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Background Morbidity has been reported as a sequelae of crystalline steroid epidural steroid injections (ESIs), and particulate steroid size, aggregation, and embolization in brain and spinal cord may be the mechanism related to these neurologic effects. Objective The objective of the study was to examine the aggregation properties of triamcinolone acetonide in commonly used local anesthetics with and without human serum. Setting This study was conducted in an academic tertiary care center. Hypothesis Triamcinolone acetonide shows different aggregation characteristics in serum compared to a non-physiologic solution. Design Triamcinolone acetonide was mixed with lidocaine 1% (first group) and bupivacaine 0.5% (second group) in a 1:1 ratio and then mixed with either distilled water (control group) or serum ex vivo. A pathologist blinded to our hypothesis inspected all solutions under light microscopy with 100× and 400× magnifications. Total number of particulate steroid aggregates and the number of particles forming each aggregate (recorded as single,1 double,2 triple,3 quadruple,4 or large [>4} crystals) were counted. Particle size and aggregate size were measured (in μm). The ratios of quadruple to total aggregates, large to total, and quadruple with large to total aggregates were calculated. Steroid-serum solutions and steroid-sterile water were then compared. Results Triamcinolone aggregates showed an increased crystal and aggregate size when compared with other steroids. Within the triamcinolone subgroup, the mixture of lidocaine 1% and serum resulted in the largest crystal aggregates. Limitations Whole blood analysis may have provided a more physiologically accurate model but was not chosen due to poor microscopic analysis. Serum donor variability may also have affected particle characteristics. Conclusion Fewer large triamcinolone aggregates were noted in the presence of serum when compared to the non-serum control groups. However, when compared to previously studied particulate steroids, it had the largest aggregates when added to serum.

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.

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.