Essentials of clinical pathology (2010)

Essentials
of
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Shirish M Kawthalkar
Associate Professor
Department of Pathology
Government Medical College
Nagpur, Maharashtra, India
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Essentials
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Clinical Pathology
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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
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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
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 Pathologyviii
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
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Chemical Pathology and
Related Studies
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COMPOSITION OF NORMAL URINE
Urinalysis is one of the most commonly performed
laboratory tests in clinical practice. Composition of
normal urine is shown in Table 1.1.
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.
COLLECTION OF URINE
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).
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.
Examination of Urine
1
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

Essentials of Clinical Pathology4
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.
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,
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.
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.

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),
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.
Box 1.3: Physical examination
• Volume • Odor
• Color • Specific gravity
• Appearance • pH
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

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, tyrosinae-
mia.
• Ammoniacal: Urinary tract infection with Escherichia
coli, old standing urine.
• Foul: Urinary tract infection
• Sulfurous: Cystinuria.
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.
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
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
Fig. 1.1: (A) Urinometer method and (B) Reagent strip
method for measuring specific gravity 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 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
(acid if pH is < 7.0; alkaline if pH is > 7.0; neutral if pH is
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 impreg-
nated 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
Fig. 1.2: Testing pH of urine with litmus paper (A) and
with reagent strip test (B)

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
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.
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
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-
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

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
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.
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%) Fig. 1.4: Principle of heat test for proteins

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
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.
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 sulpho-
salicylic 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

• 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
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).
Table 1.5: Grading of albuminuria
Condition mg/24 hr mg/L mg/g creatinine μg/min μg/mg creatinine g/mol creatinine
Normal < 30 < 20 < 20 < 20 < 30 < 2.5
Microalbuminuria 30-300 20-200 20-300 20-200 30-300 2.5-25
Overt albuminuria >300 >200 >300 >200 >300 >25

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

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,andtoxicrenaltubulardamage.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.
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.
Fig. 1.9: Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl

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.
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. 1.10: Grading of Benedict’s test (above) and reagent
strip test (below) for glucose
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

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%.
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. 1.12: Formation of ketone bodies. A small part of
acetoacetate is spontaneously and irreversibly converted to
acetone. Most is converted reversibly to β-hydroxybutyrate
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
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)

Box 1.9: Clinical and laboratory findings in bilirubinuria
• Jaundice
• Urine color: Dark yellow with yellow foam
• Elevated serum conjugated bilirubin
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).
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.
Fig. 1.14: Rothera’s tube test and reagent strip test for
ketone bodies in urine
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

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 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
Fig. 1.15: Positive Gmelin’s test for bilirubin showing
play of colors
Fig. 1.16: Positive Fouchet’s test for bilirubin in urine

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).
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
• Glomerulardiseases:Glomerulonephritis,Berger’s
disease, lupus nephritis, Henoch-Schonlein
purpura
Fig. 1.17: Ehrlich’s aldehyde test for urobilinogen

• 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
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).
Fig. 1.18: Interpretation of Watson-Schwartz test
Fig. 1.19: Principle of chemical test for red cells, hemoglobin, or myoglobin in urine

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.

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
reagent strip format that can detect significant
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
Fig. 1.21: Staining of urine sediment with Prussian blue
stain to demonstrate hemosiderin granules (blue)
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

Fig. 1.22: Different types of urinary sediment
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.
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Red Blood Cells
Normally there are no or an occasional red blood cell in
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. 1.23: Preparation of urine sediment for
microscopic examination
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
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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
Fig. 1.25: Organisms in urine: (A) Bacteria, (B) Yeasts,
(C) Trichomonas, and (D) Egg of Schistosoma haematobium
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 >103 colony-
forming units/ml of urine in a suprapubic aspiration
sample.
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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
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
Fig. 1.26: Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts
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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
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
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
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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.
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
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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%
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.
3. Bilirubin crystals: These are small (5 μ), brown crystals
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.
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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.
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. (20thEd). 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

Renal Function Tests
2
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.
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.
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
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vip.persianss.ir

Essentials of clinical pathology (2010)

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