Analytical Variability and Agreement of Leukocyte Quantification Methods in Inland Bearded Dragons (Pogona vitticeps)

Abstract

Hematology is a common diagnostic assay for evaluation of health in veterinary species. Several manual methods exist to evaluate the leukocyte component of reptile complete blood counts; however, there is no single method that has been proven reliable for all reptiles. To investigate the analytical variability and agreement of commonly used leukocyte quantification methods, blood was collected from ten clinically healthy bearded dragons (Pogona vitticeps) and analyzed in triplicate using Natt-Herricks (NH), eosinophilic leukopet (LO), and estimated white blood cell counts on peripheral blood smears made from fresh whole blood or blood anticoagulated with lithium heparin (LH). To investigate inter-observer variability, blood film white blood cell estimates were generated by both a novice veterinary student and a boarded clinical pathologist and analyzed for agreement. Analytical variability was highest using the LH blood film estimate by a boarded clinical pathologist (21.0%), followed by Natt-Herricks method (student) (20.8%), fresh whole blood film estimate from the clinical pathologist (16.6%), eosinophilic leukopet (student) (8.7%), and blood film estimates using fresh whole blood (6.5%) and LH blood (4.5%) read by the student. Bland Altman plots revealed there was agreement between LO and NH methods and between the two blood film estimate methods, and Passing-Bablok regression analysis revealed constant and proportional error between LO and both estimate methods and between NH and both estimate methods. There was agreement between the veterinary student and clinical pathologist for both blood smear leukocyte estimates. These results highlight the importance of considering which leukocyte quantification method is used when clinically assessing reptile patients.