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

2021, 28 July

| 2 min read

Publisher: ASM Journals mSphere

Authors: J. Waldock, L. Zheng, E.J. Remarque, A. Civet, B. Hu, S. Lartey Jalloh, R. Jane Cox, S. Ho, K. Hoschler, T. Ollinger, C.M. Trombetta, O.G. Engelhardt, C.Caillet

Date: 28 July 2021




The hemagglutination inhibition (HAI) assay is an established technique for assessing influenza immunity, through measurement of antihemagglutinin 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.

Patients and methods

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.