Principle
The principle behind the manual hemoglobin test using Drabkin’s solution is based on the conversion of hemoglobin to cyanmethemoglobin and its colorimetric detection:
Hemoglobin is a protein present inside red blood cells that binds and carries oxygen. It contains iron atoms that give blood its red color.
Drabkin’s solution contains potassium ferricyanide and potassium cyanide. Ferricyanide oxidizes the iron in hemoglobin from ferrous (Fe2+) to ferric (Fe3+) state.
The ferric iron then binds to cyanide, forming cyanmethemoglobin. This is a very stable colored pigment that has a maximal light absorption at 540 nm wavelength.
Cyanmethemoglobin color intensity is directly proportional to the total hemoglobin concentration in the blood sample.
When the converted sample is placed in a spectrophotometer, the instrument measures light absorbance at 540 nm.
The higher the hemoglobin concentration, more light will be absorbed. By comparing absorbance against a blank control, the actual hemoglobin level can be determined.
A pre-plotted standard calibration curve is used to correlate the absorbance with total hemoglobin concentration. This allows quantification of hemoglobin in the unknown sample.
In summary, the conversion of hemoglobin to colored cyanmethemoglobin, followed by spectrophotometric determination of color intensity at 540 nm forms the basis for manual hemoglobin estimation using Drabkin’s method.
Equipments
1. 10 – 100 ul + 100 – 100 ul Automatic Pipette
2. Large glss tubes
3. Drabkin’s Solution
4. yellow and blue tips
5. Colorimeter or Spectrophotometer
6. Lancet for finger prick
7. Cuvette in case of using colorimeter
Sample Type
EDTA Anticoagulated Sample or Direct Fingerprick using lancet
Procedure
1. Prepare the patient’s finger for blood collection using an alcohol swab in case of collecting finger blood. Allow to air dry and Use a sterile lancet to prick the side of the finger and collect 20μl of blood using a capillary tube or Pipette, “in case of EDTA only collect blood in tube using syringe and mix the sample well”.
2. in clean dry Large glass tube pour 5ml from Drabkin’s So;ution
3. Add the 20μl blood sample to 5ml of Drabkin’s solution in a test tube.
| Blank (B) | STandard (STD) | Test (T) | |
|---|---|---|---|
| Drabkin’s Solution | 5,000 μl (5ml) | 5,000 μl (5ml) | 5,000 μl (5ml) |
| Standard | – | 20 μl | – |
| Sample | – | – | 20 μl |
Procedure
4. Mix well by inverting gently.
5. Incubate for 10 minutes at room temperature. This allows conversion of hemoglobin to cyanmethemoglobin.
6. Prepare a blank tube containing Drabkin’s solution only.
7. After Zeroing the device read the absorption of Test Against STD
Normal Range
M. Sulieman
mohammad@mlsgaate.com
Precise volume of blood and Drabkin’s solution is essential
Allow adequate time for hemoglobin conversion
Measure against a blank control for accuracy
Follow universal precautions for safety
من الضروري قياس حجم الدم ومحلول درابكين بدقة
يجب السماح بوقت ٍ
كاف لتحول الهيموجلوبين
اتبع الاحتياطات العالمية للسلامة
