title: Automated Imaging and Analysis of the Hemagglutination Inhibition Assay.
abstract: The hemagglutination inhibition (HAI) assay quantifies the level of strain-specific influenza virus antibody present in serum and is the standard by which influenza vaccine immunogenicity is measured. The HAI assay endpoint requires real-time monitoring of rapidly evolving red blood cell (RBC) patterns for signs of agglutination at a rate of potentially thousands of patterns per day to meet the throughput needs for clinical testing. This analysis is typically performed manually through visual inspection by highly trained individuals. However, concordant HAI results across different labs are challenging to demonstrate due to analyst bias and variability in analysis methods. To address these issues, we have developed a bench-top, standalone, high-throughput imaging solution that automatically determines the agglutination states of up to 9600 HAI assay wells per hour and assigns HAI titers to 400 samples in a single unattended 30-min run. Images of the tilted plates are acquired as a function of time and analyzed using algorithms that were developed through comprehensive examination of manual classifications. Concordance testing of the imaging system with eight different influenza antigens demonstrates 100% agreement between automated and manual titer determination with a percent difference of ≤3.4% for all cases.
authors.affiliation: Sanofi Pasteur VaxDesign, Orlando, FL, USA Michael.Nguyen@sanofipasteur.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Nguyen
authors.affiliation: Sanofi Pasteur Global Clinical Immunology, Swiftwater, PA, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Fries
authors.affiliation: Sanofi Pasteur Global Clinical Immunology, Swiftwater, PA, USA.
authors.forename: Rawia
authors.initials: R
authors.lastname: Khoury
authors.affiliation: Sanofi Pasteur Global Clinical Immunology, Swiftwater, PA, USA.
authors.forename: Lingyi
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Sanofi Pasteur Global Clinical Immunology, Swiftwater, PA, USA.
authors.forename: Branda
authors.initials: B
authors.lastname: Hu
authors.affiliation: Sanofi Pasteur Global Clinical Immunology, Swiftwater, PA, USA.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Hildreth
authors.affiliation: Sanofi Pasteur VaxDesign, Orlando, FL, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Parkhill
authors.affiliation: Sanofi Pasteur VaxDesign, Orlando, FL, USA.
authors.forename: William
authors.initials: W
authors.lastname: Warren
mesh_terms: D000914:Antibodies, Viral / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D057205:Automation, Laboratory / Q000379:methods*; D006385:Hemagglutination Inhibition Tests / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D061848:Optical Imaging / Q000379:methods*; D009975:Orthomyxoviridae / Q000276:immunology; D015203:Reproducibility of Results
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral
keywords: automation; hemagglutination inhibition assay; high-throughput; image analysis; influenza