Blood is mixed with a solution of potassium ferricyanide, potassium cyanide and a non-ionic detergent (Drabkin’s solution). Erythrocytes are lysed producing an evenly distributed hemoglobin solution. Potassium ferricyanide converts hemoglobin to methemoglobin, and methemoglobin combines with potassium cyanide to form cyanm-ethemoglobin (hemiglobincyanide). All forms of hemoglobin present in blood are completely converted to a single compound, cyanmethemo-globin. When the reaction is completed, absorbance of the solution is measured in a spectro-photometer at 540 nm. At this wavelength, cyanmethemoglobin has a broad absorbance peak. To obtain the amount of hemoglobin in the unknown sample, its absorbance is compared with that of the standard cyanmethemoglo-bin solution (the hemoglobin concentration of which is known) by using a formula or a previously prepared graph/table. The reaction is linear up to 20 gm/dl.
(1) Photoelectric colorimeter or spectrophotometer
(2) Sahli’s pipette marked at 20 μl (20 cmm).
(3) Pipette 5 ml.
Non-ionic detergent 1 ml
Distilled water to 1000 ml
(1) In a test tube, take 5 ml of Drabkin’s solution and to it add 20 μl of blood (1:251 dilution). Stopper the tube, mix by inverting several times, and allow to stand for atleast 5 minutes. This time is adequate for conversion of hemoglobin to cyanmethemoglo-bin.
(2) Transfer the test sample to a cuvette. Read the absorbance in a spectrophotometer at 540 nm or in a photoelectric colorimeter using a yellow-green filter. Also take the absorbance of the standard solution. Absorbance should be read against reagent blank (Drabkin’s solution).
(3) Hemoglobin value is derived from the formula given below or from the previously prepared graph or table.
Absorbance of standard
Diluted cyanmethemoglobin standards are available commer-cially for preparation of a calibration graph. Alternatively, standard cyanmethemoglobin solution is diluted serially with Drabkin’s solution. On a linear graph paper, hemoglobin concen-tration (horizontal axis) in each dilution is plotted against the absorbance (vertical axis). A straight line joining the points and passing through the origin is obtained. From this graph, a table can be prepared relating absorbance to hemoglobin concentration.
(1) Lipemic blood (hypertriglyceridemia), high total leukocyte count (> 25,000/μl), or abnormal plasma proteins (e.g. in multiple myeloma, Waldenström’s macroglobulinemia) can cause erroneous results.
(2) Cyanmethemoglobin solution is stable so that delay in taking the reading of absorbance does not affect the result.