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).

Contribution of biology to the diagnosis and follow-up of VITT: A French multicentre experience

Background: Vaccine-induced immune thrombocytopenia (VITT) is a rare but serious complication of SARS-COV- 2 vaccination and its diagnosis is based on both clinical and biological criteria. Aim(s): To evaluate the performance of these criteria in a prospective cohort of patients with suspected VITT. Method(s): Among 105 consecutive patients (Table 1), 79 had received one (n = 71) or two (n = 8) injections of adenoviral vector vaccine, and 26 one (n = 18) or two (n = 8) doses of an mRNA vaccine. Most patients presented thrombosis either isolated (n = 40), or associated with thrombocytopenia (T+T = 45). Other patients had isolated thrombocytopenia (n = 15), or variable clinical manifestations. ELISA (PVS/PF4 HAT45G) detected anti-PF4 antibodies, and when present VITT diagnosis was confirmed using PF4 serotonin release assay. Result(s): VITT was confirmed in 26 patients (both PVS/PF4 ELISA and PF4/SRA were positive) and probable in one case (PVS/PF4 ELISA OD = 2.4 but PF4/SRA negative). VITT was excluded in all patients who had received an mRNA vaccine, or in those with isolated thrombocytopenia or thrombosis or with other symptoms. In contrast, the presence of both thrombocytopenia and thrombosis 5 to 30 days after injection was highly suggestive of VITT with a specificity and PPV of 85% and 69%, respectively, these values increasing to 93.6% and 85% if patients had received adenoviral vector vaccine. Importantly, the PPV reached in these cases 100% when OD > 1 in ELISA. (Table) Conclusion(s): The occurrence of thrombocytopenia and thromboses, 5 to 30 days after vaccination with an adenoviral vector is highly predictive of VITT. PF4/PVS ELISA allows to rule out VITT when OD is < 0.4 (NPV 100%) and to confirm the diagnosis when OD is > 1 without necessitating a platelet activation test. Moreover, the follow-up of patients with ELISA is mandatory since anti-PF4 antibodies may remain several weeks after VITT. (Table Presented).

Long-term persistence of anti-PF4- antibodies following vaccine-induced thrombotic thrombocytopenia after first ChAdOx1 vaccination

Background: Vaccine-induced Thrombotic Thrombocytopenia (VITT) is a rare but potentially life-threatening complication of the ChAdOx1 COVID-vaccine, which involves binding of IgG antibodies against platelet factor 4 (PF4) to the platelet Fc-gamma receptor. This causes platelet activation with thrombosis and thrombocytopenia. Because of the similarity with heparin-induced thrombocytopenia (HIT), heparin is avoided in the acute treatment of VITT. There is limited information about the long-term persistence of anti-PF4- antibodies and their clinical relevance. Aim(s): To describe long-term clinical and serological outcomes after VITT. Method(s): A case series of patients from Leuven University Hospitals with confirmed VITT and at least 6 months of follow-up. All patients provided informed consent. Anti-PF4 antibodies were measured via chemiluminescence (HemosIL AcuStar HIT-IgG( PF4-H), Werfen) and enzyme-linked immunosorbent assay (ELISA) with immobilised polyvinylsulfonate/PF4 complexes (PF4-IgG Immucor, GTI Diagnostics);with an optical density (OD) cut-off of 0.4. Aggregation of platelets after exposure to patient plasma with 0, 1 or 200 IU/ml of heparin was measured by whole-blood impedance aggregometry (HIMEA) (Roche multiplate analyser). Result(s): Three middle-aged women presented with thrombosis with thrombocytopenia and a positive anti-PF4 ELISA 9 to 16 days after first ChAdOx1 vaccination. Their clinical presentation, lab results and treatment are summarised in Table 1. All patients recovered rapidly after non-heparin anticoagulation with (case 2-3) or without (case 1) intravenous immunoglobulins. All patients received subsequent COVID-vaccination with an mRNA-based vaccine without thrombocytopenia or symptoms. Anti-PF4- antibodies remained elevated in two patients after 3 months and in one out of three after more than 6 months, but HIMEA results for all follow-up tests became negative (Figure 1). Conclusion(s): We report good short-and long-term outcomes of three cases of VITT, including successful subsequent vaccination with an mRNA vaccine. Anti-PF4- antibodies can persist for at least several months. In contrast with the initial presentation, these persistent anti-PF4- antibodies did not trigger platelet activation in our patients. (Table Presented).

Influence of C-reactive protein on thrombin generation assay

Background : C-reactive protein (CRP) is an acute phase reactant plasma protein considered as biomarker representative of the burden of inflammation also linked to the development of prothrombotic states. In moderate to severe COVID-19 patients, persistent elevated levels of CRP were observed contributing to the risk of venous thromboembolic events. CRP is known to artefactually interfere with certain coagulation assays. As the use of global coagulation test like thrombin generation assay (TGA) may be relevant to characterize the evolution of the disease or identify its severity, it is interesting to investigate the impact of CRP on TGA. Aims : This study aimed to assess how CRP impacts TGA on the automated ST Genesia system, using the STG-ThromboScreen-TM as triggering reagent. A comparison with the Calibrated Automated Thrombogram (CAT) system was performed. Methods : Normal pooled plasma (NPP) constituted of 50 healthy individuals was used as the matrix. Human CRP (Merk, Germany) was spiked in NPP at five relevant plasma concentrations (0 [=Phosphate buffer saline], 50, 100, 200 and 350 mg/L). Both TGA methods were performed in duplicate and assessed by 3 independent runs. Results : Based on mean values, no statistically significant difference was observed between the five tested concentrations ( P -value >0.05), regardless of the platform used. On the other hand, the comparison between both analyzers showed significant differences for lag-time and time-to-peak, which were significantly higher when TGA was performed on the CAT system. These differences are possibly associated with the specific algorithm of each platform. Conclusions : This study demonstrated that CRP levels up to 350 mg/L did not impact significantly thrombin generation performed either on CAT or on ST Genesia system. Therefore, TGA could be an efficient test to assess the hemostatic function of patients with elevated CRP, like those in sepsis and suffering from chronic or acute inflammatory conditions, such as COVID-19.

Is viscoelastometric testing a good tool to assess hemostasis of COVID-19 patients?

Background : Infection by SARS-CoV-2 is associated with a high risk of thrombosis. The laboratory documentation of hypercoagulability and impaired fibrinolysis remains a challenge. Aims : Our main aim was to assess the potential usefulness of viscoelastometric testing (VET) to predict thrombotic events in COVID-19 patients according to the literature. Our secondary aims were: (i) to analyze the impact of anticoagulation and the methods used to neutralize heparin, (ii) to see whether maximal clot mechanical strength brings more information than Clauss fibrinogen, and (iii) to point out results from studies with enhanced fibrinolysis modified tests. Methods : We performed a systematic search in PubMed and Scopus databases, until December 31st, 2020 (Figure 1). VETs methods and parameters, as well as patients ' features and outcomes were extracted. Results : VET was performed for 1063 patients (893 ICU and 170 non-ICU, n = 44 studies). There was a huge heterogeneity concerning study design, the VET device (ROTEM, TEG, Quantra and ClotPro) and reagents (with non-systematic use of heparin neutralization by heparinase and/or polybrene), timing of assay, and definition of hypercoagulable state. The common findings were an increased clot mechanical strength mainly due to an excessive fibrinogen component with impaired to absent fibrinolysis, more conspicuous in the presence of an added plasminogen activator. This profile was associated with an uncontrolled thrombin generation despite a standard thromboprophylaxis. However, only 4 studies out of 16 that addressed this point found an association of VETs with thrombotic events. Functional fibrinogen assessed by VET showed a variable correlation with Clauss fibrinogen. Abnormal VET pattern tended to normalization after an enhancement in thromboprophylaxis. Notably, only 4 studies out of 25 using ROTEM reported data where heparin is neutralized by heparinase (HEPTEM). Conclusions : The heterogeneity among VET studies and small sample sizes do not permit to point out an association between the illdefined hypercoagulable state and thrombotic events.

Analytical and clinical evaluation of four commercial SARS-CoV-2 serological immunoassays in hospitalized patients and ambulatory individuals.

BACKGROUND: This study aimed to compare four anti-SARS-CoV-2 immunoassays in populations presenting different clinical severity levels. METHODS: Three populations were included: "severe-to-critical" ICU-hospitalized patients (n = 18), "mild-to-moderate" hospitalized patients (n = 16) and non-hospitalized symptomatic patients (n = 24). Four commercial immunoassays were analyzed and validated: anti-IgG ARCHITECT® (Abbott), anti-Total antibodies (Ab) VITROS® (Ortho Clinical Diagnostics), anti-IgG NovaLisa® (NovaTec Immundiagnostica) and Healgen® IgM and IgG (Zhejiang Orient Gene Biotech). Sensitivities were evaluated according to days post-symptoms onset (pso). Specificities were evaluated on SARS-CoV-2-negative control sera collected before January 2020. RESULTS: A majority of severe-to-critically ill patients showed detectable Ab already at day 14 and sensitivities reached 100 % after 22 days pso. For patients with "mild-to-moderate" illness, sensitivities increased by at least 5-fold from day 0 to day 14 pso. Non-hospitalized symptomatic individuals already seroconverted at day 14 days pso with 100 % sensitivities for Total Ab VITROS®. Specificities were evaluated at 97 % for ARCHITECT® and NovaLisa®, 98 % for VITROS® and at 94 % for Healgen® combined IgM and IgG. Five "severe-to-critically" ill patients presented high positive Ab levels for at least 16 weeks pso. CONCLUSION: The Ab levels and the evaluated sensitivities, representing the true positive rate, increased overtime and were related to the COVID-19 severity. Automated Total Ab immunoassay showed better sensitivities and specificity for immunological surveillance and vaccine evaluation.

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.