Estimation of Hemoglobin by the Cyanmethemoglobin Method

 1. Clinical Significance

To estimate the hemoglobin concentration in a blood sample using the cyanmethemoglobin method and a spectrophotometer.

2. Principle

Hemoglobin in the blood reacts with Drabkin's reagent. Potassium ferricyanide oxidizes hemoglobin to methemoglobin, which then reacts with potassium cyanide to form cyanmethemoglobin (hemiglobincyanide). The resulting colored compound is measured spectrophotometrically at 540 nm. Its absorbance is proportional to the hemoglobin concentration in the sample.

3. Requirements

  • Spectrophotometer
  • Clean test tubes
  • Micropipette (20 µL)
  • Pipette for 5 mL reagent
  • Drabkin's reagent
  • EDTA-anticoagulated blood sample
  • Appropriate cuvettes
  • Gloves and laboratory protective equipment

4. Procedure

  1. Switch on the spectrophotometer and allow it to stabilize for 20 mins.
  2. Label a clean test tube with the sample identification.
  3. Add 5.0 mL of Drabkin's reagent to the test tube.
  4. Using a micropipette, add 20 µL of well-mixed EDTA-anticoagulated blood to the reagent.
  5. Mix gently to ensure complete mixing.
  6. Allow the mixture to stand for the 5 mins.
  7. Set the spectrophotometer to 540 nm.
  8. Use the appropriate reagent blank to zero the spectrophotometer, following the instrument and reagent instructions.
  9. Transfer the prepared sample into a clean cuvette, avoiding bubbles.
  10. Measure and record the absorbance of the sample at 540 nm.

5. Calculation

Calculate the hemoglobin concentration using the factor specified by the reagent manufacturer.

Formula:

Hemoglobin concentration (g/dL) = Absorbance of the sample × 36.80

6. Quality Control

  • Use a properly mixed, non-clotted blood sample.
  • Ensure accurate pipetting of 20 µL of blood and 5 mL of reagent.
  • Use the correct wavelength of 540 nm.
  • Zero the spectrophotometer with the appropriate reagent blank.
  • Check the reagent's expiry date and storage conditions.
  • Run appropriate hemoglobin quality-control material according to the laboratory's quality-control protocol.
  • Confirm that the factor of 36.80 is appropriate for the specific reagent, sample volume, optical path length, and manufacturer's instructions.

7. Precautions

  • Wear gloves and appropriate laboratory protective equipment throughout the procedure.
  • Avoid hemolysis caused by improper specimen collection or handling.
  • Do not use clotted or inadequately mixed blood.
  • Avoid bubbles, fingerprints, and contamination on the cuvette's optical surfaces.
  • Handle Drabkin's reagent with care because cyanide-containing formulations are hazardous. Follow the safety data sheet (SDS), institutional chemical-safety procedures, and applicable waste-disposal requirements. Never mix cyanide-containing reagent or waste with acids.
  • Dispose of reagent waste according to institutional hazardous-chemical waste procedures.

8. Result

________ g/dL.

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