| Objective: The determination of hemoglobin concentration in human blood is oneof the most frequently performed determinations in the clinical pathology laboratory.Numerous different methods can be used in the determination. These methods oftenyield widely different results so that the results must be traceable to the referencemethod to assure the accuracy and comparability.In 1964 the Expert Panel on Haemoglobinometry of ICSH drew uprecommendations for haemoglobinometry in human blood that were published in1967. The method had been revised three times; the apparatus, reagent and procedurehad been changed. We have established the reference method in our laboratoryaccording to the requirements of ICSH. Its methodology characteristics need to bevalidated whether the method is fit for charaterisation of haemiglobincyanidereference material.Haemiglobincyanide reference material is one of the cornerstones of thestandardization of haemoglobinometry. As it's difficult to obtain the internationalprimary standard, many nations and organizations prepared their own referencematerials, e.g. BCR CRM 522. There are secondary reference materials provided by two internal orgnizations several years ago through literature retrieval, but they areunavailable now. Therefore we intend to prepare and certificate a candidatehaemiglobincyanide reference material to standardize the haemoglobinometry.Methods: The specifications of ultraviolet-visible spectrophotometers wereaffirmed to ensure that they can meet the requirements of the reference method, andthe reference measurement procedure was established. According to the guidelines ofmethodology validation in ICH and AOAC, we chose the selectivity, linearity,precision and accuracy to evaluate. The linearity and precision were validatedaccording to NCCLS EP6-P2 and EP5-A. The accuracy was verified by determinationof WHO international standard haemiglobincyanide.The preparation of candidate haemiglobincyanide reference material was mainlyreferred to ICSH document. Then the candidate haemiglobincyanide referencematerial was certificated according to ISO Guide 35, including homogeneity study,stability study and Characterization.Results: The precision showed coefficient variation (CV) of 0.12～0.32％. Thecorrelation coefficient of least squares regression equation between measured andexpected value was 1.000 (n=6). The relative deviations between the assayed resultsby U-3000 and U-3310 and the assigned value of WHO international standard were—0.01％and—0.35％, respectively.The candidate reference material's uncertainty of between-bottle homogeneity,which was calculated according to ISO Guide 35, was 0.0004 g/L. The CV ofbetween-bottle homogeneity was 0.09％, which was equal to the CV of WHOinternational standard's between-bottle homogeneity. The uncertainty of long-termstability was 0.0006 g/L for a shelf life of one year; the mean of means ofcharacterization was 0.6159 g/L, wich is corresponding to the haemoglobin of 154.7g/L, with uncertainty of 0.0004 g/L. Therefore the combined uncertainty could beworked out at 0.0009 g/L and the expanded uncertainty was 0.0018 g/L if the coverfactor k=2. If calculated as the WHO formulation of international standard haemiglobincyanide, the uncertainty of candidate reference material was 0.0012 g/L(the uncertainty of WHO international standard is 0.0013 g/L).Conclusion: Coefficient variation of the reference method is within the acceptablelimit(＜0.5％) in NCCLS H15-A3. There is a linearity between measured value andexpected value within 70～270 g/L haemoglobin concentration. The determined valueof WHO international standard is within the uncertainty limits of certified value.The results mentioned above show that the candidate reference material haspreferable homogeneity and stability, and the characterization is 0.6159±0.0018 g/L.
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