Written by Monday, 07 August 2017 18:11
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 (Figure 822.1). Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and to 10-25 mg/dl of acetone.
Figure 822.1 Principles of Rothera Test in Urine
Figure 822.1 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
  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 (Figure 822.2).
False-positive test can occur in the presence of L-dopa in urine and in phenylketonuria.
Figure 822.2 Rotheras tube test and reagent strip test for ketone bodies in urine
Figure 822.2 Rothera’s tube test and reagent strip test for ketone bodies in urine
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.
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.
Reagent strips tests are modifications of nitroprusside test (Figures 822.1 and 822.2). 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. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.


Written by Monday, 07 August 2017 13:36
This test is based on the principle that proteins get precipitated when boiled in an acidic solution.
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 (Figure 821.1). 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.
Figure 821.1 Principle of heat test for proteins
Figure 821.1 Principle of heat test for proteins
False-positive test occurs with tolbutamide and large doses of penicillins.
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 (Figures 821.2 and 821.3). The principle is known as “protein error of indicators”.
Figure 821.2 Principle of reagent strip test for proteins
Figure 821.2 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
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.
Figure 821.3 Grading of proteinuria with reagent strip test
Figure 821.3 Grading of proteinuria with reagent strip test (above) and sulphosalicylic acid test (below)
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. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
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).
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 (Figure 821.3).
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 821.1.
Table 821.1 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
Indications for quantitative estimation of proteins in urine are:
  • Diagnosis of nephrotic syndrome
  • Detection of microalbuminuria or early diabetic nephropathy
  • To follow response to therapy in renal disease
Proteinuria >1500 mg/ 24 hours indicates glomerular disease; proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria; in tubular, hemodynamic and post renal diseases, proteinuria is usually < 1500 mg/24 hours.
Grading of albuminuria is shown in Table 821.2. 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.
Table 821.2 Grading of albuminuria
Condition mg/24 hr mg/L mg/g creatinine μg/min μg/mg creatinine g/mol creatinine
Normal < 30 < 20 < 20 < 20 < 30 < 2.5
Microalbuminuria 30-300 20-200 20-300 20-200 30-300 2.5-25
Overt albuminuria > 300 > 200 > 300 > 200 > 300 > 25
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.
This is defined as urinary excretion of 30 to 300 mg/24 hours (or 2-20 mg/dl) of albumin in urine.
Significance of Microalbuminuria
  1. Microalbuminuria is considered as the earliest sign of renal damage in diabetes mellitus (diabetic nephropathy). It indicates increase in capillary permeability to albumin and denotes microvascular disease. Microalbuminuria precedes the development of diabetic nephropathy by a few years. If blood glucose level and hypertension are tightly controlled at this stage by aggressive treatment then progression to irreversible renal disease and subsequent renal failure can be delayed or prevented.
  2. Microalbuminuria is an independent risk factor for cardiovascular disease in diabetes mellitus.
Detection of Microalbuminuria: Microalbuminuria cannot be detected by routine tests for proteinuria. Methods for detection include:
  • Measurement of albumin-creatinine ratio in a random urine sample
  • Measurement of albumin in an early morning or random urine sample
  • Measurement of albumin in a 24 hr sample
Test strips that screen for microalbuminuria are available commercially. Exact quantitation can be done by immunologic assays like radioimmunoassay or enzyme linked immunosorbent assay.
Bence Jones 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 (Figure 821.4).
Figure 821.4 Urine protein electrophoresis showing heavy Bence Jones proteinuria
Figure 821.4 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 821.5.
Figure 821.5 Evaluation of proteinuria
Figure 821.5 Evaluation of proteinuria.
Note: Quantitation of proteins and creatinine clearance are done in all patients with persistent proteinuria


Written by Saturday, 05 August 2017 23:28
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 (Figure 820.1). The extent of reduction depends on the concentration of the reducing substance. This test, however, is not specific for glucose.
Figure 820.1 Principle of Benedict’s qualitative test for sugar in urine
Figure 820.1 Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl
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.
  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 (Figure 820.2):
  • 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.
Figure 820.2 Grading of Benedicts test
Figure 820.2 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.
This test is specific for glucose and is therefore preferred over Benedict’s and Clinitest methods. It is based on glucose oxidase-peroxidase reaction. Reagent area of the strips is impregnated with two enzymes (glucose oxidase and peroxidase) and a chromogen. Glucose is oxidized by glucose oxidase with the resultant formation of hydrogen peroxide and gluconic acid. Oxidation of chromogen occurs in the presence of hydrogen peroxide and the enzyme peroxidase with resultant color change (Figure 820.3). Nature of chromogen and buffer system differ in different strips. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
The strip is dipped into the urine sample and color is observed after a specified time and compared with the color chart provided (Figure 820.2).
Figure 820.3 Principle of reagent strip test for glucose in urine
Figure 820.3 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
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).


Written by Saturday, 05 August 2017 17:22
The parameters to be examined on physical examination of urine are listed below.
  • Volume
  • Color
  • Appearance
  • Odor
  • Specific Gravity
  • pH
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.
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 819.1.
Table 819.1 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
Normal, freshly voided urine is clear in appearance. Causes of cloudy or turbid urine are listed in Table 819.2. Foamy urine occurs in the presence of excess proteins or bilirubin.
Table 819.2 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
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.
This is also called as relative mass density. It depends on amount of solutes in solution. It is basically a comparison of density of urine against the density of distilled water at a particular temperature. Specific gravity of distilled water is 1.000. Normal SG of urine is 1.003 to 1.030 and depends on the state of hydration. SG of normal urine is mainly related to urea and sodium. SG increases as solute concentration increases and decreases when temperature rises (since volume expands with rise in temperature).
SG of urine is a measure of concentrating ability of kidneys and is determined to get information about this tubular function. SG, however, is affected by proteinuria and glycosuria.
Causes of increase in SG of urine are diabetes mellitus (glycosuria), nephrotic syndrome (proteinuria), fever, and dehydration.
Causes of decrease in SG of urine are diabetes insipidus (SG consistently between 1.002-1.003), chronic renal failure (low and fixed SG at 1.010 due to loss of concentrating ability of tubules) and compulsive water drinking.
Methods for measuring SG are urinometer method, refractometer method, and reagent strip method.

1. Urinometer method: This method is based on the principle of buoyancy (i.e. the ability of a fluid to exert an upward thrust on a body placed in it). Urinometer (a hydrometer) is placed in a container filled with urine (Figure 819.1A). 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).
Figure 819.1 A. Urinometer method and B. Reagent strip method for measuring specific gravity of urine
Figure 819.1 (A) Urinometer method and (B) Reagent strip method for measuring specific gravity of urine
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. If not, then the difference needs to be adjusted in test readings taken subsequently.
The method is as follows:
  1. Fill a measuring cylinder with 50 ml of urine.
  2. Lower urinometer gently into the urine and let it float freely.
  3. Let urinometer settle; it should not touch the sides or bottom of the cylinder.
  4. Take the reading of SG on the scale (lowest point of meniscus) at the surface of the urine.
  5. Take out the urinometer and immediately note the temperature of urine with a thermometer.
Correction for temperature: Density of urine increases at low temperature and decreases at higher temperature. This causes false reading of SG. Therefore, SG is corrected for difference between urine temperature and calibration temperature. Check the temperature of calibration of the urinometer To get the corrected SG, add 0.001 to the reading for every 3°C that the urine temperature is above the temperature of calibration. Similarly subtract 0.001 from the reading for every 3°C below the calibration temperature.
Correction for dilution: If quantity of urine is not sufficient for measurement of SG, urine can be appropriately diluted and the last two figures of SG are multiplied by the dilution factor.
Correction for abnormal solute concentration: High SG in the presence of glycosuria or proteinuria will not reflect true kidney function (concentrating ability). Therefore it is necessary to nullify the effect of glucose or proteins. For this, 0.003 is subtracted from temperature-corrected SG for each 1 gm of protein/dl urine and 0.004 for every 1 gm of glucose/dl urine.
2. Refractometer method: SG can be precisely determined by a refractometer, which measures the refractive index of the total soluble solids. Higher the concentration of total dissolved solids, higher the refractive index. Extent of refraction of a beam of light passed through urine is a measure of solute concentration, and thus of SG. The method is simple and requires only 1-2 drops of urine. Result is read from a scale or from digital display.
3. Reagent strip method: Reagent strip (Figure 819.1B) 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). Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
The pH is the scale for measuring acidity or alkalinity (acid if pH is < 7.0; alkaline if pH is > 7.0; neutral if pH is 7.0). On standing, urine becomes alkaline because of loss of carbon dioxide and production of ammonia from urea. Therefore, for correct estimation of pH, fresh urine should be examined.
There are various methods for determination of reaction of urine: litmus paper, pH indicator paper, pH meter, and reagent strip tests.
  1. Litmus paper test: A small strip of litmus paper is dipped in urine and any color change is noted. If blue litmus paper turns red, it indicates acid urine. If red paper turns blue, it indicates alkaline urine (Figure 819.2A).
  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 (Figure 819.2B) contains polyionic polymer bound to H+; on reaction with cations in urine, H+ is released causing change in color of the pH-sensitive dye. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
Figure 819.2 A. Testing pH of urine with litmus paper and B. with reagent strip test
Figure 819.2 Testing pH of urine with litmus paper (A) and with reagent strip test (B)
Normal pH range is 4.6 to 8.0 (average 6.0 or slightly acidic). Urine pH depends on diet, acid base balance, water balance, and renal tubular function.
Acidic urine is found in ketosis (diabetes mellitus, starvation, fever), urinary tract infection by Escherichia coli, and high protein diet. Alkaline urine may result from 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.


Written by Saturday, 05 August 2017 15:23
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.
Ehrlich’s reagent (pdimethylaminobenzaldehyde) 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).
Fig. 818.1 Ehrlichs aldehyde test for urobilinogen
Figure 818.1 Ehrlich’s aldehyde test for urobilinogen
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 paradimethylaminobenzaldehyde 2 gm). Allow to stand at room temperature for 5 minutes. Development of pink color indicates normal amount of urobilinogen. Darkred color means increased amount of urobilinogen (Figure 818.1).
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 (Figure 818.2).
Figure 818.2 Interpretation of Watson Schwartz test
Figure 818.2 Interpretation of Watson-Schwartz test
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).
This method is specific for urobilinogen. Test area is impregnated with either p-dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.


Written by Thursday, 03 August 2017 18:15
Porphyrias (from Greek porphura meaning purple pigment; the name is probably derived from purple discoloration of some body fluids during the attack) are a heterogeneous group of rare disorders resulting from disturbance in the heme biosynthetic pathway leading to the abnormal accumulations of red and purple pigments called as porphyrins in the body. Heme, a component of hemoglobin, is synthesized through various steps as shown in Figure 817.1. Each of the steps is catalyzed by a separate enzyme; if any of these steps fails (due to hereditary or acquired cause), precursors of heme (porphyrin intermediates) accumulate in blood, get deposited in skin and other organs, and excreted in urine and feces. Depending on the site of defect, different types of porphyrias are described with varying clinical features, severity, and the nature of accumulated porphyrin.
Porphyria has been offered as a possible explanation for the medieval tales of vampires and werewolves; this is because of the number of similarities between the behavior of persons suffering from porphyria and the folklore (avoiding sunlight, mutilation of skin on exposure to sunlight, red teeth, psychiatric disturbance, and drinking of blood to obtain heme).
Porphyrias are often missed or wrongly diagnosed as many of them are not associated with definite physical findings, screening tests may yield false-negative results, diagnostic criteria are poorly defined and mild disorders produce an enzyme assay result within ‘normal’ range.
Heme is mainly required in bone marrow (for hemoglobin synthesis) and in liver (for cytochromes). Therefore, porphyrias are divided into erythropoietic and hepatic types, depending on the site of expression of disease. Hepatic porphyrias mainly affect the nervous system, while erythropoietic porphyrias primarily affect the skin. Porphyrias are also classified into acute and nonacute (or cutaneous) types depending on clinical presentation (Table 817.1).
Table 817.1 Various classification schemes for porphyrias
Classification based on predominant clinical manifestations
Classification based on site of expression of disease
Classification based on mode of clinical presentation
1. Acute intermittent porphyria
1. ALA-dehydratase porphyria
1. ALA-dehydratase porphyria (Plumboporphyria)
2. ALA-dehydratase porphyria (Plumboporphyria)
2. Acute intermittent porphyria
2. Acute intermittent porphyria
Cutaneous (Photosensitivity)
3. Hereditary coproporphyria
3. Hereditary coproporphyria
1. Congenital erythropoietic porphyria
4. Variegate porphyria
4. Variegate porphyria
2. Porphyria cutanea tarda
Erythropoietic porphyria
Non-acute (cutaneous)
3. Erythropoietic protoporphyria
1. Congenital erythropoietic porphyria
1. Porphyria cutanea tarda
Mixed (Neuropsychiatric and cutaneous)
2. Erythropoietic protoporphyria
2. Congenital erythropoietic porphyria
1. Hereditary coproporphyria
3. Erythropoietic protoporphyria
2. Variegate porphyria
1. Porphyria cutanea tarda
Inheritance of porphyrias may be autosomal dominant or recessive. Most acute porphyrias are inherited in an autosomal dominant manner (i.e. inheritance of one abnormal copy of gene). Therefore, the activity of the deficient enzyme is 50%. When the level of heme falls in the liver due to some cause, activity of ALA synthase is stimulated leading to increase in the levels of heme precursors up to the point of enzyme defect. Increased levels of heme precursors cause symptoms of acute porphyria. When the heme level returns back to normal, symptoms subside.
Accumulation of porphyrin precursors can occur in lead poisoning due to inhibition of enzyme aminolevulinic acid dehydratase in heme biosynthetic pathway. This can mimick acute intermittent porphyria.
Clinical features of porphyrias are variable and depend on type. Acute porphyrias present with symptoms like acute and severe abdominal pain/vomiting/constipation, chest pain, emotional and mental disorders, seizures, hypertension, tachycardia, sensory loss, and muscle weakness. Cutaneous porphyrias present with photosensitivity (redness and blistering of skin on exposure to sunlight), itching, necrosis of skin and gums, and increased hair growth over the temples (Table 817.2).
Table 817.2 Clinical characteristics of porphyrias
Porphyria Deficient enzyme Clinical features Inheritance Initial test
1. Acute intermittent porphyria (AIP)* PBG deaminase Acute neurovisceral attacks; triggering factors+ (e.g. drugs, diet restriction) Autosomal dominant Urinary PBG; urine becomes brown, red, or black on standing
2. Variegate porphyria Protoporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
3. Hereditary coproporphyria Coproporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
4. Congenital erythropoietic porphyria Uroporphyrinogen cosynthase Onset in infancy; skin fragility, bullae; extreme photosensitivity with mutilation; red teeth and urine (pink red urinestaining of diapers) Autosomal recessive Urinary/fecal total porphyrins; ultraviolet fluorescence of urine, feces, and bones
5. Porphyria cutanea tarda* Uroporphyrinogen decarboxylase Skin fragility, bullae Autosomal dominant (some cases) Urinary/fecal total porphyrins
6. Erythropoietic protoporphyria* Ferrochelatase Acute photosensitivity Autosomal dominant Free erythrocyte protoporphyrin
Disorders marked with * are the three most common porphyrias. PBG: Porphobilinogen
Symptoms can be triggered by drugs (barbiturates, oral contraceptives, diazepam, phenytoin, carbamazepine, methyldopa, sulfonamides, chloramphenicol, and antihistamines), emotional or physical stress, infection, dieting, fasting, substance abuse, premenstrual period, smoking, and alcohol. Autosomal dominant porphyrias include acute intermittent porphyria, variegate porphyria, porphyria cutanea tarda, erythropoietic protoporphyria (most cases), and hereditary coproporphyria. Autosomal recessive porphyrias include: congenital erythropoietic porphyria, erythropoietic protoporphyria (few cases), and ALAdehydratase porphyria (plumboporphyria).
Porphyria can be diagnosed through tests done on blood, urine, and feces during symptomatic period. Timely and accurate diagnosis is required for effective management of porphyrias. Due to the variability and a broad range of clinical features, porphyrias are included under differential diagnosis of many conditions. All routine hospital laboratories usually have facilities for initial investigations in suspected cases of porphyrias; laboratory tests for identification of specific type of porphyrias are available in specialized laboratories.
In suspected acute porphyrias (acute neurovisceral attack), a fresh randomly collected urine sample (10-20 ml) should be submitted for detection of excessive urinary excretion of porphobilinogen (PBG) (see Figure 817.2). In AIP, urine becomes red or brown on standing (see Figure 817.3). In suspected cases of cutaneous porphyrias (acute photosensitivity without skin fragility), free erythrocyte protporphyrin or FEP in EDTA blood (for diagnosis of erythrocytic protoporphyria) and for all other cutaneous porphyrias (skin fragility and bullae), examination of fresh, random urine (10-20 ml) and either feces (5-10 g) or plasma for excess porphyrins are necessary (see Figure 817.4 and Table 817.2).
Figure 817.2 Evaluation of acute neurovisceral porphyria
 Figure 817.2 Evaluation of acute neurovisceral porphyria
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
Figure 817.4 Evaluation of cutaneous porphyrias
Figure 817.4 Evaluation of cutaneous porphyrias
Apart from diagnosis, the detection of excretion of a particular heme intermediate in urine or feces can help in detecting site of defect in porphyria. Heme precursors up to coproporphyrinogen III are water-soluble and thus can be detected in urine. Protoporphyrinogen and Protoporphyrin are insoluble in water and are excreted in bile and can be detected in feces. All samples should be protected from light.
Samples required are
  1. 10-20 ml of fresh random urine sample without any preservative;
  2. 5-10 g wet weight of fecal sample, and
  3. blood anticoagulated with EDTA.
Test for Porphobilinogen in Urine
Ehrlich’s aldehyde test is done for detection of PBG. Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts with PBG in urine to produce a red color. The red product has an absorption spectrum with a peak at 553 nm and a shoulder at 540 nm. Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. Urobilinogen can be removed by solvent extraction. (See Watson-Schwartz test). Levels of PBG may be normal or near normal in between attacks. Therefore, samples should be tested during an attack to avoid false-negative results.
Test for Total Porphyrins in Urine
Total porphyrins can be detected in acidified urine sample by spectrophotometry (Porphyrins have an intense absorbance peak around 400 nm). Semiquantitative estimation of porphyrins is possible.
Test for Total Porphyrins in Feces
Total porphyrins in feces can be determined in acidic extract of fecal sample by spectrophotometry; it is necessary to first remove dietary chlorophyll (that also absorbs light around 400 nm) by diethyl ether extraction.
Tests for Porphyrins in Erythrocytes and Plasma
Visual examination for porphyrin fluorescence, and solvent fractionation and spectrophotometry have now been replaced by fluorometric methods.
Further Testing
If the initial testing for porphyria is positive, then concentrations of porphyrins should be estimated in urine, feces, and blood to arrive at specific diagnosis (Tables 817.3 and 817.4).
Table 817.3 Diagnostic patterns of concentrations of heme precursors in acute porphyrias
Porphyria Urine Feces
Acute intermittent porphyria PBG, Copro III
Variegate porphyria PBG, Copro III Proto IX
Hereditary coproporphyria PBG, Copro III Copro III
PBG: Porphobilinogen; Copro III: Coproporphyrinogen III; Proto IX: Protoporphyrin IX
Table 817.4 Diagnostic patterns of concentrations of heme precursors in cutaneous porphyrias
Porphyria Urine Feces Erythrocytes
Congenital erythropoietic porphyria Uro I, Copro I Copro I
Porphyria cutanea tarda Uroporphyrin Isocopro
Erythropoietic protoporphyria Protoporphyrin
Uro I: Uroporphyrinogen I; Copro I: Coproporphyrinogen I; Isocopro: Isocoproporphyrinogen
In latent porphyrias and in patients during remission, porphyrin levels may be normal; in such cases, enzymatic and DNA testing is necessary for diagnosis.
If porphyria is diagnosed, then it is necessary to investigate close family members for the disorder. Positive family members should be counseled regarding triggering factors.


Written by Wednesday, 02 August 2017 09:18
Because diagnoses and treatment plans are made based on laboratory findings, it is imperative that the equipment utilized in the lab be in excellent working order, serviced at regular intervals, calibrated and cleaned as recommended by the manufacturer, and used properly. In addition to properly functioning equipment, there are things the technician can do to improve the accuracy of their test results:
  1. Follow manufacturer directions precisely.
  2. Become familiar with normal and abnormal findings.
  3. Log all activity of equipment, including daily, weekly, and monthly servicing.
  4. Save enough sample to perform tests more than once to verify accuracy of findings.


Remember, all laboratory equipment and its results are only as reliable as the human operating the equipment!


Written by Monday, 31 July 2017 18:06
Routine care and proper maintenance of microscope will ensure good performance over the years. In addition to this, a properly maintained and clean microscope will always be ready for use at any time. Professional cleaning and maintenance should be considered when routine techniques fail to produce optimal performance of the microscope.
Cleaning and maintenance supplies
Dust cover: When not in use, a microscope should be covered to protect it from dust, hair, and any other possible sources of dirt. It is important to note that a dust cover should never be placed over a microscope while the illuminator is still on.
Lens tissue: Lint-free lens tissues are delicate wipes that would not scratch the surface of the oculars or objective. Always ensure that you are using these types of tissues. Never substitute facial tissue or paper towel, as they are too abrasive.
Lens cleaner: Lens cleaning solution assists in removing fingerprints and smudges on lenses and objectives. Apply the lens cleaner to the lens tissue paper and clean/polish the surface.
Compressed air duster: Using compressed air to rid the microscope of dust particles is far superior to using your own breath and blowing onto the microscope. Compressed air is clean, and avoids possible contamination of saliva particles.
Maintenance tips
  1. Whenever the microscope is not in use, turn off the illuminator. This will greatly extend the life of the bulb, as well as keep the temperature down during extended periods of laboratory work.
  2. When cleaning the microscope, use distilled water or lens cleaner. Avoid using other chemicals or solvents, as they may be corrosive to the rubber or lens mounts.
  3. After using immersion oil, clean off any residue immediately. Avoid rotating the 40× objective through immersion oil. If this should occur, immediately clean the 40× objective with lens cleaner before the oil has a chance to dry.
  4. Do not be afraid to use many sheets of lens tissue when cleaning. Use a fresh piece (or a clean area of the same piece) when moving to a different part of the microscope. This avoids tracking dirt/oil/residue to other areas of the microscope.
  5. Store the microscope safely with the stage lowered and the smallest objective in position (4× or 10×). This placement allows for the greatest distance between the stage and the objective. If the microscope is bumped, the likelihood of an objective becoming damaged by the stage surface will be greatly minimized.


Written by Tuesday, 25 July 2017 15:42
The microscope is the most important piece of equipment in the clinic laboratory. The microscope is used to review fecal, urine, blood, and cytology samples on a daily basis (see Figure). Understanding how the microscope functions, how it operates, and how to care for it will improve the reliability of your results and prolong the life of this valuable piece of equipment.

Parts and functions of a compound microscope

Compound Microscope(A) Arm: Used to carry the microscope.
(B) Base: Supports the microscope and houses the light source.
(C) Oculars (or eyepieces): The lens of the microscope you look through. The ocular also magnifies the image. The total magnification can be calculated by multiplying the objective power by the ocular power. Oculars come in different magnifications, but 10× magnification is common.
(D) Diopter adjustment: The purpose of the diopter adjustment is to correct the differences in vision an individual may have between their left and right eyes.
(E) Interpupillary adjustment: This allows the oculars to move closer or further away from one another to match the width of an individual’s eyes. When looking through the microscope, one should see only a single field of view. When viewing a sample, always use both eyes. Using one eye can cause eye strain over a period of time.
(F) Nosepiece: The nosepiece holds the objective lenses. The objectives are mounted on a rotating turret so they can be moved into place as needed. Most nosepieces can hold up to five objectives.
(G) Objective lenses: The objective lens is the lens closest to the object being viewed, and its function is to magnify it. Objective lenses are available in many powers, but 4×, 10×, 40×, and 100× are standard. 4× objective is used mainly for scanning. 10× objective is considered “low power,” 40× is “high power” and 100× objective is referred to as “oil immersion.” Once magnified by the objective lens, the image is viewed through the oculars, which magnify it further. Total magnification can be calculated by multiplying the objective power by the ocular lens power.
For example: 100× objective lens with 10× oculars = 1000× total magnification.
(H) Stage: The platform on which the slide or object is placed for viewing.
(I) Stage brackets: Spring-loaded brackets, or clips, hold the slide or specimen in place on the stage.
(J) Stage control knobs: Located just below the stage are the stage control knobs. These knobs move the slide or specimen either horizontally (x-axis) or vertically (y-axis) when it is being viewed.
(K) Condenser: The condenser is located under the stage. As light travels from the illuminator, it passes through the condenser, where it is focused and directed at the specimen.
(L) Condenser control knob: Allows the condenser to be raised or lowered.
(M) Condenser centering screws: These crews center the condenser, and therefore the beam of light. Generally, they do not need much adjustment unless the microscope is moved or transported frequently.
(N) Iris diaphragm: This structure controls the amount of light that reaches the specimen. Opening and closing the iris diaphragm adjusts the diameter of the light beam.
(O) Coarse and fine focus adjustment knobs: These knobs bring the object into focus by raising and lowering the stage. Care should be taken when adjusting the stage height. When a higher power objective is in place (100× objective for example), there is a risk of raising the stage and slide and hitting the objective lens. This can break the slide and scratch the lens surface. Coarse adjustment is used for finding focus under low power and adjusting the stage height. Fine adjustment is used for more delicate, high power adjustment that would require fine tuning.
(P) Illuminator: The illuminator is the light source for the microscope, usually situated in the base. The brightness of the light from the illuminator can be adjusted to suit your preference and the object you are viewing.


Written by Tuesday, 25 July 2017 13:43
What is Kohler illumination?

Kohler illumination is a method of adjusting a microscope in order to provide optimal illumination by focusing the light on the specimen. When a microscope is in Kohler, specimens will appear clearer, and in more detail.

Process of setting Kohler
Materials required
  • Specimen slide (will need tofocus under 10× power)
  • Compound microscope.
Kohler illumination
  1. Mount the specimen slide onthe stage and focus under 10×.
  2. Close the iris diaphragm completely.
  3. If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
  4. Using the condenser adjustment knobs, raise or lower the condenser until the edges of the field becomes sharp (see Figure 797.1 and Figure 797.2).
  5. Open the iris diaphragm until the entire field is illuminated.
Note the blurry edges of the unfocused light
Figure 797.1 Note the blurry edges of the unfocused light
Adjusting the condenser height sharpens the edges of the ball of light
Figure 797.2 Adjusting the condenser height sharpens the edges of the “ball of light.”
When should you set/check Kohler?
  • During regular microscope maintenance
  • After the microscope is moved/transported
  • Whenever you suspect objects do not appear as sharp as they could be.
Further Reading:

Useful Sites

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