Carvalho Marcelo P N, Queiroz-Hazarbassanov Nicolle G T, Massoco Cristina O, Rossi Silmara, Sant'Anna Sávio S, Catão-Dias José L, Grego Kathleen F
Laboratory of Wildlife Comparative Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
Laboratory of Applied Pharmacology and Toxicology, Department of Pathology, School of Veterinary Medicine and Animal Science, and , University of São Paulo, São Paulo, Brazil.
Vet Clin Pathol. 2016 Jun;45(2):271-80. doi: 10.1111/vcp.12358. Epub 2016 May 23.
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.
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).
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.
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.
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.
爬行动物的免疫系统由先天性、体液性和细胞介导的机制组成，涉及不同类型的血液白细胞。需要开发优化的方法来深入研究爬行动物血液白细胞的起源和功能。
本研究的目的是优化从蛇外周血样本中分离白细胞的密度梯度分离方案，并通过流式细胞术根据大小和内部复杂性对回收的细胞进行表征，以对一条蟒蛇（红尾蚺）和两种蝰蛇（巴西矛头蝮、三色矛头蝮）的白细胞群体进行定性和半定量评估。
将来自30条蛇（每种10条，5雄5雌）的血样收集到含有肝素钠的试管中。新鲜血液用ficoll-paque PLUS或Percoll密度梯度离心以分离白细胞。基于大小（前向散射[FSC]）和内部复杂性（侧向散射[SSC]）定义流式细胞术白细胞门。将每种蛇的一条蛇中在这些门中分选细胞后的相对白细胞分类计数与未分选的分离白细胞的传统光学显微镜分类计数进行比较。
通过ficoll或Percoll分离的样本之间，包括嗜中性粒细胞、嗜天青粒细胞以及小淋巴细胞和大淋巴细胞在内的相对白细胞群体没有统计学差异。根据它们在FSC/SSC细胞图中的位置确定了四个白细胞门。单只动物分选后的相对白细胞分类计数与未分选细胞的光学显微镜分类计数有一定的一致性。
基于FSC和SSC，在从蟒蛇和蝰蛇外周血中通过ficoll或Percoll密度梯度分离的白细胞中发现了4个不同的白细胞群体。该技术的进一步优化应能进行功能测定。
