Chemical examination of urine
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This document provides information on chemical examination of urine, including tests to detect various analytes like protein, glucose, ketone bodies, bilirubin, urobilinogen, bile salts, and blood. It describes the principle, requirements, procedure, and interpretation for common tests to identify these substances, which can indicate underlying renal or metabolic conditions. Causes for abnormalities in the various analytes are also outlined.
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Chemical examination of urine
- 1. CHEMICAL EXAMINATION OF URINE P SUNIL KUMAR Haematology & Transfusion medicine 12/13/2017 1SUNIL KUMAR P
- 2. • 1.Protein/ Albumin • 2.Glucose • 3.Ketone bodies • 4.Urobilinogen • 5.Bile salts • 6.Bile pigments • 7.Blood 12/13/2017 2SUNIL KUMAR P
- 3. PROTEINURIA • The presence of detectable protein in the urine is known as proteinuria. • It indicates glomerular injury. • If turbid, filter or centrifuge the urine before testing. 12/13/2017 3SUNIL KUMAR P
- 4. TESTS FOR PROTEIN • A. Heat and acetic acid test • B. Sulfosalicylic acid test • C. Heller’s test • D. Dipstick method 12/13/2017 4SUNIL KUMAR P
- 5. Heat and Acetic Acid Test • Principle : • Heat induced coagulation of proteins and precipitation. Coagulation can further enhanced when drops of acetic acid are added. 12/13/2017 5SUNIL KUMAR P
- 6. PROCEDURE Fill three –fourth ¾ of a test tube with clear urine. Heat the upper part (1/3) of the urine . (lower part of the urine acts as a control) Add a few drops of (3-5) 10% glacial acetic acid 12/13/2017 6SUNIL KUMAR P
- 8. • Interpretation : • If turbidity persists it is due to proteins • Depending upon the amount of precipitate, results are interpreted below……. 12/13/2017 8SUNIL KUMAR P
- 9. Interpretation : 12/13/2017 9SUNIL KUMAR P
- 10. 12/13/2017 SUNIL KUMAR P 10
- 11. SULFOSALICYLIC ACID TEST • This test detects all types of proteins (albumin, globulin, glycoproteins and Bence Jones Protein). • Principle : Cold precipitation of proteins by a strong acid 12/13/2017 11SUNIL KUMAR P
- 12. PROCEDURE Take 2.5 mL of urine in a small test tube Slowly pour 2.5 mL of Sulfosalicylic acid Wait for 5 Min 12/13/2017 12SUNIL KUMAR P
- 13. INTERPRETATION • Presence of a cloudy precipitate indicates the presence of proteins in urine. • However, it also precipitates mucus and Bence jones protein. • Caution : Filter urine if turbid, as it interferes with the final reading 12/13/2017 13SUNIL KUMAR P
- 15. DIPSTICK METHOD 12/13/2017 15SUNIL KUMAR P
- 16. CAUSES OF PROTEINURIA 12/13/2017 16SUNIL KUMAR P
- 17. REDUCING SUBSTANCES IN THE URINE • Reducing substances are those compounds which reduce cupric ions ( from copper sulphate in Benedict’s reagent) in an alkaline solution to cuprous ions ( cuprous oxide) . • Such substances may be sugar or non sugar. 12/13/2017 17SUNIL KUMAR P
- 18. Sugars : These include : • 1. Glucose • 2.Fructose • 3.Pentose • 4.Galactose • 5.Lactose • 6.Maltose • 7.Sucrose 12/13/2017 18SUNIL KUMAR P
- 19. Non Sugars : • 1.Ascorbic acid • 2.Uric acid • 3.Urates • 4.Glucuronides • 5.Choloroform • 6.Homogentisic acid 12/13/2017 19SUNIL KUMAR P
- 20. TESTS FOR REDUCING SUBSTANCES SUGARS & NON SUGAR • 1. Benedict’s qualitative test • 2.Dipstick method 12/13/2017 20SUNIL KUMAR P
- 21. Benedict’s Qualitative Test (Semiquantitative Test) • This test detects the presence of reducing substances in urine and is not specific for glucose. 12/13/2017 21SUNIL KUMAR P
- 22. PRINCIPLE • The copper sulphate present in the Benedict’s reagent reacts with the reducing substances in the urine which convert cupric sulphates to cuprous oxide in hot alkaline media. 12/13/2017 22SUNIL KUMAR P
- 23. 12/13/2017 SUNIL KUMAR P 23
- 24. PROCEDURE Take 5 mL of Benedict’s qualitative reagent in a test tube Boil to exclude presence of reducing substance in reagent Add 8 drops (0.5 mL) of protein free urine. Boil the mixture for 5 min. Allow to cool ( under running tapwater) 12/13/2017 24SUNIL KUMAR P
- 25. • The ratio of 5 mL Benedict’s reagent and 8 drops (0.5 mL) of urine ratio is important because it is a Semiquantitative test. • Interpretation : The change of color from blue to green, yellow orange / red depends on the amount of sugar present. 12/13/2017 25SUNIL KUMAR P
- 26. 12/13/2017 26SUNIL KUMAR P
- 27. 12/13/2017 27SUNIL KUMAR P
- 28. 12/13/2017 28SUNIL KUMAR P
- 29. Causes of Glycosuria 12/13/2017 29SUNIL KUMAR P
- 30. KETONE BODIES • Ketone bodies are three water soluble molecules ( Acetoacetic acid , beta-hydroxy butyric acid and acetone) that are produced by the liver from fatty acids during low food intake , carbohydrate restrictive diets , starvation. 12/13/2017 30SUNIL KUMAR P
- 31. Tests for Ketone bodies • 1. Rothera’s test • 2.ferric chloride test • 3.Hart’s test • 4.Dipstick method 12/13/2017 31SUNIL KUMAR P
- 32. ROTHERA’S TEST • Principle : – Acetoacetic acid and acetone react with sodium nitroprusside in presence of an alkali to form a purple color compound 12/13/2017 32SUNIL KUMAR P
- 33. PROCEDURE Take 4 mL of urine in a test tube Add a few crystals of sodium nitroprusside saturate the urine with ammonium sulphate By mixing vigorously Overlay with few drops of liquor ammonia along the wall of tube 12/13/2017 33SUNIL KUMAR P
- 34. • INTERPRETATION • Development of purple ring indicates the presence of Acetoacetic acid / acetone or both. • A brown or red colour is of no significance. 12/13/2017 34SUNIL KUMAR P
- 35. 12/13/2017 35SUNIL KUMAR P
- 36. 12/13/2017 36SUNIL KUMAR P
- 37. Causes of ketonuria 12/13/2017 37SUNIL KUMAR P
- 38. BILIRUBIN (Bile Pigment) • Tests for Bilirubin in urine provides information concerning metabolic or systemic disorders, especially liver function. • Bilirubin is a breakdown product of hemoglobin and is normally is not present in urine. • Bilirubinuria causes yellow-brown to greenish- brown urine and forms yellow foam on shaking. 12/13/2017 SUNIL KUMAR P 38
- 39. Tests for Bilirubin • A. Fouchet’s test • B. dipstick method 12/13/2017 SUNIL KUMAR P 39
- 40. Fouchet’s test • Principle : • Fouchet’s reagent contains trichloroacetic acid and ferric chloride. In acetic medium ferric chloride oxidizes Bilirubin to produce a dark green colored bileverdin. 12/13/2017 SUNIL KUMAR P 40
- 41. • Requirements : • 1.Test tubes • 2.pasteur pipettes • 3.What man No.1 filter paper • 4. 10% Barium chloride • 5.Fouchets reagent 12/13/2017 SUNIL KUMAR P 41
- 42. PROCEDURE Take 10mL of urine in a test tube add 3 mL of 10% barium chloride solution Mix the two and filter the mixture through filter paper (Bilirubin along with barium salt remains on filter paper) Add a few drops of Fouchet’s reagent onto the filter paper 12/13/2017 SUNIL KUMAR P 42
- 43. Interpretation Green or blue color Indicates Bilirubinuria Precaution : test to be done on fresh urine sample 12/13/2017 SUNIL KUMAR P 43
- 44. Causes of Bilirubinuria • Obstructive Jaundice : Urine shows Bilirubin with out urobilinogen • Hepatocellular jaundice. 12/13/2017 SUNIL KUMAR P 44
- 45. UROBILINOGEN • Urobilinogen is a colorless by-product of bilirubin reduction. • It is formed in the intestines by bacterial action on bilirubin. • Urobilinogen is normally present in urine in trace amount (1-12 mg/dl) and is insufficient to cause a significant positive reaction. 12/13/2017 SUNIL KUMAR P 45
- 46. UROBILINOGEN • Whenever the liver is unable to efficiently remove the reabsorbed urobilinogen from the portal circulation ( e.g. Liver disease, hemolytic anaemia ) • more urobilinogen than normal is routed through the kidney and hence excreted in the urine. 12/13/2017 SUNIL KUMAR P 46
- 47. Tests for urobilinogen • A. Ehrlich’s test • B. Dipstick method 12/13/2017 SUNIL KUMAR P 47
- 48. Ehrlich’s Test • Principle : • Ehrlich’s reagent reacts with urobilinogen and forms a pink colored aldehyde complex. • Reagent : Paradimethyl amino benzaldehyde 12/13/2017 SUNIL KUMAR P 48
- 49. • Requirements : • 1.Test tubes • 2.Pasteur pipette • 3.Ehrlichs reagent • 4.Sodium acetate • 5.Urine sample 12/13/2017 SUNIL KUMAR P 49
- 50. PROCEDURE Take 2.5 mL urine in a test tube Add 2.5 mL of Ehrlich’s aldehyde reagent Mix well by Inversion Add 10 mL of saturated sodium acetate solution and mix 12/13/2017 SUNIL KUMAR P 50
- 51. Interpretation • Pink to cherry red color indicates presence of urobilinogen 12/13/2017 SUNIL KUMAR P 51
- 52. Causes of increased urobilinogen in urine • Hemolytic anaemias • A. Thalassaemia • B.Sickle cell anaemia • C. hereditary spherocytosis • Liver Diseases • A. Drug or toxic hepatitis • B.cirrhosis 12/13/2017 SUNIL KUMAR P 52
- 53. Causes of decreased / absent urobilinogen in urine • In obstructive Jaundice. 12/13/2017 SUNIL KUMAR P 53
- 54. Bile salts • Bile salts are composed of mixture of bile acids and glycine or taurine. • Two important bile salts are sodium and potassium salts of glycocholates. • Normally bile salts are not present in urine 12/13/2017 SUNIL KUMAR P 54
- 55. • Requirements : • 1.Test tubes 10 mL • 2.Suphur powder • 3.Pasteur pipette 12/13/2017 SUNIL KUMAR P 55
- 56. Tests for bile salts • Hay’s Sulfur test : • Principle : Bile salts have unusual property of lowering the surface tension of urine markedly even when present in small concentrations. 12/13/2017 SUNIL KUMAR P 56
- 57. Procedure Take 10 mL urine in a wide bore test tube or small beaker Sprinkle sulfur powder over its surface, watch for 5 min 12/13/2017 SUNIL KUMAR P 57
- 58. Interpretation • Sulfur powder sinks to the bottom of test tube in the presence for bile salts in urine • Causes : Hepatocellular and obstructive Jaundice 12/13/2017 SUNIL KUMAR P 58
- 59. Tests for Blood in Urine • These tests detects • hematuria, • hemoglobinuria or • myoglobinuria 12/13/2017 SUNIL KUMAR P 59
- 60. • Hematuria : presence of red cells in urine E.g. Renal stones • Hemoglobinuria : Presence of free hemoglobin in urine E.g. Intravascular hemolysis • Myoglobinuria : presence of myoglobin in urine . E.g. Crush injury to muscle. 12/13/2017 SUNIL KUMAR P 60
- 61. Tests for blood • A. Benzidine test • B. Orthotoludine test • C. Dipstick method 12/13/2017 SUNIL KUMAR P 61
- 62. Benzidine test • Principle : • The test depends upon the ability of heme compounds derived from hemoglobin to catalyze the oxidation of bezidine by hydrogen peroxide. 12/13/2017 SUNIL KUMAR P 62
- 63. • Requirements: • 1.Test tubes • 2.Pasteur pipette • 3.Benzedine reagent • 4.3%Hydrogen peroxide • 5. Glacial acetic acid 12/13/2017 SUNIL KUMAR P 63
- 64. Procedure Dissolve a small amount of Benzidine in 2 mL of glacial acetic acid and equal volume of 3 % hydrogen peroxide From the above, take 2 mL in another test tube and add 2 mL of previously boiled and cooled urine and mix 12/13/2017 SUNIL KUMAR P 64
- 65. Interpretation The appearance of blue color indicates the presence of blood. 12/13/2017 SUNIL KUMAR P 65
- 66. Causes of hematuria • 1.Renal causes a. Acute glomerulonephritis b. Bacterial hypertension c. Malignant hypertension 2. Non glomerular causes a. polycystic kidney b. Renal cell carcinoma c. Renal stones 3. Blood Disorders a. Bleeding disorders 12/13/2017 SUNIL KUMAR P 66