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How much haemoglobin is there in the blood?

Especially interesting for
  • laboratory medicine
  • haematologists

The haemoglobin concentration in the blood (Hb) is one of the most important laboratory parameters for the diagnosis and classification of diverse anomalies of the haemoglobin metabolism, e.g. anaemia. To improve accuracy and reliability, PTB has investigated two methods with regard to their suitability as reference measurement procedures. Both methods are based on the determination of the haemoglobin content in the blood by means of photometry. In order to establish a representative comparison of these two procedures, the absorbance values measured with the photometer must be traceable. A corresponding procedure which is specifically suited for haemoglobin measurements has been successfully applied.

Absorption spectrum of a diluted blood sample after conversion of the haemoglobin variants into HiCN. The expanded measurement uncertainties (95 % level of confidence) include the contributions of the traceability, the preparation and the repeatability. The photo shows six different dilutions for Hb measurement in the back row and the photometric cuvettes with the conversion solution required for the differential measurement.

The total Hb concentration in the blood is measured by transforming each of the different variants of haemoglobin (e.g. oxyhaemoglobin or carboxyhaemoglobin) into a uniform, stable end product using specific conversion reagents and by determining the spectral absorbance of the solution by means of a spectral photometer. The first procedure is the frequently applied HiCN method, where a conversion into the end product cyano methaemoglobin takes place. This method has certain disadvantages: it is too expensive for non-industrialized countries; there is no pure calibration material; and its application is not allowed in all countries due to its toxicity. The second investigated method (the AHD method) – based on a conversion into a complex composed of alkaline haematin and non-ionic dispersant – is, thus, of particular interest. After conversion into the respective end product, for both methods a dilution series is prepared and the spectral absorbances are determined at the wavelengths 540 nm (HiCN) (see figure) and 574 nm (AHD). In order to derive the “true” (or the so-called “conventional quantity”) values from the spectral absorbance values, which are measured relative to the conversion solution, they have to be traceable by measuring the characteristic line of the spectral photometer used for the Hb determination. This characteristic curve is obtained by comparing the absorbance values of neutral-density glass filters with the reference measurement values of the spectral absorbance of these filters determined with the national reference instrument at PTB. These experiments for the traceability of the photometer have shown that the measured absorbance of the filters is in very good agreement with the reference measurement values.

The uncertainty of measurement of the “true” absorbance values is higher by approx. one order of magnitude compared to that of the measured absorbance values, which corresponds to the repeatability in the case of filter measurements and of Hb measurements. This indicates that the measurement uncertainties of the filter absorbance values used for the traceability contribute considerably to the combined measurement uncertainty of the haemoglobin determination. Hence, a reduction of this measurement uncertainty would improve Hb measurement all in all. The good agreement of the haemoglobin concentrations determined with these two conversion procedures proves that both methods are suitable as higher-order methods for the determination of reference values.

The work was partly supported by the funding programme “Messen, Normen, Prüfen und Qualitätssicherung” (MNPQ) of the Federal Ministry of Economic Affairs and Energy and by the European Union within the European Metrology Research Programme (EMRP) HLT-05 2012 “Metrology for metalloproteins”.

Scientific publication

K. Witt, H.U. Wolf, C. Heuck, M. Kammel, A. Kummrow, J. Neukammer: Establishing traceability of photometric absorbance values for accurate measurements of the haemoglobin concentration in blood. Metrologia 50, 539-548 (2013)