title: Flow cytometric characterization of peripheral blood leukocyte populations of 3 neotropical snake species: Boa constrictor, Bothrops jararaca, and Crotalus durissus.
abstract: BACKGROUND
  The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed.
  
  
  OBJECTIVE
  The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus).
  
  
  METHODS
  Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes.
  
  
  RESULTS
  There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells.
  
  
  CONCLUSIONS
  Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays.
authors.affiliation: Laboratory of Wildlife Comparative Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
authors.forename: Marcelo P N
authors.initials: MP
authors.lastname: Carvalho
authors.affiliation: Laboratory of Applied Pharmacology and Toxicology, Department of Pathology, School of Veterinary Medicine and Animal Science, and , University of São Paulo, São Paulo, Brazil.
authors.forename: Nicolle G T
authors.initials: NG
authors.lastname: Queiroz-Hazarbassanov
authors.affiliation: Laboratory of Applied Pharmacology and Toxicology, Department of Pathology, School of Veterinary Medicine and Animal Science, and , University of São Paulo, São Paulo, Brazil.
authors.forename: Cristina O
authors.initials: CO
authors.lastname: Massoco
authors.affiliation: Luiz de Queiroz College of Agriculture and Center of Nuclear Energy in Agriculture, University of São Paulo, São Paulo, Brazil.
authors.forename: Silmara
authors.initials: S
authors.lastname: Rossi
authors.affiliation: Laboratory of Wildlife Comparative Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.|Laboratory of Herpetology, Butantan Institute, São Paulo, Brazil.
authors.forename: Sávio S
authors.initials: SS
authors.lastname: Sant'Anna
authors.affiliation: Laboratory of Wildlife Comparative Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
authors.forename: José L
authors.initials: JL
authors.lastname: Catão-Dias
authors.affiliation: Laboratory of Herpetology, Butantan Institute, São Paulo, Brazil.
authors.forename: Kathleen F
authors.initials: KF
authors.lastname: Grego
mesh_terms: D000818:Animals; D017821:Boidae / Q000097:blood*; D017837:Bothrops / Q000097:blood*; D017839:Crotalus / Q000097:blood*; D005260:Female; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D007962:Leukocytes; D008297:Male
keywords: Hematology; ficoll; forward scatter, side scatter; immune system; percoll