Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.
Keywords: epidemiology; histologic grading; immunohistochemistry; method validation; observational studies; pathology; quantitative PCR; reproducibility of results; research design; robustness.