Pilot study for the standardization of assays and the analysis of microvesicles in samples from individuals diagnosed with Bothrops atrox envenomations treated at a reference center in the Brazilian Amazon

Introduction: Bothrops envenomations are common in the Brazilian Amazon and result in local and systemic complications. In response to the envenomation, immune cells release vesicular structures derived from the cell membrane, which are known as microvesicles (MVs). The MVs are present in biological fluids and may be associated with the severity of pathological processes; however, the role of MVs during Bothrops envenomations is poorly explored and may contribute to the formulation of protocols of adjuvant treatments during serotherapy. Methods: A pilot study for the standardization of assays and the analysis of microvesicles in samples from individuals diagnosed with Bothrops atrox (B. atrox) envenomations was conducted at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD) in the Brazilian Amazon. The assay was based on the MV immunophenotyping protocol of the Integrated Biomarker Research Group (René Rachou Institute - FIOCRUZ Minas). Samples of one patient who had suffered a B. atrox snakebite were used and were evaluated before antivenom administration, at 24 hours and at 48 hours afterwards, with the inclusion of the one participant as a healthy control. The samples were collected in a tube with sodium citrate anticoagulant and then centrifuged at 1,500×g/5 min to obtain platelet-free plasma (PFP). Subsequently, the PFP was diluted in a citrate buffer solution containing heparin, centrifuged for 1,500 xg/90 min, and the MV-rich sediment was resuspended in Annexin-V buffer. The suspension containing the MVs was transferred to tubes, which were incubated at room temperature (20 ºC to 25 ºC) for 30 min in the presence of the following fluorescent-labeled specific anti-human cell surface monoclonal antibodies: Annexin-V (MVs), CD235a (erythrocytes), CD41a (platelets), CD51 (endothelial cells), CD45 (leukocytes), CD66b (neutrophils), CD14 (monocytes), CD3 (T-lymphocytes) and CD19 (B-lymphocytes). After 30 min of incubation, Annexin-V buffer was added, and the sample acquisition was performed on the CytoFLEX flow cytometer. In addition, the evaluation of the MV size ranges was carried out in the FlowJo software (v10) using calibration microspheres (Megamix). Results: We observed the presence of all the MVs derived from the cells in the panel, with an increase in MV levels in relation to the control. Before treatment, the size profile in the MV population varied between 100 nm and 240 nm, which in the literature is associated to MV with a greater capacity for inflammatory activity. Between 24 h and 48 h after envenomation administration, the size profile of the MV population was between 300 nm and 900 nm, which is associated with the regulation of inflammatory processes. Conclusion: The findings demonstrate that, before treatment, patients present an increase in the inflammatory response, with resolution after treatment. Furthermore, the data is in line with the studies previously published by our group. As such, the standardization and execution of this technique can help to elucidate the mechanisms associated with the development of an inflammatory response after envenomation, as well as the prediction of the severity of B. atrox snakebites.