Schistocyte quantitation, thrombotic microangiopathy and acute kidney injury in Australian snakebite coagulopathy [ASP28]

Abstract Introduction The major systemic manifestation of hemotoxicity in human snakebite envenoming is venom‐induced consumption coagulopathy (VICC). A subset of patients with VICC develop thrombotic microangiopathy (TMA), in which acute kidney injury (AKI) occurs. We aimed to investigate the association between schistocytosis in snakebite patients with VICC and AKI, compared to non‐envenomed patients. Methods Serial blood films collected from a prospective cohort of snakebite patients (Australian Snakebite Project) were examined. Cases were classified a priori as non‐envenomed snakebites (normal controls), envenomed without VICC, partial VICC without AKI, complete VICC without AKI, and VICC with AKI based on defined clinical and laboratory criteria. The percentage of schistocytes between groups was compared and correlated by Kendall's tau b test. Results Seven hundred and eighty blood films from 234 snakebite cases were analyzed. There was a statistically significant correlation (τ = .69, SE .03, P < .001) for schistocytosis between the ordered groups of non‐envenomed snakebites, envenomed without VICC, partial VICC without AKI, complete VICC without AKI, and VICC with AKI groups. Patients with VICC and AKI had a platelet nadir median of 42 × 109/L (interquartile range [IQR] :25‐130 × 109/L), hemoglobin nadir of median 107 g/L (IQR 66‐122 g/L), and maximum LDH median of 1128 U/L (IQR 474‐3255 U/L). A 1.0% threshold for schistocytosis yielded 90% sensitivity (95% CI: 67%‐98%) and 71% specificity (95% CI: 62%‐79%) for predicting AKI in patients with VICC. Conclusion Schistocyte quantitation has good diagnostic utility in snakebite patients with VICC. A definition of snakebite TMA as MAHA with ≥1.0% schistocytes and thrombocytopenia, would appear to be appropriate.

presenting with schistocytes on the peripheral blood film. 7 Multiple small observational pathological studies of snakebite and AKI have reported snakebite cases with schistocytes on the peripheral blood film, together with histological findings of TMA in the kidney. 6,7,11,12 TMAs more broadly are a diverse range of disorders defined by clinicopathological criteria, including blood film examination for red cell schistocytes, which are the red cell morphological hallmark of TMA. Schistocytes form due to mechanical damage to red blood cells, and they are defined morphologically by sharp rectilinear fracture or tear lines and sharp angles. The occurrence of schistocytosis has been best studied in well elucidated TMA disorders including thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). A schistocytosis threshold of 1% has been cited as a robust cut-off for the presence of TMA on a blood film, and in TTP and HUS it is often much higher than this. 13,14 Normal blood films show only very rare schistocytes, typically less than 0.2%-0.5%. 13,15,16 There are no comprehensive studies examining blood film findings in snakebite VICC or TMA, nor a standardized or consistent definition of snakebite-associated TMA. Laboratory features of snakebite-associated TMA including blood film schistocytes have to date been reported in mostly case reports, small case series and occasional larger observational studies. 5,6,[17][18][19][20] None have systematically examined blood film findings in a quantitative or comparative way between clinical toxin syndromes of snakebite. Confounding this, the scientific literature both VICC and TMA in snakebite have historically been described by different and sometimes unclear or erroneous nomenclature, including defibrination syndrome, intravascular hemolysis, or disseminated intravascular coagulation (DIC); variably in association with AKI. 17,21,22 VICC and snakebite associated TMA are distinctly different from DIC with respect to etiology, presenting features, and outcomes. DIC is an acquired thrombo-hemorrhagic syndrome associated with widespread activation of the tissue factor/factor VIIa pathway. 23,24 It is strongly associated with the systemic inflammatory response syndrome in sepsis, severe trauma, burns, and obstetric calamities. Compared to the hemorrhagic risk in VICC, and the renal end-organ injury of snakebite-associated TMA, DIC is marked by multiorgan dysfunction syndrome, which confers a high mortality rate. 17 Much of the existing literature referencing snakebite and "defibrination syndrome," "intravascular hemolysis," or DIC is difficult to interpret given the ill-defined nomenclature typically used. 17 We report a prospective cohort of snakebites from the Australian Snakebite Project (ASP), with quantitation of schistocytes on serial blood films using the International Council for Standardization in Haematology (ICSH) method. Our primary aim was to investigate the association between blood film schistocyte counts in snakebite patients, and VICC with and without AKI, compared to non-envenomed patients. In addition, we aimed to determine a schistocyte cut-off suggestive of TMA in snakebite patients.

| MATERIAL S AND ME THODS
The ASP is a multicenter prospective cohort study, enrolling patients with suspected or definite snakebites at time of presenta- Serial peripheral blood films collected from ASP cases from 2005 to 2014 at initial presentation, 2 hours, 6 hours, and then 24 hourly, were examined and a quantitation of red cell schistocytes was performed. Blood films were prepared from EDTA blood samples and spread by the attending hospital and then forwarded to the research center. Films were stained via an automated slide stainer using routine Romanowsky method. Blood films were examined in random order blinded to patient identity and diagnosis.
For each blood film, 1000 erythrocytes were counted and examined at high power by oil immersion (100× objective, magnification ×1000). All blood films were examined by the first author (TN).
A total schistocyte percentage was calculated as per the ICSH method. 28 Quality control was ensured by exclusion criteria for rejection of a slide if poor film quality was present, defined by either insufficient working area due to an improperly or poorly spread film such that 1000 cells could not be counted in the working area of the slide; unacceptable morphology within working area (for example presence of artefact such as crenation of cells, staining or water artefact from poor fixation); or the presence of significant global red cell poikilocytosis (for example thalassemia major, other haemoglobinopathies, significant dysplasia) making counting of schistocytes difficult or invalid.
Schistocyte quantitation was presented for the different clinical toxin syndromes by Tukey box plots for median, interquartile range (IQR), 95% percentiles and outliers. Statistical analysis for correlation and comparison between ordered groups (non-envenomed, envenomed with no VICC, partial VICC with no AKI, complete VICC with no AKI, and VICC with AKI, including AKI KDIGO 1, 2 and 3 ordinal groups) was performed using Kendall's tau b test for nonparametric data. Sensitivity and specificity were calculated for VICC with AKI vs VICC with no AKI (negative controls) at the ICSH recommended cut-off of 1.0% for significant schistocytosis. A P value of <.05 was set for significance. Receiver Operating Characteristic (ROC) curves were plotted as follows. Sensitivity and specificity of the results using quantitations for schistocytes using the ICSH method were performed for a range of different cut-off thresholds including around the previously reported cut-off of 1.

| RE SULTS
Seven-hundred-and-eighty peripheral blood films were examined for 234 snakebite patients in total. Forty-one blood films were excluded from the analysis due to poor quality or inadequate patient identifiers.
This left 739 blood films for analysis, which included 102 blood films from 19 cases with VICC (partial or complete) and AKI; 304 blood films from 73 cases with complete VICC but no AKI; 98 from 31 cases with partial VICC; 82 from 34 cases with envenoming but no VICC or TMA and 153 films from 77 non-envenomed cases. (Figure 1).

| D ISCUSS I ON
We found evidence that a schistocyte count ≥1.0% using the ICSH method is sensitive and specific for differentiating VICC with AKI vs VICC alone. Together with our finding of no AKI in the nonenvenomed and envenomed non-VICC groups, this supports a hypothesis that TMA is a predominant etiology of AKI in snakebite.
This finding is particularly true for Australian elapid envenoming, in which AKI has only rarely been associated with severe myotoxicity and rhabdomyolysis, such as seen with tiger snake (Notechis scutatus). 25 For patients with snakebite VICC we propose schistocytosis ≥1.0% with anemia and thrombocytopenia as diagnostic criteria for TMA in snakebite. This recommendation is consistent with an existing international consensus on schistocyte quantitation threshold for TMA, and with the standardization of terminology for TMA more broadly. 28,29 If this definition is applied, a small number of outliers will occur, with MAHA and no thrombocytopenia, or thrombocytopenia without anemia, whom are at a much lesser risk of AKI. Similar outliers have been reported in other TMA disorders, such as hemolytic uremic syndrome. 30

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.