1. Factitiously low automated WBCs occasionally occur as a consequence of leucocyte
agglutination, prolonged sample storage, or abnormally fragile cells (e.g., in leukaemia).
Factitiously high counts are more common and usually result from failure of lysis of red cells.
With certain instruments this may occur with the cells of neonates or be consequent on uraemia
or on the presence of an abnormal haemoglobin such as haemoglobin S or haemoglobin C;
high counts may also be the result of microclots, platelet clumping, or the presence of a
cryoglobulin.
HAEMIGLOBINCYANIDE (CYANMETHAEMOGLOBIN) METHOD
The haemiglobincyanide (cyanmethaemoglobin) method is the internationally recommended
method for determining the haemoglobin[2]
concentration of blood.[*]
The basis of the method is
dilution of blood in a solution containing potassium cyanide and potassium ferricyanide.
Haemoglobin, Hi, and HbCO, but not SHb, are converted to HiCN. The absorbance of the
solution is then measured in a spectrometer at a wavelength of 540 nm or a photoelectric
colorimeter with a yellow–green filter (e.g., Ilford 625, Wratten 74, Chance 0 Gr1)
Diluent
The original (Drabkin's) reagent had a pH of 8.6. The following modified solution, Drabkin-type
reagent, as recommended by the International Committee for Standardization in Haematology,[2]
has a pH of 7.0–7.4. It is less likely to cause turbidity from precipitation of plasma proteins and
requires a shorter conversion time (3–5 min) than the original Drabkin's solution, but it has the
disadvantage that the detergent causes some frothing:
Potassium ferricyanide (0.607 mmol/l) 200 mg
Potassium cyanide (0.768 mmol/l) 50 mg
Potassium dihydrogen phosphate (1.029 mmol/l) 140 mg
Nonionic detergent 1 ml
Distilled or deionized water To 1 litre
The pH should be 7.0–7.4 and must be checked with a pH meter at least once a month. The
diluent should be clear and pale yellow in colour. When measured against water as a blank in a
spectrometer at a wavelength of 540 nm, absorbance must be zero. If stored at room
temperature in a brown borosilicate glass bottle, the solution keeps for several months. If the
ambient temperature is higher than 30°C, the solution should be stored in the refrigerator but
brought to room temperature before use. It must not be allowed to freeze. The reagent must be
discarded if it becomes turbid, if the pH is found to be outside the 7.0–7.4 range, or if it has an
absorbance other than zero at 540 nm against a water blank.
Inherent errors result from uneven distribution of cells in the counting chamber, and no amount of
mixing will minimise this inherent variation in numbers between areas. Inherent error can only be
reduced by counting more cells in a preparation.