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Preparation of a certified reference material of haemiglobincyanide for standardization of blood haemoglobin measurement


Measurement of haemoglobin concentration in blood is an essential test for healthcare and diagnosis of anaemia. In the past many methods and arbitrary standards have been used, resulting in confusion and lack of comparability. To overcome this the International Council for Standardization in Haematology (ICSH) established an accurately defined, and reasonably stable standard of a haemoglobin derivative haemiglobincyanide (HiCN). The specifications were based on the molar decadic absorbance coefficient and molecular mass of haemoglobin which had been established from experimental studies, and the haemoglobin concentration was calculated from the spectrometric measurement of HiCN absorbance at 540 nm.. The ICSH preparation was accepted by World Health Organization as an International Haemoglobin Reference Standard.

The existing batch of the ICSH/WHO preparation was almost exhausted and also near the limit of its stability. The objective of this project was to prepare a new batch of a stable preparation of haemiglobincyanide to serve as a primary reference standard in order to assign values to secondary standards, calibrators and comparators used in laboratories in the EU. It was also intended to offer material to WHO for a new International Standard to ensure its continuing availability to national health authorities world-wide. It was decided to use bovine instead of human blood as it has the advantages of better stability, availability and biosafety. The preparation was required to conform to the original ICSH specifications: molar absorbance coefficient of 11.0, mass concentration calculated from spectrometric absorbance at 540 nm, purity demonstrable by ratio A540/A504 of ò1.59 ó1.63, and no turbidity by measurement ó 0.003 at A750.
Mean absorbance at 540 nm was 0.5457 (95% confidence interval 0.5448 - 0.5465). Purity was shown by A540/A504 ratio of 1.613. The material was designated as certified reference material CRM522 with assigned value for mass concentration of 800.3 (ñ 1.3) mg/L; substance concentration 49.61(ñ 0.08) æmol/L. Homogeneity studies on 30 ampoules taken at random from the batch gave a batch CV of 0.09%. Thermal degradation studies showed by extrapolation that the product should remain stable for at least six years and probably much longer when stored at 4oC. The batch is undergoing annual monitoring: after four years the value was 800.1 mg/L, with A540/A504 ratio of 1.60 and no evidence of turbidity. The CRM has been described in laboratory text books, and information has been provided to haematologists, manufacturers of in-vitro diagnostic devices and to organizers of external quality assessment schemes.
Values were assigned by a collaborative study in 13 expert laboratories in Europe together with three ICSH Expert laboratories in the USA. The measurements were made by calibrated spectrophotometers, traceable to primary and metrological standards and to the original ICSH/WHO standard.

Funding Scheme

CSC - Cost-sharing contracts


Royal Postgraduate Medical School
150,Ducane Road
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United Kingdom