Eastern brown envenomation venom induced consumptive coagulopathy prospective study

Subjects: 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.本研究经昆士兰大学动物伦理委员会（Animal Ethics Committee）批准（项目编号：2021/AE001005），于2022年10月X日至2023年2月X日在澳大利亚昆士兰大学兽医学院加顿分校的UQ兽医医院（UQ Vets, School of Veterinary Science, University of Queensland, Gatton, Australia）开展。本研究共纳入17只犬，所有犬只入组前均获得犬主书面知情同意。 犬只需满足以下东部棕蛇咬伤中毒（eastern brown snake envenomation, EBSE）诊断纳入标准方可入组：①有目击蛇咬伤史，且就诊时有可识别棕蛇的临床表现或影像证据，同时存在下运动神经元体征和/或凝血功能异常；②蛇毒检测试剂盒（snake venom detection kit, SVDK）检测棕蛇抗原呈阳性；③在已知有棕蛇分布的区域，且于每年9月至3月期间就诊的犬或猫，实验室检查证实存在凝血功能异常（表现为凝血酶原时间（prothrombin time, PT）及活化部分凝血活酶时间（activated partial thromboplastin time, aPTT）延长，同时伴有下运动神经元体征）。 排除标准包括：已知存在既往凝血功能异常、定期服用任何药物、体重<5kg的犬只；仅存在下运动神经元体征或凝血功能异常其中一项，且未获得明确诊断的疑似东部棕蛇咬伤中毒病例也予以排除。 研究设计：本研究入组时，在获得犬主知情同意并将犬只收治入院后，为每只犬在前肢头静脉留置20号导管（Terumo Medical Corporation, NJ, USA）。在初始采样时间点（0小时，T0），通过导管采集10ml血液，分别转移至1ml肝素锂采血管、0.5ml EDTA采血管（Greiner Bio-One GmbH, Austria）、0.5ml MAX-ACT采血管（Helena Laboratories, TX, USA）、4支1.8ml 3.2%柠檬酸钠采血管（Becton, Dickinson and Company, NJ, USA）及2支毛细管采血管，确保采血管充盈至合适比例以保证抗凝剂与血液的配比准确。随后由临床医师自主决定治疗方案。T0时间点纳入的所有犬只（n=17）构成研究A组（图1）。 满足以下额外纳入标准的犬只将进一步纳入研究B组：收治入院接受住院管理、接受抗蛇毒血清治疗、体重>15kg、血流动力学稳定且性情适宜采样导管留置，共纳入9只犬。为该组犬只在隐静脉留置20号导管，经导管置入导丝，移除导管后沿导丝置入8cm长的18号采样管路（Promanec, Casablanca city, Morocco），随后移除导丝，并将采样管路缝合固定。在采样导管置管过程中出现烦躁不安的犬只（n=1），或因置管部位出血形成血肿导致置管失败的犬只（n=2）被排除出B组，最终B组纳入6只犬。 B组为纵向研究队列，在入院接受抗蛇毒血清治疗后8小时（T8）、16小时（T16）及24小时（T24）重复采集上述T0阶段描述的静脉血样。所有病例使用的抗蛇毒血清均含有不少于4050单位棕蛇抗蛇毒血清（虎蛇/多棕蛇抗蛇毒血清，Summerland Serums, Australia）。 血液样本分析：针对每个时间点的样本，在将血液采集至MAX-ACT采血管后立即启动计时，将采血管置于37℃水浴中轻柔混匀30秒，随后每5秒将采血管倾斜至水平位置，通过观察磁体移动情况判断是否形成血凝块，记录磁体停止移动（即血凝形成终点）的时间（秒）。若240秒内未观察到血凝形成，则报告为无血凝形成。犬血凝形成的正常参考范围为55~80秒（See等，2009）。 毛细管采血管（Jorvet, CO, USA）用封蜡密封，离心后进行手工红细胞压积（%）及总蛋白（g/L）检测。采集至EDTA采血管的样本于采集后24小时内送至当地兽医实验室服务中心（昆士兰大学加顿校区兽医实验室服务中心）进行自动化血细胞分析（Sysmex XN-1000血液分析仪，Sysmex Corporation, Kobe, Japan）及手工血小板计数；若采集时间晚于下午5点，则将样本冷藏至次日送检。 从3.2%柠檬酸钠采血管中取出0.2ml样本，使用IDEXX Coag Dx分析仪（IDEXX Laboratories, Westbrook, Maine, USA）进行室内PT及aPTT检测。柠檬酸钠抗凝血样本在进行粘弹性检测前置于室温（20~25℃）保存。ClotPro®（Heamonetics Corporation, Boston MA, USA）为新一代床旁血栓弹力图设备，本研究使用其进行粘弹性检测。使用柠檬酸钠抗凝血样本，在采集后30~60分钟内并行开展FIB-检测、EX-检测及IN-检测，所有检测均严格遵循ClotPro用户手册7中规定的标准检测流程。简要操作如下：将患者信息录入ClotPro电子系统后，将每个检测杯和针置于37℃预热的仪器台检测位上。将适配的检测吸头安装至电子移液器，从采血管中吸取340μl柠檬酸钠抗凝血至检测杯中。为确保充分混匀，将血液重新吸入移液器后再回注至检测杯。随后将针与检测杯连接以启动检测。每项检测运行60分钟，检测参数由ClotPro设备自动测量并以图形化方式展示（检测参数说明见表2）。本研究预先确定了该设备的机构特异性参考区间（REFERENCE US）。 将剩余的3.2%柠檬酸钠采血管以2500g离心15分钟，吸取上清液至15ml离心管（Eisco labs, NY, USA），再次以2500g离心15分钟后，将上清液吸取至1.5ml Eppendorf管（Eppendorf South Pacific Pty. Ltd., NSW, AUS）中，分装为0.5~1.5ml每管。这些乏血小板血浆样本随后保存于-80℃以备后续批量检测。在样本采集后7个月内完成PT、aPTT、纤维蛋白原及血清蛇毒水平的批量检测。 STA R Max Stago凝血检测方法：将冻存样本于37℃解冻6分钟。所有PT、aPTT及纤维蛋白原样本均使用Stago STA R Max（Stago, Asnières sur Seine, France）进行三次重复检测。PT检测：取50μl样本、50μl高岭素磷脂试剂（Stago, Asnières sur Seine, France）混合后于37℃孵育240秒，随后加入50μl 0.025M氯化钙溶液（Stago, Asnières sur Seine, France）并启动检测。PT的可报告范围为3~120秒。aPTT检测：取50μl样本于37℃孵育240秒，随后加入100μl Neoplastine试剂（Stago, Asnières sur Seine, France）并启动检测。aPTT的可报告范围为10~140秒。纤维蛋白原浓度检测：采用改良Clauss功能法，取150μl以Owren-Koller缓冲液按1:20稀释的样本，于37℃孵育240秒，加入50μl凝血酶（Stago, Asnières sur Seine, France）后启动检测。纤维蛋白原的可报告范围为0.4~12g/L。 犬纤维蛋白原参考标准品由正常犬柠檬酸钠抗凝血血浆制备，并与商业兽医诊断实验室（Gribbles Veterinary Pathology, Clayton, Australia）的标准品进行校准。通过对正常犬（n=4）和猫（n=2）采集的柠檬酸钠抗凝血血浆样本进行检测，确定上述凝血参数的正常参考区间，样本采集后1小时内冻存，随后于37℃水浴解冻（表1）。按照制造商说明书绘制标准曲线，纤维蛋白原浓度以g/L表示。 棕蛇毒抗原ELISA检测方法：采用双抗体夹心ELISA法（Padula & Leister, 2017）。临床样本（血清及血浆）初始用ELISA缓冲液（含PBS-T20 + 0.1% BSA + 1%正常犬血清）按50%比例稀释后进行检测。将96孔高结合力酶标板（Greiner）用6μg/ml兔抗棕蛇毒IgG（特异性针对澳洲东部棕蛇（Pseudonaja textilis））包被，包被液为pH9.6的碳酸盐缓冲液，密封后于2~8℃孵育24小时。检测前即刻用PBS-T20洗涤酶标板3次。每块板均设置复孔标准曲线，标准品为澳洲东部棕蛇（P. textilis）毒液（浓度梯度为100、50、25、12.5、6.3、3.1、1.6及0.8ng/mL），以ELISA缓冲液配制；每孔加入100μl待测样本及校准品，每块板上进行八倍倍比稀释。每块板均设置仅含ELISA缓冲液的空白对照孔。向每孔加入100μl 1:800稀释的兔抗棕蛇毒IgG-过氧化物酶结合物（ELISA缓冲液配制）。将酶标板置于37℃、600rpm摇床孵育30分钟，随后按前述方法洗涤。每孔加入100μl四甲基联苯胺（TMB）底物溶液以显色，室温孵育15分钟后，加入100μl 10% H2SO4终止反应。使用酶标仪（Tecan Sunrise, Austria）在450nm波长下读取吸光度，参比波长为620nm。使用计算机软件（Tecan Magellan v7.3, Tecan, Australia）扣除空白孔吸光度，对标准曲线吸光度值进行对数转换后拟合线性回归曲线，进而推算待测样本浓度。所有酶标板的曲线拟合效果极佳（r>0.998），标准品复孔的变异系数均小于10%。本检测方法对澳洲东部棕蛇毒抗原的定量下限为0.78ng/mL（较ELISA缓冲液空白孔吸光度高3倍标准差）。由于临床样本初始稀释比例为50%，因此未经稀释的样本（纯血清/血浆）中蛇毒抗原的定量下限为1.56ng/mL。 数据管理：将所有患者个体特征、就诊时及住院期间记录的临床症状、转归数据与所有实验室数据整理并录入Microsoft Excel，存储于昆士兰大学研究数据管理系统中。