Abstract
Standardization of the hemagglutination inhibition (HAI) assay for influenza serology is challenging. Poor reproducibility of HAI results from one laboratory to another is widely cited, limiting comparisons between candidate vaccines in different clinical trials and posing challenges for licensing authorities. In this study, we standardized HAI assay materials, methods, and interpretive criteria across five geographically dispersed laboratories of a multidisciplinary influenza research network and then evaluated intralaboratory and interlaboratory variations in HAI titers by repeatedly testing standardized panels of human serum samples. Duplicate precision and reproducibility from comparisons between assays within laboratories were 99.8% (99.2% to 100%) and 98.0% (93.3% to 100%), respectively. The results for 98.9% (95% to 100%) of the samples were within 2-fold of all-laboratory consensus titers, and the results for 94.3% (85% to 100%) of the samples were within 2-fold of our reference laboratory data. Low-titer samples showed the greatest variability in comparisons between assays and between sites. Classification of seroprotection (titer ≥ 40) was accurate in 93.6% or 89.5% of cases in comparison to the consensus or reference laboratory classification, respectively. This study showed that with carefully chosen standardization processes, high reproducibility of HAI results between laboratories is indeed achievable.