Essentials of Clinical Pathology Shirish M Kawthalkar
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1Chemical Pathology and Related Studies2

Examination Of Urinechapter 1

 
COMPOSITION OF NORMAL URINE
Urinalysis is one of the most commonly performed laboratory tests in clinical practice. Composition of normal urine is shown in Table 1.1.
 
INDICATIONS FOR URINALYSIS
  1. Suspected renal diseases like glomerulonephritis nephrotic syndrome, pyelonephritis, and renal failure
  2. Detection of urinary tract infection
  3. Detection and management of metabolic disorders like diabetes mellitus
  4. Differential diagnosis of jaundice
  5. Detection and management of plasma cell dyscrasias
  6. Diagnosis of pregnancy.
 
COLLECTION OF URINE
There are various methods for collection of urine. Method of collection to be used depends on the nature of investigation (Boxes 1.1 and 1.2).
 
Time of Collection
  1. A single specimen: This may be a first morning voiding, a random specimen, or a post-prandial specimen.
The first voided specimen in the morning is the most concentrated and has acidic pH in which formed elements (cells and casts) are well preserved. This specimen is used for routine examination, fasting glucose, proteins, nitrite, microscopic analysis for cellular elements, pregnancy test, orthostatic proteinuria, and bacteriological analysis.
Table 1.1   Composition of normal urine (24 hour) in adults
Parameters
Values
1. Volume
600–2000 ml
2. Specific gravity
1.003–1.030
3. Osmolality
300–900 mOsm/kg
4. pH
4.6–8.0
5. Glucose
<0.5 gm
6. Proteins
<150 mg
7. Urobilinogen
0.5–4.0 mg
8. Porphobilinogen
0–2 mg
9. Creatinine
14–26 mg/kg (men), 11–20 mg/kg (women)
10. Urea nitrogen
12–20 gm
11. Uric acid
250–750 mg
12. Sodium
40–220 mEq
13. Potassium
25–125 mEq
14. Chloride
110–250 mEq
15. Calcium (low calcium diet)
50–150 mg
16. Formiminoglutamic acid (FIGlu)
<3 mg
17. Red cells, epithelial cells, and white blood cells
<1–2/high power field
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The random specimen is a single specimen collected at any time of day. It is used for routine urine examination.
Post-prandial specimen (collected 2 hours after a meal in the afternoon) is sometimes requested for estimation of glucose (to monitor insulin therapy in diabetes mellitus) or of urobilinogen.
  1. 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 ina 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. Subsequent stream is collected which is from the urinary bladder.
  2. Clean-catch specimen: This is recommended for bacteriologic culture. In men, glans penis is sufficiently exposed and cleaned with soap and water. In women urethral opening should be exposed, washed with soapy cotton balls, rinsed with water-saturated cotton, and holding the labia apart, the initial urine is allowed to pass into the toilet and the remaining is voided into the bottle (amount 20–100 ml). This method avoids contamination of urine with the vaginal fluids.
  3. Catheter specimen: This is used for bacteriological study or culture in bedridden, ill patients or in patients with obstruction of urinary tract. It is usually avoided in ambulatory patients since it carries the risk of introduction of infection.
  4. Infants: In infants, a clean plastic bag can be attached around the baby's genitalia and left in place for some time. For bacteriologic examination, urine is aspirated from bladder by passing a needle just above symphysis pubis.
 
Changes which Occur in Standing Urine at Room Temperature
If urine is left standing at room temperature for long after collection, following changes occur:
  • Increase in pH due to production of ammonia from urea by urease-producing bacteria.
  • Formation of crystals due to precipitation of phosphates and calcium (making the urine turbid)
  • Loss of ketone bodies, since they are volatile.
  • Decrease in glucose due to glycolysis and utilization of glucose by cells and bacteria.
  • Oxidation of bilirubin to biliverdin causing falsenegative test for bilirubin
  • Oxidation of urobilinogen to urobilin causing falsenegative test for urobilinogen
  • Bacterial proliferation
  • Disintegration of cellular elements, especially in alkaline and hypotonic urine.
 
Preservation of Urine Sample
The urine sample should ideally be examined within 1–2 hours of voiding. If delay in examination is expected, 5then 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 preseratives can be added to the 24-hour urine sample:
  • Hydrochloric acid: It is used for preservation of a 24-hour urine sample for adrenaline, noradrenaline, vanillylmandelic acid, and steroids.
  • Toluene: It forms a thin layer over the surface and acts as a physical barrier for bacteria and air. It is used for measurement of chemicals.
  • Boric acid: A general preservative.
  • Thymol: It inhibits bacteria and fungi.
  • Formalin: It is an excellent chemical for preservation of formed elements.
 
PHYSICAL EXAMINATION
The parameters to be examined on physical examination of urine are shown in Box 1.3.
 
Volume
Volume of only the 24-hr specimen of urine needs to be measured and reported. The average 24-hr urinary output in adults is 600–2000 ml. The volume varies according to fluid intake, diet, and climate. Abnormalities of urinary volume are as follows:
  • Polyuria means urinary volume > 2000 ml/24 hours. This is seen in diabetes mellitus (osmotic diuresis), diabetes insipidus (failure of secretion of antidiuretic hormone), chronic renal failure (loss of concentrating ability of kidneys) or diuretic therapy.
  • Oliguria means urinary volume < 400 ml/24 hours. Causes include febrile states, acute glomerulonephritis (decreased glomerular filtration), congestive cardiac failure or dehydration (decreased renal blood flow).
  • Anuria means urinary output < 100 ml/24 hours or complete cessation of urine output. It occurs in acute tubular necrosis (e.g. in shock, hemolytic transfusion reaction), acute glomerulonephritis, and complete urinary tract obstruction.
 
Color
Normal urine color in a fresh state is pale yellow or amber and is due to the presence of various pigments collectively called urochrome. Depending on the state of hydration urine may normally be colorless (over hydration) or dark yellow (dehydration). Some of the abnormal colors with associated conditions are listed in Table 1.2.
Table 1.2   Different colors of urine
Colors
Conditions
Colorless
Dilute urine (diabetes mellitus, diabetes insipidus, overhydration)
Red
Hematuria, Hemoglobinuria, Porphyria, Myoglobinuria
Dark brown or black
Alkaptonuria, Melanoma
Brown
Hemoglobinuria
Yellow
Concentrated urine
Yellow-green or green
Biliverdin
Deep yellow with yellow foam
Bilirubin
Orange or orange- brown
Urobilinogen Porphobilinogen
Milky-white
Chyluria
Red or orange fluorescence with UV light
Porphyria
Note: Many drugs cause changes in urine color; drug history should be obtained if there is abnormal coloration of urine
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Appearance
Normal, freshly voided urine is clear in appearance. Causes of cloudy or turbid urine are listedin Table 1.3. Foamy urine occurs in the presence of excess proteins or bilirubin.
 
Odor
Freshly voided urine has a typical aromatic odor due to volatile organic acids. After standing, urine develops ammoniacal odor (formation of ammonia occurs when urea is decomposed by bacteria). Some abnormal odors with associated conditions are:
  • Fruity: Ketoacidosis, starvation
  • Mousy or musty: Phenylketonuria
  • Fishy: Urinary tract infection with Proteus, tyrosinaemia.
  • Ammoniacal: Urinary tract infection with Escherichia coli, old standing urine.
  • Foul: Urinary tract infection
  • Sulfurous: Cystinuria.
 
Specific Gravity (SG)
This is also called as relative mass density. It depends on amount of solutes in solution. It isbasically a comparison of density of urine against the density of distilled water at a particular temperature. Specific gravity of distilled water is 1.000. Normal SG of urine is 1.003 to 1.030 and depends on the state of hydration. SG of normal urine is mainly related to urea and sodium. SG increases as solute concentration increases and decreases when temperature rises (since volume expands with rise in temperature).
SG of urine is a measure of concentrating ability of kidneys and is determined to get information about this tubular function. SG, however, is affected by proteinuria and glycosuria.
Causes of increase in SG of urine are diabetes mellitus (glycosuria), nephrotic syndrome (proteinuria), fever, and dehydration.
Causes of decrease in SG of urine are diabetes insipidus (SG consistently between 1.002–1.003), chronic renal failure (low and fixed SG at 1.010 due to loss of concentrating ability of tubules) and compulsive water drinking.
Methods for measuring SG are urinometer method, refractometer method, and reagent strip method.
  1. Urinometermethod: This method is based on the principle of buoyancy (i.e. the ability of a fluid to exert an upward thrust on a body placed in it). Urinometer (a hydrometer) is placed in a container filled with urine (Fig. 1.1A). When solute concentration is high, upthrust of solution increases and urinometer is pushed up (high SG). If solute concentration is low, urinometer sinks further into the urine (low SG).
Accuracy of a urinometer needs to be checked with distilled water. In distilled water, urinometer should show SG of 1.000 at the temperature of calibration.
zoom view
Fig. 1.1: (A) Urinometer method and (B) Reagent strip method for measuring specific gravity of urine
Table 1.3   Causes of cloudy or turbid urine
Cause
Appearance
Diagnosis
1. Amorphous phosphates
White and cloudy on standing in alkaline urine
Disappear on addition of a drop of dilute acetic acid
2. Amorphous urates
Pink and cloudy in acid urine
Dissolve on warming
3. Pus cells
Varying grades of turbidity
Microscopy
4. Bacteria
Uniformly cloudy; do not settle at the bottom following centrifugation
Microscopy, Nitrite test
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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-correctedSG for each 1 gm of protein/dl urine and 0.004 for every 1 gm of glucose/dl urine.
  1. Refractometer method: SG can be precisely determined by a refractometer, which measures the refractive index of the total soluble solids. Higher the concentration of total dissolvedsolids, higher the refractive index. Extent of refraction of a beam of light passed through urine is a measure of solute concentration, and thus of SG. The method is simple and requires only 1–2 drops of urine. Result is read from a scale or from digital display.
  2. 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 ifpH is > 7.0; neutral if pH is 7.0). On standing, urine becomes alkaline because of loss of carbon dioxide and production of ammonia from urea. Therefore, for correct estimation of pH, fresh urine should be examined.
There are various methods for determination of reaction of urine: litmus paper, pH indicator paper, pH meter, and reagent strip tests.
  1. Litmus paper test: A small strip of litmus paper is dipped in urine and any color change is noted. If blue litmus paper turns red, it indicates acid urine. If red paper turns blue, it indicates alkaline urine (Fig. 1.2A).
  2. pH indicator paper: Reagent area (which is impregnated with bromothymol blue and methyl red) of indicator paper strip is dipped in urine sample and the color change is compared with the color guide provided. Approximate pH is obtained.
  3. pH meter: An electrode of pH meter is dipped in urine sample and pH is read off directly from the digital display. It is used if exact pH is required.
  4. Reagent strip test: The test area (Fig. 1.2B) contains polyionic polymer bound to H+ on reaction with cations in urine, H+ is released causing change in color of the pH-sensitive dye.
Normal pH range is 4.6 to 8.0 (average 6.0 or slightly acidic). Urine pH depends on diet, acid base balance, water balance, and renal tubular function.
Acidic urine is found in ketosis (diabetes mellitus, starvation, fever), urinary tract infection by Escherichia coli, and high protein diet.
zoom view
Fig. 1.2: Testing pH of urine with litmus paper (A) and with reagent strip test (B)
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Alkaline urine may result from urinary tract infection by bacteria that split urea to ammonia (Proteus or Pseudomonas), severe vomiting, vegetarian diet, old ammoniacal urine sample and chronic renal failure.
Determining pH of urine helps in identifying various crystals in urine. Altering pH of urine may be useful in treatment of renal calculi (i.e. some stones form only in acid urine e.g. uric acid calculi; in such cases urine is kept alkaline); urinary tract infection (urine should be kept acid); and treatment with certain drugs (e.g. streptomycin is effective in urinary tract infection if urine is kept alkaline). In unexplained metabolic acidosis, measurement of urine pH is helpful in diagnosing renal tubular acidosis; in renal tubular acidosis, urine pH is consistently alkaline despite metabolic acidosis.
 
CHEMICAL EXAMINATION
The chemical examination is carried out for substances listed in Box 1.4.
 
Proteins
Normally, kidneys excrete scant amount of protein in urine(up to 150 mg/24 hours). These proteins include proteins from plasma (albumin) and proteins derived from urinary tract (Tamm-Horsfall protein, secretory IgA, and proteins from tubular epithelial cells, leucocytes, and other desquamated cells); this amount of proteinuria cannot be detected by routine tests. (Tamm-Horsfall protein is a normal mucoprotein secreted by ascending limb of the loop of Henle).
Proteinuria refers to protein excretion in urine greater than 150 mg/24 hours in adults.
 
Causes of Proteinuria
Causes of proteinuria can be grouped as shown in Box 1.5.
  1. Glomerular proteinuria: Proteinuria due to increased permeability of glomerular capillary wall is called as glomerular proteinuria.
There are two types of glomerular proteinuria: selective and nonselective. In early stages of glomerular disease, there is increased excretion of lower molecular weight proteins like albumin and transferrin. When glomeruli can retain larger molecular weight proteins but allow passage of comparatively lower molecular weight proteins, the proteinuria is called as selective. With further glomerular damage, this selectivity is lost and larger molecular weight proteins (globulins) are also excreted along with albumin; this is called as nonselective proteinuria.
Selective and nonselective proteinuria can be distinguished by urine proteinelectrophoresis. In selective proteinuria, albumin and transferrin bands are seen, while in nonselective type, the pattern resembles that of serum (Fig. 1.3).
Causes of glomerular proteinuria are glomerular diseases that cause increased permeability of glomerular basement membrane. The degree of glomerular proteinuria correlates with severity of disease and prognosis.
zoom view
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
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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).
  1. Tubular proteinuria: Normally, glomerular membrane, although impermeable to high molecular weight proteins, allows ready passage to low molecular weight proteins like β2-microglobulin, retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light chains. These low molecular weight proteins are actively reabsorbed by proximal renal tubules. In diseases involving mainly tubules, these proteins are excreted in urine while albumin excretion is minimal.
Urine electrophoresis shows prominent α- and 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.
  1. Overflow proteinuria: When concentration of a low molecular weight protein rises in plasma, it “overflows” from plasma into the urine. Such proteins are immunoglobulin light chains or Bence Jones proteins (plasma cell dyscrasias), hemoglobin (intravascular hemolysis), myoglobin (skeletal muscle trauma), and lysozyme (acute myeloid leukemia type M4 or M5).
  2. 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%) and is probably due to lordotic posture that causes inferior venacaval compression between the liver and vertebral column. The condition disappears in adulthood. Amount of proteinuria is <1000 mg/day. First-morning urine after rising is negative for proteins, while another urine sample collected after patient performs normal activities is positive for proteins. In such patients, periodic testing for proteinuria should be done to rule out renal disease.
  1. Post-renal proteinuria: This is caused by inflammatory or neoplastic conditions in renal pelvis, ureter, bladder, prostate, or urethra.
 
Tests for Detection of Proteinuria
  1. Heat and acetic acid test (Boiling test): This test is based on the principle that proteins get precipitated when boiled in an acidic solution.
Method: Urine should be clear; if not, filter or use supernatant from a centrifuged sample.
Urine should be just acidic (check with litmus paper); if not, add 10% acetic acid drop by drop until blue litmus paper turns red.
A test tube is filled 2/3rds with urine. The tube is inclined at an angle and the upper portion is boiled over the flame. (Only the upper portion is heated so that convection currents generated by heat do not disturb the precipitate and the upper portion can be compared with the lower clear portion). Compare the heated part with the lower part. Cloudiness or turbidity indicates presence of either phosphates or proteins (Fig. 1.4). A few drops of 10% acetic acid are added and the upper portion is boiled again. Turbidity due to phosphates disappears while that due to proteins does not.
zoom view
Fig. 1.4: Principle of heat test for proteins
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False-positive test occurs with tolbutamide and large doses of penicillins.
  1. 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 “proteinerror of indicators ”.
The reagent area is impregnated with bromophenol blue indicator buffered to pH 3.0 with citrate. When the dye gets adsorbed to protein, there is change in ionization (and hence pH) of the indicator that leads to change in color of the indicator. The intensity of the color produced is proportional to the concentration of protein. The test is semi-quantitative.
Reagent strip test is mainly reactive to albumin. It is false-negative in the presence of Bence Jones proteins, myoglobin, and hemoglobin. Overload (Bence Jones) proteinuria and tubular proteinuria may be missed entirely if only reagent strip method is used. This test should be followed by sulphosalicylic acid test, which is a confirmatory test. Highly alkaline urine, gross hematuria, and contamination with vaginal secretions can give false-positive reactions.
  1. Sulphosalicylic acid test: Addition of sulphosalicylic acid to the urine causes formation of a white precipitate if proteins are present (Proteins are denatured by organic acids and precipitate out of solution).
zoom view
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
zoom view
Fig. 1.6: Grading of proteinuria with reagent strip test (above) and sulphosalicylic acid test (below)
Take 2 ml of clear urine in a test tube. If reaction of urine is neutral or alkaline, a drop of glacial acetic acid is added. Add 2–3 drops of sulphosalicylic acid (3 to 5%), and examine for turbidity against a dark background (Fig. 1.6).
This test is more sensitive and reliable than boiling test.
False-positive test may occur due to gross hematuria, highly concentrated urine, radiographic contrast media, excess uric acid, tolbutamide, sulphonamides, salicylates, and penicillins.
False-negative test can occur with very dilute urine.
The test can detect albumin, hemoglobin, myoglobin, and Bence Jones proteins.
Comparison of reagent strip test and sulphosalicylic acid test is shown in Table 1.4.
 
Quantitative Estimation of Proteins
Indications for quantitative estimation of proteins in urine are:
  • Diagnosis of nephrotic syndrome
Table 1.4   Comparison of two tests for proteinuria
Parameter
Reagent strip test
Sulphosalicylic acid test
1. Principle
Colorimetric
Acid precipitation
2. Proteins detected
Albumin
All (albumin, Bence Jones proteins, hemoglobin, myoglobin)
3. Sensitivity
5–10 mg/dl
20 mg/dl
4. Indicator
Color change
Turbidity
5. Type of test
Screening
Confirmatory
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Table 1.5   Grading of albuminuria
Condition
mg/24 hr
mg/L
mg/g creatinine
μ g/min
μ g/mg creatinine
g/mol creatinine
Normal
< 30
< 20
< 20
< 20
< 30
< 2.5
Microalbuminuria
30s–300
20–200
20–300
20–200
30–300
2.5–25
Overt albuminuria
> 300
> 200
> 300
> 200
> 300
> 25
  • Detection of microalbuminuria or early diabetic nephropathy
  • To follow response to therapy in renal disease
Proteinuria >1500 mg/ 24 hours indicates glomerular disease; proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria; in tubular, hemodynamic and post renal diseases, proteinuria is usually < 1500 mg/24 hours.
Grading of albuminuria is shown in Table 1.5.
There are two methods for quantitation of proteins: (1) Estimation of proteins in a 24-hour urine sample, and (2) Estimation of protein/creatinine ratio in a random urine sample.
  1. Quantitative estimation of proteins in a 24-hour urine sample: Collection of a 24-hour sample is given earlier. Adequacy of sample is confirmed by calculating expected 24-hour urine creatinine excretion. Daily urinary creatinine excretion depends on muscle mass and remains relatively constant in an individual patient. In adult males creatinine excretion is 14–26 mg/kg/24 hours, while in women it is 11–20 mg/kg/24 hours. Various methods are available for quantitative estimation of proteins: Esbach's albuminometer method, turbidimetric methods, biuret reaction, and immunologic methods.
  2. Estimation of protein/creatinine ratio in a random urine sample: Because of the problem of incomplete collection of a 24-hour urine sample, many laboratories measure protein/creatinine ratio in a random urine sample. Normal protein/creatinine ratio is < 0.2. In low-grade proteinuria it is 0.2–1.0; in moderate, it is 1.0–3.5; and in nephrotic- range proteinuria it is > 3.5.
 
Microalbuminuria
This is defined as urinary excretion of 30 to 300 mg/24 hours (or 2–20 mg/dl) of albumin in urine.
Significance of microalbuminuria
  1. Microalbuminuria is considered as theearliest sign of renal damage in diabetes mellitus (diabetic nephropathy). It indicates increase in capillary permeability to albumin and denotes microvascular disease. Microalbuminuria precedes the development of diabetic nephropathy by a few years. If blood glucose level and hypertension are tightly controlled at this stage by aggressive treatment then progression to irreversible renal disease and subsequent renal failure can be delayed or prevented.
  2. Microalbuminuria is an independent risk factor for cardiovascular disease in diabetes mellitus.
Detection of microalbuminuria: Microalbuminuria cannot be detected by routine tests for proteinuria. Methods for detection include:
  • Measurement of albumin-creatinine ratio in a random urine sample
  • Measurement of albumin in an early morning or random urine sample
  • Measurement of albumin in a 24 hr sample
Test strips that screen for microalbuminuria are available commercially. Exact quantitation can be done by immunologic assays like radioimmunoassay or enzyme linked immunosorbent assay.
 
Bence Jones Proteinuria
Bence Jones proteins are monoclonal immunoglobulin light chains (either κ or λ) that are synthesized by neoplastic plasma cells. Excess production of these light chains occurs in plasma cell dyscrasias like multiple myeloma and primary amyloidosis. Because of their low molecular weight and high concentration they are excreted in urine (overflow proteinuria).
Bence Jones proteins have a characteristic thermal behaviour. When heated, Bence Jones proteins precipitate at temperatures between 40°C to 60°C (other proteins precipitate between 60–70°C), and precipitate disappears on further heating at 85–100°C (while precipitate of other proteins does not). When cooled (60–85°C), there is reappearance of precipitate of Bence Jones proteins. This test, however, is not specific for Bence Jones proteins and both false-positive and -negative results can occur. This test has been replaced by protein electrophoresis of concentrated urine sample (Fig. 1.7).12
zoom view
Fig. 1.7: Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine
Further evaluation of persistent overt proteinuria is shown in Figure 1.8.
 
Glucose
The main indication for testing for glucose in urine is detection of unsuspected diabetes mellitus or follow-up of known diabetic patients.
Practically all of the glucose filtered by the glomeruli is reabsorbed by the proximal renal tubules and returned to circulation. Normally a very small amount of glucose is excreted in urine (< 500 mg/24 hours or <15 mg/dl) that cannot be detected by the routine tests. Presence of detectable amounts of glucose in urine is called as glucosuria or glycosuria (Box 1.7). Glycosuria results if the filtered glucose load exceeds the capacity of renal tubular reabsorption. Most common cause is hyperglycemia from diabetes mellitus.
 
Causes of Glycosuria
  1. Glycosuria with hyperglycemia:
    • Endocrine diseases: diabetes mellitus, acromegaly, Cushing's syndrome, hyperthyroidism, pancreatic disease
    • Non-endocrine diseases: central nervous system diseases, liver disorders
    • Drugs: adrenocorticotrophic hormone, corticosteroids, thiazides
    • Alimentary glycosuria (Lag-storage glycosuria): After a meal, there is rapid intestinal absorption of glucose leading to transient elevation of blood glucose above renal threshold. This can occur in persons with gastrectomy or gastrojejunostomy and in hyperthyroidism. Glucose tolerance test reveals a peak at 1 hour above renal threshold (which causes glycosuria); the fasting and 2-hour glucose values are normal.
  2. Glycosuria without hyperglycemia
    • Renal glycosuria: This accounts for 5% of cases of glycosuria in general population. Renal threshold is the highest glucose level in blood at which glucose appears in urine and which is detectable by routine laboratory tests.
      zoom view
      Fig. 1.8: Evaluation of proteinuria
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      The normal renal threshold for glucose is 180 mg/dl. Threshold substances need a carrier to transport them from tubular lumen to blood. When the carrier is saturated, the threshold is reached and the substance is excreted. Up to this level glucose filtered by the glomeruli is efficiently reabsorbed by tubules. Renal glycosuria is a benign condition in which renal threshold is set below 180 mgs/dl but glucose tolerance is normal; the disorder is transmitted as autosomal dominant. Other conditions in which glycosuria can occur with blood glucose level remaining below 180 mgs/dl are renal tubular diseases in which there is decreased glucose reabsorption like Fanconi's syndrome, and toxic renal tubular damage. During pregnancy, renal threshold for glucose is decreased. Therefore it is necessary to estimate blood glucose when glucose is first detected in urine.
 
Tests for Detection of Glucose in Urine
1. Copper reduction methods
A. Benedict's qualitative test: When urine is boiled in Benedict's qualitative solution, blue alkaline copper sulphate is reduced to red-brown cuprous oxide if a reducing agent is present (Fig. 1.9). The extent of reduction depends on the concentration of the reducing substance. This test, however, is not specific for glucose. Other carbohydrates (like lactose, fructose, galactose, pentoses), certain metabolites (glucuronic acid, homogentisic acid, uric acid, creatinine), and drugs (ascorbic acid, salicylates, cephalosporins, penicillins, streptomycin, isoniazid, para-aminosalicylic acid, nalidixic acid, etc.) also reduce alkaline copper sulphate solution.
 
Method
  1. Take 5 ml of Benedict's qualitative reagent in a test tube (composition of Benedict's qualitative reagent: copper sulphate 17.3 gram, sodium carbonate 100 gram, sodium citrate 173 gram, distilled water 1000 ml).
  2. Add 0.5 ml (or 8 drops) of urine. Mix well.
  3. Boil over a flame for 2 minutes.
  4. Allow to cool at room temperature.
  5. Note the color change, if any.
Sensitivity of the test is about 200 mg reducing substance per dl of urine. Since Benedict's test gives positive reaction with carbohydrates other than glucose, it is also used as a screening test (for detection of galactose, lactose, fructose, maltose, and pentoses in urine) for inborn errors of carbohydrate metabolism in infants and children. For testing urine only for glucose, reagent strips are preferred (see below).
The result is reported in grades as follows (Fig. 1.10):
Nil: no change from blue color
Trace: Green without precipitate
1+ (approx. 0.5 grams/dl): Green with precipitate
2+ (approx. 1.0 grams/dl): Brown precipitate
3+ (approx. 1.5 grams/dl: Yellow-orange precipitate
4+ (<2.0 grams/dl): Brick- red precipitate.
zoom view
Fig. 1.9: Principle of Benedict's qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl
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zoom view
Fig. 1.10: Grading of Benedict's test (above) and reagent strip test (below) for glucose
B. Clinitest tablet method (Copper reduction tablet test): This is a modified form of Benedict's test in which the reagents are present in a tablet form (copper sulphate, citric acid, sodium carbonate, and anhydrous sodium hydroxide). Sensitivity is 200 mgs/dl of glucose.
2. Reagent strip method This test is specific for glucose andis therefore preferred over Benedict's and Clinitest methods. It is based on glucose oxidase-peroxidasereaction. Reagent area of the strips is impregnated with two enzymes (glucose oxidase and peroxidase) and a chromogen. Glucose is oxidized by glucose oxidase with the resultant formation of hydrogen peroxide and gluconic acid. Oxidation of chromogen occurs in the presence of hydrogen peroxide and the enzyme peroxidase with resultant color change(Fig. 1.11). Nature of chromogen and buffer system differ in different strips.
The strip is dipped into the urine sample and color is observed after a specified time and compared with the color chart provided (Fig. 1.10).
This test is more sensitive than Benedict's qualitative test and specific only for glucose. Other reducing agents give negative reaction.
Sensitivity of the test is about 100 mg glucose/dl of urine.
False positive test occurs in the presence of oxidizing agent (bleach or hypochlorite used to clean urine containers), which oxidizes the chromogen directly.
False-negative test occurs in the presence of large amounts of ketones, salicylates, ascorbic acid, and severe Escherichia coli infection (catalase produced by organisms in urine inactivates hydrogen peroxide).
 
Ketones
Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric acid, and acetone) in urine is called as ketonuria. Ketones are breakdown products of fattyacids and their presence in urine is indicative of excessive fatty acid metabolism to provide energy.
 
Causes of Ketonuria
Normally ketone bodies are not detectable in the urine of healthy persons. If energy requirements cannot be met by metabolism of glucose (due to defective carbohydrate metabolism, low carbohydrate intake, or increased metabolic needs), then energy is derived from breakdown of fats. This leads to the formation of ketone bodies (Fig. 1.12).
  1. Decreased utilization of carbohydrates
    1. Uncontrolled diabetes mellitus with ketoacidosis: In diabetes, because of poor glucose utilization, there is compensatory increased lipolysis. This causes increase in the level of free fatty acids in plasma. Degradation of free fatty acids in the liver leads to the formation of acetoacetyl CoA which then forms ketone bodies. Ketone bodies are strong acids and produce H+ ions, which are neutralized by bicarbonate ions; fall in bicarbonate (i.e. alkali) level produces ketoacidosis. Ketone bodies also increase the plasma osmolality and cause cellular dehydration. Children and young adults with type 1 diabetes are especially prone to ketoacidosis during acute illness and stress.
      zoom view
      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
      15
      zoom view
      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
      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.
    2. Glycogen storage disease (von Gierke's disease)
  2. Decreased availability of carbohydrates in the diet:
    1. Starvation
    2. Persistent vomiting in children
    3. Weight reduction program (severe carbohydrate restriction with normal fat intake)
  3. Increased metabolic needs:
    1. Fever in children
    2. Severe thyrotoxicosis
    3. Pregnancy
    4. Protein calorie malnutrition
 
Tests for Detection of Ketones in Urine
The proportion of ketone bodies in urine in ketosis is variable: β-hydroxybutyric acid 78%, acetoacetic acid 20%, and acetone 2%.
No method for detection of ketonuria reacts with all the three ketone bodies. Rothera's nitroprusside method and methods based on it detect acetoacetic acid and acetone (the test is 10–20 times more sensitive to acetoacetic acid than acetone). Ferric chloride test detects acetoacetic acid only. β-hydroxybutyric acid is not detected by any of the screening tests.
Methods for detection of ketone bodies in urine are Rothera's test, Acetest tablet method, ferric chloride test, and reagent strip test.
1. Rothera's' test (Classic nitroprusside reaction) Acetoacetic acid or acetone reacts with nitroprusside in alkaline solution to form a purple-colored complex (Fig. 1.13). Rothera's test is sensitive to 1–5 mg/dl of acetoacetate and to 10–25 mg/dl of acetone.
 
Method
  1. Take 5 ml of urine in a test tube and saturate it with ammonium sulphate.
  2. Add a small crystal of sodium nitroprusside. Mix well.
  3. Slowly run along the side of the test tube liquor ammonia to form a layer.
  4. Immediate formation of a purple permanganate colored ring at the junction of the two fluids indicates a positive test (Fig. 1.14).
False-positive test can occur in the presence of L-dopa in urine and in phenylketonuria.
2. Acetest tablet test This is Rothera's test in the form of a tablet. The Acetest tablet consists of sodium nitroprusside, glycine, and an alkaline buffer. A purplelavender discoloration of the tablet indicates the presence of acetoacetate or acetone (5 mg/dl). A rough estimate of the amount of ketone bodies can be obtained by comparison with the color chart provided by the manufacturer. The test is more sensitive than reagent strip test for ketones.
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Fig. 1.13: Principles of Rothera's test and reagent strip test for ketone bodies in urine. Ketones are detected as acetoacetic acid and acetone but not β-hydroxybutyric acid
16
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Fig. 1.14: Rothera's tube test and reagent strip test for ketone bodies in urine
3. Ferric chloride test (Gerhardt's): Addition of 10% ferric chloride solution to urine causes solution to become reddish or purplish if acetoacetic acid is present. The test is not specific since certain drugs (salicylate and L-dopa) give similar reaction. Sensitivity of the test is 25–50 mg/dl.
4. Reagent strip test: Reagent strips tests are modifications of nitroprusside test (Figs 1.13 and 1.14). Their sensitivity is 5–10 mg/dl of acetoacetate. If exposed to moisture, reagent strips often give false-negative result. Ketone pad on the strip test is especially vulnerable to improper storage andeasily 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 urobilinogen) is helpful in the differential diagnosis of jaundice (Table 1.6).
Table 1.6   Urine bilirubin and urobilinogen in jaundice
Urine test
Hemolytic jaundice
Hepatocellular jaundice
Obstructive jaundice
1. Bilirubin
Absent
Present
Present
2. Urobilinogen
Increased
Increased
Absent
In acute viral hepatitis, bilirubin appears in urine even before jaundice is clinically apparent. In a fever of unknown origin bilirubinuria suggests hepatitis.
 
Tests for Detection of Bilirubin in Urine
Bilirubin is converted to non-reactive biliverdin on exposure to light (daylight or fluorescent light) and on standing at room temperature. Biliverdin cannot be detected by tests that detect bilirubin. Therefore fresh sample that is kept protected from light is required.
Findings associated with bilirubinuria are shown in Box 1.9.
Methods for detection of bilirubin in urine are foam test, Gmelin's test, Lugol iodine test, Fouchet's test, Ictotest tablet test, and reagent strip test.
  1. Foam test: About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also obtained with proteins and highly concentrated urine. In normal urine, foam is white.
  2. Gmelin's test: Take 3 ml of concentrated nitric acid in a test tube and slowly place equal quantity of urine over it. The tube is shaken gently; play of colors (yellow, red, violet, blue, and green) indicates positive test (Fig. 1.15).
  3. Lugol iodine test: Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test tube and add 4 drops of urine. Mix by shaking. Development of green color indicates positive test.
    17
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    Fig. 1.15: Positive Gmelin's test for bilirubin showing play of colors
  4. Fouchet's test: This is a simple and sensitive test.
    1. 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).
    2. Filter to obtain the precipitate on a filter paper.
    3. 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).
    4. Immediate development of blue-green color around the drop indicates presence of bilirubin (Fig. 1.16).
  5. Reagent strips or tablets impregnated with diazo reagent: These tests are based on reaction of bilirubin with diazo reagent; color change is proportional to the concentration of bilirubin. Tablets (Ictotest) detect 0.05–0.1 mg of bilirubin/dl of urine; reagent strip tests are less sensitive (0.5 mg/dl).
 
Bile Salts
Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).
Bile salts along with bilirubin can be detected in urine in cases of obstructive jaundice. In obstructive jaundice, bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their detection is Hay's surface tension test. The property of bile salts to lower the surface tension is utilized in this test.
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Fig. 1.16: Positive Fouchet's test for bilirubin in urine
Take some fresh urine in a conical glass tube. Urine should be at the room temperature. Sprinkle on the surface particles of sulphur. If bile salts are present, sulphur particles sink to the bottom because of lowering of surface tension by bile salts. If sulphur particles remain on the surface of urine, bile salts are absent.
Thymol (used as a preservative) gives false positive test.
 
Urobilinogen
Conjugated bilirubin excreted into the duodenum through bile is converted by bacterial action to urobilinogen in the intestine. Major part is eliminated in the feces. A portion of urobilinogen is absorbed in blood, which undergoes recycling (enterohepatic circulation); a small amount, which is not taken up by the liver, is excreted in urine. Urobilinogen is colorless; upon oxidation it is converted to urobilin, which is orange-yellow in color. Normally about 0.5–4 mg of urobilinogen is excreted in urine in 24 hours. Therefore, a small amount of urobilinogen is normally detectable in urine.
Urinary excretion of urobilinogen shows diurnal variation with highest levels in afternoon. Therefore, a 2-hour post-meal sample is preferred.
 
Causes of Increased Urobilinogen in Urine
  1. Hemolysis: Excessive destruction of red cells leads to hyperbilirubinemia and therefore increased formation of urobilinogen in the gut. Bilirubin, being of unconjugated type, does not appear in urine. Increased urobilinogen in urine without bilirubin is 18typical 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 urobilinogen in urine are Ehrlich's aldehyde test and reagent strip test.
1. Ehrlich's aldehyde test: Ehrlich's reagent (p-dimethylaminobenzaldehyde) reacts with urobilinogen in urine to produce a pink color. Intensity of color developed depends on the amount of urobilinogen present. Presence of bilirubin interferes with the reaction, and therefore if present, should be removed. For this, equal volumes of urine and 10% barium chloride are mixed and then filtered. Test for urobilinogen is carried out on the filtrate. However, similar reaction is produced by porphobilinogen (a substance excreted in urine in patients of porphyria).
Method: Take 5 ml of fresh urine in a test tube. Add 0.5 ml of Ehrlich's aldehyde reagent (which consists of hydrochloric acid 20 ml, distilled water 80 ml, and para-dimethylaminobenzaldehyde 2 gm). Allow to stand at room temperature for 5 minutes. Development of pink color indicates normal amount of urobilinogen. Dark red color means increased amount of urobilinogen (Fig. 1.17).
Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. For this,Watson-Schwartz test is used. Add 1–2 ml of chloroform, shake for 2 minutes and allow to stand. Pink color in the chloroform layer indicates presence of urobilinogen, while pink coloration of aqueous portion indicates presence of porphobilinogen. Pink layer is then decanted and shaken with butanol. A pink color in the aqueous layer indicates porphobilinogen (Fig. 1.18).
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Fig. 1.17: Ehrlich's aldehyde test for urobilinogen
False-negative reaction can occur in the presence of (i) urinary tract infection (nitrites oxidize urobilinogen to urobilin), and (ii) antibiotic therapy (gut bacteria which produce urobilinogen are destroyed).
2. Reagent strip method: This method is specific for urobilinogen. Test area is impregnated with either p-dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate.
 
Blood
The presence of abnormal number of intact red blood cells in urine is called as hematuria. It implies presence of a bleeding lesion in the urinary tract. Bleeding in urine may be noted macroscopically or with naked eye (gross hematuria). If bleeding is noted only by microscopic examination or by chemical tests, then it is called as occult, microscopic or hidden hematuria.
 
Causes of Hematuria
  1. Diseases of urinary tract
    • Glomerular diseases: Glomerulonephritis, Berger's disease, lupus nephritis, Henoch-Schonlein purpura19
      zoom view
      Fig. 1.18: Interpretation of Watson-Schwartz test
    • Nonglomerular diseases: Calculus, tumor, infection, tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney disease, trauma, after strenuous physical exercise, diseases of prostate (benign hyperplasia of prostate, carcinoma of prostate).
  2. Hematological conditions: Coagulation disorders, sickle cell disease
    Presence of red cell casts and proteinuria along with hematuria suggests glomerular cause of hematuria.
 
Tests for Detection of Blood in Urine
  1. Microscopic examination of urinary sediment: Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test.
  2. Chemical tests: These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Fig. 1.19). Intensity of color produced is proportional to the amount of hemoglobin present.
Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.
  • Benzidine test: Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive.
  • Orthotoluidine test: In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test.
  • Reagent strip test: Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).
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Fig. 1.19: Principle of chemical test for red cells, hemoglobin, or myoglobin in urine
20
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Fig. 1.20: Evaluation of positive chemical test for blood in urine
Causes of false-positive tests:
  • Contamination of urine by menstrual blood in females
  • Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.
Causes of false-negative tests:
  • Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
  • Use of formalin as a preservative for urine
Evaluation of positive chemical test for blood is shown in Figure 1.20.
 
Hemoglobin
Presence of free hemoglobin in urine is called as hemoglobinuria.
 
Causes of Hemoglobinuria
  1. Hematuria with subsequent lysis of red blood cells in urine of low specific gravity.
  2. Intravascular hemolysis: Hemoglobin will appear in urine when haptoglobin (to which hemoglobin binds in plasma) is completely saturated with hemoglobin. Intravascular hemolysis occurs in infections (severe falciparum malaria, clostridial infection, E. coli septicemia), trauma to red cells (march hemoglobinuria, extensive burns, prosthetic heart valves), glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant drugs, immune hemolysis (mismatched blood transfusion, paroxysmal cold hemoglobinuria), paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and disseminated intravascular coagulation.
 
Tests for Detection of Hemoglobinuria
Tests for detection of hemoglobinuria are benzidine test, orthotoluidine test, and reagent strip test.
 
Hemosiderin
Hemosiderin in urine (hemosiderinuria) indicates presence of free hemoglobin in plasma. Hemosiderin appears as blue granules when urine sediment is stained with Prussian blue stain (Fig. 1.21). Granules are located inside tubular epithelial cells or may be free if cells have disintegrated. Hemosiderinuria is seen in intravascular hemolysis.
 
Myoglobin
Myoglobin is a protein present in striated muscle (skeletal and cardiac) which binds oxygen. Causes of myoglobinuria include injury to skeletal or cardiac muscle, e.g. crush injury, myocardial infarction, dermatomyositis, severe electric shock, and thermal burns.21
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Fig. 1.21: Staining of urine sediment with Prussian blue stain to demonstrate hemosiderin granules (blue)
Chemical tests used for detection of blood or hemoglobin also give positive reaction with myoglobin (as both hemoglobin and myoglobin have peroxidase activity). Ammonium sulfate solubility test is used as a screening test for myoglobinuria (Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while hemoglobin is insoluble and is precipitated. A positive chemical test for blood done on supernatant indicates myoglobinuria).
Distinction between hematuria, hemoglobinuria, and myoglobinuria is shown in Table 1.7.
 
Chemical Tests for Significant Bacteriuria (Indirect tests for urinary tract infection)
In addition to direct microscopic examination of urine sample, chemical tests are commercially available in a reagent strip format that can detect significant bacteriuria: nitrite test and leucocyte esterase test. These tests are helpful at places where urine microscopy is not available. If these tests are positive, urine culture is indicated.
  1. Nitrite test: Nitrites are not present in normal urine; ingested nitrites are converted to nitrate and excreted in urine. If gram-negative bacteria (e.g. E. coli, Salmonella, Proteus, Klebsiella, etc.) are present in urine, they will reduce the nitrates to nitrites through the action of bacterial enzyme nitrate reductase. Nitrites are then detected in urine by reagent strip tests. As E. coli is the commonest organism causing urinary tract infection, this test is helpful as a screening test for urinary tract infection.
    Some organisms like Staphylococci or Pseudomonas do not reduce nitrate to nitrite and therefore in such infections nitrite test is negative. Also, urine must be retained in the bladder for minimum of 4 hours for conversion of nitrate to nitrite to occur; therefore, fresh early morning specimen is preferred. Sufficient dietary intake of nitrate is necessary. Therefore a negative nitrite test does not necessarily indicate absence of urinary tract infection.
    The test detects about 70% cases of urinary tract infections.
  2. Leucocyte esterase test: It detects esterase enzyme released in urine from granules of leucocytes. Thus the test is positive in pyuria. If this test is positive, urine culture should be done. The test is not sensitive to leucocytes < 5/HPF.
 
MICROSCOPIC EXAMINATION
Microscopic examination of urine is also called as the “liquid biopsy of the urinary tract”.
Urine consists of various microscopic, insoluble, solid elements insuspension. These elements are classified as organized or unorganized.
Table 1.7   Differentiation between hematuria, hemoglobinuria, and myoglobinuria
Parameter
Hematuria
Hemoglobinuria
Myoglobinuria
1. Urine color
Normal, smoky, red, or brown
Pink, red, or brown
Red or brown
2. Plasma color
Normal
Pink
Normal
3. Urine test based on peroxidase activity
Positive
Positive
Positive
4. Urine microscopy
Many red cells
Occasional red cell
Occasional red cell
5. Serum haptoglobin
Normal
Low
Normal
6. Serum creatine kinase
Normal
Normal
Markedly increased
22
Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. The unorganized substances are crystalline and amorphous material. These elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; therefore they are known as urinary deposits or urinary sediments. Examination of urinary deposit is helpful in diagnosis of urinary tract diseases as shown in Table 1.8. Different types of urinary sediments are shown in Figure 1.22. The major aim of microscopic examination of urine is to identify different types of cellular elements and casts. Most crystals have little clinical significance.
Specimen: The cellular elements are best preserved in acid, hypertonic urine; they deteriorate rapidly in alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is the most concentrated. The specimen should be examined within 2 hours of voiding because cells and casts degenerate upon standing at room temperature. If preservative is required, then 1 crystal of thymol or 1 drop of formalin (40%) is added to about 10 ml of urine.
Method: A well-mixed sample of urine (12 ml) is centrifuged in a centrifuge tube for 5 minutes at 1500 rpm and supernatant is poured off. The tube is tapped at the bottom to resuspend the sediment (in 0.5 ml of urine). A drop of this is placed on a glass slide and covered with a cover slip (Fig. 1.23). The slide is examined immediately under the microscope using first the low power and then the high power objective. The condenser should be lowered to better visualize the elements by reducing the illumination.
 
Cells
Cellular elements in urine are shown in Figure 1.24.
Table 1.8   Urinary findings in renal diseases
Condition
Albumin
RBCs/HPF
WBCs/HPF
Casts/LPF
Others
1. Normal
0–trace
0–2
0–2
Occasional (Hyaline)
2. Acute glomerulonephritis
1–2+
Numerous; dysmorphic
0-few
Red cell, granular
Smoky urine or hematuria
3. Nephrotic syndrome
<4+
0-few
0-few
Fatty, hyaline, Waxy, epithelial
Oval fat bodies, 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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Fig. 1.22: Different types of urinary sediment
23
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Fig. 1.23: Preparation of urine sediment for microscopic examination
 
Red Blood Cells
Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9–10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of >80% of dysmorphic red cells is strongly suggestive of glomerular pathology.
The quantity of red cells can be reported as number of red cells per high power field.
Causes of hematuria have been listed earlier.
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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
24
 
White Blood Cells (Pus Cells)
White blood cells are spherical, 10–15 μ in size, granular in appearance in which nuclei may be visible. Degenerated 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, tubulointerstitial 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 (pyelonephritis).
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 distinguish from pus cells in unstained preparations.
 
Squamous Epithelial Cells
Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus.
 
Transitional Epithelial Cells
Transitional cells line renal pelvis, ureters, urinary bladder, and upper urethra. These cells are large, and diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization and in transitional cell carcinoma.
 
Oval Fat Bodies
These are degenerated renal tubular epithelial cells filled with highly refractile lipid (cholesterol) droplets. Under polarized light, they show a characteristic “Maltese cross” pattern. They can be stained with a fat stain such as Sudan III or Oil Red O. They are seen in nephrotic syndrome in which there is lipiduria.
 
Spermatozoa
They may sometimes be seen in urine of men.
Telescoped urinary sediment: This refers to urinary sediment consisting of red blood cells, white blood cells, oval fat bodies, and alltypes of casts in roughly equal proportion. It occurs in lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis.
 
Organisms
Organisms detectable in urine are shown in Figure 1.25.
 
Bacteria
Bacteria in urine can be detected by microscopic examination, reagent strip tests for significant bacteriuria (nitrite test, leucocyte esterase test), and culture.
Method of collection for bacteriologic examination is given earlier in Box 1.2.
Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a cleancatch midstream sample, >104 colony forming units/ml of urine in catheterized sample, and >103 colonyforming units/ml of urine in a suprapubic aspiration sample.
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Fig. 1.25: Organisms in urine: (A) Bacteria, (B) Yeasts,(C) Trichomonas, and (D) Egg of Schistosoma haematobium
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 Gramnegative 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 immunocompromised state, vaginal candidiasis, or diabetes mellitus. Usually pyuria is present if there is infection by Candida. Candida may also be a contaminant in the sample and therefore urine sample must be examined in a fresh state.
 
Trichomonas vaginalis
These are motile organisms with pear shape, undulating membrane on one side, and four flagellae. They cause vaginitis in females and are thus contaminants in urine. They are easily detected in fresh urine due to their motility.
 
Eggs of Schistosoma haematobium
Infection by this organism is prevalent in Egypt.
 
Microfilariae
They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel.
 
Casts
Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts. They take the shape and diameter of the lumina (molds or ‘casts’) of the renal tubules. They have parallel sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein). Since casts form only in renal tubules their presence is indicative of disease of the renal parenchyma. Although there are several types of casts, all urine casts are basically hyaline; various types of casts are formed when different elements get deposited on the hyaline material (Fig. 1.26). Casts are best seen under low power objective (×10) with condenser lowered down to reduce the illumination.
zoom view
Fig. 1.26: Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts
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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 TammHorsfall protein. They occur transiently after strenuous muscle exercise in healthy persons and during fever. Increased numbers are found in conditions causing glomerular proteinuria.
Granular casts: Presence of degenerated cellular debris in a cast makes it granular in appearance. These are cylindrical structures with coarse or fine granules (which represent degenerated renal tubular epithelial cells) embedded in Tamm-Horsfall protein matrix. They are seen after strenuous muscle exercise and in fever, acute glomerulonephritis, and pyelonephritis.
Waxy cast: These are the most easily recognized of all casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have homogenous, smooth glassy appearance, cracked or serrated margins and irregular broken-off ends. The ends are straight and sharp and not rounded as in other casts. They are light yellow in color. They are most commonly seen in end-stage renal failure.
Fatty casts: These are cylindrical structures filled with highly refractile fat globules (triglycerides and cholesterol esters) in Tamm-Horsfall protein matrix.
zoom view
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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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 interstitium 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
  1. Uric acid crystals: These are variable in shape (diamond, rosette, plates), and yellow or red-brown in color (due to urinary pigment). They are soluble in alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid crystals may be mistaken for cysteine crystals that also form in acid urine.
  2. Calcium oxalate crystals: These are colorless, refractile, and envelope-shaped. Sometimes dumbbell-shaped or peanut-like forms are seen. They are soluble in dilute hydrochloric acid. Ingestion of certain foods like tomatoes, spinach, cabbage, asparagus, and rhubarb causes increase in their numbers. Their increased number in fresh urine (oxaluria) may also suggest oxalate stones. A large number are seen in ethylene glycol poisoning.
  3. Amorphous urates: These are urate salts of potassium, magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are soluble in alkali or saline at 60°C.
Crystals present in alkaline urine:
  1. Calcium carbonate crystals: These are small, colorless, and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve.
  2. Phosphates: Phosphates may occur as crystals (triple phosphates, calcium hydrogen phosphate), or as amorphous deposits.
    • Phosphate crystals
      • Triple phosphates (ammonium magnesium phosphate): They are colorless, shiny, 3–6 sided prisms with oblique surfaces at the ends (“coffin-lids”), or may have a feathery fern-like appearance.
      • Calcium hydrogen phosphate (stellar phosphate): These are colorless, and of variable shape (star-shaped, plates or prisms).
    • Amorphous phosphates: These occur as colorless small granules, often dispersed.
      All phosphates are soluble in dilute acetic acid.
  3. Ammonium urate crystals: These occur as cactus-like (covered with spines) and called as ‘thornapple’ crystals. They are yellow-brown and soluble in acetic acid at 60°C.
 
Abnormal Crystals
They are rare, but result from a pathological process. These occur in acid pH, often in large amounts. Abnormal crystals should not be reported on microscopy alone;additional chemical tests are done for confirmation.28
zoom view
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
  1. Cysteine crystals: These are colorless, clear, hexagonal (having 6 sides), very refractile plates in acid urine. They often occur in layers. They are soluble in 30% hydrochloric acid. They are seen in cysteinuria, an inborn error of metabolism. Cysteine crystals are often associated with formation of cysteine stones.
  2. Cholesterol crystals: These are colorless, refractile, flat rectangular plates with notched (missing) corners, and appear stacked in a stair-step arrangement. They are soluble in ether, chloroform, or alcohol. They are seen in lipiduria e.g. nephrotic syndrome and hypercholesterolemia. They can be positively identified by polarizing microscope.
  3. Bilirubin crystals: These are small (5 µ), brown crystals of variable shape (square, bead-like, or fine needles). Their presence can be confirmed by doing reagent strip or chemical test for bilirubin. These crystals are soluble in strong acid or alkali. They are seen in severe obstructive liver disease.
    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
  • Appearance: Clear
  • Odor: Aromatic
  • Specific gravity: 1.003–1.030
  • Osmolality: 300–900 mOsm/kg of water
  • pH: 4.6–8.0 (Average: 6.0)
  • Proteins: Qualitative test: Negative
  • Quantitative test: < 150 mg/24 hours
  • Albumin: < 30 mg/24 hours
  • Glucose: Qualitative test: Negative
    Quantitative test: < 500 mg/24 hours (< 15 mg/dl)
  • Ketones: Qualitative test: Negative
  • Bilirubin: Negative
  • Bile salts: Negative
  • Occult blood: Negative
  • Urobilinogen: 0.5–4.0 mg/24 hours
  • Myoglobin(Ammonium sulphate solubility test): Negative
  • Microscopy: 1–2 red cells, pus cells, or epithelial cells/HPF; occasional hyaline cast/LPF; Phosphate, oxalate, or urate crystals depending on urine pH.
 
CRITICAL FINDINGS
  • Strongly positive test for glucose and ketone bodies
  • Positive test for reducing sugar in an infant
  • Hemoglobinuria
  • Red cell casts or >50% dysmorphic red cells on microscopic examination
  • Abnormal crystals like cysteine, leucine, or tyrosine.
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  1. Wallach J. Interpretation of diagnostic tests. (7th Ed). Lippincott Williams and Wilkins,  Philadelphia:  2000.
  1. World Health Organization. Manual of basic techniques for a health laboratory (2nd Ed). World Health Organization,  Geneva;  2003.