Abstract
BackgroundThe hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development. It is unknown if the results can be influenced by sample type and anticoagulants. The purpose of this study was to evaluate the influence of different sample collection methods, in particular different anticoagulants, and choice of plasma or serum, on influenza virus serological assays.MethodsBlood samples from thirty donors previously immunized against influenza viruses were collected using six different types of blood collection tubes, two of which collect serum and four of which contain various anticoagulants for collecting plasma. Serum: (1) serum separator tubes (SST); and (2) Plus Plastic serum “red-top serum” tubes. Plasma: (3) spray-coated K2 ethylenediaminetetraacetic acid (EDTA) tubes: (4) Sodium Heparin tubes; (5) Citrate tubes with 3.2% sodium citrate solution; and (6) Glass Blood Collection tubes with acid citrate dextrose. Samples were tested against three different influenza viruses (A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012) for hemagglutination inhibition titer and virus neutralization titer via a microneutralization (MN) assay, and data compared to that obtained for standard serum sample collected in SST.ResultsHAI and MN titers against type A viruses were within two dilutions compared to SST collection method over 96% of the time irrespective of sample type or anticoagulant. However, HAI titers for type B virus were more variable across different collection methods. EDTA plasma samples were greater than two dilutions higher than SST serum samples 70% (21 of 30 samples) of the time. In contrast, MN titers were within two dilutions over 96% of the time, with the highest deviation noted in acid citrate dextrose plasma samples (3 of 30 samples tested, 10%).ConclusionsThese data provide useful guidelines for sample collection and serology testing when screening: (i) influenza vaccine immunogenicity antibody response; (ii) antibody responses to newly emerging viral strains; and (iii) clinical samples for anti-influenza antibody activity.
Highlights
The hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development
Despite difference in starting material we found that only 2% (6 out of 300) of samples deviated from the serum separator tubes (SST) reference for the MN assay
In the present study we found that RT sera, acid citrate dextrose (ACD) plasma, heparin plasma, and 3.2% sodium citrate solution-plasma resulted in generally lower HAI and MN titer values (17/20 had a negative bias compared to SST), but statistically similar, compared to SST sera sample collection
Summary
The hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development It is unknown if the results can be influenced by sample type and anticoagulants. A variety of additional serological assays to assess functional anti-influenza antibody titers exist [9,10,11], the MN and, in particular, the HAI assays remain gold-standard assays to evaluate immunogenicity of influenza vaccines and to measure serological responses to natural infection [12]. There is increasing awareness of the variability of influenza serological assay results [12] and the need to improve inter-laboratory agreement on serological assay standards [13] This is especially relevant for: (i) assessment of influenza vaccine immunogenicity; (ii) epidemiological studies seeking to catalog newly emerging viral strains in an affected region over time; and (iii) assessment of clinical samples for anti-influenza Ab activity
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