Clin Path Lab 7

CLINICAL PATHOLOGY LABORATORY 7 USTMED ’07 Sec C – AsM

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Detection of Urobilinogen (Ehrlich’s test) Principle: Ehrlich’s reagent reacts w/ urobilinogen chromogens to form a colored urobilinogen-aldehyde

and

other

Reagents: Erhlich’s reagent (para-dimethyl-aminobenzaldehyde in conc HCl) 10% calcium chloride A.

Qualitative Test • add 0.5 mL of Ehrlich’s reagent to 5 mL of urine. • Mix. • A cherry red color appears, if positive.

B.

Quantitative Test 1. arrange 6 test tubes in a rack 2. place 1 ml urine and 9 ml o water in tube 1. mix 3. place 5 ml of water in tubes 2, 3, 4, 5 and 6 4. transfer 5 ml from tube 1 to tube 2. mix 5. transfer 5 ml from tube 2 to tube 3. mix. Do the same with tubes 4 to 6. After mixing, discard 5 ml from tube 6(?) 6. add 0.5 ml of Ehrlich’s reagent and mix. Allow to stand for 5 mins. 7. examine test tubes by viewing through the mouth against a white background. The highest dilution which shows the slightest pink color is reported positive up to that particular dilution. Normal value is up to 1:20 dilution.

NV = 0.5 to 2.5 mg/day Note: if bilirubin is present, it is removed by adding one part of 10% calcium chloride for every 4 parts of urine. After filtering, the filtrate is then tested as described above. Quantitative test for Bilirubin (van den Bergh reaction) Preparation of Diazo reagent: 1. Solution A – dissolve 1 gm of suphanilic acid in 15 ml of con HCl in water and then dilute to 1000 mL 2. Soltuion B – Dissolve 0.4 gm of sodium nitrite in water and dilute to 100 ml 3. Diazo Reagent – take 5 mL of Solution A and add 3 drops of Solution B. Prepare fresh for each determination Procedure: Overlay 0.5 mL of Diazo reagent to 1ml of serum Interpretation: A violet ring at the point of contact is positive for direct bilirubin Detection of Bile in Urine (Harrison Spot Test) Princple: The test for bile pigments are based upon their oxidation by acids with the formation of a series of colored derivatives like biliverdin (green), bilicyanine (blue) and choletelin (yellow). Reagents: Alcoholic tincture of iodine, conc yellow nitric acid, Fouchet’s reagent (ferric chloride with 25% trichloroacetic acid), 10% Barium chloride Procedure: 1. add 5 ml of barium chloride to 5 ml urine. Filter 2. place 2 drops of Fouchet’s reagent to the precipitate on the filter paper Interpretation: If bilirubin is present, a green color is produced Barium chloride precipitates bilirubin which is then oxidized to biliverdin by ferric chloride.