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
- Hemoglobin (Drabkin’s) solution can be prepared in the laboratory.
- Drabkin’s solution reagents needed are:
- Potassium ferricyanide = 200 mg
- Potassium cyanide = 50 mg
- Potassium dihydrogen phosphate = 140 mg
- Non-ionic detergent = 1 ml
- Distal water = Make up to 1000 ml (1 L)
Precautions to keep the Drabkin’s solution
- Keep the solution in a dark-colored bottle and in the dark to protect it from the light.
- 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.
- Spectrophotometer cells should be free of fingerprints; otherwise, the reading will be high.
- 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:
- Whole blood is diluted 1 into 200 dilutions with Drabkin’s solution, containing potassium ferricyanide and potassium cyanide.
- RBCs are hemolyzed by the Drabkin’s solution.
- Now hemoglobin is oxidized, and its derivatives, except sulfhemoglobin, 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):
- Take 20 microliter of blood + Drabkin 4 mL = 1 : 200 dilution.
- OR take 20 microliter of blood + Drabkin 5 mL = 1 : 250 dilution.
- Now mix well.
- Read within 6 hours of mixing the blood with Drabkin’s solution.
- Read on a spectrophotometer at filter 540.
- Read against the blank of Drabkin’s solution (Drabkin solution can be used as blank).
- Also, read the standard solution (12 G/dL) with the same dilution as the test sample.
- Read by the spectrophotometer; reading is called optical density (OD).
- Adult male = 14 to 18 g/dL
- Adult female = 12 to 16 g/dL
- 10 years old child = 12 to 14.5 g/dL
- 3 months old infants = 9 to 14 g/dL
- Newborn = 17 to 23 g/dL
Physiological variation of Hb:
- Strenuous physical exercise.
- There is a diurnal variation, with the highest level in the morning and low in the evening.
- High altitude increases the Hb concentration.
False causes of raised Hb:
- Hemoconcentration due to dehydration and burns.
- Immediately after hemorrhage.
- If taken during the I/V infusion, if it contains iron.
Question 1: What is the end result of KCN reaction for hemoglobin?.
Methemoglobin is the colored stable compound to measure.
Question 2: What is the physiological variation in the hemoglobin?
These are strenuous exercise, diurnal variation and high altitude.