Alkaline haematin D-575, a new tool for the determination of haemoglobin as an alternative to the cyanhaemiglobin method. II. Standardisation of the method using pure chlorohaemin.

Chlorohaemin with high purity (greater than 99%), a stable and well-defined compound, can be used as a primary standard for the standardisation of a haemoglobin assay based on alkaline haematin D-575 [6]. Dissolved in a solution of 25 g Triton X-100 per litre of 0.1 mol/l NaOH ('AHD solution'), the millimolar absorbance coefficient of the end product (alkaline haematin D-575) is 6.960 +/- 0.046 [l X mmol-1 X cm-1] at 575 nm. Within the range of haemoglobin concentrations of 5 to 25 g/100 ml there is a strong linear relation between chlorohaemin concentration and absorbance with a deviation of less than or equal to 2% from the theoretical values. As compared to the conventional cyanhaemiglobin standard solutions, standardisation with pure chlorohaemin is the method of choice because of the simplicity of the preparation of standard solutions, which can be done in every laboratory, and the stability of both the solid compound chlorohaemin and its solutions in alkaline Triton X-100. For the first time a real standard for quality control in haemoglobinometry is recommended: a concentrated solution which behaves like blood, i.e. the simulation of all steps in haemoglobin determination (dilution and photometry) is possible.