This retrospective observational study included adults aged more than 18 years with venomous snakebite who were consecutively treated for over a 51-month period (from 2011 May to 2015 July) from the time when the DNI checks were implemented. In this study, the emergency department (ED) was located in a single urban, tertiary-care hospital, which has an annual visit volume of over 45,000 and is staffed 24 hours per day by board-certified emergency physicians.

All patients with the word “snake,” “venomous,” or “envenoming/envenomation” in the ED discharge codes registered in computerized hospital records were initially considered. The diagnostic criteria for venomous snakebite included one or more of the following: 1) a confirmed two-fang wound, 2) a bite from a snake with a triangular-shaped head, 3) venomous snakebite confirmation based on the patient’s experience, and 4) presence of abnormal physical and laboratory findings, such as shock, tachypnea, bleeding, thrombocytopenia, DIC, or diplopia, despite other insufficient findings [14]. Antivenin was administered when the patients had systemic signs, including collapse, convulsions, weakness, paralysis, respiratory failure, shock, or bleeding, or severe local symptoms associated with snakebite. The dose was repeated if the symptoms persisted.

The exclusion criteria were as follows: 1) use of granulocyte colony-stimulating factors, glucocorticoids, or other immunosuppressants that could affect the DNI values; 2) presence of hematological abnormalities and evidence of wound infection, such as redness, localized heat sensation, or purulent discharge, and major trauma that may influence the DNI values; 3) use of anticoagulants and presence of liver cirrhosis, which could affect the coagulation profiles, such as prothrombin time (PT); 4) presentation more than 24 hours after snakebite; 5) transfer of patient from other hospitals after antivenin use, which may exert effects on the DNI that could not be excluded; and 6) transfer of patient to another hospital regardless of ED admission or not.

Patients were categorized as having DIC or none. The DIC group included patients with consumptive coagulopathy through activation of the entire clotting pathway, such as DIC. We used a diagnostic algorithm for DIC that was proposed by the International Society on Thrombosis and Haemostasis. The algorithm parameters include the platelet count (>100,000/μL=0, <100,000/μL=1, <50,000/μL=2); fibrin degradation product, such as d-dimer (no increase=0, moderate increase=2, strong increase=3); prolonged PT (<3 sec=0, >3 sec=1, >6 sec=2); and fibrinogen level (>1.0 g/dL=0, <1.0 g/dL=1). A score of 5 or higher is consistent with the presence of DIC, and this algorithm has been reported to have a sensitivity and specificity of 91% and 97%, respectively, for DIC diagnosis [15,16]. We reclassified patients with DIC into early DIC (DIC within 24 hours after snakebite) and late DIC (DIC beyond 24 hours after snakebite) groups.

The data were collected through a retrospective review of the patients’ electronic medical records by two emergency physicians who were blinded to the study objectives and hypothesis. The abstractors were blinded to the patient group categorization, which was accomplished by another emergency physician. Additionally, the abstractors were trained prior to data collection to reduce the possible bias from the data collection procedure. We used explicit case report forms. The chart abstractors and study coordinators periodically met to resolve any disputes and review coding rules. The study coordinators monitored the abstractors’ performance. Considering that the study was performed retrospectively and observationally, the patients’ records and information were anonymously processed prior to analysis. The following information was obtained from the medical records: age, sex, site of the bite, time from snakebite to ED arrival, underlying diseases, initial systolic blood pressure, severity as determined by a traditional snakebite severity grading scale (Table 1) [17], antivenin use, and initial symptoms and signs, including both local and systemic manifestations. Systemic complications, including hematological (anemia [18], thrombocytopenia, PT/partial thrombin time [PTT] prolongation, and bleeding), neurological (blurred vision, diplopia, and dizziness), and other complications (rhabdomyolysis, acute kidney injury [AKI] [19,20], and shock [systolic blood pressure <90 mmHg]), occurring during hospitalization were investigated.

Blood samples for the analyses of serum white blood cell (WBC) counts, C-reactive protein levels, DNI, platelet counts, and fibrinogen, PT, PTT, d-dimer, and antithrombin III levels were obtained through venipuncture within the first hour of ED admission. An automatic cell analyzer (ADVIA 120/2120, Siemens Healthcare Diagnostics) was used to measure the DNI. The technique is specific to the technology found in the ADVIA unit manufactured by Siemens Healthcare Diagnostics. This flow cytometry-based hematological analyzer utilizes two independent WBC analysis methods by using myeloperoxidase and lobularity/nuclear density channels. The DNI was calculated in leukocyte differentials using the following formula: DNI = (leukocyte subfraction assayed in the myeloperoxidase channel via cytochemical reaction) – (leukocyte subfraction counted in the nuclear lobularity channel via reflected light beam) [7,12,13].

The primary outcome of this study was the difference in the initial DNI measured in the ED in relation to the presence of DIC in patients with venomous snakebites. The secondary outcome was the predictive ability of the initial DNI on the presence of DIC upon presentation at the ED. This study was approved by the Institutional Review Board of our medical college (CR316051). The informed consent was waived due to retrospective design.

Categorical variables are presented as frequencies and percentages, whereas continuous variables are presented as means and standard deviations or medians and interquartile ranges. Chi-square or Fisher’s exact tests were used to compare categorical variables, whereas two-sample t-tests or Mann-Whitney U-tests were utilized to compare continuous variables. To compare three groups, we performed the chi-square test (Fisher exact test), analysis of variance, or Kruskal-Wallis test, as appropriate. We also analyzed multiple comparisons using Bonferroni correction, Sheffé’s method, and Dunn’s procedure, as appropriate. Normality was first assessed using the Shapiro-Wilk test. Correlation coefficients (r) were calculated via the Spearman’s method. Logistic regression analysis was used to identify the factors that predicted the presence of DIC. The results were expressed as odds ratio (OR) with 95% confidence intervals (CIs). The area under the receiver operating characteristic curve was utilized to evaluate the DNI’s predictive ability in classifying DIC. P-values less than 0.05 were considered statistically significant, and all statistical analyses were conducted using IBM SPSS Statistics ver. 23.0 (IBM Corp., Armonk, NY, USA).

A total of 170 patients with venomous snakebites were consecutively assessed during the study period. Among these patients, 56 were excluded based on the following criteria: ED arrival with wound infection (one patient), presentation beyond 24 hours following bite (20 patients), transferred from other hospital after antivenin use (20 patients), transferred to other hospital regardless of ED admission or not (six patients), discharged against medical advice (two patients), and insufficient data (seven patients). After exclusion, we included a total of 114 patients with venomous snakebites.

Of the 114 analyzed patients, 76 (66.7%) were men with a median age of 56 years (range, 18 to 93 years). The median time from snakebite to ED arrival was 3 hours. Hypertension (28 patients, 25.7%) was the most common underlying disease. Patients with bite severity grade of II (56 patients, 50.5%) were most common, and 81 patients (71.1%) were treated using intravenous antivenin. The common local symptoms at initial presentation included pain (107 patients, 99.1%) and edema (90 patients, 86.5%), whereas the common systemic symptom was nausea (seven patients, 6.3%). Among the hematological complications, anemia (42 patients, 36.8%) was the most common.

Thirty patients (26.3%) developed DIC. Of these patients, nine developed early DIC (DIC within 24 hours after snakebite), and 21 developed late DIC (DIC beyond 24 hours after snakebite). Tables 2 and 3 show the comparisons of baseline characteristics and inflammatory markers. Patients in the no DIC and DIC groups differed significantly in the proportion of males (57.1% vs. 93.3%, P<0.001). The DIC group had significantly longer time from snakebite to ED arrival than the no DIC group (5.0 vs. 2.0 hours, P=0.002). Furthermore, the DIC group had significantly higher final bite severity (P<0.001) and more frequent multidose antivenin administrations (P<0.001) than the no DIC group. All hematological complications, including anemia, thrombocytopenia, PT/PTT prolongation, and bleeding, were significantly more common in the DIC group than in the no DIC group. However, no significant differences in neurological complications were observed between the two groups. Patients with DIC had significantly higher rhabdomyolysis and AKI incidences than those with no DIC (P=0.033 and 0.019, respectively).

The DIC group had significantly higher median initial serum WBC and DNI values than the no DIC group (7,635 vs. 9,800, P=0.017; 0 vs. 0.2, P<0.001, respectively). The initial serum C-reactive protein levels did not differ significantly between groups. When the DIC group was divided into the early and late groups, the mean DNI value of the former was significantly higher than that of the latter and no DIC group. The late DIC group had significantly higher DNI values than the no DIC group. Additionally, the early DIC group has significantly higher WBC counts than the no DIC group (Table 4 and Fig. 1). DNI was positively correlated with DIC score (r=0.548, P<0.001).

Multiple logistic regression analyses were conducted to identify the early predictors related to DIC development, such as male sex, time from snakebite to ED arrival, serum WBC, and DNI. Male sex (OR, 5.785; 95% CI, 1.155 to 28.964; P=0.033), time from snakebite to ED arrival (OR, 1.110; 95% CI, 1.036 to 1.189; P= 0.003), and initial DNI (OR, 4.449; 95% CI, 1.738 to 11.388; P= 0.002) were found to be statistically associated with DIC development. The area under the curve based on the initial DNI’s receiver operating characteristic curve was 0.724 (Table 5 and Fig. 2).