Observational Study Design in Veterinary Pathology, Part 2: Methodology

Abstract

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.

Validating Immunohistochemistry Assay Specificity in Investigative Studies: Considerations for a Weight of Evidence Approach

“…35 While a manufacturer's datasheet can provide some information about the expected cross-species reactivity, this information also needs to be interpreted based on the quality of the data. 5 Protein sequence alignments can provide information on the likelihood of cross-species immunoreactivity, especially if the cognate epitope is known. 5 This approach was used in evaluating an anti-human CD30 antibody in feline tissues.…”

“…5 Protein sequence alignments can provide information on the likelihood of cross-species immunoreactivity, especially if the cognate epitope is known. 5 This approach was used in evaluating an anti-human CD30 antibody in feline tissues. 4 Unfortunately, an antibody that works well in one species, will not always perform similarly in a different species.…”

“…The use of a second antibody that detects a different epitope on the target protein can provide evidence that IHC labeling pattern is specific. 5 In principle, if both antibodies are specific, they should have identical labeling patterns despite detecting different epitopes. However, this approach has theoretical complications if one epitope is unavailable in select cell populations, perhaps due to being engaged in protein complexes.…”

Observational Study Design in Veterinary Pathology, Part 1: Study Design

“…Finally, we consider the need for careful critique of the study design and approaches to adding value and rigor. The second article of the series 8 addresses methodology and validation of methods.…”

Ensuring Observational Studies Have Validity and Impact

“…The previous and current issues of Veterinary Pathology , September and November 2018, include 2 commentaries on designing and conducting observational studies in veterinary pathology. 1,2 In this editorial, I offer 10 suggestions and critiques of current practices. These reflections arise from assessing or editing about 1400 manuscripts submitted to Veterinary Pathology over the past 5 years.…”