Methods comparison

Abstract

Abstract. The current report discusses the process in which a methods comparison study in the National Animal Health Laboratory Network is performed. Specific details are provided for designing and analyzing studies intended to evaluate analytical sensitivity, efficiency, analytical specificity, cross-contamination, repeatability, operator variability, and to compare the performance of methods using diagnostic samples. As an example, a case study is presented comparing the performance of a candidate reverse transcription polymerase chain reaction (RT-PCR) chemistry to the current RT-PCR chemistry in use when the assay was originally validated. The present study revealed that, for all of the validation factors evaluated, the candidate method performed at least as well and generally better than the current method. The candidate method was, therefore, deemed fit for the original intended purpose of the current method and rendered acceptable for use. A discussion of the case study is intended to further motivate consideration of the study designs chosen.

Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories

“…Method comparability analytical characteristics. An analytical sensitivity study is recommended using up to 3 reference strains, 32 which is a reduction in resources from the requirement to test all major strains in a de novo assay. The 3 reference strains selected should span the sequence diversity.…”

“…A single reference strain at 1 log 10 more concentrated than the LOD. † 5 replicates over 6 d. 24,32 Accuracy Schematic in one chapter 57 starting with nucleic acid. Here we give an example starting at the extraction step.…”

“…Candidate method should be comparable or more sensitive than the reference method. † Calculate standard deviation32 or coefficient of variation 24. ‡ Diagnostic samples should be comparable to or more sensitive using the candidate method.…”

Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories

“…Laboratories using pooling protocols should be aware that often the pooling potential is predicted from the Ct level of individual samples and is not empirically derived, and such predictions do not take into account the increased possibility of more inhibitors given the diversity and complexity of biological samples. A method comparability study 55,73,89 is appropriate before adopting a pooling strategy.…”

“…Suitability testing is performed in addition to the verification required for introduction of a new assay to the repertoire of tests for a laboratory. 55,73,89…”