Eastern brown envenomation venom induced consumptive coagulopathy prospective study

This study was approved by the Animal Ethics Committee of the University of Queensland (Project ID: 2021/AE001005) and conducted between October X, 2022 and Feb X, 2023 and conducted at a single veterinary hospital (UQ Vets, School of Veterinary Science, University of Queensland, Gatton, Australia). Seventeen dogs were enrolled in the study. Written informed owner consent was obtained prior to inclusion of dogs in the study. Dogs were eligible for inclusion if they met the following inclusion criteria for diagnosis of eastern brown snake envenomation: a witnessed snake envenomation with presentation or photographic evidence allowing identification of a brown snake, and evidence of lower motor neuron signs and/or a coagulopathy; a positive urine or blood snake venom detection kit (SVDK) for brown snake antigen; laboratory evidence of a coagulopathy as demonstrated by a prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) and lower motor neuron signs in a dog or cat in a known brown snake locality in the months between September - March. Exclusion criteria included any dogs known to have previous coagulopathy, on any regular medication, <5kg bodyweight. Cases where there was suspicion of EBSE but only one of lower motor neuron signs or a coagulopathy, without a definitive diagnosis, were also excluded. Study design: On enrolment to the study, collection of informed client consent, and admission to hospital, a 20-gauge catheter (Terumo Medical Corporation, NJ, USA) was placed in the cephalic vein of each dog. At this initial sampling point (time point 0 hours (T0)), 10ml of blood was drawn from the catheter and transferred to a 1ml lithium heparin tube, a 0.5ml EDTA tube (Greiner Bio-One GmbH, Austria), a 0.5ml MAX-ACT tube (Helena Laboratories, TX, USA), four 1.8ml 3.2% sodium citrate tubes (Becton, Dickinson and Company, NJ, USA) and two capillary tubes, ensuring proper filling of collection tubes for correct ratios of anticoagulant to blood. Dogs were then treated at the discretion of the clinician. All dogs included at T0 (n = 17) comprised Part A of the study (Figure 1). Dogs that met additional inclusion criteria including admission to hospital for inpatient management, administration of antivenom, weighing over 15 kg, deemed haemodynamically stable and of appropriate temperament for placement of a sampling catheter were then also enrolled in Part B (n = 9) of the study. A 20-gauge catheter was placed into the lateral saphenous vein of these subjects, a guidewire was passed through this, the catheter was removed and replaced with an 8cm 18-gauge sampling line (Promanec, Casablanca city, Morocco) inserted over the guidewire (which was subsequently removed), and the sampling line was sutured into place. Dogs that became distressed during sampling catheter placement (n = 1), or in which sampling catheter placement failed due to haemorrhage from the placement site and haematoma formation (n = 2) were excluded from Part B, leaving six dogs included. Part B constituted a longitudinal analysis in which repeat venous blood samples as described above for T0 were obtained at 8 (T8), 16 (T16), and 24 (T24) hours following admission and antivenom administration. Antivenom administered to all cases contained no less than 4050 units of brown snake antivenom (Tiger/multi-brown snake antivenom, Summerland Serums, Australia). Blood sample analysis: For each sample at each time point, following collection of blood into the MAX-ACT tube, a timer was set, the tube was gently mixed in a 37°C water bath for 30 seconds, then checked every 5 seconds for clot formation by tipping the tube into a horizontal position to observe for movement of the magnet. The time at which no magnet movement, and thus the endpoint of clot formation was attained, was recorded in seconds. If no clot formation was observed by 240 seconds, the sample was reported as having no clot formation. The reported normal range for clot formation in dogs is 55 – 80 seconds (See et al., 2009). The capillary tubes (Jorvet, CO, USA) were sealed with sealing wax and manual packed cell volume (%) and total protein (g/L) were performed following centrifugation. The samples collected into EDTA tubes were sent to the local veterinary laboratory service (Veterinary Laboratory Services, Gatton Campus, University of Queensland) for automated (Sysmex XN-1000 Hematology Analyzer, Sysmex Corporation, Kobe, Japan)and manual platelet count within 24 hours of collection, and were refrigerated if collected after 5pm until delivery to the laboratory the following morning. The 3.2% citrate tube had 0.2 mL removed to perform the in-house PT and aPTT using an IDEXX Coag Dx Analyzer (IDEXX, IDEXX Laboratories, Westbrook, Maine). The citrated samples were kept at room temperature (20 – 25°C) until viscoelastic testing was performed. The ClotPro® (Heamonetics Corporation, Boston MA, USA) is a newer generation point-of-care thromboelastometry device, utilised for viscoelastometry measurement in this study. Using the citrated blood, the FIB-test, EX-test and IN-test were run in parallel between 30 to 60 minutes after sample collection. All tests were performed according to the standard test protocol outlined in the ClotPro User Manual7. Briefly, following entry of the patient details into the ClotPro electronic system, each cup and pin were loaded into the test positions on the 37°C prewarmed instrument bench. The appropriate test tip was loaded onto the electronic pipette, and 340 uL of citrated blood was transferred from the collection tube into the cup. To ensure adequate mixing, the blood was then redrawn into the pipette and replaced back into the test cup. The pin was then connected to the cup to initiate the test. Each test was run for 60 minutes, and parameters were automatically measured and graphically displayed by the ClotPro device (see Table 2 for description of parameters measured). Institution specific reference intervals for this machine were predetermined (REFERENCE US). The remaining 3.2% citrate tubes were then centrifuged at 2500 g for 15 minutes, the supernatant was pipetted into a 15ml centrifuge tube (Eisco labs, NY, USA), this tube was then centrifuged for a subsequent 15minutes at 2500 g and then the supernatant was pipetted into 1.5ml Eppendorf tubes (Eppendorf South Pacific Pty. Ltd., NSW, AUS) into aliquots of 0.5 – 1.5ml each. These platelet poor plasma samples were then stored at -80°C for later batch analysis. Batch analysis of PT, aPTT, fibrinogen and serum venom levels occurred within 7 months of collection. STA R Max Stago coagulation test methods: Frozen samples were thawed at 37°C for 6 minutes. All samples for PT, aPTT and fibrinogen were analysed in triplicate using the Stago STA R Max (Stago, Asnières sur Seine, France). To measure PT, 50uL of sample, 50uL of kaolin and phospholipid reagent (Stago, Asnières sur Seine, France) were combined and incubated at 37°C for 240 seconds before 50uL of 0.025M CaCl2 (Stago, Asnières sur Seine, France) was then added and the test run. The reportable range for PT was 3 – 120 seconds. To measure aPTT, 50uL of sample was incubated at 37°C for 240 seconds before 100uL of Neoplastine (Stago, Asnières sur Seine, France) was then added and the test run. The reportable range for aPTT was 10 – 140 seconds. To determine fibrinogen concentration, a modified function Clauss assay was performed by combining 150uL of sample diluted 1:20 in Owren-Koller buffer (Stago, Asnières sur Seine, France), incubating at 37°C for 240 seconds, adding 50uL of thrombin (Stago, Asnières sur Seine, France) and allowing the test to run. The reportable range for fibrinogen was 0.4 – 12 g/L. A reference standard for canine fibrinogen was prepared from a normal dog citrated plasma and calibrated against a standard from a commercial diagnostic veterinary laboratory (Gribbles Veterinary Pathology, Clayton, Australia). The normal reference ranges for these coagulation parameters were determined by assaying citrated plasma samples collected from normal canines (n 1⁄4 4) and felines (n 1⁄4 2), frozen within 1 h and subsequently thawed in a water bath at 37 C (Table 1). A calibration curve was prepared as per manufacturer instructions with fibrinogen concentrations expressed in g/L. The reportable range for fibrinogen was 0.4–12 g/L. Brown Snake Venom Antigen ELISA Method A simultaneous sandwich ELISA format was used (Padula & Leister, 2017). Individual clinical samples (serum & plasma) were initially diluted 50% in ELISA buffer consisting of PBS-T20 + 0.1% BSA + 1 % normal dog serum before assaying. 96 well high binding microplates (Greiner) were coated with 6 µg/mL rabbit anti-BSV-IgG (specific for Pseudonaja textilis) in carbonate buffer pH 9.6, sealed and incubated for 24 hours at 2-8°C. Immediately prior to assay, plates were washed three times with PBS-T20. A standard curve was run in duplicate on every plate consisting of P. textilis venom (100, 50, 25, 12.5, 6.3, 3.1, 1.6, & 0.8 ng/mL) in ELISA buffer; 100 µL of test samples and calibrator were doubly diluted eight-fold on each plate. Blank wells were included on every plate containing ELISA buffer only. To each well, 100 µL of rabbit anti-BSV-IgG-peroxidase (1:800) in ELISA buffer was also added. Plates were incubated at 37°C with 600 rpm shaking for 30 minutes before washing as above. To visualise the bound enzyme activity 100 µL of TMB was added to each well, and plates incubated at RT for 15-minutes at which time the reaction was stopped by 100 µL of 10% H2S04. Colour intensity was read at 450nm (Tecan Sunrise, Austria) with a reference wavelength of 620nm. Computer software (Tecan Magellan v7.3, Tecan, Australia) was used to subtract blank well absorbances, a linear regression line was fitted (log transformed x and y data) to the standard curve absorbances, and unknown values interpolated. Curve fit was excellent for all plates (r>0.998) and standard curve replicates had CVs less than 10%. The lower limit of P. textilis venom antigen quantification for all runs was 0.78 ng/mL (3 x SD higher than ELISA buffer blank). Because clinical samples were initially diluted 50% in ELISA buffer, the lower limit of quantification in undiluted samples (neat serum/plasma) was therefore 1.56 ng/mL. Data management: Individual patient characteristics, clinical signs reported at presentation and during hospitalisation, and outcome data was recorded and collated with all laboratory data in Microsoft Excel and was stored on the University of Queensland Research Data Manager.