e. a criterion based on a ratio rather than a difference. This has the opposite disadvantage: higher false positive probability in plates with low background counts. For example, if the criterion is a four-fold ratio, and the negative control has two spots, an experimental well will be considered positive if it has ≥ eight spots, and this is much more likely to occur by chance than a value of 800 spots where the control well has 200. These

considerations have led many groups to apply a combination of absolute and fold difference ( Larsson et al., 1999, Russell et al., 2003 and Jeffries et al., 2006). For example, the T-SPOT manual recommends a difference of at least 6 if the negative control has 5 or fewer spots, and a ratio of at least 2 when it has 6 or more ( Oxford Immunotec, 2006). Additionally, a threshold value (e.g. at least 11 SFU/106 PBMC in Decitabine ic50 the experimental well) is also sometimes applied to provide a threshold of responsiveness that is considered to have biological significance. Similarly, an upper limit on the number of spots in the negative control well may be imposed, e.g. 10 in the case of T-SPOT and IAVI (International AIDS Vaccine Initiative)( Gill et al., 2010). These cut-offs and thresholds are often defined with

reference to ELISpot responses in a known negative population and are therefore often referred to as empirical NVP-BEZ235 methods ( Moodie et al., 2006). By contrast, statistical methods have been developed which use the variation between Calpain replicate control wells to define positivity thresholds ( Hudgens et al., 2004 and Moodie et al., 2006). However,

when a wide range of peptides is being examined it may be impractical to include replication of the peptide and negative control wells. In the current paper we develop a positivity criterion for such plate layouts, in the context of a study of cell mediated immunological response to influenza. We present a method which uses within-plate differences between test and control wells, and a positivity threshold based on their statistical distribution over plates. The method relies on the principle that pools can only be reliably declared positive when the test counts tend to be larger than the negative control ones. The method is illustrated using data from a cohort study in Vietnam (Horby et al., 2012). The cohort study included 932 individuals aged between 5 and 90 years. PBMC samples were taken to measure the prevalence of T-cell responsiveness to seasonal and avian influenza peptides in order to determine the protective effect of pre-existing T-cell responses. Institutional review boards in the United Kingdom and Vietnam approved the study and all subjects provided written informed consent.