Ebook Essentials of clinical pathology Part 1

Essentials
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Clinical Pathology

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Essentials
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Clinical Pathology

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Associate Professor
Department of Pathology
Government Medical College
Nagpur, Maharashtra, India

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Shirish M Kawthalkar

®

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

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Published by
Jitendar P Vij
Jaypee Brothers Medical Publishers (P) Ltd

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4838/24 Ansari Road, Daryaganj, New Delhi - 110002, India,
Phone: +91-11-43574357, Fax: +91-11-43574314

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Essentials of Clinical Pathology
© 2010, Shirish M Kawthalkar

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First Edition: 2010
ISBN 978-93-80704-19-7
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Preface

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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
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
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
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.
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
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.
However, scope of this book does not allow microbiology and genetics to be included in this book.
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
Medical College, Akola, Dr Smt Deepti Dongaonkar, Dean, Government Medical College, Nagpur, Dr BB Sonawane,
Professor and Head, Department of Pathology, Government Medical College, Akola, and Dr WK Raut, Professor
and Head, Department of Pathology, Government Medical College, Nagpur, for encouraging me in undertaking
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
undertaking to publish this book, being patient with me during the preparation of the manuscript, and bringing it
out in an easy-to-read and reader-friendly format.
Although I have made every effort to avoid any mistakes and errors, some may persist and feedback in this
regard will be highly appreciated.
Shirish M Kawthalkar

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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

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Essentials of Clinical Pathology

Section 3
Practical Blood Transfusion
33.
34.
35.
36.
37.
38.
39.

Blood Group Systems ............................................................................................................................................
Blood Grouping ......................................................................................................................................................
Collection of Donor Blood, Processing and Storage ........................................................................................
Screening Tests for Infections Transmissible by Transfusion ......................................................................
Compatibility Test (Cross-match) .......................................................................................................................
Adverse Effects of Transfusion ............................................................................................................................
Blood Components .................................................................................................................................................

329
336
341
347
352
354
359

General References ...................................................................................................................................................... 365
Index ........................................................................................................................................................................... 367

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Chemical Pathology and
Related Studies

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1

Examination of Urine

COMPOSITION OF NORMAL URINE

COLLECTION OF URINE

Urinalysis is one of the most commonly performed
laboratory tests in clinical practice. Composition of
normal urine is shown in Table 1.1.

There are various methods for collection of urine. Method
of collection to be used depends on the nature of
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

1.
2.

3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.

Parameters

Values

Volume
Specific gravity
Osmolality
pH
Glucose
Proteins
Urobilinogen
Porphobilinogen
Creatinine
Urea nitrogen

Uric acid
Sodium
Potassium
Chloride
Calcium (low calcium diet)
Formiminoglutamic acid (FIGlu)
Red cells, epithelial cells, and white blood cells

600-2000 ml
1.003-1.030
300-900 mOsm/kg
4.6-8.0
<0.5 gm
<150 mg
0.5-4.0 mg
0-2 mg
14-26 mg/kg (men), 11-20 mg/kg (women)
12-20 gm
250-750 mg
40-220 mEq
25-125 mEq
110-250 mEq
50-150 mg
< 3 mg
<1-2/high power field


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Essentials of Clinical Pathology

Box 1.1: Collection of urine sample

Box 1.2: Collection of urine for routine and culture
examination

• 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.

Collection for routine urinalysis
For routine examination of urine, a wide-mouthed glass bottle
of 20-30 ml capacity, which is dry, chemically clean, leakproof, and with a tight fitting stopper is used. About 15 ml
of midstream sample is cleanly collected.

The random specimen is a single specimen collected
at any time of day. It is used for routine urine examination.
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.

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.

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. Subsequent stream is collected which is from the urinary
bladder.
2. Clean-catch specimen: This is recommended for

bacteriologic culture. In men, glans penis is sufficiently 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.

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.
•
•
•
•

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 phosphates 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 falsenegative test for bilirubin
• Oxidation of urobilinogen to urobilin causing falsenegative 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
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 preservatives can be added to the 24-hour urine sample:
• Hydrochloric acid: It is used for preservation of a 24hour 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.

5

Box 1.3: Physical examination
• Volume
• Color
• Appearance

• Odor
• Specific gravity
• 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 glomerulonephritis (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

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),

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
Red
Dark brown or black
Brown
Yellow
Yellow-green or
green

Deep yellow with
yellow foam
Orange or orangebrown
Milky-white
Red or orange
fluorescence with
UV light

Dilute urine (diabetes mellitus, diabetes insipidus, overhydration)
Hematuria, Hemoglobinuria, Porphyria, Myoglobinuria
Alkaptonuria, Melanoma
Hemoglobinuria
Concentrated urine
Biliverdin
Bilirubin
Urobilinogen
Porphobilinogen
Chyluria
Porphyria

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
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, tyrosinaemia.
• Ammoniacal: Urinary tract infection with Escherichia
coli, old standing urine.
• Foul: Urinary tract infection
• Sulfurous: Cystinuria.

Causes of decrease in SG of urine are diabetes insipidus
(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,
and dehydration.

Fig. 1.1: (A) Urinometer method and (B) Reagent strip
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
alkaline urine

Disappear on addition of a drop of
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
following centrifugation

Microscopy, Nitrite test

Examination of Urine
show SG of 1.000 at the temperature of calibration. If not,
then the difference needs to be adjusted in test readings
taken subsequently.
The method is as follows:
1. Fill a measuring cylinder with 50 ml of urine.
2. Lower urinometer gently into the urine and let it float
freely.
3. Let urinometer settle; it should not touch the sides or
bottom of the cylinder.
4. Take the reading of SG on the scale (lowest point of
meniscus) at the surface of the urine.
5. Take out the urinometer and immediately note the
temperature of urine with a thermometer.
Correction for temperature: Density of urine increases at
low temperature and decreases at higher temperature.
This causes false reading of SG. Therefore, SG is corrected
for difference between urine temperature and calibration
temperature. Check the temperature of calibration of the
urinometer To get the corrected SG, add 0.001 to the
reading for every 3°C that the urine temperature is above
the temperature of calibration. Similarly subtract 0.001
from the reading for every 3°C below the calibration
temperature.
Correction for dilution: If quantity of urine is not sufficient
for measurement of SG, urine can be appropriately
diluted and the last two figures of SG are multiplied by
the dilution factor.
Correction for abnormal solute concentration: High SG in the
presence of glycosuria or proteinuria will not reflect true

kidney function (concentrating ability). Therefore it is
necessary to nullify the effect of glucose or proteins. For
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 determined 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 concentration, 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).

7

7.0). On standing, urine becomes alkaline because of loss
of carbon dioxide and production of ammonia from urea.
Therefore, for correct estimation of pH, fresh urine
should be examined.
There are various methods for determination of
reaction of urine: litmus paper, pH indicator paper, pH
meter, and reagent strip tests.
1. Litmus paper test: A small strip of litmus paper is
dipped in urine and any color change is noted. If blue

litmus paper turns red, it indicates acid urine. If red
paper turns blue, it indicates alkaline urine (Fig. 1.2A).
2. pH indicator paper: Reagent area (which is impregnated with bromothymol blue and methyl red) of
indicator paper strip is dipped in urine sample and
the color change is compared with the color guide
provided. Approximate pH is obtained.
3. pH meter: An electrode of pH meter is dipped in urine
sample and pH is read off directly from the digital
display. It is used if exact pH is required.
4. Reagent strip test: The test area (Fig. 1.2B) contains
polyionic polymer bound to H+; on reaction with
cations in urine, H+ is released causing change in color
of the pH-sensitive dye.
Normal pH range is 4.6 to 8.0 (average 6.0 or slightly
acidic). Urine pH depends on diet, acid base balance,
water balance, and renal tubular function.
Acidic urine is found in ketosis (diabetes mellitus,
starvation, fever), urinary tract infection by Escherichia
coli, and high protein diet. Alkaline urine may result from

Reaction and pH
The pH is the scale for measuring acidity or alkalinity
(acid if pH is < 7.0; alkaline if pH is > 7.0; neutral if pH is

Fig. 1.2: Testing pH of urine with litmus paper (A) and
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
Glucose

Ketones
Bilirubin
Bile salts

•
•
•
•
•

Urobilinogen
Blood
Hemoglobin
Myoglobin
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 distinguished 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
permeability of glomerular capillary wall is called as
glomerular proteinuria.
There are two types of glomerular proteinuria:
selective and nonselective. In early stages of glomerular

Fig. 1.3: Glomerular and tubular proteinuria. Upper figure shows
normal serum protein electrophoresis pattern. Lower part shows
comparison of serum and urine electrophoresis in (1) selective
proteinuria, (2) non-selective proteinuria, and (3) tubular
proteinuria


Examination of Urine
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 membrane, 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 βbands (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 “overflows” 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%)

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 inflammatory 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 bromophenol 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.
It is false-negative in the presence of Bence Jones
proteins, myoglobin, and hemoglobin. Overload
(Bence Jones) proteinuria and tubular proteinuria

may be missed entirely if only reagent strip method
is used. This test should be followed by sulphosalicylic acid test, which is a confirmatory test. Highly
alkaline urine, gross hematuria, and contamination
with vaginal secretions can give false-positive
reactions.
3. Sulphosalicylic acid test: Addition of sulphosalicylic
acid to the urine causes formation of a white
precipitate if proteins are present (Proteins are

Fig. 1.5: Principle of reagent strip test for proteins. The principle
is called as ‘protein error of indicators’ meaning that one color
appears if protein is present and another color if protein is
absent. Sensitivity is 5-10 mg/dl. The test does not detect Bence
Jones proteins, hemoglobin, and myoglobin

Fig. 1.6: Grading of proteinuria with reagent strip test
(above) and sulphosalicylic acid test (below)

denatured by organic acids and precipitate out of
solution).
Take 2 ml of clear urine in a test tube. If reaction of
urine is neutral or alkaline, a drop of glacial acetic acid is
added. Add 2-3 drops of sulphosalicylic acid (3 to 5%),
and examine for turbidity against a dark background
(Fig. 1.6).
This test is more sensitive and reliable than boiling
test.
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.
Quantitative Estimation of Proteins
Indications for quantitative estimation of proteins in
urine are:
• Diagnosis of nephrotic syndrome

Table 1.4: Comparison of two tests for proteinuria
Parameter

Reagent strip test

Sulphosalicylic acid test

1. Principle
2. Proteins detected

Colorimetric
Albumin

3. Sensitivity
4. Indicator
5. Type of test

5-10 mg/dl
Color change

Screening

Acid precipitation
All (albumin, Bence Jones proteins,
hemoglobin, myoglobin)
20 mg/dl
Turbidity
Confirmatory


Examination of Urine

11

Table 1.5: Grading of albuminuria
Condition

mg/24 hr

mg/L

mg/g creatinine

μg/min

μg/mg creatinine

g/mol creatinine

Normal

Microalbuminuria
Overt albuminuria

< 30
30-300
>300

< 20
20-200
>200

< 20
20-300
>300

< 20
20-200
>200

< 30
30-300
>300

< 2.5
2.5-25
>25

• 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 laboratories 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

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 precipitate 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
glucosuria or glycosuria (Box 1.7). Glycosuria results if
the filtered glucose load exceeds the capacity of renal
tubular reabsorption. Most common cause is hyperglycemia from diabetes mellitus.
Causes of Glycosuria

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

1. Glycosuria with hyperglycemia:
• Endocrine diseases: diabetes mellitus, acromegaly,
Cushing’s syndrome, hyperthyroidism, pancreatic disease
• Non-endocrine diseases: central nervous system
diseases, liver disorders
• Drugs: adrenocorticotrophic hormone, corticosteroids, 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

Note: Quantitation of proteins and creatinine clearance are done in all patients with persistent proteinuria
Fig. 1.8: Evaluation of proteinuria


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, homogentisic acid, uric acid, creatinine), and drugs (ascorbic
acid, salicylates, cephalosporins, penicillins, streptomycin, 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
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

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 peroxidase 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.

Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric 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 breakdown 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 bicarbonate 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. 1.11: 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

Examination of Urine

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%.

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 nitroprusside, glycine, and an alkaline buffer. A purplelavender 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.

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)

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
Table 1.6: Urine bilirubin and urobilinogen in jaundice
Urine test

Hemolytic
jaundice

Hepatocellular Obstructive
jaundice
jaundice

1. Bilirubin

Absent

Present

Present

Increased

Absent

2. Urobilinogen Increased

In acute viral hepatitis, bilirubin appears in urine
even before jaundice is clinically apparent. In a fever
of unknown origin bilirubinuria suggests hepatitis.

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 watersoluble, is filtered by the glomeruli, and therefore appears
in urine.
Detection of bilirubin in urine (along with urobilinogen) is helpful in the differential diagnosis of
jaundice (Table 1.6).

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


Examination of Urine

17

Fig. 1.16: Positive Fouchet’s test for bilirubin in urine

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,

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 urobilinogen 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 urobilinogen 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