Drabkin’s Solution for Hemoglobin Estimation, Preparation of Drabkin’s

Drabkin’s Solution for hemoglobin estimation

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

Sample for hemoglobin estimation

• Whole blood is needed for hemoglobin estimation.

Manual Method for preparation of Drabkin’s solution

1. Hemoglobin (Drabkin’s)  solution can be prepared in the laboratory.
2. 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
      1. https://patents.google.com/patent/US4341527A/en
   5. Distal water = Make up to 1000 ml (1 L)

Precautions to keep the Drabkin’s solution

1. Keep the solution in a dark-colored bottle and in the dark to protect it 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 fingerprints; otherwise, the reading will be high.
4. Drabkin’s solution is a pale yellow clear fluid; when it is cloudy or lost color should not be used.

The principle of Drabkin’s solution:

1. Whole blood is diluted 1 into 200 dilutions with Drabkin’s solution, containing potassium ferricyanide and potassium cyanide.
2. RBCs are hemolyzed by the Drabkin’s solution.
3. Now hemoglobin is oxidized, and its derivatives, except sulfhemoglobin, form methemoglobin in the presence of alkaline K- ferricyanide. 
4. The methemoglobin reacts with K-cyanide to form a very stable compound, cyanmethemoglobin, and this complex has maximum absorption at 540 nm.

Drabkin’s solution principle

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

1. Take 20 microliter 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 the blood with Drabkin’s solution.
5. Read on a spectrophotometer at filter 540.
6. Read against the blank of Drabkin’s solution (Drabkin solution can be used as blank).
7. Also, read the standard solution (12 G/dL) with the same dilution as the test sample.
8. Read by the spectrophotometer; reading is called optical density (OD).
   

   Drabkin’s solution procedure for hemoglobin estimation

Hemoglobin calculation formula (OD = optical density)

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.