Assay Harmonization and Use of Biological Standards To Improve the Reproducibility of the Hemagglutination Inhibition Assay: a FLUCOP Collaborative Study

Joanna Waldock, Lingyi Zheng, Edmond J. Remarque, Alexandre Civet, Branda Hu, Sarah Lartey Jalloh, Rebecca Jane Cox, Sammy Ho, Katja Hoschler, Thierry Ollinger, Claudia Maria Trombetta, Othmar G. Engelhardt, Catherine Caillet*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)
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The hemagglutination inhibition (HAI) assay is an established technique for assessing influenza immunity, through measurement of anti-hemagglutinin antibodies. Improved reproducibility of this assay is required to provide meaningful data across different testing laboratories. This study assessed the impact of harmonizing the HAI assay protocol/reagents and using standards on interlaboratory variability. Human pre- and postvaccination sera from individuals (n =30) vaccinated against influenza were tested across six laboratories. We used a design of experiment (DOE) method to evaluate the impact of assay parameters on interlaboratory HAI assay variability. Statistical and mathematical approaches were used for data analysis. We developed a consensus protocol and assessed its performance against in-house HAI testing. We additionally tested the performance of several potential biological standards. In-house testing with four reassortant viruses showed considerable interlaboratory variation (geometric coefficient of variation [GCV] range of 50% to 117%). The age, concentration of turkey red blood cells, incubation duration, and temperature were key assay parameters affecting variability. Use of a consensus protocol with common reagents, including viruses, significantly reduced GCV between laboratories to 22% to 54%. Pooled postvaccination human sera from different vaccination campaigns were effective as biological standards. Our results demonstrate that the harmonization of protocols and critical reagents is effective in reducing interlaboratory variability in HAI assay results and that pools of postvaccination human sera have potential as biological standards that can be used over multiple vaccination campaigns. Moreover, the use of standards together with in-house protocols is as potent as the use of common protocols and reagents in reducing interlaboratory variability.

IMPORTANCE The hemagglutination inhibition (HAI) assay is the most commonly used serology assay to detect antibodies from influenza vaccination or influenza virus infection. This assay has been used for decades but requires improved standardization of procedures to provide meaningful data. We designed a large study to assess selected parameters for their contribution to assay variability and developed a standard protocol to promote consistent HAI testing methods across laboratories. The use of this protocol and common reagents resulted in lower levels of variability in results between participating laboratories than achieved using in-house HAI testing. Human sera sourced from vaccination campaigns over several years, and thus including antibody to different influenza vaccine strains, served as effective assay standards. Based on our findings, we recommend the use of a common protocol and/or human serum standards, if available, for testing human sera for the presence of antibodies against seasonal influenza using turkey red blood cells.

Original languageEnglish
Article numberARTN e00567-21
Pages (from-to)1-18
Number of pages18
Issue number4
Early online date28 Jul 2021
Publication statusPublished - Aug 2021

Bibliographical note

Funding Information: We thank Damien Friel from GSK for his participation and support during the development of this study and Marie-Clotilde Bernard and Sylvie Commandeur from Sanofi Pasteur for the preparation of the HI video. This study was funded by the Innovative Medicines Initiative Joint Undertaking (IMIJU) under grant agreement 115672, with financial contribution from the European Union Seventh Framework Programme (FP/2007-2013) and EFPIA companies? in-kind contribution. Medical writing support was funded by Sanofi Pasteur. Conceptualization and study design: J.W., L.Z., E.J.R., B.H., O.G.E., C.C.; laboratory work: J.W., S.L.J., R.J.C., S.H., K.H., T.O., C.M.T.; data analysis: L.Z., E.J.R., A.C.; writing and editing: J.W., L.Z., E.J.R., C.C.; critical review of the manuscript and approval of the final draft: all authors. T.O. is an employee of the GSK group of companies. L.Z. and C.C. are employees of Sanofi Pasteur and hold stock in the company. B.H. was employed by Pasteur at the time this work was generated and may/may not hold stock in the company. A.C. was employed by Quinten at the time this work was generated. J.W. and O.G.E. are employees of NIBSC/MHRA and report funding from IFPMA outside the area of this work. C.M.T., R.J.C., S.L.J., S.H., K.H., and E.J.R. have nothing to disclose.

Open Access: This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license

Publisher Copyright: © 2021. Waldock et al.

Citation: n Waldock J, Zheng L, Remarque EJ, Civet A, Hu B, Jalloh SL, Cox RJ, Ho S, Hoschler K, Ollinger T, Trombetta CM, Engelhardt OG, Caillet C. 2021. Assay harmonization and use of biological standards to improve the reproducibility of the hemagglutination
inhibition assay: a FLUCOP collaborative study. mSphere 6:e00567-21.



  • hemagglutination inhibition assay
  • influenza
  • serology
  • standardization


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