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Licensed Unlicensed Requires Authentication Published by De Gruyter March 13, 2020

Total haemoglobin – a reference measuring system for improvement of standardisation

  • Denis Grote-Koska EMAIL logo , Rainer Klauke , Patricia Kaiser , Udo Kramer , Rainer Macdonald , Dietmar Lerche , Antje Staaden , Korbinian Brand and Gerhard Schumann



Total haemoglobin (Hb) concentration in blood belongs to the most requested measurands, and the HiCN method (hemiglobincyanide) is accepted as a reference. Although the reaction principle is clearly characterised, measurement conditions and settings are not consistently defined, some of them influencing the results. An improvement of standardisation is the object.


After method optimization, measurement results between different calibration laboratories (CL) were compared with each other and also with results of the National Metrology Institute of Germany (PTB), with target values of certified reference material, within the RELA scheme, and to >1500 results from routine laboratories.


Overall deviations between three CLs were ≤0.5% (n = 24 samples) in a measurement range of 20 g/L to 300 g/L. A CV of 0.4% was determined in pooled blood (1 year long-term imprecision, 99.0%–101.1% recovery of the mean). For selected measurements (n = 4 samples) the PTB participated without significant differences to three CLs, and no significant differences were observed comparing CLs to certified values of reference materials. The expanded measurement uncertainty (probability 95%) was estimated as 1.1%.


A reference measuring system, comprising measuring instruments and other devices, including reagents and supply, to generate reference measurement values for total Hb concentration of high accuracy and low measurement uncertainty is presented. Measurement parameters are investigated and defined. The reference measuring system is ready to offer service to EQA providers and to the IVD industry for certifying control materials or calibrators.

Corresponding author: Dr. Denis Grote-Koska, Medizinische Hochschule Hannover, Institute of Clinical Chemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: Laboratory A: Reference Institute for Bioanalytics, Foundation for Pathobiochemistry and Molecular Diagnostics, Bonn, Germany.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.


1. Lewis SM, Garvey B, Manning R, Sharp SA, Wardle J. Lauryl sulphate haemoglobin: a non-hazardous substitute for HiCN in haemoglobinometry. Clin Lab Haemat 1991;13:279–90.10.1111/j.1365-2257.1991.tb00283.xSearch in Google Scholar

2. Lema OE, Carter JY, Arube PA, Munafu CG, Wangai MW, Rees PH. Evaluation of the alkaline haematin D-575 method for haemoglobin estimation in east Africa. Bull World Health Organ 1994;72:937–41.Search in Google Scholar

3. Moharram NM, El Aouad R, Al Busaidy S, Fabricius A, Heller S, Wood WG, et al. International collaborative assessment study of the AHD575 method for the measurement of blood haemoglobin. East Mediterranean Health J 2006;12:722–34.Search in Google Scholar

4. Shah VB, Shah BS, Puranik GV. Evaluation of non-cyanide methods for haemoglobin estimation. Indian J Pathol Microbiol 2011;54:764–8.Search in Google Scholar

5. Whitehead RD, Mei Z, Mapango C, Jefferds ME. Methods and analyzers for hemoglobin measurement in clinical laboratories and field settings. Ann N Y Acad Sci 2019;1450:147–71.10.1111/nyas.14124Search in Google Scholar

6. Heuck CC, Reinauer H, Wood WG. The alkaline haematin detergent (AHD575) method for the determination of haemoglobin in blood – a candidate reference measurement procedure. Clin Lab 2008;54:255–72.Search in Google Scholar

7. Drabkin DL, Austin J H. Spectrophotometric studies: I. Spectrophotometric constants for common haemoglobin derivates in human, dog, and rabbit blood. J Biol Chem 1932;98:719–33.10.1016/S0021-9258(18)76122-XSearch in Google Scholar

8. Zwart A, Van Assendelft OW, Bull BS, England JM, Lewis SM, Zijlstra WG. Recommendations for reference method for haemoglobinometry in human blood (ICSH standard 1995) and specifications for international haemiglobincyanide standard (4th edition). J Clin Pathol 1996;49:271–4.10.1136/jcp.49.4.271Search in Google Scholar

9. Bull BS, Houwen B, Koepke JA, Simson E, van Assendelft OW. H15-A3: reference and selected procedures for the quantitative determination of haemoglobin in blood. Wayne, PA: Clinical Laboratory Standards Institute, 2000.Search in Google Scholar

10. BS 3985:2003. Haemiglobincyanide (cyanmethaemoglobin) preparation as a standard for spectrophotometric haemoglobinometry. London: British Standards Institution, 2003.Search in Google Scholar

11. DIN 58931:2010. Haematology – determination of haemoglobin concentration in blood – reference method. Berlin: Beuth, 2010.Search in Google Scholar

12. Davis BH, Jungerius B. International Council for Standardization in Haematology technical report 1-2009: new reference material for haemiglobincyanide for use in standardization of blood haemoglobin measurements. Int J Lab Hematol 2010;32:139–41.10.1111/j.1751-553X.2009.01196.xSearch in Google Scholar

13. ISO/DIS 17511:2019. In vitro diagnostic medical devices – requirements for establishing metrological traceability of values assigned to calibrators, trueness control materials and human samples. Geneva: ISO, 2019.Search in Google Scholar

14. Siekmann L, Bonora R, Burtis CA, Ceriotti F, Clerc-Renaud P,Ferrero CA, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 C, Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes. Clin Chem Lab Med 2002;40:635–4.10.1515/CCLM.2002.109Search in Google Scholar

15. Zijlstra WG, Kampen EJ. Standardization of haemoglobinometry, I. The extinction coefficient of hemiglobincyanide. Clin Chim Acta 1960;5:719–26.10.1016/0009-8981(60)90014-0Search in Google Scholar

16. IFCC-DGKL-RELA. Accessed: 30 Oct 2019.Search in Google Scholar

17. ISO 15195:2018. Laboratory medicine – requirements for the competence of calibration laboratories using reference measurement procedures. Geneva: ISO, 2018.Search in Google Scholar

18. ISO/IEC 17025:2017. General requirements for the competence of testing and calibration laboratories. Geneva: ISO, 2017.Search in Google Scholar

19. Frank C, Brauckmann C, Palos M, Arsene CG, Neukammer J, Del Castillo Busto ME, et al. Comparison of potential higher order reference methods for total haemoglobin quantification – an interlaboratory study. Anal Bioanal Chem 2017;409:2341–51.10.1007/s00216-016-0176-7Search in Google Scholar PubMed

20. ISO 13528:2005. Statistical methods for use in proficiency testing by interlaboratory comparisons. Geneva: ISO, 2005.Search in Google Scholar

21. ISO/IEC Guide 98-3 2008. Uncertainty of measurement – part 3: Guide to the expression of uncertainty in measurement (GUM: 1995). JCGM member organizations (BIPM, IEC, IFCC, ILAC, ISO, IUPAC, IUPAP and OIML), 1st ed, 2008. Accessed: 4 Mar 2020.Search in Google Scholar

22. Witt K, Wolf HU, Heuck C, Kammel M, Kummrow A, Neukammer J. Establishing traceability of photometric absorbance values for accurate measurements of the haemoglobin concentration in blood. Metrologia 2013;50:539–48.10.1088/0026-1394/50/5/539Search in Google Scholar

23. OIML R 135:2004. Spectrophotometers for medical laboratories. International Recommendation, International Organization of Legal Metrology, Paris, 2004. Accessed: 4 Mar 2020.Search in Google Scholar

24. Mielenz KD, Eckerle KL. Spectrophotometer linearity testing using the double-aperture method. Appl Opt 1972;11:2294–303.10.1364/AO.11.002294Search in Google Scholar PubMed

25. JCGM 200:2012. International vocabulary of metrology – basic and general concepts and associated terms (VIM), 3rd ed. Accessed: 30 Oct 2019.Search in Google Scholar

26. De la Salle B. Survey material choices in haematology EQA: a confounding factor in automated counting performance assessment. Biochem Med (Zagreb) 2017;15:63–72.10.11613/BM.2017.008Search in Google Scholar PubMed PubMed Central

27. Zijlstra WG. Standardisation of haemoglobinometry: history and new challenges. Comp Haematol Int 1997;1:125–32.10.1007/BF02652590Search in Google Scholar

28. Zierk J, Hirschmann J, Toddenroth D, Arzideh F, Haeckel R, Bertram A, et al. Next-generation reference intervals for pediatric hematology. Clin Chem Lab Med 2019;57:1595–607.10.1515/cclm-2018-1236Search in Google Scholar PubMed

29. William WJ. Clinical evaluation of the patient. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, editors. Hematology, 4th ed. New York: Mc Graw Hill, 1990:10.Search in Google Scholar

30. Revision of the “Guideline of the German Medical Association on Quality Assurance in Medical Laboratory Examinations – Rili-BAEK” (unauthorized translation). J Lab Med 2015;39:26–69.10.1515/labmed-2014-0046Search in Google Scholar

Supplementary Material

The online version of this article offers supplementary material (

Received: 2019-11-14
Accepted: 2020-02-11
Published Online: 2020-03-13
Published in Print: 2020-07-28

©2020 Walter de Gruyter GmbH, Berlin/Boston

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