Solutions:- Part 4 – Haemoglobin Solution (Drabkin’s Solution) Preparation

• There are commercially available kits, which is easy to make the solution.
• When blood runs in the hematology analyzer, these instruments also do hemoglobin.

Manual Method

• Hemoglobin solution can be prepared in the laboratory. This is called Drabkin’s solution.
  1. Drabkin’s solution reagents needed are:
     1. Potassium ferricyanide = 200 mg
     2. Potassium cyanide = 50 mg
     3. Potassium dihydrogen phosphate = 140 mg
     4. Non-ionic detergent = 1 ml
     5. Distal water = Make up to 1000 ml (1 L)

Precautions

1. Keep the solution in a dark-colored bottle and in the dark to protect from the light.
2. If the solution is cloudy after adding the blood, in that case, centrifuge before the reading, this may be due to nonhemolyzed RBCs or globulins.
3. Spectrophotometer cells should be free of the fingerprints otherwise result will be high

The principle of Drabkin’s solution:

• This is based on the oxidation of Hb and its derivatives except sulfhemoglobin to form methemoglobin in the presence of alkaline K- ferricyanide.
• The methemoglobin reacts with K-cyanide to form a very stable compound, cyanmethemoglobin, and this complex has maximum absorption at 540 nm. 

How to estimate hemoglobin by Drabkin’s solution (Procedure):

1. Take 20 microlit. of blood + Drabkin 4 mL = 1 : 200 dilution.
2. OR take 20 microliter of blood + Drabkin 5 mL = 1 : 250 dilution.
3. Now mix well.
4. Read within 6 hours of mixing on filter 540.
5. Read against blank of Drabkin solution (Drabkin solution can be used as blank).
6. Also, read the standard solution (12 G/dL) with the same dilution as the test sample.
   

   Drabkin’s solution procedure

Normal hemoglobin:

1. Adult male = 14 to 18 g/dL
2. Adult female = 12 to 16 g/dL
3. 10 years old child = 12 to 14.5 g/dL
4. 3 months old infants = 9 to 14 g/dL
5. Newborn = 17 to 23 g/dL

Physiological variation of Hb:

1. Strenuous physical exercise.
2. There is a diurnal variation with the highest level in the morning and low in the evening.
3. High altitude increases the Hb concentration.

False causes of raised Hb:

1. Hemoconcentration due to dehydration, and burns.
2. Immediately after hemorrhage.
3. If taken during the I/V infusion if it contains iron.

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