marrow aspirate, the culture is incubated
at 22 to 28°C, and examined weekly for promastigote
forms for upto 4 weeks.
In vitro culture of infected tissue is not required for
diagnosis in clinical practice. Parasite culture is necessary
if other methods of diagnosis are negative in the presence
of strong clinical suspicion, and to obtain sufficient
quantity of antigen for direct agglutination test.
Animal Inoculation Studies
Laboratory animals (especially golden hamsters) are
inoculated with infected tissue sample via intra-
peritoneal, intrasplenic, or intradermal route. In positive
cases, parasite can be demonstrated in cutaneous lesions,
liver, or spleen.
The test becomes positive after several weeks or
several months and therefore this test is not used for
routine diagnosis.
DNA Diagnosis
Molecular methods based on polymerase chain reaction
have been described for diagnosis of VL. In this test, DNA
specific for Leishmania is identified. This test can be used
for diagnosis, species identification, and in assessing
response to treatment. Currently this test is largely a
research tool.
BIBLIOGRAPHY
1. British Committee for Standards in Hematology. The
laboratory diagnosis of malaria. Clin Lab Haem 1997;
19:165-70.
2. Cheesbrough M. District Laboratory Practice in Tropical
Countries. 1998, Cambridge University Press.
3. Chatterjee KD. Parasitology (Protozoology and
Helminthology). 9th ed. Calcutta. Published by the
author, 1973.
4. Herwaldt BL. Leishmaniasis. Lancet 1999;354:1191-9.
5. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
Hematology. (9th ed). London: Churchill Livingstone,
2001.
 Diagnosis of Malaria and Other Parasites in Blood
6. Moody A. Rapid diagnostic tests for malaria parasites.
Clin Microbiol Reviews 2002;15:66-78.
7. Palmer CJ, et al. Evaluation of the OptiMal test for rapid
diagnosis of P. vivax and P. falciparum malaria. J Clin
microbial 1998;36:203-6.
8. Sundar S, Rai M. Laboratory diagnosis of visceral
Leishmaniasis. Clinical and Diagnostic Laboratory
Immunology 2002; 9:951-8.
9. Warhurst DC, Williams JE. Laboratory diagnosis of
malaria. J Clin Pathol 1996;49:533-98.
10. Warrell DA, Gilles HM (Eds): Essential Malariology. (4th
Ed). 2002. London. Arnold.
243
11. Weil GJ, Lammie PJ, and Weiss N. The ICT Filariasis
Test: A rapid-format antigen test for diagnosis of
bancroftian filariasis. Parasitology Today 1997;13:
401-4.
12. WHO information Fact sheets: The Leishmaniases and
Leishmania/HIV co-infections. Fact sheet no. 116.
Revised May 2000. Geneva. World Health Organization.
13. World Health Organization. New perspectives in
malaria diagnosis. World Health Organization. 2000.
Geneva, Switzerland. WHO/MAL/2000.1091.
14. World health Organization. Basic laboratory methods
in medical parasitology. 1991. Geneva. World Health
Organization.
 27

Laboratory Tests in Anemia

Anemia is defined as a reduction in hemoglobin
concentration below the level, which is expected for
healthy persons of same age and sex, and in the same
environment. Adequate oxygen cannot be delivered to
various organs and tissues due to low oxygen carrying
capacity of blood.
The normal hemoglobin ranges, as proposed by
World Health Organization (WHO), are given at the end
of this chapter under “Reference Ranges”.
Anemia may occur without symptoms and may be
detected incidentally during medical examination. When
severe enough, clinical features due to anemia result from
hypoxia such as fatigue, weakness, dizziness, fainting,
and mental confusion. Pallor of skin, mucous mem-
branes, and conjunctiva is present. Hyperdynamic
circulation causes palpitations and heart murmurs, and
in severe cases congestive cardiac failure can develop,
particularly in elderly. Anginal pain can result from
myocardial hypoxia.
Anemia is an objective sign of disease and needs
further evaluation to determine the underlying cause and
appropriate treatment.
CLASSIFICATION OF ANEMIAS
There are two ways of classifying anemia:
• Etiological classification
• Morphological classification
1. Etiological classification: Anemia can result from a
variety of causes (Table 27.1).

Table 27.1: Etiological classification of anemia

I. Anemia due to decreased production of red blood cells

• Nutritional deficiencies: Iron deficiency anemia, megaloblastic anemia due to deficiency of folate or vitamin B12
• Anemia of chronic disease
• Sideroblastic anemia
• Aplastic anemia
• Anemia due to infiltration of bone marrow by malignant cells
• Anemia of chronic renal failure
II. Anemia due to increased destruction of red blood cells (Hemolytic anemia)
A. Hereditary:
• Defect in red cell membrane: Hereditary spherocytosis
• Defect in hemoglobin: Sickle cell disease, thalassemia, hemoglobin E disease
• Defect in red cell enzymes: Glucose-6-phosphate dehydrogenase deficiency
B. Acquired:
• Autoimmune hemolytic anemia
• Paroxysmal nocturnal hemoglobinuria
• Hemolytic transfusion reaction
• Hemolytic disease of newborn
• Mechanical hemolytic anemia
• Hypersplenism
• Malaria
III. Anemia due to acute blood loss: Hemorrhage due to trauma, massive gastrointestinal bleeding, or child delivery.

2. Morphological classification: This classification is
based on red cell size and hemoglobin content (red
cell indices) in a case of anemia. This classification is
given later in Table 27.16.
ANEMIAS DUE TO DECREASED
PRODUCTION OF RED BLOOD CELLS
Iron Deficiency Anemia
Iron deficiency is the most common cause of anemia
worldwide, and is a public health problem in developing
countries. Pregnant women, women in reproductive age
group, and children under 5 years of age are particularly
susceptible for nutritional deficiency. In men and
postmenopausal women, blood loss (especially from the
gastrointestinal tract) is the main cause. Common causes
are listed in Table 27.2.
Table 27.2: Causes of iron deficiency anemia
• Nutritional deficiency due to poor diet or increased
requirements: infants and children (6 months-2 years),
women in reproductive age group, pregnancy
• Blood loss:
a. Gastrointestinal: Esophageal varices, peptic ulcer,
carcinoma of stomach, hookworm infestation,
colorectal carcinoma, hemorrhoids
b. Genitourinary tract: Menorrhagia, hematuria
• Malabsorption: Celiac disease
Clinical Features
Apart from nonspecific clinical manifestations of anemia,
iron deficiency can cause koilonychia (flattened or spoon-
shaped nails), angular stomatitis, and glossitis.
Stages of Iron Deficiency
There are three stages of iron deficiency as shown in Box
27.1.
Box 27.1: Stages of iron deficiency
• Stage 1 (Iron depletion): Depletion of storage iron (low
serum ferritin), normal transport iron (serum iron, total
iron binding capacity), normal hemoglobin
• Stage 2 (Low transport iron): Depletion of storage iron,
low transport iron, normal hemoglobin
• Stage 3: (Low hemoglobin production): Depletion of
storage iron, low transport iron, low hemoglobin
Laboratory Tests in Anemia 245
Laboratory Features
1. Low hemoglobin and low packed cell volume
2. Low mean cell volume, mean cell hemoglobin, and mean
cell hemoglobin concentration; red cell distribution
width is increased.
3. Blood smear shows microcytic, hypochromic red cells
and pencil cells (Fig. 27.1).
4. Serum ferritin is less than 15 μg/dl. The best
determinant of storage iron is serum ferritin. Serum
ferritin >100 μg/dl rules out diagnosis of iron
deficiency in most cases. Serum ferritin is a storage
form of iron and is reduced only in iron deficiency
anemia. However, it is also an acute phase reactant.
If iron deficiency anemia is associated with infla-
mmatory, neoplastic, or liver disease, its concen-
tration is raised; therefore, in these conditions,
estimation of serum ferritin will not be helpful for
diagnosis of iron deficiency. In the presence of an
inflammatory disorder, assay for soluble transferrin
receptor is preferable for diagnosis as it is not affected
by inflammation.
5. Serum iron, total iron binding capacity (TIBC), and
transferrin saturation: Serum iron is a measure of iron
bound to transferrin (transport protein for iron).
Serum iron level is affected by diurnal variation, iron-
containing medications, hemolysis, etc. Total iron
binding capacity refers to the iron-binding sites of all
the circulating transferrin. Transferrin saturation is
the ratio of serum iron to transferrin. Typically, serum
iron is low, TIBC is increased, and transferrin
Fig. 27.1: Blood smear in iron deficiency anemia showing
microcytic hypochromic red cells and “pencil” cells
246 Essentials of Clinical Pathology

saturation is <10% in iron deficiency anemia. Free
erythrocyte protoporphyrin is increased.
6. Increased soluble transferrin receptor (sTfR) in serum: This
assay is useful in cases with associated inflammation.
The transferrin receptor is a specific receptor for
transferrin-iron complex and is located on cell
membranes. A soluble form also exists in circulation,
which is derived from proteolysis of cell membrane
during erythrocyte maturation. Concentration of
serum transferrin receptors correlates with the
number of cellular receptors and varies with the rate
of erythropoiesis. In iron deficiency, its level increases
due to increased expression on cell membranes.
However, increased levels are also found in any
condition associated with increased erythropoietic
activity like hemolytic anemias and myeloprolife-
rative disorders.
7. Bone marrow iron stain: This is the gold standard for
diagnosis of iron deficiency and shows lack of
stainable iron in the bone marrow. The test assesses
storage iron in bone marrow (confined mainly within
macrophages; small amount in erythroblasts). This
test is expensive, and not required for routine
diagnosis.
8. Response to oral iron therapy: Increased reticulocyte
count beginning around 3rd day and reaching
maximum on 5th-10th day after starting oral iron
therapy indicates optimal response. Hemoglobin rises
at the rate of 0.5-1.0 gm/dl/week.
Iron deficiency anemia should be differentiated from
other causes of microcytic hypochromic anemia such as
anemia of chronic disease, thalassemia, and sideroblastic
anemia (Table 27.3).
Diagnosis of iron deficiency anemia is straight
forward. It is more important to determine the cause of
iron deficiency, especially in adults since a serious
underlying disorder (like carcinoma of gastrointestinal
tract) may be present. To uncover the causative factor,
apart from complete clinical examination, it may be
necessary to perform stool examination for parasites and
for occult blood, urine examination for occult hematuria,
radiologic and endoscopic workup of gastrointestinal
tract, and in females, pelvic ultrasonography.
Megaloblastic Anemia
Megaloblastic anemia results from deficiency of either
folate or vitamin B12 (Table 27.4). Folate and vitamin B12
are essential for synthesis of deoxyribonucleic acid

Table 27.3: Differential diagnosis of microcytic

hypochromic anemia
Parameter Iron deficiency Anemia of β thalassemia Sideroblastic
anemia chronic disease minor anemia

1. Mean cell volume Low Normal or low Markedly low Low

2. Red cells on Microcytic Normocytic Marked Dimorphic
blood smear hypochromic normochromic; anisopoikilocytosis,
rarely microcytic basophilic stippling
hypochromic
3. Serum iron Low Low Normal Increased
4. Serum ferritin Low Normal or increased Normal Increased
5. TIBC Increased Low Normal Normal
6. Storage iron in marrow Absent Normal or increased Normal Normal
7. Hemoglobin electrophoresis Normal Normal Increased HbA 2 Normal
8. Iron in erythroblasts Absent Present Present Ringed sideroblasts
9. Serum soluble Increased Normal Normal or increased Normal or
transferrin receptor increased

TIBC: Total iron binding capacity

 Laboratory Tests in Anemia 247

Table 27.4: Causes of megaloblastic anemia

Mechanism Folate deficiency Vitamin B12 deficiency

Inadequate intake Poor diet, chronic alcoholism Strict vegetarians

Inadequate absorption
Increased demand
Malabsorption syndromes like
celiac disease, tropical sprue;
Crohn’s disease
Pregnancy, chronic
hemolysis, neoplasia
Pernicious anemia*,
gastrectomy, resection of ileum,
Crohn’s disease, fish tapeworm infestation
–

*Autoimmune disorder characterized by gastric atrophy and loss of production of intrinsic factor in stomach that is
necessary for absorption of vitamin B12

(DNA); in case of deficiency, therefore, there is defective

synthesis of DNA in all the proliferating cells including
bone marrow cells. Vitamin B12 is also required for
neurological functions.
Anemia, mild jaundice (due to ineffective erythro-
poiesis or premature destruction of erythroid precursors
in bone marrow), and glossitis are common to both folate
and vitamin B12 deficiency. Neurologic features like
symmetric paresthesiae especially in lower limbs,
reduced vibration sense, ataxia, loss of memory, and
personality change occur in vitamin B12 deficiency but
not in folate deficiency. Untreated cases may develop
subacute combined degeneration of the spinal cord.
Neurological damage due to vitamin B12 deficiency can
occur in the absence of anemia.

Laboratory Features Fig. 27.2: Blood smear in megaloblastic anemia showing

oval macrocytes and a hypersegmented neutrophil
• Macrocytic anemia (mean cell volume >100 fl in
adults). Elevation of mean cell volume is an early sign
and precedes the onset of anemia. Pancytopenia is
common.
• Blood smear shows oval macrocytosis, basophilic
stippling, Howell-Jolly bodies, and hyperseg-
mentation of neutrophils (>5% of neutrophils
showing 5 or more lobes) (Fig. 27.2).
• Reticulocyte count is normal or low.
• Bone marrow shows megaloblasts, erythroid hyper-
plasia, and giant metamyelocytes and bands
(Fig. 27.3).
• Vitamin assays: See Table 27.5.

Identification of cause It is necessary to distinguish

between folate and vitamin B12 deficiency and to Fig. 27.3: Bone marrow smear showing megaloblasts and
determine the cause for appropriate treatment a giant metamyelocyte
248 Essentials of Clinical Pathology

Fig. 27.4 Evaluation of suspected megaloblastic anemia. (*: Measurement of serum methylmalonic acid is said to be
more sensitive than measurement of vitamin B12 and an earlier marker for detection of vitamin B12 deficiency)

Table 27.5: Differences between folate and vitamin B12 deficiency

Parameter Folate deficiency Vitamin B12 deficiency

1. Usual mechanism of deficiency Inadequate intake Inadequate absorption

2. Neurologic features Absent May be present
3. Serum vitamin B12 Normal Low
4. Serum folate Low Normal or increased
5. Red cell folate Low Low
6. Serum homocysteine Increased Increased
7. Serum methylmalonic acid Normal Increased
8. Therapeutic trial Optimal response to folate Optimal response to vitamin B12

(Table 27.5, and Fig. 27.4). Treatment only with folate in
vitamin B12 deficiency can worsen the neurological
abnormalities.
In vitamin B 12 deficiency, test for vitamin B 12
absorption (Schilling test) can be carried out. In this test,
urinary excretion of oral dose of radio-labeled vitamin
B 12 is compared with the excretion of oral dose of
radiolabeled vitamin B12 bound to intrinsic factor. In
pernicious anemia, deficient absorption is corrected with
addition of intrinsic factor.
Anemia of Chronic Disease
Anemia of chronic disease is the most common form of
anemia amongst hospitalized patients. The three disease
categories associated with anemia of chronic disease are
chronic infection, inflammation, and malignancy
(Table 27.6). There is a block in release of storage iron
from macrophages for erythropoiesis that is mediated
by inflammatory cytokines.
Anemia is mild (9-10 gm/dl) and non-progressive.
Clinical features reflect underlying disease. The main
differential diagnosis is iron deficiency anemia.

Table 27.6: Diseases associated with anemia of chronic disease
1. Chronic infections: Tuberculosis, urinary tract infection, bronchiectasis, osteomyelitis, subacute bacterial
endocarditis
2. Chronic inflammation: Rheumatoid arthritis, systemic lupus erythematosus.
3. Malignancy
Laboratory Features
• Normocytic normochromic anemia (70% cases) or
microcytic hypochromic anemia (30% cases).
• Decreased serum iron, decreased total iron binding
capacity, and normal or raised serum ferritin. Serum
transferrin receptor level is normal.
• Increased marrow storage iron
• Erythrocyte sedimentation rate is increased out of
proportion to the degree of anemia.
Sideroblastic Anemia
In sideroblastic anemia, heme synthesis is deficient. There
is a mitochondrial defect that leads to the failure of
incorporation of iron into heme. Iron accumulates in
mitochondria that surround the nucleus of erythroblasts
forming ringed sideroblasts. Sideroblastic anemia is
characterized by:
• Dimorphic anemia (i.e. blood smear shows dual
population of cells: normocytic normochromic, and
microcytic hypochromic) (Fig. 27.5).
• Ringed sideroblasts in bone marrow (see Fig. 27.5).
Fig. 27.5: Blood smear (on left) in sideroblastic anemia showing
dimorphic red cells, basophilic stippling, and a polychromatic
red cell. Bone marrow smear stained with iron stain (on right)
shows ringed sideroblasts
Laboratory Tests in Anemia 249
Sideroblastic anemia may be hereditary (X-linked) or
acquired. Most cases are acquired and causes include:
(i) drugs: isoniazid, chloramphenicol, cytotoxic drugs,
(ii) alcoholism, (iii) lead poisoning, (iv) myelodysplastic
syndrome, and (v) acute myeloid leukemia.
Aplastic Anemia
Aplastic anemia is characterized by pancytopenia in
peripheral blood (reduction of red cells, leukocytes, and
platelets in peripheral blood) and decreased cellularity
in bone marrow. Aplastic anemia can occur at any age
and clinical presentation is related to pancytopenia
(anemia, risk of infections, and bleeding manifestations).
Organomegaly is typically absent.
Causes of aplastic anemia are listed in Table 27.7.
Investigations in a Suspected Case of
Aplastic Anemia
• Tests for confirmation of aplastic anemia: Complete blood
count (to demonstrate pancytopenia, low hemo-
globin, and low reticulocyte count) and bone marrow
examination (to demonstrate depletion of hema-
topoietic precursors and hypocellularity).
Table 27.7: Causes of aplastic anemia
Acquired
Idiopathic
Drugs:
Idiosyncratic: antibacterials (chloramphenicol,
sulfonamides), nonsteroidal anti-inflammatory drugs
(phenylbutazone, indomethacin, piroxicam, diclofenac),
antithyroid drugs, furosemide, phenothiazines, allopurinol,
oral antidiabetics, Dose-related: Cytotoxic drugs
Chemicals (e.g. benzene) or radiation
Infections: Hepatitis; Epstain Barr virus, Mycobacteria
Paroxysmal nocturnal hemoglobinuria
Systemic lupus erythematosus
Graft vs. host disease
Inherited
Fanconi’s anemia, Dyskeratosis congenita
250 Essentials of Clinical Pathology

• Tests for diagnosis of underlying cause: Viral studies
(hepatitis A antibody, hepatitis B surface antigen,
hepatitis C antibody), Ham’s test and/or flow
cytometry for lack of CD55 and CD59 (for paroxysmal
nocturnal hemoglobinuria), detailed drug history,
antinuclear antibody test (for systemic lupus
erythematosus), and cytogenetic analysis (for Fanconi
anemia). Etiology remains unknown in 50% cases of
aplastic anemia.
Severity of Aplastic Anemia
polypeptide chain with abnormal structure (Table 27.8).
The inherited disorders of hemoglobin have achieved a
high frequency due to the selective advantage afforded
to the heterozygotes of these genetic variations against
infection by Plasmodium falciparum.
Thalassemias
The thalassemias are inherited disorders characterized
by reduced or absent synthesis of α or β globin
polypeptide chains.

1. Severe aplastic anemia: Aplastic anemia is considered to Classification

be severe when following features are present (Camitta
et al, 1976):
• Bone marrow cellularity is either < 25% of normal or
is 25-50% of normal with <30% of hematopoietic cells,
AND
• Two out of three of the following:
– Reticulocytes <1%
– Neutrophils <500/cmm
– Platelets <20000/cmm
2. Very severe aplastic anemia (Bacigalupo et al, 1988):
Criteria are as above, except neutrophil count is < 200/
cmm.
3. Non-severe aplastic anemia: This is present when criteria
do not meet requirements for severe or very severe
aplastic anemia.
HEREDITARY DISORDERS OF
HEMOGLOBIN
Hereditary disorders of hemoglobin result either from
(i) reduced synthesis of one globin polypeptide chain,
leading to chain imbalance, or (ii) synthesis of a
There are two main forms of thalassemias:
• α thalassemias (deficient synthesis of α globin chains).
• β thalassemias (deficient synthesis of β globin chains).
Distribution
• α thalassemias: Southeast Asia, Eastern Mediterranean
region, Middle East.
• β thalassemias: Mediterranean region, Africa, Middle
East, India, Pakistan, Southeast Asia.
In India, β thalassemia is more common in commu-
nities like Punjabis, Sindhis, Bengalis, Gujaratis,
Bhanushalis, and Jains.
β thalassemias: Normally, there are two β globin genes,
one on each member of chromosome 11 (genotype β/β).
β thalassemias result from a single base substitution
(point mutation) in β globin gene. More than 150
mutations of β globin gene have been reported to cause
β thalassemia. There are two main forms of β
thalassemias—β0 and β+. β0 thalassemia is characterized
by complete absence of β chain synthesis, while in β+
thalassemia β chain synthesis is reduced.

Table 27.8: Classification of hereditary disorders of hemoglobin

Disorder Distribution Basic defect Examples

1. Hemoglobinopathies Varied; In India, HbS, Structural alteration of a HbS, HbD, HbC

2. Thalassemias
HbD, and HbE are common
Mediterranean region,
Africa, Middle east,
India, Pakistan,
Southeast Asia
globin polypeptide chain
Reduced synthesis of one α-thalassemia,
globin polypeptide chain β-thalassemia
 Laboratory Tests in Anemia 251

There are three clinical forms of β thalassemias:

thalassemia major, thalassemia intermedia, and
thalassemia minor.
1. β thalassemia major (Homozygous β thalassemia, Cooley’s
anemia): β thalassemia major results when both the β
globin genes are defective, e. g. genotype β°/β° or
β° /β+ . It is characterized by severe anemia, and
requires regular blood transfusion therapy for
survival.
Clinical features of β thalassemia major are severe
hemolytic anemia that develops around 6 months of age,
jaundice and progressive splenomegaly, severe growth
retardation and delayed development, skeletal defor-
mities (frontal bossing, malar prominence), susceptibility
to infections and to folate deficiency, and transfusion- Fig. 27.6: Blood smear in β thalassemia major showing marked
dependence for survival. anisopoikilocytosis, microcytosis, and hypochromia
In untreated patients, death usually occurs before 5
years of age. In individuals who receive regular blood • Blood smear shows less severe abnormalities than
transfusion therapy, iron overload gradually develops thalassemia major
during adolescence. Unless iron chelation therapy is • Reticulocytosis (5-10%)
given, such individuals will die prematurely from • Varible elevation of hemoglobin F on electrophoresis
damage to organs like heart, pancreas, and liver. 3. β thalassemia minor (thalassemia trait, asymptomatic
thalassemia): Usually individuals having one normal
Characteristic laboratory features are:
and one abnormal β globin gene have β thalassemia
• Severe anemia (hemoglobin < 7.0 gm/dl).
minor (e.g.β/β+ or β/β°). In this heterozygous carrier
• Blood smear shows marked variation in size and
state, anemia is either mild or absent. This condition
shape of red cells, red cell fragments, hypochromia,
is often detected during the course of routine
target cells, basophilic stippling, and nucleated red
hematological investigations. β thalassemia trait
cells (Fig. 27.6).
should be distinguished from iron deficiency anemia
• Reticulocytosis (5-15%).
as iron therapy is not required in the former. Serum
• Low mean cell volume, mean cell hemoglobin, and
iron and serum ferritin are low in iron deficiency
mean cell hemoglobin concentration.
anemia, while normal in β thalassemia minor.
• Markedly elevated hemoglobin F on electrophoresis
(Fig. 27.28, later). Laboratory features are:
2. β thalassemia intermedia: This results from homozygous • Hemoglobin slightly low or normal (> 10 gm/dl)
inheritance of mild β+ thalassemia (e.g. genotype β+/ • Low mean cell volume and mean cell hemoglobin;
β+). It is characterized by moderate degree of anemia normal mean cell hemoglobin concentration
which does not require regular blood transfusion • Blood smear shows microcytic hypochromic red cells,
target cells, and basophilic stippling (Fig. 27.7)
therapy. Worsening of anemia occurs during
• Normal serum ferritin
infections and pregnancy.
• Hemoglobin electrophoresis shows elevated hemo-
Thalassemia intermedia presents at a later age (2-5
globin A2 (> 3.5%); this is a diagnostic test for β
years) than thalassemia major. These patients have
thalassemia minor.
chronic hemolytic anemia, splenomegaly, and skeletal
In Indian communities with a high prevalence of
changes.
β thalassemia gene, spouses of persons with β thala-
Laboratory features are: ssemia trait should be screened for β thalassemia trait
• Moderate degree of anemia (hemoglobin 7-10 and hemoglobin E trait to avoid birth of a child with
gm/dl). homozygous β thalassemia.
252 Essentials of Clinical Pathology

There are three clinical forms of α thalassemia: Hb

Bart’s hydrops fetalis syndrome (--/--), Hb H disease (--
/-α), and α thalassemia carrier state (-α/αα, -α/-α, or --
/αα) (Fig. 27.8).
Hb Bart’s hydrops fetalis syndrome is the most severe
form and infants are either stillborn prematurely or die
soon after birth. They have severe anemia, generalized
edema, and hepatosplenomegaly. Mothers of such
infants usually have toxemia of pregnancy. Blood smear

Fig. 27.7: Blood smear in β thalassemia minor showing

microcytes with hypochromia, target cells, polychromatic cells,
and basophilic stippling

β thalassemia major and minor are compared in Table

27.9.
α thalassemias: Normally, there are four α globin genes,
two on each member of chromosome 16 (genotype αα/
αα). α thalassemias usually results from gene deletions.
Since α chains are present in both fetal and adult
hemoglobin, α thalassemia manifests in both fetal and Fig. 27.8: Genotypes in α thalassemias. Normal genotype is
represented as αα/αα. The effective genotypes of various
adult lives. In α° thalassemia, both α globin genes on
thalassemia syndromes are represented as: Hb Bart’s hydrops
one chromosome are lost, while in α+ thalassemia, one α fetalis syndrome: --/--; Hb H disease: α-/--; and α thalassemia
globin gene of the pair is lost. carrier state: αα/-- or αα/α-

Table 27.9: Comparison of two main forms of β thalassemias

Parameter β thalassemia major β thalassemia minor

1. Genotype β0/β+, β0/β 0 β/β+, β/β0

2. Blood smear
3. Hemoglobin electrophoresis
Marked anisopoikilocytosis, Microcytosis, hypochromia, target
microcytosis, hypochromia, cells, basophilic stippling
target cells, basophilic
stippling, nucleated red cells
β0/β0: HbA: 0, HbF: 95-98%, HbA: 90-95%, HbF: 0.5-4%,
HbA2: 2-5%; β0/β+: HbA: 10-30%, HbA2: >3.5%
HbF: 70-90%, HbA2: 2-5%

Note: β0 indicates no production of β globin chain; β+ indicates diminished but some production of β globin chain;
β indicates normal β chain production

shows marked variation in size and shape of red cells,
microcytosis, hypochromia, and nucleated red cells.
Hemoglobin electrophoresis reveals predominance of Hb
Bart’s and absence of Hb A.
Patients with HbH disease have moderate anemia
(hemoglobin 7-10 gm/dl), jaundice, and hepato-
splenomegaly. Typical laboratory features are microcytic
hypochromic red cells, Hb H inclusions in red cells, and
variable amount of HbH on electrophoresis.
α thalassemia carriers are asymptomatic. Microcytic
hypochromic red cells may or may not be present.
Hemoglobin electrophoresis is normal. Determining
ethnic origin, family studies, globin chain analysis, or
genetic analysis may be needed for definitive diagnosis.
Sickle Cell Disorders
Sickle cell disorders are characterized by the presence of
hemoglobin S (HbS) in red cells due to a point mutation
A→T in the 6th codon of β globin gene, which results in
substitution of valine for glutamic acid at position 6 of β
polypeptide chain (β6glu→val). Upon deoxygenation, HbS
polymerizes in red cells leading to the formation of sickle-
shaped red cells; such red cells are rigid, occlude the
microvasculature, and cause painful vaso-occlusive
crises. Sickle cells adhere to vascular endothelium due
to increased expression of adhesion molecules. Deformed
red cells are removed from the circulation and destroyed
prematurely, leading to chronic hemolytic anemia.
Disease manifests in individuals with homozygous sickle
cell disease; heterozygotes are usually asymptomatic.
Sickling disorders (Table 27.10) are prevalent in
Africa, Middle East, Mediterranean region, and central
and southern parts of India. It is thought that the disease
has achieved high frequency due to evolutionary
selection for resistance against falciparum malaria.
Table 27.10: Sickle cell disorders
Disorder Genotype
1. Sickle cell anemia βS/βS
2. Sickle cell trait βS/β
3. Double heterozygous states
• Hemoglobin S-C disease βS/βC
• Hemoglobin S-D disease βS/βD
• Sickle cell-β thalassemia βS/β0
Laboratory Tests in Anemia 253
Mode of inheritance is autosomal recessive. If both the
parents have sickle cell trait, sickle cell anemia will develop
in the offspring (1:4 chance). Clinical features of sickle cell
anemia are highly variable, and range from infrequent
and mild to frequent and severe. Manifestations include
chronic hemolytic anemia, vaso-occlusive crises (acute
episodes of severe pain in chest, abdomen, back, or
extremities), aplastic crisis (sudden aggravation of anemia
due to infection by parvovirus), hemolytic crisis
(exacerbation of hemolysis due to infection or associated
glucose-6-phosphate dehydrogenase deficiency), splenic
sequestration crisis (sudden enlargement of spleen due to
pooling of blood leading to circulatory failure), risk of
infections (especially Streptococcus pneumonia, Hemophilus
influenzae, Neisseria meningitidis, Escherichia coli) and
retarded growth and development.
In sickle cell anemia, blood smear shows sickled red
cells and target cells (Fig. 27.9), solubility test is positive,
and hemoglobin electrophoresis shows mostly HbS with
no HbA. In sickle cell trait, blood smear is normal or
shows target cells, solubility test is positive, and
hemoglobin electrophoresis shows HbA (60%) and HbS
(40%).
Although blood smear shows sickle cells in sickle cell
anemia, and both slide test and solubility test are positive
in sickle cell disorders, hemoglobin electrophoresis is
Fig. 27.9: Blood smear in sickle cell anemia.
254 Essentials of Clinical Pathology

Table 27.11: Hemoglobin electrophoresis patterns in sickle cell disorders

Disorder Hemoglobin Parents’ phenotype

electrophoresis

1. Sickle cell anemia SF Both parents: AS

2. Sickle cell trait AS One parent: AA; Other parent: AS
3. Sickle cell-β0 thalassemia SF; A2 increased One parent: AS; Other parent: β thalassemia minor
4. Sickle cell-β+ thalassemia SAF; A2 increased One parent: AS; Other parent: β thalassemia minor

essential for diagnosis (Fig. 27.28, later). This is to

differentiate between sickle cell trait and sickle cell
anemia, and for detection of double heterozygous states
like sickle cell-β thalassemia or sickle cell-HbD disease
(Table 27.11).

Hemoglobin D
In India, hemoglobin D is observed in Punjab. Hetero-
zygotes (A/D) are asymptomatic, while homozygotes
(D/D) have mild anemia. Blood smear shows hypo-
chromia, microcytosis, and target cells. On hemoglobin
electrophoresis at alkaline pH, hemoglobin D co-migrates
with HbS. Slide test for sickling, however, is negative in
Hb D disease.

HEREDITARY SPHEROCYTOSIS
This is the most common type of hereditary hemolytic
anemia in North Europeans. Hereditary spherocytosis
(HS) is characterized by an inherited defect in the red
cell cytoskeleton leading to the formation of spherocytes.
Normally, the lipid bilayer is anchored to the underlying
cytoskeleton by interactions of (i) spectrin, protein 4.1,
actin, and glycophorin C, and (ii) spectrin, ankyrin, and
band 3. In most cases of HS, there is a deficiency of
ankyrin so that membrane becomes unstable with
fragmentation of part of membrane, loss of surface area,
and spherocyte formation. Spherocytic red cells are rigid,
less deformable than normal red cells, and are destroyed
prematurely in spleen. Mode of transmission is usually
autosomal dominant.
Usual clinical manifestations are presentation in
childhood with mild to moderate anemia, intermittent
jaundice, and splenomegaly. Pigment gallstones are
frequent. Similar history is obtained in a close relative.
Clinical presentation, however, is markedly variable.
Fig. 27.10: Blood smear in hereditary spherocytosis showing
spherocytes, a polychromatic cell, and a nucleated red cell
Anemia, reticulocytosis, and microspherocytes on
blood smear are the usual findings (Fig. 27.10). Micro-
spherocytes are small and dense red cells lacking central
area of pallor. (They are also observed in autoimmune
hemolytic anemia, ABO hemolytic disease of newborn,
burns, microangiopathic hemolytic anemia, and
hemolytic transfusion reaction).
The usual screening test for HS is osmotic fragility
(OF) test (Figs 27.31 and 27.32, later). This test assesses
the ability of the red cells to withstand osmotic stress
when they are suspended in decreasing concentrations
of hypotonic saline solutions. Spherocytes have reduced
surface area to volume ratio and are osmotically fragile.
Incubated variant of OF test is more sensitive. Auto-
hemolysis test shows markedly increased hemolysis that is
partially corrected by addition of glucose (Fig. 27.33, later).
 Laboratory Tests in Anemia 255

Direct antiglobulin test is carried out to exclude immune

hemolysis.

GLUCOSE-6-PHOSPHATE
DEHYDROGENASE DEFICIENCY

Glucose-6-phosphate dehydrogenase (G6PD) deficiency

is an X-linked disorder characterized by reduced activity
of G6PD enzyme in red cells and occurrence of hemolysis
on exposure to oxidant stress. In India, G6PD deficiency
is especially common in Parsees and Vataliya Prajapatis.
Numerous (>400) biochemical variants of G6PD enzyme
have been reported due to point mutations or deletions
in the gene. In India, G6PD Mediterranean, G6PD Kerala-
Kalyan, and G6PD Orissa variants are more common.
G6PD deficiency leads to inability of red cells to
remove toxic hydrogen peroxide (H2O2), an oxidative Fig. 27.11: Blood smear during acute hemolysis in glucose
metabolite. Accumulated H2 O2 leads to oxidation of 6 phosphate dehydrogenase deficiency showing a bite cell
hemoglobin with subsequent denaturation and precipi- and hemi-ghost cells
tation of globin chains. Precipitated globin form
inclusions attached to red cell membrane (Heinz bodies).
(due to intravascular hemolysis). Heinz bodies can be
Red cells containing Heinz bodies are destroyed in spleen
demonstrated by methyl violet staining. The screening
(extravascular hemolysis). Oxidant damage also causes tests used for G6PD deficiency are fluorescent spot test,
peroxidation of membrane lipids of red cells leading to methemoglobin reduction test, and dye decolorization
intravascular hemolysis. test.
G6PD deficiency is usually asymptomatic. It can
cause (i) neonatal jaundice, (ii) acute hemolytic anemia IMMUNE HEMOLYTIC ANEMIAS
on exposure to oxidant stress (Table 27.12), or (iii) chronic
Immunological destruction of red cells occurs when
hemolytic anemia.
antibody and/or complement bind to red cell membrane.
Examination of blood smear during hemolytic
Hemolysis may be extra- or intravascular. Classification
episode shows polychromasia, fragmented red cells,
of immune hemolytic anemias is shown in Table 27.13.
spherocytes, bite cells (red cells having bitten out
Warm antibody type autoimmune hemolytic anemia
margins) and half-ghost cells (one half of the red cell is
occurs mainly in persons over 50 years of age. Mild
empty and the other half is filled with hemoglobin)
jaundice and splenomegaly are common. Red cells coated
(Fig. 27.11). Biochemical abnormalities include increased
with IgG are recognized by Fc receptors on macrophages
serum bilirubin, hemoglobinemia, and hemoglobinuria
and phagocytosed in spleen. In many cases, phagocytosis
is incomplete with formation of spherocytes (Fig. 27.12).
Red cells coated with IgG are detected by direct
Table 27.12: Causes of hemolysis in glucose-6- antiglobulin test (Fig. 27.34, later).
phosphate dehydrogenase deficiency
Cold agglutinin disease is characterized by acro-
1. Bacterial and viral infections cyanosis (cyanosis of fingers, toes, nose, and ears) due to
2. Drugs: the presence of cold agglutinins that cause agglutination
• Antimalarials: Primaquine, pamaquine of erythrocytes on exposure to cold. The antibody is IgM,
• Antibacterials: Sulfonamides, nalidixic acid, cold-reactive, and after binding to red cells activates
nitrofurantoin, dapsone complement. The disease usually occurs in older
• Antipyretics and analgesics
individuals. Blood smear shows autoagglutination of red
3. Chemicals: Naphthalene balls cells (Fig. 27.13); preparation of blood smear after
256 Essentials of Clinical Pathology

Table 27.13: Classification of immune hemolytic anemias

1. Autoimmune hemolytic anemia
• Warm antibody type
– Primary or idiopathic
– Secondary: infections, autoimmune disorders, lymphoma, chronic lymphocytic leukemia
• Cold antibody type
– Cold agglutinin disease
– Paroxysmal cold hemoglobinuria
2. Alloimmune hemolytic anemia
• Hemolytic disease of newborn
• Hemolytic transfusion reaction
3. Drug-induced hemolytic anemia

Fig. 27.12: Blood smear in warm antibody type autoimmune Fig. 27.13: Blood smear in cold agglutinin disease
hemolytic anemia showing spherocytes, polychromasia, and showing large clusters of red cells (autoagglutination)
late normoblast

warming of blood sample causes disappearance of
autoagglutination. Direct antiglobulin test is positive due
to the presence of complement on red cells.
HEMOLYTIC DISEASE OF NEWBORN
Hemolytic disease of newborn (HDN) is characterized
by destruction of red cells of the fetus or neonate due to
antibodies produced by the mother. Allo-antibodies
develop in the mother against foreign red cell antigens
of the fetus inherited from the father. Leakage of fetal
red cells during pregnancy or delivery into the maternal
circulation stimulates formation of antibodies. Passage
of IgG maternal antibodies across placenta into the fetal
circulation causes hemolysis of fetal red cells.
The two main red cell antigens responsible for HDN
are Rh and ABO. In Rh HDN, antibodies develop against
anti-D and less commonly against anti-C or anti-E. In
case of anti-D, mother is Rh D-negative and father is Rh
D-positive. Rh HDN develops during second or
subsequent pregnancies if the fetus is Rh D-positive.
Clinical presentation is variable. Rh HDN may
manifest with (i) mild anemia and jaundice, (ii) icterus
gravis neonatorum with kernicterus, or (iii) hydrops
fetalis with intrauterine fetal death.
The usual clinical presentation of ABO HDN is mild
anemia and jaundice.
Antenatal investigations consist of:
1. Maternal: These are ABO and Rh grouping, antibody
screening, antibody identification, and antibody
titration: If antibody is clinically significant, titer is
determined. Titer >1:32 or a rising titer showing
increase of 2 dilutions or more is significant and
amniocentesis should be performed to determine
severity of disease (Fig. 27.14).
 Laboratory Tests in Anemia 257

Fig. 27.14: Approach to the management of hemolytic disease of newborn

2. Fetal: This is amniocentesis or cordocentesis for

assessment of severity of hemolysis by determining
bilirubin level. Liley’s graph is used for predicting
severity of HDN and management of the fetus. Liley’s
graph plots degree of absorption at 450 nm versus
gestational age in weeks on a semilogarithmic graph
paper.
After delivery, Kleihauer-Betke acid elution test
(Fig. 27.15) is used to identify number of fetal red cells in
maternal circulation, and calculate the number of vials
of Rh immune globulin to be infused to prevent maternal
immunization (Fig. 27.16).

Investigations in newborn consist of:

1. Blood grouping
2. Blood smear (Fig. 27.17)
3. Direct antiglobulin test (DAT) on cord red cells:
Positive DAT (Fig. 27.34, later) can result from coating
of red cells of the newborn with immune anti-A or
anti-B from group O mother, or immune anti-D from
Rh-negative mother.
4. Estimation of bilirubin and hemoglobin
Differences between Rh and ABO HDN are presented
in Table 27.14.
Fig. 27.15: Acid elution or Kleihauer-Betke test. Maternal blood
sample is withdrawn within 2 hours of birth, a blood smear
is prepared and flooded with acid. Hemoglobin in adult red
cells (HbA) is washed out by the acid solution, while hemoglobin
in fetal red cells (HbF) is not. After counterstaining with safranin,
red cells containing HbA appear pale, while cells containing
HbF appear dark. Acid elution test is performed to assess
amount of fetomaternal hemorrhage and to calculate the dose
of Rh immune globulin
258 Essentials of Clinical Pathology

Fig. 27.16: Use of Kleihauer-Betke test to determine the dose of anti-D following delivery by a Rh D-negative woman
of a Rh D-positive baby. The maternal sample should be taken within 2 hours of delivery or any other sensitizing event

PAROXYSMAL NOCTURNAL
HEMOGLOBINURIA
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare,
acquired clonal stem cell disorder in which red cells are
abnormally sensitive to the hemolytic action of the
complement. PNH red cells are deficient in several cell
membrane proteins including CD 55 (decay accelerating
factor) and CD 59 (membrane inhibitor of reactive lysis or
MIRL). Normally, small amount of complement is being
continuously generated via alternate complement
pathway. CD 55 inactivates C3 convertase while CD 59
inhibits the formation of membrane attack complex and
protects the red cells against complement-mediated attack.
Fig. 27.17: Blood smear in Rh hemolytic disease of
In a typical case of PNH, red cell destruction occurs newborn showing erythroblasts and polychromatic cells
at night so that hemoglobinuria (passage of reddish-
brown urine) is noticed after getting up in the morning.
However, presentation is often variable and may be in • Demonstration of increased sensitivity of red cells to
the form of pancytopenia, aplastic anemia, chronic complement: Sucrose hemolysis test, Ham’s test.
intravascular hemolysis, or recurrent venous thromboses. • Flow cytometric analysis of blood cells for lack of
Some cases of PNH evolve to acute myeloid leukemia. CD55 or CD59.
Laboratory diagnosis of PNH is based on:
• Evidence of intravascular hemolysis: Hemoglo- MICROANGIOPATHIC HEMOLYTIC ANEMIA
binuria, hemosiderinuria, methemalbuminemia, and This is a form of mechanical hemolytic anemia resulting
increased indirect serum bilirubin. from intravascular fragmentation and lysis of red cells
 Laboratory Tests in Anemia 259

Table 27.14: Comparison of Rh and ABO hemolytic disease of newborn

Parameter Rh HDN ABO HDN

1. Blood group of mother D-negative O

2. Blood group of fetus D-positive A or B
3. Pregnancy usually affected Second or subsequent First
4. Hydrops fetalis Common Uncommon
5. Stillbirth Common Uncommon
6. Severe anemia Usual Rare
7. Blood smear Erythroblastosis Spherocytes
8. Direct antiglobulin test Strongly positive Weakly positive or negative
9. Prevention Rh immune globulin None

diagnosis of anemia are estimation of hemoglobin and

packed cell volume.

Fig. 27.18: Blood smear in microangiopathic hemolytic
anemia showing many fragmented red cells
due to vascular endothelial abnormalities. Common
causes include thrombotic thrombocytopenic purpura,
hemolytic uremic syndrome, disseminated intravascular
coagulation, eclampsia, disseminated malignancy, and
generalized vasculitis. Blood film shows numerous
fragmented red cells (schistocytes) (Fig. 27.18).
APPROACH TO DIAGNOSIS OF ANEMIA
Evaluation of a case of anemia consists of:
• Establishing the presence and severity of anemia, and
• Determining the cause of anemia.
Establishing the Presence and
Severity of Anemia
Clinical features of anemia are non-specific and their
assessment is often subjective. Laboratory methods for
Estimation of Hemoglobin
Cyanmethemoglobin method is the method of choice.
Recently introduced WHO hemoglobin color scale is a
simple and inexpensive method and well suited for
under-resourced laboratories (see Chapter 18: Estimation
of Hemoglobin).
Depending on hemoglobin concentration, anemia is
graded as mild (hemoglobin less than lower limit of
normal but above 10.0 gm/dl), moderate (hemoglobin
7-10 gm/dl), and severe (hemoglobin < 7 gm/dl).
Measured hemoglobin level depends on amount of
hemoglobin in red blood cells and blood volume.
Pseudoanemia or spurious anemia results from relative
increase in plasma volume in pregnancy, splenomegaly,
congestive cardiac failure, and paraproteinemias.
Therefore, hemoglobin level should be interpreted in the
light of clinical features.
Estimation of Packed Cell Volume (PCV)
PCV is the volume of packed red cells obtained after
centrifugation of a sample of anticoagulated blood in a
Wintrobe tube or a capillary tube (see Chapter 19 : Packed
Cell Volume). Hematocrit is the equivalent measurement
derived on automated blood cell analyzer from the red
cell indices. Although not entirely synonymous, the terms
PCV and hematocrit are used interchangeably in clinical
practice.
PCV is about three times the value of hemoglobin
concentration and thus can be used to cross-check the
hemoglobin value.
Sometimes, additional information can be gained
from the observation of color of plasma and thickness of
260 Essentials of Clinical Pathology

buffy coat layer. Normal plasma is straw-colored; it is

colorless in iron deficiency anemia, pink in hemo-
globinemia, and yellow in hemolysis. A thick buffy coat
indicates leukocytosis and/or thrombocytosis.

Determining the Cause of Anemia

Determining the cause of anemia depends on evaluation

of clinical and laboratory studies (Fig. 27.19).

Evaluation of Clinical Features in Anemia

Clinical features in a case of anemia depend on (i) severity

of anemia, (ii) cause of anemia, and (iii) rapidity of
development of anemia. Anemia may be acute or chronic.
Acute anemia may be due to either acute blood loss or
acute hemolysis, and symptoms are often due to loss of
circulatory volume. Chronic anemia is tolerated well due Fig. 27.19: Approach to diagnosis of anemia
to compensatory mechanisms. Chronic anemia may be
symptomatic or may be detected incidentally during the
Basic Laboratory Studies in Anemia
course of investigations for some other disease. Clinical
features, which are related to the cause of anemia, are Peripheral blood smear: In majority of cases, if correlated
shown in Table 27.15. with clinical features, blood smear findings suggest the

Table 27.15: Clinical features and the cause of anemia

Clinical feature Probable cause of anemia

1. Pregnancy, females of reproductive age group, Nutritional deficiency

growing children
2. Chronic blood loss Iron deficiency
3. Chronic alcoholism Folate deficiency, gastrointestinal blood loss,
sideroblastic anemia
4. Ingestion of drugs:
• Prolonged intake of aspirin Iron deficiency from gastric bleeding
• Cytotoxic drugs Aplastic anemia
• Alpha methyl dopa, rifampicin Autoimmune hemolytic anemia
5. Family history of anemia, jaundice, gallstones, Inherited hemolytic anemia
or splenectomy
6. Organomegaly (hepatomegaly, splenomegaly, Hematologic malignancy
lymphadenopathy)
7. Community:
• Sindhis, Lohanas Thalassemia
• Parsees, Vataliya Prajapatis Glucose 6 phosphate dehydrogenase deficiency
• Tribal groups Sickle cell disease
8. Deformities of skull and facial bones like Thalassemia
malar prominence, frontal bossing, depressed
bridge of nose
 Laboratory Tests in Anemia 261

probable cause of anemia. Apart from morphological type
of anemia and abnormality of red cells, blood smear also
provides information regarding disorders of white cells
(e.g. leukemias), and platelets (thrombocytopenia). (See
Chapter 22: Blood Smear).
Reticulocyte count: It is a measure of the capacity of the
bone marrow to produce red cells. Reticulocyte count is
mainly useful for diagnosis of anemia due to decreased
red cell production or ineffective erythropoiesis (in which
reticulocyte count is low). Increased reticulocyte count
appropriate for degree of anemia indicates effective red
cell production. Causes of low reticulocyte count are
megaloblastic anemia, aplastic anemia, bone marrow
infiltration, and alcoholism. Causes of increased
reticulocyte count are response to hematinic therapy in
nutritional anemia, hemolysis, and bleeding (see Chapter
21: Reticulocyte Count). If reticulocyte count is increased,
then tests for hypoproliferative anemias are generally not
required (such as iron studies, vitamin B12 and folate
assays, bone marrow examination).
Red cell indices: Among the red cell indices, mean cell
volume is the most useful for classification of anemia
into normocytic, microcytic, and macrocytic types
(Table 27.16). Red cell distribution width is used to
distinguish between iron deficiency anemia and
thalassemia (see Chapter 23: Red Cell Indices).
Microcytic Hypochromic Anemia
An approach for evaluation for microcytic hypochromic
anemia is shown in Figure 27.20. The distinction between
different causes of microcytic anemia is shown in Table
27.3 earlier.
Macrocytic Anemias
Evaluation of macrocytic anemia is presented in Figure
27.21.
Normocytic Normochromic Anemia
Evaluation of normocytic anemia is shown in Figure
27.22.

Table 27.16: Morphological classification of anemia

Microcytic hypochromic anemia
(MCV < 80 fl; MCHC < 30 gm/dl)
• Iron deficiency anemia
• Thalassemia
• Anemia of chronic disease
• Sideroblastic anemia
Macrocytic anemia*
(MCV > 100 fl; MCHC 30-35 gm/dl)
• Megaloblastic • Non-megaloblastic
– Folate or vitamin B12 deficiency – Alcoholism
– Liver disease
– Hemolytic anemia
– Myelodysplastic syndrome
– Hypothyroidism

Normocytic and normochromic anemia

(MCV 80-100 fl; MCHC 30-35 gm/dl)
• Reticulocyte count increased • Reticulocyte count low
– Recent blood loss – Aplastic anemia
– Hemolysis – Chronic renal failure
– Anemia of chronic disease
– Anemia due to infiltration of marrow

*Another way of classification of macrocytic anemia: (1) Round macrocytosis: alcoholism, liver disease, hypothyroidism;
and (2) oval macrocytosis: folate or vitamin B12 deficiency, myelodysplastic syndrome. Abbreviations: MCV: mean cell
volume; MCHC: mean cell hemoglobin concentration
262 Essentials of Clinical Pathology

Fig. 27.20: Evaluation of microcytic hypochromic anemia

Fig. 27.21: Evaluation of macrocytic anemia


Fig. 27.22: Evaluation of normocytic normochromic anemia
Hemolytic Anemia
Increased rate of red cell destruction which cannot be
compensated by increased red cell production by the
bone marrow leads to hemolytic anemia. Normally, red
cells have a life span of about 120 days after which they
are destroyed. In hemolytic anemia, red cell life-span is
shortened. Causes of hemolytic anemia are listed earlier
in Table 27.1.
Apart from general features of anemia, hemolytic
anemia may manifest clinically with jaundice, and in
chronic cases with gallstones and splenomegaly. Family
history may be positive in hereditary hemolytic anemias,
and occurrence in a particular ethnic group may be
suggestive of a particular disorder (e.g. thalassemia,
sickle cell disease, glucose-6-phosphate dehydrogenase
deficiency). In acquired hemolytic anemias, evidence of
an underlying disorder may be present, e.g. malaria,
septicemia, drug ingestion, lymphoproliferative disorder,
connective tissue disease, disseminated cancer, etc.
Laboratory Tests in Anemia 263
Approach to the diagnosis of hemolytic anemia
consists of determining (i) presence of hemolysis, (ii)
whether hemolysis is intravascular or extravascular, and
(iii) cause of hemolysis.
1. Establishing the presence of hemolysis: Laboratory
features of excessive red cell destruction are:
• Increased indirect serum bilirubin
• Increased urobilinogen in urine
• Decreased or absent plasma haptoglobin (protein
that binds free hemoglobin in plasma)
• Appearance of free hemoglobin in plasma or urine
• Increased serum lactate dehydrogenase
Laboratory features of increased red cell production by
the bone marrow (in response to hemolysis) are:
• Increased reticulocyte count
• Polychromatic cells and nucleated red cells on blood
smear
• Erythroid hyperplasia in bone marrow.
264 Essentials of Clinical Pathology
The three typical laboratory features indicative of
hemolytic anemia are low hemoglobin, increased
reticulocyte count, and raised indirect serum bilirubin.
2. Whether hemolysis is intravascular or extravascular: In
intravascular hemolysis, red cell destruction occurs
within circulation (Fig. 27.23), while in extravascular
hemolysis, red cells are destroyed by macrophages
of the reticuloendothelial system in spleen and liver
(Fig. 27.24).
Main causes of intravascular hemolysis are hemolytic
transfusion reaction, glucose-6-phosphate dehydro-
genase deficiency, blackwater fever in falciparum
malaria, septicemia, autoimmune hemolytic anemia
(some types), and paroxysmal nocturnal hemoglobinuria.
Laboratory features of intravascular hemolysis are:
• Hemoglobinemia
• Decreased or absent plasma haptoglobin
• Hemosiderinuria
• Methemalbuminemia: Methemalbumin has a
characteristic absorption band at 558 nm (Schumm’s
test).
Fig. 27.23: Catabolism of hemoglobin following intravascular hemolysis.
In extravascular hemolysis, unconjugated bilirubin in
serum and urobilinogen in urine are increased. However,
hemoglobinemia, hemoglobinuria, hemosiderinuria, and
methemalbuminemia are absent.
3. Defining cause of hemolysis: Usually, clinical features
and morphology of red cells on blood smear suggest
the probable cause of hemolytic anemia (Fig. 27.25).
Specific laboratory tests are then carried out for
definitive diagnosis.
Specific laboratory studies to establish the cause of
hemolytic anemia are:
a. Tests in hereditary hemolytic anemias:
• Hemoglobin disorders: sickling test, hemoglobin
electrophoresis, high performance liquid
chromatography, estimation of hemoglobin A2,
estimation of hemoglobin F, red cell inclusions.
• Red cell enzyme defects: test for glucose-6-
phosphate dehydrogenase deficiency
• Red cell membrane disorders: osmotic fragility
test, autohemolysis test

Fig. 27.24: Catabolism of hemoglobin following
extravascular hemolysis
Fig. 27.25: Evaluation of hemolytic anemia
Laboratory Tests in Anemia 265
b. Tests in acquired hemolytic anemias:
• Immune hemolytic anemias: antiglobulin
(Coombs’) test
• Paroxysmal nocturnal hemoglobinuria: sucrose
hemolysis test, Ham’s acidified serum lysis test,
flow cytometric analysis.
Specific Laboratory Studies in Anemia
Vitamin B12 and Folate
Vitamin B12 and folate are measured in patients with (i)
MCV>100 fl (macrocytosis), (ii) macrocytic anemia, or
(iii) neurological and psychiatric abnormalities. Some
investigators also recommend measurement of serum
methylmalonic acid and serum homocysteine due to
interpretive difficulties associated with vitamin assays.
Iron Studies
Iron studies for diagnosis of iron deficiency anemia
include serum iron, total iron binding capacity,
transferrin saturation, and serum ferritin. Before testing,
causes of iron deficiency and presence of any chronic
disease should be assessed. Serum ferritin is superior to
other tests for diagnosis of iron deficiency (< 15 μg/dl is
diagnostic of iron deficiency, while >100 μg/dl excludes
iron deficiency).
266 Essentials of Clinical Pathology
Bone Marrow Examination
If facilities for iron studies and vitamin assays are not
available, examination of bone marrow is indicated to
(i) distinguish between causes of microcytic hypochromic
anemia by assessing storage iron and ringed sideroblasts,
and (ii) distinguish between megaloblastic and non-
megaloblastic macrocytosis. Bone marrow biopsy is
essential for diagnosis of aplastic anemia.
Tests for Hemoglobin S
Two tests are available for detection of Hb S:
• Sickle cell slide test
• Solubility test for hemoglobin S
1. Sickle cell slide test: When red cells containing Hb S
are deprived of oxygen, they become sickle-shaped.
Reducing agent that is used to remove oxygen from
red cells is 2% sodium metabisulphite.
A drop of capillary or anticoagulated venous blood
is mixed on a glass slide with a drop of 2% sodium
metabisulphite. A coverslip is placed over the mixture
and sealed with petroleum jelly-paraffin wax. The
preparation is examined under the microscope after
30 minutes. If sickle cells are not seen, examine the
slide again after 2 hours, and 24 hours. The test is
reported as negative if the red cells remain round, and
as positive if red cells become sickle shaped (crescent-
shaped with pointed ends) or holly-leaf shaped
(Fig. 27.26).
Fig. 27.26: Sickle cell slide test
A positive test indicates presence of Hb S. The
test cannot differentiate between sickle cell trait and
sickle cell disease. However, if the test is positive, then
on the basis of blood smear examination, a probable
diagnosis of sickle cell trait (target cells) or sickle cell
disease (presence of sickle cells and target cells) may
be suggested. It is necessary to perform hemoglobin
electrophoresis for confirmation of Hb S and to
differentiate between various sickle cell disorders
(sickle cell trait, sickle cell anemia, sickle cell-β
thalassemia, sickle cell-DPunjab disease, etc.).
False-negative test can occur if the reagent is outdated
or not freshly prepared, concentration of Hb S is low
(e.g. in infants below 6 months, following recent blood
transfusion), or if there is severe anemia.
False-positive test can occur if the concentration of
sodium metabisulphite is excessive or if there is
drying of the wet preparation.
2. Solubility test for hemoglobin S: In this test,
anticoagulated blood is added to the reagent solution
consisting of phosphate buffer, saponin, and sodium
dithionite. Red cells are hemolyzed and Hb S, if
present, is reduced by dithionite. Hb S forms tactoids
which refract light and the solution appears turbid
(Fig. 27.27). The solution remains clear with other
hemoglobins like Hb A, Hb F, Hb C, Hb D, Hb G,
and Hb O-Arab. The test cannot differentiate between
various sickle cell disorders.
Causes of false-positive test include polycythemia,
marked leukocytosis, paraproteinemias, and hyper-
lipidemia.
Fig. 27.27: Solubility test for sickle hemoglobin. In a negative
test, black lines kept behind the test tube are visible. In a positive
test, black lines are not visible because the solution becomes
turbid

Causes of false-negative test are old or outdated
reagent, low concentration of Hb S (e.g. infants <6
months, severe anemia), and recent blood transfusion.
If the test is positive, hemoglobin electrophoresis
should be performed for confirmation of Hb S, and to
distinguish between various sickle cell disorders.
Hemoglobin Electrophoresis
There are different forms of normal (e.g. HbA, Hb F, Hb
A 2 ) and abnormal (e.g. Hb S, Hb C, Hb D, Hb G)
hemoglobins. The proportions of different hemoglobins
in common disorders of hemoglobin are shown in Table
27.17. Various hemoglobins differ in their amino acid
composition, and hence in their charge when placed in
an electric field and migrate at different rates. Electro-
phoresis can detect only those abnormalities of hemo-
globin that alter the charge. These hemoglobins are
identified by positions they occupy when they migrate
from their point of origin. A control sample consisting
of known normal and abnormal hemoglobins is also run
along with the test sample so as to compare and identify
locations of hemoglobins in the test sample.
Hemoglobin electrophoresis is used for:
• Detection of abnormal hemoglobins like Hb S, Hb C,
Hb D, Hb G, etc. and determining the phenotype by
assessing relative proportions of Hb A, Hb F, and the
abnormal hemoglobin.
• Detection of elevated HbF or Hb A2 in thalassemias.
• Quantitation of Hb A2 for diagnosis of β thalassemia
trait.
Two types of hemoglobin electrophoresis are commonly
used:
• Cellulose acetate electrophoresis at alkaline pH
• Citrate agar electrophoresis at acid pH.
Table 27.17: Proportions of different hemoglobins in normal individuals and in hemoglobin disorders
Condition HbA
1. Normal adults 97%
2. Normal newborn 25%
3. Sickle cell trait 56-60%
4. Sickle cell anemia 0 5-10%
5. β thalassemia minor 90-95%
6. β thalassemia major
0
0 95-98%
Laboratory Tests in Anemia 267
Cellulose acetate electrophoresis at alkaline pH is the
first-line test in the investigation of hemoglobinopathies.
The procedure in short is as follows:
1. Hemolysates are prepared from EDTA antico-
agulated blood (both control and patient’s samples)
to obtain hemoglobin solutions. The control consists
of hemoglobins A, F, S, and C.
2. Samples are applied near one end of the cellulose
acetate strip (point of origin) in separate lanes.
3. Cellulose acetate strips are placed in the electro-
phoresis chamber containing the Tris-EDTA-borate
buffer (pH 8.5) with point of origin towards the
cathode.
4. The chamber is connected to the power supply and
electric current is applied till adequate separation of
hemoglobins is obtained.
5. The strips are removed from the chamber and results
are read visually. If a permanent record is required,
strips are stained with a protein stain.
Relative mobilities of some hemoglobins after
electrophoresis at alkaline pH are shown in Figure 27.28.
At alkaline pH, some hemoglobins have the same net
charge so that they migrate to the same position, e.g.
hemoglobins S, D, and G occupy the same position, and
A2 , C, E, and O run together. To differentiate these
hemoglobins from each other, citrate agar electrophoresis
should next be carried out at acid pH (see Fig. 27.28).
Citrate agar electrophoresis at acid pH (6.0) provides
separation of hemoglobins that migrate to the same
position on cellulose acetate at pH 8.5. In addition, it is
also well-suited for neonatal screening of sickle cell
anemia since clear separation between hemoglobins A,
F, and S is obtained.
HbF HbA2 HbS
<1% 1-3% 0
75% <1% 0
0 1-3% 40%
1-3% 90-95%
0-5% 3.5-7% 0
2-5% 0
268 Essentials of Clinical Pathology
Fig. 27.28: Diagrammatic representation of hemoglobin electrophoresis
(Control is a mixture of hemoglobins A, F, C, and S)
High Performance Liquid Chromatography
In this chromatographic technique, blood sample is
introduced in a matrix column. Mixture of hemoglobins
gets adsorbed onto the matrix and each hemoglobin is
eluted at a different time. Absorbance of eluted
hemoglobins is measured (Fig. 27.29).
Fig. 27.29: Diagrammatic representation of chromatograms
obtained with high performance liquid chromatography
Estimation of Hemoglobin A2 (Hb A2)
Measurement of HbA2 is carried out for diagnosis of β
thalassemia carriers; in these cases HbA2 is characteris-
tically raised to 3.5-7.0%.
There are two methods for estimation of Hb A2:
• Cellulose acetate electrophoresis at alkaline pH
followed by elution and comparing absorbance of Hb
A2 eluate against absorbance of eluates from other
hemoglobin bands.
• High performance liquid chromatography.
Estimation of Hemoglobin F (Hb F)
Fetal hemoglobin (Hb F) is the predominant form of
hemoglobin during fetal life, and at birth constitutes 75%
of all hemoglobins. After birth, Hb F gradually declines
and is about 5% at 6 months and less than 2% at 1 year.
In adults, Hb F is about 0.5%.
Markedly raised Hb F level occurs in:
• β thalassemia major
• Hereditary persistence of fetal hemoglobin (a form
of mild thalassemia)
• Sickle cell diseases.
Hb F is commonly measured by alkali denaturation
test.
Red Cell Inclusions
In thalassemias, red cell inclusions can be demonstrated
by staining with methyl violet. Hb H inclusions represent
 Laboratory Tests in Anemia 269

Fig. 27.31: Osmotic fragility test

Fig. 27.30: Heinz bodies in red cells in glucose 6

phosphate dehydrogenase deficiency

precipitated tetramers of β globin chains in red cells in α

thalassemias. They appear as multiple, small, ragged
inclusions. α chain inclusions are seen in β thalassemia in
erythroblasts in bone marrow and appear as single
ragged structures closely attached to the nucleus.
Heinz bodies represent precipitated denatured
hemoglobin and are seen in unstable hemoglobin disease
and glucose-6-phosphate dehydrogenase deficiency.
They are deep purple and attached to the cell membrane
(Fig. 27.30).

Osmotic Fragility Test

In this test, red cells are suspended in decreasing
concentrations of hypotonic saline solutions to determine
the ability of the red cells to withstand osmotic stress. In
hypotonic solutions, water enters red cells causing
cellular swelling, and at one point, cell lysis occurs.
Normal red cells are biconcave and disc-shaped, have
high surface area to volume ratio, and therefore can
increase their volume upto 70% before they are lysed.
Spherocytes have decreased surface area to volume ratio
and therefore they can take up less water than normal
red cells and lyse earlier (i.e. at relatively higher saline
concentration than normal red cells).
With normal red cells, hemolysis usually starts at
saline concentration of 0.5 gm/dl and is complete at 0.30
gm/dl (Figs 27.31 and 27.32).
Fig. 27.32: Osmotic fragility curve. Percent lysis is plotted on
vertical axis and concentration of saline is plotted on horizontal
axis. In hereditary spherocytosis (red), curve is shifted to the
right of the normal, while in thalassemia (blue), curve is shifted
to the left of the normal
In the presence of spherocytosis, red cells show
beginning of hemolysis at 0.6 to 0.8 gm/dl (i.e. increased
osmotic fragility). Increased number of spherocytes (and
consequently increased osmotic fragility) is seen in
hereditary spherocytosis, warm antibody type auto-
immune hemolytic anemia, ABO hemolytic disease of
newborn, and burns.
Osmotic fragility is decreased in the presence of target
cells (e.g. in thalassemia) since target cells have increased
surface volume.
270 Essentials of Clinical Pathology

This test may be negative if only a few spherocytes are Antiglobulin (Coombs’) Test
present in peripheral blood. Sensitivity of the test can be
Antiglobulin or Coomb’s test is of two types: direct
increased by incubating the red cells at 37°C for 24 hours
(Fig. 27.34) and indirect (Fig. 27.35).
before performing the test (osmotic fragility test after
incubation). Direct antiglobulin test (DAT): This test detects antibodies
or complement or both attached to red cells. DAT is used
Autohemolysis Test
in the investigation of acquired immune hemolytic
In autohemolysis test, blood is incubated at 370 C for 48
hours and the amount of spontaneous hemolysis is noted.
The test is carried out with and without addition of
glucose. In hereditary spherocytosis, autohemolysis is
markedly increased, which is corrected partially
following addition of glucose (Fig. 27.33).

Tests for Glucose-6-Phosphate

Dehydrogenase Deficiency
Definitive diagnosis depends on demonstration of G6PD
deficiency by fluorescent spot test, methemoglobin
reduction test, or dye decolorization test.
Recommended test is fluorescent spot test. G6PD is
required for conversion of NADP to NADPH. Under
ultraviolet light, NADPH fluoresces while NADP does
not. In the absence of G6PD, NADPH will not form and
there will be no fluorescence. Presence of fluorescence
indicates normal G6PD activity.

Fig. 27.34: Principle of direct antiglobulin (Coombs’) test

Fig. 27.33: Autohemolysis test. Left two tubes are control

samples. Glucose has been added to the second normal tube
leading to partial correction of hemolysis. Two tubes on the
right are test samples from a patient with hereditary sphero-
cytosis. The first tube from the patient is showing marked
hemolysis. After addition of glucose, hemolysis is partially
corrected Fig. 27.35: Principle of indirect antiglobulin test
 Laboratory Tests in Anemia 271

anemia and determines whether hemolysis is of immune Applications of IAT

type. In principle, red cells of the patient are washed with
normal saline (to remove unbound antibodies) and
antihuman globulin (AHG) reagent is added.
Agglutination of red cells indicates positive test. (AHG
sera are either polyspecific, i.e. they contain antibodies to
both human IgG and C3d, or monospecific, i.e. they contain
antibodies to either IgG or C3b-C3d).
Causes of positive test:
1. Autoimmune hemolytic anemia (coating of red cells
with autoantibodies).
2. Hemolytic disease of newborn (coating of red cells of
fetus with maternal antibodies).
3. Hemolytic transfusion reaction (coating of red cells
of donor with recipient antibodies).
4. Drug-induced hemolysis, e.g. methyldopa, L-dopa,
cephalothin, penicillin, etc.
Indirect antiglobulin test (IAT): This test detects presence
of antibodies in serum which are directed against red
cell antigens. In this test, patient’s serum is incubated
with red cells (donor or screening) to allow binding of
antibodies in serum to red cell antigens. After washing
of red cells in saline (to remove unbound antibodies),
antihuman globulin reagent is added. Agglutination of
red cells denotes positive test.
1. Cross matching (compatibility testing) before blood
transfusion.
2. Antibody screening and identification.
3. Phenotyping of red cell antigens using known
antisera.
4. Titration of anti-Rh antibodies.
Tests for Paroxysmal Nocturnal Hemoglobinuria
1. Sucrose hemolysis test: This is the screening test. It is
highly sensitive but less specific than Ham’s test. Red
cells from the patient are mixed with fresh ABO-
compatible normal serum and isotonic sucrose
solution (a low ionic strength medium which activates
complement). Hemolysis >10% indicates PNH.
2. Ham’s acidified serum lysis test: Acidified serum lysis
test or Ham’s test is the definitive test for the diagnosis
of PNH. Patient’s red cells are mixed with acidified
normal serum (acidification activates complement).
Hemolysis occurs if red cells are abnormally sensitive
to complement action (Fig. 27.36). This test may also
be positive in a rare form of congenital
dyserythropoietic anemia called as hereditary
erythroblast multinuclearity with positive acidified
serum. Examination of bone marrow for abnormal
erythroblasts can differentiate between these two
conditions.

Fig. 27.36: Ham’s acidified serum lysis test for paroxysmal nocturnal hemoglobinuria. Tube 1: Fresh normal serum + Patient’s
red cells; Tube 2: Fresh normal serum + Hydrochloric acid + Patient’s red cells; Tube 3: Patient’s serum + Hydrochloric acid
+ Patient’s red cells; Tube 4: Heat-inactivated normal serum + Hydrochloric acid + Patient’s red cells; Tube 5: Fresh normal
serum + Normal red cells; Tube 6: Fresh normal serum + Hydrochloric acid + Normal red cells; Tube 7: Heat-inactivated
normal serum + Hydrochloric acid + Normal red cells. Tube 1 is showing trace hemolysis, tube 2: marked hemolysis;
tube 3: slight hemolysis; tubes 4 to 7: No hemolysis
272 Essentials of Clinical Pathology
3. Flow cytometric analysis: This can detect deficiency of
CD 55 or CD 59 proteins by using monoclonal
antibodies.
REFERENCE RANGES
Hemoglobin
• Adult males: 13.0-17.0 gm/dl.
• Adult females (non-pregnant): 12.0-15.0 gm/dl.
• Adult females (pregnant): 11.0-14.0 gm/dl.
• Children, 6-12 years: 11.5-15.5 gm/dl.
• Children, 6 months to 6 years: 11.0-14.0 gm/dl.
• Infants, 2-6 months: 9.5-14.0 gm/dl.
• At birth (full term): 13.6-19.6 gm/dl.
Packed Cell Volume
• Adult males: 40-50%
• Adult females (non-pregnant): 38-45%.
• Adult females (pregnant): 36-42%
• Children, 6-12 years: 37-46%
• Children, 6 months to 6 years: 36-42%
• Infants, 2 – 6 months: 32-42%
• At birth (full term): 44-60%
Red Cell Count
• Adult males: 4.5-5.5 million/cmm
• Adult females: 3.8-4.8 million/cmm
Red Cell Indices
• Mean cell volume (MCV): 80-100 fl
• Mean corpuscular hemoglobin (MCH): 27-32 pg
• Mean corpuscular hemoglobin concentration
(MCHC): 32-36 g/dl
• Red cell distribution width: 9-14.5
Osmotic Fragility of Red Cells
• Starts at 0.5% of sodium chloride or lower
• Complete at 0.3% of sodium chloride
Iron Studies
• Serum iron: 50-150 μg/dl
• Total iron binding capacity (TIBC): 300-400 μg/dl
• Percent transferrin saturation: 20-55%
• Free erythrocyte protoporphyrin (FEP): <80 μg/dl
• Serum transferrin receptor: 2.8-8.5 μg/L
• Serum ferritin: 15-300 μg/L
Vitamin B12 and Folate Studies
• Serum vitamin B12: 150-700 ng/L
• Serum folate: 3-20 µg/L
• Red cell folate: 150-700 µg/L
Serum Haptoglobin
0.8-2.7 g/L
CRITICAL VALUES
• Hemoglobin <7 g/dl or >20 g/dl
• Packed cell volume <20% or >60%
• Presence of sickle cells on blood smear
• New diagnosis of aplastic anemia
BIBLIOGRAPHY
1. Bacigalupo A, Hows JM, Gluckman E, Nissen C, Marsh
J, Van Lint MT, Congiu M, De Planque MM, Ernst P,
McCann S. Bone marrow transplantation (BMT) versus
immunosuppression for the treatment of severe aplastic
anemia (SAA): a report of the EBMT SAA Working
Party. Br J Haemat 1988;70:177-82.
2. Camitta BM, Thomas ED, Nathan DG, Santos G,
Gordon-Smith EC, Gale RP, Rappeport JM, Storb R.
Severe aplastic anemia: a prospective study of the effect
of early marrow transplantation on acute mortality.
Blood 1976;48:63-9.
3. Clarke GM, Higgins TN. Laboratory investigation of
hemoglobinopathies and thalassemias: Review and
update. Clin Chem 2000;46:1284-90
4. Evatt BL, Gibbs WN, Lewis SM, McArthur JR. Funda-
mental diagnostic hematology. Anemia (2nd ed). US
Department of Health and Human Services, Georgia
and World Health Organization, Switzerland, 1992.
5. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
6. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
Hematology. (9th Ed). London: Churchill Livingstone, 2001.
7. Smellie WSA, Wilson D, McNulty CAM, et al. Best
practice in primary care pathology: review 1. J Clin
Pathol 2005;58:1016-24.
8. Tripathy V, Reddy BM. Present status of understanding
on the G6PD deficiency and natural selection. J Postgrad
Med 2007;53:193-202.
9. Ward PCJ. Modern approaches to the investigation of
vitamin B12 deficiency. Clin Lab Med 2002;22:435-45.
10. Working party of the BCSH Blood Transfusion and
General Hematology Task Forces: The estimation of
fetomaternal hemorrhage. Transf Med 1999;9:87-92.
11. Working party of the General Hematology Task Force
of the British Committee for Standards in Hematology:
The laboratory diagnosis of hemoglobinopathies. Br J
Haemat 1998;101:783-92.
 28
Laboratory Tests in
Hematological Malignancies

ACUTE LEUKEMIAS Clinically, patients with acute leukemia present with

Acute leukemias are malignant clonal hematopoietic
stem cell disorders characterized by proliferation of blast
cells in the bone marrow and rapidly progressive fatal
course if untreated. Malignant cells in acute leukemias
are primitive cells with very little differentiation into
functioning mature cells and are called as blast cells.
Acute leukemias are primary disorders of the bone
marrow and arise from malignant transformation of a
single hematopoietic progenitor cell followed by
proliferation and accumulation of the abnormal clone.
Proliferating leukemic blast cells replace normal bone
marrow and subsequently enter into the peripheral
blood. In the bone marrow, blast cells inhibit the growth
and proliferation of normal hematopoietic cells. After
entering the peripheral blood, leukemic cells can involve
any organ system but lymph nodes, liver, spleen, central
nervous system, and skin are commonly affected.
(i) features due to suppression of normal hematopoiesis
like anemia (pallor, weakness, fatigue), neutropenia
(infections), and thrombocytopenia (purpura and
mucosal bleeding), (ii) features due to accumulation of
blast cells in marrow (bony pain and tenderness), and
(iii) features due to infiltration of various organs by blast
cells (lymphadenopathy, splenomegaly, hepatomegaly,
swelling of gums, central nervous system manifestations,
and skin infiltrations).
Classification of Acute Leukemias
The most widely used classification of acute leukemias
is French-American-British (FAB) Co-operative Group
classification, which was originally proposed in 1976.
This classification is based on morphology and cyto-
chemistry. Acute leukemias are divided into two major
types: acute myeloid leukemia (AML) and acute
lymphoblastic leukemia (ALL). Each type is further
subclassified (Table 28.1).

Table 28.1: French-American-British (FAB) classification of acute leukemias

Acute myeloid leukemia (AML)

M0: Acute myeloid leukemia, minimally differentiated
M1: Acute myeloid leukemia without maturation
M2: Acute myeloid leukemia, with maturation
M3: Acute promyelocytic leukemia M3v: Hypo- or microgranular promyelocytic leukemia
M4: Acute myelomonocytic leukemia M4Eo: Acute myelomonocytic leukemia with bone marrow eosinophilia
M5: Acute monocytic leukemia M5a: Undifferentiated (monoblastic) M5b: Well-differentiated (promonocytic-monocytic)
M6: Acute erythroleukemia
M7: Acute megakaryocytic leukemia

Acute lymphoblastic leukemia (ALL)

L1
L2
L3
274 Essentials of Clinical Pathology

Recently, World Health Organization (WHO) classifi-
cation of hematopoietic and lymphoid neoplasms has
been proposed (2001). WHO classification (2001) of acute
myeloid leukemias is shown in Table 28.2. This
classification integrates clinical, morphologic, genetic, and
immunophenotypic features to define specific disease
categories.
In WHO classification, acute lymphoblastic leukemias
are placed under lymphoid neoplasms. ALL and
lymphoblastic lymphoma or Burkitt’s lymphoma are
considered as biologically the same disease with different
clinical presentations. The term ALL is used for leukemic
phase of precursor neoplasms of B or T cell type. Burkitt
cell leukemia is the counterpart of Burkitt lymphoma and
corresponds with FAB L3 subtype of ALL. ALL includes
following categories in WHO classification:
• Precursor B cell ALL
• Precursor T cell ALL
• Burkitt cell leukemia
Salient features of WHO classification are outlined in
Table 28.2.
According to WHO criteria, blast count should be 20%
or more in either peripheral blood smear or bone marrow
for diagnosis of AML (Earlier FAB recommendation was
30%). The term ‘blast’ includes myeloblasts, promyelo-
cytes (for acute promyelocytic leukemia), monoblasts and
promonocytes (for acute myelomonocytic and acute
monocytic leukemia), and megakaryoblasts (for acute
megakaryocytic leukemia).
Myeloid cell lines are erythroid, granulocytic,
monocytic, and megakaryocytic. Myeloid nature of blasts
is established by morphology (Auer rods), cytochemistry
(myeloperoxidase, nonspecific esterase), and immuno-
phenotyping (myeloid related antigens like CD 117, CD
13, CD 33).
In contrast to FAB classification, WHO classification
recognizes AML with recurrent cytogenetic abnormalities,
AML with multilineage dysplasia, and therapy-related
AML as distinct entities.
Patients with clonal, recurrent cytogenetic abnor-
malities listed in Table 28.2 are considered to have AML
irrespective of the percentage of blasts in blood or bone
marrow. These patients have characteristic clinical and
morphological features and a favorable response to
therapy.

Table 28.2: WHO classification of acute myeloid leukemia (AML), 2001

1. Acute myeloid leukemia with recurrent genetic abnormalities

• AML with t (8; 21) (q22; q22), (AML1/ETO)
• AML with abnormal bone marrow eosinophils and inv (16) (p13q22) or t (16; 16) (p13; q22), (CBFβ /MYH11)
• Acute promyelocytic leukemia with t(15;17) (q22; q12), (PML/RARα ) and variants
• AML with 11q23 (MLL) abnormalities
2. Acute myeloid leukemia with multilineage dysplasia
• Following myelodysplastic syndrome (MDS)
• Without prior myelodysplastic syndrome
3. AML and MDS, therapy related
• Alkylating agent related
• Topoisomerase II inhibitor related
• Others
4. AML, not otherwise categorized
• AML, minimally differentiated
• AML without maturation
• AML with maturation
• Acute myelomonocytic leukemia
• Acute monoblastic and monocytic leukemia
• Acute erythroid leukemia
• Acute megakaryoblastic leukemia
• Acute basophilic leukemia
• Acute panmyelosis with myelofibrosis
• Myeloid sarcoma
 Laboratory Tests in Hematological Malignancies 275

Diagnosis of AML with multilineage dysplasia
requires either (i) prior history of myelodysplastic
syndrome for atleast 6 months before the onset of overt
AML, or (ii) ≥ 50% of dysplastic cells in two or more
myeloid lineages. This category of AML is associated
with poor outcome.
Features of alkylating therapy related AML are (i)
development 4-7 years following therapy, (ii) charac-
teristic cytogenetic abnormalities involving chromo-
somes 5 or 7, (iii) associated and preceding multilineage
dysplasia, and (iv) adverse prognosis.
Topoisomerase II inhibitor therapy-related AML
develops 6 months to 5 years after starting therapy, and
is also associated with characteristic cytogenetic
alterations affecting 11q23. There is no preceding
myelodysplasia. This form of therapy can also cause acute
lymphoblastic leukemia.
Cases of AML which do not fit into any of the above
three major categories are placed under the category of
AML, not otherwise categorized.
According to WHO classification, FAB terms L1, L2,
and L3 are not distinct disease categories since they do
not have clinical, immunophenotypic, or genetic
correlates, and therefore these terms should no longer
be used. L3 subtype of ALL, however, correlates with
Burkitt’s lymphoma.
In WHO classification, the term ALL corresponds
with leukemic phase of precursor neoplasms of B or T
lymphocytic lineage. For diagnosis of ALL, blast count
in marrow should be greater than 25%.
Laboratory Diagnosis of Acute Leukemias
Currently, diagnosis of leukemias is based on combi-
nation of clinical features, microscopic examination of
peripheral blood and bone marrow, cytochemistry,
immunophenotyping by flow cytometry, cytogenetics,
and molecular analysis.
1. Morphology: Initial step in the diagnosis of acute
leukemias is examination of Romanowsky-stained
smears of peripheral blood and marrow aspirate.
In a typical case, bone marrow suppression leads
to normocytic normochromic anemia, thrombo-
cytopenia, and neutropenia. Total leukocyte count is
usually elevated; however, it may be normal or low.
In subleukemic leukemia, leukocyte count is normal or
decreased, but blasts are demonstrable in peripheral
blood. In aleukemic leukemia, blasts are not demonst-
rable in peripheral blood, but present in bone marrow.
In AML (M0, M1), leukemic hiatus occurs, i.e.
peripheral blood shows blast cells and mature cells,
while intermediate cells (like promyelocytes,
myelocytes, and metamyelocytes) are not seen.
Bone marrow is usually hypercellular due to
infiltration by monomorphic population of leukemic
cells; normal hematopoietic cells are reduced.
Blast percentage should be derived from 200-cell
differential leukocyte count on a blood smear or 500-
cell differential count on a marrow aspirate smear.
Presence of Auer rods (needle- or rod-shaped
inclusion in cytoplasm composed of azurophil
granules) in blast cells is diagnostic of AML.
However, they are seen in only 10-20% cases of AML.
Morphological features of ALL and AML are shown
in Tables 28.3 and 28.4 and Figures 28.1 and 28.2.
Features of AML and ALL are compared in Table 28.5.
2. Cytochemistry: Cytochemistry comprises of techni-
ques for identification of enzymes, fats, or certain
other substances in the cytoplasm of blood cells. In
acute leukemia, cytochemistry is mainly useful for
identifying various subtypes of AML. In lymphoid
leukemias, cytochemistry has been replaced by
immunophenotyping for characterization of different
subtypes. The results of cytochemistry should always
be interpreted along with conventional morphology

Table 28.3: Morphological features of ALL (FAB classification)

FAB classification Morphological features
1. ALL L1 Predominantly small cells with scanty cytoplasm; nuclear membrane
regular; nucleoli indistinct
2. ALL L2 Large cells, heterogeneous in size with moderate amount of cytoplasm;
nuclear membrane is irregular with clefting; nucleoli 1-2 and prominent
3. ALL L3 Large cells with moderate amount of deeply basophilic cytoplasm;
prominent cytoplasmic vacuoles; regular nuclear membrane; 1-2
prominent nucleoli
276 Essentials of Clinical Pathology

Table 28.4: Laboratory features of acute myeloid leukemias

Type Morphology Cytochemistry Immunophenotyping Other features

1. AML M0 Large, agranular blasts MPO – (+ on EM) CD13+, CD 33+, –

CD34+, HLADR+
2. AML M1 Minimal maturation (≥ 90% MPO + (>3% CD13+, CD33+, CD34+, t (9;22)
type I and II blasts in marrow); blasts) HLADR+
remaining cells maturing
granulocytes or monocytes
3. AML M2 20-89% non-erythroid cells MPO+ CD13+, CD33+, t(8;21)
are myeloblasts; type II blasts HLADR+
common; Auer rods frequent;
full range of granulocyte
maturation
4. AML M3 Abnormal promyelocytes MPO+ CD13+, CD33+ t(15;17),
predominant (>50%); in (strong) disseminated
hypergranular type, nucleus is intravascular
reniform and there are abundant coagulation
cytoplasmic granules and common
numerous Auer rods; in
microgranular variant, granules
are small and few, nucleus is
convoluted, and TLC is very high
5. AML M4 Both myeloblasts and monoblasts MPO+ ≥20% CD13+, CD33+, Inv(16) in M4
are ≥20% of nonerythroid cells blasts; CD14+, CD64+, with eosinophilia;
NSE+ ≥20% cells HLADR+ increased lysozyme
in blood and urine
6. AML M5 >80% cells monocytic NSE+ CD14+, CD64+, t(9;11)
HLADR+
7. AML M6 >20% of nonerythroid cells are PAS+ Glycophorin A+, CD71+
myeloblasts; >50 of all nucleated
cells are erythroblasts; marked
dyserythropoiesis
8. AML M7 >20% of blasts that may be Platelet CD41+, CD61+,
small, lymphoblast-like or large peroxidase+ HLADR+
with fine chromatin and prominent (EM)
nucleoli and cytoplasmic blebs

MPO: Myeloperoxidase; EM: Electron microscopy; Type I blasts: No azurophil granules in cytoplasm; Type II blasts: Few
azurophil granules; NSE: Non-specific esterase; PAS: Periodic Acid Schiff; TLC: Total leucocyte count

and immunophenotyping. The important cyto-
chemical studies in acute leukemias are (Fig. 28.3):
• Myeloperoxidase (identifies an enzyme present
in primary granules of granulocytic cells)
• Non-specific esterase (identifies an enzyme
present in large amounts in monocytic cells)
• Periodic acid Schiff (identifies intracellular
glycogen).
Myeloperoxidase (MPO)
• MPO enzyme is located in primary (azurophil) and
secondary granules in all stages of neutrophil series
(Fig. 28.4).
• MPO stain is positive in AML M1, AML M2, AML M3,
and AML M4 (only in myeloblasts). In AML M0, MPO
activity is visible only by electron microscopy.
• Lymphoblasts are negative for MPO.
Nonspecific Esterase (NSE)
NSE activity is strongly positive in monocytic series
(monoblasts, promonocytes, and monocytes). It allows
diagnosis of AML M4 and AML M5 (Fig. 28.5).
Sodium fluoride may be added that renders monocytic
cells negative.
 Laboratory Tests in Hematological Malignancies 277

Fig. 28.1: (A) AML M0; (B) AML M1; (C) AML M2; (D) AML M3; (E) AML M4; (F) AML M5; (G) AML M6; (H) AML M7

Fig. 28.2: (A) ALL L1; (B) ALL L2; (C) ALL L3
278 Essentials of Clinical Pathology

Table 28.5: Differences between acute lymphoblastic leukemia and acute myeloid leukemia
Parameter Acute lymphoblastic leukemia Acute myeloid leukemia
1. Predominant age Children (peak 3-4 years) Adults
2. Morphology of blasts
• Size of blasts Small Large
• Cytoplasm Scanty Moderate
• Granules in cytoplasm Absent May be present
• Nuclear chromatin Coarse Fine
• Nucleoli 0-2 >3
• Auer rods Absent Pathognomonic, if present
3. Cytochemistry
• Myeloperoxidase Negative Positive
• Periodic acid Schiff Block-like Diffuse
4. Immunophenotyping B or T lymphoid markers Myeloid markers
5. Prognosis Curable in majority of children Curable in minority of adults

Fig. 28.3: Principle cytochemical reactions in acute leukemias

Periodic Acid Schiff (PAS) 3. Immunophenotyping: This technique consists of

• PAS reagent stains glycogen in the cytoplasm.
• Lymphoblasts show block-like positivity (Fig. 28.6).
• PAS is positive in 70% of ALL L1 and ALL L2 cases.
Lymphoblasts in ALL L3 are negative.
Another stain that may be used is Sudan black B that dentifies
phospholipids and other neutral fats in membranes of both
primary and specific granules in granulocytes.
identification of antigens present on leukemic cells
in blood or bone marrow with fluorescently-labeled
monoclonal antibodies. As blood and bone marrow
cells are in fluid suspension, flow cytometery is the
method of choice. Cell surface antigens are named
according to the cluster of differentiation (CD) system.
Specific antigens are expressed on cells of different
lineages at different stages of development. A panel

Fig. 28.4: Myeloperoxidase (MPO) stain. MPO is a marker
for primary azurophil granules and is the routine initial cyto-
chemical stain in all acute leukemias. Dark brown granules
in the cytoplasm of blasts differentiate AML from ALL. However,
negative MPO reaction does not exclude AML
Fig. 28.5: Nonspecific esterase reaction. This stain is used
for demonstration of monocytic differentiation in acute myeloid
leukemia
consisting of a combination of specific antibodies is
employed to determine the immunophenotype and
diagnostic classification of leukemias. It is especially
Laboratory Tests in Hematological Malignancies 279
Fig. 28.6: Periodic acid Schiff reaction. Coarsely granular
(‘block-like’) perinuclear staining pattern is observed in some
cases of acute lymphoblastic leukemia. Negative PAS staining
is not helpful for differentiating AML from ALL
helpful if morphology and cytochemistry are ambi-
guous. A large number of antigens can be assessed
simultaneously on a single specimen. The immuno-
logic profiles of cell lineages are follows:
• Primitive stem cell markers: CD34, CD117, TdT
• Myeloid lineage: CD13, CD33, cytoplasmic myelo-
peroxidase
• Monocytic lineage: CD14, CD64
• Erythroid lineage: Glycophorin A
• Megakaryocytic lineage: CD41, CD42, CD61
• B-ALL: CD19, CD20, CD21, CD22, CD79a, CD10
(common ALL antigen or CALLA), cytoplasmic
μ chain, surface immunoglobulin
• T-ALL: CD2, CD3, CD5, CD7, cytoplasmic CD3.
For further subclassification of B-ALL (into pro-B
ALL, common ALL, Pre-B ALL, mature B ALL) or T-
ALL (into pro-T ALL, pre-T ALL, cortical T ALL, mature
T ALL), another set of antibodies is needed.
Terminal deoxynucleotidyl transferase (TdT) is
positive in all forms of ALL except L3 type.
Immunophenotyping is essential for diagnosis of
AML M0 in which myeloperoxidase is negative on light
microscopy, myeloid-specific antigens (CD13, CD33,
cytoplasmic myeloperoxidase, CD117) are positive, and
T or B lymphoid markers are absent. CD14 and anti-
lysozyme are strongly expressed in AML with significant
280 Essentials of Clinical Pathology

monocytic component (AML M4 and M5). In acute • Detection of minimal residual disease: Relapse
erythroleukemia (AML M6), glycophorin A and in acute following therapy is due to the persistence of viable
megakaryocytic leukemia (AML M7), platelet glyco- leukemic cells following cytotoxic chemotherapy.
protein antigens (CD41, CD42, and CD61) are demons- Leukemic cells on the background of normal cells can
trated. be detected by morphology, cytogenetics, immuno-
Immunophenotyping is also used for detection of phenotyping, and molecular methods.
minimal residual disease. Detection of minimal residual • To establish clonality i.e. determining whether a cell
disease (MRD) consists of identification of a small population is derived from a single cell (monoclonal)
population of leukemic cells among a large population or from multiple cells (polyclonal); it is helpful mainly
of normal cells after therapy to determine whether in B or T cell lymphomas.
residual disease is present that may later cause a relapse. • To detect chromosomal disorders which predispose
MRD can also be detected by morphology, cytogenetic to acute leukemia, e.g. trisomy 21.
analysis, and molecular analysis. 5. Molecular genetic analysis: This is used for:
4. Cytogenetic analysis: Structural or numerical • Detection of clonality by gene rearrangement
abnormalities of chromosomes are detected by studies (by polymerase chain reaction or Southern
cytogenetic analysis or karyotyping (Table 28.6). With blot analysis).
cytogenetic analysis, a variety of gross alterations can • Detection of minimal residual disease: Molecular
be detected such as translocations, deletions, and methods like polymerase chain reaction can be
duplications. In contrast to molecular methods, helpful in detecting a small submicroscopic
cytogenetic analysis is more widely available. In acute population of residual leukemic cells when
leukemias, cytogenetic abnormalities are linked to the peripheral blood and bone marrow examinations
pathogenesis of the disease. appear normal.
Applications of cytogenetic analysis in acute leukemia are • Diagnosis of specific types of acute leukemias by
• Diagnosis of specific types of AML: WHO classi- specific molecular probes: Molecular methods are
fication of AML recognizes distinct entities, which used for detection of chromosomal translocations
have clonal, recurrent cytogenetic abnormalities. that generate fusion transcripts and chimeric
Their identification is necessary as they have a proteins. The commonly used methods are reverse
correlation with response to therapy. transcription-polymerase chain reaction (RT-PCR)
• Prediction of prognosis: Certain cytogenetic abnor- and fluorescent in situ hybridization (FISH). The
malities are associated with poor prognosis and their technique detects t(15;17) in acute promyelocytic
detection may affect treatment strategies. leukemia that generates PML/RARα fusion gene,
and t(9;22) in B-ALL that generates bcr/abl fusion
Table 28.6: Chromosomal abnormalities in gene. Detection of these translocations is also
acute leukemias helpful for determining prognosis and response
Chromosomal abnormality Prognosis to treatment.
• Detection of opportunistic pathogens in immuno-
Acute lymphoblastic leukemia compromised patients.
1. t(9;22)(q34;q11.2) Poor
2. t(4;11)(q21;q23) Poor
CHRONIC LEUKEMIAS
3. t(1;19)(q23;p13.3) Average
4. t(12;21)(p13;q22) Good
Chronic leukemias are heterogeneous disorders
5. Hyperdiploidy>50 Good
6. Hypodiploidy Poor characterized by neoplastic proliferation of mature-
looking cells of myeloid or lymphoid lineage. General
Acute myeloid leukemia differences between acute and chronic leukemias are
1. t(8;21)(q22;q22) Good outlined in Table 28.7.
2. t(15;17)(q22;q12) Good Chronic leukemias are of two main types:
3. inv(16) Good
• Chronic myeloid leukemia
4. Monosomy 7 Poor
• Chronic lymphoid leukemias (Table 28.8).
 Laboratory Tests in Hematological Malignancies 281

Table 28.7: General differences between acute and chronic leukemias

Parameter Acute leukemias Chronic leukemias
Clinical presentation Usually sudden and fulminant Usually incidental or insidious onset
Hematological features in Immature blast cells Mature and differentiated cells
marrow and blood
Course Aggressive Indolent

Table 28.8: Chronic lymphoid leukemias

B cell type T cell type
1. Chronic lymphocytic leukemia 1. Prolymphocytic leukemia
2. Prolymphocytic leukemia 2. Large granular lymphocytic leukemia
3. Waldenström macroglobulinemia 3. Adult T cell leukemia/lymphoma
4. Hairy cell leukemia 4. Sezary syndrome
5. Plasma cell leukemia
6. Leukemic phase of non-Hodgkin’s lymphoma

Chronic Myeloid Leukemia
Chronic myeloid leukemia (CML) is a chronic myelo-
proliferative neoplasm originating from a pleuripotent
hematopoietic stem cell and characterized by predomi-
nant proliferation of granulocytic cells.
There are three phases of CML: chronic phase (3-5
years), accelerated phase (6-12 months), and blast crisis
(2-4 months).
CML is associated with a characteristic cytogenetic
abnormality called Philadelphia chromosome that results
from t(9;22)(q34;q11) (Fig. 28.7). This leads to juxtaposition
of c-abl gene from chromosome 9 to bcr gene on chromosome
22. The chimeric gene bcr-abl codes a tyrosine kinase that
affects cell proliferation.
Chronic phase: The average age at diagnosis is 45 years. In
chronic phase, the usual presenting features include
weakness, weight loss, abdominal fullness, easy
bruisability, and splenomegaly.
Blood smear in chronic phase of CML shows marked
leukocytosis, immature white blood cells, basophilia,
eosinophilia, anemia, and thrombocytosis (Fig. 28.8).

Fig. 28.8: Blood smear in chronic phase of chronic myeloid

Fig. 28.7: Translocation between chromosomes 9 and 22 leukemia showing all forms of immature white cells and a
causing formation of Philadelphia chromosome basophil
282 Essentials of Clinical Pathology
Fig. 28.9: Diagrammatic representation of fluorescence in situ
hybridization (FISH) in interphase nuclei. (A) Normal interphase
nucleus showing two red dots (located on abl genes on two
chromosomes number 9) and two green dots (located on bcr
genes on two chromosomes number 22). (B) Interphase
nucleus of a leukemic cell in chronic myeloid leukemia showing
one red dot (abl on normal chromosome 9), one green dot
(bcr on normal chromosome 22), and a combined dot on fused
bcr-abl gene on Philadelphia chromosome. This fusion causes
a fluorescent color change (red+green = yellow) which is not
shown in the figure
Bone marrow shows markedly increased myeloid to
erythroid ratio, < 10% blasts, and increased reticulin or
fibrosis. Diagnostic feature is Philadelphia chromosome
on cytogenetic analysis, and presence of a bcr-abl fusion
gene on fluorescence in situ hybridization (FISH)
(Fig. 28.9). Principle of FISH is shown in Box 28.1.
Leukocyte alkaline phosphatase (LAP) score: This cytochemical
stain is used to demonstrate presence and amount of the
enzyme alkaline phosphatase within neutrophils. Blood
smear is prepared from finger stick, air-dried, fixed, and
stained. Enzyme activity is indicated by the presence of
bright blue granules in neutrophils; nuclei are stained red
(Fig. 28.10). In chronic myeloid leukemia, LAP is either
absent or low. In leukemoid reaction and in other
myeloproliferative neoplasms (polycythemia vera,
essential thrombocythemia, and myelofibrosis), LAP score
is increased. Therefore, LAP score is useful in
differentiating CML from these disorders. Depending on
the intensity of staining, LAP score is graded in each
neutrophil as follows:
• 0: Negative; No granules
• 1: Positive with very few granules
• 2: Positive with few to moderate number of granules
• 3: Strongly positive with numerous granules
• 4: Very strongly positive with cytoplasm crowded
with granules.
Box 28.1: Fluorescence in situ hybridization (FISH)
This is a combination of cytogenetic and molecular
techniques used to identify and localize the presence or
absence of specific chromosomes or chromosomal regions
through hybridization of fluorescent probes that bind
specifically to its complementary target sequence. Fluores-
cent microscope is used to detect the presence or absence
of fluorescent signals. FISH can be used in metaphase
chromosomes (FISH-metaphase) or in interphase cells
(FISH-interphase), and is a very versatile procedure.
The technique consists of (1) Preparation of fluorescent
probes: Probes are short sequences of single-stranded DNA
that are complementary to the portion of gene of interest;
probes are labeled with a fluorescent dye, (2) Preparation
of metaphase chromosomes or interphase cells that are fixed
on a microscope slide, (3) Hybridization: Fluorescently-
labeled probe is applied to the denatured chromosomal DNA
and incubated. Attachment or hybridization occurs between
the probe and the complementary DNA sequence. Non-
hybridized probes are removed by washing, (4) Detection:
This consists of observation of hybridization under fluorescent
lighting. Fluorescent signals indicate hybridization or
presence of complementary sequence of DNA, while absence
of fluorescent signals indicates absence of complementary
sequence of DNA. Each probe can be labeled with a different
coloured fluorescent dye, so that a number of different probes
can be simultaneously detected in the same preparation.
In interphase FISH, probes are directly introduced into
the cell. In contrast to conventional cytogenetic anlysis that
requires 7-10 days for detection of chromosomal abnor-
malities, interphase FISH provides rapid diagnosis within
1-2 days. Unlike, metaphase FISH, actual chromosomes
cannot be visualized with interphase FISH and the location
of abnormality on the chromosome cannot be seen.
Fig. 28.10: Leukocyte alkaline phosphatase reaction: three
neutrophils with score: 0, two neutrophils with a score of 2,
and one neutrophil with score of 3

LAP score in an individual smear is the sum of the
scores of 100 consecutive neutrophils. Normal range is
40-100.
Chronic phase of CML should be differentiated from
leukemoid reaction in which myeloid cells increase in
number in peripheral blood secondary to infections,
inflammation, and other disorders (See Table 22.1 in
Chapter 22: Blood Smear).
Accelerated phase: During accelerated phase, basophils
and blast cells increase in number and patient increa-
singly becomes resistant to therapy.
Blast crisis: Blast crisis results when blast cells become
> 20% in blood or bone marrow. Additional cytogenetic
abnormalities develop in accelerated and blast phases.
Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia (CLL) is a neoplastic
disorder characterized by monoclonal proliferation of
immunologically incompetent, slowly dividing, mature
B lymphocytes. Most patients are adults >60 years.
Clinical features include insidious onset of weakness,
weight loss, susceptibility to infections, lymphadeno-
pathy, and splenomegaly. About 25% of patients are
asymptomatic.
Laboratory features include absolute lymphocytosis
(> 5000/cmm) consisting of small mature lymphocytes,
smudge cells, and typical immunophenotype (weak
Fig. 28.11: Blood smear in chronic lymphocytic leukemia
showing small mature-looking lymphocytes and a smudge cell
Laboratory Tests in Hematological Malignancies 283
surface membrane immunoglobulin, single light chain,
CD19, CD20, and T-associated antigen CD5). The
neoplastic lymphocytes are more fragile than normal cells
and are often disrupted while spreading blood smear
producing smudge (or basket) cells (Fig. 28.11).
Complications include infections, autoimmune
hemolytic anemia, and transformation to prolymphocytic
leukemia or large cell lymphoma (Richter’s syndrome).
PLASMA CELL DYSCRASIAS
Plasma cell dyscrasias are a group of disorders
(Table 28.9) characterized by clonal proliferation of
plasma cells or plasmacytoid lymphocytes and mono-
clonal production of immunoglobulins.
Features of plasma cell dyscrasias are summarized
in Table 28.10.
Laboratory investigations in plasma cell dyscrasias
are shown in Box 28.2.
The laboratory findings that are suspicious of a
plasma cell dyscrasia are raised erythrocyte sedimen-
tation rate, rouleaux formation on blood smear, increased
plasma cells in bone marrow (Fig. 28.12), renal impair-
ment with bland urinary sediment, unexplained lytic bone
lesions, anemia associated with renal failure and bone
Table 28.9: Plasma cell dyscrasias
1. Multiple myeloma
2. Plasmacytoma
3. Waldenström macroglobulinemia
4. Amyloidosis
5. Heavy chain disease
6. Monoclonal gammopathy of undetermined
significance (MGUS)
Table 28.10: Characteristics of plasma cell dyscrasias
Multiple myeloma: Osteolytic lesions, bone pain, pathological
fractures, raised erythrocyte sedimentation rate, hyper-
calcemia, anemia, renal disease, serum M protein >3 g/dl,
urinary M protein, plasma cells >10% in bone marrow
Solitary plasmacytoma: Single tumor in bone, no urine or
serum protein abnormalities, no myeloma cells in marrow
Waldenström macroglobulinemia: Organomegaly, hyper-
viscocity, Serum IgM >3 g/dl, infiltration of bone marrow with
plasmacytoid lymphocytes
Monoclonal gammopathy of undetermined significance:
No osteolytic lesions/hypercalcemia/anemia/renal disease,
serum M protein <3 g/dl, plasma cells <10% in bone marrow,
no urinary M protein
284 Essentials of Clinical Pathology

Box 28.2: Laboratory investigations in

plasma cell dyscrasias

• Serum and urine protein electrophoresis: Screening,

diagnosis, and monitoring the course of disease and
response to therapy
• Immunoelectrophoresis: Diagnosis and identification of
type of M protein
• Serum/urine immunofixation: Diagnosis, identification of
clonal nature by immunotyping
• Quantitation of M protein by radial immunodiffusion:
Diagnosis and follow-up; may help in differentiating benign
from malignant process (e.g. MGUS from multiple
myeloma)
• Serum viscosity: Diagnosis of hyperviscosity syndrome; this
is especially common in Waldenström macroglobulinemia
(due to elevated IgM) and uncommon in IgG-secreting multiple
myeloma. Hyperviscosity symptoms become clinically
apparent when viscosity exceeds 3.0 centipoise.
Fig. 28.12: Bone marrow smear in myeloma showing
plasma cells

Fig. 28.13: Principle of serum protein electrophoresis. (A) On agar gel electrophoresis, serum proteins get separated into
5 regions or bands. Each region or band contains 1 or more proteins, knowledge of which aids in interpretation. (B) Densitometric
scan of serum protein electrophoresis reveals 5 peaks; elevation or depression of the peak(s) can reflect concentrations
of constituent proteins. If abnormal pattern is identified (e.g. M protein), further testing is done to identify the nature of M
protein and quantification of M protein

pain, Bence Jones proteinuria, and hypergamma-
globulinemia with immune deficiency.
1. Serum protein electrophoresis: The most common
indication for serum protein electrophoresis is
suspected plasma cell dyscrasia (especially multiple
myeloma). In this technique, patient’s serum is placed
on the supporting medium (like agar gel, cellulose,
etc.) which is then placed in an electrophoresis
chamber and exposed to the electric current. Serum
proteins get separated into five components: albumin,
α1-globulins, α2-globulins, β-globulins, and
γ-globulins (Fig. 28.13). The last four zones are
 Laboratory Tests in Hematological Malignancies 285

Table 28.11: Components of serum protein electrophoresis

Component Proteins Common abnormalities

1. Albumin Albumin ↓ in liver disease, malnutrition,

nephrotic syndrome
2. α1-globulins • α1-antitrypsin ↓ in α1-antitrypsin deficiency
• α1-glycoprotein (orosomucoid)
• Thyroid-binding globulin
3. α2-globulins • α2-macroglobulin ↑ in acute phase response
• Haptoglobin
• Ceruloplasmin
4. β-globulins • Transferrin
• C-reactive protein
• β-lipoprotein
5. γ-globulins Immunoglobulins ↑ severe infection, chronic liver disease,
autoimmune disorders, plasma cell dyscrasias;
↓ hypogammaglobulinemia
↓: Decreased; ↑: Increased

Table 28.12: Comparison of polyclonal and monoclonal gammaglobulinemias

Parameter Polyclonal gammaglobulinemia
1. Pathogenesis Production of immunoglobulins from
single
different clones of plasma cells
2. Causes Chronic liver disease, collagen
vascular disease, chronic infections,
metastatic carcinoma
3. Protein electrophoresis A wide band with indistinct borders
4. Densitometer scan A broad-based peak
5. Nature of light chains Both κ and λ light chains
Monoclonal gammaglobulinemia
Production of immunoglobulins from a
neoplastic or potentially neoplastic clone of
plasma cells
Multiple myeloma, Waldenström
macroglobulinemia, amyloidosis,
monoclonal gammopathy
of undetermined significance, heavy chain
disease
A localized dense band with sharp borders
A narrow, tall, sharp spike
Either κ or λ light chain

composed of a mixture of proteins (Table 28.11).
Majority of plasma proteins are synthesized in the liver,
except immunoglobulins that are produced by plasma
cells.
The bands are inspected visually for any
qualitative abnormality. For quantitation, membrane
is then run through an instrument called as densito-
meter. This records absorbance of each protein band,
generates a tracing, and quantitates percentage
fraction of each band. If total serum proteins have been
measured earlier, concentration of each protein band
can be obtained from the percentage fraction.
If plasma cell dysrasia is strongly suspected and
serum protein electrophoresis is normal, immuno-
fixation should be performed as it is more sensitive
for detection of small amounts of monoclonal protein.
The amount of M protein corresponds directly with
tumour burden.
Comparison of polyclonal vs. monoclonal
gammaglobulinemia is presented in Table 28.12.
2. Immunoelectrophoresis: This technique combines
separation by electrophoresis and identification by
double immunodiffusion. Antigenic mixture (placed
in a well cut in agar gel) is separated by electro-
286 Essentials of Clinical Pathology
phoresis. A trough is cut parallel to the electrophoretic
migration, filled with antibody, and then kept for
immunodiffusion in a moist chamber for 24 hours.
The resulting immunoprecipitates form arcs that
assume different shapes and varying mobilities
(Figs 28.14 and 28.15).
This technique is most useful for recognition of
abnormal immunoglobulins in paraproteinemias.
3. Immunofixation electrophoresis: This technique is
used on serum or urine to identify the nature of M
protein. In this type of electrophoresis, monospecific
antibodies are used to identify specific types of
immunoglobulins and light chains.
Patient’s serum sample is subjected to agarose gel
electrophoresis in 5 different lanes. Different
monospecific antibodies (IgG, IgM, IgA, κ light chain,
λ light chain) are then applied directly over the
surface of gel. The antibody reacts with the corres-
Fig. 28.14: Principle of immunoelectrophoresis
ponding immunoglobulin protein to form a complex
that becomes fixed in the gel. The gel is washed to
remove the unbound proteins, leaving only the fixed
protein that is then visualized with staining (Fig.
28.16). The test is sensitive and can detect small
amounts of M protein.
4. Single radial immunodiffusion (SRID): This is a
quantitative method used for measuring concen-
tration of serum proteins (M protein in parapro-
teinemias). In this technique, antibody is incorporated
into the gel. A soluble antigen is placed in the well
cut in the agar gel. Following the radial diffusion of
the antigen into the antibody-incorporated gel,
circular precipitates develop. The diameter of the
circular precipitates is directly proportional to the
antigenic concentration, provided that the mono-
specific antibody is evenly distributed in the uniformly
thick gel, and the well size and the volume of the
 Laboratory Tests in Hematological Malignancies
Fig. 28.15: Imunoelectrophoresis. (A) Two wells and central
trough pattern. This is used for comparing immunoelectro-
phoretic pattern of normal serum with that of patient’s serum.
Top well is filled with normal serum while bottom well is filled
with patient’s serum. After electrophoresis, central trough is
charged with monospecific antiserum to IgG. (B) Thickening
and bowing of IgG arc from patient’s serum indicates IgG
paraproteinemia. Normal IgG arc is shown for comparison
BIBLIOGRAPHY
Fig. 28.16: Immunofixation showing monoclonal protein
IgG λ. SPE: Serum protein electrophoresis
antigen are kept constant. The concentration can be
read from the reference graph prepared earlier using a
set of standards (Fig. 28.17).
287
Fig. 28.17: Principle of single radial immunodiffusion
1. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for
the classification of the acute leukemias: French-
American-British (FAB) Cooperative Group. Br J
Hematol 1976;33:451-8.
2. Bennett JM, Catovsky D, Daniel MT, et al. Proposal for
the recognition of minimally differentiated acute
myeloid leukemia (AML-M0). Br J Hematol 1991;
78:325-9.
3. Bennett JM, Catovsky D, Daniel MT, et al. Proposed
revised criteria for the classification of acute myeloid
leukemia. Ann Intern Med 1985;103:620-5.
4. Bennett JM, Catovsky D, Daniel MT, et al. The
morphological classification of acute lymphoblastic
leukemia: concordance among observers and clinical
correlations. Br J Hematol 1981;47:553-61.
5. Jaffe ES, Harris NL, Stein H, Vardiman JW (Eds). World
Health Organization Classification of Tumours.
Pathology and Genetics of Tumours of Hematopoietic
and Lymphoid Tissues. Lyon; IARC Press, 2001.
6. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers and Medical Publishers (P) Ltd, 2006.
 29
Laboratory Tests in
Bleeding Disorders

PHYSIOLOGY OF HEMOSTASIS
Hemostasis is the normal physiologic mechanism for
keeping the blood in fluid state in vascular system and for
prevention of hemorrhage by complex interaction of blood
vessel walls, platelets, and plasma proteins. Following
injury, initially vessel wall and platelets interact to control
hemorrhage by forming a platelet plug at the site of injury;
this is called as primary hemostasis. This is followed by
activation of coagulation factors by a series of enzymatic
reactions to form a stable fibrin clot (platelet plug
enmeshed by fibrin); this is secondary hemostasis.
Dissolution of the clot will eventually occur by fibrinolysis
when healing is complete. Hemostatic equilibrium
requires normal blood vessels, normal platelets, normal
coagulation factors, normal fibrinolysis, and normal
coagulation inhibitor system.
Role of the three main components of hemostasis is
outlined below.
Blood Vessel Wall
Transient constriction of blood vessels occurs at the site
of injury which helps to control blood loss. Endothelial
cells synthesize von Willebrand factor (vWF), tissue factor,
and platelet activating factor, which promote hemostasis.
Also, following injury, subendothelial collagen is exposed
which provides site for attachment of platelets (adhesion).
Endothelial cells synthesize prostacycline (inhibits platelet
aggregation), protein S (a cofactor for protein C, which is
an inhibitor of coagulation), and tissue plasminogen
activator (activates fibrinolysis).
Platelets
Platelets are produced by cytoplasmic fragmentation of
megakaryocytes in bone marrow. Life-span of platelets is
about 7-10 days. Normal platelet count in peripheral blood
is 1.5-4.0 lac/µl. About 2/3rd of platelets in the body are
circulating in peripheral blood, while 1/3rd are pooled in
spleen.
Main functions of platelets in hemostasis are adhesion,
release reaction, and aggregation (Fig. 29.1).
Platelets attach to exposed subendothelium following
injury; this adhesion is mediated by vWF, which binds
to GpIb receptor on platelets. This initiates activation of

Fig. 29.1: Platelet adhesion and aggregation

 Laboratory Tests in Bleeding Disorders 289

platelets, which change shape from a disc to a sphere and
release their contents to the exterior (release reaction).
These are mainly adenosine diphosphate (ADP),
serotonin, and thromboxane A 2 (TxA 2 ). Platelet
aggregation refers to sticking of platelets to each other;
platelet agonists such as collagen, ADP, thrombin, and
TxA2 mediate aggregation. Binding of ADP to platelets
causes exposure of GpIIb/IIIa receptors which bind
fibrinogen. Fibrinogen binding to multiple platelets
causes formation of large platelet aggregates.
Activated platelets also provide phospholipid surface
for certain coagulation reactions (platelet factor 3 or
platelet procoagulant activity).
Plasma Proteins
Plasma proteins, which regulate hemostasis, are
coagulation factors, coagulation inhibitors, and proteins
of fibrinolytic system.
Table 29.1: Coagulation factors
Coagulation System
Normally, coagulation factors (Table 29.1) are circulating
in an inactive form. Except for thromboplastin and
calcium, all the coagulation factors are proteins.
Coagulation factors have been divided into thee groups
depending on similarities in structural and functional
properties: (1) Fibrinogen group: I, V, VIII, XIII; (2)
Vitamin-K dependent: II, VII, IX, X; and (3) Contact
group: XI, XII, high molecular weight kininogen,
prekallikrein. When activated, coagulation factors
interact with each other in a sequential manner to
ultimately form a fibrin clot and arrest bleeding. Blood
coagulation occurring in vitro is divided into three
pathways: extrinsic, intrinsic, and common (Fig. 29.2).
This division is helpful in understanding the principles
of common screening tests of coagulation.
Extrinsic pathway: The extrinsic pathway is initiated when
F VII combines with tissue factor (released after tissue

Factor Synonym Site of synthesis Significant features

I Fibrinogen Liver Normal: 200-400 mg/dl; absent in serum

II Prothrombin Liver Vitamin K-dependent; multiple actions; absent in
adsorbed plasma
III Tissue factor; Various tissues Activates coagulation through
thromboplastin extrinsic pathway
IV Calcium Obtained from Acts as a cofactor in various
diet and bones coagulation reactions
V Labile factor Liver, platelets Cofactor in conversion of
prothrombin to thrombin; absent in aged plasma
VI There is no factor VI
VII Stable factor Liver Vitamin K-dependent; absent in
adsorbed plasma; sensitive to
oral anticoagulant therapy
VIII Antihemophilic globulin; Liver Two subunits: VIII:C and VIII:vWF; cofactor in the
antihemophilic factor conversion of F X to F Xa; absent in aged plasma
IX Christmas factor Liver Vitamin-K dependent; absent in adsorbed plasma
X Stuart-Prower factor Liver Vitamin-K dependent; absent in adsorbed plasma
XI Plasma thromboplastin Liver Contact factor
antecedent
XII Hageman factor; Liver Contact factor; activates coagulation in vitro;
Contact factor deficiency does not cause bleeding
XIII Fibrin stabilizing factor; Liver, platelets Stabilizes fibrin clot by cross-linking fibrin
Laki-Lorand factor monomers
Prekallik- Fletcher factor Liver Contact factor; has many functions
rein
High Fitzgerald factor Liver Contact factor; has many functions
molecular
weight
kininogen

Aged plasma: Stored plasma that is deficienct in factors V and VIII. Adsorbed plasma: Plasma adsorbed with ammo-
nium hydroxide that is deficient in factors II, VII, IX, and X (vitamin K-dependent factors)
290 Essentials of Clinical Pathology
Fig. 29.2: Normal coagulation mechanism. HMWK = high molecular weight kininogen; PL = phospholipid; Ca = calcium;
TF: tissue factor; solid arrow: conversion; Dotted arrow: action; subscript a: activated form of the coagulation factor
injury) in the presence of calcium ions. F VII-tissue factor
complex in turn activates F X to F Xa.
Intrinsic pathway: Intrinsic system is initiated when F XII
is activated in vitro ( e.g. by glass). This activation requires
presence of high molecular weight kininogen (HMWK)
and prekallikrein. The roles of F XII, HMWK, and
prekallikrein in coagulation in vivo are not clear since
their deficiencies are not associated with bleeding.
Activated F XII activates F XI, which in turn activates F
IX to F IXa. F IXa combines with F VIII, phospholipid,
and calcium to activate F X to F Xa.
Common pathway: Activation of F X to F Xa, by either
extrinsic or intrinsic mechanism, marks the beginning
of common pathway. F Xa binds FV, phospholipid, and
calcium and converts prothrombin to thrombin.
Thrombin splits off fibrinopeptides A and B from
fibrinogen to form fibrin monomers, which sponta-
neously polymerize. F XIII stabilizes fibrin polymers by
introducing covalent bonds between adjacent poly-
peptide chains.
Inhibitors of Coagulation
Several physiologic inhibitors of coagulation are present
in circulation to ensure that the fibrin clot remains
localized to the site of injury and activation of coagulation
is short-lived. Important inhibitors of coagulation are
tissue factor pathway inhibitor (released from blood
vessels; neutralizes VII-tissue factor complex), anti-
thrombin III (most important physiological inhibitor;
synthesized in liver; heparin-like substances on
endothelial cells promote activity; inhibits mainly
thrombin and F Xa), and protein C (activated by thrombin
in the presence of thrombomodulin on the surface of
endothelial cells; causes proteolysis of activated forms
of F V and F VIII; protein S acts as a cofactor in this
reaction) (Fig. 29.3).
Fibrinolytic System
Fibrinolysis is the process of dissolution of blood clot to
ensure free flow of blood in vascular system. During
coagulation process, plasminogen binds to fibrin
 Laboratory Tests in Bleeding Disorders 291

Fig. 29.3: Natural inhibitors of coagulation and their actions. – indicates inhibition. Antithrombin III mainly inhibits F Xa and
thrombin, activated protein C degrades activated forms of FV and F VIII, while tissue factor pathway inhibitor inhibits F VII-
tissue factor complex

Fig. 29.4: Mechanism of fibrinolysis

network. Endothelial cells secrete tissue plasminogen
activator, which activates plasminogen to plasmin.
Plasmin cleaves fibrin strands to release fibrin
degradation products. Plasmin also digests fibrinogen
(Fig. 29.4). Fibrinogen/fibrin degradation products
interfere with action of thrombin and with platelet
aggregation. FDPs are cleared from the circulation by
macrophages.
Fibrinolysis is inhibited by plasminogen activator
inhibitor (PAI) type 1, α2-antiplasmin and α2-macro-
globulin.
BLEEDING DISORDERS
Bleeding disorders are the result of a generalized defect in
hemostasis due to abnormalities of blood vessels, platelets,
or coagulation factors. Common bleeding disorders are
listed in Table 29.2.
292 Essentials of Clinical Pathology

Table 29.2: Bleeding disorders

Disorders of blood vessels

Acquired Hereditary
Allergic purpura Hereditary hemorrhagic telangiectasia
Infections: Gram-negative septicemia, viral infections
Scurvy
Senile purpura
Disorders of platelets
Thrombocytopenia Defective platelet function
Increased destruction of platelets Acquired
Idiopathic thrombocytopenic purpura Drugs: aspirin, other nonsteroidal anti-inflammatory drugs,
antibiotics, xylocaine
Infections: malaria, dengue, septicemia, subacute Myeloproliferative disorders
bacterial endocarditis, rubella, infectious mononucleosis
Drugs: quinidine, quinine, heparin, procainamide Uremia
Heparin induced thrombocytopenia Paraproteinemia
Disseminated intravascular coagulation Hereditary
Massive blood transfusion Storage pool deficiency
Thrombotic thrombocytopenic purpura Bernard-Soulier syndrome
Alcohol Glanzmann’s thrombasthenia
Toxemia of pregnancy
Increased pooling of platelets in spleen
Hypersplenism
Decreased production of platelets in bone marrow
Aplastic anemia
Bone marrow infiltration: leukemias, lymphomas, myeloma
Megaloblastic anemia
Drugs
Infections
Radiation
Hereditary disorders
Disorders of coagulation
Hereditary Acquired
Hemophilia A (F VIII deficiency) Disseminated intravascular coagulation
von Willebrand’s disease Liver disease
Hemophilia B (F IX deficiency) Vitamin K deficiency
Disorders of fibrinogen Massive blood transfusion
Heparin or oral anticoagulant therapy
Renal disease
Paraproteinemia
Inhibitors of coagulation

Disorders of Blood Vessels present if platelet count is < 1,50,000/cmm. Causes of

Vascular disorders can result in easy bruising and muco-
cutaneous type of bleeding. Vascular disorders may be
inherited or acquired (Table 29.2) and defect may be in
the connective tissue lining (collagen or elastic tissue) or
in endothelial cells.
Disorders of Platelets
Disorders of platelets can be divided into disorders of
platelet number or of function. Thrombocytopenia is
thrombocytopenia are listed in Table 29.2. Bleeding
manifestations include petechie, purpura, ecchymoses,
and mucous membrane bleeding.
Immune Thrombocytopenic Purpura (ITP)
ITP is of two types: acute and chronic. Acute ITP has a
sudden onset, occurs usually in children, and often
follows a viral infection. Platelet count is very low.

Complete remission is usual. Chronic ITP has a gradual
onset, occurs usually in adults (especially young females),
and platelet count is moderately decreased. Cause is
unknown. It is characterized by remissions and
exacerbations over a long duration. Bone marrow
examination in ITP shows increased number of mega-
karyocytes. Diagnosis of ITP requires exclusion of all
other causes of thrombocytopenia.
Thrombotic Thrombocytopenic Purpura
There is formation of hyaline microthrombi in micro-
circulation due to systemic clumping of platelets because
of unusually large multimers of von Willebrand factor.
This causes thrombocytopenia. The characteristic pentad
of signs includes: bleeding secondary to severe thrombo-
cytopenia, microangiopathic hemolytic anemia (with
production of schistocytes), fever, neurological signs, and
renal abnormalities.
Hemolytic Uremic Syndrome
It is caused by ingestion of food contaminated with
verotoxin-producing Escherichia coli and is commonly
Fig. 29.5: Sites of defects in disorders of platelet function
Laboratory Tests in Bleeding Disorders 293
seen in children. It is characterized by a triad of acute
renal failure, thrombocytopenia, and microangiopathic
hemoloytic anemia.
Post-Transfusion Purpura
This is allo-antibody induced thrombocytopenia. There is
a sudden onset of severe thrombocytopenia in some adult
maltiparous women 1-10 days following blood
transfusion. It is due to sensitization to platelet antigen
HPA-1a (platelet antigen A1 or PlA1) during previous
pregnancy by foetal platelets.
Disorders of Platelet Function
Disorders of platelet function are characterized by
prolonged bleeding time and normal platelet count. They
may be inherited or acquired. Acquired disorders are
more common. Sites of defects in platelet function
disorders are shown in Figure 29.5.
Bernard Soulier syndrome: This is a rare, autosomal
recessive disorder with severe bleeding manifestations.
There is a congenital absence of platelet membrane
glycoprotein complex GpIb/IX leading to failure of
294 Essentials of Clinical Pathology

adhesion of platelets to subendothelium via von
Willebrand factor. Blood smear shows giant platelets.
Platelet aggregation studies reveal impaired aggregation
with ristocetin and normal aggregation with other
agonists.
Glanzmann’s thrombasthenia: In this very rare autosomal
recessive disorder, platelet aggregation is defective due
to congenital absence of platelet membrane glycoproteins
GpIIb/IIIa on platelet surface. Laboratory features
include discrete, small platelets on blood smear, poor clot
retraction, and defective platelet aggregation with ADP,
epinephrine, and collagen, and normal aggregation with
ristocetin.
Aspirin inhibits the enzyme cyclo-oxygenase leading to
failure of synthesis of thromboxane A2 that is required
for platelet aggregation. The inhibitory effect of aspirin
on platelets lasts for 7-10 days (i.e. lifespan of platelets).
Inherited Disorders of Coagulation
Deficiencies of all the coagulation factors have been
reported. Out of these, the three relatively common
disorders are hemophilia A (F VIII deficiency), hemo-
philia B (F IX deficiency), and von Willebrand disease
(Table 29.3).
Hemophilia A
(Classical Hemophilia, F VIII Deficiency)
It is caused by hereditary deficiency or dysfunction of F
VIII due mainly to point mutations or deletions of F VIII
gene. It is an X-linked recessive disorder primarily affecting
males; females are carriers but do not manifest the disease
(Fig. 29.6). Hemophilia A is classified into three types
based on the level of F VIII level in plasma: mild, moderate,
and severe (Table 29.4).
In severe hemophilia, hemarthroses lead to crippling
deformities; intramuscular hematomas can compress
vital structures; intracranial hemorrhage can occur
following minor trauma; operative and post-traumatic
bleeding can be life-threatening; infections like hepatitis
and acquired immunodeficiency syndrome can be
transmitted through blood products. Screening tests for
hemostasis show normal bleeding time, platelet count,
and prothrombin time. Activated partial thromboplastin
time is prolonged. Diagnosis is made by one-stage F VIII
assay.
Hemophilia B (Christmas Disease, F IX Deficiency)
This is clinically indistinguishable from hemophilia A.
Diagnosis requires F IX assay.
von Willebrand Disease (VWD)
vWD is a markedly heterogeneous congenital bleeding
disorder characterized by deficiency or functional defect
of von Willebrand factor (vWF). Mode of inheritance is
autosomal dominant or recessive, with overall prevalence
in the general population being 1%. There are three main

Table 29.3: Inheritance and incidence of inherited bleeding disorders

Disorder Inheritance Incidence
von Willebrand Autosomal dominant 1:100
disease or recessive
Factor VIII deficiency X-linked recessive 1:10000
Factor IX deficiency X-linked recessive 1:60000
Factor VII deficiency Autosomal recessive 1:500,000
Fibrinogen deficiency Autosomal recessive 1:1 million
Factor V deficiency Autosomal recessive 1:1 million
Factor X deficiency Autosomal recessive 1:1 million
Factor XI deficiency Autosomal recessive 1:1 million
Factor XIII deficiency Autosomal recessive 1:2 million
Prothrombin deficiency Autosomal recessive 1:2 million
Note: Deficiencies of F XII, high molecular weight kininogen, and prekallikrein are not associated with bleeding
 Laboratory Tests in Bleeding Disorders 295

Fig. 29.6: Typical pedigree in X-linked recessive trait (hemophilia A or B). Females are
heterozygous (carriers) and only males are affected

Table 29.4: Classification of hemophilia

Type F VIII level Clinical features
Mild >5% Excess bleeding only after major trauma or surgery
Moderate 1-5% Excess bleeding after mild to moderate trauma; occasional
hemarthrosis; spontaneous bleeding infrequent
Severe <1% Frequent and spontaneous deep tissue hematomas and
hemarthroses

Table 29.5: Comparison of three main inherited bleeding disorders

Feature Von Willebrand disease Hemophilia A Hemophilia B
1. Incidence 1:100 1:10,000 1:60,000
2. Inheritance AD or AR XR XR
3. Sex affected Male or female Male Male
4. Nature of bleeding Superficial (Mucocutaneous) Deep (muscles, joints) Deep (muscles, joints)
5. Screening tests
• BT Increased Normal Normal
• PC Normal or low Normal Normal
• PT Normal Normal Normal
• APTT Normal or increased Increased Increased
6. Specific test Platelet aggregation with F VIII assay F IX assay
for diagnosis ristocetin

AD: Autosomal dominant; AR: Autosomal recessive; XR: X-linked recessive; BT: Bleeding time; PC: Platelet count;
PT: Prothrombin time; APTT: Activated partial thromboplastin time

types: I, II, and III. Type I (partial deficiency of vWF) is the
most common in which all types of vWF multimers (small,
intermediate, and large) are mildly deficient; bleeding
manifestations are slight. In type II vWD (qualitative
defects in vWF) there is a qualitative abnormality of vWF
with absence of large vWF multimers. Type III (complete
vWF deficiency) is a rare severe bleeding disorder in which
there is a severe deficiency of all forms of vWF multimers.
Screening tests for hemostasis reveal prolonged bleeding
time and activated partial thromboplastin time. Platelet
count and prothrombin time are normal. Ristocetin-
induced platelet aggregation is deficient. One stage F VIII
assay shows reduced F VIII activity.
The three main inherited bleeding disorders are
compared in Table 29.5.
296 Essentials of Clinical Pathology

Acquired Disorders of Coagulation

Disseminated Intravascular Coagulation (DIC)

This is an acquired thrombohemorrhagic disorder
occurring as a secondary complication in a wide
spectrum of disorders, and characterized by (i) activation
of coagulation with formation of microthrombi in
microcirculation, (ii) activation of fibrinolysis, and (iii)
consumption of platelets, coagulation factors, and fibrin
leading to bleeding diathesis.
Important causes of DIC include sepsis or severe
infections, massive crush injury, obstetric complications
(amniotic fluid embolism, placental abruption, intra-
uterine retention of dead fetus, eclampsia, septic
abortion), malignancy (disseminated mucin-secreting
adenocarcinoma, acute promyelocytic leukemia), snake
bite, shock, heat stroke, severe liver disease, and acute
intravascular hemolysis. DIC can be initiated due to Fig. 29.7: Blood smear in acute disseminated
above causes by: (i) release of tissue factor or thrombo- intravascular coagulation showing fragmented red cells
plastic substances in circulation, or (ii) endothelial cell
damage.
newborn, and in adults with poor dietary intake,
There are two main presentations: acute and chronic.
malabsorption , obstructive jaundice, and drugs like oral
Acute or uncompensated DIC is a severe disease with
anticoagulants.
significant bleeding manifestations. Chronic DIC is a
mild, protracted disease characterized mainly by venous Hemorrhagic disease of newborn Normally, vitamin K-
thrombosis. Laboratory features in DIC are shown in Box dependent factors are low at birth; vitamin K deficiency
29.1. exaggerates this deficit and causes bleeding. In classic
cases, hemorrhage manifests around 2-4 days of life.
Screening tests reveal normal platelet count and
Box 29.1: Laboratory features in disseminated
intravascular coagulation prolongation of both prothrombin time and activated
partial thromboplastin time.
• Acute DIC: Platelet count: Low, Prothrombin time:
Prolonged, Activated partial thromboplastin time: Pro-
Liver Disease
longed, Blood smear: Fragmented red cells (Fig. 29.7),
Fibrinogen level: Low, Fibrinogen/fibrin degradation Pathophysiology of hemostatic defect in liver disease is
products: Increased, D-dimer: Increased
complex: (i) deficient synthesis of coagulation factors in
• Chronic DIC: Platelet count, Prothrombin time, Activated hepatocellular disease, (ii) deficient synthesis of vitamin
partial thromboplastin time: Normal; Fibrinogen/fibrin
degradation products, D-dimer: Increased. K-dependent factors in biliary obstruction, (iii) synthesis
of dysfunctional fibrinogen (dysfibrinogenemia), (iv)
decreased clearance of activated coagulation factors, (v)
Vitamin K Deficiency defective platelet function due to raised fibrinogen/fibrin
Vitamin K is a fat-soluble vitamin necessary for the degradation products, or (vi) disseminated intravascular
synthesis of coagulation factors II, VII, IX, and X, and also coagulation.
two natural anticoagulant proteins C and S. Vitamin K is
required for gamma carboxylation of glutamic acid Acquired Inhibitors of Coagulation
residues of the above coagulation factors. Vitamin K, (Circulating Anticoagulants)
being fat-soluble, requires bile salts for absorption. Acquired inhibitors of coagulation are of two main types:
Vitamin K deficiency occurs in hemorrhagic disease of specific and non-specific. Most common specific inhibitor

is an antibody directed against F VIII. It develops in multi-
transfused patients of F VIII deficiency. Inhibitors render
F VIII replacement therapy ineffective. Autoantibodies
against F VIII can also develop in old age, rheumatoid
arthritis, and postpartum females.
Non-specific inhibitors are lupus inhibitors that
develop in some patients with systemic lupus erythe-
matosus, other autoimmune disorders, neoplasia, and
human immunodeficiency virus infection. These are
antiphospholipid antibodies which interfere with
coagulation reactions that need phospholipid surface.
Patients usually have recurrent venous thromboses,
repeated spontaneous abortions, and intrauterine fetal
death.
Coagulation inhibitors cause prolongation of APTT
and are detected by mixing experiment (see later). F VIII
inhibitor is measured by Bethesda inhibitor assay. For
detection of lupus anticoagulant, various tests are
available like kaolin clotting time, dilute Russell’s viper
venom time, and platelet neutralization assay.
APPROACH TO THE DIAGNOSIS OF
BLEEDING DISORDERS
A systematic approach, consisting of both clinical and
laboratory evaluation, is necessary for diagnosis of
bleeding disorders (Fig. 29.8).
Fig. 29.8: General approach for investigation of a
bleeding disorder
Laboratory Tests in Bleeding Disorders 297
Clinical Evaluation
Significant bleeding may result from a local cause or a
bleeding disorder (generalized hemostatic defect). A
bleeding disorder is suggested by the presence of easy
bruising, spontaneous bleeding, bleeding from multiple
sites, repeated episodes of excessive bleeding, restart of
bleeding hours or days following injury, similar past or
family history, history of poor wound healing or when
bleeding is out of proportion to the degree of trauma.
Nature of bleeding can help in differentiating
between primary and secondary disorders of hemostasis:
• Petechia: Tiny pinpoint areas of red to purple
hemorhage (< 2 mm in diameter) on skin or mucous
membranes due to a primary hemostatic disorder.
• Purpura: Areas of hemorrhage in skin or mucous
membrane, red to purple in color, non-blanching and
>3 mm but <1 cm in diameter. They occur in disorders
of primary hemostasis.
• Ecchymosis (Bruise): An area of extravasated blood in
skin > 1 cm in diameter. It may be due to defective
hemostasis or trauma.
• Hematoma: A swelling resulting from a large area of
hemorrhage in subcutaneous tissue or muscle. It
occurs in coagulation disorders.
• Hemarthrosis: This refers to bleeding into a joint. It
results from a coagulation disorder, especially
hemophilia.
Bleeding manifestations typical of some bleeding
disorders are:
• Muco-cutaneous bledding or bleeding from skin and
mucous membranes (purpura, petechia, bleeding from
gums, epistaxis, gastrointestinal/rectal bleeding,
menorrhagia): Platelet disorders, von Willebrand
disease.
• Cephalhematomas, hemarthrosis, intramuscular
hematoma, intracerebral bleeding, retroperitoneal
hemorrhage: hemophilia, von Willebrand disease,
afibrinogenemia
• Umbilical stump bleeding: afibrinogenemia, F XIII
deficiency
• Defective wound healing: F XIII deficiency
• Recurrent severe epistaxis: hereditary hemorrhagic
telangiectasia
Detailed family history should be elicited and a family
tree constructed. History of similar abnormality in close
relatives is suggestive of a hereditary disorder. From the
pattern of inheritance, diagnostic possibilities can be
narrowed and investigations can be directed accordingly.
298 Essentials of Clinical Pathology

History of bleeding only in males and positive family Laboratory Evaluation

history on maternal side spanning many generations is
suggestive of an X-linked disorder (e.g. F VIII or F IX
deficiency). In autosomal recessive diseases (e.g.
afibrinogenemia, deficiency of FV or FX, von Willebrand
disease), both males and females are affected (in the same
generation) and history of consanguinity in parents is
common. In autosomal dominant disorders (e.g. some
types of von Willebrand disease), bleeding manifests in
both sexes, in one parent and also in older generations.
Various acquired conditions can cause a generalized
hemostatic defect. These include diseases of liver, renal
diseases, autoimmune disorders, malabsorption,
malignancy, infections, and administration of drugs (e.g.
heparin, oral anticoagulants, aspirin and other nonsteroi-
dal anti-inflammatory drugs). Important features to look
for on physical examination include fever, splenomegaly,
hepatomegaly, lymphadenopathy, and icterus.
A bleeding disorder can be placed into the category
of either vascular/platelet or coagulation abnormality
on the basis of clinical features (Table 29.6).
Initial tests, which should be performed in a suspected
bleeding disorder, are complete blood count including
blood smear, platelet count, bleeding time, clotting time,
prothrombin time, and activated partial thromboplastin
time. Depending on the results of these screening tests,
one or more specific tests are carried out for definitive
diagnosis (e.g. platelet function studies, assays of
coagulation factors, and test for fibrin degradation
products) (Table 29.7). Abnormalities of blood vessels
are usually not detectable by laboratory tests for
hemostasis, and their diagnosis requires correlation of
clinical and other investigations.
Screening Tests for Hemostasis
Complete Blood Count including Blood Smear
A complete blood count and a blood smear can provide
information in the form of:
• Presence of cytopenia (anemia, leukopenia,
thrombocytopenia)

Table 29.6: Differences between coagulation and platelet/vascular disorders

Parameter Coagulation disorder Platelet/vascular disorder
1. Sex More common in males More common in females
2. Family history Often positive Often negative
3. Petechie, bleeding gums, Rare Common
nose bleeds, melena
4. Skin hemorrhages Large and few Small and many
5. Deep hematomas (muscle Common Not seen
bleeding), hemarthrosis
(joint bleeding)
6. Delayed bleeding Characteristic (12-24 hours Not seen
(recurrence of bleeding hours after injury)
or days following injury)

Table 29.7: Laboratory evaluation of bleeding disorders

Screening tests Specific tests

• Complete blood count and blood smear • Platelet aggregation studies

• Platelet count • Coagulation factor assays
• Bleeding time • Estimation of fibrinogen
• Platelet function analyzer-100 • Test for fibrinogen/fibrin degradation products
• Clotting time • Test for D-dimer
• Prothrombin time • Platelet glycoprotein analysis
• Activated partial thromboplastin time
• Thrombin time
 Laboratory Tests in Bleeding Disorders 299

• Red cell abnormalities (especially fragmented red 2. Charge the improved Neubauer counting chamber as
cells which may indicate disseminated intra- described for total leukocyte count.
vascular coagulation) 3. Place the mounted counting chamber inside a moist
• White cell abnormalities (like abnormal cells in chamber (which consists of a covered Petri dish with
leukemias) a dampened filter paper at the bottom) and leave it
• Abnormalities of platelets: thrombocytopenia undisturbed for 20 minutes. This allows platelets to
(normally there is 1 platelets per 500-1000 red settle and prevents drying of fluid.
cells), giant platelets (seen in myeloproliferative
4. Place the charged counting chamber on the stage of
disorders and Bernard-Soulier syndrome), and
the microscope. With the illumination reduced to give
isolated discrete platelets without clumping in
sufficient contrast, bring the central large square
finger-prick smear (seen in uremia, Glanzmann’s
under the focus of the low power (×10) objective.
thrombasthenia).
Changing to ×40 objectives, count the total number
of platelets in five smaller squares (Fig. 29.9). Platelets
Platelet Count
appear as bluish, round or oval, small, brightly
Platelets can be counted manually under a microscope refractile fragments with one or more fine dendritic
or by means of an automated hematology analyzer. processes.
Platelets are difficult to count by manual method since
5. Calculation:
they are small in size (2-4 μm) and difficult to distinguish
from dirt particles and cell debris. Platelet count/μl of blood =
Number of platelets counted × Correction for dilution × Correction
Manual Method for volume
Platelet count/L of blood = Number of platelets counted × 109
Principle: Whole blood sample is mixed with a diluent
(1% ammonium oxalate) in which red cells are lysed. An Note:
improved Neubauer counting chamber is filled with the 1. For counting platelets, phase-contrast microscope is
mixture, and platelets are counted under the microscope. preferable to ordinary light microscope. This is
Result is expressed as number platelets per μl or per liter. because platelets are easily distinguished from dirt
Equipment: Similar to that required for total leukocyte
count (see Chapter 20: Total Leukocyte Count).
Reagent: This is 1% ammonium oxalate. It is prepared by
dissolving 1.0 gm of ammonium oxalate in 100 ml of
distilled water. It should be stored in a refrigerator. Since
small particles can be mistaken for platelets, reagent
should be filtered immediately before use.
Specimen: Venous blood anticoagulated with EDTA is
preferable. Capillary blood should be avoided since
platelets adhere to the skin puncture site resulting in a
erroneously lower platelet count. Platelet count from skin
puncture blood is also not reproducible.

Method
1. Take 0.38 ml of 1% ammonium oxalate in a test tube.
To this add 20 µl of well-mixed anticoagulated
venous blood and mix thoroughly. Dilution of blood Fig. 29.9: Area (P) for counting platelets in
is 1:20. Neubauer chamber
300 Essentials of Clinical Pathology
particles or debris using phase-contrast. A flat-bottom,
thin, phase-contrast hemocytometer with Neubauer
ruling is preferable.
2. Platelet count done on blood obtained by skin
puncture is significantly lower due to adhesion of
platelets to the puncture site. Therefore, anticoagu-
lated venous blood should be used (If capillary blood
is used, a free flowing blood drop should be obtained
after wiping away the first drop of blood).
3. Blood and the anticoagulant should be mixed
thoroughly by inverting the tube for 20 times. Venous
blood sample anticoagulated with EDTA should not
contain any clots.
4. All the glassware must be scrupulously clean and the
diluent fluid must be filtered just before use. This is
to prevent erroneous counting of dirt particles as
platelets.
5. A well-spread blood smear should always be
examined simultaneously to check the direct platelet
count on hemocytometer. Normally there is one
platelet per 500-1000 red cells on a blood smear. A
rough estimate about number of platelets (adequate,
low, or increased) thus can be obtained.
Fig. 29.10: Evaluation of thrombocytopenia
Clinical significance: Platelet count is usually obtained if
there is a suspicion of a bleeding disorder. Thrombo-
cytopenia between 1,50,000-50,000/μl is generally not
associated with bleeding. Platelet count between 50,000-
20,000/μl causes excess bleeding following surgery or
mild degree of spontaneous bleeding. Platelet count
below 20,000/μl is usually associated with spontaneous,
severe hemorrhage. Bleeding is often serious if platelets
are below 5,000/μl. Thrombocytosis (platelet count
> 4,00,000/μl) in chronic myeloproliferative disorders is
sometimes associated with thrombosis and bleeding
manifestations.
Causes of thrombocytopenia: Thrombocytopenia is defined
as platelet count below 1,50,000/μl. Causes of thrombo-
cytopenia are listed earlier in Table 29.2. Evaluation of
thrombocytopenia is shown in Figure 29.10.
Causes of thrombocytosis: Thrombocytosis is defined as
platelet count greater than 4,00,000/μl. Its causes are (i)
primary: chronic myeloproliferative disorders like
chronic myeloid leukemia, essential thrombocythemia,
idiopathic myelofibrosis, and polycythemia vera; (ii)
secondary (reactive): disseminated malignancy,
hemorrhage, splenectomy, chronic inflammation, and
iron deficiency anemia with bleeding.

Automated Method
Automated hematology analyzers more precisely count
platelets. However, these are expensive and have high
running costs. Platelet counting is usually based on the
principle of aperture impedance. A smaller aperture tube
is required for platelets and a threshold level needs to be
set to exclude larger red cells and smaller debris. False
elevation of platelet count by this method can result from
the presence of fragments of red or white cells,
microspherocytes, and elevated cryoglobulins. Causes of
pseudothrombocytopenia (falsely low platelet count) are:
clumping of platelets in EDTA-anticoagulated venous
blood sample (due to the presence of EDTA-dependent
platelet antibody in some patients which is active only
in vitro), platelet satellitism (adherence of platelets to
neutrophils in EDTA-anticoagulated sample), platelet
clumping due to the presence of platelet cold agglutinins
in blood, and presence of giant platelets (which are not
counted as platelets by electronic cell analyzers).
Some analyzers can measure platelet distribution width
(PDW), which is a measure of degree of variation of
platelet size present in a blood sample. High PDW is seen
in myeloproliferative disorders. In secondary thrombo-
cytosis, PDW is normal.
Mean platelet volume (MPV) is increased when
thrombocytopenia is due to peripheral platelet
destruction and is normal or low when thrombo-
cytopenia is due to defective platelet production.
Young platelets with residual RNA are called as
reticulated platelets. These can be identified by some
analyzers and flow cytometers. Increased numbers are
seen in idiopathic thrombocytopenic purpura and lower
numbers in aplastic anemia.
Bleeding Time
The bleeding time test assesses primary hemostasis
(vascular and platelet components) and is dependent on
adequate functioning of platelets and blood vessels.
In this test, a superficial skin puncture or incision is
made and the time required for bleeding to stop is
measured. Three methods are commonly used: Duke’s,
Ivy’s, and template. Duke’s method, which measures
bleeding time following ear lobe puncture, is not
advocated since it cannot be standardized and can cause
a large local hematoma. In Ivy’s method, 3 punctures
are made on the volar surface of the forearm with a lancet
(cutting depth 2-2.5 mm) under standardized venous
Laboratory Tests in Bleeding Disorders 301
pressure (40 mm Hg). A disadvantage with this method is
closure of puncture wound before cessation of bleeding.
Template method uses a special surgical blade, which
makes a larger cut (5 mm long and 1 mm deep). Although
template method is better, it can produce a large scar and
even a keloid in predisposed individuals. Ivy’s method is
described below.
Ivy’s method
Principle: Three standard punctures are made with a
lancet on the volar surface of the forearm under standard
pressure, and the average time required for bleeding to
cease from the puncture sites is measured.
Equipment
1. Sphygmomanometer
2. Sterile disposable lancets (2-2.5 mm blade with
shoulder, which limits the depth of penetration).
3. Stopwatch
4. Filter paper
Method
1. A sphygmomanometer cuff is wrapped around the
upper arm and inflated to 40 mm of Hg.
2. The dorsal surface of the forearm is cleansed with
70% ethanol and allowed to dry.
3. Three punctures are made (about 5 cm apart) in quick
succession with a lancet (Superficial veins, and scars
or bruises should be avoided).
4. A stopwatch is started as soon as a puncture is made.
One stopwatch is needed for each puncture.
5. Blood oozing from the puncture wound is gently
blotted with a filter paper at 15 seconds intervals.
Avoid directly touching the edges of the wound.
6. The timer is stopped when blood no longer stains the
filter paper.
7. Time required for bleeding to cease from all the three
puncture wounds is noted. The average time is
reported as the bleeding time.
8. Sterile adhesive strip is applied over the puncture.
Reference range: 2-7 minutes. Majority of individuals have
bleeding time less than 4 minutes. It should be reported
in minutes or nearest half minute. If bleeding continues
beyond 20 minutes, BT should be reported as >20 minutes
and the test is discontinued.
Causes of prolongation of bleeding time
1. Thrombocytopenia: If platelet count is less than
1,00,000/ml, bleeding time should not be performed,
as it will be prolonged. With a very low platelet count,
bleeding may be difficult to control.
302 Essentials of Clinical Pathology
Fig. 29.11: Evaluation of prolonged bleeding time
2. Disorders of platelet function
3. von Willebrand disease
4. Disorders of blood vessels
Evaluation of prolonged bleeding time is shown in
Figure 29.11.
Platelet Function Analyzer (PFA-100)
This is a newly introduced screening test for platelet
function that assesses both platelet adhesion and
aggregation. This method uses an instrument called as
PFA-100 in which anticoagulated whole blood is passed
at a high shear rate through small membranes that have
been coated with either collagen and epinephrine or
collagen and ADP. Platelets adhere to each membrane
and gradually occlude an aperture at the centre of the
membrane. The time required for complete occlusion of
the aperture is called as closure time. Normal closure
time is 1-3 minutes. The PFA-100 test is performed
initially with the collagen/epinephrine membrane; if
closure time is normal, there is no significant platelet
function defect. If closure time with collagen/epine-
phrine is prolonged, test with collagen/ADP is carried
out; if normal, aspirin-induced platelet dysfunction is the
probable cause; if prolonged, other platelet function
defect (acquired or inherited) is likely.
This test is more sensitive than bleeding time to assess
primary hemostasis, sensitive for detection of von
Willebrand disease and easy to perform. However, in
the presence of thrombocytopenia and anemia, closure
time is prolonged. Also, in the presence of a strong clinical
suspicion of a platelet function defect and normal PFA-
100 result, further testing is still necessary.
Clotting Time
This is a crude test and is now replaced by activated
partial thromboplastin time. Clotting time measures the
time required for the blood to clot in a glass test tube
kept at 37°C. Prolongation of clotting time only occurs
in severe deficiency of a clotting factor and is normal in
mild or moderate deficiency.
Prothrombin Time (PT)
PT assesses coagulation factors in extrinsic pathway (F
VII) and common pathway (F X, F V, prothrombin, and
fibrinogen) (Fig. 29.12).
Principle: Tissue thromboplastin and calcium are added
to plasma and clotting time is determined. The test
determines the overall efficiency of extrinsic and
common pathways.
Equipment
1. Water bath at 37°C
2. Test tubes (75 × 12 mm)
3. Stopwatch
Reagents
1. Thromboplastin reagent: This contains tissue factor
and phospholipids and is available commercially.
2. Calcium chloride 0.025 mol/liter.
Specimen: Platelet-poor citrated plasma (Box 29.2).
tahir99 - UnitedVRG
vip.persianss.ir

Fig. 29.12: Principle of prothrombin time
Box 29.2: Collection of blood for coagulation studies
Venous blood is collected from antecubital fossa with a
plastic, siliconized glass, or polypropylene syringe and a
large bore needle (20 or 21 G in adults, 22 or 23 G in infants).
Blood should never be collected from indwelling intravenous
lines, as these often contain heparin. Glass syringe should
not be used for collection since it activates coagulation. The
blood is drawn gently but quickly after a single, smooth
venepuncture. The needle is detached from the syringe, and
the sample is passed gently into the plastic container. After
capping the container, the blood and the anticoagulant are
mixed immediately by gentle inversion 5 times. The
anticoagulant used for coagulation studies is trisodium citrate
(3.2%), with anticoagulant to blood proportion being 1:9.
Most coagulation studies require platelet poor plasma (PPP).
To obtain PPP, blood sample is centrifuged at 3000-4000
revolutions per minute for 15-30 minutes. Coagulation studies
are carried out within 2 hours of collection of sample.
Method
1. Deliver 0.1 ml of plasma in a glass test tube kept in
water bath at 37°C.
2. Add 0.1 ml of thromboplastin reagent and mix.
3. After 1 minute, add 0.1 ml of calcium chloride
solution. Immediately start the stopwatch and record
the time required for clot formation.
Normal range: 11-16 seconds.
Causes of prolongation of PT
1. Treatment with oral anticoagulants
2. Liver disease
3. Vitamin K deficiency
4. Disseminated intravascular coagulation
5. Inherited deficiency of factors in extrinsic and
common pathways.
Laboratory Tests in Bleeding Disorders 303
Uses of PT
1. To monitor patients who are on oral anticoagulant therapy:
PT is the standard test for monitoring treatment with
oral anticoagulants. Oral anticoagulants inhibit
carboxylation of vitamin K-dependent factors
(Factors II, VII, IX, and X) and make these factors
inactive.
In patients receiving oral anticoagulants, PT
should be reported as a ratio of PT of patient to PT of
control; it should not be reported as percentage.
Various types of thromboplastin reagents obtained
from different sources (like ox brain, rabbit brain, or
rabbit lung) are available for PT test. These differ in
their responsiveness to deficiency of vit. K-dependent
factors. Technique of PT is also different in different
laboratories. For standardization and to obtain
comparable results, it is recommended to report PT
(in persons on oral anticoagulants) in the form of an
International Normalized Ratio (INR).
ISI
PT of patient
INR =
PT of control
International Sensitivity Index (ISI) of a particular
tissue thromboplastin is derived (by its manufacturer)
by comparing it with a reference thromboplastin of
known ISI.
INR should be maintained in the therapeutic range
for the particular indication (INR of 2.0-3.0 for
prophylaxis and treatment of deep venous throm-
bosis; INR of 2.5-3.5 for mechanical heart valves).
Therapeutic range provides adequate anticoagulation
for prevention of thrombosis and also checks excess
dosage, which will cause bleeding.
2. To assess liver function: Liver is the site of synthesis of
various coagulation factors, including vitamin K-
dependent proteins. Therefore PT is a sensitive test
for assessment of liver function.
3. Detection of vitamin K deficiency: PT measures three of
the four vitamin K-dependent factors (i.e. II, VII, and
X).
4. To screen for hereditary deficiency of coagulation factors
VII, X, V, prothrombin, and fibrinogen.
Activated Partial Thromboplastin Time (APTT)
APTT is a measure of coagulation factors in intrinsic
pathway (F XII, F XI, high molecular weight kininogen,
prekallikrein, F IX, and F VIII) and common pathway (F
X, F V, prothrombin, and fibrinogen) (Fig. 29.13).
tahir99 - UnitedVRG
vip.persianss.ir
304 Essentials of Clinical Pathology
Fig. 29.13: Principle of activated partial thromboplastin time
Principle: Plasma is incubated with an activator (which
initiates intrinsic pathway of coagulation by contact
activation). Phospholipid (also called as partial thrombo-
plastin) and calcium are then added and clotting time is
measured.
Equipment: This is same as for PT.
Reagents
1. Kaolin 5 gm/liter: This is a contact activator.
2. Phospholipid: Various APTT reagents are available
commercially, which contain phospholipids.
3. Calcium chloride 0.025 mol/liter.
Specimen: Citrated platelet poor plasma (Box 29.2).
Method
1. Mix equal volumes of phospholipid reagent and
calcium chloride solution in a glass test tube and keep
in a waterbath at 37°C.
2. Deliver 0.1 ml of plasma in another test tube and add
0.1 ml of kaolin solution. Incubate at 37°C in the
waterbath for 10 minutes.
3. After exactly 10 minutes, add 0.2 ml of phospholipid-
calcium chloride mixture, start the stopwatch, and
note the clotting time.
Normal range: 30-40 seconds.
Causes of prolongation of APTT
1. Hemophilia A or B.
2. Deficiencies of other coagulation factors in intrinsic
and common pathways.
3. Presence of coagulation inhibitors
4. Heparin therapy
5. Disseminated intravascular coagulation
6. Liver disease
Uses of APTT
1. Screening for hereditary disorders of coagulation: Since
deficiencies of F VIII (hemophilia A) and F IX
(hemophilia B) are relatively common, APTT is the
most important screening test for inherited coagu-
lation disorders. APTT detects deficiencies of all
coagulation factors except F VII and F XIII.
PT is also performed along with APTT. Prolongation
of both PT and APTT is indicative of deficiency of
coagulation factors in common pathway. Normal PT
with prolongation of APTT is indicative of intrinsic
pathway deficiency (particularly of F VIII or IX).
2. To monitor heparin therapy: Heparin potentiates the
action of natural anticoagulant antithrombin III which
tahir99 - UnitedVRG
vip.persianss.ir
 Laboratory Tests in Bleeding Disorders 305

is an inhibitor of thrombin and activated factors IX, X, Thrombin Time (TT)

and XI. Full dose heparin therapy needs monitoring
Thrombin time assesses the final step of coagulation i.e.
by APTT to maintain the dose in the therapeutic range
conversion of fibrinogen to fibrin by thrombin
(1.5 to 2.5 times the upper reference limit of APTT).
(Fig. 29.15).
3. Screening for circulating inhibitors of coagulation: APTT
is prolonged in the presence of specific inhibitors Principle: Thrombin is added to patient’s plasma and time
(which are directed against specific coagulation required for clot formation is noted.
factors) and non-specific inhibitors (which interfere Equipment: Same as for PT.
with certain coagulation reactions).
Reagent: Thrombin solution.
Mixing experiment for detection of inhibitors: Mixing studies
Specimen: Citrated platelet poor plasma (Box 29.2).
are used to distinguish between factor deficiencies and
factor inhibitors (specific coagulation factor inhibitor or Method: Take 0.1 ml of buffered saline in a test tube and
non-specific inhibitor such as lupus anticoagulant). If add 0.1 ml of plasma. Note clotting time after addition
APTT is prolonged, patient’s plasma is mixed with an of 0.1 ml of thrombin solution.
equal volume of normal plasma (called as a 50:50 mix) Normal range: ± 3 seconds of control.
and APTT is repeated. In coagulation factor deficiency,
prolongation of APTT gets corrected by more than 50%
of the difference between the clotting times of control
and test plasma. In the presence of lupus anticoagulant,
there is no such correction. With lupus anticoagulant,
APTT remains prolonged after mixing and for 2 hours
following incubation. With F VIII inhibitor (which is time-
and temperature-dependent), prolonged APTT gets
immediately corrected after mixing, but becomes
prolonged after incubation (Fig. 29.14).
Fig. 29.15: Principle of thrombin time

Fig. 29.14: Interpretation of mixing experiment. Lupus inhibitor is an immediate-acting inhibitor,

while F VIII inhibitor is a time-dependent inhibitor

tahir99 - UnitedVRG
vip.persianss.ir
306 Essentials of Clinical Pathology
Causes of Prolongation of TT
1. Disorders of fibrinogen: Prolongation of TT occurs in
afibrinogenemia (virtual absence of fibrinogen),
hypofibrinogenemia (fibrinogen less than 100 mgs/dl),
and dysfibrinogenemia (dysfunctional fibrinogen).
2. Heparin therapy: Heparin inhibits action of thrombin.
3. Presence of fibrin degradation products (FDPs): These
interfere with fibrin monomer polymerization. TT is
repeated using a mixture of normal plasma and
patient’s plasma. If TT remains prolonged, then FDPs
are present (provided patient is not receiving
heparin).
Interpretation of Screening Tests
In a patient with a bleeding disorder, results of all the
screening tests should be interpreted together (Fig. 29.16).
1. Selective thrombocytopenia: If low platelet count is
the only abnormality on hemostasis screen, blood
smear and bone marrow examinations are required
mainly to exclude underlying hematologic disease
like aplastic anemia, leukemia, lymphoma, or
myelodysplastic syndrome. In the absence of a
hematologic disorder, normal or increased numbers
of megakaryocytes in bone marrow is indicative of
peripheral destruction of platelets (e.g. idiopathic
thrombocytopenic purpura, drugs, infections,
collagen disorders).
Fig. 29.16: Evaluation of a suspected bleeding disorder. BT: bleeding time; PC: platelet count;
PT: prothrombin time; APTT: activated partial thromboplastin time
2. Selective prolongation of bleeding time: This occurs
in disorders of platelet function, von Willebrand
disease, and vascular disorders. For definitive
diagnosis of platelet function defects, platelet
aggregation studies are required. In vascular
disorders, associated clinical and laboratory features
are often suggestive of diagnosis.
3. Selective prolongation of APTT: Isolated prolon-
gation of APTT suggests deficiency of coagulation
factors in intrinsic pathway, especially that of F VIII
(hemophilia A) or F IX (hemophilia B). Definitive
diagnosis is based on specific coagulation factor assay.
Deficiency of F XII, high molecular weight kininogen,
or prekallikrein is not associated with clinical
bleeding.
Acquired causes of prolongation of APTT are heparin
therapy and circulating inhibitors of coagulation.
4. Prolongation of APTT and bleeding time: This
combination is observed in von Willebrand disease.
Platelet aggregation studies are required for definitive
diagnosis.
5. Selective prolongation of PT: Isolated prolongation
of PT is suggestive of F VII deficiency. Acquired
causes, which cause prolongation of only PT, are early
vitamin K deficiency and start of oral anticoagulant
therapy.
6. Prolongation of both PT and APTT: This indicates
deficiency of one or more factors in common pathway
tahir99 - UnitedVRG
vip.persianss.ir

(i.e. F X, F V, prothrombin, or fibrinogen), or
dysfibrinogenemia. Other causes are heparin therapy,
liver disease, vit K deficiency (in which prolongation
of PT is more as compared to APTT), and oral
anticoagulant therapy.
7. All screening tests abnormal: Combination of low
platelet count and prolongation of both PT and APTT
is seen in acute disseminated intravascular coagu-
lation (DIC) and liver disease. DIC should be
suspected when a patient with some underlying
condition (like sepsis, major trauma, malignancy,
snake bite, obstetric problem, etc.) develops acute
bleeding manifestations. Other laboratory abnor-
malities in DIC are fragmented red cells on blood
smear, and raised levels of FDPs, and D-dimer. In
liver disease, in contrast to DIC, fragmented red cells
are absent, D-dimer test is negative, liver function
tests are abnormal, and F VIII level is normal.
8. All screening tests normal: If, in a patient suspected
of having a bleeding disorder, all the screening tests
are normal, possibilities include mild forms of von
Willebrand disease, some platelet function defects,
vascular disorder, mild coagulation factor deficiency,
or F XIII deficiency. Investigations in such cases
include blood smear, platelet function studies,
workup for von Willebrand disease, and clot
solubility test for F XIII deficiency.
Specific Tests for Hemostasis
1. Platelet aggregation studies: Platelet aggregation
tests are carried out in specialized hematology
Fig. 29.17: Normal platelet aggregation curves
Laboratory Tests in Bleeding Disorders 307
laboratories if platelet dysfunction is suspected. These
tests are usually indicated in patients presenting with
mucocutaneous type of bleeding and in whom
screening tests reveal normal platelet count, prolon-
ged bleeding time, normal prothrombin time, and
normal activated partial thromboplastin time.
Platelet aggregation studies are carried out on
platelet-rich plasma using aggregometer. When a
platelet aggregating agent is added to platelet-rich
plasma, platelets form aggregates and optical density
falls (or light transmission increases); this is recorded
by a chart recorder on a strip chart. Commonly used
platelet aggregating agents are ADP (adenosine 5-
diphosphate), epinephrine (adrenaline), collagen,
arachidonic acid, and ristocetin. ADP (low dose) and
epinephrine induce primary and secondary waves
of aggregation (biphasic curve). Primary wave is due
to the direct action of aggregating agent on platelets.
Secondary wave is due to thromboxane A2 synthesis
and secretion from platelets. Collagen, arachidonic
acid and ristocetin induce a single wave of aggre-
gation (monophasic curve) Normal aggregation curve
is shown in Figure 29.17. Aggregation patterns in
various platelet function defects are shown in Figures
29.18 to 29.20, and in Table 29.8.
2. Factor VIII assay: One stage F VIII assay based on
APTT is usually performed for diagnosis of F VIII
deficiency. Coagulation factor assays are based on the
ability of patient’s plasma to correct specific factor-
deficient plasma. The abilities of the dilutions of
standard plasma (normal plasma) and patient’s
plasma to correct the APTT of the plasma known to
tahir99 - UnitedVRG
vip.persianss.ir
308 Essentials of Clinical Pathology

Fig. 29.18: Platelet aggregation curves in von Willebrand disease and Bernard-Soulier syndrome (absent aggregation
with ristocetin, normal aggregation with ADP, epinephrine, and arachidonic acid)

Fig. 29.19: Platelet aggregation curves in storage pool defect (absent second wave of aggregation with ADP and
epinephrine, absent or greatly diminished aggregation with collagen, and normal ristocetin aggregation)

Fig. 29.20: Platelet aggregation curves in Glanzmann’s thrombasthenia

(absent aggregation with all agonists except ristocetin)

tahir99 - UnitedVRG
vip.persianss.ir
 Laboratory Tests in Bleeding Disorders 309

Table 29.8: Laboratory features of platelet function disorders

Disorder
Bernard Soulier syndrome
Glanzmann’s thrombasthenia
Storage pool defect
Aspirin-like defect
von Willebrand disease
Platelet aggregation pattern
Normal with ADP, epinephrine,
collagen, and arachidonic acid;
deficient with ristocetin
Deficient with ADP, epinephrine,
collagen, and arachidonic acid;
normal with ristocetin
Primary wave with ADP and
epinephrine, normal with
arachidonic acid, deficient with
collagen, normal with ristocetin
Primary wave with ADP and
epinephrine, deficient with
arachidonic acid, deficient with
collagen, normal with ristocetin
Normal with ADP, epinephrine,
collagen, and arachidonic acid;
deficient with ristocetin
Other features
Autosomal recessive; severe bleeding; giant
platelets
Autosomal recessive; severe bleeding;
small and discrete platelets; defective clot
retraction
Defects of platelet granules; platelet dense
granules are decreased with deficient
release of ADP, ATP, calcium, and serotonin
Autosomal dominant/recessive; abnormality in
aggregation corrected with cryoprecipitate

be completely deficient in F VIII are compared. The

results are plotted (clotting times in seconds vs.
percent factor activity) on a log/log graph paper. The
1:10 dilution is considered as 100% activity. F VIII
activity of 50-150% is considered as normal.
3. Detection of fibrinogen/fibrin degradation products
(FDPs): FDPs are fragments produced by proteolytic
digestion of fibrinogen or fibrin by plasmin.
For determination of FDPs, blood is collected in a tube
containing thrombin (to remove all fibrinogen by
converting it into a clot) and soybean trypsin inhibitor
(to inhibit plasmin and thus prevent in vitro
breakdown of fibrin).
A suspension of latex particles linked to anti-
fibrinogen antibodies (or fragments D and E) is mixed
with dilutions of patient’s serum on a glass slide. If
FDPs are present, agglutination of latex particles Fig. 29.21: Principle of latex agglutination test for
occurs (Fig. 29.21). The highest dilution of serum at fibrinogen/fibrin degradation products
which agglutination is detected is used to determine
concentration of FDPs. microparticles coated with monoclonal antibody to D-
Increased levels of FDPs occur in fibrinogenolysis or dimer are mixed with patient’s sample and observed
fibrinolysis. This occurs in disseminated intravascular for microparticle agglutination. As the particle is small,
coagulation, deep venous thrombosis, severe turbidometric endpoint can be determined in auto-
pneumonia, and recent myocardial infarction. mated instruments.
4. Detection of D-dimers: D-dimer is derived from the D-dimer and FDPs are raised in disseminated
breakdown of fibrin by plasmin and D-dimer test is intravascular coagulation, intravascular thrombosis
used to evaluate fibrin degradation. Blood sample can (myocardial infarction, stroke, venous thrombosis,
be either serum or plasma. Latex or polystyrene pulmonary embolism), and during postoperative

tahir99 - UnitedVRG
vip.persianss.ir
310 Essentials of Clinical Pathology
period or following trauma. D-dimer test is commonly
used for exclusion of thrombosis and thrombotic
tendencies.
5. Estimation of fibrinogen: Fibrinogen is commonly
measured by Clauss method that consists of modifi-
cation of thrombin time by diluting plasma; thrombin
time of diluted plasma is inversely proportional to
concentration of fibrinogen. Fibrinogen can also be
estimated by immunological method; in dysfibrino-
genemia, fibrinogen estimated by functional assay
(Clauss method) is abnormal while immunological
assay is normal.
6. Platelet glycoprotein analysis: This is done by flow
cytometric analysis for detection of lack of GpIb/IX
in Bernanrd Soulier syndrome (deficiency of CD42),
and lack of GpIIb/IIIa in Glanzmann’s thromb-
asthenia (deficiency of CD41, CD61).
REFERENCE RANGES
• Bleeding time: Ivy method: 2-7 minutes; Template
method: 2.5-9.5 minutes
• Prothrombin time: 11-16 seconds
• Activate partial thromboplastin time: 30-40 seconds
• Thrombint time: ±3 seconds of control
• Plasma fibrinogen: 200-400 mg/dl
• Fibrinogen/fibrin degradation products: < 10 μg/ml
• D-dimer: Qualitiative: Negative; Quantitative: < 200
mg/L
• Factors II, V, VII, VIII, IX, X, XI, XII: 50-150%
• vWF:Ag: 50-150%
CRITICAL VALUES
• Prothrombin time: > 30 seconds or > 3 times control
value
• Activated partial thromboplastin time: ≥ 75 seconds
• Platelet count < 20,000/cmm or > 1 million/cmm
• D-dimer: Positive
• Plasma fibrinogen: < 100 mg/dl
BIBLIOGRAPHY
1. Evatt BL, Gibbs WN, Lewis SM, McArthur JR.
Fundamental Diagnostic Hematology: The Bleeding
and Clotting Disorders (2nd ed), 1992. US Dept. of
health and Human Services, Atlanta, Georgia and
World Health Organization, Geneva, Switzerland.
2. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
3. Lewis SM, Bain BJ, bates I (Eds). Dacie and Lewis
Practical Hematology (9th ed). London: Churchill
Livingstone, 2002.
4. Provan D, Krentz A. Oxford Handbook of Clinical and
Laboratory Investigations (2002). Oxford university
Press. Oxford.
5. Shrikhande AV, Warhadpande MS, Kawthalkar SM.
A laboratory manual of coagulation (1994). Dept. of
Pathology. Govt. Medical College, Nagpur.
6. Wallach J. Interpretation of Diagnostic Tests (7th ed).
Philadelphia: Lippincott Williams and Wilkins, 2000.
tahir99 - UnitedVRG
vip.persianss.ir
 30
Laboratory Tests in
Thrombophilia
Thrombophilia is a hemostatic disorder in which there
is a predisposition to thrombosis due to an inherited or
acquired condition (Table 30.1). It results from alteration
in hemostatic constituents of blood.
INHERITED THROMBOPHILIA
1. Factor V Leiden (Activated protein C resistance):
Factor V Leiden (so-called because of its discovery in
Leiden, Netherlands) is the most common inherited
cause of thrombophilia. Mutation in FV gene
(transmitted through autosomal dominant inheri-
tance) leads to replacement of arginine by glutamine
at position 506. This substitution renders activated
form of FV resistant to degradation by activated
protein C. More than 95% cases of activated protein
C resistance are due to FV Leiden (other causes of
activated protein C resistance being pregnancy, oral
contraceptive use, malignancy, and other FV
mutations). The terms FV Leiden and activated
protein C resistance are not synonymous. Failure of
Table 30.1: Conditions associated with increased risk of thrombosis
Inherited (Primary)
1. Factor V Leiden (Activated protein C resistance), i.e.
Abnormal F V that resists degradation by protein C
2. Prothrombin gene mutation
3. Hyperhomocysteinemia
4. Deficiency or decreased activity of protein C
5. Deficiency or decreased activity of protein S
6. Deficiency or decreased activity of antithrombin III
7. Dysfibrinogenemia
removal of F Va leads to increased prothrombinase
complex (F Xa-Va-PL-Ca) activity, and greater
thrombin generation. It is primarily associated with
increased risk of venous thrombosis. Heterozygotes
have 2-6% increased risk, while homozygotes have
50-100 times risk. Risk of thrombosis is greatly
increased during pregnancy and following oral
contraceptive use.
Laboratory tests for FV Leiden include activated
protein C resistance assay and genetic analysis. In
activated protein C resistance assay, activated partial
thromboplastin time (APTT) on the patient’s sample
is compared with APTT done after addition of
activated protein C to plasma sample. The ratio is
called as activated protein C sensitivity ratio. In
normal subjects, addition of activated protein C to
the plasma sample will prolong APTT because
activated protein C inhibits F V and F VIII. In F V
Leiden, APTT shows only slight prolongation. Low
activated protein C ratio indicates more resistance to
Acquired (Secondary)
1. Oral contraceptive therapy
2. Pregnancy
3. Antiphospholipid syndrome
4. Paroxysmal nocturnal hemoglobinuria
5. Malignancy
6. Disseminated intravascular coagulation
7. Heparin-induced thrombocytopenia
8. Thrombotic thrombocytopenic purpura
9. Nephrotic syndrome
10. Myeloproliferative disorders
tahir99 - UnitedVRG
vip.persianss.ir
312 Essentials of Clinical Pathology
activated protein C. If the screening test is positive for
activated protein C resistance, genetic testing is
performed. Genetic analysis by polymerase chain
reaction can detect heterozygous and homozygous
states.
2. Prothrombin gene G20210A mutation: A G→A
mutation at position 20210 of prothrombin gene is
the second most common cause of inherited thrombo-
philia. This mutation, by some unknown mechanism,
leads to a rise in the concentration of prothrombin,
thus making available large amounts of prothrombin
for conversion to thrombin. It is implicated in
causation of both arterial and venous thrombosis and
pregnancy-associated thrombosis. Diagnosis is based
on genetic analysis.
3. Deficiency of protein C and S: Protein C, when
activated by thrombin, inactivates factors V and VIII.
Protein S serves as a cofactor in this reaction. In
protein C and S deficiency, thrombin generation is
increased, producing hypercoagulability. Both
protein C and protein S deficiencies primarily cause
venous thrombosis. Diagnosis of protein C deficiency
requires quantification of protein C concentration.
Distinction between deficiency and dysfunction of
protein C is based on functional assay for protein C.
Protein S deficiency is detected by quantification of
protein S (both free and bound forms).
4. Deficiency of antithrombin III: Antithrombin III is
the natural inhibitor of thrombin, F Xa, IXa, XIa, and
XIIa. Deficiency state is associated with venous
thrombosis. Homozygous state is incompatible with
life.
5. Hyperhomocysteinemia: Increased levels of the
amino acid homocysteine can occur in various
inherited disorders like homocystinuria, cystathione
synthase deficiency, and C677T gene polymorphism
in the methyl tetrahydrofolate reductase (MTHFR) gene.
Acquired causes of hyperhomocysteinemia are vitamin
B12 deficiency, folate deficiency, and vitamin B6
deficiency. Hyperhomocysteinemia is associated with
increased risk of both arterial and venous thrombosis.
Laboratory tests are assay for homocyseine or genetic
analysis of MTHFR gene.
ACQUIRED THROMBOPHILIA
1. Oral contraceptive therapy and pregnancy: Increa-
sed predisposition to thrombosis results from
increased synthesis of coagulation factors in liver and
decreased synthesis of antithrombin III.
2. Antiphospholipid syndrome: Antiphospholipid
antibodies are autoantibodies directed against
antigens composed of phospholipids. They are of two
types: lupus anticoagulant (so named because it was
first detected in a patient with systemic lupus
erythematosus) and anticardiolipin antibodies. Lupus
anticoagulant is detected by prolongation of phos-
pholipid-dependent coagulation tests such as
activated partial thromboplastin time (APTT) and
dilute Russell’s viper venom time. If APTT is
prolonged, the test is repeated after mixing the sample
50:50 with normal plasma. If not corrected, it suggest
presence of lupus anticoagulant (Box 30.1). Anti-
cardiolipin antibodies are detected by enzyme linked
immunosorbent assay (ELISA). Antiphospholipid
antibodies are associated with arterial and venous
thrombosis, spontaneous, recurrent abortions, and
thrombocytopenia. Antiphospholipid syndrome is
present if antiphospholipid antibodies (lupus
anticoagulant or anticardiolipin antibodies or both)
are present along with an episode of arterial or venous
thrombosis, thrombocytopenia, or frequent second
trimester abortions. Antiphospholipid antibodies
may occur without any underlying disorder
(primary) or in association with systemic lupus
erythematosus, Sjogren’s syndrome, rheumatoid
arthritis, human immunodeficiency virus infection or
malignancy (secondary).
3. Heparin-induced thrombocytopenia: This compli-
cation occurs in 1-3% of patients receiving any type
of heparin. Platelet count should be checked every
alternate day in patients receiving heparin. The
condition should be suspected when patient is not
Box 30.1: Criteria for diagnosis of lupus anticoagulant
(International Society of Thrombosis and Hemostasis)
• Prolongation of a phospholipid-dependent screening test
for coagulation like activated partial thromboplastin time
(APTT) or dilute Russell’s viper venom time
• Mixing study with APTT using 50:50 mixture of patient’s
and normal plasma shows no correction, indicating that
prolongation is due to an inhibitor.
• Demonstration of antiphospholipid nature of antibodies
by neutralizing them with high concentration of platelets
(platelet neutralization test)
• No other cause for thrombosis
tahir99 - UnitedVRG
vip.persianss.ir

responding to heparin, and develops thrombo-
cytopenia (50% below baseline level) and thrombosis.
These patients are at risk of developing acute
myocardial infarction, stroke, peripheral arterial
thrombosis and deep venous thrombosis. The
pathogenesis consists of binding of IgG-heparin-
platelet factor 4 complexes to Fc receptors on platelets
that causes activation and aggregation of platelets as
well as clearance of coated platelets by macrophages.
This leads to thrombosis and thrombocytopenia.
LABORATORY TESTING IN THROMBOPHILIA
Laboratory studies will identify the underlying cause in
majority of patient presenting with thrombosis. The
screening tests for thrombophilia are not readily
available. Also, testing for thrombophilia is expensive,
rarely helps in acute patient management, and results
may not be interpreted correctly, leading to improper
treatment. Concentration solely on thrombophilia
detection can overlook a serious underlying disorder.
Testing for thrombophilia should be ordered in selected
patients in whom such complex and costly testing will
be of significant benefit for management.
Indications for Testing for Thrombophilia
1. Thrombosis at young age with no obvious risk factors.
2. Unexplained recurrent thrombosis.
3. Thrombosis at unusual locations.
4. Strong family history of thrombosis (in first degree
relative).
Laboratory Tests in Thrombophilia 313
5. Recurrent abortions (≥ 3)
6. Pregnancy-associated thrombosis.
Laboratory Tests
The tests should be performed atleast 4-6 weeks after
the acute thrombotic event and discontinuation of
warfarin, as acute states cause elevation of acute phase
reactants (that interfere with testing and cause false
positive results).
1. For inherited thrombophilia:
• DNA analysis for F V Leiden
• DNA analysis for prothrombin gene mutation
• Test for homocysteine level.
• Testing for protein C, protein S, and antithrombin
III deficiency or dysfunction
2. For acquired thrombophilia:
• Exclusion of diabetes mellitus, hyperlipidemia,
myeloproliferative disorders, paroxysmal noctur-
nal hemoglobinuria
• Test for lupus anticoagulant and anticardiolipin
antibodies.
BIBLIOGRAPHY
1. Brandt JT, Triplett DA, Alving B, et al. Criteria for the
diagnosis of lupus anticoagulants: an update. Thromb
Hemost 1995;74:1185-90.
2. Van Cott EM, Laposata M. Laboratory evaluation of
hypercoagulable states. Hematology/Oncology Clinics
of North America 1998;12:1141-66.
3. Whiteman T, Hassouna HI. Hypercoagulable states.
Hematology/Oncology Clinics of North America 2000;
14:355-77.
 31
Laboratory Tests in
Porphyrias

Porphyrias (from Greek porphura meaning purple
pigment; the name is probably derived from purple
discoloration of some body fluids during the attack) are
a heterogeneous group of rare disorders resulting from
disturbance in the heme biosynthetic pathway leading
to the abnormal accumulations of red and purple
pigments called as porphyrins in the body. Heme, a
component of hemoglobin, is synthesized through
various steps as shown in Figure 31.1. Each of the steps is
catalyzed by a separate enzyme; if any of these steps fails
(due to hereditary or acquired cause), precursors of heme
(porphyrin intermediates) accumulate in blood, get
deposited in skin and other organs, and excreted in urine
and feces. Depending on the site of defect, different types
of porphyrias are described with varying clinical features,
severity, and the nature of accumulated porphyrin.

Fig. 31.1: Figure on left shows steps in the biosynthesis of heme. Some steps (first and last three) occur in mitochondria,
while some occur in cytosol. Figure on right shows deficiency state associated with each enzyme. Deficiency of ALA synthase
is associated with sideroblastic anemia, and deficiencies of other enzymes cause porphyria
 Laboratory Tests in Porphyrias 315

Porphyria has been offered as a possible explanation
for the medieval tales of vampires and werewolves; this
is because of the number of similarities between the
behavior of persons suffering from porphyria and the
folklore (avoiding sunlight, mutilation of skin on
exposure to sunlight, red teeth, psychiatric disturbance,
and drinking of blood to obtain heme).
Porphyrias are often missed or wrongly diagnosed
as many of them are not associated with definite physical
findings, screening tests may yield false-negative results,
diagnostic criteria are poorly defined and mild disorders
produce an enzyme assay result within ‘normal’ range.
Heme is mainly required in bone marrow (for hemo-
globin synthesis) and in liver (for cytochromes).
Therefore, porphyrias are divided into erythropoietic and
hepatic types, depending on the site of expression of
disease. Hepatic porphyrias mainly affect the nervous
system, while erythropoietic porphyrias primarily affect
the skin. Porphyrias are also classified into acute and non-
acute (or cutaneous) types depending on clinical
presentation (Table 31.1).
Inheritance of porphyrias may be autosomal domi-
nant or recessive. Most acute porphyrias are inherited in
an autosomal dominant manner (i.e. inheritance of one
abnormal copy of gene). Therefore, the activity of the
deficient enzyme is 50%. When the level of heme falls in
the liver due to some cause, activity of ALA synthase is
stimulated leading to increase in the levels of heme
precursors up to the point of enzyme defect. Increased
levels of heme precursors cause symptoms of acute
porphyria. When the heme level returns back to normal,
symptoms subside.
Accumulation of porphyrin precursors can occur in
lead poisoning due to inhibition of enzyme aminolevulinic
acid dehydratase in heme biosynthetic pathway. This can
mimick acute intermittent porphyria.
CLINICAL FEATURES
Clinical features of porphyrias are variable and depend
on type. Acute porphyrias present with symptoms like
acute and severe abdominal pain/vomiting/consti-
pation, chest pain, emotional and mental disorders,
seizures, hypertension, tachycardia, sensory loss, and
muscle weakness. Cutaneous porphyrias present with
photosensitivity (redness and blistering of skin on
exposure to sunlight), itching, necrosis of skin and gums,
and increased hair growth over the temples (Table 31.2).
Symptoms can be triggered by drugs (barbiturates,
oral contraceptives, diazepam, phenytoin, carbama-
zepine, methyldopa, sulfonamides, chloramphenicol,
and antihistamines), emotional or physical stress,
infection, dieting, fasting, substance abuse, premenstrual
period, smoking, and alcohol.
Autosomal dominant porphyrias include acute
intermittent porphyria, variegate porphyria, porphyria

Table 31.1: Various classification schemes for porphyrias

Classification based on Classification based on site of Classification based on
predominant clinical expression of disease mode of clinical
manifestations presentation

Neuropsychiatric Hepatic Acute

1. Acute intermittent porphyria
2. ALA-dehydratase porphyria
(Plumboporphyria)
Cutaneous (Photosensitivity)
1. Congenital erythropoietic
porphyria
2. Porphyria cutanea tarda
3. Erythropoietic protoporphyria
Mixed (Neuropsychiatric and cutaneous)
1. Hereditary coproporphyria
2. Variegate porphyria
1. ALA-dehydratase porphyria
2. Acute intermittent porphyria
3. Hereditary coproporphyria
4. Variegate porphyria
Erythropoietic porphyria
1. Congenital erythropoietic
porphyria
2. Erythropoietic protoporphyria
Hepatic/Erythropoietic
1. Porphyria cutanea tarda
1. ALA-dehydratase porphyria
(Plumboporphyria)
2. Acute intermittent
porphyria
3. Hereditary coproporphyria
4. Variegate porphyria
Non-acute (cutaneous)
1. Porphyria cutanea tarda
2. Congenital erythropoietic
porphyria
3. Erythropoietic protoporphyria
316 Essentials of Clinical Pathology

Table 31.2: Clinical characteristics of porphyrias

Porphyria Deficient enzyme Clinical features Inheritance Initial test
1. Acute intermittent PBG deaminase Acute neurovisceral Autosomal dominant Urinary PBG; urine
porphyria attacks; triggering becomes brown,
(AIP)* factors+ (e.g. drugs, red, or black on
diet restriction) standing
2. Variegate Protoporphyrinogen Acute Autosomal Urinary PBG
porphyria oxidase neurovisceral dominant
attacks + skin
fragility, bullae
3. Hereditary Coproporphyrinogen Acute Autosomal Urinary PBG
coproporphyria oxidase neurovisceral dominant
attacks + skin
fragility, bullae
4. Congenital Uroporphyrinogen Onset in infancy; Autosomal Urinary/fecal
erythropoietic cosynthase skin fragility, recessive total porphyrins;
porphyria bullae; extreme ultraviolet
photosensitivity fluorescence of
with mutilation; urine, feces,
red teeth and urine and bones
(pink red urine-
staining of diapers)
5. Porphyria Uroporphyrinogen Skin fragility, Autosomal Urinary/fecal
cutanea tarda* decarboxylase bullae dominant total porphyrins
(some cases)
6. Erythropoietic Ferrochelatase Acute Autosomal Free erythrocyte
protoporphyria* photosensitivity dominant protoporphyrin

Disorders marked with * are the three most common porphyrias. PBG: Porphobilinogen

cutanea tarda, erythropoietic protoporphyria (most cases),
and hereditary coproporphyria. Autosomal recessive
porphyrias include: congenital erythropoietic porphyria,
erythropoietic protoporphyria (few cases), and ALA-
dehydratase porphyria (plumboporphyria).
LABORATORY DIAGNOSIS
Porphyria can be diagnosed through tests done on blood,
urine, and feces during symptomatic period. Timely and
accurate diagnosis is required for effective management
of porphyrias. Due to the variability and a broad range of
clinical features, porphyrias are included under
differential diagnosis of many conditions. All routine
hospital laboratories usually have facilities for initial
investigations in suspected cases of porphyrias; laboratory
tests for identification of specific type of porphyrias are
available in specialized laboratories.
Initial Studies
In suspected acute porphyrias (acute neurovisceral attack),
a fresh randomly collected urine sample (10-20 ml) should
be submitted for detection of excessive urinary excretion
of porphobilinogen (PBG) (Fig. 31.2). In AIP, urine becomes
red or brown on standing (Fig. 31.3). In suspected cases of
cutaneous porphyrias (acute photosensitivity without skin
fragility), free erythrocyte protporphyrin or FEP in EDTA
blood (for diagnosis of erythrocytic protoporphyria) and
for all other cutaneous porphyrias (skin fragility and
bullae), examination of fresh, random urine (10-20 ml)
and either feces (5-10 g) or plasma for excess porphyrins
are necessary (Fig. 31.4 and Table 31.2).
Apart from diagnosis, the detection of excretion of a
particular heme intermediate in urine or feces can help
in detecting site of defect in porphyria. Heme precursors
up to coproporphyrinogen III are water-soluble and thus
can be detected in urine. Protoporphyrinogen and
Protoporphyrin are insoluble in water and are excreted
in bile and can be detected in feces.
All samples should be protected from light. Samples
required are (i) 10-20 ml of fresh random urine sample
without any preservative; (ii) 5-10 g wet weight of fecal
sample, and (iii) blood anticoagulated with EDTA.
 Laboratory Tests in Porphyrias 317

Fig. 31.2: Evaluation of acute neurovisceral porphyria

Fig. 31.3: Red coloration of urine on

standing in acute intermittent porphyria

Fig. 31.4: Evaluation of cutaneous porphyrias

318 Essentials of Clinical Pathology

Table 31.3: Diagnostic patterns of concentrations of heme precursors in acute porphyrias

Porphyria Urine Feces

Acute intermittent porphyria PBG, Copro III –

Variegate porphyria PBG, Copro III Proto IX
Hereditary coproporphyria PBG, Copro III Copro III
PBG: Porphobilinogen; Copro III: Coproporphyrinogen III; Proto IX: Protoporphyrin IX

Table 31.4: Diagnostic patterns of concentrations of heme precursors in cutaneous porphyrias

Porphyria Urine Feces Erythrocytes

Congenital erythropoietic porphyria Uro I, Copro I Copro I –

Porphyria cutanea tarda Uroporphyrin Isocopro –
Erythropoietic protoporphyria – – Protoporphyrin
Uro I: Uroporphyrinogen I; Copro I: Coproporphyrinogen I; Isocopro: Isocoproporphyrinogen

Test for Porphobilinogen in Urine
Ehrlich’s aldehyde test is done for detection of PBG.
Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts
with PBG in urine to produce a red color. The red product
has an absorption spectrum with a peak at 553 nm and a
shoulder at 540 nm. Since both urobilinogen and
porphobilinogen produce similar reaction, further testing
is required to distinguish between the two. Urobilinogen
can be removed by solvent extraction. (See Watson-
Schwartz test in Chapter 1: Examination of Urine).
Levels of PBG may be normal or near normal in
between attacks. Therefore, samples should be tested
during an attack to avoid false-negative results.
Test for Total Porphyrins in Urine
Total porphyrins can be detected in acidified urine
sample by spectrophotometry (Porphyrins have an
intense absorbance peak around 400 nm). Semi-
quantitative estimation of porphyrins is possible.
Test for Total Porphyrins in Feces
Total porphyrins in feces can be determined in acidic
extract of fecal sample by spectrophotometry; it is
necessary to first remove dietary chlorophyll (that also
absorbs light around 400 nm) by diethyl ether extraction.
Tests for Porphyrins in Erythrocytes and Plasma
Visual examination for porphyrin fluorescence, and
solvent fractionation and spectrophotometry have now
been replaced by fluorometric methods.
Further Testing
If the initial testing for porphyria is positive, then
concentrations of porphyrins should be estimated in
urine, feces, and blood to arrive at specific diagnosis
(Tables 31.3 and 31.4)
In latent porphyrias and in patients during remission,
porphyrin levels may be normal; in such cases, enzymatic
and DNA testing is necessary for diagnosis.
If porphyria is diagnosed, then it is necessary to
investigate close family members for the disorder.
Positive family members should be counseled regarding
triggering factors.
BIBLIOGRAPHY
1. Deacon AC, Elder GH: Frontline tests for the investi-
gation of suspected porphyria. J Clin Pathol 2001; 54;
500-7.
2. Crook MA: Clinical chemistry and metabolic medicine.
Seventh edition. London. Edward Arnold, 2006.
3. Thadani H, Deacon A, Peters T: Diagnosis and
management of porphyria. BMJ 2000; 320; 1647-51.
 32
Automation in Hematology
AUTOMATED HEMATOLOGY ANALYZER
Automation is a process of replacement of tasks hitherto
performed by humans by computerized methods.
Until recently, hematological tests were performed
only by manual methods. These methods, though still
performed in many peripheral laboratories, are labor-
intensive, and involve use of hemocytometers (counting
chambers), centrifuges, Wintrobe tubes, photometers,
and stained blood smears. Hematology cell analyzers can
generate the blood test results rapidly and also perform
additional tests not possible by manual technology.
Both manual and automated laboratory techniques
have advantages and disadvantages, and it is unlikely
that one will completely replace the other.
Advantages of Automated Hematology Analyzer
• Speed with efficient handling of a large number of
samples
• Accuracy and precision in quantitative blood tests
• Ability to perform multiple tests on a single platform
• Significant reduction of labor requirements
• Invaluable for accurate determination of red cell
indices.
Disadvantages of Automated
Hematology Analyzer
• Flags: Flagging of a laboratory test result demands
labour-intensive manual examination of a blood
smear
• Comments on red cell morphology cannot be
generated. Abnormal red cell shapes (such as
fragmented cells) cannot be recognized.
• Erroneously increased or decreased results due to
interfering factors
• Expensive with high running costs.
Automated hematology analyzers are of two main types:
• Semi-automated: Some steps like dilution of blood
sample are performed by the technologist; can
measure only a few parameters
• Fully automated: Require only anticoagulated blood
sample; measure multiple parameters.
PRINCIPLES OF WORKING
Automated hematology analyzers work on different
principles:
• Electrical impedance
• Light scatter
• Fluorescence
• Light absorption
• Electrical conductivity.
Most analyzers are based on a combination of
different principles.
1. Electrical impedance: This is the classic and time-
tested technology for counting cellular elements of
blood. As this method of cell counting was first
developed by Coulter Electronics, it is also called as
Coulter principle (Fig. 32.1). Two electrodes placed
in isotonic solutions are separated by a glass tube
having a small aperture. A vacuum is applied and as
a cell passes through the aperture, flow of current is
impeded and a voltage pulse is generated.
The requisite condition for cell counting by this
method is high dilution of sample so that minimal
numbers of cells pass through the aperture at one
point of time. There are two electrodes on either side
of the aperture; as the solution in which the cells are
suspended is an electrolyte solution, an electric
current is generated between the two electrodes.
When a cell passes through this narrow aperture
across which a current is flowing, change in electrical
320 Essentials of Clinical Pathology
Fig. 32.1: Coulter principle of electrical impedance
resistance (i.e. momentary interruption of electrical
current between the two electrodes) occurs. A small
pulse is generated due to a temporary increase in
impedance. This pulse is amplified, measured, and
counted. The height of the pulse is proportional to
cell volume. The width of the pulse corresponds with
the time required for the cell to traverse the aperture.
Cells that do not pass through the center of the
aperture generate a distorted pulse that is not
representative of the cell volume. Some analyzers use
hydrodynamic focusing to force the cells through the
central path so that all cells take the same path for
volume measurement.
An anticoagulated whole blood sample is aspirated
into the system, divided into two portions, and mixed
with a diluent. One dilution is passed to the red cell
aperture bath (for red cell and platelet counting), and
the other is delivered to the WBC aperture bath
(where a reagent is added for lysis of red cells and
release hemoglobin; this portion is used for leukocyte
counting followed by estimation of hemoglobin).
Particles between 2-20 fl are counted as platelets,
Table 32.1: Parameters measured by hematology analyzers
Parameters measured by most analyzers
• RBC count
• Hemoglobin
• Mean cell volume
• Mean cell hemoglobin
• Mean cell hemoglobin concentration
• WBC count
• WBC differential
• Platelet count
while those between 36-360 fl are counted as red cells.
Hemoglobin is estimated by light transmission at 535
nm.
2. Light scatter: Each cell flows in a single line through
a flow cell. A laser device is focused on the flow cell;
as the laser light beam strikes a cell it is scattered in
various directions. One detector captures the forward
scatter light (forward angle light scatter or FALS) that
is proportional to cell size and a second detector
captures side scatter (SS) light (90°) that corresponds
to the nuclear complexity and granularity of
cytoplasm. This simultaneous measurement of light
scattered in two directions is used for distinguishing
between granulocytes, lymphocytes, and monocytes.
3. Fluorescence: Cellular fluorescence is used to
measure RNA (reticulocytes), DNA (nucleated red
cells), and cell surface antigens.
4. Light absorption: Concentration of hemoglobin is
measured by absorption spectrophotometry, after
conversion of hemoglobin to cyanmethemoglobin or
some other compound. In some analyzers, peroxidase
cytochemistry is used to classify leukocytes; the
peroxidase activity is determined by absorbance.
5. Electrical conductivity: Some analyzers use
conductivity of high frequency current to determine
physical and chemical composition of leucocytes for
their classification.
PARAMETERS MEASURED BY
HEMATOLOGY ANALYZERS
Parameters measured by hematology analyzers and their
derivation are shown in Tables 32.1 and 32.2. Most
automated hematology analyzers measure red cell count,
red cell indices (mean cell volume, mean cell hemoglobin,
mean cell hemoglobin concentration), hemoglobin,
hematocrit, total leukocyte count, differential leukocyte
count (three-part or five-part), and platelet count.
Parameters measured by some analyzers
• Red cell distribution width
• Reticulocyte count
• Reticulocyte hemoglobin content
• Mean platelet volume
• Platelet distribution width
• Reticulated platelets
 Automation in Hematology 321

Table 32.2: Parameters reported by hematology analyzers

Parameters measured directly or Parameters measured through

derived through histogram calculation

• RBC count • Hematocrit

• Mean cell volume (Derived from RBC histogram) • Mean cell hemoglobin
• Red cell distribution width (Derived from RBC histogram) • Mean cell hemoglobin concentration
• Hemoglobin
• Reticulocyte count
• WBC count
• Differential WBC count (Derived through WBC histogram)
• Platelet count
• Mean platelet volume (Derived from platelet histogram)

Estimation of Hemoglobin
Hemoglobin is measured directly by a modification of
cyanmethemoglobin method (all hemoglobins are
converted to cyanmethemoglobin by potassium
ferricyanide; cyanmethemoglobin has a broad absor-
bance peak at 540 nm). Some analyzers use a non-
hazardous reagent such as sodium lauryl sulphate. A
non-ionic detergent is added for rapid red cell lysis and
to minimize turbidity caused by cell membranes and
plasma lipids.
Fig. 32.2: Diagrammatic representation of red cell histogram
Estimation of Red Blood Cell Count and obtained by aperture impedance. The analyzer counts cells
Mean Cell Volume (MCV) between 36 fl and 360 fl as red cells. Although leukocytes
are present and counted along with red cells in the diluting
Red cell count and cell volume are directly measured by fluid, their number is not statistically significant. Only if
aperture impedance or light scatter analysis. In a red cell leukocyte count is markedly elevated (>50,000/μl), histogram
histogram, cell numbers are plotted on Y-axis, while cell and the red cell count will be affected. Area of the peak
volume is indicated on X-axis (Fig. 32.2). The analyzer between 60 fl and 125 fl is used for calculation of mean
counts those cells as red cells volume of which ranges cell volume and red cell distribution width. Abnormalities in
between 36 fl and 360 fl. red cell histogram include: (1) Left shift of the curve in
MCV is used for morphological classification of microcytosis, (2) Right shift of the curve in macrocytosis, and
anemia into microcytic, macrocytic, and normocytic (3) Bimodal peak of the curve in double (dimorphic)
types. population of red cells

Estimation of MCH, MCHC, and Hematocrit

Mean cell volume (fl)
These parameters are obtained indirectly through Hematocrit (%) = ———————————
calculations. Red cell count (106/μl)
Hemoglobin (g/l)
Mean cell hemoglobin (pg) = ——————————— Estimation of Red Cell Distribution Width (RDW)
Red cell count (106/μl) RDW is a quantitative measure of variation in sizes of
red cells and is expressed as coefficient of variation of
Hemoglobin (g/dl) red cell size distribution. It is equivalent to anisocytosis
Mean cell hemoglobin = ——————————— observed on blood smear. It is derived from red cell
concentration (g/dl) Hematocrit (%) histogram in some analyzers. RDW is usually elevated
322 Essentials of Clinical Pathology

in iron deficiency anemia, but not in β-thalassemia minor
and anemia of chronic disease (other causes of microcytic
anemia). However, this distinction is not absolute and
there is a significant overlap between values among
patients. Raised RDW requires examination of blood
smear.
Among the red cell values generated by the analyzer
(red cell count, hemoglobin, hematocrit, MCV, MCH,
MCHC, and RDW), most important for decision-
making are hemoglobin, hematocrit, and MCV.
WBC Differential
Hematology analyzers can either generate a 3-part
differential (differential count reported as lymphocytes,
monocytes, and granulocytes) or a 5-part differential
(lymphocytes, monocytes, neutrophils, eosinophils, and
basophils). The 3-part differential counting is based on
electrical impedance volume measurement of leukocytes.
In volume histogram for WBCs, approximate numbers
of cells are plotted on Y-axis and cell size on X-axis. Those
cells with volume 35-90 fl are designated as lymphocytes,
cells with volume 90-160 fl as mononuclear cells, and
cells with volume 160-450 fl as neutrophils (Fig. 32.3).
Any deviation from the expected histogram is flagged
by the analyzer, mandating review of blood smear. A
large proportion of 3-part differential counts are ‘flagged’
to avoid missing abnormal cells.
Instruments measuring a 5-part differential work on a
combination of different principles, e.g. light scatter,
impedance, and electrical conductivity, a combination
of light scatter, peroxidase staining, and resistance of
basophils to lysis in acid buffer, etc.
Platelet Count
Platelets are difficult to count because of their small size,
marked variation in size, tendency to aggregation, and
overlapping of size with microcytic red cells, cellular
fragments, and other debris. In hematology analyzers,
this difficulty is addressed by mathematical analysis of
platelet volume distribution so that it corresponds to log-
normal distribution. Platelets are counted by electrical
impedance method in the RBC aperture, and a histogram
is generated with platelet volume on X-axis and relative
cell frequency on Y-axis (Fig. 32.4). Normal platelet
histogram consists of a right-skewed single peak.
Particles greater than 2 fl and less than 20 fl are classified
as platelets by the analyzer.
Two other platelet parameters can be obtained from
platelet histogram using computer technology: mean
platelet volume (MPV) and platelet distribution width
(PDW). Some analyzers can generate another parameter
called as reticulated platelets.
MPV refers to the average size of platelets and is
obtained from mathematical calculation. Normal MPV

Fig. 32.3: Diagrammatic representation of WBC histogram. WBC histogram analysis shows relative numbers of cells on
Y-axis and cell size on X-axis. The lytic agent lyses the cytoplasm that collapses around the nucleus causing differential
shrinkage. The analyzer sorts the WBCs according to the nuclear size into 3 main groups (3-part differential): Cells with
35-90 fl volume are designated as lymphocytes, cells with 90-160 fl volume are designated as monocytes, and cells with
160-450 fl volume are designated as neutrophils. Abnormalities in WBC histogram include: (1) Peak to the left of lymphocyte
peak: Nucleated red cells, (2) Peak between lymphocytes and monocytes: Blast cells, eosinophilia, basophilia, plasma
cells, and atypical lymphocytes, and (3) Peak between monocytes and neutrophils: Left shift

Fig. 32.4: Diagrammatic representation of normal platelet
histogram: Counting and sizing of platelets by electrical
impedance method occurs in the RBC aperture. The counter
designates particles between sizes 2 fl and 20 fl as platelets.
Abnormalities in platelet histogram result from interferences
such as cytoplasmic fragments (peak at left end of histogram)
or severely microcytic red cells and giant platelets (peak at
right end of histogram)
is 7-10 fl. Increased MPV (> 10 fl) results from presence of
immature platelets in circulation; peripheral destruction
of platelets stimulates megakaryocytes to produce such
platelets (e.g. in idiopathic thrombocytopenic purpura).
Decreased MPV (< 7 fl) is due to presence of small platelets
in circulation (in conditions associated with reduced
production of platelets in bone marrow).
PDW is analogous to RDW and is a measure of variation
in size of platelets (normal <20%). Increased PDW is
observed in megaloblastic anemia, chronic myeloid
leukemia, and after chemotherapy.
Some analyzers measure reticulated platelets or
young platelets that contain RNA (similar to reticulo-
cytes). Increased numbers of reticulated platelets are seen
in thrombocytopenia due to peripheral destruction of
platelets.
Reticulocyte Count
Various fluorescent dyes can combine with RNA of
reticulocytes; the fluorescence then is counted in a flow
cytometer. More immature reticulocytes fluoresce more
strongly as they contain more RNA.
Reticulocyte hemoglobin content is a parameter that
estimates hemoglobinization of most recently produced
red cells. It is a predictor of iron deficiency.
WBC Cytogram (Scattergram)
In the scattergram, each dot represents a cell of a given
volume and density, and the positions of dots in the
Automation in Hematology 323
graph are determined by the degree of side scatter, degree
of forward scatter, light absorption by the cell, and
cytochemical staining (if used). The forward angle light
scatter (FALS) is represented on Y-axis, and the side
scatter (SS) is represented on X-axis. Low FALS and low
SS are indicative of lymphocytes; with subsequent
increasing FALS and SS, monocytes, neutrophils, and
lastly eosinophils are designated in the graph. Counting
of basophils is based on a different technology.
FLAGGING
‘Flags’ are signals that occur when an abnormal result is
detected by the analyzer. Flags are displayed to reduce
false-positive and false-negative results by mandating a
review of blood smear examination.
CAUSES OF ERRONEOUS RESULTS
(INTERFERENCES CAUSING ABNORMAL
RESULT)
These are listed in Table 32.3.
FLOW CYTOMETRY
Flow cytometry is a procedure used for measuring
multiple cellular and fluorescent properties of cells when
they flow as a single cell suspension through a laser beam
by a specialized instrument called as a flow cytometer.
Flow cytometry can analyze numerous cells in a short
time and multiple parameters of a single cell can be
analyzed simultaneously. From the measured para-
meters, specific cell populations are defined. Cells or
particles with size 0.2-150 μm are suitable for flow
cytometer analysis.
Flow cytometry can provide following information
about a cell (Box 32.1):
• Cell size (forward scatter)
• Internal complexity or granularity (side scatter)
• Relative fluorescence intensity.
A flow cytometer consists of three main components
or systems: fluidics, optics, and electronics.
1. Fluidics: The function of the fluidics system is to
transport cells in a stream to the laser beam for
interrogation. Cells (fluorescence-tagged) are
introduced into the cytometer (injected into the sheath
fluid within the flow chamber) and made to flow in a
single file past a laser (light amplification by
stimulated emission of radiation) beam. The stream
324 Essentials of Clinical Pathology

Table 32.3: Causes of erroneous results with hematology analyzer

Parameter Interfering factors

Erroneous increase Erroneous decrease

1. All parameters — • Clotted sample
1. WBC count • Nucleated red cells • Clotted sample
• Large platelet clumps
• Unlysed red cells (some abnormal
red cells resist lysing)
• Cryoglobulins
2. RBC count • Very high WBC* • Clotted sample
• Large numbers of giant platelets • Microcytic red cells
• Autoagglutination
3. Hemoglobin • Very high WBC • Clotted sample
• Hyperlipidemia
• High bilirubin
4. MCV • Very high WBC • Cryoglobulins
• Hyperglycemia
• Autoagglutination (cold agglutinins)
5. MCHC • Hyperlipidemia • Very high WBC
• Autoagglutination (cold agglutinins)
6. Platelets • Microcytic red cells • Platelet satellitism
• WBC fragments • Platelet clumping
• Cryoglobulins
*: WBCs are counted along with RBCs, but normally their number is statistically insignificant

are then collected by appropriately positioned lenses.

Box 32.1: Properties of a cell measured by a
flow cytometer
• Relative size
• Relative granularity or internal complexity
• Relative fluorescence
transporting the cells should be positioned in the
center of the laser beam. The portion of the fluid
stream where the cells are located is called as the
sample core. Only a single cell or particle should pass
3. Electronics: The optical signals (photons) are
through the laser beam at one time. Flow cytometers
use the principle of hydrodynamic focusing (process
of centering the sample core within the sheath fluid)
for presenting cells to the laser.
2. Optics: This system consists of lasers for illumination
of cells in the sample, and filters to direct the
generated light signals to the appropriate detectors.
The light source used in most flow cytometers is
laser. The laser most commonly used in flow
cytometry is Argon-ion laser. The light signals are
generated when the laser beam strikes the cell, which
A system of optical mirrors and filters then directs
the specified wavelengths of light to the designated
detectors. Two types of light signals are generated
when the laser beam strikes cells tagged with
fluorescent molecules: fluorescence and light scatter.
The cells tagged with fluorescence emit a momentary
pulse of fluorescence; in addition, two types of light
scatter are measured: forward scatter (proportional
to cell diameter) and side scatter (proportional to
granularity of cell).
converted to corresponding electronic signals
(electrons) by the photodetectors (photodiodes and
photomultiplier tubes). The electronic signal pro-
duced is proportional to the amount of light striking
a cell. The electric current travels to the amplifier and
is converted to a voltage pulse. The voltage pulse is
assigned a digital value representing a channel by
the Analog-to Digital Converter (ADC). The channel
number is then transferred to the computer which
displays it to the appropriate position on the data plot.

TERMINOLOGY IN FLOW CYTOMETRY
Fluorescence
A fluorochrome absorbs light energy and emits excess
energy in the form of photon light (fluorescence).
Fluorescence is the property of molecules to absorb light
at one wavelength and emit light at a longer wavelength.
The fluorescent dyes commonly used in flow cytometry
are fluorescein isothiocyanate (FITC) and phycoerythrin
(PE). The fluorochrome-labeled antibodies are used for
detection of antigenic markers on the surface of cells. A
particular cell type can be identified on the basis of the
antigenic profile expressed. Multiple fluorochromes can
be used to identify different cell types in a mixed
population of cells.
Light Scatter
Light is scattered when the incident light is deflected by a
particle traversing through a beam of light. This depends
on the physical properties of the cell. Two forms of light
scatter are used to identify different cell types: forward
scatter and side scatter. Forward scatter (light scattered in
the same direction as the laser beam) is related to cell size.
Side scatter (light scattered at a 90° angle to the laser beam)
is related to internal granularity of the cell. Main
subpopulations of leukocytes are identified on the
basis of correlated measurements of forward and side
scatters.
Fig. 32.5: Principle of working of a flow cytometer
Automation in Hematology 325
When a cell passes through laser beam, side scatter
and fluorescent signals that are emitted by the cell are
directed to photomultiplier tubes, while the forward scatter
signals are directed to a photodiode. Photomultiplier tubes
and photodiodes are called as detectors. Optical filters
are placed before the detectors that allow only a narrow
range of wavelengths to reach the detectors (Fig. 32.5).
Data Analysis
The data collected and stored in the computer can be
displayed in various formats. A parameter means forward
scatter, or side scatter, or emitted fluorescence from a
particle as it passes through a laser beam. A histogram is
a data plot of a single parameter, with the parameter’s
signal value in channel numbers or relative fluorescence
intensity on X-axis (horizontal axis) and number of events
on the Y-axis. A dot plot is a two parameter data graph in
which each dot represents one event that the flow
cytometer analyzed; one parameter is displayed on the X-
axis and the other on the Y-axis. A 3-D plot represents one
parameter on X-axis, another parameter on Y-axis, and
number of events per channel on Z-axis.
Gating
A gate is a boundary that can be set to restrict the analysis
to a specific population within the sample. For example, a
gate boundary can be drawn on a dot plot or histogram to
326 Essentials of Clinical Pathology
restrict the analysis only to cells with the size of
lymphocytes. Gates can be inclusive (selection of events
that fall within the boundary) or exclusive (selection of
events that fall outside the boundary). Data selected by
the gate is then displayed in subsequent plots.
Sorting
Usually, when a cell passes through the laser beam, it is
sent to waste. Sorting consists of collecting cells of interest
(defined through criteria of size and fluorescence) for
further analysis (such as microscopy or functional or
chemical analysis). Sorting feature is available only in
some flow cytometers.
COMMON APPLICATIONS OF FLOW
CYTOMETRY IN HEMATOLOGY
1. Leukemias and lympomas: Immunophenotyping
(evaluation of cell surface markers), diagnosis,
detection of minimal residual disease, and to identify
prognostically important subgroups.
2. Paroxysmal nocturnal hemoglobinuria: Deficiency of
CD 55 and CD 59.
3. Hematopoietic stem cell transplantation: Enumeration
of CD34+ stem cells.
4. Feto-maternal hemorrhage: Detection and quantitation
of foetal hemoglobin in maternal blood sample.
5. Anemias: Reticulocyte count.
6. Human immunodeficiency virus infection: For
enumeration of CD4+ lymphocytes.
7. Histocompatibility cross matching.
BIBLIOGRAPHY
1. Henry JB. Clinical Diagnosis and Management by
Laboratory Methods (20th Ed). Philadelphia: WB
Saunders Company, 2001.
2. Houwen B. The differential count. Laboratory Hema-
tology 2001;7:89-100.
3. Ward PJ. The CBC at the turn of the millennium: an
overview. Clin Chem 2000;46:1215-20.
Practical Blood Transfusion
 33

Blood Group Systems

International Society of Blood Transfusion (ISBT) Table 33.1: Blood group systems
Working Party has organized red cell antigens into 25 ABO Dombrock
blood group systems (Table 33.1). Red cell antigens that MNS Colton
are produced by alleles (alternative forms of a specified P Landseiner-Weiner
gene) at a single gene locus or very closely linked loci Rh Chido/Rogers
constitute a blood group system. Lutheran Hh
Kell Kx
Blood group genes are inherited in a Mendelian
Lewis Gerbich
manner and are mostly located on autosomes. Most of Duffy Cromer
the blood group genes are expressed in a co dominant Kidd Knops
manner (i.e. the two allelic forms are expressed equally Diego Indian
if inherited in a heterozygous state, and no gene or Yt Ok
allele is dominant over another). The particular alleles Xg Raph
Scianna
at a specified gene locus in an individual constitute the
genotype. Phenotype is the outward expression of the
genotype. ABO SYSTEM
In blood transfusion practice, most important blood In ABO system, there are four main types of blood groups:
group systems are ABO and Rh. This is because, A, B, A, B, AB, and O. Identification of these four blood groups
and Rh D antigens are the most immunogenic (i.e. is based on presence or absence of A and/or B antigens
capable of eliciting a strong antibody response on on red cells. According to Landsteiner’s law, anti-A and/
stimulation) and their alloantibodies can cause destruc- or anti-B antibodies are always present in plasma of
tion of transfused red cells or induce hemolytic disease individuals who lack corresponding antigen(s) on their
of newborn (HDN). ABO antigens are also important in red cells (Table 33.2). ABO is the only blood group system,
organ transplantation. in which if an antigen is absent in an individual,

Table 33.2: ABO blood groups

Blood group Caucasian Indian Genotype Antigen(s) Antibody in plasma
frequency frequency on red cells

A 40% 27% AA or AO A Anti-B

B 11% 31% BB or BO B Anti-A
AB 4% 8% AB AB Nil
O 45% 34% OO Nil Anti-A and anti-B

Note: A and B genes are dominant while O gene is recessive. Individuals with AA, BB, and OO are homozygous
respectively for A, B, and O, while AO, BO, and AB are heterozygous.
330 Essentials of Clinical Pathology
corresponding antibody is always present in plasma.
There is a geographical variation in frequencies of
different ABO blood groups. However, generally, O
blood group is the most common, while AB group is the
least common in a population.
Blood Group O
This is usually the most frequently occurring blood group
in the population. Group O red cells have large amounts
of H antigen on their surface. Genotype is OO and the
antibodies in plasma are anti-A, anti-B, and anti-A,B.
These antibodies are usually IgG in nature. They can
therefore cross the placenta and induce hemolytic disease
of newborn.
Blood Group A
Genotype of group A persons is either AA or AO, and
the antigens present on the red cells are A and H. Their
plasma contains anti-B antibodies that are IgM in nature.
Blood group A is divided into two subgroups: A1 (80%)
and A2 (20%). Anti-A1 produced by A2 or A2B
individuals is weak and clinically insignificant; however,
it can cause problems during blood grouping. Some
persons with A2 and with A2B can make anti-A1. A1
and A2 red cells can be differentiated by lectins of Dolichos
biflorus that cause agglutination of A1 red cells, but not
of A2 red cells.
Blood Group B
Genotype of group B persons is either BB or BO, and the
antigens present on red cells are B and H. Their plasma
contains anti-A antibodies that are IgM in nature.
Blood Group AB
This is the least common of the ABO blood groups.
Genotype is AB, and antigens present on red cells are A,
B, and very small amount of H. The AB blood group is
further subdivided into A1B and A2B. Plasma of group
AB individuals contain no anti-A or anti-B antibodies.
Antigens of the ABO system
Antigens of the ABO system are: A (A1, A2), B, and H.
They are glycolipids, glycoproteins, or glycosphingo-
lipids. In addition to red cells, they are also expressed on
white cells, platelets, and body tissues. They are also
present in a soluble form in various body secretions (in
Box 33.1: Distribution of ABH antigens
• Red blood cells
• White blood cells (weak)
• Platelets (weak)
• Body tissues
• Body fluids (in secretors)
secretors) (Box 33.1).
ABO antigens are poorly expressed at birth, increase
gradually in strength and become fully expressed around
1 year of age. In older age, they become slightly weak.
Formation of ABH Antigens
The H gene (genotype HH or Hh) produces a transferase
enzyme, which changes precursor substance (present on
red cells) into H substance. The A and B genes (chromo-
some 9) produce specific transferase enzymes, which
convert H substance into A and B antigens respectively.
Sugar N-acetylgalactosamine is added to H antigen chain
to make the A antigen, while sugar galactose is added to
H antigen chain to produce the B antigen. Some amount
of H antigen remains unchanged on red cells. The O gene
produces an inactive transferase so that H antigen
persists unchanged on red cells (Fig. 33.1).
Some persons do not inherit the H gene (genotype
hh) and thus cannot synthesize H substance. Such persons
may inherit the A or B gene but cannot express it, as they
are unable to produce the H substance. Such individuals
are said to have Bombay phenotype or Bombay blood
group (Oh) (the genotype of Bombay phenotype is hh).
The name derives its name because it was first detected
in persons living in Bombay, India. It has also been
described in other populations. This blood group is
extremely rare. There is complete lack of H, A, and B
antigens due to the lack of H (and Se) genes. Their red
cells type as group O; however, unlike group O
individuals, Oh persons have no H antigen on their red
cells and their plasma contains strong anti-H in addition
to anti-A and anti-B. On cross-matching, all units are
incompatible (Box 33.2). Therefore Bombay group
persons should be transfused only with Oh blood.
Secretors and Non-secretors
Secretors are persons who secrete A, B, and H antigens
into body fluids (such as plasma, gastric juice, saliva,
sweat, tears, semen, milk, etc.). This ability is dependent

Fig. 33.1: Formation of antigens of the ABO system
Box 33.2: Identification of Bombay phenotype
• Blood group: O
• Cross-matching: Serum incompatible with O cells
• Red cells yield negative reaction with anti-H lectin
on presence of a dominant secretor gene (Se) on
chromosome 19. About 80% of individuals are secretors
(genotype Sese or SeSe) and remaining are non-secretors
(genotype sese). Both secretors and non-secretors express
ABO antigens on red cells.
Antigens secreted by different ABO blood groups are:
• Group A: A, H
• Group B: B, H
• Group AB: A, B, H
• Group O: H
Antigens secreted into body fluids are called as ABH
substances. Testing for ABH substances in saliva may be
helpful when red cell grouping yields uncertain results.
Determining secretor status in saliva and semen can be
helpful in resolving ABO blood group discrepancies and
in forensic studies (e.g. semen sample collected from a
rape victim revealing a soluble ABH antigen that does
Blood Group Systems 331
not match with the ABO blood group of the accused).
Inhibitor tests are used to detect the presence of soluble
blood group antigens in body secretions. If saliva contains
a soluble antigen, and if corresponding antibody is
added, the activity of the antibody is neutralized. When
red cells carrying the appropriate antigens are subse-
quently added to the mixture, there will be inhibition of
agglutination (i.e. the person is a secretor). If aggluti-
nation occurs, then the individual is a non-secretor.
Example:
1. Blood group: B
Saliva + anti-B; Add B cells: No agglutination
Interpretation: Secretor
2. Blood group: O
Saliva + Anti-A; Add A cells: Agglutination; Saliva +
Anti-B; Add B cells: Agglutination; Saliva + Anti-H;
Add O cells: No agglutination.
Interpretation: Secretor
3. In non-secretors, no soluble antigens are present in
secretions. Thus antibodies in the reagent are free to
bind to red cells of respective group. There will be
agglutination reaction.
332 Essentials of Clinical Pathology
Antibodies of the ABO System
The most important antibodies in transfusion practice are
anti-A and anti-B. They are also called as naturally
occurring antibodies because they arise without immune
stimulation by red cells by relevant blood group antigens.
They are regularly-occurring, i.e. if an antigen is absent
the corresponding antibody is always present. They are
not detectable in the blood of newborn infants due to
their underdeveloped immune system and appear
around 3-6 months of life. It is thought that they are
produced in response to A- and B-like antigens of
bacteria, which are present in the intestine and certain
foods. If anti-A and/or anti-B are present at birth, they
are of maternal origin (IgG). Anti-A and anti-B antibodies
are usually of IgM class. Immune or IgG antibodies (anti-
A or anti-B) can be stimulated by exposure to red cells,
white cells, or platelets.
IgM antibodies are large molecules and can bind up
to ten antigens; therefore they can cause direct
agglutination of red cells and cause visible agglutination.
IgM antibodies can efficiently fix the complement.
Naturally occurring ABO antibodies can cause:
• Hemolytic transfusion reaction in a case of ABO-
mismatched blood transfusion,
• Acute graft rejection in case of ABO-incompatible
solid organ transplantation, and
• Hemolysis of donor red cells following ABO-
incompatible bone marrow transplantation.
Less commonly, some individuals have large
amounts of ABO antibodies of immune nature. Usually,
group O individuals following immune stimulation by
transfusion, pregnancy, or injection of certain vaccines
or toxoids (that contain bacterial A- and B-like antigens)
produce them in large amounts. These antibodies are of
IgG class, of high titer, and cannot be neutralized by
soluble blood group antigens. If blood of such group O
individuals (called dangerous universal group O donors)
is transfused to group A or B individuals, serious
hemolysis of recipient’s red cells can occur. Therefore
group O donors should not be employed as universal
donors. In addition to causing hemolytic transfusion
reaction, these IgG antibodies can cross the placenta and
induce hemolytic disease of newborn.
Concepts of Universal Donor and Recipient
Red cells of group O donors are devoid of A and B
antigens and cannot be agglutinated by anti-A and anti-
B antibodies. Therefore, group O persons are traditionally
considered as universal donors.Group AB persons are
considered as universal recipients. This, however, is an
oversimplification because only the reaction between
recipient’s plasma (antibodies) and the donor’s red cells
is considered; a small amount of antibodies in donor’s
plasma can cause destruction of recipient’s red cells. In
addition, antigens other than A, B, or RhD can bind to
corresponding antibodies or immunize the recipient.
THE Rh SYSTEM
When Rhesus monkey red cells were injected into rabbits
and guinea pigs (by Landsteiner and Weiner in 1940),
antibody, which was raised, was found to react with
Rhesus monkey red cells as well as with 85% of human
red cells (White residents of New York city). The antigen
involved was called as Rh factor. Subsequently it was
shown that the original antibody was different from anti-
D antibody discovered later. The name of the antigen,
however, has remained as Rhesus. Apart from
Landsteiner and Weiner, credit for discovery of Rh
system also goes to Levine and Stetson who discovered
in 1939 the antibody (actually anti-D) that caused
hemolytic disease of newborn.
The Rh system is only next in importance to ABO
system in transfusion practice. The importance of this
system lies in the high immunogenicity of Rh D antigen,
which readily induces formation of anti-D antibodies in
50-70% of Rh D-negative individuals. Anti-D antibodies
can cause hemolytic transfusion reaction or, in pregnant
women, Rh hemolytic disease of newborn.
The Rh system was discovered independently by:
(1) Stetson and Levine in 1939 and (2) by Landsteiner
and Weiner in 1940.
Antigens of the Rh System
Rh system is highly complex and consists of about 40
antigens. The important antigens of the Rh system are
C, D, E, c, and e. Antigen d does not exist. D antigen is
the most immunogenic. There are various nomenclature
systems for Rh antigens. Fisher-Race or CDE nomen-
clature system and Weiner system are popular.
According to Fisher and Race, three closely linked
genes are inherited together on one chromosome
(haplotype) from each parent. Allelic forms of these genes

are C and c, D and d, and E and e with eight possible
haplotypes: Cde, cde, cDE, cDe, cdE, Cde, CDE, and CdE.
As an individual inherits one haplotype from each
parent, 36 genotypes are possible such as Cde/cde, Cde/
cDe, CDE/cde, etc (Fig. 33.2). The presence of D in either
homozygous (D/D) or heterozygous (D/d) state makes
that individual Rh positive, while Rh negative persons
are homozygous for d (d/d). It was thought that d gene
was an amorph.
According to the theory by Dr. Alexander Weiner, a
single Rh gene is inherited from each parent; this single
Rh gene, however, has multiple alleles (Fig. 33.3). The
Weiner system uses Rh-Hr nomenclature. The major
difference between Fisher-Race and Weiner systems is
that according to Fisher-Race, there are three closely
linked genes that are inherited from each parent, while
according to Weiner, a single gene with multiple alleles
is inherited from each parent.
Results of current genetic studies suggest that both
Fisher-Race and Weiner systems are partially correct. It
has been found that the RH locus is located on
chromosome 1 and consists of two closely linked genes-
RHD and RHCE (Fig. 33.4). The alleles of RHCE are CE,
Ce, ce, and cE.
Fig. 33.2: Fisher-Race system of nomenclature of Rh blood group inheritance
Blood Group Systems 333
In Rh negative persons, deletions, point mutations, or
partial mutations of D gene have been found. Rh antigens
are expressed only on red cells and not on any other
tissues. They are also not secreted in body fluids. In
contrast to ABO antigens, Rh antigens are fully expressed
on red cells before birth and also on red cells of early
fetuses.
Depending on the presence or absence of antigen D
on red cells, a person is grouped either as Rh positive
(when red cells express antigen D) or Rh negative (when
D antigen is absent on red cells). Frequency of D antigen
varies in different populations. In India, approx. 95% of
the people express D antigen on their red cells (Rh D
positive), while 5% are Rh D negative. The frequency
in Caucasians is 85% Rh positive and 15% Rh negative.
Other forms of D antigen are weak D and partial D
(Fig. 33.5). Red cells having weak D antigen were
formerly called as Du cells which react weakly with anti-
D reagent. There is a quantitative reduction in the
number of D antigen sites on such red cells. Du recipients
do not make anti-D antibodies following stimulation by
D antigen (e.g. following D positive blood transfusion).
Du donors should be considered as Rh positive and their
blood should not be transfused to Rh negative donors.
334 Essentials of Clinical Pathology

Fig. 33.3: Weiner theory of Rh system inheritance

Fig. 33.4: According to current genetic studies, there are two genes on
short arm of chromosome 1: RHCE and RHD

Fig. 33.5: Diagrammatic representation of Fig. 33.6: Relative immunogenicity of Rh antigens. D antigen
variations of Rh antigen is the most immunogenic while e antigen is the weakest
 Blood Group Systems 335

Table 33.3: Blood group systems other than ABO and Rh

Blood group Antigens Antibodies Comment
system

1. Lewis Lea, Leb Natural, IgM Lewis antigens are passively absorbed from plasma on red cells;
Lewis antibodies are rarely of clinical significance
2. Kell About 20 IgG Anti-K antibodies can cause hemolytic transfusion reaction and
(KEL1, hemolytic disease of newborn; Some individuals do not have
KEL2, etc) precursor substance on their red cells from which K antigens are
produced; such red cells have short life, acanthocytic features, and
express Kell antigens weakly (MacLeod phenotype).
3. Duffy Fya, Fyb IgG Antibodies can cause hemolytic transfusion reaction. Plasmodium
vivax enters the red cells at the Duffy antigen site. The Fy(a-b-)
phenotype in blacks confers resistance against Plasmodium vivax
infection
4. Kidd JKa, JKb IgG Antibodies cause delayed transfusion reaction and mild hemolytic
disease of newborn
5. MNSs M, N, S, s M and N antigens are important in paternity testing
k
6. P P, P1, P IgM, IgG Auto-anti-P occurs in paroxysmal cold hemoglobinuria

In red cells having partial D antigen, parts of D antigen
are missing. Variants of partial D antigen exist.
Individuals with DVI variant are able to produce anti-D
antibody against the missing part of the antigen. Such
recipients should be considered as Rh negative, while
donors should be regarded as Rh positive. However, in
practice, individuals with partial D antigen are typed as
D negative and are identified only after they have
produced anti-D antibodies.
Complete absence of all Rh antigens on red cells
(Rh null cells) is associated with stomatocytosis and
compensated hemolysis.
be transfused only with Rh-negative blood. During
pregnancy, IgG anti-D can cross the placenta and induce
hemolytic disease of newborn by causing immune
hemolysis of fetal red cells. Rh hemolytic disease of
newborn can be prevented by prophylactic adminis-
tration of Rh immune globulin to all Rh-negative women
during mid pregnancy and within 72 hours of delivery.
Anti-D and anti-c can cause severe HDN. Anti-C, anti-
E, and anti-e usually do not cause HDN or cause mild
HDN. Relative immunogenicity of Rh antigens is shown
in Figure 33.6.

OTHER BLOOD GROUP SYSTEMS

Rh Antibodies
In general, most Rh antibodies are of immune type, i.e.
they are the result of immunization by blood transfusion
or pregnancy. Most of these antibodies are of IgG class.
Rh antibodies can cause hemolytic transfusion
reaction or hemolytic disease of newborn (HDN). Since
Rh antibodies do not activate complement, hemolysis is
extravascular and predominantly occurs in spleen. Due
to the high immunogenicity of D antigen, Rh negative
persons (especially women of childbearing age) should
Salient features of some other blood group systems are
summarized in Table 33.3.
BIBLIOGRAPHY
1. Cheesbrough M. District Laboratory Practice in Tropical
Countries. Part 1 and Part 2. Cambridge: Cambridge
University Press, 1998.
2. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
3. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
Hematology (9th ed). London: Churchill Livingstone,
2001.
 34

Blood Grouping

ABO GROUPING
Box 34.1: ABO antisera
There are two methods for ABO grouping:
• Three types: Anti-A, anti-B, anti-A, B.
• Cell grouping (forward grouping): Red cells are tested
• Anti-A: Blue-colored;
for the presence of A and B antigens employing
• Anti-B: Yellow-colored.
known specific anti-A and anti-B (and sometimes
• Anti-A, B: Colorless.
anti-A, B) sera.
• Serum grouping (reverse grouping): Serum is tested for • Sodium azide is added to prevent the growth of bacteria
the presence of anti-A and anti-B antibodies by • Antisera are kept stored at 4-6°C to preserve their potency
employing known group A and group B reagent red • Routinely, anti-A and anti-B are used for blood grouping.
cells. • Indications for using anti-A, B: (1) for confirmation of cell
grouping in newborns, and (2) to resolve ABO group
Both cell and serum grouping should be done since
discrepancies.
each test acts as a check on the other.
• Antisera may be polyclonal or monoclonal. Monoclonal
There are three methods for blood grouping: tube, antisera are specific, avid, and can detect weak antigens.
microplate, and slide. Slide test is described below. Tube Monoclonal antisera are commonly used.
and microplate methods are better and are employed in
blood banks; they are outlined in short following slide
test.

SLIDE TEST
Principle
Red cells from the specimen are reacted with reagent
antisera (anti-A and anti-B). Agglutination of red cells
indicates presence of corresponding antigen (aggluti-
nogen) on red cells.

Specimen
Capillary blood from finger prick, or venous blood
collected in EDTA anticoagulant.

Reagents
ABO antisera: See box 34.1 and Figure 34.1. Fig. 34.1: Anti-A and anti-B sera used for cell grouping

Method 2. Place one drop of anti-A serum and one drop of anti-
1. A clean and dry glass slide is divided into two sections B serum in the center of the corresponding section of
with a glass marking pencil. The sections are labeled the slide. Antiserum must be taken first to ensure that
as anti-A and anti-B to identify the antisera (Fig. 34.2). no reagents are missed.
 Blood Grouping 337

Table 34.1: Interpretation of cell grouping

(forward grouping) by slide test
Anti-A Anti-B Blood group
+ – A
– + B
+ + AB
– – O

Slide test is quick and needs only simple equipment. It

can be used in blood donation camps and in case of an
emergency. However, it is not recommended as a routine
test in blood banks since weakly reactive antigens on cells
on forward grouping and low titer anti-A and anti-B on
reverse grouping may be missed. Also, drying of the
reaction mixture at the edges causes aggregation that may
be mistaken for agglutination. Results of slide test should
always be confirmed by cell and serum grouping by tube
method.

Fig. 34.2: Cell grouping by slide method TUBE METHOD

3. Add one drop of blood sample to be tested to each
drop of antiserum.
4. Mix antiserum and blood by using a separate stick or
a separate corner of a slide for each section over an
area about 1 inch in diameter.
5. By tilting the slide backwards and forwards, examine
for agglutination after exactly two minutes.
6. Result:
Positive (+): Little clumps of red cells are seen floating
in a clear liquid.
Test tube method is more reliable than slide test, but takes
longer time and more equipment. For cell grouping,
patient’s saline-washed red cells are mixed with known
antiserum in a test tube; the mixture is incubated at room
temperature, and centrifuged. For serum grouping,
patient’s serum is mixed with reagent red cells of known
group (available commercially or prepared in the
laboratory), incubated at room temperature, and
centrifuged (Table 34.2). Following centrifugation, a red
cell button (sediment) will be seen at the bottom of the
tube. Cell button is dislodged by gently tapping the base
of the tube and examined for agglutination.

Negative (–): Red cells are floating homogeneously in a Positive (+) Test
uniform suspension. Clumps of red cells suspended in a clear fluid. Aggluti-
7. Interpretation: Interpret the result as shown in the nation in tube test is graded from 1+ to 4+ and read
Table 34.1 and Figure 34.2. macroscopically (Fig. 34.3).

Table 34.2: Interpretation of forward (cell) and reverse (serum) grouping

Forward (Cell) grouping Reverse (serum) grouping Interpretation
Anti-A serum Anti-B serum A1 cells B cells Blood group
+ – – + A
– + + – B
– – + + O
+ + – – AB

+: Agglutination of red cells; –: No agglutination of red cells.

338 Essentials of Clinical Pathology
Fig. 34.3: Grading of ABO tube test. Negative: Uniform
suspension of red cells; Grade 1 (1+): Many small clumps
of red cells (fine granular appearance); Grade 2 (2+): Many
large clumps with many free red cells; Grade 3 (3+): Three
or four individual clumps with few free red cells; and Grade
4(4+): One solid clump of red cells with no free red cells
Negative (–) Test
Uniform suspension of red cells.
Separate tubes of auto-control, positive control, and
negative control should always be setup along with the
test sample tube.
Auto-control tube consists of mixture of patient’s red
cells and patient’s own serum. This is required to rule
out false-positive result due to autoantibodies in patient’s
serum causing auto-agglutination of patient’s own red
cells. Auto-control test is particularly essential when ABO
grouping is being done only by forward method and
blood group is typed as AB. If there are autoantibodies
in recipient’s serum, ABO grouping, Rh typing, antibody
screening, and cross-matching all will show positive
result.
In two positive control tubes, anti-A serum is mixed
with group A red cells and anti-B is mixed with group B
red cells respectively. In two negative control tubes, anti-
A serum is mixed with group B red cells and anti-B serum
is mixed with group A red cells respectively. These
controls are necessary to confirm that reagents are
working properly.
If forward grouping, reverse grouping, and autocontrol
tests are all positive, then these results are probably
indicative of a cold-reactive autoantibody. Before
performing forward typing, red cells should be washed
with normal saline to elute the antibody. Before
performing reverse grouping, autoantibody should be
adsorbed by washed cells till autocontrol is negative.
Test tube method of blood grouping is more reliable
than slide method. This is because centrifugation
enhances the reaction by bringing antigen and antibodies
closer together and allows detection of weaker antigen-
antibody reactions; in addition drying is avoided and
smaller amounts of reagent are required.
MICROPLATE METHOD
Microplate is a polystyrene plate consisting of 96 micro
wells of either U- or V-shape. Grouping is carried out in
micro wells. This method is sensitive and ideal for large
number of samples (Fig. 34.4).
FALSE REACTIONS IN ABO GROUPING
1. Autoagglutination: Presence of IgM autoantibodies
reactive at room temperature in patient’s serum can
lead to autoagglutination. If autocontrol is not used,
blood group in such a case will be wrongly typed as
AB. Therefore, for correct result, if autocontrol is also
showing agglutination, cell grouping should be
repeated after washing red cells with warm saline,
and serum grouping should be repeated at 37°C.
2. Rouleaux formation: Rouleux formation refers to red
cells adhering to each other like a stack of coins and
can be mistaken for agglutination. Rouleaux forma-
tion is caused by high levels of fibrinogen, immuno-
globulins, or intravenous administration of a plasma
expander such as dextran. Rouleaux formation (but
not agglutination) can be dispersed by addition of
normal saline during serum grouping.
3. False-negative result due to inactivated antisera: For
preservation of potency of antisera, they should be
kept stored at 4°-6°C. If kept at room temperature for
long, antisera are inactivated and will give false-
negative result.
4. Age: Infants start producing ABO antibodies by 3-6
months of age and serum grouping done before this
age will yield false-negative result. Elderly indivi-
duals also have low antibody levels.
Rh D GROUPING
D antigen is the most immunogenic after ABO antigens
and therefore red cells are routinely tested for D.
Individuals are called as Rh-positive or Rh-negative
depending on presence or absence of D antigen on their
red cells. Following transfusion of Rh-positive blood to
Rh-negative persons, 70% of them will develop anti Rh-
D antibodies. This is of particular importance in women
of childbearing age as anti-D antibodies can crosss the
placenta during pregnancy and destroy D-positive fetal
 Blood Grouping 339

Fig. 34.4: Microplate (96 well) method for blood grouping. Numbers 1 to 12 represent patient identification numbers. Reagents
added to the patient sample are written on left, while interpretation (blood group of patient) is written at the bottom. For understanding,
reaction patterns of the 8 possible blood groups are shown, while the last 4 columns have been kept empty. Red compact
button indicates agglutination, while uniform suspension indicates no agglutination

Table 34.3: Comparison of ABO grouping and Rh typing

Parameter ABO grouping Rh typing

1. Blood groups A, B, AB, O Positive, Negative

2. Forward grouping Yes Yes
3. Reverse grouping Yes No
4. Antisera IgM IgG or IgM
5. Dosage effect* No Yes
6. Color coding of antisera A:Blue, B: Yellow, AB: Colorless D: Colorless
7. Optimum reaction temperature Room temperature 37°C or room temperature

*In dosage effect, antibody reacts stronger with homozygous cells than with heterozygous cells

red cells and cause hemolytic disease of newborn. In other
sensitized individuals, re-exposure to D antigen can
cause hemolytic transfusion reaction.
In Rh D grouping, patient’s red cells are mixed with
anti-D reagent. Serum or reverse grouping is not carried
out because most Rh-negative persons do not have anti-
D antibodies; anti-D develops in Rh-negative individuals
only following exposure to Rh-positive red cells.
Rh typing is done at the same time as ABO grouping.
Method of Rh D grouping is similar in principle to ABO
grouping. Since serum or reverse grouping is not possible,
each sample is tested in duplicate. Dosage effect (stronger
antigen-antibody reaction in homozygous cells i.e.
stronger reaction with DD) is observed with antigens of
the Rh system. Autocontrol (patient’s red cell + patient’s
serum) and positive and negative controls are included
340 Essentials of Clinical Pathology
in every test run. Monoclonal IgM anti-D antiserum
should be used for cell grouping, which allows Rh
grouping to be carried out at the same time as ABO
grouping at room temperature. With monoclonal
antisera, most weak and variant forms of D antigen are
detected and further testing for weak forms of D antigen
(Du) is not required.
Differences between ABO and Rh grouping are
shown in Table 34.3.
BIBLIOGRAPHY
1. Cheesbrough M. District Laboratory Practice in Tropical
Countries. Part 1 and Part 2. Cambridge: Cambridge
University Press, 1998.
2. Henry JB. Clinical Diagnosis and Management by
Laboratory Methods. (20th Ed). Philadelphia: WB
Saunders Company, 2001.
3. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
 35
Collection of Donor Blood,
Processing and Storage
In India, collection of blood, processing, storage, and
preparation of blood components and derivatives are
regulated by Food and Drugs Administration (FDA).
Blood is regarded as a “drug” (under section 3(b) of the
Drugs and Cosmetics Act, 1940) by the FDA and all the
blood banks have to obtain a license from the FDA and
follow the FDA guidelines.
TYPES OF BLOOD DONORS
Blood donation is a process through which a blood donor
has a specified amount of blood drawn for the purpose
of storage in a blood bank and for subsequent blood
transfusion. Blood donation may be done in a blood bank
or in blood donation camps. The process of blood
donation involves selection of blood donors by screening,
actual donation of blood, and a brief recovery period after
Fig. 35.1: General steps before collection of donor blood
blood donation. General steps for collection of donor blood
are shown in Figure 35.1. The safe transfusion practice
starts with proper selection of blood donors; if donors are
not properly screened, blood can become a medium of
transmission of infections like human immunodeficiency
virus, hepatitis B and C virus, syphilis, and malaria.
Blood donations are of three main types: whole blood
donation (collection of one unit or 350 ml of whole blood
in an anticoagulant solution), autologous donation
(donation for subsequent transfusion to self), and
apheresis donation (removal of whole blood from a
donor, separation and retention of the desired portion,
and returning the remaining portion to the donor).
There are three main types of whole blood donors:
• Voluntary
• Professional
• Replacement.
342 Essentials of Clinical Pathology
A blood bank should function only on voluntary blood
donations. A voluntary blood donor donates blood out of
his/her own free will and on humanitarian grounds or
out of sense of duty or responsibility towards the
community. A voluntary donor is issued a voluntary donor
card which can be presented for free blood unit if blood is
required for himself or a close family member.
Paid or professional donors donate blood for money.
As payment encourages concealment of a significant
illness, high-risk behavior, or medical history, such form
of donations should be completely discouraged.
A replacement blood donor is a friend or a relative
of the recipient whose donated blood unit is credited to
the patient (predeposit donation). Blood unit that has
been donated replaces the blood unit used for the patient.
Directed donor donates blood for a specific, named
patient and is a friend or a relative of the patient. Patient
selects his or her own blood donor. Directed donations
can be less safe as the donor may hide significant medical
information due to pressure of donation. Graft vs. host
disease is possible after blood transfusion from a relative.
CRITERIA FOR SELECTION OF
BLOOD DONORS
Careful selection of blood donors is an essential
requirement for safe transfusion practice. It is necessary
to ensure safety of both the donor and the recipient. It is
essential to identify potential health problems for the
donor and to prevent transmission of infections through
transfusion to the recepient. Selection process consists
of obtaining medical history, and performing physical
examination and certain laboratory tests. Selection of
blood donors should be carried out by a qualified
physician or by a person working under his supervision.
A blood donor questionnaire and consent form needs
to be filled by the prospective donor before blood
donation. Typical questions asked to the prospective
donor are shown in Box 35.1. The prospective donor
should be assured that the personal information revealed
shall be kept confidential.
Criteria for selection of blood donors are given below
in short. Reasons for permanent and 1 year deferral are
shown in Boxes 35.2 and 35.3.
Age
Blood donor should be between 18 and 60 years of age.
Box 35.1: Typical questions in the
questionnaire asked to the prospective donor
• Have you donated blood previously? If yes, date of last
donation and discomfort during or after donation if any.
• Were you deferred as a donor?
• Did you receive any blood or blood products in the last
6 months?
• Do you currently suffer from or were suffering from any
of the following conditions: Heart disease, lung disease,
liver disease, kidney disease, cancer, diabetes, epilepsy,
tuberculosis, bleeding disorder, hepatitis, allergy, jaundice,
sexually transmitted disease, malaria (last 6 months),
typhoid (last 1 year)?
• Have you had recent weight loss, swollen glands, low
grade fever, persistent cough, or repeated diarrhea?
• Individuals with multiple sexual partners, homosexuals,
and intravenous drug abusers are likely to be infected
with AIDS virus. Do you practice any of these?
• Have you taken aspirin in last 3 days? Are you taking
any other medications?
• Have you been vaccinated recently?
• Have you had any major surgery or received blood
transfusion in the last 6 months?
• Did you have any accidental needle stick (health care
workers), tattooing, or ear piercing in the last 6 months?
• For women donors: Are you pregnant? Are you breast-
feeding a child? Did you have an abortion in the last 6
months?
Box 35.2: Reasons for permanent deferral of a
blood donor
• Positive test for HBV, HCV, HIV
• Known case of angina pectoris or myocardial infarction
• Recipient of clotting factor concentrate
• High risk behavior for HIV infection
• Gay or bisexual male
• History of viral hepatitis in adult life
• Intravenous drug abuse
• Recipients of human pituitary-derived growth hormone
• Any malignancy, endocrine disease, chronic renal disease,
chronic liver disease, bleeding disorder, diabetes mellitus
dependent on insulin, asthma
Donation Interval
To protect the donor from iron deficiency, interval
between two successive donations should be atleast 3
months.

Box 35.3: Reasons for 1 year deferral
• History of tattooing, ear-piercing, accidental needle
stick in health workers
• Rabies vaccine or hepatitis B immunoglobulin
• Rape victims
• Sexual contact with a person with hepatitis, AIDS, or
with an intravenous drug abuser
• Recipient of blood transfusion or organ transplant
Volume of Donation
A donor weighing 45 kg or more can give 350 ml of blood
along with pilot samples for processing.
Pregnancy and Lactation
Pregnant women and lactating mothers (up to 1 year
post-partum) are not accepted.
Infectious Diseases
Human Immunodeficiency Virus (HIV)
Donors with history of unexplained fever, loss of weight,
lymphadenopathy, and persistent diarrhea should be
excluded. “High risk” group donors (homosexuals;
intravenous drug abusers; or individuals having contact
with commercial sex workers, with multiple sexual
partners, or with known acquired immunodeficiency
syndrome or HIV positive persons) should be requested
not to donate blood. Purpose of this policy is elimination
of donors in early (HIV antibody-negative) period of HIV
infection.
Viral Hepatitis
An individual with history of jaundice within last 1 year
should not be accepted. HBSAg- or anti-HCV positive
subjects are permanently barred from donation.
Malaria
In countries where malaria is endemic, a donor may be
accepted after 3 months of asymptomatic period
following malarial attack and after full treatment.
Illness
Prospective donors with history of diabetes mellitus,
hypertension, heart disease, renal disease, liver disease,
lung disease, cancer, epilepsy, bleeding disorder, or
allergic disease are not permitted to donate blood.
Collection of Donor Blood, Processing and Storage 343
Medications
Many donors who are taking drugs are excluded because
of their clinical condition. Other donors are not accepted
because the drug they are taking can be potentially
harmful to the recipient, e.g. aspirin (which inhibits
platelet function), and drugs with teratogenic action like
finasteride, isotretinoin, acitretin, and etretinate, or
cytotoxic drugs (like cyclophosphamide). Recipients of
human pituitary-derived growth hormone are perma-
nently debarred due to the risk of Creutzfeldt-Jakob
disease.
Dental Treatment
Following dental treatment or tooth extraction, a 72-hour
deferral period before donation is required due to the
risk of bacteremia.
Skin Piercing
As tattooing, electrolysis, ear piercing, accidental needle
stick in health care workers, or acupuncture carry the
risk of transmission of hepatitis or HIV infection, blood
donation should be deferred for 12 months.
Blood Transfusion
A person should not be accepted as blood donor for 12
months after receiving blood transfusion or organ
transplant.
Immunization
There is no deferral for recent immunization with a killed
vaccine, recombinant vaccine, or a toxoid. Nature of
vaccine received and respective deferral period are as
follows.
• Attenuated live virus vaccine for measles, mumps,
yellow fever, oral polio: 2 weeks.
• Rubella, varicella: 4 weeks.
• Rabies vaccine following bite by a rabid animal: 1 year
• Passive immunization with animal sera: 4 weeks.
• Hepatitis B immunoglobulin: 1 year.
Physical Examination
• Donor should be in good general health.
• Weight: should be minimum 45 kg.
• Blood pressure: systolic blood pressure should be
100-180 mm of Hg and diastolic 50-100 mm of Hg.
• Pulse: Pulse rate should be 60-100/min and regular.
344 Essentials of Clinical Pathology
• Temperature: should be normal.
• Donor skin at the venepuncture site should be free of
scars of needle pricks as they may indicate drug
addiction.
Estimation of Hemoglobin
Screening of donors for anemia in blood bank is
commonly done by copper sulphate specific gravity
method. Haemoglobin value should be ≥ 12.5 g/dl.
Specific gravity of 1.053 of copper sulphate solution
is approximately equal to hemoglobin concentration of
12.5 grams/dl. A drop of fingerprick blood is allowed to
fall in this solution from a height of 1 cm. If the drop
sinks within 15 seconds time, the hemoglobin level is
acceptable. This method often underestimates the
hemoglobin value and leads to unnecessary rejection of
donors. In addition, specific gravity of whole blood does
not depend only on hemoglobin content and is also
affected by marked rise in total leukocyte count or plasma
protein level; in the presence of these conditions,
hemoglobin will be spuriously normal.
COLLECTION OF DONOR BLOOD
In the blood bank, donor blood is collected in a well
ventilated, well-lighted, and air-conditioned room. Blood
is drawn by qualified physician or by an assistant who
is well trained and is working under his supervision.
Equipments and Materials
1. Blood bag containing anticoagulant-preservative
solution: Blood from a donor is collected in a closed
system of sterile, disposable plastic bag (single,
double, or triple bag depending on component to be
prepared) with 350 ml capacity. These bags contain
49 ml of citrate phosphate dextrose adenine (CPDA)
solution. Currently, CPDA-1 solution is commonly
used in which blood can be kept stored at 2-6°C for
35 days. This solution inhibits clotting of blood and
provides nutrition for cell metabolism. In India, whole
blood transfusion is still practiced at many places, and
therefore single bag is commonly used. Double or
triple bag systems are used if facilities for separation
of components are available.
2. Sphygmomanometer, weighing scale, blood
weighing balance, sealing clips, artery forceps.
3. Iodine, spirit, sterile cotton swabs, adhesive tape.
4. Emergency drugs and equipment.
5. Pilot tubes for collection of blood for testing
(grouping, cross matching, screening for infectious
diseases).
Technique
Blood bag and pilot tubes should be labeled with the
donor identification number and date of collection before
blood collection.
The donor should be under constant observation
throughout the procedure of collection. Before blood
donation, the prospective donor should have eaten well
and taken sufficient fluids. The blood donor lies supine
on a bed. The site for venepuncture is selected in the
antecubital fossa. A sphygmomanometer cuff is applied
to the arm and inflated to 60-70 mm of Hg to make the
veins prominent. The selected area is thoroughly
cleansed with iodine and spirit and allowed to dry.
The blood collection bag is placed on a weighing
balance that has been kept below the level of the arm. A
loose knot is tied in the tubing close to the venepuncture
needle.
Phlebotomy is carried out and the blood should start
flowing freely across the tubing into the blood bag. The
time of venepuncture is noted in the donor form. The
tubing is carefully taped to the needle in place with an
adhesive tape.
The pressure is reduced to 30-40 mm of Hg. The donor
is asked to squeeze a rubber ball or a soft sponge material
slowly and continuously to quicken the speed of
donation. The blood and the anticoagulant are mixed
gently and periodically in the blood bag. The amount of
blood collected is checked on the weighing balance (1
ml of blood weighs 1.05 gm and the weight of 350 ml of
blood is 367 gm). When the weight of the blood bag is as
required (367 gm + weight of empty bag and antico-
agulant), the requisite amount of blood has been
collected. The procedure of blood collection should not
take more than 8 minutes.
The pressure cuff is deflated and the tubing is
clamped with forceps about 10 cm away from the needle.
The knot made earlier (close to the needle) is tightened
or a sealing clip is applied.
The tubing is cut between the clamp and the knot/
sealing clip. The clamp is removed from the tubing and
blood samples (for grouping, cross matching, infectious
disease screening) are collected in appropriate tubes. The
tubing is then reclamped.

Needle is removed from the vein and pressure is
applied over the puncture site with sterile cotton gauze.
The needle is discarded in a special “sharps” container.
Blood remaining in the tubing is non-anticoagulated
and is forced back into the blood bag. Bag is inverted
gently several times to mix the blood and the anticoagu-
lant. Anticoagulated blood is then allowed to run back
into the tubing. The blood bag is placed in the refrigerator
at 4-6°C immediately following collection. If platelet
concentrate is to be prepared, the bag should be kept at
room temperature till platelets are separated (within 4
hours of collection).
After cessation of bleeding, the venepuncture site is
covered with sterile gauze and an adhesive tape. After
8-10 minutes, the donor is allowed to sit up and guided
to the refreshment area. The donor is issued a donation
card and is given information about need to drink more
fluids, activities permissible, and care of venepuncture
site.
Donor Reactions
Donor reactions are rare.
1. Syncope or vasovagal attack: This is the most common
reaction. It is due to the action of the autonomic
nervous system and is induced by anxiety, site of
blood, or pain. Its features are sweating, slowing of
pulse rate, pallor, coldness of skin, sudden hypo-
tension, and sometimes fainting, vomiting, or
incontinence. In such a case, donation is stopped, and
Fig. 35.2: General procedure after collection of donor blood
Collection of Donor Blood, Processing and Storage 345
legs are elevated above the level of the head. Cold
compresses are placed on the neck and forehead. This
reaction should be distinguished from hypotensive
shock (tachycardia, hypotension, and loss of con-
sciousness). A severe vasovagal reaction is a contra-
indication for future donations.
2. Hyperventilation: This is seen in first-time donors
who are highly excited. Hyperventilation causes loss
of carbon dioxide. This may result in facial twitching
or muscular spasms. Relief can be obtained by
breathing in a paper bag.
3. Nausea and vomiting
4. Hematoma at the site of venepuncture
5. Infection at venepuncture site or thrombophlebitis
6. Inadvertent arterial puncture.
Any donor reactions should be recorded on the card
issued to the donor.
General procedure after collection of donor blood is
outlined in Figure 35.2.
PROCESSING OF DONOR BLOOD
This refers to various tests and procedures carried out
on the donor blood after collection but prior to cross
matching. Tests done on donor blood are listed in Table
35.1.
Changes Occurring during Storage
1. Loss of viability of red cells: Viability refers to the
capacity of red blood cells to survive in recipient’s
346 Essentials of Clinical Pathology
Table 35.1: Tests done on donor blood after collection
and before cross-matching
• ABO and Rh grouping
• Screening and identification of unexpected antibodies
• Screening tests for infections transmissible by transfusion
– Hepatitis B surface antigen
– Test for antibodies to hepatitis C virus
– Test for antibodies to human immunodeficiency virus
– Venereal disease research laboratory (VDRL) test for
syphilis
– Blood smear for malaria parasite
circulation after transfusion. There is progressive loss
of viability with increasing durtion of storage. Storage
conditions should be such that, after transfusion, at
least 75% of transfused red cells should survive at 24
hours in the recipient’s circulation. Shelf life of the
stored whole blood is based on this criterion. For
whole blood stored in CPDA-1 and maintained at 2°
to 6°C, shelf life is 35 days.
2. Depletion of ATP: Progressive loss of ATP with
increasing length of storage causes decreased
deformability of red cells and impairment of Na+/
K+-ATPase pump.
3. Reduction of 2, 3-diphosphoglycerate (2,3-DPG):
Progressive depletion of 2, 3-DPG with storage
increases oxygen affinity of haemoglobin and reduces
release of oxygen to the tissues. 2,3-DPG in red cells
tends to return to normal levels within 24 hours of
transfusion.
4. Loss of granulocyte function (phagocytic and
bactericidal property) occurs within 24 hours and
loss of platelet function occurs within 48 hours of
blood collection. F VIII level declines to 50% by 24
hours and F V declines to 50% by 10-14 days.
5. Decrease in pH of blood
6. Formation of microaggregates: In stored blood,
aggregates of aged platelets, leucocytes, and cold
insoluble globulin form. Their number and size
increase with increasing length of storage.
BIBLIOGRAPHY
1. Cheesbrough M. District Laboratory Practice in Tropical
Countries. Part 1 and Part 2. Cambridge: Cambridge
University Press, 1998.
2. Henry JB. Clinical Diagnosis and Management by
Laboratory Methods. 20th Ed. Philadelphia: WB
Saunders Company, 2001.
3. Kawthalkar SM. Essentials of Haematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
 36
Screening Tests for Infections
Transmissible by Transfusion

The organisms likely to be transmitted by transfusion • Concealment of relevant medical history by pros-
are usually those, which are prevalent in a particular pective donors
geographic area or population. Organisms transmissible • Specificity and sensitivity of screening tests for
by transfusion are listed in Table 36.1. infections may be poor
According to studies conducted in India, prevalence • Non-implementation of National Transfusion Policy
of transmission of infections through transfusions is • Repeat donor system is non-existent
significantly higher as compared to developed nations. • Collection of donor blood during window period
Some of the reasons include: In India, pre-transfusion testing of donor blood for
• Proportion of replacement and professional dona- agents listed in Table 36.2 is currently mandatory.
tions is high The importance of mandatory testing for infectious
organisms transmissible by transfusion is: (i) some
carriers of disease are often asymptomatic, (ii) some viral
Table 36.1: Microorganisms transmissible by transfusion
infections have long incubation periods, and (iii) it is
• Hepatitis viruses essential to safeguard the health of the recipient.
– Hepatitis A virus (rare) Infections by hepatitis B and C viruses and HIV-1 and
– Hepatitis B virus
HIV-2 are characterized by:
– Hepatitis C virus
• Human immunodeficiency virus (HIV) • Persistence of organisms in circulation for prolonged
– HIV-1 duration without necessarily causing clinical
– HIV-2 manifestations
• Human parvovirus B 19 • Persistence in high titers
• Cytomegalovirus
• Long incubation period
• Epstein Barr virus (rare)
• Human T cell leukemia virus (HTLV) • Ability to cause chronic carrier state
– HTLV-1 • Viability of organisms in blood stored at 4°-6°C.
– HTLV-2
Following principal measures can prevent trans-
Prions mission of infection through transfusion:
• Creutzfeldt-Jakob disease (CJD) and variant CJD • Blood should be collected only from voluntary, non-
Bacteria remunerated donors. All high risk persons (intra-
• Treponema pallidum (syphilis) venous drug abusers, homosexuals, prostitutes, and
• Bacterial contamination of donor unit sexual partners of such persons) and professional
• Brucellosis
donors should be excluded. Standard criteria for
Parasites selection of blood donors should be followed.
• Malaria parasites • All blood donations should be tested for infectious
• Trypanosoma cruzi
agents by screening tests; addition of further
• Toxoplasma gondii
• Babesia microti screening tests like HIV RNA and HCV RNA will
• Leishmania donovani further reduce the risk.
• Universal hepatitis B virus vaccination
348 Essentials of Clinical Pathology

Table 36.2: Mandatory infectious disease testing in blood transfusion practice in India
Disease Test
1. Syphilis Serologic test like Venereal Disease Research Laboratory (VDRL) test
2. Hepatitis B Hepatitis B surface antigen (HBsAg)
3. Human immunodeficiency Anti-HIV 1 and anti-HIV 2 antibodies*
virus (HIV) infection
4. Hepatitis C Anti-HCV antibodies•
5. Malaria Blood smear

* Window periods for HIV and HCV infections are further reduced if tests for HIV RNA and HCV RNA (nucleic acid testing or
NAT) are added. NAT, however, is not currently mandatory in India

• Observation of universal precautions in blood
collection, processing, storage, and transfusion
• Leukofiltration of blood products
• Stringent quality control measures.
VIRUSES
Hepatitis B Virus (HBV)
HBV is a partially double-stranded DNA virus of 42 nm
diameter (Fig. 36.1). It can cause acute hepatitis, chronic
hepatitis, asymptomatic carrier state, cirrhosis, or
hepatocellular carcinoma.
In India, prevalence of HBsAg carriers is reported to
be 1.5 to 4%. HBV is highly infectious and is transmitted
through all blood components and most of the blood
derivatives. According to one study in India, incidence
of post-transfusion hepatitis following a single trans-
fusion was 6.7%; this was much higher in multi-transfused
patients like patients with thalassemia and hemophilia.
Infected hepatocytes release large amounts of hepatitis
B surface antigen (HbsAg) into the bloodstream. Presence
of HbsAg indicates active infection. The serological marker
first to appear in HBV infection is HBsAg (as early as 5
days after infection).
Screening of all blood donations for HbsAg has
greatly reduced the risk of transmission of HBV through
transfusion.
Tests for screening donor blood for HbsAg are:
• Reverse passive hemagglutination assay (RPHA)
• Enzyme linked immunosorbent assay (ELISA)
• Radioimmunoassay (RIA).
Commercial test kits for detection of HbsAg are
available and the exact test procedure is provided with
each kit. General principles of these tests are outlined
below.

Reverse Passive Hemagglutination Assay

In RPHA, red cells that are coated with anti-HBs antibody
are added to the donor’s serum. If HbsAg is present in
the serum, agglutination of red cells will occur. Absence
of agglutination indicates negative test. The test is called
as ‘passive reverse agglutination’ because antibody, and
not antigen, is artificially coated on to the red cells. The
test is performed in U- or V-shaped wells of a microtiter
plate which can be centrifuged to augment agglutination.
The test is sensitive to 10-100 ng of HbsAg antigen per
ml of serum (Fig. 36.2).

Enzyme-linked Immunosorbent Assay

Fig. 36.1: Diagrammatic representation of Serum sample to be tested is incubated with anti-HbsAg
hepatitis B virus antibody which has been coated to microtitre plate. The
 Screening Tests for Infections Transmissible by Transfusion
Fig. 36.2: Principle of reverse passive hemagglutination
assay for HBsAg
test serum is incubated in the well during which binding
of antigen from serum (if present) and antibody (attached
to the well) occurs. An enzyme-linked anti-HBs antibody
(conjugate) is added which will combine with the bound
antigen. A chromogenic enzyme substrate is finally
added and the mixture is incubated in the dark. If enzyme
is present (bound to the antigen), then its action on the
substrate will lead to the colour development. A stopping
Fig. 36.3: Hepatitis C virus genome and the recombinant proteins
349
solution (an acid) is added to prevent any further reaction
between the enzyme and the substrate. The result is read
in a spectrophotometer at the specified wavelength.
Hepatitis C Virus (HCV)
HCV is a single-stranded RNA virus of flaviviridae
family. Incubation period is about 8 weeks. Most cases
of HCV infection are asymptomatic. Some patients
develop chronic hepatitis, cirrhosis, or hepatocellular
carcinoma.
In India, prevalence of HCV is reported to be 1.66%.
As the amount of HCV antigen in bloodstream is
small and cannot be detected readily, screening of donor
blood for HCV infection is done by detection of anti-HCV
antibody in serum (becomes detectable after 6-8 weeks
of infection).
The earlier tests (first generation ELISA) used
recombinant proteins complementary to the NS4 region
(c100-3) of the HCV genome. Second generation ELISA
incorporated recombinant or synthetic antigens from
NS4 as well as NS3 regions (c100-3, c22-3, c33c) of the
genome, resulting in improvement in sensitivity and
specificity. The third generation ELISA, in addition to
NS3 and NS4, also includes antigens from NS5 region
(Fig. 36.3).
Human Immunodeficiency Virus
Human immunodeficiency virus or HIV is a RNA
retrovirus (Fig. 36.4), which causes slowly progressive
350 Essentials of Clinical Pathology

Fig. 36.4: Diagrammatic representation of human

immunodeficiency virus Fig. 36.5: Principle of ELISA test for anti-HIV antibodies

immunodeficiency in infected persons. The cells most ELISA, and (ii) HIV-1 nucleic acid amplification testing
susceptible to HIV infection are CD4+ T lymphocytes. (NAT). NAT testing is based on polymerase chain reaction.
Destruction of CD4+ T lymphocytes results in slowly
progressive impairment of the immune system. The
BACTERIA
infected individual becomes susceptible to a range of Treponema pallidum
opportunistic infections and malignancies. The most Blood transfusion can transmit Treponema pallidum, the
advanced stage of HIV disease is acquired immune causative agent for syphilis. It is a spirochete that can be
deficiency syndrome or AIDS. visualized by dark ground illumination (Fig. 36.6). T.
According to the estimates of India’s National AIDS pallidum is destroyed by storage of blood at 2-8°C for 48-
Control Organization (NACO), adult prevalence of HIV 72 hours. However, fresh blood or platelet concentrates
infection is 0.7% with approximately 4 million HIV can transmit these organisms. Transfusion-transmission
infections, 90% of which are in the age group of 15-45 of syphilis is, therefore, rare. The main value of testing
years. donor blood for T pallidum is to identify and exclude
donors with high-risk behavior and thus who are at risk
Following HIV infection, viremia becomes detectable
of having sexually transmitted infections.
after a few days and lasts for several weeks. Anti-HIV
antibodies appear 6-12 weeks after infection (called as
seroconversion). Principle of ELISA test for detection of
anti-HIV antibodies is shown in Figure 36.5. Window
period is the period between the onset of HIV infection
and appearance of detectable anti-HIV antibodies in
serum; it is the infectious but seronegative period (i.e.
the test for anti-HIV antibodies is yet to become positive).
Transfusion of donor blood collected during the window
period will transmit HIV to the recipient.
According to American Association of Blood Bank
standards, HIV screening tests necessary for whole blood Fig. 36.6: Treponema pallidum observed under dark
donors are (i) Anti-HIV antibodies (HIV-1 and HIV-2) by ground illumination
 Screening Tests for Infections Transmissible by Transfusion 351

Screening of donor blood for antibody to T. pallidum is

usually carried out by rapid plasma reagin test or Venereal
Disease Research Laboratory (VDRL) test. In VDRL test
that is a non-specific test, donor serum (heated to 56°C to
inactivate the complement) is mixed with cardiolipin-
lecithin-cholesterol antigen. If flocculation is observed, the
test is reported as reactive (Fig. 36.7).

PARASITES

Plasmodium Species
Malaria parasite can be transmitted through all blood
components. For detection of malaria parasite, commonly
blood smears are examined by light microscopy.
However, the test is positive only when parasites are
> 100/μl in blood.

BIBLIOGRAPHY
1. Chatterjee K, Sen A. Step by Step Blood Transfusion
Services. A Practical Manual on the Technical and
Clinical Aspects. New Delhi: Jaypee Brothers and
Medical Publishers (P) Ltd, 2006.
Fig. 36.7: Principle of Venereal Disease Research 2. Kawthalkar SM. Essentials of Haematology. New Delhi:
Laboratory (VDRL) slide test for syphilis Jaypee Brothers Medical Publishers (P) Ltd, 2006.
 37

Compatibility Test
(Cross-match)

When the recipient’s ABO and Rh blood groups are

determined, the donor blood unit that is ABO and Rh
compatible is selected, and compatibility test is carried
out. The purpose of compatibility test is to prevent the
transfusion of incompatible red cell units and thus
avoidance of hemolytic transfusion reaction in the
recipient. Compatibility test detects (i) major ABO
grouping error, and (ii) most clinically significant
antibodies reactive against donor red cells.
There are two types of cross-match: major cross-match
(testing recipient’s serum against donor’s red cells) and
minor cross-match (testing donor’s serum against
recipient’s red cells). However, minor cross-match is
considered as less important since antibodies in donor
blood unit get diluted or neutralized in recipient’s
plasma. Also, if antibody screening and identification is
being carried out, minor cross-matching is not essential.
Therefore, only the red cells from the donor unit are
tested against the recipient’s serum and the name Fig 37.1: Immediate spin cross-match
compatibility test has replaced the term cross-matching.
For transfusion of platelets or fresh frozen plasma,
cross-matching is not required. However, fresh frozen
plasma should be ABO-compatible.
Causes of False-negative Test
A full cross-matching procedure consists of:
• Immediate spin cross-match at room temperature, and 1. A2B donor red cells and group B recipient serum.
• Indirect antiglobulin test at 37°C. 2. Rapid complement fixation of potent ABO antibodies
with bound complement interfering with aggluti-
IMMEDIATE SPIN CROSS-MATCH nation.
• The purpose of this test is to detect ABO incompati-
Causes of False-positive Test
bility. Equal volumes of 2% saline suspension of red
cells of donor and recipient’s serum are mixed, 1. Rouleaux formation
incubated at room temperature for 5 minutes, and 2. Cold-reactive antibodies: If agglutination disappears
centrifuged. Agglutination or hemolysis indicates by keeping the tube at 37°C for 10 minutes, presence
incompatibility (Fig. 37.1). of cold agglutinins is confirmed.

Fig 37.2: Indirect antiglobulin test for detection of
clinically significant IgG antibodies
INDIRECT ANTIGLOBULIN TEST
Saline-suspended red cells of the donor after being
incubated in patient’s serum are washed in saline and
antiglobulin reagent is added. Following re-centri-
fugation, examine for agglutination or hemolysis
(Fig. 37.2). This test detects most of the clinically
significant IgG antibodies.
If agglutination or hemolysis is not observed in any
of the above stages, donor unit is compatible with
Compatibility Test (Cross-match) 353
recipient’s serum. Agglutination or hemolysis at any stage
is indicative of incompatibility.
EMERGENCY CROSS-MATCH
If blood is required urgently, ABO and Rh grouping are
carried out by rapid slide test and immediate spin cross
match (i.e. the first stage of cross match) is performed
(to exclude ABO incompatibility). If the blood unit is
compatible, then after issuing it, remaining stage of the
cross-match is completed. If any incompatibility is
detected, the concerned physician is immediately
informed about the incompatibility detected.
ANTIBODY SCREENING AND
IDENTIFICATION
Screening for unexpected or irregular antibodies is done
during pre-transfusion testing in recipient’s serum and
in donor’s blood. In this test, serum of the recipient is
tested against a set of three group O screening cells of
known antigenic type. If unexpected antibodies are
detected, then they are identified and blood unit that
lacks the corresponding antigen is selected for compati-
bility test.
BIBLIOGRAPHY
1. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
2. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical
Hematology (9th Ed). London: Churchill Livingstone,
2001.
 38

Adverse Effects of
Transfusion

Blood transfusion is a life saving procedure in an
appropriate setting and there are no side-effects in
majority of cases. However, it is a potentially harmful
procedure and every recipient of transfusion is at risk of
an adverse reaction. It should be prescribed only if there
is a definite clinical indication. This is because even with
best possible blood banking standards, transmission of
infections or other complications can occur.
Adverse effects of transfusion are listed in Table 38.1.
Main causes of transfusion-related deaths are:
• Immediate acute hemolytic transfusion reaction (ABO
incompatibility)
• Pulmonary edema and congestive heart failure
(circulatory overload)
• Bacterial contamination of blood unit
• Transfusion of physically damaged red cells (e.g. by
heat, cold)
• Transfusion-associated graft vs. host disease
the recipient. It results from transfusion of ABO-
mismatched blood to the recipient due most commonly
to a clerical error. Most severe reaction occurs if group A
blood is transfused to a group O recipient.
Pathophysiology consists of antigen-antibody
reaction that leads to complement activation and
intravascular hemolysis. This causes hypotension, shock,
acute renal failure, and disseminated intravascular
coagulation (Fig. 38.1). Signs and symptoms (that appear
within minutes of starting transfusion) include fever,
pain at the infusion site, loin pain, tachycardia,
hemoglobinuria, and hypotension. In anesthetized
patients, bleeding and hypotension are the only
indications.

ACUTE HEMOLYTIC TRANSFUSION

REACTION
This is a medical emergency and results from intra-
vascular destruction of donor red cells by antibodies in

Table 38.1: Adverse effects of transfusion

Immediate Delayed
1. Acute hemolytic 1. Delayed hemolytic
transfusion reaction transfusion reaction
2. Febrile non-hemolytic 2. Transmission of infections
transfusion reaction
3. Allergic reactions 3. Iron overload
4. Anaphylactic reactions 4. Graft vs. host disease
5. Transfusion-associated 5. Post-transfusion purpura
lung injury
6. Volume overload
7. Bacterial contamination
of donor unit Fig. 38.1: Pathophysiology of acute hemolytic
transfusion reaction

Laboratory features are:
• Hemoglobinemia (pink coloration of plasma after
centrifugation of post-transfusion sample)
• Positive direct antiglobulin test
• Hemoglobinuria
• Schistocytes (fragmented red cells) and spherocytes
on blood smear
• Elevated indirect serum bilirubin
FEBRILE NON-HEMOLYTIC
TRANSFUSION REACTION
This is the most common transfusion reaction. It occurs
in about 1% of all transfusions and is defined as an
unexplained rise of temperature of atleast 1°C during or
shortly after transfusion. It is caused by the release of
pyrogenic cytokines from white cells (during storage of
blood unit or following transfusion due to reaction of
alloantibodies with white cells of donor). This reaction
is common in multiply-transfused patients. Signs and
symptoms include fever, chills, and tachycardia.
Diagnosis depends on exclusion of other causes of febrile
transfusion reaction.
Transfusion reactions presenting with fever are
shown in Figure 38.2.
BACTERIAL CONTAMINATION OF
DONOR UNIT
Transfusion of an infected blood product is more
common with platelet concentrates since platelets are
stored at a higher temperature (20-24°C) that promotes
multiplication of contaminating bacteria. Organisms
depend on the nature of blood product. Platelets are
usually contaminated with gram-positive cocci, while red
cells are contaminated with Yersinia enterocolitica,
Escherichia coli, or Pseudomonas species.
Signs and symptoms include high grade fever with
rigors, hypotension, and shock. Laboratory studies
include inspection of blood bag for discoloration, and
Fig. 38.2: Transfusion reactions presenting with fever
Adverse Effects of Transfusion 355
Gram staining and culture of blood from the blood bag
and from the recipient. Direct antiglobuin test is negative.
TRANSFUSION-ASSOCIATED LUNG INJURY
This is an acute respiratory disorder that manifests with
fever, chills, dyspnea, and dry cough. X-ray shows diffuse
pulmonary infiltrates. One probable mechanism is
reaction of anti-HLA or anti-neutrophil antibodies in
donor blood with leukocytes of the recipient leading to
the formation of leukocyte aggregates; these aggregates
deposit in pulmonary vasculature and cause increased
vascular permeability and pulmonary edema.
DELAYED HEMOLYTIC
TRANSFUSION REACTION
This is a hemolytic transfusion reaction occurring several
days or weeks after transfusion. This occurs in indi-
viduals who have been sensitized to a red cell antigen
by a previous transfusion or pregnancy so that the
antibody is present in a low titer. On re-exposure, there
is a secondary IgG immune response and mainly
exravascular hemolysis. This reaction is typically
associated with Kidd antibodies.
Signs and symptoms include fever, mild jaundice,
and mild anemia. Laboratory features include raised
indirect serum bilirubin, spherocytes on blood smear,
anemia, and positive direct antiglobulin test.
Acute and delayed hemolytic transfusion reactions
are compared in Table 38.2.
ANAPHYLACTIC REACTION
This rare reaction occurs in IgA-deficient recipients in
whom anti-IgA antibodies react with IgA in donor
plasma, leading to activation of complement and
formation of anaphylatoxins (C3a and C5a). Signs and
symptoms include development of acute hypotension,
shock, and dyspnoea after transfusion of a few drops of
blood.
356 Essentials of Clinical Pathology
Table 38.2: Comparison of acute and delayed hemolytic transfusion reactions
Parameter Acute
1. Type of antibody Anti-ABO
2. Nature of antibody Naturally-occurring
3. Time of appearance of Within minutes
clinical features after
transfusion
4. Site of hemolysis Intravascular
5. Clinical features Fever, chills, back pain,
acute renal failure, DIC
ALLERGIC REACTION
This results from type I hypersensitivity reaction to some
donor plasma proteins. It is the second most frequently
reported transfusion reaction. Signs and symptoms
include mild urticaria, rash, and pruritus.
VOLUME OVERLOAD
This occurs if transfusion rate is too rapid, or excessive,
or if cardiac or renal impairment is present. It causes
cardiac failure and lung edema.
IRON OVERLOAD
Each unit of blood contains 200 mg of iron. Patients
receiving regular transfusion therapy such as those with
thalassemia develop manifestations of parenchymal
damage due to iron accumulation.
TRANSMISSION OF INFECTIONS
Organisms transmissible by transfusion are listed in
Table 36.1 (See Chapter 36: Screening Tests for Infections
Transmissible by Transfusion).
1. Hepatitis A virus: Hepatitis A is rarely transmitted
by transfusion, as the duration of viremia is short.
Donors with hepatitis A or who are in close contact
with a hepatitis A are deferred for 1 year.
2. Hepatitis B virus: In India, prevalence of hepatitis B
virus is 1.5 to 4%, and there are 43 million carriers.
Hepatitis B virus, a DNA virus, can be transmitted
by both cellular and plasma components. Incubation
period is 2-6 months. All blood donations are tested
for HBsAg by a sensitive method that has greatly
reduced the risk of transmission. During early period
of infection, HBsAg may be undetectable; during this
window period, antibodies to the hepatitis B core
Delayed
Anti-Kidd
Immune
After several days or weeks
Extravascular
Fever, mild jaundice, anemia
antigen (anti-HBc) may be the only evidence of disease.
HBsAg-positive donors are permanently excluded from
donations.
Liver diseases caused by hepatitis B virus include:
subclinical hepatitis, acute icteric hepatitis, fulminant
hepatitis (massive hepatic necrosis), chronic hepatitis,
cirrhosis, and hepatocellular carcinoma.
3. Hepatitis C virus: In India, prevalence of hepatitis C
virus is reported to be 1.66% and there are 15 million
carriers. This RNA virus is the most common cause
of transfusion-transmitted hepatitis. It is transmitted
by both cellular and plasma components. Incubation
period is about 8 weeks. Persistent infection is the
rule. Chronic hepatitis is very common (usually mild).
Cirrhosis occurs in a minority of patients, and
hepatocellular carcinoma occurs in about 10% with
cirrhosis.
The test used for donor screening is anti-HCV
antibody test. From infection till the appearance of
anti-HCV positivity (70-80 days), HCV RNA can be
detected by PCR testing.
4. Human immunodeficiency virus (HIV): In India,
adult prevalence is 0.7%. Two genetically different
but closely related viruses are recognized: HIV-1 and
HIV-2. Both are RNA retroviruses. Natural history
of HIV infection consists of (i) acute, self-limited, “flu-
like” illness occurring 3-6 weeks after infection, (ii)
chronic phase lasting for several years that may be
asymptomatic or associated with persistent lymph-
adenopathy, and (iii) final, full-blown phase with
opportunistic infections and malignancies. HIV can
be transmitted by both cellular and plasma compo-
nents.
The test used for detection of infection is anti-
HIV-1/2 antibodies by enzyme immunoassay. To

reduce the window period from infection to appea-
rance of antibodies from about 22 days to 10 days,
nucleic acid testing (NAT) for HIV RNA is
recommended.
5. Treponema pallidum: Trasfusion-transmission of
syphilis is rare since T. pallidum does not survive in
refrigerated storage and is inactivated at 4°C after 4
days; however, fresh blood and platelet concentrates
can transmit the organism. The main value of
screening test is as a marker of high-risk behavior.
6. Malaria parasites: Malaria parasites are readily
transmitted by transfusion. In endemic areas, it is not
practical to reject all potential donors with history of
malaria in the past. In endemic areas, the only safe
prevention is administration of preventive anti-
malarial drugs to all recipients of transfusion.
COMPLICATIONS ASSOCIATED WITH
MASSIVE TRANSFUSION
Massive transfusion refers to transfusion of stored blood
equivalent to patient’s blood volume in 24 hours.
Morbidity and mortality is due to rapid blood loss
coupled with transfusion of stored blood.
Storage of blood is associated with loss of 2,3-
diphosphoglycerate, lowering of pH, loss of ATP, loss of
platelet function, and depletion of coagulation factors.
Microaggregates composed of leukocytes and platelets
Fig. 38.3: Immediate management of a suspected transfusion reaction. All reactions should be assumed to be
hemolytic and investigated accordingly
Adverse Effects of Transfusion 357
form gradually in stored blood. Rapid transfusion of large
volumes of stored blood leads to:
• Dilution of platelets and coagulation factors
• Hyperkalemia (due to release of potassium from
stored red cells)
• Hypocalcemia (due to binding of calcium by citrate)
• Hypothermia (due to rapid infusion of large amount
of cold blood)
• Adult respiratory distress syndrome due to migration
of microaggregates to lungs.
RECOGNITION AND INVESTIGATION OF A
TRANSFUSION REACTION
All reactions following blood transfusion should be
considered as hemolytic in nature and should be
investigated accordingly (Fig. 38.3).
1. Transfusion should be immediately stopped, leaving
open intravenous line with normal saline.
2. All paperwork and blood bag should be checked for
clerical error. More than 90% of hemolytic trans-
fusions result from a clerical error (i.e. a wrong unit
of blood is given to the wrong recipient).
3. Blood bank is informed immediately and the blood
bag, administration set, and post-transfusion blood
and urine samples should be sent to the blood bank.
4. Evidence of hemolysis: Obtain a post-transfusion
blood sample from the recipient, centrifuge, and
observe for pink discoloration of overlying plasma
(hemoglobinemia); this is the most rapid way of
358 Essentials of Clinical Pathology
detecting intravascular hemolysis if pre-transfusion
sample is normal. Similarly, visual examination of
patient’s urine can be done for hemoglobinuria.
Blood smear examination will show fragmented red
cells and spherocytes. Indirect serum bilirubin is
raised.
5. Evidence of blood group incompatibility: Perform
a direct antiglobulin test (DAT) on post- and pre-
transfusion blood samples. Positive DAT on post-
transfusion sample (with negative test on pre-
transfusion sample) is indicative of an immunological
hemolytic transfusion reaction. Blood group incom-
patibility will also be revealed on (i) repeat ABO
grouping on recipient’s pre- and post-transfusion
samples and on donor unit, and (ii) repeat cross-
matching of donor blood against recipient’s pre- and
post-transfusion samples.
6. Investigations for detection of complications of
hemolytic transfusion reaction:
• Tests for disseminated intravascular coagulation:
Blood smear, coagulation screen, and test for fibrin
degradation products
• Tests for acute renal failure: Blood urea, serum
creatinine, and serum electrolytes
7. Bacteriological culture if the cause of the acute
transfusion is still not clear.
BIBLIOGRAPHY
1. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
2. World Health Organization: Blood Transfusion Safety:
The Clinical Use of Blood. Geneva: World Health
Organization, 2002.
 39

Blood Components

A single whole blood donation can be separated into
different components to provide treatment to more than
one patient. One unit of whole blood can be broken down
into one unit of packed red cells, one unit of platelets,
and one unit of fresh frozen plasma/cryoprecipitate. This
practice avoids wastage of collected whole blood (each
component is stored at a temperature that is optimal for
that component), allows administration of specific
replacement therapy, and also avoids transfusion of
unnecessary blood elements that are not required by the
patient.
Terms used in transfusion therapy are shown in Table
39.1.
There are two methods for collection of blood for
preparation of blood components:
1. Single whole blood donation: Preparation of blood
components has been greatly facilitated by the
introduction of double and triple bags having closed
integral tubing. After collection of a unit of whole
blood in the primary bag, blood components can be
separated from one another by differential centrifu-
gation due to differences in their specific gravities. After
their separation, various components can be
transferred from one bag to another in a closed circuit
thus avoiding exposure to external environment and
maintaining the sterility. Blood should be processed
for component separation within 6 hours of collection
(Figs 39.1 and 39.2).
2. Apheresis: This is a procedure in which a suitable donor
is connected to an automated cell separator machine
(that is essentially designed as a centrifuge) through
which whole blood is withdrawn, the desired blood
component is retained, and the remainder of the
blood is returned back to the donor. Depending on
the component that is separated and removed, the
procedure is called as plateletpheresis, leukapheresis,
or plasmapheresis.
WHOLE BLOOD
Whole blood is one unit of donor blood collected in a
suitable anticoagulant-preservative solution (citrate
phosphate dextrose adenine or CPDA-1). Its total volume

Table 39.1: Definitions used in transfusion therapy

Blood product
A therapeutic substance prepared from human blood
Whole blood
One unit of non-separated donor blood collected in an appropriate container containing anticoagulant-preservative
solution
Blood component
A constituent separated from whole blood by differential centrifugation or that is obtained directly from donor by apheresis
Plasma derivative
Human plasma proteins obtained from multiple donor units of plasma under pharmaceutical manufacturing conditions.
These products are heat-treated or chemical-treated to inactivate lipid-enveloped viruses.

Plasma derivatives like factor concentrates and immunoglobulins can also be prepared by recombinant DNA technology
360 Essentials of Clinical Pathology

Fig. 39.1: Blood components are prepared from a unit of whole blood within 6 hours of collection. Initial light centrifugation
separates red cells from platelets and plasma. Heavy centrifugation of platelet-rich plasma separates platelets and plasma

Fig. 39.2: Principle of preparation of blood components from one unit of whole blood

is about 400 ml (350 ml of blood + 49 ml of anticoagulant).
It consists of cellular elements and plasma. Whole blood
is stored in an approved blood bank refrigerator at
4°-6°C. Shelf life of such blood (collected in CPDA
anticoagulant) is 35 days. It does not contain functionally
effective platelets and labile coagulation factors (F V and
F VIII). Transfusion of whole blood should commence
within 30 minutes of removal from the refrigerator, and
should be complete within 4 hours of starting. Trans-
fusion of one unit raises hemoglobin by 1 gm/dl or
hematocrit by 3%.
Indications and contraindications for whole blood
transfusion are given in Table 39.2.

Table 39.2: Indications and contraindications for whole
blood transfusion
Indications
• Acute blood loss with hypovolemia
• Exchange transfusion in neonates
• Non-availability of red cell concentrate or suspension
Contraindications
• Chronic anemia with compromised cardiovascular
function
BLOOD COMPONENTS
Blood components are listed in Table 39.3.
RED CELL COMPONENTS
1. Packed red cells: Packed red cells are prepared by
removing most of the plasma from one unit of whole
blood (hematocrit 70-75%). Whole blood is either
allowed to sediment overnight in a refrigerator at 2-
6°C or is spun in a refrigerated centrifuge. Super-
natant plasma is then separated from red cells in a
closed system by transferring it to the attached empty
satellite bag. Red cells and a small amount of plasma
are left behind in the primary blood bag. Packed red
cells have a high viscosity and therefore the rate of
infusion is slow. Transfusion of one unit of red cells
increases hemoglobin by 1 gm% (or increases
hematocrit by 3%).
Indications for packed red cells are shown in
Table 39.4.
2. Red cells in additive solution (Red cell suspension):
These are red cells with minimal residual plasma and
an additive solution (SAG-M which contains saline,
adenine, glucose, and mannitol). This increases shelf
life from 35 days to 42 days. After collection of whole
Table 39.3: Blood components
Cellular components
• Red cells: Packed red cells, red cells in additive
solution, leukocyte-poor red cells, washed red cells,
frozen red cells, irradiated red cells
• Platelets: Platelet concentrate, apheresis platelets
• Granulocytes: granulocyte concentrate
Plasma components
• Fresh frozen plasma
• Cryoprecipitate
Blood Components 361
Table 39.4: Indications for packed red cell transfusion
• Anemia: Chronic severe anemia, severe anemia with
congestive cardiac failure, anemia in elderly
• Acute blood loss (transfused along with a crystalloid
or a colloid solution)
blood in the primary collection bag (containing CPDA-
1), maximum amount of plasma is removed (after
centrifugation) and transferred to one satellite bag. The
additive solution from the second satellite bag is
transferred into the primary collection bag (containing
packed red cells) in a closed system.
Indications for red cells in SAG-M are similar to
those for packed red cells.
3. Leukocyte-poor red cells: Leukocyte-poor red cells
contain < 5 × 106 white cells per bag. Methods for
leukocyte depletion are (i) leukocyte-reduction filters,
and (ii) removal of buffy coat. Indications for
leucocyte-poor red cells are: (i) prevention of HLA
immunization in patients who are likely to receive
allogeneic bone marrow transplantation, (ii) preven-
tion of febrile nonhemolytic transfusion reactions in
persons receiving multiple transfusions, and (iii)
prevention of transmission of cytomegalovirus.
4. Washed red cells: Red cells can be washed with
normal saline to remove plasma proteins, white cells,
and platelets. Such red cells are used for IgA-deficient
individuals who have developed anti-IgA antibodies,
as exposure will lead to anaphylaxis.
5. Frozen red cells: If a cryoprotective agent such as
glycerol is added, red cells can be stored frozen for
upto 10 years. This method can be used for storage
for donor red cells with rare blood groups, for future
autologous transfusion, and for individuals who have
repeated febrile nonhemolytic transfusion reactions.
6. Irradiated red cells: Gamma-irradiation of red cells
inactivates lymphocytes and prevents graft vs. host
disease. Irradiated red cells are indicated for
intrauterine or premature neonate transfusions, and
in individuals with immunodeficiency, and in those
receiving blood from first-degree relative donors.
PLATELETS
Platelet concentrates can be obtained from single donor
units or by plateletpheresis.
1. Platelet concentrate (Random donor platelets prepared from
whole blood unit): One unit of whole blood is
centrifuged (light spin) to obtain platelet-rich plasma
362 Essentials of Clinical Pathology
(PRP). PRP is then transferred to the attached satellite
bag and spun (high spin) to get platelets at the bottom
and supernatant plasma. Most of the supernatant is
returned back to the primary collection bag or to
another satellite bag, leaving behind 50-60 ml of
plasma with the platelets.
Platelets are stored at 20°-24°C with continuous
agitation (in a storage device called platelet agitator).
Maximum period of storage is 5 days.
One unit of platelet concentrate contains > 45 ×
109 platelets. Transfusion of one unit will raise the
platelet count in the recipient by about 5000/μl.
The usual adult dose is 4-6 units of platelet
concentrate (or 1 unit/10 kg of body weight). These
units (which are from different donors) are pooled
into one bag before transfusion. This dose will raise
the platelet count by 20,000 to 40,000/μl.
2. Plateletpheresis (Single donor platelets): In platelet
pheresis, a donor is connected to a blood cell separator
machine in which whole blood is collected in an
anticoagulant solution, platelets are separated and
retained, and remaining components are returned
back to the donor. With this method, a large number
of platelets can be obtained from a single donor
(equivalent to 6 units of platelet concentrate). This
method is especially suitable if HLA-matched
platelets are required (i.e. if patient has developed
refractoriness to platelet transfusion due to the
formation of alloantibodies against HLA antigens).
The usual indications and contraindications for
administering platelets are shown in Table 39.5.
PLASMA COMPONENTS
The main plasma components are fresh frozen plasma
and cryoprecipitate.
1. Fresh frozen plasma (FFP): FFP is prepared from
whole blood within 6 hours of collection because after
this time labile coagulation factors are lost. Plasma is
Table 39.5: Indications and contraindications to
platelet transfusions
Indications
• Bleeding due to decreased platelet production
• Bleeding in hereditary disorders of platelet function
• Massive blood transfusion
Contraindications
• Thrombotic thrombocytopenic purpura
• Hemolytic uremic syndrome
Table 39.6: Indications for fresh frozen plasma
• Multiple coagulation factor deficiencies: liver disease,
warfarin overdose, massive blood transfusion
• Disseminated intravascular coagulation
• Inherited deficiency of a coagulation factor for which
no specific replacement therapy is available
• Thrombotic thrombocytopenic purpura
separated from whole blood by centrifugation,
expressed into the attached satellite bag, and rapidly
frozen at –20°C or at lower temperature. FFP contains
all the coagulation factors.
FFP can be stored for 1 year if temperature is
maintained below –25°C. When required for transfusion,
FFP is thawed between 30-37°C and then stored in the
refrigerator at 2-6°C. Since labile coagulation factors
rapidly deteriorate, FFP should be transfused within 2
hours of thawing.
Indications for FFP are shown in Table 39.6.
2. Cryoprecipitate: Cryoprecipitate is prepared from
plasma that has been freshly separated (within 6
hours of collection) by rapidly freezing it at -20°C or
lower and thawing it slowly at 4-6°C. A white
flocculent precipitate and plasma are obtained. The
mixture is centrifuged and supernatant plasma is
removed leaving behind sediment of cryoprecipitate
suspended in 10-20 ml of plasma. The unit is then
refrozen (-20°C or colder) and can be stored at this
temperature for 1 year. When needed, cryoprecipitate
is thawed at 30-37°C, required donations are pooled
and transfused to the patient. Cryoprecipitate
contains F VIII, von Willebrand factor, fibrinogen, F
XIII, and fibronectin. Indications for cryoprecipitate
are F VIII deficiency (if F VIII concentrate is not
available), von Willebrand disease, and deficiency of
fibrinogen.
PLASMA DERIVATIVES
Plasma derivatives are manufactured by fractionation of
large volumes of pooled human plasma. Important
plasma derivatives are listed in Table 39.7.
1. Human albumin solutions: Albumin is prepared by
cold ethanol fractionation of pooled plasma and is
sterilized during manufacture to destroy viruses and
bacteria. Albumin is used as a replacement fluid in
therapeutic plasma exchange, and for treatment of
diuretic-resistant edema of hypoproteinemia.
tahir99 - UnitedVRG
vip.persianss.ir

Table 39.7: Plasma derivatives
1. Human albumin solutions
2. F VIII concentrate
3. F IX concentrate
4. Prothrombin complex concentrate
5. Immunoglobulins
2. F VIII concentrate: Freeze-dried F VIII concentrate is
prepared by fractionation from large pools of fresh
frozen plasma. To reduce the risk of transmission of
viral infections, it is treated with heat or chemicals
during manufacturing process. F VIII concentrate is
the treatment of choice for treatment of hemophilia
A and severe von Willebrand disease.
3. Prothrombin complex concentrate (PCC): PCC
contains factors II, VII, IX, and X, and also protein C
and S. Main uses of PCC are (i) deficiency of F IX, (ii)
deficiency of F VIII with development of inhibitors
against F VIII, and (iii) inherited deficiency of factors
II, VII, and X. A serious risk of PCC is thrombotic
complications due to the presence of small amounts
of activated coagulation factors.
4. Immunoglobulins: Immunoglobulins are obtained
by cold ethanol fractionation of large pools of human
plasma. They are of two types: specific and non-
specific.
Blood Components 363
a. Non-specific immunoglobulins: These are derived
from the pooled plasma of non-selected donors.
Some indications include (i) passive prophylaxis
of viral infections like hepatitis, rubella, and
measles, (ii) treatment of hypogamma-
globulinaemia, (iii) autoimmune thrombocyto-
paenic purpura to induce a rise in platelet count,
and (iv) neonatal sepsis.
b. Specific immunoglobulins: They are obtained from
donors who have selected high titer IgG antibodies.
Anti-RhD immunoglobulin is prepared from
plasma of Rh-negative donors who have produced
anti-D following immunization; it is used for
prevention of sensitization to RhD antigen in Rh-
negative women giving birth to a Rh-positive baby.
Other specific immunoglobulins include hepatitis
B immune globulin, varicella-zoster immune
globulin, and tetanus immune globulin that are
used for passive prophylaxis of infections.
BIBLIOGRAPHY
1. Kawthalkar SM. Essentials of Hematology. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd, 2006.
2. World Health Organization. Blood Transfusion Safety:
The Clinical Use of Blood. World Health Organization.
Geneva, 2002.
tahir99 - UnitedVRG
vip.persianss.ir
 General References

1. Cheesbrough M. District laboratory practice in tropical countries. Part 1 and Part 2. Cambridge. Cambridge University
Press, 1998.
2. Crook MA. Clinical chemistry and metabolic medicine (7th Ed). London: Edward Arnold (Publishers) Ltd, 2006.
3. Wallach J. Interpretation of diagnostic tests (7th Ed). Philadelphia: Lippincott Williams and Wilkins, 2000.
4. Mitchell RN, Kumar V, Abbas AK, Fausto N. Robbins and Cotran pathologic basis of disease (7th Ed). Philadelphia:
Saunders, 2006.
5. Henry JB. Clinical diagnosis and management by laboratory methods (20th Ed). Philadelphia: WB Saunders Company,
2001.
6. Burtis CA, Ashwood ER. Tietz fundamentals of clinical chemistry (5th Ed). Philadelphia: WB Saunders Company,. 2001.
7. World Health Organization. Manual of basic techniques for a health laboratory (2nd Ed). Geneva: World Health
Organization, 2003.
8. Provan D, Krentz A. Oxford Handbook of Clinical and Laboratory Investigation. Oxford. Oxford University Press, 2002.
9. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Haematology (9th Ed). London: Churchill Livingstone, 2001.
10. Provan D, Singer CRJ, Baglin T, Lilleyman J. Oxford Handbook of Clinical Haematology (2nd Ed). Oxford: Oxford
University Press, 2004.
11. King M. A medical laboratory for developing countries. London: Oxford University Press, 1973.
12. Kawthalkar SM. Essentials of Haematology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, 2006.
13. Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart MJ, Shepherd J. Clinical biochemistry. An illustrated colour test.
3rd Ed. Edinburgh: Churchill Livingstone, 2004.
14. Hoffbrand AV, Pettit JE, Moss PAH. Essential Haematology (4th Ed). Oxford. Blackwell Science Ltd, 2001.
15. Chatterjee K, Sen A. Step by step blood transfusion services. A practical manual on the technical and clinical aspects.
New Delhi: Jaypee Brothers and Medical Publishers (P) Ltd, 2006.

tahir99 - UnitedVRG
vip.persianss.ir
 Index

A Amylase 97, 135 Basal

Abnormal Anaphylactic reaction 355 acid output 123, 125
coagulation profile 66 Anemia of chronic disease 248 body temperature 156
crystals 28 Angina pectoris 76 Basic laboratory studies in anemia 260
red cell arrangement 206 Animal inoculation studies 242 Basophilic stippling 205
ABO Antibodies of ABO system 331 Basophils 174, 203, 207
grouping 336 Antibody screening and identification Bence Jones proteinuria 11
system 329 353 Benedict’s qualitative
Abortion 148 Antigens of solution 13
Acetest tablet ABO system 330 test 13
method 15 Rh system 332 Benedict’s test 14
test 15 Antiglobulin test 270 Bentiromide test 134
Acid Antiphospholipid syndrome 312 Bernard Soulier syndrome 293
Antisperm antibodies 164 Bile
load test 37
Antithyroid antibodies 143 pigment 16
phosphatase 166
Anuria 5 salts 17
Acquired
Aplastic anemia 249 Biliary peritonitis 67
disorders of coagulation 296
Apolipoproteins 71 Bilirubin 16, 97
inhibitors of coagulation 296
Appearance of sputum 99 crystals 28
Activated
Approach to diagnosis of Bioassays 148
partial thromboplastin time 303
anemia 259 Biochemical
protein C resistance 311
bleeding disorders 297 analysis of semen 165
Acute
Apt test 118 cardiac marker 74
coronary syndrome 74
Ascaris lumbricoides 112 studies 77
hemolytic transfusion reaction 354
Ascorbic acid 13 Biosynthesis of thyroid hormones 137
hepatitis 66
Asexual cycle 229 Blast cells 208
leukemias 273
Assessment of severity of Bleeding
myeloid leukemia 9
inflammatory disorders 218 diathesis 66, 82
phase reactants 218
Atypical lymphocytes 208 disorders 291
Advantages of automated hematology
Autohemolysis test 270 Blood 18
analyzer 319
Automated ammonia 59
Adverse effects of transfusion 354
hematology analyzer 319 biochemistry 33
Albumin 96 components 359, 361
method 301
Albuminuria 35 group
Automation in hematology 319
Alcoholic liver disease 66 A 330
Alicylates 13 B AB 330
Alimentary glycosuria 12 B cell B 330
Alkaline picrate reaction 34 development 175 O 330
Allergic reaction 356 ontogeny 175 systems 329, 335
Ammonium Bacteria 24, 350 mixed CSF 83
chloride loading test 37 Bacterial smear 200
magnesium phosphate 27 contamination of donor unit 355 transfusion 343
urate crystals 27 culture 81 urea nitrogen 33
Amorphous urates 27 proliferation 4 vessel wall 288

tahir99 - UnitedVRG
vip.persianss.ir
368 Essentials of Clinical Pathology

Boiling test 9 cell count in CSF 84 Clean-catch specimen 4

Bone marrow CK 78 Clearance of radiolabeled agents 32
aspiration 222, 224, 241 MCV 213 Clearance tests 32
smears 225 serum creatinine level 34 Clinitest tablet method 14
examination 266 total T4 142 Clot formation 84
iron stain 246 urobilinogen in urine 17 Clotting time 302
trephine biopsy 223, 224, 227 ketonuria 14 Coagulation system 289
Boric acid 5 low MCV 213 Coccidia 111
Breath malabsorption 118, 127 Collection methods 4
CO2 tests 131 male infertility 151 Collection of
hydrogen test 130, 131 pleural effusion 91 blood 179
tests 134 positive test 271 cerebrospinal fluid 80
Broad casts 26 prolongation of bleeding time 301 donor blood 344
Bromosulphthalein excretion test 63 proteinuria 8 pleural fluid 91
Bromothymol blue 7 Cell 22 sample 95, 123
Brugia malayi 239 count 93, 98 semen for investigation of infertility
Bulbourethral glands 159 ontogeny 175 159
Butter fat test 129 Cellular specimen for parasites 105
casts 27 sputum 99
C elements 3 urine 3
Cabot’s rings 206 Cephalic phase 121 Colorimetric methods 183
Calcium Cephalosporins 13 Combined acidity 124
carbonate crystals 27 Cerebrospinal fluid 80, 85 Commercial automated culture
hydrogen phosphate 27 systems 102
Cervical mucus penetration test 166
oxalate crystals 27 Compatibility test 352
Charcot joints 39
Capillary tube method 238 Complete blood count including blood
Cholestasis 63
Cardiac tamponade 224 smear 298
Cholesterol 69
Cardiorespiratory compromise 82 Complications associated with massive
crystals 28
Casts 25 transfusion 357
Christmas disease 294
Catabolism of steroid hormones 52 Complications of
Chromosomal analysis 152
Catheter specimen 4 bone marrow aspiration 224
Chronic
Causes of lumbar puncture 82
leukemias 280
Composition of normal
ascites 95 liver disease 66
cerebrospinal fluid in adult 80
decreased lymphocytic leukemia 283
urine 3
CSF pressure 83 myeloid leukemia 281
Computer-assisted semen analysis 166
serum creatinine level 34 renal disease 30
Concepts of universal donor and
total T4 143 Chylomicrons 69
recipient 332
erroneous results 323 Chylothorax 94
Congo red test 126
false Classic nitroprusside reaction 15
Conjugated
negative test 20, 118, 352 Classical hemophilia 294
bilirubin 56, 58
positive test 19, 118, 352 Classification of hyperbilirubinemia 56
female infertility 154 acute leukemias 273 Contraindications to
glycosuria 12 anemias 244 gastric analysis 123
hematuria 18 diabetes mellitus 39 lumbar puncture 82
hemoglobinuria 20 intestinal parasites of humans 107 Coombs’ test 270
increased lipoprotein disorders 72 Copper reduction
ALP 61 liver function tests 54 methods 13
BUN 34 renal function tests 30 tablet test 14

tahir99 - UnitedVRG
vip.persianss.ir
 Index 369

Correction for Detection of Double oxalate 182

abnormal solute concentration 7 antigen in stool samples 111 Drugs 12
dilution 7 filarial Dubin-Johnson syndrome 56
temperature 7 antigen 239 Duffy antigen 231
Correction of TLC for nucleated red DNA 240 D-xylose absorption test 130
cells 194 infection 218
Counting cells 194 microalbuminuria 11 E
Creatine kinase 78 nucleic acid sequences of malaria Ectopic pregnancy 147
Creatinine clearance 32 parasites 236 Eggs of schistosoma haematobium 25
Crigler-Najjar syndrome 55 parasites 104 Ehrlich’s aldehyde test 18
Criteria for selection of blood donors postoperative infection 219 Electrical
342 Determination of blood group conductivity 320
Crohn’s disease 211 substances 166 impedance 319
Cryptococcus neoformans 87 Determining cause of anemia 260 Emergency cross-match 353
Cryptosporidium parvum 111 Diabetes mellitus 4, 12, 39-41, 44 Endocrine
Crystals 27 Diacetyl monoxime urea method 34 component 133
present in Diagnosis of diseases 12
acid urine 27 malaria and other parasites in blood Endocytosis of colloid droplets 138
alkaline urine 27 229 Endogenous pathway 71
CSF renal disease 30 Endometrial biopsy 155
culture 88 Diagnostic thoracentesis 91 Endoscopic biopsy of ulcer in intestine
protein electrophoresis 88 Diazo method 58 110
Culture 102 Differential leukocyte count 85, 208 Entamoeba histolytica 108
Cushing’s syndrome 12 Diffusion of iodide 137 Enterobius vermicularis 114
Cyanmethemoglobin method 185 Dilution of blood 193 Enzymatic methods 35
Cyclospora cayetanensis 111 Direct Enzyme-linked immunosorbent assay
Cystatin C clearance 32 antiglobulin test 270 348
Cysteine crystals 28 bilirubin 58 Eosinophil 174, 203, 207
Cytogenetic analysis 280 fluorescent antibody assay 111 Eosinophilia 210
Cytological examination of sputum spectroscopic estimation 58 Epididymis 159
103 tests 133 Errors in
Cytoplasmic vacuoles 208 wet mount of CSF 87 blood collection 194
Directed donor 342 filling of chamber 194
D Disadvantages of automated pipetting 194
D-dimer test 94 hematology analyzer 319 Erythrocyte sedimentation rate 215,
Decrease in glucose 4 Disintegration of cellular elements 4 219
Decreased utilization of carbohydrates Disorders of Erythrocytic schizogony 230
14 blood vessels 292 Erythropoiesis 171
Deficiency of lipids 69 Escherichia coli 6, 7
antithrombin III 312 platelet 292, 293 Esophageal disease 94
protein C and S 312 thyroid 139 Esophagogastroduodenoscopy 126
Delayed hemolytic transfusion reaction Disseminated intravascular Estimation of
355 coagulation (DIC) 296 blood glucose 45
Demonstration of Distal tubular function 36 creatinine clearance from serum
eggs of A. lumbricoides 112 DNA diagnosis 242 creatinine 33
hookworm eggs 113 Döhle inclusion bodies 207 fecal
trophozoites 110 Donation interval 342 enzymes 134
Dental treatment 343 Donor reactions 345 fat 129, 135

tahir99 - UnitedVRG
vip.persianss.ir
370 Essentials of Clinical Pathology

glucose in CSF 86 Fecal Gilbert’s syndrome 55

hemoglobin 183, 268, 321, 344 fat 129 Glanzmann’s thrombasthenia 294
progesterone in mid-luteal phase osmotic gap 119 Glitter cells 24
157 pH 119 Glomerular proteinuria 8
protein 11 Female infertility 154 Glomerulonephritis nephrotic
red blood cell count 321 Fern test 156 syndrome 3
red cell distribution width 321 Ferric chloride test 15, 16 Glucose 12
Ethylene diamine tetra-acetic acid Fibrinolytic system 290 6-phosphate dehydrogenase
(EDTA) 181 Flagging 323 deficiency 231, 255
Evaluation of Floatation techniques 107 tolerance test 12
chronic diarrhea 104 Florence test 166 Glycated hemoglobin 47
clinical features in anemia 260 Flow cytometric analysis 272 Glycosuria 6, 35, 43, 48
dysentery 104 Fluorescence 320, 325 without hyperglycemia 12
malabsorption syndromes 127 microscopy 101, 236 Gmelin’s test 16
Examination of Foam test 16 Gram’s
ascitic fluid 96 Follicle stimulating hormone 150, 151 stained smear 25
blood smear 203 Follicular cell and proteolysis 138 smear 87
bone marrow 220 Formalin 5 Granular casts 26
cerebrospinal fluid 80 Formation of crystals 4 Graves’ disease 144
feces 104 Fouchet’s test 17 Gross appearance of cerebrospinal
marrow specimens 225 Fractional fluid 83
pleural fluid 93 excretion of sodium 35
seminal fluid 160 test meal 126 H
splenic aspirate 241 Haemophilus influenzae 82, 88, 100
Free
sputum 99 Ham’s acidified serum lysis test 271
acidity 124
smear 101 Hamster egg penetration assay 166
thyroxine 143
urine 3 Hay’s surface tension test 17
Fresh frozen plasma (FFP) 362
Excretion of metabolic waste products HDL-cholesterol 74
Froin’s syndrome 84
30 Heat and acetic acid test 9
Frozen red cells 361
Excretory function 52 Heinz bodies 199, 269
Functions of
Exocrine component 131 Helminths 112
cerebrospinal fluid 80
Exoerythrocytic schizogony 230 Hematopoiesis 52, 169
liver 52
Exogenous pathway 71
Further testing 318 Hemiglobincyanide method 185
Extrahepatic biliary obstruction 66
Hemocytometer 192
Exudates 93 G Hemodynamic proteinuria 9
F Gametogony 230 Hemoglobin 20, 172
Gasometric method 183 content 204
Factor V Leiden 311
Gastric D 254
Factors affecting
analysis 121 electrophoresis 267
erythrocyte sedimentation rate 215
phase 121 Hemolysis 17
renal function 30
Gastrin 132 Hemolytic
False reactions in ABO grouping 338
Fasting Gaucher’s disease 221 anemia 261
blood glucose 45 General metabolic functions 52 disease of newborn 256
glucose 3 Generalized aminoaciduria 35 uremic syndrome 293
plasma glucose 47 Gestational Hemophilia
Fatty casts 26 diabetes mellitus 41 A 294
Febrile non-hemolytic transfusion trophoblastic disease 148 B 294
reaction 355 Giardia intestinalis 110 Hemorrhage 18, 224

tahir99 - UnitedVRG
vip.persianss.ir
 Index 371

Hemorrhagic Hypothyroidism 140 semen analysis 159

disease of newborn 296 Hysterosalpingo-contrast sonography testing for thrombophilia 313
fluid 93, 96 157 urinalysis 3
Hemosiderin 20 Hysterosalpingography (HSG) 157 Indirect
Heparin 182 antiglobulin test 271, 353
induced thrombocytopenia 312 I bilirubin 58
Hepatic Identification of tests 134
injury 59 adult worms 112 urinary tract infection 21
jaundice 55 cause of dyslipidemia 74 Induction of sputum 99
schizogony 230 larvae of S. stercoralis 114 Infants 4
Hepatitis lipid disorder 73 Infection in spinal canal 82
A virus 356 malaria parasites 232 Infectious disease 157, 343
B virus 348, 356 rotavirus 105 Infertility 150
C virus 349, 356 trophozoites and cysts on stool 108 Infiltrative diseases 63
Hepatocellular injury 63 Iliac spines 222 Inherited
Hereditary Illness 343
disorders of coagulation 294
disorders of hemoglobin 250 Immediate spin cross-match 352
thrombophilia 311
spherocytosis 254 Immune
Insulin
Herniation of brain 82 hemolytic anemias 255
hypoglycemia test 125
High thrombocytopenic purpura 292
resistance syndrome 42
density lipoprotein 71 Immunochemical tests 118
Intermediate density lipoprotein (IDL)
fever 9 Immunoelectrophoresis 285
70
performance liquid Immunofixation electrophoresis 286
Interpretation of
chromatography 268 Immunological assays 148
liver function tests 63
Histoplasma capsulatum 212, 226 Immunophenotyping 278
screening tests 306
Histoplasmosis 221 Importance of HLA antigens 177
Intrahepatic cholestasis 56
Hodgkin’s disease 211 Increase in pH 4
Intraperitoneal hemorrhage 67
Hollander’s test 125 Increased
Intravascular hemolysis 9, 264
Hookworms 113 destruction in peripheral blood 210
Inulin clearance 32
Hormonal studies 152 sequestration in spleen 210
Investigation of
Howell-Jolly bodies 199, 205 soluble transferrin receptor 246
male infertility 152
Human testosterone 157
pyrexia of unknown origin 221
albumin solutions 362 Indications and limitations of liver
function test 53 Iron
chorionic gonadotropin 146
Indications for deficiency anemia 245
cycle 229
abdominal paracentesis 95 overload 356
immunodeficiency virus 343, 349,
bone marrow staining of bone marrow aspiration
356
aspiration 221 smears 226
leukocyte antigens 175
biopsy 221 studies 265
Hydatid cyst of liver 67
examination 221 Irradiated red cells 361
Hydrochloric acid 5, 122
gastric analysis 122 Isomorphic red cells 23
Hyperglycemia 43
Hyperhomocysteinemia 312 hemoglobin 183 Isoniazid 13
Hyperosmolar hyperglycemic state lumbar puncture 81 Isospora belli 111
(HHS) 44 measurement of erythrocyte Ivy’s method 301
Hypersegmented neutrophils 208 sedimentation 216
Hyperthyroidism 139 renal J
Hypertonic urine 22 biopsy 37 Jaffe’s reaction 34
Hypo-osmotic swelling of flagella 166 function tests 30 Jaundice 54

tahir99 - UnitedVRG
vip.persianss.ir
372 Essentials of Clinical Pathology

K Life cycle of Mediastinitis 224

Kala azar 221 Leishmania donovani 240 Megaloblastic anemia 246
Karyopyknotic index (KI) 156 malaria parasites 229 Membrane filtration 238
Kawamoto technique 236 Light Metabolic
Ketones 14 absorption 320 actions of insulin 39
Ketosis 43 scatter 325 alterations in diabetes mellitus 43
Kidney function 30 Lipoproteins 69 syndrome 42
Klebsiella pneumoniae 100 Liquid biopsy of urinary tract 21 Metabolism of thyroid hormones 138
Litmus paper test 7 Method of gastric analysis 123
L Liver Methods for estimation of
Laboratory diagnosis of biopsy 64 BUN 34
acute leukemias 275 disease 296 erythrocyte sedimentation 216
visceral leishmaniasis 241 function tests 52 hemoglobin 183
cerebrospinal fluid 82 transplantation 66 serum creatinine 34
Laboratory testing in thrombophilia Loeffler’s syndrome 210 Methods for HLA antigen typing 177
313 Loop of Henle 8 Micro method 189
Laboratory tests for Loss of ketone bodies 4 Microalbuminuria 11, 35, 49
diagnosis of diabetes mellitus 44 Low-density lipoprotein (LDL) 71 Microangiopathic hemolytic anemia
human chorionic gonadotropin 148 Lugol iodine test 16 258
lipoprotein disorders 73 Lundh meal 133 Microangiopathy 44
screening of diabetes mellitus 47 Lung diseases 210 Microcytic hypochromic anemia 221,
Laboratory tests in Lupus pleuritis 94 261
anemia 244 Luteinizing hormone 150 Microfilariae 25
bleeding disorders 288 Lymphatic filariasis 237
Microhematocrit tube 238
hematological malignancies 273 Lymphocytes 175, 207
Microplate method 338
management of acute metabolic Lympocytotoxicity test 177
Microscopic examination of
complication of diabetes mellitus 50 Lysed
blood for demonstration 238
porphyrias 314 capillary blood method 238
urinary sediment 19
thrombophilia 311 venous blood method 238
Microsporidia 111
Laboratory tests to assess
M Microvascular disease 44
glycemic control 47
Midstream specimen 4
long-term risks 49 Macro method 188
Miliary tuberculosis 221
Lactate dehydrogenase 97 Macroangiopathy 44
Macrocytic anemias 261 Mixed lymphocyte
Lactation 343
Macrophages 52 culture 177
Lactose tolerance test 130
Macrovascular disease 44 reaction 177
Laparoscopic liver biopsy 67
Malabsorption syndromes 127 Modes of transmission 231
Laparoscopy and dye hydrotubation
Malaria 229, 343 Molecular genetic
test 157
Male infertility 150 analysis 280
Latex agglutination tests 87, 88
Manual method 192, 299 techniques 177
LDL-cholesterol 74
Mastalgia 155 Molecular methods 102
Leishman stain 202
Maximum acid output (MAO) 123 Monocytes 174, 203, 207
Leishmania donovani 211, 226
Mean cell Moraxella catarrhalis 100
Leucine crystals 29
hemoglobin 213 Morphological classification of anemia
Leucocyte esterase test 21
Leukemoid reaction 209 concentration 214 213
Leukocyte-poor red cells 361 volume 213 Morphology 275

tahir99 - UnitedVRG
vip.persianss.ir
 Index 373

Morphology of Oral Phosphates 27

abnormal leukocytes 207 contraceptive therapy and
microfilariae on Romanowsky pregnancy 312
stained 238 glucose tolerance test 45
normal leukocytes 207 Organ transplantation 177
Mosquito cycle 230 Organisms 24, 211
Mycobacterium tuberculosis 87, 101 Orthostatic proteinuria 3
Myeloperoxidase (MPO) 276 Osmotic fragility test 269
Myocardial infarction 219 Ounting chamber with cover glass 192
Oval fat bodies 24
Myoglobin 20, 78
Overflow proteinuria 9
N Oxidation of
Naegleria fowleri 87 bilirubin to biliverdin 4
urobilinogen to urobilin 4
Nalidixic acid 13
Oxyhemoglobin method 186
NBT-PABA test 134
Neimann-Pick disease 221
P
Neisseria meningitidis 82
P. falciparum 231
Neonatal screening for hypothyroidism
P. ovale 231
144
P. vivax 231
Nephropathy 39
Packed
Nephrotic syndrome 6
cell volume 188
Neurogenic phase 121 red cells 361
Neutropenia 210 Pancreas 131
Neutrophilia 209 Pancreatic
Neutrophils 174, 203 disease 12, 94
Nitrite test 21 function tests 131
Nitroprusside test 16 Pancreolauryl test 134
Non-cellular casts 26 Pappenheimer bodies 199, 205
Non-endocrine diseases 12 Para-aminosalicylic acid 13
Non-specific esterase (NSE) 276 Parasite culture 242
Normal Parasites 203, 351
Paroxysmal nocturnal hemoglobinuria
absorption of carbohydrates 127
258
bilirubin metabolism 52
bone marrow 220 Peak acid output 123, 125
Pelger-Huet cells 208
crystals 27
Penicillins 13
gastric anatomy and physiology 121
Percutaneous
pregnancy 146
blind liver biopsy 66
Normocytic normochromic anemia 261
guided liver biopsy 67
Numerical abnormalities of leukocytes
plugged liver biopsy 67
209
Periodic acid Schiff (PAS) 278
Peripheral
O blood smear 260
Obstructive jaundice 18 neuropathy 39
OGTT in gestational diabetes mellitus Peritoneal fluid 95
46 pH
Oligoclonal bands 89 indicator paper 7
Oliguria 5 meter 7
Physiologic jaundice of newborn 56
Physiology of hemostasis 288
Plasma
cell dyscrasias 9, 283
components 362
derivatives 362
proteins 289
Plasmodium species 351
Platelet 203, 211, 288, 361
concentrate 361
count 299, 322
function analyzer 302
glycoprotein analysis 310
pheresis 362
Pleural
biopsy 94
effusion 91
fluid 91
Polychromatic cells 203
Polymerase chain reaction 87, 88, 177
Polymorphonuclear neutrophil 207
Polyuria 5
Postcoital test 165
Posthepatic jaundice 56
Postprandial blood glucose 45
Post-puncture headache 82
Post-renal proteinuria 9
Post-transfusion purpura 293
Postural proteinuria 9
Prediabetes 42
Pre-erythrocytic schizogony 230
Pregnancy 343
test 3, 146
Prehepatic jaundice 55
Preparation of
blood smear 200
slides 106
Preservation of urine sample 4
Primary
biliary cirrhosis 56, 66
sclerosing cholangitis 56, 66
Primed lymphocyte typing 177
Processing of
donor blood 345
marrow specimens 224
Production of vit. D3 30
Professional donors 342

tahir99 - UnitedVRG
vip.persianss.ir
374 Essentials of Clinical Pathology

Prostate 159 Reduction of intestinal bacterial flora Self-monitoring of blood glucose

Proteins 8, 97 18 (SMBG) 48
Proteinuria 6 Refractometer method 7, 58 Semen 159
Prothrombin Regular cycles 155 analysis 152, 159
complex concentrate 363 Regulation of blood pressure 30 Seminal
time 59, 302 Renal fluid 159
Protozoa 107 biopsy 37 vesicles 159
Pseudochylous effusion 93 failure 3 Sensitivity of test 13
Pseudomonas aeruginosa 100 function tests 30 Sequence of filling of tubes 182
Pus cells 23 transplantation 30 Serologic
Pyelonephritis 3 tubular epithelial cell 24, 27 methods 237
Pyloric part 121 Replacement blood donor 342 tests 110, 87, 88
Pyrexia 66 Response to oral iron therapy 246 Serological testing 177
Restriction fragment length Serum
Q polymorphism 177 albumin 59
Qualitative tests 147 Reticulocyte count 196261, 323 alkaline phosphatase 61
Quantitative Retinopathy 39 aminotransferases 60
buffy coat 236 Reverse passive hemagglutination bilirubin 56
estimation of assay 348 creatinine 34, 35
fecal fat 118 Reye’s syndrome 59 ferritin 245
proteins 10 Rh iron 245
antibodies 335 lipase 135
tests 147
D grouping 338 protein electrophoresis 59, 284
Queckenstedt’s test 81
system 332 triglycerides 73
R Rheumatoid effusion 94 Sexual cycle 230
Radioactive iodine uptake test 144 Role of Sickle cell
Random genetic factors in malaria 231 disorders 253
laboratory tests in diabetes mellitus slide test 266
blood glucose 45
44 Sideroblastic anemia 249
donor platelets 361
Rothera’s Significance of
Reaction and pH 7
nitroprusside method 15 erythrocyte sedimentation rate 215
Reagent strip
test 15 microalbuminuria 11
method 7, 14, 18
Rouleaux formation 338 Sims-Huhner test 165
test 7, 10, 15, 16
Round cells 163 Single
Red blood cells 22, 171
Roundworm 112 donor platelets 362
Red cell 203
radial immunodiffusion 286
casts 27 S Sites for bone marrow aspiration 222
components 361 Sahli’s acid hematin method 184 Skeletal muscle trauma 9
distribution width 214 Scattergram 323 Skin
enzymes 172 Schizogony 229 piercing 343
inclusions 205, 268 Screening tests for puncture 179
indices 213, 261 hemostasis 298 test 242
membrane 172 infections transmissible by Slide test 336
suspension 361 transfusion 347 Small lymphocytes 203
Red cells in additive solution 361 Secondary lipoprotein diseases 72 Solubility test for hemoglobin S 266
Red cells with abnormal Secretin-cerulin test 133 Sources of error in manual blood cell
shape 205 Sedimentation count 194
size 204 rate 215 Specific gravity 6
staining 204 techniques 107 method 186

tahir99 - UnitedVRG
vip.persianss.ir
 Index 375

Specific tests for hemostasis 307 T pancreatic function 133

Sperm T cell paroxysmal nocturnal
count 162 development 175 hemoglobinuria 271
function tests 165 ontogeny 175 porphyrins in erythrocytes and
morphology 162 Taenia plasma 318
motility 161 saginata 116 Thalassemias 250
viability 161 solium 115 Therapeutic thoracentesis 92
vitality 161 Tallqvist hemoglobin chart 184 Thick
Spermatozoa 24, 159 Tamm-Horsfall protein 8, 25 blood smear 238
Sperms 159 Telescoped urinary sediment 24 exudative fluid 93
Spinnbarkeit test 156 Tense ascites 67 viscous CSF 84
Spinous processes of lumbar vertebra Terminology in flow cytometry 324 Thoracentesis 91
222 Test for Thoracoscopy 95
Sporogony 230 fructose 165 Thrombin time (TT) 305
Spot check of gastric pH 126 malabsorption of fat 118 Thrombopoiesis 178
Squamous epithelial cells 24 occult blood in stools 117 Thrombotic thrombocytopenic
Stages of pancreatic arylesterase 134 purpura 293
erythrocyte sedimentation rate 215 porphobilinogen in urine 318 Thymol 5
iron deficiency 245 reducing sugars 119 Thyroid
Staining of blood smear 202 total porphyrins in function tests 137, 142
Staphylococcus aureus 100 feces 318 hormones 137
Statistical error 194 urine 318 scintiscanning 144
Stellar phosphate 27 urobilinogen in feces 119 stimulating hormone 142
Sterile’ pyuria 25 Testes 159 Thyrotropin releasing hormone
Sternum 222 Testicular biopsy 152 stimulation test 143
Straw-colored transudative fluid 93 Testing for ketone bodies 51 Tibia 222
Streptococcus Tests for detection of Time of collection 3
pneumoniae 100 anti-leishmania antibodies 241 Titration 124
pyogenes 100 bilirubin in urine 16 Toluene 5
Streptomycin 13 blood in urine 19 Total
Strongyloides stercoralis 113 glucose in urine 13 acidity 124
Subarachnoidal epidermal cyst 82 hemoglobinuria 20 cholesterol 73
Subdural hematoma 82 ketones in urine 15 iron binding capacity 245
Sucrose hemolysis test 271 occult blood in feces 117 leukocyte count 84, 192
Sulfonamide crystals 29 urobilinogen in urine 18 serum proteins 58
Sulphosalicylic acid test 10 Tests for thyroxine 142
Suspected evaluate Toxic granules 207
acute leukemia 221 glomerular function 30 Transferrin saturation 245
chronic lymphoid leukemias 221 tubular function 35 Transfusion associated lung injury 355
infections 221 gastric analysis 125 Transitional epithelial cells 24
myelodysplastic syndrome 221 glucose-6-phosphate Transvaginal ultrasonography 157
myeloproliferative disorders 221 dehydrogenase deficiency 270 Transvenous liver biopsy 67
plasma cell dyscrasia 221 hemoglobin S 266 Treponema pallidum 350, 357
storage disorder 221 malabsorption and pancreatic Trichomonas vaginalis 25
Synthesis of erythropoietin 30 function 127 Trichuris trichiura 113
Synthetic function 52 ovulation 155 Triglycerides 69

tahir99 - UnitedVRG
vip.persianss.ir
376 Essentials of Clinical Pathology

Triple phosphates 27 Urinometer method 6 Waxy cast 26

Trisodium citrate 182 Urobilinogen 17 WBC cytogram 323
Troponins 78 Uses of Wedge method 200
True chylous effusion 93 C-reactive protein 218 Westergren method 216, 219
Trypanosomes 87 PCV 188
White
Tube method 337 red cell indices 213
blood cells 23, 174
Tubeless gastric analysis 126
V cell 203, 206
Tuberculous pleural effusion 94
Vagina 166 casts 27
Tubes for collection of blood 182
Tubular proteinuria 9, 35 Vaginal cytology 156 count 96
Turk solution 84 Vas deferens 159 WHO hemoglobin color scale 185
Types of Vasectomy 166 Wilson’s disease 66
blood donors 341 Venous blood collection 179 Wintrobe
liver biopsy 65 Very low-density lipoproteins (VLDL) method 188, 218
Tyrosine crystals 29 70 mixture 182
Viral hepatitis 343 Wuchereria bancrofti 238
U Viruses 348
Ultrasonography (USG) 156 Visceral leishmaniasis 240 X
Unconjugated bilirubin 58 Viscosity 161 Xanthochromia 84
Unexplained cytopenia 221 Vitamin
Unstable angina 76 B12 and folate 265 Y
Urea clearance 33 K deficiency 296
Yeast cells 25
Urease-berthelot reaction 34 Volume of donation 343
Yersinia pestis 100
Uric acid crystals 27 von Willebrand disease (vWD) 294
Urinary Z
albumin excretion 49 W
Zeta sedimentation ratio (ZSR) 218
concentration of sodium 35 Washed red cells 361
Water Ziehl-Neelsen
Urine
bilirubin and urobilinogen 58 deprivation test 36 smear 87
osmolality 36 loading antidiuretic hormone technique 101
specific gravity 36 suppression test 36 Zollinger-Ellison syndrome 122

tahir99 - UnitedVRG
vip.persianss.ir