Ebook Essentials of clinical pathology Part 1

(BQ) Part 1 book Essentials of clinical pathology presentation of content: Examination of urine, renal function tests, diabetes mellitus, liver function tests, examination of cerebrospinal fluid, examination of sputum, examination of feces, gastric analysis, thyroid function tests,... and other contents.

of Clinical Pathology

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Shirish M Kawthalkar

Associate ProfessorDepartment of PathologyGovernment Medical CollegeNagpur, Maharashtra, India

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Essentials

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Essentials of Clinical Pathology

The major aims of this book are discussion of (i) use of laboratory tests in the investigation and management ofcommon diseases, and (ii) basic biochemical and pathological principles underlying the application of laboratorytests The book has been written keeping in mind mainly the curricula of undergraduate students of pathology Itshould also prove to be appropriate for postgraduate residents and students of medical laboratory technology Thelaboratory tests that are demonstrated to and performed by medical students in pathology practical class and duringuniversity examination are given in more detail To keep pace with new knowledge and advances, principles ofcurrently performed techniques in clinical laboratory practice have also been outlined Most of the chapters arefollowed by reference ranges and critical values for ready access Critical values or action values are those laboratoryresults that require immediate attention of the treating clinician While interpreting results of laboratory tests, it isnecessary to follow two fundamental rules of laboratory medicine: (i) diagnosis should never be made from a singleabnormal 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 isconcerned 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 pathologyare 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 twochildren, Ameya and Ashish during preparation of this book I am thankful to Dr HT Kanade, Dean, GovernmentMedical 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, Professorand Head, Department of Pathology, Government Medical College, Nagpur, for encouraging me in undertakingthis 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 forundertaking to publish this book, being patient with me during the preparation of the manuscript, and bringing itout 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 thisregard will be highly appreciated

Shirish M Kawthalkar

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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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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 ofinvestigation (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-prandialspecimen

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.

16 Formiminoglutamic acid (FIGlu) < 3 mg

17 Red cells, epithelial cells, and white blood cells <1-2/high power field

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 inpatients with obstruction of urinary tract It is usuallyavoided in ambulatory patients since it carries therisk 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 sometime For bacteriologic examination, urine is aspiratedfrom bladder by passing a needle just abovesymphysis pubis

Changes which Occur in Standing Urine at Room Temperature

If urine is left standing at room temperature for long aftercollection, 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 2hours of collection to get the correct results

Preservation of Urine Sample

The urine sample should ideally be examined within 1-2hours 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, proof, and with a tight fitting stopper is used About 15 ml

leak-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 antidiuretichormone), chronic renal failure (loss of concentratingability of kidneys) or diuretic therapy

• Oliguria means urinary volume < 400 ml/24 hours.

Causes include febrile states, acute nephritis (decreased glomerular filtration), congestivecardiac failure or dehydration (decreased renal bloodflow)

glomerulo-• Anuria means urinary output < 100 ml/24 hours or

complete cessation of urine output It occurs in acutetubular necrosis (e.g in shock, hemolytic transfusionreaction), acute glomerulonephritis, and completeurinary tract obstruction

Color

Normal urine color in a fresh state is pale yellow or amberand is due to the presence of various pigmentscollectively called urochrome Depending on the state

of hydration urine may normally be colorless (overhydration) or dark yellow (dehydration) Some of theabnormal colors with associated conditions are listed inTable 1.2

Box 1.3: Physical examination

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 renalfailure (low and fixed SG at 1.010 due to loss ofconcentrating ability of tubules) and compulsive waterdrinking

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 withurine (Fig 1.1A) When solute concentration is high,upthrust of solution increases and urinometer ispushed up (high SG) If solute concentration is low,urinometer sinks further into the urine (low SG).Accuracy of a urinometer needs to be checked withdistilled water In distilled water, urinometer should

Table 1.3: Causes of cloudy or turbid urine

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

2 Amorphous urates Pink and cloudy in acid urine Dissolve on warming

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

Examination of Urine 7

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 ofreaction of urine: litmus paper, pH indicator paper, pHmeter, 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 bluelitmus paper turns red, it indicates acid urine If redpaper 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) ofindicator paper strip is dipped in urine sample andthe color change is compared with the color guideprovided 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 digitaldisplay 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 withcations 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 slightlyacidic) 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 molecularweight proteins like albumin and transferrin Whenglomeruli can retain larger molecular weight proteinsbut allow passage of comparatively lower molecularweight proteins, the proteinuria is called as selective.With further glomerular damage, this selectivity is lostand larger molecular weight proteins (γ globulins) arealso excreted along with albumin; this is called asnonselective proteinuria

Selective and nonselective proteinuria can be guished by urine protein electrophoresis In selective

distin-proteinuria, albumin and transferrin bands are seen,while in nonselective type, the pattern resembles that ofserum (Fig 1.3)

Causes of glomerular proteinuria are glomerulardiseases that cause increased permeability of glomerularbasement membrane The degree of glomerular proteinu-

Box 1.4: Chemical examination of urine

• Proteins • Urobilinogen

• Ketones • Hemoglobin

• Bilirubin • Myoglobin

• Bile salts • Nitrite or leukocyte esterase

Box 1.5: Causes of proteinuria

Examination of Urine 9 Box 1.6: Nephrotic syndrome

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 precipitatedwhen boiled in an acidic solution

Method: Urine should be clear; if not, filter or usesupernatant from a centrifuged sample

Urine should be just acidic (check with litmus paper);

if not, add 10% acetic acid drop by drop until blue litmuspaper turns red

A test tube is filled 2/3rds with urine The tube isinclined at an angle and the upper portion is boiled overthe flame (Only the upper portion is heated so thatconvection currents generated by heat do not disturb theprecipitate and the upper portion can be compared withthe lower clear portion) Compare the heated part withthe 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 isboiled again Turbidity due to phosphates disappearswhile 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

hemoglobin, myoglobin)

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 ofsolution)

Take 2 ml of clear urine in a test tube If reaction ofurine is neutral or alkaline, a drop of glacial acetic acid isadded 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 boilingtest

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 sulphosalicylicacid test is shown in Table 1.4

Quantitative Estimation of Proteins

Indications for quantitative estimation of proteins inurine are:

• Diagnosis of nephrotic syndrome

Examination of Urine 11

• 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 microvasculardisease Microalbuminuria precedes the development

of diabetic nephropathy by a few years If bloodglucose level and hypertension are tightly controlled

at this stage by aggressive treatment then progression

to irreversible renal disease and subsequent renalfailure can be delayed or prevented

2 Microalbuminuria is an independent risk factor forcardiovascular disease in diabetes mellitus

Detection of microalbuminuria: Microalbuminuria cannot

be detected by routine tests for proteinuria Methods fordetection include:

• Measurement of albumin-creatinine ratio in a randomurine sample

• Measurement of albumin in an early morning orrandom urine sample

• Measurement of albumin in a 24 hr sampleTest strips that screen for microalbuminuria areavailable commercially Exact quantitation can be done

by immunologic assays like radioimmunoassay orenzyme linked immunosorbent assay

Bence Jones Proteinuria

Bence Jones proteins are monoclonal immunoglobulinlight chains (either κ or λ) that are synthesized byneoplastic plasma cells Excess production of these lightchains occurs in plasma cell dyscrasias like multiplemyeloma and primary amyloidosis Because of their lowmolecular weight and high concentration they areexcreted in urine (overflow proteinuria)

Bence Jones proteins have a characteristic thermalbehaviour When heated, Bence Jones proteins precipi-tate at temperatures between 40°C to 60°C (other proteinsprecipitate between 60-70°C), and precipitate disappears

on further heating at 85-100°C (while precipitate of otherproteins does not) When cooled (60-85°C), there isreappearance of precipitate of Bence Jones proteins Thistest, however, is not specific for Bence Jones proteins andboth false-positive and -negative results can occur Thistest has been replaced by protein electrophoresis ofconcentrated urine sample (Fig 1.7)

Table 1.5: Grading of albuminuria

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 renaltubular 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 systemdiseases, liver disorders

• Drugs: adrenocorticotrophic hormone, steroids, thiazides

cortico-• Alimentary glycosuria (Lag-storage glycosuria):After a meal, there is rapid intestinal absorption

of glucose leading to transient elevation of bloodglucose above renal threshold This can occur inpersons with gastrectomy or gastrojejunostomyand in hyperthyroidism Glucose tolerance testreveals a peak at 1 hour above renal threshold(which causes glycosuria); the fasting and 2-hourglucose values are normal

2 Glycosuria without hyperglycemia

• Renal glycosuria: This accounts for 5% of cases ofglycosuria 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

Examination of Urine 13

is the highest glucose level in blood at which

glucose appears in urine and which is detectable

by routine laboratory tests The normal renal

threshold for glucose is 180 mg/dl Threshold

substances need a carrier to transport them from

tubular lumen to blood When the carrier is

saturated, the threshold is reached and the

substance is excreted Up to this level glucose

filtered by the glomeruli is efficiently reabsorbed

by tubules Renal glycosuria is a benign condition

in which renal threshold is set below 180 mgs/dl

but glucose tolerance is normal; the disorder is

transmitted as autosomal dominant Other

conditions in which glycosuria can occur with

blood glucose level remaining below 180 mgs/dl

are renal tubular diseases in which there is

decreased glucose reabsorption like Fanconi’s

syndrome, and toxic renal tubular damage During

pregnancy, renal threshold for glucose is

decreased Therefore it is necessary to estimate

blood glucose when glucose is first detected in

urine

Tests for Detection of Glucose in Urine

1 Copper reduction methods

A Benedict’s qualitative test: When urine is boiled in

Benedict’s qualitative solution, blue alkaline copper

sulphate is reduced to red-brown cuprous oxide if a

reducing agent is present (Fig 1.9) The extent of

reduction depends on the concentration of the reducing

substance This test, however, is not specific for glucose

Other carbohydrates (like lactose, fructose, galactose,pentoses), certain metabolites (glucuronic acid, homo-gentisic acid, uric acid, creatinine), and drugs (ascorbicacid, 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 testtube (composition of Benedict’s qualitative reagent:copper sulphate 17.3 gram, sodium carbonate 100gram, sodium citrate 173 gram, distilled water 1000ml)

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 ofgalactose, lactose, fructose, maltose, and pentoses inurine) for inborn errors of carbohydrate metabolism ininfants 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 precipitate1+ (approx 0.5 grams/dl): Green with precipitate2+ (approx 1.0 grams/dl): Brown precipitate3+ (approx 1.5 grams/dl: Yellow-orange precipitate4+ (> 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 ofurine

False positive test occurs in the presence of oxidizingagent (bleach or hypochlorite used to clean urinecontainers), which oxidizes the chromogen directly.False-negative test occurs in the presence of largeamounts 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, butyric acid, and acetone) in urine is called as ketonuria.Ketones are breakdown products of fatty acids and theirpresence in urine is indicative of excessive fatty acidmetabolism to provide energy

β-hydroxy-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 carbohydratemetabolism, low carbohydrate intake, or increasedmetabolic 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: Indiabetes, because of poor glucose utilization, there iscompensatory increased lipolysis This causesincrease in the level of free fatty acids in plasma.Degradation of free fatty acids in the liver leads tothe formation of acetoacetyl CoA which then formsketone bodies Ketone bodies are strong acids andproduce H+ ions, which are neutralized by bicar-bonate ions; fall in bicarbonate (i.e alkali) levelproduces ketoacidosis Ketone bodies also increasethe 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

Examination of Urine 15

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 allthe three ketone bodies Rothera’s nitroprusside methodand methods based on it detect acetoacetic acid andacetone (the test is 10-20 times more sensitive toacetoacetic acid than acetone) Ferric chloride test detectsacetoacetic acid only β-hydroxybutyric acid is notdetected by any of the screening tests

Methods for detection of ketone bodies in urine areRothera’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 alkalinesolution to form a purple-colored complex (Fig 1.13).Rothera’s test is sensitive to 1-5 mg/dl of acetoacetateand to 10-25 mg/dl of acetone

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 prusside, glycine, and an alkaline buffer A purple-lavender discoloration of the tablet indicates the presence

nitro-of acetoacetate or acetone (≥ 5 mg/dl) A rough estimate

of the amount of ketone bodies can be obtained bycomparison with the color chart provided by themanufacturer.The test is more sensitive than reagent striptest 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 conjugatedhyperbilirubinemia (obstructive or hepatocellularjaundice) This is because only conjugated bilirubin iswater-soluble Bilirubin in urine is absent in hemolyticjaundice; this is because unconjugated bilirubin iswater-insoluble

Tests for Detection of Bilirubin in Urine

Bilirubin is converted to non-reactive biliverdin onexposure to light (daylight or fluorescent light) and onstanding at room temperature Biliverdin cannot bedetected by tests that detect bilirubin Therefore freshsample that is kept protected from light is required.Findings associated with bilirubinuria are shown inBox 1.9

Methods for detection of bilirubin in urine are foamtest, 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 andhighly concentrated urine In normal urine, foam iswhite

2 Gmelin’s test: Take 3 ml of concentrated nitric acid

in a test tube and slowly place equal quantity of urineover it The tube is shaken gently; play of colors(yellow, red, violet, blue, and green) indicates positivetest (Fig 1.15)

3 Lugol iodine test: Take 4 ml of Lugol iodine solution

(Iodine 1 gm, potassium iodide 2 gm, and distilledwater to make 100 ml) in a test tube and add 4 drops

of urine Mix by shaking Development of green colorindicates 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

1 Bilirubin Absent Present Present

2 Urobilinogen Increased Increased Absent

Examination of Urine 17

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 bloodfrom biliary canaliculi (due to increased intrabiliarypressure) and are excreted in urine The test used for theirdetection is Hay’s surface tension test The property ofbile salts to lower the surface tension is utilized in thistest

Take some fresh urine in a conical glass tube Urineshould be at the room temperature Sprinkle on thesurface 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 positivetest

Urobilinogen

Conjugated bilirubin excreted into the duodenumthrough 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, whichundergoes recycling (enterohepatic circulation); a smallamount, which is not taken up by the liver, is excreted inurine Urobilinogen is colorless; upon oxidation it isconverted to urobilin, which is orange-yellow in color.Normally about 0.5-4 mg of urobilinogen is excreted inurine in 24 hours Therefore, a small amount of urobili-nogen is normally detectable in urine

Urinary excretion of urobilinogen shows diurnalvariation with highest levels in afternoon Therefore, a2-hour post-meal sample is preferred

Causes of Increased Urobilinogen in Urine

1 Hemolysis: Excessive destruction of red cells leads

to hyperbilirubinemia and therefore increasedformation 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 chloroformlayer indicates presence of urobilinogen, while pinkcoloration of aqueous portion indicates presence ofporphobilinogen Pink layer is then decanted and shakenwith butanol A pink color in the aqueous layer indicatesporphobilinogen (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 whichproduce urobilinogen are destroyed)

2 Reagent strip method: This method is specific for

urobilinogen Test area is impregnated with either

p-dimethylaminobenzaldehyde or benzene diazonium tetrafluoroborate

Causes of Hematuria

1 Diseases of urinary tract

• Glomerular diseases: Glomerulonephritis, Berger’sdisease, lupus nephritis, Henoch-Schonleinpurpura

Fig 1.17: Ehrlich’s aldehyde test for urobilinogen

Examination of Urine 19

• 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 inhemoglobin act as peroxidase, which reduceshydrogen peroxide to water This process needs ahydrogen donor (benzidine, orthotoluidine, orguaiac) Oxidation of hydrogen donor leads todevelopment of a color (Fig 1.19) Intensity of colorproduced is proportional to the amount of hemo-globin present

Chemical tests are positive in hematuria, globinuria, and myoglobinuria

hemo-• 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 ofurine If green or blue color develops within 5minutes, the test is positive

• Orthotoluidine test: In this test, instead of benzidine,orthotoluidine is used It is more sensitive thanbenzidine test

• Reagent strip test: Various reagent strips arecommercially available which use differentchromogens (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

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 deficiencyfollowing exposure to oxidant drugs, immunehemolysis (mismatched blood transfusion, paroxy-smal cold hemoglobinuria), paroxysmal nocturnalhemoglobinuria, hemolytic uremic syndrome, anddisseminated 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) indicatespresence of free hemoglobin in plasma Hemosiderinappears as blue granules when urine sediment is stainedwith Prussian blue stain (Fig 1.21) Granules are locatedinside tubular epithelial cells or may be free if cells havedisintegrated Hemosiderinuria is seen in intravascularhemolysis

Myoglobin

Myoglobin is a protein present in striated muscle (skeletaland cardiac) which binds oxygen Causes of myoglo-binuria include injury to skeletal or cardiac muscle, e.g.crush injury, myocardial infarction, dermatomyositis,severe electric shock, and thermal burns

Examination of Urine 21

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 significantbacteriuria: nitrite test and leucocyte esterase test Thesetests are helpful at places where urine microscopy is notavailable If these tests are positive, urine culture isindicated

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 theaction of bacterial enzyme nitrate reductase Nitrites

are then detected in urine by reagent strip tests As E.

coli is the commonest organism causing urinary tractinfection, this test is helpful as a screening test forurinary tract infection

Some organisms like Staphylococci or Pseudomonas do

not reduce nitrate to nitrite and therefore in suchinfections nitrite test is negative Also, urine must beretained in the bladder for minimum of 4 hours forconversion of nitrate to nitrite to occur; therefore, freshearly 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 tractinfections

2 Leucocyte esterase test: It detects esterase enzyme

released in urine from granules of leucocytes Thusthe 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, solidelements 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

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

peroxidase activity

4 Urine microscopy Many red cells Occasional red cell Occasional red cell

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 andcasts degenerate upon standing at room temperature Ifpreservative is required, then 1 crystal of thymol or 1drop 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 1500rpm and supernatant is poured off The tube is tapped

at the bottom to resuspend the sediment (in 0.5 ml ofurine) A drop of this is placed on a glass slide andcovered with a cover slip (Fig 1.23) The slide is examinedimmediately under the microscope using first the lowpower and then the high power objective The condensershould be lowered to better visualize the elements byreducing the illumination

Cells

Cellular elements in urine are shown in Figure 1.24

Table 1.8: Urinary findings in renal diseases

(Hyaline)

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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Examination of Urine 23

Red Blood Cells

Normally there are no or an occasional red blood cell inurine 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 ofred cells through the damaged glomeruli Presence of

> 80% of dysmorphic red cells is strongly suggestive ofglomerular 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: (C) Trichomonas, and (D) Egg of Schistosoma haematobiumOrganisms in urine: (A) Bacteria, (B) Yeasts,

diamond- or pear-shaped (caudate cells) Large numbers

or sheets of these cells in urine occur after catheterizationand in transitional cell carcinoma

Oval Fat Bodies

These are degenerated renal tubular epithelial cells filledwith highly refractile lipid (cholesterol) droplets Underpolarized light, they show a characteristic “Maltesecross” pattern They can be stained with a fat stain such

as Sudan III or Oil Red O They are seen in nephroticsyndrome in which there is lipiduria

Spermatozoa

They may sometimes be seen in urine of men

Telescoped urinary sediment: This refers to urinarysediment consisting of red blood cells, white blood cells,oval fat bodies, and all types of casts in roughly equalproportion It occurs in lupus nephritis, malignanthypertension, rapidly proliferative glomerulonephritis,and diabetic glomerulosclerosis

Method of collection for bacteriologic examination

is given earlier in Box 1.2

Significant bacteriuria exists when there are >10 5

bacterial colony forming units/ml of urine in a catch midstream sample, >10 4 colony forming units/ml

clean-of urine in catheterized sample, and >10 3 forming units/ml of urine in a suprapubic aspiration sample.

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Examination of Urine 25

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, undulatingmembrane on one side, and four flagellae They causevaginitis in females and are thus contaminants in urine.They are easily detected in fresh urine due to theirmotility

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 microscopicstructures that form in distal renal tubules and collectingducts They take the shape and diameter of the lumina(molds or ‘casts’) of the renal tubules They have parallelsides and rounded ends Their length and width may bevariable Casts are basically composed of a precipitate of

a protein that is secreted by tubules (Tamm-Horsfallprotein) Since casts form only in renal tubules theirpresence is indicative of disease of the renal parenchyma.Although there are several types of casts, all urine castsare basically hyaline; various types of casts are formedwhen different elements get deposited on the hyalinematerial (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 causingglomerular proteinuria

Granular casts: Presence of degenerated cellular debris in

a cast makes it granular in appearance These arecylindrical structures with coarse or fine granules (whichrepresent degenerated renal tubular epithelial cells)embedded in Tamm-Horsfall protein matrix They areseen after strenuous muscle exercise and in fever, acuteglomerulonephritis, and pyelonephritis

Waxy cast: These are the most easily recognized of allcasts They form when hyaline casts remain in renaltubules for long time (prolonged stasis) They havehomogenous, smooth glassy appearance, cracked orserrated margins and irregular broken-off ends The endsare straight and sharp and not rounded as in other casts.They are light yellow in color They are most commonlyseen in end-stage renal failure

Fatty casts: These are cylindrical structures filled withhighly 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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Examination of Urine 27

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 inalkali, and insoluble in acid Increased numbers arefound in gout and leukemia Flat hexagonal uric acidcrystals may be mistaken for cysteine crystals that alsoform 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 indilute hydrochloric acid Ingestion of certain foodslike tomatoes, spinach, cabbage, asparagus, andrhubarb causes increase in their numbers Theirincreased number in fresh urine (oxaluria) may alsosuggest oxalate stones A large number are seen inethylene glycol poisoning

c Amorphous urates: These are urate salts of potassium,

magnesium, or calcium in acid urine They are usuallyyellow, fine granules in compact masses They aresoluble 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 acidand give off bubbles of gas when they dissolve

b Phosphates: Phosphates may occur as crystals (triple

phosphates, calcium hydrogen phosphate), or asamorphous deposits

• Phosphate crystals

 Triple phosphates (ammonium magnesiumphosphate): They are colorless, shiny, 3-6 sidedprisms 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 colorlesssmall 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 aceticacid 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 Theyare soluble in ether, chloroform, or alcohol They areseen in lipiduria e.g nephrotic syndrome and hyper-cholesterolemia They can be positively identified bypolarizing 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 reagentstrip or chemical test for bilirubin These crystals aresoluble in strong acid or alkali They are seen in severeobstructive liver disease

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Examination of Urine 29

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

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

2 Carroll MF, Temte JL Proteinuria in adults: A diagnostic

approach Am Fam Physician 2000;62:1333-40.

3 Cheesbrough M District laboratory practice in tropical countries Part 1 and Part 2 Cambridge; Cambridge University Press, 1998.

4 Grossfeld GD, Wolf JS, Litwin MS, et al Asymptomatic microscopic hematuria in adults: Summary of the AUA

best policy recommendations Am Fam Physician 2001;

63:1145-54.

5 Henry JB (Ed): Clinical diagnosis and management by laboratory methods (20th Ed) Philadelphia; WB Saunders Company, 2001.

6 King M A medical laboratory for developing countries London Oxford University Press, 1973.

7 Mathieson PW The cellular basis of albuminuria Clinical

Science 2004;107:533-8.

8 Simerville JA, Maxted WC, Pahira JJ Urinalysis: A comprehensive review Am Fam Physician 2005;71: 1153-62.

9 Wallach J Interpretation of diagnostic tests (7th Ed) Philadelphia Lippincott Williams and Wilkins, 2000.

10 World Health Organization Manual of basic techniques for a health laboratory (2nd Ed) Geneva; World Health Organization, 2003.

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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 D 3 (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

Renal function tests can be classified as shown in Table2.1

In practice, the commonly performed renal functiontests are routine urinalysis, estimation of serumcreatinine, blood urea nitrogen (BUN), BUN/Serumcreatinine ratio, creatinine clearance test (or estimation

of GFR from serum creatinine value by a predictionequation), and estimation of urine concentrating ability(water deprivation test) Urine examination is the firsttest performed in patients suspected of having renaldisease It is the simplest and the least expensive renalfunction test In urine examination parameters that canassess renal function are urine volume in 24 hours,specific gravity, osmolality, proteinuria, and microscopicexamination of urinary sediment

Tests to Evaluate Glomerular Function

The best test to assess overall kidney function isestimation 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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Renal Function Tests 31

Normal GFR in young adults is 120-130 ml/min per 1.73

m2 of body surface area GFR declines progressively with

age (due to arteriolosclerosis of glomeruli) After 40 years

of age, there is a steady and progressive fall in the GFR

at the rate of 1 ml/minute/year because of reduction in

the number of glomeruli due to arteriolosclerosis

GFR is measured to (i) detect suspected incipient

kidney disease (i.e early detection), (ii) monitor course

of established kidney disease, (iii) plan renal replacement

therapy in advanced renal disease, and (iv) adjust dosage

of certain drugs which are nephrotoxic

Based on GFR, chronic kidney disease is divided into

following stages (US National Kidney Foundation

Kidney Disease Quality Outcomes Initiative

Classifi-cation of Chronic Kidney Disease, 2002):

• Stage 1: Kidney damage with normal or increased

in blood or urine tests or imaging studies Symptomsusually develop at or after stage 3 GFR <60 ml/min per1.73 m2 indicates loss of ≥ 50% of kidney function GFR

<15 ml/min per 1.73 m2 is associated with kidney failureand uremia Following methods are used to measureGFR: (1) Clearance tests and (2) Prediction equations

Clearance Tests to Measure Glomerular Filtration Rate (GFR)

Glomerular filtration rate refers to the rate in ml/min atwhich a substance is cleared from the circulation by theglomeruli The ability of the glomeruli to filter a substancefrom the blood is assessed by clearance studies If asubstance is not bound to protein in plasma, is completelyfiltered by the glomeruli, and is neither secreted norreabsorbed by the tubules, then its clearance rate is equal

to the glomerular filtration rate (GFR) Clearance of asubstance refers to the volume of plasma, which iscompletely cleared of that substance per minute; it iscalculated from the following formula:

UVClearance = ——

Pwhere, U = concentration of a substance in urine inmg/dl; V = volume of urine excreted in ml/min; and P

= concentration of the substance in plasma in mg/dl.Since U and P are in the same units, they cancel eachother and the clearance value is expressed in the sameunit as V i.e ml/min All clearance values are adjusted

to a standard body surface area i.e 1.73 m2

Table 2.1: Classification of renal function tests

Tests to evaluate glomerular function Tests to evaluate tubular function

1 Clearance tests to measure glomerular 1 Tests to assess proximal tubular

filtration rate: Inulin clearance, 125 I-iothalamate function:

clearance, 51 Cr-EDTA clearance, Cystatin C • Glycosuria, phosphaturia, uricosuria

clearance, Creatinine clearance, and Urea • Generalized aminoaciduria

2 Calculation of creatinine clearance from • Fractional sodium excretion

prediction equations 2 Tests to assess distal tubular function:

3 Blood biochemistry: Serum creatinine, • Specific gravity and osmolality of urine

Blood urea nitrogen (BUN), and BUN/serum creatinine ratio • Water-deprivation test and water-loading test

4 Microalbuminuria and albuminuria • Ammonium chloride loading test

Box 2.2: Glomerular filtration rate (GFR)

• Best test for assessment of excretory renal function

• Varies according to age, sex, and body weight of an

individual; a normal GFR also depends on normal

renal blood flow and pressure.

• Normal GFR in young adults is 120-130 ml/min per

1.73 m 2

• Creatinine clearance is commonly used as a measure

of GFR Equations can be used to estimate GFR

from serum creatinine value.

• GFR declines with age (due to glomerular

arteriolo-sclerosis)

• GFR <60 ml/min per 1.73 m 2 indicates loss of ≥50%

of kidney function.

• Fall in GFR leads to accumulation of waste products

of metabolism in blood GFR <15 ml/min per 1.73 m 2

is associated with uremia.

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The agents used for measurement of GFR are:

• Exogenous: Inulin, Radiolabelled ethylenediamine

tetraacetic acid (51Cr- EDTA), 125I-iothalamate

• Endogenous: Creatinine, Urea, Cystatin C

The agent used for measurement of GFR should have

following properties: (1) It should be physiologically inert

and preferably endogenous, (2) It should be freely filtered

by glomeruli and should be neither reabsorbed nor

secreted by renal tubules, (3) It should not bind to plasma

proteins and should not be metabolized by kidneys, and

(4) It should be excreted only by the kidneys However,

there is no such ideal endogenous agent

Clearance tests are cumbersome to perform,

expensive, and not readily available One major

problem with clearance studies is incomplete urine

collection.

Abnormal clearance occurs in: (i) pre-renal factors:

reduced blood flow due to shock, dehydration, and

congestive cardiac failure; (ii) renal diseases; and

(iii) obstruction to urinary outflow

Inulin Clearance

Inulin, an inert plant polysaccharide (a fructose polymer),

is filtered by the glomeruli and is neither reabsorbed nor

secreted by the tubules; therefore it is an ideal agent for

measuring GFR A bolus dose of inulin (25 ml of 10%

solution IV) is administered followed by constant

intravenous infusion (500 ml of 1.5% solution at the rate

of 4 ml/min) Timed urine samples are collected and

blood samples are obtained at the midpoint of timed

urine collection This test is considered as the ‘gold

standard’ (or reference method) for estimation of GFR

However, this test is rarely used because it is time

consuming, expensive, constant intravenous infusion of

inulin is needed to maintain steady plasma level, and

difficulties in laboratory analysis Average inulin

clearance for males is 125 ml/min/1.73 m2 and for

females is 110 ml/min/1.73 m2 In children less than 2

years and in older adults, clearance is low This test is

largely limited to clinical research

Clearance of Radiolabeled Agents

Urinary clearance of radiolabeled iothalamate (125

I-iothalamate) correlates closely with inulin clearance

However, this method is expensive with risk of exposure

to radioactive substances Other radiolabelled substances

used are 51Cr-EDTA and 99Tc-DTPA

Cystatin C Clearance

This is a cysteine protease inhibitor of MW 13,000, which

is produced at a constant rate by all the nucleated cells

It is not bound to protein, is freely filtered by glomeruli

and is not returned to circulation after filtration It is amore sensitive and specific marker of impaired renalfunction than plasma creatinine Its level is not affected

by sex, diet, or muscle mass It is thought that cystatin C

is a superior marker for estimation of GFR than creatinineclearance It is measured by immunoassay

A 24-hour urine sample is preferred to overcome theproblem of diurnal variation of creatinine excretion and

to reduce the inaccuracy in urine collection

After getting up in the morning, the first voided urine

is discarded Subsequently all the urine passed iscollected in the container provided After getting up inthe next morning, the first voided urine is also collectedand the container is sent to the laboratory A bloodsample for estimation of plasma creatinine is obtained

at midpoint of urine collection Creatinine clearance iscalculated from (1) concentration of creatinine in urine

in mg/ml (U), (2) volume of urine excreted in ml/min(V) (this is calculated by the formula: volume of urinecollected/collection time in minutes e.g volume of urinecollected in 24 hours ÷ 1440), and (3) concentration ofcreatinine in plasma in mg/dl (P) Creatinine clearance

in ml/min per 1.73 m2 is then derived from the formulaUV/P

Because of secretion of creatinine by renal tubules,the above formula overestimates GFR by about 10% Inadvanced renal failure, secretion of creatinine by tubules

is increased and thus overestimation of GFR is even more.Jaffe’s reaction (see later under serum creatinine) usedfor estimation of creatinine measures creatinine as well

as some other substances (non-creatinine chromogens)

in blood and thus gives slightly higher result Thus effect

of tubular secretion of creatinine is somewhat balanced

by slight overestimation of serum creatinine by Jaffe’sreaction

To provide values closer to the actual GFR, cimetidine(which blocks secretion by renal tubules) can beadministered before commencing urine collection(cimetidine-enhanced creatinine clearance)

Creatinine clearance is not an ideal test for estimation

of GFR because of following reasons:

1 A small amount of creatinine is secreted by renaltubules that increase even further in advanced renalfailure

2 Collection of urine is often incomplete

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