Estimation of Hemoglobin by the Cyanmethemoglobin Method

Introduction

Hemoglobin (Hb) is an iron-containing protein present in red blood cells and is responsible for the transport of oxygen from the lungs to tissues and carbon dioxide from tissues to the lungs. Estimation of hemoglobin concentration is one of the most important routine investigations in hematology, as it aids in the diagnosis of anemia, polycythemia, and other hematological disorders.
Among the various methods available, the cyanmethemoglobin method (also known as the Drabkin’s method) is considered the reference method for hemoglobin estimation due to its accuracy, reliability, and international standardization.

Principle

The cyanmethemoglobin method is based on the conversion of hemoglobin into cyanmethemoglobin, a stable colored compound.
When blood is added to Drabkin’s reagent, the following reactions occur:

1. Potassium ferricyanide oxidizes hemoglobin (Hb) to methemoglobin.

2. Methemoglobin reacts with potassium cyanide to form cyanmethemoglobin.

The intensity of the color formed is directly proportional to the hemoglobin concentration in the blood. The absorbance is measured using a spectrophotometer at a wavelength of 540 nm and compared with a standard solution.

Reagents

Drabkin’s Reagent, which contains:

• Potassium ferricyanide

• Potassium cyanide

• Potassium dihydrogen phosphate

• Non-ionic detergent

• Distilled water

Standard Cyanmethemoglobin Solution

Specimen

• Capillary blood or venous blood

• Anticoagulant: EDTA (for venous samples)

  
  

Equipment

• Spectrophotometer or colorimeter

• Test tubes

• Micropipette

• Cuvettes

Procedure

1. Label test tubes for blank, standard, and test sample.

2. Add 5 mL of Drabkin’s reagent into each tube.

3. Add 20 µL of blood to the test tube containing Drabkin’s reagent.

4. Mix thoroughly and allow the mixture to stand for 10 minutes at room temperature for complete conversion.

5. Set the spectrophotometer to 540 nm using the reagent blank.

6. Measure the absorbance of the test sample and the standard.

Calculation

$$\text{Hemoglobin (g/dL)} = \frac{\text{Absorbance of Test}}{\text{Absorbance of Standard}} \times \text{Concentration of Standard}$$

Normal Values

• Adult males: 13–17 g/dL

• Adult females: 12–15 g/dL

• Children: 11–14 g/dL

Advantages

• Internationally accepted reference method

• Measures all forms of hemoglobin except sulfhemoglobin

• High accuracy and reproducibility

• Suitable for automation

Disadvantages

• Use of cyanide requires careful handling and disposal

• Turbidity due to lipemia or high leukocyte count may interfere

Precautions

• Handle cyanide-containing reagents with care

• Use clean and dry glassware

• Ensure accurate pipetting

• Dispose of reagents according to laboratory safety guidelines

Conclusion

The cyanmethemoglobin method is a reliable, accurate, and standardized technique for hemoglobin estimation in clinical laboratories. Due to its precision and reproducibility, it remains the gold standard for hemoglobin measurement and plays a crucial role in the diagnosis and management of hematological disorders.

Sample Collection for Hemoglobin Estimation

(Cyanmethemoglobin Method)

Type of Sample

• Capillary blood or venous blood

Preferred Anticoagulant (for venous blood)

• EDTA

A. Capillary Blood Collection (Finger-prick Method)

1. Explain the procedure to the patient and obtain consent.

2. Clean the fingertip (usually ring or middle finger) with 70% alcohol and allow it to dry.

3. Prick the fingertip using a sterile lancet.

4. Wipe away the first drop of blood (to avoid tissue fluid contamination).

5. Allow a large drop of blood to form naturally without squeezing.

6. Collect 20 µL of blood using a micropipette or hemoglobin pipette.

7. Immediately add the blood to Drabkin’s reagent and mix well.

B. Venous Blood Collection

1. Apply a tourniquet and select a suitable vein (usually antecubital vein).

2. Clean the puncture site with antiseptic and allow it to dry.

3. Draw 2–3 mL of venous blood using a sterile syringe or vacutainer.

4. Transfer blood into an EDTA tube and mix gently by inversion.

5. Using a micropipette, take 20 µL of blood for the test.

6. Add it to Drabkin’s reagent and mix thoroughly.

Important Precautions During Sample Collection

• Avoid hemolysis due to rough handling.

• Do not squeeze the finger excessively during capillary sampling.

• Ensure accurate pipetting of blood volume.

• Mix EDTA samples gently to prevent clotting.

• Analyze the sample as soon as possible.

Sample Storage

• Fresh samples are preferred.

• EDTA blood can be stored at 2–8°C for up to 24 hours if immediate testing is not possible.

Rejection Criteria

• Clotted blood samples

• Hemolyzed samples

• Improperly labeled samples

• Insufficient sample volume