Statistical Evaluation of the Orthogonal Pooling Strategy for High-Throughput Screening

Abstract

In high-throughput screening (HTS), compounds can be tested in mixtures using the orthogonal pooling strategy. However, several experimental studies that used two-way orthogonal pooling have shown that the rate of false negative testing errors is still not negligible. Because replicate screening is prohibited due to the large number of compounds that need to be tested, the false negative results eventually lead to loss of active compounds. This article generalizes the two-way orthogonal pooling strategy to a three-way pooling and develops statistical methods to evaluate the false negative rate (FNR) and the false positive rate (FPR) for the two-way and three-way pooling strategies. Calculations show that the three-way pooling strategy decreases the FNR but increases the FPR to approximately three times that of the two-way pooling strategy. In terms of strategy selection, three-way pooling requires 50% more tests than two-way pooling but gains benefit when the testing technique has low sensitivity and high specificity, while two-way pooling may be preferred when the technique has high sensitivity and low specificity.