Traceability of photometric absorbance values for the accurate determination of the total haemoglobin concentration in blood

One of the analytes that is most frequently measured in laboratory medicine is the total haemoglobin concentration (Hb) in blood. It is based on transforming (by means of appropriate conversion reagents) the different haemoglobin variants into stable end products whose spectral extinction is determined subsequently by means of a spectral photometer. In order to determine reference values, two procedures are used at PTB: conversion of the haemoglobin variants into haemiglobincyanide (HiCN method), and transformation of the haemoglobin variants into a complex of alkaline haematin and non-ionic detergent (AHD complex).

In both methods, the total haemoglobin concentration in blood is determined by subsequently measuring the spectral extinction of the reaction product at the wavelengths λ = 540 nm (HiCN) and λ = 574 nm (AHD), respectively. These wavelengths are located at the maximum of the corresponding Q bands (see Figure 1).

Figure 1: Absorption spectra of HiCN and AHD. The wavelengths of 540 nm and 574 nm, respectively, which are used to determine the total Hb concentration, are plotted as vertical lines.

To achieve the traceability of the photometric absorption values – from which the concentration of the total Hb is determined – the characteristic curve of the spectral photometer used for Hb determination was measured. This characteristic curve is obtained by comparing the absorption values of neutral-density filters with the reference values of the spectral absorbance measured with the national reference instrument at PTB in Braunschweig (WG 4.24).

Figure 2 shows the absorption spectra that are obtained for the HiCN method (Fig. 2a) and the AHD method (Fig. 2b), respectively. The conventional quantity value A of the spectral absorbance is represented as a function of the wavelength. The traceability measurements show that the measured absorption values of the filters are in very good agreement with the reference values, i.e. the deviations observed between the measured spectral extinction a and the conventional quantity value of the spectral absorbance A are less than 10^-3. This is shown in Figs. 2c and 2d. The measurement uncertainties of the quantity A are, however, larger than the reproducibility of the measured quantity values a (see Figs. 2e and 2f) by more than one order of magnitude. Hence, the measurement uncertainties due to the assignment of traceable absorbance values to the filters contribute significantly to the combined measurement uncertainty of the haemoglobin concentration. We conclude that the assignment of reference values for the absorbance of the neutral-density filters with lower measurement uncertainties would lead to lower measurement uncertainties for the measurement of the total Hb.

The haemoglobin concentrations determined with the aid of these two conversion methods are in excellent agreement and prove that both methods are suitable as higher-order methods for the determination of reference values.

Figure 2: Absorption spectra of diluted blood samples after conversion in HiCN (a) and AHD (b). Differences between the measured absorbance and the conventional quantity value of the absorption value for HiCN (c) and AHD (d). Expanded measurement uncertainties ku(A) and ku(a) of the conventional quantity values A and of the measured quantity values a for the two different conversion methods HiCN (e) and AHD (f), respectively.

Witt K, Wolf HU, Kammel M, Kummrow A, Neukammer J (2013): Establishing traceability of photometric absorbance values for accurate measurement of the haemoglobin concentration in blood; Metrologia 50: 539-548

http://iopscience.iop.org/0026-1394/50/5/539/article

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