key: cord-1003949-nttp2rod authors: Ferrara, Michael J.; MacArthur, Taleen A.; Butenas, Saulius; Mann, Kenneth G.; Immermann, Joseph M.; Spears, Grant M.; Bailey, Kent R.; Kozar, Rosemary A.; Heller, Stephanie F.; Loomis, Erica A.; Stephens, Daniel; Park, Myung S. title: Exploring the utility of a novel point‐of‐care whole blood thrombin generation assay following trauma: A pilot study date: 2021-03-08 journal: Res Pract Thromb Haemost DOI: 10.1002/rth2.12483 sha: 0427e819e7c414a515bcfd038cc589365ca0359a doc_id: 1003949 cord_uid: nttp2rod INTRODUCTION: Plasma thrombin generation kinetics as measured by the calibrated automated thrombogram (CAT) assay is a predictor of symptomatic venous thromboembolism after trauma. We hypothesized that data from a new prototype assay for measurement of thrombin generation kinetics in fresh whole blood (near patient testing of thrombin generation), will correlate with the standard CAT assay in the same patients, making it a potential tool in the future care of trauma patients. METHODS: Patients were enrolled from June 2018 to February 2020. Within 12 hours of injury, blood samples were collected simultaneously for both assays. Variables compared and correlated between assays were lag time, peak height, time to peak, and endogenous thrombin potential. Data are presented as median with interquartile range (IQR). Spearman and Pearson correlations were estimated and tested between both assays; a P value of <0.05 was considered to be significant. RESULTS: A total of 64 trauma patients had samples analyzed: injury severity score = 17 (IQR), 10‐26], hospital length of stay = 7.5 (IQR), 2‐18) days, age = 52 (IQR, 35‐63) years, 71.9% male, and 42.2% of patients received a transfusion within 24 hours of injury. Thrombin generation parameters between plasma and whole blood were compared and found that all parameters of the two assays correlate in trauma patients. CONCLUSION: In this pilot study, we have found that a novel point‐of‐care whole blood thrombin generation assay yields results with modest but statistically significant correlations to those of a standard plasma thrombin generation assay. This finding supports studying this device in a larger, adequately powered study. • A novel device has been developed to measure thrombin generation kinetics in whole blood. • Accelerated thrombin generation is a predictor of venous thromboembolism after trauma. • Thrombin generation values correlate between plasma calibrated automated thrombogram assay and the novel whole blood assay. • Novel whole blood thrombin generation assay should continue to be explored as a future tool. Traumatic injury remains a leading cause of morbidity and mortality worldwide. In addition to physical and psychological trauma, these patients also face potential morbidity associated with traumainduced coagulopathy (TIC). [1] [2] [3] [4] While effective resuscitation can treat the hemorrhagic effects of TIC, many of these patients suffer persistent hypercoagulability following trauma, even after hospital discharge. Prior work from Dr Park's lab has shown that up to 40% of symptomatic venous thromboembolisms (VTEs), including deep vein thrombosis (DVT) and pulmonary embolism (PE), occur after hospital discharge, and that this risk remains elevated up to 3 months after trauma. 5 Understanding an individual's coagulation profile is essential in the care of trauma patients including initial resuscitation efforts, chemoprophylaxis administration, and postdischarge monitoring. Laboratory tests that rapidly and accurately quantify TIC have the potential to augment care for trauma patients. Plasma-based assays, such as the calibrated automated thrombogram (CAT), have been developed that assess thrombin generation kinetics in real time. 6, 7 This assay is able to quantify an individual's plasma thrombin generation profile in response to tissue factor (TF) and procoagulant phospholipid. Plasma-based CAT describes several parameters of thrombin generation that will be discussed here: lag time (LT), which is the time (minutes) to the start of thrombin generation; peak height (PH), which is the maximum thrombin concentration (nM) at a given time point during the assay; time to peak (ttPeak), which is the time (minutes) to peak rate of thrombin generation; and endogenous thrombin potential (ETP), which is the total thrombin that can be generated during the assay (nM × minute). Prior studies have shown enhanced thrombin generation after trauma and that certain derangements in an individual's plasma thrombin generation profile may be independent predictors of VTE after trauma. [8] [9] [10] Unfortunately, plasma thrombin generation assays are not amenable to point-of-care (POC) use because they are typically run in batches of several patient samples at a time. Additionally, the absence of platelets and other cellular components in a plasma-based assay potentially limits the approximation of physiologic conditions. A novel method of measuring thrombin generation kinetics in fresh whole blood has been developed that allows for near patient testing of thrombin generation (NPT-TG). This POC assay has the potential to provide information about an individual's coagulation profile that could be applied at the bedside. Additionally, the testing of whole blood rather than plasma includes platelets and other cellular components involved in clot formation. The novel whole blood assay measures thrombin generation kinetics in a manner similar to the standard plasma-based assay. A TF agonist initiates a clotting reaction in the presence of a fluorogenic substrate, which changes as thrombin is generated. The results are standardized against a calibrator of known activity, and a curve is produced showing thrombin activity over time. In this pilot study, our main objective was to compare thrombin generation parameters from this novel whole blood assay with those of the well-established plasma-based assay. We hypothesized that thrombin generation kinetics measured in whole blood will correlate with plasma thrombin generation parameters. We anticipate that these correlations will likely be modest due to the presence of platelets and cellular components in whole blood, with these two assays offering complementary views of an individual's coagulation profile. This study was approved by the Mayo Clinic Institutional Review Board and conducted using a waiver of informed consent. Adult patients presenting to the Mayo Clinic Emergency Department as trauma activations, from June 2018 through February 2020 were considered for study inclusion. Exclusion criteria included age <18 years, ongoing systemic anticoagulation at presentation (eg, heparin, warfarin, or novel oral anticoagulants) other than antiplatelet agents, known preexisting coagulopathy, cirrhosis, active malignancy, sepsis, renal failure requiring dialysis, burn injuries, or recent major surgery or another significant trauma in the past year, as well as pregnant women or prisoners. Trauma patients or their legal authorized representative (LAR) gave consent for this study after the collection of one or more blood samples. If the patient or LAR could not be consented for study participation or declined consent, the sample was destroyed and the patient was excluded. The time of injury (TOI) was determined by the prehospital medical providers based on information at the injury scene. We collected demographic and clinical characteristics for each patient from the electronic medical record. Blood samples were collected from both trauma patients and healthy volunteers, who were recruited as outpatients specifically for this study. Healthy volunteers provided full written informed consent before any sample collection. Two separate samples were obtained from each subject: one for the whole blood thrombin generation assay and one for plasma thrombin generation, as described below. Blood was collected simultaneously for both assays through venipuncture or from indwelling catheters. Samples were collected upon patient arrival, up to 12 hours from the TOI for the trauma patients. A total of 4.5 mL of whole blood was collected by venipuncture or via existing indwelling catheters into citrated Vacutainer tubes Whole blood was collected in 4-mL evacuated tubes containing both sodium citrate (3.2%) and corn trypsin inhibitor (CTI; 100 µg/mL; Hematologic Technologies, Essex Junction, VT, USA). The whole blood thrombin generation assay was run within 30 minutes of the blood draw. If the whole blood assay was unable to be run within 30 minutes of the blood draw, the sample was discarded. A dedicated software program, Thrombinoscope (Thrombinoscope BV) was used to calculate thrombin activity over time. The parameters derived were LT, PH, ttPeak, and ETP. Thrombin generation kinetics in whole blood was measured using the NPT-TG, a prototype machine designed by Stago (Diagnostica Stago, Asnières-sur-Seine, France) specifically for this purpose. This Categorical variables were summarized as n (%). Continuous variables were summarized as median with interquartile ranges (IQRs) and compared using the Kruskal-Wallis test. Pearson correlations and Spearman rank correlations were estimated and tested for significance between plasma and the corresponding whole blood variables. The assessment of association was based on the more robust Spearman correlation, while Pearson correlations were estimated primarily for descriptive purposes. A P value of <0.05 was considered to be statistically significant. Trauma patients were also analyzed with and without a subgroup of patients taking antiplatelet medications, and both Pearson and Spearman rank correlations were estimated between the plasma and whole blood parameters for each of those groups. All analysis was performed using SAS, version 9.4 (SAS Institute, Cary, NC, USA). Eighty-nine patients were screened for participation, 5 patients met exclusion criteria, 17 patients declined consent, and 3 patients were excluded due to technical errors with the whole blood thrombin generation assay (one instance of blood collected in the wrong tube, one error preparing reagents, and one mechanical error with machine after the assay had begun). The remaining cohort of 64 trauma patients had a median age of 52 (IQR, 35-63), median injury severity score of 17, 10,24 71.9% were male, and 96.9% had blunt mechanism of injury. As described in Table 1 Trauma patients were compared with 13 healthy volunteers, who had a median age of 35 2539 and were 61.5% female ( Table 1) . All of the female controls were premenopausal and 3 of 8 (37.5%) were on the Nexplanon (etonogestrel 68 mg) hormonal contraceptive implant at the time of sample collection. As described in Table 2, healthy volunteers had significantly shorter LT and ttPeak as compared to trauma patients in the whole blood assay. However, in the plasma thrombin generation assay, the healthy volunteers showed no difference in LT and had a significantly longer ttPeak compared to the trauma patients. Healthy volunteers also had significantly lower ETP than trauma patients using the whole blood assay, but this difference was not significant with the plasma thrombin generation assay. When the healthy volunteers were evaluated by sex, there were no differences in the thrombin generation profiles between males and females. Among the trauma cohort, the absolute values for each thrombin generation parameter were different between the whole blood-and plasma-based assays as described in Table 2 . However, there was statistically significant correlation between the two assays for each thrombin generation parameter (Table 3) . Additionally, when select patients with outlier values for individual thrombin generation parameters ( Figure 1A-1D) were removed from the analysis, the correlation between the two assays was persistent. Six patients within the trauma cohort developed symptomatic VTE during hospitalization, with two PEs and four DVTs. Thrombin generation characteristics of these patients are described in When reviewed separately from the rest of the cohort, there were no statistically significant correlations between the plasma and whole blood thrombin generation parameters for these 13 patients. However, when the remaining trauma patients who are not on any antiplatelet agents (n = 50) were analyzed alone, the correlation coefficients between plasma and whole blood thrombin generation increased (Table 3) In this pilot study, we sought to compare and correlate thrombin generation parameters from the well-established plasma thrombin generation assay with a novel POC prototype assay for whole blood thrombin generation. We found a modest but statistically significant correlation between the plasma and whole blood assays for all thrombin generation parameters in trauma patients. The raw values for each thrombin generation parameter from these two assays differ, as anticipated, given that they employ different tools, methodologies, and sample types (whole blood vs plasma) to measure the same pathway. Some of the differences in these values may be explained by the presence of platelets and cellular components Note: Spearman and Pearson correlations between plasma and whole blood thrombin generation parameters for the trauma patients who are not on any antiplatelet agents (n = 50) presented in the bottom half of the table. Bold values indicate a P-value of < .05 and are considered significant. When the trauma cohort was analyzed with the 13 patients who were taking antiplatelet agents removed, correlations between the plasma and whole blood thrombin generation assays increased (Table 3) . This emphasizes the important role of platelets in thrombin generation that is captured in the whole blood assay. The important role of platelets is also supported by the fact that the patients taking antiplatelet agents had lower PH than the rest of the trauma patients. This indicates that these patients may not make as much thrombin, likely due to thrombin generation being reduced by those medications. Prior work from our lab showed that plasma ttPeak is an independent predictor of VTE development up to 92 days after trauma. 10 As shown in Figure 1 , among the 5 VTE patients who have thrombin generation data available, the majority appear to have shorter LT and ttPeak than trauma patients who did not develop VTE. This should continue to be examined in a larger cohort to evaluate if the whole blood thrombin generation assay has a similar predictive value for VTE after trauma to the plasma thrombin generation assay. Interestingly, the healthy volunteers in this study showed more accelerated thrombin generation than trauma patients using the whole blood assay. In this small pilot study, we had only 13 healthy volunteers, the majority of which were premenopausal females, while the trauma cohort was predominantly middle-aged males. This is because enrollment of healthy controls had to be paused early due to the coronavirus disease 2019 pandemic. Though data are limited, F I G U R E 1 A, Linear regression models for individual plasma and whole blood thrombin generation parameters. Trauma patients who developed venous thromboembolism (VTE; n = 5) are shown in the filled-in circles, non-VTE trauma patients (n = 58) are shown in the open circles. One trauma patient who developed a VTE is not shown due to lack of plasma thrombin generation data prior studies have shown that reproductive-age females have more accelerated thrombin generation than males and that this can be influenced both by menstrual cycle stage and use of hormonal contraceptives. [17] [18] [19] [20] Additionally, prior studies have shown an acceleration of thrombin generation with age in healthy adults using the plasma CAT assay. 21 The impact of age on the whole blood thrombin generation assay has not yet been established, and it may be that varying levels of cellular components and hormones with age may impact this assay differently. This study has several additional limitations. First, given that this is a pilot study with a small number of patients, selection bias is certainly a concern. Additionally, because blood samples were collected which could also impact thrombin generation results in patients who received blood product transfusion before sample collection. [22] [23] [24] Additionally, as described in the methods section, samples for the whole blood assay were collected in 3.2% citrated tubes containing 100 µg/mL CTI, while the samples from the plasma-based assay were collected in 3.2% citrated tubes that did not contain CTI. Because of this difference in collection technique, the samples used in the plasma assay could have had the intrinsic pathway stimulated before addition of CTI to plasma at the time of the assay. This may be one reason to explain why the correlations between the two assays were not more robust. We observed that when patients taking antiplatelet medications were removed from the trauma cohort, correlation coefficients improved between the plasma and whole blood thrombin generation assays. These results should be interpreted cautiously as these patients were heterogeneous in terms of what medications and dosages they were taking, and we do not know to what extent they were taking these medications as prescribed. As such, these results may reflect varying degrees of platelet inhibition. The novel POC prototype whole blood thrombin generation assay has potential clinical utility in the care of trauma patients, given that it can be run near the patient as opposed to the standard plasmabased thrombin generation assay, which is not amenable to bedside use. Our pilot study shows that in trauma patients, the plasma and whole blood thrombin generation assays significantly correlate. A larger cohort is needed to assess if the novel whole blood thrombin generation assay is comparable to the plasma-based assay in predicting VTE and hemorrhagic complications, as we have shown previously in our laboratory. 25 Taleen A. MacArthur @TaleenMacarthur Acute traumatic coagulopathy Cause of trauma-induced coagulopathy Advances in the understanding of trauma-induced coagulopathy Coagulopathy of trauma Risk factors for venous thromboembolism after acute trauma: a case-cohort study Recollections on thrombin generation Thrombin generation, a function test of the haemostatic-thrombotic system Thrombin generation and procoagulant microparticle profiles after acute trauma: a prospective cohort study Quantification of hypercoagulable state after blunt trauma: microparticle and thrombin generation are increased relative to injury severity while standard markers are not Thrombin generation profiles as predictors of symptomatic venous thromboembolism after trauma: a prospective cohort study Standardization of preanalytical variables in plasma microparticle determination: results of the International Society on Thrombosis and haemostasis SSC Collaborative workshop The new metric to define large-volume hemorrhage: results of a prospective study of the critical administration threshold Platelets and thrombin generation Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation The role of the red cell membrane in thrombin generation Whole blood thrombin generation is distinct from plasma thrombin generation Endogenous thrombin potential is higher during the luteal phase than during the follicular phase of a normal menstrual cycle Comparison by sex between thrombin generation and fibrin network characteristics in a healthy population The influence of oral contraceptives on the time-integral of thrombin generation (thrombin potential) Thrombin generation measurement using the ST Genesia Thrombin Generation System in a cohort of healthy adults: normal values and variability Agedependency of thrombin generation measured by means of calibrated automated thrombography (CAT) Transfusion of stored red blood cells in trauma patients is not associated with increased procoagulant microparticles Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation Elevated levels of thrombingenerating microparticles in stored red blood cells Thrombin generation kinetics are predictive of rapid transfusion in trauma patients meeting critical administration threshold Exploring the utility of a novel point-of-care whole blood thrombin generation assay following trauma: A pilot study