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Qus : Write down the test procedure of urine free haemoglobin?

Ans : Principle : Peroxide in heam of haemoglobin liberate oxygen from hydrogen peroxide with oxidies benzidine in acidic medium and change it to a blue colour compound.

              
Method: Benzidine test

Reagent:

1. Glacial acitic acid

2. Benzidine powder

3. Hydrogen peroxide (H₂O₂)

               

Reagent preparation: Take 1.0 ml of glacial acitic acid in a test tube. Make saturated solution by benzidine powder.Then add 1.0 ml of hydrogen peroxide and mix well.

Sample preparation: Take 5.0 ml fresh urine in a test tube and then centrifuge.After centrifuge separate the supernatant from deposit.

Procedure: Take 1-2 ml of supernatant urine in a test tube and add few drops of benzidine reagent and observe.A deep blue colour indicates present of free haemoglobin otherwise absent.

Indication:

A.      Haematuria : Haematuria is the presence  of red cells in urine.

a.       Gross haematuria : Appears smoky or red on naked eye examination and  plenty red cells on microscopic examination.

b.       Microscopic  haematuria : Red cells are detectable under microscope due to small number.

Causes :

1. Renal causes: The blood is intimately mixed with urine.

a.       Acute glomerulonephritis

b.       Calculi and crystals

c.        Infections –Severe acute pyelonephritis, papillary necrosis, tuberculosis

d.       Malignant hypertension, infraction, right heart failure

e.        Renal cell carcinoma, tumours of renal pelvis

f.        Polycystic kidney, solitary cyst.

2.       Post-renal causes : Blood coming from ureter is intimately mixed with urine. Blood coming from urinary bladder appears in the last part of micturition, or is intimately mixed with urine. Blood coming from urethra appears chiefly in the fast part of micturition.

a.       Pelvis, ureter and urinary bladder, Pyogenic infection tuberculosis, calculi, tumours, schistosomiasis.

b.       Urethra-Impaction of stone and tumours.

c.        Secondary involvement of urinary tract by tumours os prostate, uterus, rectum.

3.       Pre-renal causes (Systemic diseases) : Blood is intimately mixed with urine.

a.       Haemorrhagic disorders

b.       Septicemia, particularly meningococcal

c.        Scurvy

d.       Overdose of anticoagulant therapy.

B.      Haemoglobinuria : Haemoglobin may appear in urine when intravascular red cell breakdown is in excess. Occult blood test is positive.

a.       Incompatible blood transfusion

b.       ‘Black water fever’ in malignant malaria.

c.        Paroxysmal noctumal haemoglobinuria

d.       Paroxysmal cold haemoglobinuria.