Abstract
Leukogram evaluation provides valuable information about inflammation, infection, and stress in free-living and zoo-maintained wildlife. While multiple protocols for quantifying leukocytes are available in reptiles, agreement between methods is infrequently described and analytical variability (including repeatability and reproducibility) has not been critically evaluated. This study addresses these knowledge gaps for two hematological methods in eastern box turtles (Terrapene carolina carolina): Avian Leukopet™ (LO) and total white blood cell (WBC) estimates from blood films (EST). The objectives of this study were (1) to evaluate agreement in total WBC and individual leukocyte counts between the LO and EST methods, (2) to document repeatability (intra-assay variability) and reproducibility (inter-assay variability) for the LO method, and (3) to investigate whether biological drivers of WBC counts differ between quantification methods. Box turtles (n = 120) were sampled from five study sites in Illinois during the 2018 active season. The LO method produced significantly higher WBC counts than the EST method, and constant and proportional error was variable for each leukocyte type. The LO method demonstrated an intra-assay variability of 8.2% and an inter-assay variability of 12%, independent of biological variation. WBC counts were significantly affected by age class using both LO and EST methods, but WBC differences between locations and sexes were only observed using the LO method. These findings emphasize the importance of considering leukocyte determination method when analyzing reptilian hematology results. The inherent variability in currently available methods creates uncertainty in resulting data and highlights the need of a gold standard for reptilian WBC quantification.
Highlights
Monitoring health is critical to the conservation of any wild species, as the viability of a population is inseparable from its health status [1]
This study addressed the following specific objectives using blood samples from free-living eastern box turtles (Terrapene carolina carolina): [1] Determine level of agreement between the estimates from blood films (EST) and the Leukopet kit (LO) methods, [2] Determine repeatability and reproducibility of the LO method, and [3] Assess whether biological drivers of leukocyte counts are similar between hematologic methods
This study assessed analytical variability in the Avian LeukopetTM, compared leukocyte values resulting from two commonlyused quantification methods, and investigated whether methodological differences affected conclusions about biological variation and the health of free-living eastern box turtles
Summary
Monitoring health is critical to the conservation of any wild species, as the viability of a population is inseparable from its health status [1]. Baseline health data including clinical pathology values (e.g., hematology, plasma biochemistries, protein electrophoresis), pathogen prevalence, and contaminant concentrations are infrequently available for free-living animals. This lack of information can complicate the design and interpretation of comprehensive health studies. Establishing baseline clinical pathology data facilitates monitoring for trends in overall health status and can inform more effective conservation management strategies [2]. Manual leukocyte counting methods used in reptiles include total white blood cell (WBC) estimates from blood films (EST), Natt and Herrick’s (NH) direct counts, and indirect leukocyte quantification using the Avian Leukopet kit (LO). The analytical variability of these methods has not yet been assessed in reptiles; limiting our understanding of the random error inherent in these approaches
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