PAWG pilot study on quantification of total haemoglobin in blood

Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P201, was organised. Five National Metrology Institutes (NMIs) participated in this pilot study and provided a total of eleven measurement results using six different methods based on three different measurement principles. The purpose of this pilot study was to develop measurement capabilities for the accurate quantification of intact haemoglobin (Hb) whereby measurement results could be made directly traceable to the SI. The choice of protein resulted from its ability to be quantified via different measurement principles providing different routes of traceability. Hb also provided a model of an intact protein with a complex tetrameric structure which is often measured via quantification of a specific subunit or the incorporated haem group. To achieve appropriate results for the chosen measurand, it was necessary to distinguish between the whole protein and fragments of the tetrameric structure, such as dimers or monomers. The participants were encouraged to investigate several of the measurement methods available. This was considered particularly advantageous in this instance as various methods applied to the same analyte may assist in identifying possible previously unknown biases or interferences of the various methods. Hb is being used as a model system to build capacity in methods for the quantification of proteins with a molar mass ≤ 100 kDa in biological fluids in a concentration range > 1∙106 pmol/g. This was the first pilot study for the quantification of an inherent protein in blood with all the natural variations that protein shows in a clinical sample. The different methods applied by the participants included isotopic dilution (ID) organic mass spectrometry (MS) of specific peptides after tryptic digestion, species-specific ID inductively coupled plasma mass spectrometry (ICP-MS), post-column ID ICP-MS, quantification via total iron after chromatographic isolation of Hb and acid digestion as well as two optical methods. One of the latter was the reference method proposed by the International Committee for Standardisation in Haematology (ICSH) based on the conversion of Hb to hemiglobincyanide (HiCN), the other used the conversion of Hb with alkaline haematin detergent both followed by the measurement of spectroscopic absorbance. Considering the use of very different measurement techniques for the quantification of total Hb, the results are in good agreement with a reference value with corresponding expanded uncertainty of (120.6 ± 1.2) mg/g calculated as DerSimonian-Laird mean. However, the observed differences between the results are not completely reflected in the reported measurement uncertainties. The calculated interlaboratory standard deviation or Tau value (dark uncertainty) was approximately 3 % whilst the average reported measurement uncertainty was approximately half this (1.4 %). A possible reason for this large difference may lie in the underestimation of any necessary conversion factors and their uncertainty contributions, between the target analyte measured and the requested measurand. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).