Hemoglobin estimation (cyanmethemoglobin method)

Principle

• Blood is diluted in Drabkin's reagent. Potassium ferricyanide oxidises haemoglobin iron (Fe2+ to Fe3+) forming methaemoglobin, which reacts with potassium cyanide to form stable cyanmethaemoglobin (HiCN).
• Absorbance of HiCN is read at 540 nm and is proportional to Hb concentration.
• Measures all common forms (oxy-, deoxy-, carboxy-, methaemoglobin) except sulfhaemoglobin. It is the ICSH reference method.

Specimen

• EDTA venous blood or capillary blood; well mixed.

Reagents and equipment

• Drabkin's reagent (potassium ferricyanide, potassium cyanide, dihydrogen potassium phosphate, non-ionic detergent), stored in a brown bottle.
• Calibrated pipette, spectrophotometer or colorimeter, HiCN reference standard.

Procedure

• Add 20 µL blood to 5.0 mL reagent (1 in 251 dilution) and mix.
• Allow to stand for the time stated by the reagent (commonly 3–10 min) for full conversion.
• Read absorbance at 540 nm against a reagent blank.
• Calculate Hb from a calibration curve or factor prepared with the HiCN standard.

Normal values

• Typical adults: men about 13–17 g/dL, women about 12–15 g/dL; newborns higher. Local reference ranges apply.

Quality control and pitfalls

• Turbidity falsely raises results: lipaemia, very high WBC count, abnormal plasma proteins, and poorly lysed cells (e.g. some HbS or HbC samples). Centrifuge the diluted sample or correct with a plasma blank.
• Pipetting errors are the main source of imprecision.
• Reagent is light sensitive and should be discarded if turbid or discoloured.
• Contains cyanide: never mouth pipette; dispose of per local safety rules.

Clinical use

• Diagnosis and grading of anaemia and polycythaemia; reference method for calibrating analysers.