Test Used for Differentiation of Hemoglobinuria and Myoglubinuria

Mayoglubinuria:
Myoglobinuria is the presence of myoglobin in the urine, which usually results from
rhabdomyolysis or muscle injury. Myoglobin is a red coloured haem protein present in
muscle cells as a reserve of oxygen.

Heamoglobinuria:
Heamoglobinuria is a condition in which the oxygen transport protein hemoglobin is found
in abnormally high concentrations in the urine.

Sample:
1. It can be done with fresh, random urine.
2. A morning sample is preferred.
3. Examine immediately.
4. If examined later on say for one hour then refrigerate the urine in the fridge to avoid
microbial growth.
5. Mix urine well before testing the urine for Hb.

Indications

1. To find the cause of hemoglobinuria due to various conditions:

1. Hemolytic anemia.
2. other conditions are given below.

Precautions

1. Examine immediately.
2. Can refrigerate if delayed.
3. Mix well before testing.

Lab Diagnosis:
• Complete blood count (CBC) and serum biochemical panel specifically looking at
creatine kinase levels and evaluating for hemolysis.
• Urine Dipstick test may diagnose myoglobinuria or heamoglobinuria.
• An ammonium sulfate precipitation test may differentiate myoglobinuria from
heamoglobinuria.
• Benzidine test is sensitive test for the presence of blood.
To Differ Myoglobinuria from Heamoglobinuria:
To rule out hemoglobinuria from myoglobinuria, following test can be performed:

Benzidine Test:
Benzidine test is performed after 40 fold dilution of urine. The positive results indicating
the presence of haemoglobin. It gives no colour with myoglobinuria.

Principle:
This test is based on the production of a blue color upon contact with a solution of
benzidine, hydrogen peroxide, and glacial acetic acid. H2O2 reduce benzidine solution. In
this solution, the iron of haemoglobin gives blue colour complex.

Results:
• Blue colour with heamoglobinuria (+ve test)
• No colour change with myoglobinuria (-ve test)

Ammonium Sulfate precipitation:

Principle:
When high concentrations of small, highly charged ions such as ammonium sulfate are
added, they compete with the proteins to bind to the water molecules. This removes the
water molecules from the protein and decreases its solubility, resulting in precipitation.

Method:
• To 5 ml urine sample 2.8 g ammonium sulfate is added slowly with thorough
mixing to obtain 80 % saturation of ammonium sulphate in urine.
• Pprecipite obtained at 80 % saturation is of haemoglobin which can be removed
by centrifugation for 10 min at 3000 rpm.
• Red brown colour persisted in the supernatant indicates myoglobin.

Precipitation with Ammonium sulfate at different concentrations indicates hemoglobin is

precipitated at 80 % saturation of Ammonium sulfate, whereas, myoglobin gets
precipitated only on full saturation.

Results:
• Precipitation at 80% saturation of Ammonium sulfate indicates
heamoglobinuria.
• Precipitation at 100% saturation of Ammonium sulfate indicates
myoglobinuria.