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. 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.
Keywords: hemagglutination inhibition assay; influenza; serology; standardization.