D-dimer testing: A narrative review.

D-dimer containing species are soluble fibrin degradation products derived from plasmin-mediated degradation of cross-linked fibrin, i.e., 'D-dimer'. D-dimer can hence be considered a biomarker of in vivo activation of both coagulation and fibrinolysis, the leading clinical application in daily practice of which is ruling out venous thromboembolism (VTE). D-dimer has been further evaluated for assessing the risk of VTE recurrence and helping define optimal duration of anticoagulation treatment in VTE, for diagnosing disseminated intravascular coagulation (DIC), and for screening those at enhanced risk of VTE. D-dimer assays should however be performed as intended by regulatory agencies, as their use outside these indications might make them a laboratory-developed test (LDT). This narrative review is aimed at: (1) reviewing the definition of D-dimer, (2) discussing preanalytical variables affecting D-dimer measurement, (3) reviewing and comparing the assays performance and some postanalytical variables (e.g., different units and age-adjusted cutoffs), and (4) discussing the interest of D-dimer measurement across different clinical settings, including pregnancy, cancer, and coronavirus disease 2019 (COVID-19).

Serial thrombin generation, while on LMWH prophylaxis in critically ill COVID-19 patients from the prospective MaastrICCht cohort

Background: COVID-19 associated coagulopathy (CAC) has been associated with an increase in venous thromboembolic events. Most guidelines advise heparins in the management of this coagulopathy. However, the relative effectiveness of this strategy on the coagulation potential of COVID-19 patients remains unclear. Aim(s): We aim to describe the development of COIVID-19 associated coagulopathy in patients treated with either low molecular weight heparins (LMWH) or unfractionated heparin (UFH) during the course of disease in an intensive care unit population. Additionally we evaluate the anticoagulant treatment in these patients in-vitro. Method(s): We included 33 patients with confirmed COVID-19 admitted at the ICU. Calibrated automated thrombinography (CAT) was measured at least twice over the course of 6 weeks after admission. Thrombin generation results were subsequently correlated with an extensive database of clinical and laboratory measurements. Result(s): Anti-Xa levels of all patients remained within their therapeutic range throughout the follow-up. The mean (SE) endogenous thrombin potential (ETP) was 1727 (170) nM min and 1620 (460) nM min for ellagic acid (EA) and tissue factor (TF) respectively at inclusion. In line with this we found a mean (SE) PH of 352.9 (44.9) nM and 263.5 (95.7) nM for EA and TF. Despite adequate treatment thrombin generation parameters remained highly elevated. In vitro assessment alternative anticoagulants showed promising aspects of direct thrombin inhibition. Conclusion(s): We showed that, in a cohort of critically ill COVID-19 patients on low molecular weight heparins, despite apparent adequate anti-coagulation doses evaluated by antiXa levels, thrombin generation potential remains high. Thrombin generation potential persists over the course of ICU admission and is independent of age, sex, body mass index, APACHE II score, cardiovascular disease and smoking status. The observations could, for a small part at the most, be explained by (anti)coagulation and thrombosis, inflammation and multi-organ failure. Direct thrombin inhibition might be a promising alternative anticoagulant therapy in COVID-19. (Figure Presented).

Rotational thromboelastometry in heparin monitoring in critical COVID-19 disease

Background: Critically-ill COVID-19 patients on UFH present with heparin resistance (defined as >35.000 IU/day) to reach target aPTT or aXa. APTT is the conventional method as aXa is time consuming and not widely available. APTT or aXa relate differently to doses of UFH, which questions their validity in COVID-19 disease. Whole-blood rotational thromboelastometry (ROTEM) INTEM/HEPTEM clotting time (CT) ratio could be a more functional alternative. Aim(s): Observational study comparing ROTEM INTEM/HEPTEM CT ratio, aPTT and anti-Xa in mechanically ventilated COVID-19 patients on UFH/LMWH. Method(s): We studied a subcohort (n = 106/232) of mechanically ventilated COVID-19 patients of the MaastrICCht cohort. We measured ROTEM, aPTT and aXa on UFH (n = 18) or therapeutic LMWH ( n = 88). Patients received UFH (ECMO/renal replacement therapy) or therapeutic LMWH (venous thromboembolism/cardiac arrhythmia). ROC and Linear regression analyses were assessed for diagnostic characteristics and associations. Result(s): INTEM/HEPTEM CT ratio was significantly higher in UFH-patients (1.4 [1.3-1.4]) compared to LMWH-patients (1.0 [1.0-1.1], p < 0.001) (Table 1). Furthermore, ROC analysis showed an AUC of 0.935 for INTEM/HEPTEM CT ratio, 0.775 for aPTT and 0.632 for aXa, suggesting that INTEM/HEPTEM CT ratio detects presence of UFH more accurately than aPTT and aXa. At an INTEM/HEPTEM CT cut-off of 1.1, sensitivity was 87.5% and specificity was 86.3% for the detection of UFH. Both APTT and aXa were positively associated with INTEM/HEPTEM CT ratio (beta(95%CI) 0.006(0.004-0.008) and 0.153 (0.071-0.234) respectively, Table 2).Only, the association between aXa and INTEM/HEPTEM CT ratio was significantly stronger (p = 0.006) in UFH-patients (beta(95%CI) 0.310 (0.001-0.619), compared to LMWH-patients (beta(95%CI) 0.094 (0.053-0.135). Conclusion(s): ROTEM INTEM/HEPTEM CT ratio can detect the presence of UFH opposed to the presence of therapeutic LMWH in mechanically ventilated COVID-19 patients. INTEM/HEPTEM CT ratio was dependent on aPTT, irrespective of UFH or LMWH use. The association between aXa and INTEM/HEPTEM CT ratio was significantly stronger in patients on UFH than LMWH. (Table Presented).

Sustained endothelial, coagulation and inflammatory cytokine activation without macrovascular dysfunction at 3 months after COVID-19: A reflection on SARS-CoV-2 induced thrombo-inflammation

Background: Endothelial damage caused by COVID-19 may imperil the cardiovascular health of millions. More than a year since WHO declared the COVID-19 pandemic, information on the lasting effects of this infection on the cardiovascular system beyond the acute phase is still lacking. Purpose: To study macrovascular endothelial dysfunction and activation, coagulation and inflammation, 3 months after resolution of acute COVID- 19 symptoms. Methods: A cross-sectional observational cohort study was conducted including 203 patients with PCR confirmed COVID-19 disease, 6-20 weeks after acute COVID-19. The primary endpoint was macrovascular endothelial function, assessed by the carotid artery reactivity (CAR) test. The CAR measures the carotid artery diameter in response to hand in icewater immersion. A historic cohort of 313 subjects served as controls. Propensity score matching was used to correct for baseline differences. Plasma endothelin-1 (ET-1), interleukin (IL)-1ra, IL-6, IL-18 were measured by ELISA. ET-1 levels were also measured in a partially overlapping COVID-19 cohort of which plasma samples were available during the acute phase. Coagulation enzyme:inhibitor complexes for thrombin:antithrombin (TAT), factor (F) IXa:AT, FVIIa:AT, FXIa:AT, FXIa:alpha 1 antitrypsin (a1AT), FXIa:C1 esterase inhibitor (C1inh), kallikrein(PKa):C1inh and von Willebrand Factor:antigen (vWF:Ag), were assessed by in house developed ELISA. Results: After propensity score matching, the prevalence of macrovascular dysfunction did not differ between the COVID-19 (22.5%) versus the historical control cohort (18.6%, RD -3.92%, 95%-CI -15 to 7.19, p=0.49). Plasma concentrations of markers for endothelial activation were elevated (>1 SD above normal);ET-1 (64.9%), and vWF:Ag (80.8%). In controls, ET- 1 levels were significantly lower as compared to COVID-19 patients during the acute phase and after 3 months. ET-1 levels were significantly higher 3 months after COVID-19 as compared to the acute phase. Cytokines were high in a majority of patients: IL-18 (73.9%), IL-6 (51.2%), and IL- 1ra (48.9%). TAT and FIXa:AT, reflecting a prothrombotic state, were high in 48.3% and 29.6% of the patients, respectively. FVIIa:AT, as marker of the extrinsic pathway, was elevated (35%). Markers of contact activation were also increased: PKa:C1inh (16.3%), FXIa:AT (16.3%), FXIa:a1AT (20.7%), and FXIa:C1inh (17.7%) (picture 1). Conclusions: At 3 months after acute COVID-19 there was no indication of macrovascular dysfunction as compared to matched historic controls;there was evidence, however, of sustained thrombo-inflammation, indicated by high circulating concentrations of ET-1, vWF:Ag, proinflammatory cytokines, and markers of coagulation (picture 2). Elevated IL-18 levels could potentially induce arterial inflammation and subsequent atherogenesis. Our data highlight the importance of further studies on SARS-CoV-2 related thrombo-inflammation, as well as longer follow-ups in recovered patients. (Figure Presented).

Sustained inflammation, coagulation activation and elevated endothelin-1 levels without macrovascular dysfunction at 3 months after COVID-19.

INTRODUCTION: Endothelial damage and thrombosis caused by COVID-19 may imperil cardiovascular health. More than a year since the WHO declared COVID-19 pandemic, information on its effects beyond the acute phase is lacking. We investigate endothelial dysfunction, coagulation and inflammation, 3 months post-COVID-19. MATERIALS AND METHODS: A cohort study was conducted including 203 patients with prior COVID-19. Macrovascular dysfunction was assessed by measuring the carotid artery diameter in response to hand immersion in ice-water. A historic cohort of 312 subjects served as controls. Propensity score matching corrected for baseline differences. Plasma concentrations of endothelin-1 were measured in patients post-COVID-19, during the acute phase, and in matched controls. Coagulation enzyme:inhibitor complexes and inflammatory cytokines were studied. RESULTS AND CONCLUSIONS: The prevalence of macrovascular dysfunction did not differ between the COVID-19 (18.6%) and the historic cohort (22.5%, RD -4%, 95%CI: -15-7, p = 0.49). Endothelin-1 levels were significantly higher in acute COVID-19 (1.67 ± 0.64 pg/mL) as compared to controls (1.24 ± 0.37, p < 0.001), and further elevated 3 months post-COVID-19 (2.74 ± 1.81, p < 0.001). Thrombin:antithrombin(AT) was high in 48.3%. Markers of contact activation were increased in 16-30%. FVIIa:AT (35%) and Von Willebrand Factor:antigen (80.8%) were elevated. Inflammatory cytokine levels were high in a majority: interleukin(IL)-18 (73.9%), IL-6 (47.7%), and IL-1ra (48.9%). At 3 months after acute COVID-19 there was no indication of macrovascular dysfunction; there was evidence, however, of sustained endothelial cell involvement, coagulation activity and inflammation. Our data highlight the importance of further studies on SARS-CoV-2 related vascular inflammation and thrombosis, as well as longer follow-up in recovered patients.

Pre-admission anticoagulant therapy and mortality in hospitalized COVID-19 patients: A covid predict retrospectivecohort study

Background : A high incidence of venous thromboembolism (VTE) is observed in patients with COVID-19. Furthermore, several studies show that hypercoagulability is associated with mortality. Aims : To investigate whether pre-admission anticoagulant therapy is associated with a lower risk of all-cause mortality in hospitalized COVID-19 patients. Methods : Retrospective data from 1,851 consecutive patients with PCR-confirmed SARS-CoV-2 infection hospitalized in eight Dutch centres between February 27 th and August 1 st 2020 were used. During this period, Dutch guidelines recommended routine thromboprophylaxis for all hospitalized COVID-19 patients. After 1:1 propensity score nearest-neighbour matching based on age, sex, and 17 comorbidities, the association between pre-admission anticoagulant therapy for VTE, atrial fibrillation, or other indications (i.e. direct oral anticoagulants or vitamin K antagonists) and all-cause mortality and intensive care unit (ICU) admission was evaluated. A secondary analysis was performed with a broader definition of antithrombotic therapy including anticoagulants and antiplatelet drugs. Results : Mean age was 66.4 years (SD, 14.8) and 39% were women. Pre-admission, 678 patients (37%) were using anticoagulant and/or antiplatelet therapy of whom 287 (16%) used anticoagulant therapy only, 408 (22%) antiplatelet therapy only, and 17 both anticoagulant and antiplatelet therapy. 253 anticoagulant users and 253 patients not using therapeutic anticoagulation were matched. During a median follow-up of 21 days [IQR: 9.8-21.0], anticoagulant therapy was neither associated with all-cause mortality (hazard ratio [HR], 0.95;95%-CI, 0.70-1.27;Figure 1) nor with ICU admission (HR, 1.0;95%-CI, 0.59-1.70). Results did not materially change in the secondary analysis of anticoagulant and/or antiplatelet therapy (HR for mortality, 1.18 [95%-CI, 0.87-1.59] and HR for ICU admission, 2.98 [95%-CI, 0.60-1.39]). Conclusions : In this retrospective cohort study, pre-admission anticoagulant use was not associated with a lower risk of mortality or ICU admission in hospitalized COVID-19 patients. Further data from randomized controlled trials are needed to determine the riskbenefit ratio of initiating anticoagulant therapy during admission for COVID-19.

Native rotational thromboelastometry observations in critically ill COVID-19 patients

Background : COVID-19 patients show profound hemostatic abnormalities and are at increased risk for thrombosis. Native rotational thromboelastometry (NATEM-ROTEM) is a viscoelastic assay in which the patient sample is solely re-calcified in absence of an activator of hemostasis and hence the activation originates from the sample itself. NATEM might better reflect in vivo hemostatic abnormalities in COVID-19 patients. Aims : We aimed to evaluate the applicability of NATEM to detect hemostatic abnormalities in critically ill COVID-19 patients and the association with contact activation biomarkers. Methods : NATEM was performed in whole blood(WB) and platelet poor plasma(PPP) with and without a thermostable inhibitor of contact activation(TICA) in 13 COVID-19 patients. All patients were admitted to the ICU and received prophylactic ( n = 6) or therapeutic ( n = 7) LMWH. Prolonged clotting time(CT), in WB and PPP, was expected due to LMWH administration[Scharbert, 2012]. Healthy volunteers served as NATEM controls. Activated coagulation factor inhibitor complexes (thrombin:antithrombin(T:AT), FXIa:AT, FXIaa1AT, FXIa:C1Inh, and FIXa:AT) were measured by ELISA to evaluate in vivo coagulation status in COVID-19 patients. Results : CT did not differ in WB between COVID-19 patients and controls, but was prolonged in PPP NATEM(Table 1). In contrast, Maximum clot firmness(MCF) was increased in WB in COVID-19 patients compared to controls, but PPP did not differ between the groups. Addition of TICA resulted in prolonged CT values. Contact activation biomarkers(FXIa:inhibitor complexes, FIXa:AT) were generally within the normal range and did not correlate with NATEM parameters or TICA-mediated changes;T:AT levels showed a negative correlation with CT(ρ = -0.76, P < 0.01). Conclusions : NATEM shows a hypercoagulable phenotype in WB samples from COVID-19 patients, illustrated by increased MCF and a lack of LMWH-mediated CT prolongation, that is absent in PPP. Contact activation biomarkers did not explain the observed results, suggesting other factors are important for NATEM clot development. This might suggest a role for endogenous tissue factor expression, leukocytes or platelets in COVID-19-associated hypercoagulability.

Excess mortality in a population of vitamin k antagonist users during the first wave of the COVID-19 pandemic in thenetherlands compared with excess mortality in the general dutch population

Background : Excess mortality has been observed in the general population during the COVID-19 pandemic, but it is unknown whether preexisting anticoagulant treatment affects survival, given that COVID-19 associated hypercoagulability adversely impacts prognosis. Aims : To investigate whether preexisting vitamin K antagonist (VKA) treatment is associated with lower excess mortality during the first wave of the COVID-19 pandemic in the Netherlands when compared with excess mortality in the general population. Methods : All atrial fibrillation (AF) patients (≥60 years) receiving long-term VKA therapy before week 11, 2020 were included from three Dutch anticoagulation clinics. The corresponding patient population managed by the same clinics in 2019 (i.e., all AF patients (≥60 years) receiving long-term VKA therapy before week 11, 2019) was enrolled as a control cohort. Difference in survival within 9 weeks (i.e., week 11 to 19) between the two cohorts was evaluated by Cox regression analysis. This was compared with the difference in survival during the same time frame of the general elderly (≥60 years) Dutch populations in 2020 versus 2019. Results : The study included 22,730 VKA users for the cohort in 2019 and 19,476 for the cohort in 2020, of which baseline characteristics were comparable. The cumulative incidences for all-cause mortality of the VKA users and the general population are presented in Table 1. Conclusions : Elderly patients with AF receiving long-term VKA therapy in the Netherlands appeared to have a lower excess mortality during the first wave of the COVID-19 pandemic when compared to the general elderly population.

Elso registry analysis-longitudinal trends in bleeding complications on extracorporeal life support (ECLS) over the past twenty years

Background: Extracorporeal life support (ECLS) is increasingly used worldwide over the past two decades and new indications are emerging, including extracorporeal cardiopulmonary resuscitation, trauma and COVID-19 cases. A frequent and remained feared complication is bleeding, and it is associated with high morbidity and mortality. However, trends of bleeding complications and outcomes have been poorly investigated. Methods: Veno-venous (V-V) and veno-arterial (V-A) ECLS patients from the Extracorporeal Life Support Organization (ELSO) Registry database between 2000 and 2020 were included. Bleeding complication and mortality trends were analyzed. Bleeding complications included surgical site, cannulation site, gastrointestinal, pulmonary central nervous system and tamponade bleeding. Risk factors for bleeding complications were identified with multivariable analysis. Results: The analysis included 50.444 patients with single ECLS runs, 30.696 patients with V-A ECLS and 19.748 with V-V ECLS. Bleeding complications were reported in 13.534 patients (26.8%) and occurred more often in V-A ECLS compared to V-V ECLS patients (30.0% versus 21.9%). Bleeding patients showed lower hospital survival rates in both groups. Over the past twenty years bleeding complications showed a decreasing trend with a coefficient of -1.124 and -1.661 for V-V and V-A ECLS respectively. Surgical and cannulation site bleeding showed highest negative trend in both ECLS groups. Conclusions: The decrease in bleeding complications, especially cannulation and surgical site related bleeding, over the past two decades suggest improvement in anticoagulation management and possible equipment development. However, the persistent high rates of bleeding complications and association with mortality reinforces the need to understand bleeding complications more thoroughly during ECLS.

Incidence of thrombotic complications and overall survival in hospitalized patients with COVID-19 in the second and first wave.

INTRODUCTION: In the first wave, thrombotic complications were common in COVID-19 patients. It is unknown whether state-of-the-art treatment has resulted in less thrombotic complications in the second wave. METHODS: We assessed the incidence of thrombotic complications and overall mortality in COVID-19 patients admitted to eight Dutch hospitals between September 1st and November 30th 2020. Follow-up ended at discharge, transfer to another hospital, when they died, or on November 30th 2020, whichever came first. Cumulative incidences were estimated, adjusted for competing risk of death. These were compared to those observed in 579 patients admitted in the first wave, between February 24th and April 26th 2020, by means of Cox regression techniques adjusted for age, sex and weight. RESULTS: In total 947 patients with COVID-19 were included in this analysis, of whom 358 patients were admitted to the ICU; 144 patients died (15%). The adjusted cumulative incidence of all thrombotic complications after 10, 20 and 30 days was 12% (95% confidence interval (CI) 9.8-15%), 16% (13-19%) and 21% (17-25%), respectively. Patient characteristics between the first and second wave were comparable. The adjusted hazard ratio (HR) for overall mortality in the second wave versus the first wave was 0.53 (95%CI 0.41-0.70). The adjusted HR for any thrombotic complication in the second versus the first wave was 0.89 (95%CI 0.65-1.2). CONCLUSIONS: Mortality was reduced by 47% in the second wave, but the thrombotic complication rate remained high, and comparable to the first wave. Careful attention to provision of adequate thromboprophylaxis is invariably warranted.

Management of the thrombotic risk associated with COVID-19: guidance for the hemostasis laboratory.

Coronavirus disease 2019 (COVID-19) is associated with extreme inflammatory response, disordered hemostasis and high thrombotic risk. A high incidence of thromboembolic events has been reported despite thromboprophylaxis, raising the question of a more effective anticoagulation. First-line hemostasis tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and D-dimers are proposed for assessing thrombotic risk and monitoring hemostasis, but are vulnerable to many drawbacks affecting their reliability and clinical relevance. Specialized hemostasis-related tests (soluble fibrin complexes, tests assessing fibrinolytic capacity, viscoelastic tests, thrombin generation) may have an interest to assess the thrombotic risk associated with COVID-19. Another challenge for the hemostasis laboratory is the monitoring of heparin treatment, especially unfractionated heparin in the setting of an extreme inflammatory response. This review aimed at evaluating the role of hemostasis tests in the management of COVID-19 and discussing their main limitations.