Circulating Microparticles in Snake Bite Patients with Microangiopathy.
Authors: Enjeti Anoop , Scorgie Fiona , Lincz Lisa , Brown Simon , Isbister Geoff , Seldon Michael 
Published: April 2020

Abstract
Aim: To investigate circulating microparticles (MP) in snake bite patients with microangiopathy
Methods: This study used samples from patients recruited to the Australian Snakebite Project (ASP) with bites from the elapid group of snakes (including brown snake, tiger snake and taipan envenoming). Severe venom induced consumption coagulopathy (VICC) was defined as patients with an international normalised ratio [INR] &gt; 3. Microangiopathy haemolytic anaemia (MAHA) was defined as the occurrence of thrombocytopaenia and red cell fragmentation in addition to VICC. Citrated plasma samples were collected from each patient at various time points over 0.5 to 6 days after the bite. All samples were processed and stored according to a national standardized protocol. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer concentrations were measured on a Behring Coagulation System (BCS; Dade Behring, Marburg, Germany). Full blood count was measured as per routine laboratory protocol and blood films were examined by an independent pathologist. Flow cytometry was done on BD FACS Canto flow cytometer (BD Biosciences,CA,USA) with combinations of CD41a-PE (Clone HIP8, BD Biosciences, CA, USA), CD62e-APC (Clone 68-5H11, BD Biosciences, CA, USA), CD45-PE-Cy5, (Clone HI30, BD Biosciences, CA, USA) and Glycophorin-APC( Clone HIR2, BD Biosciences, CA, USA) or appropriate isotype controls in a final volume of 500 μl of PBS. The results were analysed using BD software and appropriate statistical tools.
Results: A total of 14 envenomed patient-samples with MAHA were compared to 14 envenomed patient-samples without MAHA and 45 normal healthy controls. All envenomed patient-samples with envenoming had evidence for VICC. The results for circulating MP levels in the three groups are shown in the table below:
Samples (number) Hb G/L Platelet count (10^9/L) Platelet MP/μL (CD41a) Endothelial MP/μL (CD62e) Red cell MP/μL (glycophorin) Healthy controls (45) 144±9.3 265 ±85 1259±959 360±104 &lt;50 SB with MAHA (14) 85±16 35 ± 13 2815±963 &lt;50 883±326 SB without MAHA 136±15 141 ± 75 2447±1252 &lt;50 104±102
The red cell micropartciples in snake bite subjects with MAHA were significantly higher than those without MAHA (p=0.005) while there was no significant difference in platelet microparticle levels between the envenomed with and without MAHA (p=0.27). Interestingly, the endothelial microparticles were reduced in all snake bite samples compared to the controls. The time trend in envenomed samples with MAHA showed that marked elevation in red cell MP occurred prior to a major drop in platelet count.
Discussion: Increase in levels of red cell microparticles in envenomed patients with MAHA is consistent with haemolysis. The marked increase in red cell MP which occurs prior to a drop in platelet count needs to be evaluated further. If this is a consistent finding then measuring MP at presentation could be useful as a predictive marker for MAHA in patients with snake bites. Patients with MAHA may benefit from more intensive intervention such as using fresh frozen plasma. The lack of increase and actually a decrease in endothelial microparticles in all snake bite patient-samples was an unexpected finding which is being further investigated. This suggests an alternative mechanism to microangiopathy other than endothelial damage due to the venom toxin. Thus, circulating microparticles could potentially be used to guide treatment in patients with snake bites.