Thromboelastometry demonstrates endogenous coagulation activation in nonsevere and severe COVID-19 patients and has applicability as a decision algorithm for intervention

In patients with severe forms of COVID-19, thromboelastometry has been reported to display a hypercoagulant pattern. However, an algorithm to differentiate severe COVID-19 patients from nonsevere patients and healthy controls based on thromboelastometry parameters has not been developed. Forty-one patients over 18 years of age with positive qRT-PCR for SARS-CoV-2 were classified according to the severity of the disease: nonsevere (NS, n = 20) or severe (S, n = 21). A healthy control (HC, n = 9) group was also examined. Blood samples from all participants were tested by extrinsic (EXTEM), intrinsic (INTEM), non-activated (NATEM) and functional assessment of fibrinogen (FIBTEM) assays of thromboelastometry. The thrombodynamic potential index (TPI) was also calculated. Severe COVID-19 patients exhibited a thromboelastometry profile with clear hypercoagulability, which was significantly different from the NS and HC groups. Nonsevere COVID-19 cases showed a trend to thrombotic pole. The NATEM test suggested that nonsevere and severe COVID-19 patients presented endogenous coagulation activation (reduced clotting time and clot formation time). TPI data were significantly different between the NS and S groups. The maximum clot firmness profile obtained by FIBTEM showed moderate/elevated accuracy to differentiate severe patients from NS and HC. A decision tree algorithm based on the FIBTEM-MCF profile was proposed to differentiate S from HC and NS. Thromboelastometric parameters are a useful tool to differentiate the coagulation profile of nonsevere and severe COVID-19 patients for therapeutic intervention purposes.

Correlation of Dengue Warning Signs during Febrile Phase with Rotational Thromboelastometry, Cortisol and Feritin

Dengue mortality remains high despite monitoring against warning signs (WS). The associations of WS at febrile phase (FP) and hemorrhage at defervescence with the levels and kinetics of ROTEM, platelet count, cortisol, and ferritin were analyzed. Patients with confirmed dengue serology and WS in two centers were screened (n = 275) and 62 eligible patients were recruited prospectively over 9 months. “Vomiting” was the commonest WS (62.9%), with shortened clotting time (CT) INTEM (p = 0.01). “Hematocrit increase” showed significant prolonged CT INTEM, EXTEM, and FIBTEM (p < 0.05). “Platelet decrease” showed reduced platelet function and reduced clot amplitude at 10 min (A10) and maximum clot firmness (MCF) in INTEM and EXTEM (p < 0.001). The kinetics were reduced in platelet count, CT EXTEM, and cortisol (p < 0.05) but increased in CT INTEM (p = 0.03). At FP, “vomiting”, “hematocrit increase”, and “platelet decrease” demonstrated impaired CT, clot strengths A10/MCF and platelet functions. Majority (60/62, 96.7%) had non-severe outcomes, consistent with increase in cortisol kinetics. In conclusion, “vomiting”, “hematocrit increase” and “platelet decrease” at FP correlated with ROTEM. No conclusion could be made further regarding ferritin and cortisol. Larger study is required to study “hematocrit increase” with ROTEM as a potential marker for hemorrhage.

Vaccine-Induced Thrombotic Thrombocytopenia: Novelty Testing in the Diagnostic Work-up?

COVID-19 vaccination campagnies with several vaccines types are currently undeway. Recently, the ASTRA ZENECA vaccine has raised public alarm with concerns regarding the development of thrombotic events known as vaccine-induced thrombotic thrombocytopenia (VITT). Early and limited studies have implicated an antibody-mediated platelet activation as the mechanism of the clotting events. Aim of this study was to investigate the platelet and coagulation activation using specialized tests. In this study we enrolled 60 patients (40 men, 20 women; mean age 55±10 years) without cardiovascular risk factors or a history of thrombosis who reported having poplitea deep vein thrombosis (35/60) and pulmonary embolism (25/60) revealed with lower-limb ultrasonography and computed tomography (CT) angiography, respectively, 7 days after vaccination with ASTRA ZENECA. All patients were evaluated for initial testing such as platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimer (DD). Platelets were measured by automated analyzer, PT and APTT by coagulometric test, Fib using Clauss method, and DD using ELISA. Complete blood hemostasis was studied by platelet function assay (PFA-100) on Collagen/ADP (CT-ADP) and Collagen/Epinephrine (CT-EPI) cartridges and Thromboelastometry method on Clotting Time (CT), Clotting Formation Time (CFT), Maximum Clot Firmness (MCF), and clot lysis at 30 minutes (LY-30). All patients had thrombocytopenia (60±5x109/L), longer PT (28±10 s) and PTT (50±10 s), lower Fib (80±20 mg/dl), higher DD ((550±100 mg/l). All patients had shorter C/ADP and C/EPI (C/ADP, n.v. 68-121 s (42±10 s) and C/EPI n.v. 84-160 s (38±5 s) and shorter CT (CT, unit: s. n.v. 100-240 s) (INTEM 30±20 s, EXTEM 18±10 s), shorter CFT (CFT, unit: s, n.v. 30-160 s (INTEM 11±10 s, EXTEM 19±10 s), longer MCF (MCF, unit: mm, n.v. 50-72 mm (INTEM 128±10 mm, EXTEM 110±10 mm), and lower LY-30 (LY-30, %: v.n. 15% (INTEM 0.8%, EXTEM 0.7%). These interesting findings may be the novelty in the diagnostic work-up of the VITT. If these tests may aid in the diagnosis of VITT deserve to be confirmed and need reproducing in other studies. Disclosures No relevant conflicts of interest to declare.

Donor and Storage Duration Impact Cold-Stored Platelet Function More Than Handling Protocols

Cold storage of platelets has the potential to mitigate the logistical and financial burdens associated with platelet inventory management. In addition, cold-stored platelets (CS-PLT, stored at 2-6°C) have better preserved hemostatic function than room temperature (RT)-stored platelets (RT-PLT), suggesting that CS-PLT may provide improved hemostatic resuscitation in actively bleeding patients as compared to RT-PLT when transfused. With CS-PLT clinical use on the rise, questions regarding CS-PLT inventory management have been raised - specifically how should CS-PLT be stored and handled over the course of storage (after collection and prior to transfusion). RT-PLT are currently stored flat on specialized porous racks and agitated at 18-22°C, which allows for oxygenation. These products are stored out to 5 days, with 7 days allowed for large volume delayed sampling collections. In contrast, refrigerated blood products (i.e., packed red blood cells and whole blood) are stored vertically in pull out drawers, with the goal of reducing harmful oxidizing damage to red blood cells. Notably, refrigeration is known to induce aggregation of platelets, a feature historically associated with bacterial contamination in RT-PLT. In order to diminish concerns regarding aggregate formation, it may be beneficial to massage CS-PLT briefly each day to reduce aggregate formation, yet how this may impact hemostatic function is unknown. To this end, optimal storage and handling conditions for CS-PLT remain to be determined. The objective of our study was to measure CS-PLT hemostatic function in response to two major storage and handling variables: agitation and massage to reduce aggregation in the bag. Single donor apheresis platelet units (single Trima collection in plasma, split equally into two bags to control for donor variability; n=20 donors in 40 bags) were purchased from our regional blood center and delivered to our lab on day of collection. Upon arrival, units were spiked and sampled under sterile conditions for baseline (day 0) profiling, assigned to one of the study groups, and stored accordingly. Our study groups were as follows: "Agitated" (n=10 units), "Not Agitated" (n=10 units, paired with Agitated donors), "Massaged Daily", (massaged 60s daily, n=10 units), and "Massaged at Sampling" (massaged only at sampling, n=10 units, paired with Massaged Daily donors). "Agitated" platelet units were stored in a custom refrigerated shaker (courtesy Helmer Scientific). For the other groups, platelet units were stored in a walk-in cold room. All units were stored on the same style perforated agitator rack, with agitation either powered on or off as assigned. Units from all groups were sampled under sterile conditions at days 2, 5, 7, 14, and 21 of storage, using 8 mL draws at each time point to ensure equal volume removal over the course of storage. Hemostatic function was assayed using light transmission aggregometry (LTA; ADP, collagen, epinephrine agonists), rotational thromboelastometry (ROTEM; ExTEM, InTEM agonists), and thrombin generation in response to 5 pM tissue factor. Platelet counts (x10 3/µL) were obtained using a hematology analyzer. During the first week of storage, there were no significant differences in the hemostatic profiles between study groups. While platelet counts, endogenous thrombin potential, and ROTEM clot formation time and maximum clot firmness were fairly stable over the first 7 days of storage across all groups, there was a &gt; 50% reduction from baseline in aggregation responses to both ADP and collagen across all 4 groups, suggesting that platelet aggregation, as detected by LTA, may not be the best representative of CS-PLT function. By day 21 of storage, there was a robust increase in endogenous thrombin potential in all study groups when compared to baseline (Agitated, 22%; Not Agitated, 70%; Massaged Daily, 51%; Massaged at Sampling, 47%). Despite this increase in thrombin generation, after extrinsic activation day 21 CS-PLT in all groups had two-fold increased clot formation times compared to baseline (Agitated, 185%; Not Agitated, 223%; Massaged Daily, 236%; Massaged at Sampling, 190%), and reduced maximum clot firmness compared to baseline (Agitated, -44%; Not Agitated, -36%; Massaged Daily, -54%; Massaged at Sampling, -43%). These data suggest that storage duration, and not agitation nor massaging to reduce aggregates, has the most impact on CS-PLT hemostatic function. Disclosures Spinella: Cerus Corporation: Consultancy, Research Funding; Secure Transfusion Services: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Thromboelastometry in Neonates with Respiratory Distress Syndrome: A Pilot Study

Background: Although respiratory distress syndrome (RDS) constitutes a postnatal risk factor for bleeding and thromboembolic events in neonates, few studies have addressed this issue. We aimed to evaluate the hemostatic profile of neonates with RDS using rotational thromboelastometry (ROTEM). Methods: An observational study was conducted from November 2018 to November 2020 in the NICU of General Hospital of Nikaia “Aghios Panteleimon”. Preterm and term neonates with RDS hospitalized in the NICU were included and EXTEM (tissue factor-triggered extrinsic pathway), INTEM (ellagic acid activated intrinsic pathway), and FIBTEM (with platelet inhibitor cytochalasin D) assays were performed at the onset of the disease. Results: A hypocoagulable profile was noted in neonates with RDS compared to controls, expressed as significant prolongation of EXTEM CT (clotting time) and CFT (clot formation time), lower EXTEM A10 (amplitude at 10 min), MCF (maximum clot firmness), and LI60 (lysis index). Furthermore, prolongation of INTEM CFT and FIBTEM CT, and decreased INTEM and FIBTEM A10 and MCF were found in neonates with RDS. Multivariable logistic regression analysis showed that RDS is an independent factor for the recorded alterations in ROTEM variables. Conclusions: RDS is associated with a hypocoagulable profile and greater hyperfibrinolytic potential compared to healthy neonates.

Using Rotational Thromboelastometry to Identify Early Allograft Dysfunction after Living Donor Liver Transplantation

Background: Diagnostic tests for early allograft dysfunction (EAD) after living donor liver transplantation (LDLT) vary widely. We aimed to evaluate the predictive value of rotational thromboelastometry (ROTEM)-derived parameters in EAD. Materials and Methods: A total of 121 patients were reviewed. The definition of EAD proposed by Olthoff et al. included the presence of any of the following at postoperative day 7: bilirubin level ≥ 10 mg/dL, INR ≥ 1.6, or serum AST or ALT levels > 2000 IU/L. All patients underwent ROTEM assay, which consisted of an extrinsically activated thromboelastometric test (EXTEM) before and 24 h after LDLT. Results: The 1-year/2-year OS were 68.%8/64.5% and 94.4%/90.8% for the EAD and non-EAD groups, respectively (p = 0.001). Two independent risks were identified for EAD, the postoperative clotting time (CT, p = 0.026) and time to maximum clot firmness (maximum clot firmness (MCF)-t, p = 0.009) on the EXTEM. CT yielded a specificity of 82.0% and negative predictive value of 83.0%, and MCF-t displayed a specificity of 76.4% and negative predictive value of 81.9% in diagnosing EAD. The use of the 24 h post-LDLT ROTEM increased the effectiveness of predicting overall survival (OS) compared to using the Olthoff’s EAD criteria alone (p < 0.001). Conclusion: We conclude that CT and MCF on EXTEM were independent predictors of EAD. The 24 h post-LDLT ROTEM can be used with conventional laboratory tests to diagnose EAD. It increases the effectiveness of predicting OS.

Pathological findings in rotation thromboelastometry associated with thromboembolic events in COVID-19 patients

Background Severe thromboembolic events are one of the major complications associated with COVID-19 infection, especially among critically ill patients. We analysed ROTEM measurements in COVID-19 patients with a severe disease course and in patients with severe sepsis. Methods In this study, data obtained by extended analysis of haemostasis with standard laboratory tests and thromboelastometry of 20 patients with severe course of COVID-19 were retrospectively analysed and compared with similar data from 20 patients with severe sepsis but no COVID-19. Results The thromboelastometry values obtained from 20 sepsis patients contained a maximum clot firmness above the normal range but among COVID-19 patients, hypercoagulability was much more pronounced, with significantly higher maximum clot firmness (FIBTEM: 38.4 ± 10.1 mm vs. 29.6 ± 10.8 mm; P = 0.012; EXTEM: 70.4 ± 10.4 mm vs. 60.6 ± 14.8 mm; P = 0.022). Additionally, fibrinogen levels were significantly higher among COVID-19 patients (757 ± 135 mg/dl vs. 498 ± 132 mg/dl, P < 0.0001). Furthermore, thromboelastometry showed fibrinolysis shutdown among COVID-19 patients with significantly lower maximum of lysis than among sepsis patients (EXTEM: 0.6 ± 1.2 % vs. 3.3 ± 3.7 %; P = 0.013). Seven of 20 COVID-19 patients experienced thromboembolic events, whereas no patient in the sepsis group experienced such events. Conclusions ROTEM analysis showed significantly different pathological findings characterized by hypercoagulability and fibrinolysis shutdown among COVID-19 patients with a severe disease course compared to patients with severe sepsis. These abnormalities seem to be associated with thromboembolic events.

Prevalence of Acute Traumatic Coagulopathy in Acutely Traumatized Dogs and Association with Clinical and Laboratory Parameters at Presentation

Objective The aim of this study was to determine the prevalence of acute traumatic coagulopathy (ATC) and identify associated clinical and laboratory parameters including rotational thromboelastometry. Study Design Dogs presenting within 6 hours after trauma were allocated to the ATC or non-ATC group based on thromboelastometry analysis (ex-tem S, in-tem S, fib-tem S). ATC was defined as ≥2 hypocoagulable parameters in 1 profile and ≥ 1 hypocoagulable parameter in an additional profile. Parameters used were ex-tem and in-tem clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), maximum lysis and fib-tem MCF. Clinical and laboratory parameters at presentation, animal trauma triage (ATT) score, transfusion requirement and outcome were compared. Logistic regression was used to identify independent factors associated with ATC. Results Eleven of 33 dogs presented with ATC and showed ex-tem CT and CFT prolongation and reduced MCF amplitude in all profiles (all p < 0.001). pH (p = 0.043) and potassium concentration (p = 0.022) were significantly lower and bleeding (p = 0.027) and plasma transfusions (p = 0.001) more common in dogs with ATC. Time after trauma (p = 0.040) and Animal Trauma Triage score (p = 0.038, including haematocrit as confounding factor) were associated with the presence of ATC. Conclusion Acute traumatic coagulopathy is more common in traumatized dogs than previously reported. Acute traumatic coagulopathy was associated with acidosis, Animal trauma triage score, time after trauma and higher transfusion needs. Coagulation abnormalities include ex-tem CT and CFT prolongations and decreased clot strength.

Greater Fibrinolysis Resistance but No Greater Platelet Aggregation in Critically Ill COVID-19 Patients

Background The hemostatic balance in patients with coronavirus disease 2019 (COVID-19) seems to be shifted toward a hypercoagulable state. The aim of the current study was to assess the associated coagulation alterations by point-of-care-diagnostics, focusing on details of clot formation and lysis in these severely affected patients. Methods The authors’ prospective monocentric observational study included critically ill patients diagnosed with COVID-19. Demographics and biochemical data were recorded. To assess the comprehensive hemostatic profile of this patient population, aggregometric (Multiplate) and viscoelastometric (CloPro) measures were performed in the intensive care unit of a university hospital at a single occasion. Coagulation analysis and assessment of coagulation factors were performed. Data were compared to healthy controls. Results In total, 27 patients (21 male; mean age, 60 yr) were included. Impedance aggregometry displayed no greater platelet aggregability in COVID-19 in comparison with healthy controls (area under the curve [AUC] in adenosine diphosphate test, 68 ± 37 U vs. 91 ± 29 U [−27 (Hodges–Lehmann 95% CI, −48 to −1); P = 0.043]; AUC in arachidonic acid test, 102 ± 54 U vs. 115 ± 26 U [−21 (Hodges–Lehmann 95% CI, −51 to 21); P = 0.374]; AUC in thrombin receptor activating peptide 6 test, 114 ± 61 U vs. 144 ± 31 U [−31 (Hodges–Lehmann 95% CI, −69 to −7); P = 0.113]). Comparing the thromboelastometric results of COVID-19 patients to healthy controls, the authors observed significant differences in maximum clot firmness in fibrin contribution to maximum clot firmness assay (37 ± 11 mm vs. 15 ± 4 mm [21 (Hodges–Lehmann 95% CI, 17 to 26); P &lt; 0.001]) and lysis time in extrinsic activation and activation of fibrinolysis by tissue plasminogen activator assay (530 ± 327 s vs. 211 ± 80 s [238 (Hodges–Lehmann 95% CI, 160 to 326); P &lt; 0.001]). Conclusions Thromboelastometry in COVID-19 patients revealed greater fibrinolysis resistance. The authors did not find a greater platelet aggregability based on impedance aggregometric tests. These findings may contribute to our understanding of the hypercoagulable state of critically ill patients with COVID-19. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New