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Published in Clinical Pathology
Thursday, 10 August 2017 23:29
The chemical examination is carried out for substances in urine are listed below:
  • Proteins
  • Glucose
  • Ketones
  • Bilirubin
  • Bile salts
  • Urobilinogen
  • Blood
  • Hemoglobin
  • Myoglobin
  • Nitrite or leukocyte esterase

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

Box 826.1: Causes of proteinuria

• Glomerular proteinuria
• Tubular proteinuria
• Overflow proteinuria
• Hemodynamic (functional) proteinuria
• Post-renal proteinuria
Causes of proteinuria can be grouped as shown in Box 826.1.
  • Glomerular proteinuria: Proteinuria due to increased permeability of glomerular capillary wall is called as glomerular proteinuria.

    There are two types of glomerular proteinuria: selective and nonselective. In early stages of glomerular disease, there is increased excretion of lower molecular weight proteins like albumin and transferrin. When glomeruli can retain larger molecular weight proteins but allow passage of comparatively lower molecular weight proteins, the proteinuria is called as selective. With further glomerular damage, this selectivity is lost and larger molecular weight proteins (γ globulins) are also excreted along with albumin; this is called as nonselective proteinuria.

    Selective and nonselective proteinuria can be distinguished by urine protein electrophoresis. In selective proteinuria, albumin and transferrin bands are seen, while in nonselective type, the pattern resembles that of serum (Figure 826.1).

    • Massive proteinuria (>3.5 gm/24 hr)
    • Hypoalbuminemia (<3.0 gm/dl)
    • Generalised edema
    Hyperlipidemia (serum cholesterol >350 mg/dl)
    • Lipiduria
    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. Serial estimations of urinary protein are also helpful in monitoring response to treatment. Most severe degree of proteinuria occurs in nephrotic syndrome (Box 826.2).

  • Tubular proteinuria: Normally, glomerular membrane, although impermeable to high molecular weight proteins, allows ready passage to low molecular weight proteins like β2-microglobulin, retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light chains. These low molecular weight proteins are actively reabsorbed by proximal renal tubules. In diseases involving mainly tubules, these proteins are excreted in urine while albumin excretion is minimal.

    Urine electrophoresis shows prominent α- and β-bands (where low molecular weight proteins migrate) and a faint albumin band (Figure 826.1).

    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.

  • 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).

  • 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.

  • Post-renal proteinuria: This is caused by inflammatory or neoplastic conditions in renal pelvis, ureter, bladder, prostate, or urethra.
Figure 826.1 Glomerular and tubular proteinuria
Figure 826.1 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
The main indication for testing for glucose in urine is detection of unsuspected diabetes mellitus or follow-up of known diabetic patients.
Box 826.3: Urine glucose
• Urine should be tested for glucose within 2 hours of collection (due to lowering of glucose by glycolysis and by contaminating bacteria which degrade glucose rapidly)
• Reagent strip test is a rapid, inexpensive, and semi-quantitative test
• In the past this test was used for home-monitoring of glucose; the test is replaced by glucometers.
• Urine glucose cannot be used to monitor control of diabetes since renal threshold is variable amongst individuals, no information about level of blood glucose below renal threshold is obtained, and urinary glucose value is affected by concentration of urine.
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 826.3). 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. 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.
Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric acid, and acetone) in urine is called as ketonuria. Ketones are breakdown products of fatty acids and their presence in urine is indicative of excessive fatty acid metabolism to provide energy.
Causes of Ketonuria
Box 826.4: Urine ketones in diabetes
Indications for testing
• At diagnosis of diabetes mellitus
• At regular intervals in all known cases of diabetes, and in gestational diabetes
• In known diabetic patients during acute illness, persistent hyperglycemia (>300 mg/dl), pregnancy, clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain)
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 (Figure 826.2).
  1. Decreased utilization of carbohydrates:
    a. Uncontrolled diabetes mellitus with ketoacidosis: In diabetes, because of poor glucose utilization, there is compensatory increased lipolysis. This causes increase in the level of free fatty acids in plasma. Degradation of free fatty acids in the liver leads to the formation of acetoacetyl CoA which then forms ketone bodies. Ketone bodies are strong acids and produce H+ ions, which are neutralized by bicarbonate ions; fall in bicarbonate (i.e. alkali) level produces ketoacidosis. Ketone bodies also increase the plasma osmolality and cause cellular dehydration. Children and young adults with type 1 diabetes are especially prone to ketoacidosis during acute illness and stress. If glycosuria is present, then test for ketone bodies must be done. If both glucose and ketone bodies are present in urine, then it indicates presence of diabetes mellitus with ketoacidosis (Box 826.4).
    In some cases of diabetes, ketone bodies are increased in blood but do not appear in urine.
    Presence of ketone bodies in urine may be a warning of impending ketoacidotic coma.
    b. Glycogen storage disease (von Gierke’s disease)
  2. Decreased availability of carbohydrates in the diet:
    a. Starvation
    b. Persistent vomiting in children
    c. Weight reduction program (severe carbohydrate restriction with normal fat intake)
  3. Increased metabolic needs:
    a. Fever in children
    b. Severe thyrotoxicosis
    c. Pregnancy
    d. Protein calorie malnutrition
Figure 826.2 Formation of ketone bodies
Figure 826.2 Formation of ketone bodies. A small part of acetoacetate is spontaneously and irreversibly converted to acetone. Most is converted reversibly to β-hydroxybutyrate
Bilirubin (a breakdown product of hemoglobin) is undetectable in the urine of normal persons. Presence of bilirubin in urine is called as bilirubinuria.
There are two forms of bilirubin: conjugated and unconjugated. After its formation from hemoglobin in reticuloendothelial system, bilirubin circulates in blood bound to albumin. This is called as unconjugated bilirubin. Unconjugated bilirubin is not water-soluble, is bound to albumin, and cannot pass through the glomeruli; therefore it does not appear in urine. The liver takes up unconjugated bilirubin where it combines with glucuronic acid to form bilirubin diglucuronide (conjugated bilirubiun). Conjugated bilirubin is watersoluble, is filtered by the glomeruli, and therefore appears in urine.
Detection of bilirubin in urine (along with urobilinogen) is helpful in the differential diagnosis of jaundice (Table 826.1).
Table 826.1 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.
Presence of bilirubin in urine indicates conjugated hyperbilirubinemia (obstructive or hepatocellular jaundice). This is because only conjugated bilirubin is water-soluble. Bilirubin in urine is absent in hemolytic jaundice; this is because unconjugated bilirubin is water-insoluble.
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).
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 typical of hemolytic anemia. This also occurs in megaloblastic anemia due to premature destruction of erythroid precursors in bone marrow (ineffective erythropoiesis).
  2. Hemorrhage in tissues: There is increased formation of bilirubin from destruction of red cells.
Causes of Reduced Urobilinogen in Urine
  1. Obstructive jaundice: In biliary tract obstruction, delivery of bilirubin to the intestine is restricted and very little or no urobilinogen is formed. This causes stools to become clay-colored.
  2. Reduction of intestinal bacterial flora: This prevents conversion of bilirubin to urobilinogen in the intestine. It is observed in neonates and following antibiotic treatment.
Testing of urine for both bilirubin and urobilinogen can provide helpful information in a case of jaundice (Table 826.1).
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 purpura
  • 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.
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 in urine (hemosiderinuria) indicates presence of free hemoglobin in plasma. Hemosiderin appears as blue granules when urine sediment is stained with Prussian blue stain (Figure 826.3). Granules are located inside tubular epithelial cells or may be free if cells have disintegrated. Hemosiderinuria is seen in intravascular hemolysis.
Figure 826.3 Staining of urine sediment with Prussian blue stain
Figure 826.3 Staining of urine sediment with Prussian blue stain to demonstrate hemosiderin granules (blue)
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.
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 826.2
Table 826.2 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
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.


Published in Clinical Pathology
Thursday, 10 August 2017 00:34
Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).
Bile salts along with bilirubin can be detected in urine in cases of obstructive jaundice. In obstructive jaundice, bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their detection is Hay’s surface tension test. The property of bile salts to lower the surface tension is utilized in this test.
Take some fresh urine in a conical glass tube. Urine should be at the room temperature. Sprinkle on the surface particles of sulphur. If bile salts are present, sulphur particles sink to the bottom because of lowering of surface tension by bile salts. If sulphur particles remain on the surface of urine, bile salts are absent.
Thymol (used as a preservative) gives false positive test.


Published in Clinical Pathology
Wednesday, 09 August 2017 12:21
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 listed below.

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 (Figure 823.1).
  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.
  4. Fouchet’s test: This is a simple and sensitive test.
    i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride, and mix well. A precipitate of sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex).
    ii. Filter to obtain the precipitate on a filter paper.
    iii. To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent. (Fouchet’s reagent consists of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml).
    iv. Immediate development of blue-green color around the drop indicates presence of bilirubin (Figure 823.2).
  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).
Figure 823.1 Positive Gmelins test for bilirubin showing play of colors
Figure 823.1 Positive Gmelin’s test for bilirubin showing play of colors
Figure 823.2 Positive Fouchets test for bilirubin in urine
Figure 823.2 Positive Fouchet’s test for bilirubin in urine


Published in Clinical Pathology
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.


Published in Clinical Pathology
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


Published in Clinical Pathology
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).

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