Comparison of Total Leukocyte Quantification Methods in Wild Galapagos Tortoises (Chelonoidis nigra)
Reptile hematologic data are important for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis nigra), yet difficult to attain due to lack of automated cell counters and reported discrepancies between leukocyte quantification methods.1 Two manual leukocyte quantification methods commonly used in reptiles, the Natt-Herricks (NH)a and the Eopette (EO)b, were compared to the white blood cell (WBC) estimate from blood film evaluation.3 Total leukocyte counts were performed using these three methods on blood samples collected from 42 adult, free-living, female Galapagos tortoises. Additionally, total leukocyte counts and differentials were performed on blood films prepared both upon collection in the field and 18–23 hours later in the laboratory to investigate the significance of delay in sample processing. Passing-Bablok method comparison analyses revealed that the NH was in agreement with the WBC estimate (regression slope=0.925), while the EO was not (regression slope=2.65).2 The WBC estimates obtained from field and laboratory-prepared blood films were in agreement (regression slope=0.82), while differential results were not. Additionally, blood film quality and cell morphology were superior in field-prepared blood films. In conclusion, the NH method (which stains all blood cell types and relies on differentiation of morphologically similar cells) was superior to the EO method (which stains only granulocytes and relies on an accurate differential to calculate total leukocytes). Our study indicates that immediate sample processing in field studies and use of the NH leukocyte quantification method with a confirmatory WBC estimate provides the highest-quality reptile hematologic data.
a. Natt-Herricks-TIC® 1:200 plus (Bioanalytic GmbH, Waldmatten 10-13, Umkirch/Freiburg, Germany)
b. Eopette™ (Exotic Animal Solutions, Inc., Hueytown, AL)
The authors thank the University of California-Davis School of Veterinary Medicine Students in Advanced Training and Research Program and the UC Davis School of Veterinary Medicine Global Programs International Externship Travel Fund for financially supporting the primary author’s travel to the Galapagos Islands to complete this project. The authors also thank Jamie Palmar of the Saint Louis Zoo’s Institute for Conservation Medicine for logistical and material support; Freddy Villamar and Walter Ernest for assisting in the field; Bioanalytic GmbH for donating the Natt-Herricks-TIC® kits; and the National Science Foundation for funding the Galapagos Tortoise Movement Ecology Programme. The authors also thank Phillip Kass, BS, DVM, MPVM, MS, PhD for statistical assistance.
1. Arnold J. White blood cell count discrepancies in Atlantic loggerhead sea turtles: Natt-Herrick vs. eosinophil Unopette. Proc Assoc Zoo Vet Tech; 1994:5–22.
2. Bilić-Zulle L. Comparison of methods: passing and Bablok regression. Biochemia Medica. 2011;21(1):49–52.
3. Strik NI, Alleman AR, Harr KE. Circulating inflammatory cells. In: Jacobson ER, ed. Infectious Diseases and Pathology of Reptiles: Color Atlas and Text. Gainesville, FL: Taylor and Francis; 2007:167–218.