Super Formula for Diagnosing Disseminated Intravascular Coagulation Using Soluble C-Type Lectin-like Receptor 2

The scoring systems for disseminated intravascular coagulation (DIC) criteria require several adequate cutoff values, vary, and are complicated. Accordingly, a simpler and quicker diagnostic method for DIC is needed. Under such circumstances, soluble C-type lectin-like receptor 2 (sCLEC-2) received attention as a biomarker for platelet activation. Materials and Methods: The diagnostic usefulness of sCLEC-2 and several formulas, including sCLEC-2xD-dimer, sCLEC-2/platelet count (sCLEC-2/PLT), and sCLEC-2/PLT × D-dimer (sCLEC-2xD-dimer/PLT), were evaluated among 38 patients with DIC, 39 patients with pre-DIC and 222 patients without DIC or pre-DIC (non-DIC). Results: Although the plasma level of sCLEC-2 alone was not a strong biomarker for the diagnosis of DIC or pre-DIC, the sCLEC-2xD-dimer/PLT values in patients with DIC were significantly higher than those in patients without DIC, and in a receiver operating characteristic (ROC) analysis for the diagnosis of DIC, sCLEC-2xD-dimer/PLT showed the highest AUC, sensitivity, and odds ratio. This formula is useful for the diagnosis of both pre-DIC and DIC. sCLEC-2xD-dimer/PLT values were significantly higher in non-survivors than in survivors. Conclusion: The sCLEC-2xD-dimer/PLT formula is simple, easy, and highly useful for the diagnosis of DIC and pre-DIC without the use of a scoring system.

As there is no gold standard for the diagnosis of DIC, many diagnostic criteria for DIC have been established by the Japanese Ministry of Health, Labour and Welfare (JMHLW) [12], the International Society of Thrombosis Haemostasis (ISTH) [13], the Japanese Association for Acute Medicine (JAAM) DIC [14], and the Japanese Society of Thrombosis Hemostasis [15]. Most diagnostic criteria for DIC are based on scoring systems for global coagulation tests, such as fibrin-related markers (FRMs), prothrombin time (PT), platelet count, and fibrinogen [15][16][17][18]. FRMs, including fibrinogen and fibrin degradation products (FDPs), D-dimer or soluble fibrin (SF), and D-dimer, require standardization [19][20][21]. The use of scoring systems for DIC is complicated, and multiple cutoff values for the parameters of DIC vary among the four diagnostic criteria for DIC [12][13][14][15]. Four DIC groups that were diagnosed using each of the diagnostic criteria showed variation in their disease severity and outcomes [22].
Simpler diagnostic criteria are required to facilitate the early treatment of DIC in the emergency room (ER) or intensive care unit (ICU). Therefore, the sepsis-induced coagulopathy (SIC) score [23], which includes the platelet count and sequential organ failure assessment score [24] or quick DIC score, which includes the D-dimer levels, platelet count, PT ratio, and underlying disease [25], which were recently developed, may be simpler and easier scoring systems and may be useful for diagnosing DIC or coagulopathy in patients with sepsis [26]. However, these scoring systems need to use adequate cutoff values and cannot be compared to other diagnostic criteria. Accordingly, these scoring systems are still complicated. In addition, there is no useful biomarker for platelet activation, which plays an important role in the progression of various pathogenic states [27,28]. Recently, elevated soluble C-type lectin-like receptor 2 (sCLEC-2) levels have been reported in patients with DIC [29], thrombotic microangiopathy (TMA) [30], acute myocardial infarction [31,32], acute cerebral infarction [33], and coronavirus disease 2019 [34].
In this study, we developed a simple formula for the diagnosis of DIC using sCLEC-2, platelet count, and D-dimer to examine the agreement with the JMHLW diagnostic criteria.

Materials and Methods
The study population included patients with the following conditions who were managed at Mie Prefectural General Medical Center from 1 September 2019 to 28 December 2022: infectious diseases (n = 215), solid cancer (n = 27), aortic aneurysm (n = 37), hematological disorders (n = 11), trauma (n = 41), cardiopulmonary arrest (n = 25), and unidentified clinical syndrome (n = 75) ( Table 1). DIC was diagnosed using the Japanese Ministry of Health Labour and Welfare criteria for DIC [12]. Patients with a DIC score of ≥ 7points, 5 or 6 points, and ≤4 points were diagnosed with DIC, pre-DIC, and non-DIC, respectively.

Statistical Analyses
The data are expressed as median (25th-75th percentiles). The significance of differences between groups was examined using the Mann-Whitney U-test. The cutoff values, areas under the curve (AUCs), sensitivity, specificity, and odds ratios were determined by a receiver operating characteristic (ROC) analysis; p-values < 0.05 were considered to indicate a statistically significant difference. All statistical analyses were performed using the Stat-Flex software program (version 7; Artec Co., Ltd., Osaka, Japan).

Results
The mortality rate was highest in CPA and was > 10.0% in cases with solid cancer and infection and 0% in cases with hematological malignancy and UCS ( Table 1). The APTT was significantly longer in cases with infection and CPA than in UCS, and the PT was significantly higher in cases with solid cancer, aortic aneurysm, infection, and CPA than UCS. Platelet counts were significantly lower in cases with aortic aneurysm, infection, and CPA than in those with UCS. In all underlying diseases, the DIC score, FDP, D-dimer, SF, and sCLEC-2 were significantly higher than in UCS.
Regarding evaluation using the JMHLW diagnostic criteria, 38, 39, and 222 patients were diagnosed with DIC, pre-DIC, and non-DIC, respectively. (Table 2). FDP, D-dimer, SF PT-INR, and sCLEC-2 levels were significantly higher in DIC and pre-DIC than in non-DIC, platelet counts were significantly lower in DIC and pre-DIC than in non-DIC, and APTT was significantly longer in DIC and pre-DIC than in non-DIC.  In the ROC analysis (Table 3), the cutoff values for FDP and D-dimer showed the highest AUC and sensitivity (both DIC vs. non-DIC and pre-DIC vs. non-DIC). With regard to the PT-INR, APTT, platelet count, and SF, the AUC for DIC was ≥0.89 but the AUC for pre-DIC was ≤0.75. Using sCLEC-2, the AUCs for DIC (0.801) and pre-DIC (0.748) were not significantly different.

Discussion
sCLEC-2 has been introduced as a new biomarker of platelet activation [35]. Elevated sCLEC-2 levels have been reported without thrombocytopenia in patients with acute coronary syndrome [31,32] or acute cerebral infarction [33], suggesting that sCLEC2 may reflect platelet activation in atherosclerotic thrombosis. In addition to reports related to platelet activation [34] such as DIC [29], TMA [30], and hypercoagulability such as nephrotic syndrome [36] and colorectal cancer [37], it was reported that CLEC-2 regulates inflammatory reactions [38][39][40] and CLEC-2 may be related to cancer progression with platelet activation and hypercoagulability [37,41]. In particular, the plasma sCLEC-2 levels in patients with COVID-19 infections were significantly higher than those in patients with other infections and reflected the progression of the severity of COVID-19 infections [34]. In particular, the sCLEC-2/platelet ratio is useful for evaluating the severity of COVID-19 infections. Furthermore, the plasma sCLEC-2 levels in patients with mild-stage COVID-19 infections were similar to those in patients with severe other pneumonia [34,42].
As there is no gold standard and many diagnostic criteria are used for DIC, this study evaluated hemostatic biomarkers for the diagnosis of DIC based on the diagnostic criteria of the JMHLW [12]. This is because JMHLW is the most frequently used and famous diagnostic criterion for DIC in Japan. The mortality rate of DIC diagnosed using the JMHLW criteria was higher in comparison to DIC diagnosed using the JAAM criteria [14] or SIC criteria [23], suggesting that the severity of pre-DIC in patients diagnosed according to the JMHLW criteria may be similar to the severity of that in patients diagnosed according to the JAAM or SIC criteria [22]. In addition, most of the previous diagnostic criteria for DIC have involved complicated scoring systems that require adequate cutoff values of biomarkers and scoring systems [12][13][14][15]. Biomarkers, especially D-dimer, strongly require standardization [19].
sCLEC-2 alone was not more useful than FDP, D-dimer, or PT-INR in the diagnosis of DIC or pre-DIC. In this study, the most useful diagnostic markers for DIC were FDP and Ddimer. These findings may depend on the underlying diseases, as this study included many patients with infectious diseases with non-DIC who had low FDP or D-dimer levels [22]. Hematological malignancy patients without DIC had relatively high FDP or D-dimer levels, suggesting that the usefulness of FDP or D-dimer for the diagnosis of DIC may be decreased in hematological malignancy [22]. Although the AUC of FDP was markedly high for both DIC and pre-DIC, the AUC of the platelet count and PT-INR was markedly high for DIC but not for pre-DIC. The AUC of sCLEC-2 was moderately high for both DIC and pre-DIC, suggesting that sCLEC-2 may be useful for the diagnosis of pre-DIC and early-stage DIC.
Regarding the formula of sCLEC-2, sCLEC-2/PLT or sCLEC-2xD-dimer increased the diagnostic for DIC or pre-DIC in comparison to sCLEC-2 alone, and sCLEC-2xD-dimer/PLT was the most useful for the diagnosis of both DIC and pre-DIC. As thrombocytopenia is frequently observed in DIC, the levels of plasma sCLEC-2 released from decreased platelets may not be sufficiently elevated in patients with DIC. Accordingly, the sCLEC-2/PLT ratio has been reported to be useful for the diagnosis of DIC [43]. An elevated sCLEC-2/PLT ratio was also reported in postoperative glioma patients with venous thromboembolism [44]. As D-dimer and FDP were the most useful biomarkers for DIC in this study [45], sCLEC-2xD-dimer, which reflects the activation of both platelets and coagulation, showed high diagnostic ability for DIC. In particular, the sCLEC-2xD-dimer/PLT ratio was more useful for the diagnosis of pre-DIC in comparison to FDP or D-dimer because the treatment of early phase DIC (e.g., pre-DIC or SIC) is recommended [23,26].
Regarding the outcome, the DIC score, FDP, sCLEC-2xD-dimer/PLT, and PT-INR were useful biomarkers for predicting a poor outcome. The complication of DIC is considered to be associated with poor outcomes [22]. The outcomes of DIC diagnosed using the JMHLW criteria were poorer in comparison to DIC diagnosed using JAAM [22], suggesting that the diagnostic criteria for the early phase of DIC cannot sufficiently predict a poor outcome. ROC analysis showed that the cutoff value of the JMHLW DIC score for a poor outcome was 3.3. These findings suggest that the treatment of DIC for the improvement of an outcome should start at a DIC score of four points before the diagnosis of DIC (DIC score of seven points). In addition, the outcome generally depends on underlying diseases; thus, survival should be examined in a large-scale study.
Although the comparison of the accuracy of the JMHLW diagnostic criteria and the super formula using sCLEC-2 in the diagnosis of DIC requires a gold standard definition for DIC, the super formula using sCLEC-2 is simpler and easier to apply than JMHLW diagnostic criteria. In addition, the present study demonstrates that the concordance between the JMHLW diagnostic criteria and the super formula using sCLEC-2 is significantly high and that the super formula using sCLEC-2 can be used in place of the JMHWD diagnostic criteria in the diagnosis of DIC.

Conclusions
sCLEC-2xD-dimer/PLT, which can be diagnosed without a complicated scoring system, is a simple and useful diagnostic formula for the diagnosis of pre-DIC as well as overt-DIC.

Institutional Review Board Statement:
The study protocol (2022-S25) was approved by the Human Ethics Review Committee of Mie Prefectural General Medical Center, and informed consent was obtained from each patient.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.

Conflicts of Interest:
The measurements of sCLEC-2 and D-dimer levels were partially supported by LSI Medience. There are no other conflict of interest.