Opportunities and challenges when pooling milk samples using ELISA.

Testing large quantities of samples in order to detect one or more test-positive sample(s) is expensive and time-consuming. It is possible to optimize this process by pooling samples. Two frameworks to produce different hierarchical and non-hierarchical pooling schemes were tested and compared to standard pooling. Their efficiency and the potential savings were determined as a function of prevalence and the number of pooled samples. The potential benefit of pooling samples is dependent upon the changes in the analytical sensitivity and specificity of the test used when diluting test-positive samples by pooling. To illustrate this, the sensitivity of antibody ELISA on pooled samples of bovine milk for Salmonella Dublin, Mycobacterium avium spp. paratuberculosis, and bovine virus diarrhea was tested. For these milk assays, the analytical sensitivity decreased rapidly with increasing pool sizes. The efficiency of pooling is usually only measured by the number of tests performed, yet real savings depend on all the costs involved in the pooling process. These may differ between laboratories depending on the available equipment and the salaries of the technicians, among other factors. Therefore, several cost parameters were introduced to describe the total cost and thereby calculate the total savings. In terms of overall savings, both tested schemes were potentially optimal depending on the prevalence, possible pool size, and the cost of retesting. For the pool sizes of interest in this study, the three-stage hierarchical pooling scheme was often marginally more efficient in terms of the total number of tests. However, if the price of re-pooling was high, the two-stage scheme performed better in terms of total savings. In addition, for low prevalences and the possibility of pooling a large number of samples, the two-stage non-hierarchical test may be more efficient, both in terms of number of tests and overall cost. In order to apply these results in different laboratory settings, a free Shiny WebApp was developed, to compare several pooling schemes with different cost parameters.