Examination of Urine - Biochemistry and Pathology for Science Students

This is a presentation made by a MBBS professor on the various types of Urine and it's properties and a clinical examination!

1. Must Know Indications of Urine Analysis Collection of Urine Method of Urine Collection Time of Urine Collection Preservation of Urine Examination of Urine Physical Examination Chemical Examination Microscopy

2. Functions of kidney Water balance Electrolyte balance Osmotic pressure Acid base balance Excretion -- M etabolic waste, Nitrogen, sulphur , drugs, toxins Retains – P rotiens , AA, glucose Secretes – R enin, Erythropoietin

3. URINE EXAMINATION INDICATIONS 1] Detect kidney & urinary tract diseases 2] Detect diseases outside urinary tract like -Diabetes mellitus, jaundice, acidosis. 3] Detect urinary tract infections. 4] Detection of pregnancy

4. COLLECTION OF URINE SAMPLE Clean , dry & preferably sterilized container & labelled properly Should be fresh Should be examined immediately [in 2 hrs ] On keeping at room temperature – Reaction may change Casts disintegrate Crystals precipitate Bacteria grow .

5. If delay in urine examination than preservation of urine sample is done by 1] keep sample in refrigerator [2-8 C] 2] chemicals added as preservatives Boric acid – 0.5 gm / 60ml Formaldehyde – 1 drop / 15 ml Thymol – 0.1gm/ 100ml

6. Types of urine samples 1] Early morning sample most concentrated uses -- detection of presence of chemicals -- microscopic examination -- pregnancy test

7. 2] Mixed 24 hrs urine sample– Patient empties bladder at 8a.m. -- discarded. After that all urine voided is collected in a wide mouthed clean container till next day 8 a.m. He empties bladder at this time & sample is mixed 150-200ml submitted . Uses – 1] quantitative estimation of chemicals . -- 2] TB bacilli detection & culture.

8. 3] Mid-stream urine sample – All aseptic precautions in a sterile container – no preservative added Genitalia cleaned with soap & water, antiseptic applied , first portion of urine voided is discarded , next portion of urine voided is directly collected in container Use – bacteriological examination

9. PHYSICAL EXAMINATION 1.VOLUME N ormal – 600-2,000 ml /24 hr [adults] -- 300-600 ml / 24 hr [infants] Abnormal – 1] Polyuria – > 2000ml/24 hr . 2] O liguria – < 500ml/24 hr . 3] Anuria – <100ml/ 24 hr. 4] N octuria – night > day [renal insufficiency]

10. 2 . Colour Normal – pale yellow [ urochrome , urobilinogen ] Abnormal : Colourless [diluted, DI] Dark yellow [concentrated, fever, jaundice, MV ] Yellowish green [ biliverdin ] Smoky red [blood] Brown or coca coloured [hemoglobin]. Black on standing [ alkaptonuria – homogentisic acid] Red orange [ rifampicin ]

11. Specific gravity— Normal – 1.003 – 1.030 in 24hrs urine SG = conc. solutes volume Abnormal – SG↑ volume ↑-- DM SG ↓ volume↓-- GN SG↑ volume N – Nephrotic syndrome SG ↓ volume ↑ -- DI

12. Measured by – 1] urinometer – 2] refractrometer – Refractive index of urine , few drops of urine required 3] dipstix method

13. Specific Gravity Calibrated at 20ºC fill 3/4 th of container with urine & float urinometer in urine Urinometer should not touch the walls of the container Correction – for every 3ºC rise of room temperature 0.001 is added to recorded reading & for every 3ºC fall 0.001 is substracted .

14. Reaction / pH – Normal –- 5.5 - 6.8 Abnormal – Alkaline Acidic Measured by – Litmus paper -- acidic urine [blue to red], alkaline urine [red to blue] pH indicator paper strips – impregnated with methyl red [alkaline urine], bromothymol blue [acidic urine] Strip multistix method

15. Reagent strips- uristix & multistix – strips of plastic – 5-8mm wide & 10cm long; impregnated with reagents for testing glucose, protiens , pH, S.G, hemoglobin, ketone bodies, urobilinogen , bilirubin .

16. Chemical Examination Glucose Ketone Bodies Protein Blood Bile Salts Bile pigments

17. History 1 A 40yrs old man had been having polyphagia and polydipsia for past 10 years. Now presented with vomiting, mental confusion and dehydration. Glucose Ketone Bodies Diagnosis - Diabetic Ketoacidosis

18. Physical Examination Volume – 25ml Color – Pale Yellow/ Colorless Appearance – Clear/Turbid P– Acidic Specific Gravity – Increased Odour - Fruity

19. Test for Glucose Benedicts Test / Semiquantitative Test Principle The copper sulphate present in the Benedict’s reagent reacts with the reducing substances in the urine which convert cupric sulphate to cuprous oxide in hot alkaline media. Thus, this test is based on the reduction of cupric ions in Benedict’s solution to cuprous ions. In the absence of reducing substances in urine, the color of the reagent remains blue.

20. Procedure Take 5 mL of Benedict’s reagent in a test tube. Boil to exclude presence of reducing substance in reagent . Add 8 drops of urine . Boil the mixture for 5 minutes and allow to cool. The ratio of 5 mL Benedict’s reagent and 8 drops (0.5 mL) of urine is important because it is a semiquantitative test.

21. Interpretation The change of color from blue to green, yellow, and orange/red depends on the amount of sugar present

22. Test for Ketone Bodies – Rothera’s Test Principle Acetoacetic acid ( diacetic acid) and acetone react with sodium nitroprusside in presence of an alkali to form a purple color compound. Procedure Take 4 mL of urine in a test tube. Saturate the urine with ammonium sulphate by mixing vigorously. Add a few crystals of sodium nitroprusside Overlay with few drops of liquor ammonia along the wall of the tube.

23. Test for Ketone Bodies Interpretation: Development of a purple ring indicates the presence of acetoacetic acid/acetone or both.

24. History 2 A 10yrs old boy presented with edema , oliguria. On examination patient also had hypertension Protein Blood Acute Glomerulonephritis

25. Test for Protein - Heat Coagulation Test Principle Heat induced coagulation of proteins and precipitation. Coagulation can be further enhanced when drops of acetic acid are added.

26. Procedure Fill three-fourth of a test tube with clear urine. Heat the upper part (1/3) of the urine ( the lower part of the urine acts as a control for checking turbidity in the heated upper part). The development of turbidity may be due to coagulation of proteins or due to phosphates. Add a few (3–5) drops of 10% glacial acetic acid and if turbidity persists it is due to proteins (phosphates will dissolve).

27. Test for Blood in urine - Benzidine Test Principle The test depends upon the ability of heme compounds derived from hemoglobin to catalyze the oxidation of benzidine by hydrogen peroxide.

28. Procedure Dissolve a small amount (knife-point full) of benzidine in 2 mL of glacial acetic acid and add an 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. Interpretation The appearance of blue color indicates the presence of blood.

29. History 3 A 45yrs old female admitted with pain in right hypochondrium, itching and yellowish discoloration of eyes and urine. Bile pigments Bile salts Obstructive / Hepatocellular Jaundice

30. Test for Bilirubin – Fouchet’s test Principle Fouchet’s reagent contains trichloroacetic acid and ferric chloride. In an acidic medium ferric chloride oxidizes bilirubin to produce a dark green colored biliverdin.

31. Procedure: Take 10 mL of urine in a test tube and add 3 mL of 10% barium chloride solution. Mix the two and filter the mixture through a filter paper. Bilirubin along with barium salt remains on filter paper. Add a few drops of Fouchet’s reagent onto the filter paper. Interpretation: Green or blue color indicates bilirubinuria

32. Test for Bile salts - Hay’s Sulfur Test Two important bile salts are sodium and potassium salts of glycocholates and taurocholates. Normally, bile salts are not present in urine. Principle Bile salts have unusual property of lowering the surface tension of urine markedly even when present in small concentrations. This property is made use of in the Hay’s test.

33. Procedure Take 10 mL urine in a wide bore test tube (2 cm diameter or more) or a small beaker. Sprinkle sulfur powder over its surface, watch for 5 minutes. Interpretation Sulfur powder sinks to the bottom of test tube in the presence of bile salts in urine.