Serotonin Release Assay: Functional Assay for Heparin- and Vaccine-Induced (Immune) Thrombotic Thrombocytopenia.

The serotonin release assay (SRA) has been the gold-standard assay for detection of heparin-dependent platelet-activating antibodies and integral for the diagnosis for heparin-induced thrombotic thrombocytopenia (HIT). In 2021, a thrombotic thrombocytopenic syndrome was reported after adenoviral vector COVID-19 vaccination. This vaccine-induced thrombotic thrombocytopenic syndrome (VITT) proved to be a severe immune platelet activation syndrome manifested by unusual thrombosis, thrombocytopenia, very elevated plasma D-dimer, and a high mortality even with aggressive therapy (anticoagulation and plasma exchange). While the platelet-activating antibodies in both HIT and VITT are directed toward platelet factor 4 (PF4), important differences have been found. These differences have required modifications to the SRA to improve detection of functional VITT antibodies. Functional platelet activation assays remain essential in the diagnostic workup of HIT and VITT. Here we detail the application of SRA for the assessment of HIT and VITT antibodies.

Prevalence of thrombosis in patients with cancer and SARS-CoV-2 infection.

BACKGROUND: Covid-19 infection and cancer are associated with an increased risk of thrombotic events. The aim of our study is to analyze the cumulative incidence of thrombosis in oncological patients with Covid-19 and detect differences with the non-cancer Covid-19 population. METHODS: We retrospectively reviewed 1127 medical records of all admitted patients to ward of the Hospital Universitario Infanta Leonor (Madrid, Spain), including 86 patients with active cancer between March 5th, 2020 to May 3rd, 2020. We analyzed cumulative incidence of thrombosis and risk factors associated to the cancer patient's cohort. RESULTS: We diagnosed 10 thrombotic events in 8 oncological patients with a cumulative incidence of 9.3%. A statistically significant association was found regarding thrombosis and history of obesity (p=0.009). No differences related to cumulative incidence of thrombosis between both groups were detected (9.8% vs 5.80%) in our hospital (p=0.25). CONCLUSION: No significant differences were observed in the cumulative incidence of thrombosis in the two study groups. The thrombotic effect of Covid-19 is not as evident in cancer patients and does not seem to be added to its prothrombotic activity.

The problem with coagulopathy .

Coagulopathy is inextricably linked to the field of hemostasis and thrombosis. In the same manner hemostasis and thrombosis may deal with bleeding and clot formation, coagulopathy also may be connected to bleeding or thrombosis, depending on the circumstances. However, when the same designation, "coagulopathy," may represent two clinically distinct presentations, it can create confusion. It is our obligation as hemostasis and thrombosis specialists to scotch this dubiety and direct the correct usage of coagulopathy.

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system.

BACKGROUND: Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. METHODS: Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. RESULTS: Our long COVID cohort's symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. CONCLUSION: Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Quantitative interpretation of PF4/heparin-EIA optical densities in predicting platelet-activating VITT antibodies.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a prothrombotic, heparin-induced thrombocytopenia (HIT)-mimicking, adverse reaction caused by platelet-activating anti-platelet factor 4 (PF4) antibodies that occurs rarely after adenovirus vector-based COVID-19 vaccination. Strength of PF4-dependent enzyme immunoassay (EIA) reactivity-judged by optical density (OD) measurements-strongly predicts platelet-activating properties of HIT antibodies in a functional test. Whether a similar relationship holds for VITT antibodies is unknown. OBJECTIVES: To evaluate probability for positive platelet activation testing for VITT antibodies based upon EIA OD reactivity; and to investigate simple approaches to minimize false-negative platelet activation testing for VITT. METHODS: All samples referred for VITT testing were systematically evaluated by semiquantitative in-house PF4/heparin-EIA (OD readings) and PF4-induced platelet activation (PIPA) testing within a cohort study. EIA-positive sera testing PIPA-negative were retested following 1/4 to 1/10 dilution. Logistic regression was performed to predict the probability of a positive PIPA per magnitude of EIA reactivity. RESULTS: Greater EIA ODs in sera from patients with suspected VITT correlated strongly with greater likelihood of PIPA reactivity. Of 61 sera (with OD values >1.0) testing negative in the PIPA, a high proportion (27/61, 44.3%) became PIPA positive when tested at 1/4 to 1/10 dilution. CONCLUSIONS: VITT serology resembles HIT in that greater EIA OD reactivity predicts higher probability of positive testing for platelet-activating antibodies. Unlike the situation with HIT antibodies, however, diluting putative VITT serum increases probability of a positive platelet activation assay, suggesting that optimal complex formation depends on the stoichiometric ratio of PF4 and anti-PF4 VITT antibodies.

Arteriovenous fistula thrombosis in hemodialysis patients with COVID-19: epiphenomenon or marker of severe clinical disease?

Background: Pandemic condition due to Coronavirus disease (COVID-19) caused a fastest augmentation of hospitalization, impairing the healthcare organization. As a consequence, diagnostic and therapeutic delays have been showed. COVID-19-associated coagulopathy is an endothelial disease related to SARSCoV-2 infection. Our study evaluated the thrombosis of arteriovenous fistula (AVF) as risk marker of mortality. Methods: the analysis included 24 dialysis-dependent patients admitted in a period between March 2020 and June 2021. Patients were divided based on AVF thrombosis: the A group without AVF thrombosis (13 patients), and the B group with AVF thrombosis events (11 patients). Pearson or Spearman' correlation tests were performed to detect possible confounding variable to include in multivariate models. Kaplan Meier and Cox regression analysis were performed to compute mortality analysis. Results: Delta D-dimer (Rho: 0.613, p=0.007), over-infections (Rho 0.456; p= 0,026), C-reactive Protein (CRP) (Rho=0.417, p=0.043), death (Rho=0.492, p=0.027), positive pulmonary imaging (Rho 0.388, p=0.074), and high OLT (0.408, p=0.047) were related to AVF thrombosis, using Pearson or Spearman correlation tests. Kaplan Meier test showed a death average of 19 days in group B compared to a global average of 38 days (p=0.029), and Cox analysis showed an HR of 5.01, 95% CI 1.01-24.99, p=0.049. Furthermore, AVF thrombosis explained about the 68% of the mortality, evaluated through the Harrel's C test. Conclusion: We can speculate that AVF thrombosis in hemodialysis patients with COVID-19 could be an early marker of both pro-coagulative process and severe clinical disease and it could be used to stratify patients and identify the ones that can be considered "frail".

Detection of Platelet-Activating Antibodies Associated with Vaccine-Induced Thrombotic Thrombocytopenia by Flow Cytometry: An Italian Experience.

Rare cases of thrombocytopenia and thrombosis after anti-COVID-19 adenovirus-associated mRNA vaccines (VITT) due to platelet-activating anti-platelet-factor 4 (PF4)/polyanion antibodies have been reported. VITT laboratory diagnosis, similarly to heparin-induced thrombocytopenia (HIT) diagnosis, requires immunoassays for anti-PF4/polyanion antibodies identification, such as ELISA assays and platelet-activating functional tests, such as heparin-induced platelet activation test (HIPA), to confirm their pathogenicity. We compared the flow cytometry (FC) measurement of platelet p-selectin exposure to the gold standard functional test HIPA for diagnosis confirmation in 13 patients with a clinical VITT syndrome (6M/7F; median age 56 (33-78)) who resulted positive to anti-PF4/polyanion antibodies ELISA assays (12/13). FC and HIPA similarly identified three different patterns: (1) a typical non-heparin-dependent VITT pattern (seven and six patients by FC and HIPA, respectively); (2) low/no platelet activation in patients under IvIg therapy (five out of five and two out of four patients by FC and HIPA, respectively); (3) a HIT pattern. Antibodies investigated by FC became negative after 7, 17, and 24 days of therapy in three patients. FC measurement of P-selectin exposure was as sensitive as HIPA but simpler to detect anti-PF4/polyanion antibodies in VITT patients. FC could reliably discriminate VITT from HIT, thus helping for the choice of the anticoagulant.

Anti-PF4 testing for vaccine-induced immune thrombocytopenia and thrombosis (VITT): Results from a NEQAS, ECAT and SSC collaborative exercise in 385 centers worldwide.

BACKGROUND: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine is a well recognized clinical phenomenon. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, raised D-dimer levels and positivity for immunoglobulin G (IgG) anti-platelet factor 4 (PF4) antibodies. OBJECTIVES: A collaborative external quality assessment (EQA) exercise was carried out by distributing five lyophilized samples from subjects with VITT and one from a healthy subject to 500 centers performing heparin-induced thrombocytopenia (HIT) testing. METHODS: Participating centers employed their locally validated testing methods for HIT assays, with some participants additionally reporting results for VITT modified assays. RESULTS: A total of 385 centers returned results for anti-PF4 immunoassay and functional assays. The ELISA assays used in the detection of anti-PF4 antibodies for the samples distributed had superior sensitivities compared with both the functional assays and the non-ELISA methods. CONCLUSION: ELISA-based methods to detect anti PF4 antibodies have a greater sensitivity in confirmation of VITT compared with functional assays regardless of whether such functional assays were modified to be specific for VITT. Rapid immunoassays should not be employed to detect VITT antibodies.

Arterial mesenteric thrombosis in COVID-19 positive patient: A case report.

COVID-19 has resulted in the death of a number of people around the world. Complications of COVID-19 including coagulopathy may contribute to the development of arterial ischemic events. Mesenterial thrombosis is a late complication of the disease. This clinical case presented the role of hypercoagulation in the clinical picture of the COVID-19 patients, which increased the risk of death.

The left atrial appendage thrombosis of in patients with persistent atrial fibrillation after the novel coronavirus infection.

Aim      To evaluate the incidence and features of left atrial appendage (LAA) thrombosis in patients with persistent atrial fibrillation (AF) after novel coronavirus infection (COVID-19).Material and methods  Percutaneous echocardiography (pcEchoCG) was performed for 128 patients with persistent AF prepared for cardioversion, 36 (28.1 %) of whom had had COVID-19. In 3 (8.3 %) patients, the lung lesion area was 50-75 %; in 31 (86.1 %) patients, 25-50 %; in 1 (2.8 %) patient, less than 25 %. One patient had no lung lesion. Median time from the onset of COVID-19 to the patient enrollment in the study was 76.5 days. At the time of enrollment, the polymerase chain reaction test for SARS-CoV-2 was negative in all patients.Results Patients after COVID-19 and those who had not had COVID-19 were comparable by age (62.5±9.2 and 62.4±9.1 years, respectively; р=0.956), gender (men 52.8 and 59.8 %, respectively; р=0.471), and risk of stroke (score 2.19±1.28 and score 1.95±1.35, respectively; р=0.350). Duration of the last arrhythmia episode was longer for patients after COVID-19 than for the comparison group (76.5 and 45.0 days, respectively; р=0.011). All patients received oral anticoagulants. 55.6 % of COVID-19 patients received rivaroxaban, whereas 62.0% of patients who had not had COVID-19 were treated with apixaban. Median duration of the anticoagulant treatment was longer for COVID-19 patients than for the comparison group (61.5 and 32.0 days; р=0.051). LAA thrombus was detected in 7 (19.4 %) patients after COVID-19 and in 6 (6.5 %) patients of the comparison group (р=0.030). In COVID-19 patients, the thrombus adhered to LAA wall over the entire thrombus length whereas in patients who had not have COVID-19, the thrombus had a free part that formed a sharp angle with LAA walls. In the presence of LAA thrombus, the LAA blood flow velocity was considerably higher for COVID-19 patients than for the comparison group (31.0±8.9 and 18.8±4.9 cm/sec, respectively; p=0.010). At the follow-up examination performed at 24.0 days on the average, the thrombus was found to be dissolved in 80 and 50% of patients after and without COVID-19, respectively (р=0.343).Conclusion      In patients with persistent AF after the novel coronavirus infection, LAA thrombosis was detected more frequently than in patients who had never had COVID-19; it was characterized by mural localization and was not associated with a decrease in LAA blood flow velocity.

COVID-19 infection as a new risk factor for penile Mondor disease.

BACKGROUND: Penile Mondor disease is a superficial dorsal vein thrombophlebitis of the penis, which mainly affects young and middle-aged men. It generally manifests as a visible painful cord located along the dorsal surface of the penis with signs of skin inflammation. The condition is usually self-limiting, but in severe cases a surgical procedure may be necessary in addition to pharmacological treatment. Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is associated with a frequent incidence of thrombophilia; therefore, such a prothrombotic state during infection may be a significant risk factor for penile Mondor disease. CASE PRESENTATION: The 34-year-old patient reported moderate pain felt on the surface of the penis. During the medical interview, the patient did not admit significant risk factors for Mondor Disease, apart from the previous, a month earlier COVID-19 disease. Examination revealed swelling erythema and a thick indurated cord on the surface of the penis. Color Doppler ultrasound was performed to confirm assumptions and exclude thrombosis of other penile vessels. Based on visible clots in the course of the superficial penile vein and after exclusion of vasculitis due to autoimmune disease the diagnosis of penile Mondor disease was made. Pharmacological therapy was implemented to further break down the clot and prevent rethrombosis in the penile vessels. The patient did not report any treatment complications and returned for a control visit, which revealed complete clot dissolution on ultrasound; therefore, complete recovery was stated. CONCLUSIONS: This case report presents the correlation between SARS-Cov-2 infection and penile Mondor disease, based on the confirmed influence of COVID-19 on the pathophysiology of thrombosis. It can be concluded that COVID- 19 is a risk factor for Mondor disease, as in the presented case the virus was the only prothrombotic risk factor for the patient. Consequently, the possibility of developing thrombosis in the form of penile Mondor disease should be taken into account among patients with post-COVID-19 and active SARS-Cov-2 infection.

Thrombosis and thrombocytopenia in COVID-19 and after COVID-19 vaccination.

Thrombosis that occurs in coronavirus disease 19 (COVID-19) is a serious complication and a critical aspect of pathogenesis in the disease progression. Although thrombocytopenia is uncommon in the initial presentation, it may also reflect disease severity due to the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to activate platelets. This occurs directly through the spike protein-angiotensin converting enzyme 2 (ACE2) interaction and indirectly by coagulation and inflammation activation. Dysregulation in both innate and adaptive immune systems is another critical factor that causes thrombosis and thrombocytopenia in COVID-19. Vaccination is the most potent and effective tool to mitigate COVID-19; however, rare side effects, namely vaccine-induced immune thrombotic thrombocytopenia (VITT)/thrombosis with thrombocytopenia syndrome (TTS) can occur following adenovirus-vectored vaccine administration. VITT/TTS is rare, and thrombocytopenia can be the clue to detect this serious complication. It is important to consider that thrombocytopenia and/or thromboembolism are not events limited to post-vaccination with vectored vaccine, but are also seen rarely after vaccination with other vaccines. Various conditions mimic VITT/TTS, and it is vital to achieving the correct diagnosis at an earlier stage. Antiplatelet factor 4 (PF4) antibody detection by the enzyme-linked immunosorbent assay (ELISA) is used for diagnosing VITT/TTS. However, false-positive rates also occur in vaccinated people, who do not show any thrombosis or thrombocytopenia. Vaccinated people with messenger RNA vaccine can show positive but low density and non-functional in terms of platelet aggregation, it is vital to check the optical density. If anti-PF4 ELISA is not available, discriminating other conditions such as antiphospholipid syndrome, thrombotic thrombocytopenic purpura, immune thrombocytopenic purpura, systemic lupus erythematosus, and hemophagocytic syndrome/hemophagocytic lymphohistiocytosis is critical when the patients show thrombosis with thrombocytopenia after COVID-19 vaccination.

Laboratory testing for platelet factor 4 antibodies: differential utility for diagnosis/exclusion of heparin induced thrombocytopenia versus suspected vaccine induced thrombotic thrombocytopenia.

Platelet factor 4 (PF4), a protein stored in the alpha-granules of platelets and released upon activation, forms cationic tetramers that bind with various polymeric anions, including heparin. Some individuals develop antibodies against PF4 in complex with heparin (PF4/H), which potentially lead to the onset of heparin induced thrombocytopenia (HIT). In some patients, this may cause activation and aggregation of platelets, promoting pathological thrombosis, in a process called heparin induced thrombocytopenia with thrombosis ('HITT'). Laboratories can assess for the presence of these antibodies using many PF4 antibody tests, including by enzyme linked immunosorbent assay (ELISA), latex immunoassay (LIA), chemiluminescence immunoassay (CLIA) and even rapid nanoparticle based lateral flow immunoassays. All these assays can identify such antibodies with high sensitivity, but methods may have variable specificity. For example, several studies have shown CLIA assays to have higher specificity to HITT than ELISA assays. Very recently, a new 'HITT-like' syndrome has been described in some individuals receiving adenovirus based COVID-19 (coronavirus disease 2019) vaccines. This condition has been given several names, including vaccine induced thrombotic thrombocytopenia (VITT) and thrombosis with thrombocytopenia syndrome (TTS), and also involves a mechanism mediated by antibodies formed against PF4. These antibodies can also be detected by PF4 antibody tests, but detection sensitivity appears to favour ELISA assays, with most other tests (including CLIA and LIA) not generally capable of detecting such antibodies. Additional functional assays assessing for PF4 mediated platelet activation may also be performed. The current review is focussed on laboratory testing for PF4 antibodies, in particular to distinguishing patterns in HITT versus VITT.

The Use of Total Thrombus Formation Analysis System as a Tool to Assess Platelet Function in Bleeding and Thrombosis Risk-A Systematic Review.

BACKGROUND: Today there are many devices that can be used to study blood clotting disorders by identifying abnormalities in blood platelets. The Total Thrombus Formation Analysis System is an automated microchip flow chamber system that is used for the quantitative analysis of clot formation under blood flow conditions. For several years, researchers have been using a tool to analyse various clinical situations of patients to identify the properties and biochemical processes occurring within platelets and their microenvironment. METHODS: An investigation of recent published literature was conducted based on PRISMA. This review includes 52 science papers directly related to the use of the Total Clot Formation Analysis System in relation to bleeding, surgery, platelet function assessment, anticoagulation monitoring, von Willebrand factor and others. CONCLUSION: Most available studies indicate that The Total Thrombus Formation Analysis System may be useful in diagnostic issues, with devices used to monitor therapy or as a significant tool for predicting bleeding events. However, T-TAS not that has the potential for diagnostic indications, but allows the direct observation of the flow and the interactions between blood cells, including the intensity and dynamics of clot formation. The device is expected to be of significant value for basic research to observe the interactions and changes within platelets and their microenvironment.