COVID-19 Vaccine-Associated Immune Thrombosis and Thrombocytopenia (VITT): Diagnostic Discrepancies and Global Implications.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) has been reported in association with the coronavirus disease 2019 preventative adenovirus vector-based vaccines ChAdOx1 nCoV-19 (Oxford/AstraZeneca) and Ad26.COV2.S (Janssen/Johnson & Johnson) in hundreds of recipients across the globe. VITT is characterized by thrombosis, typically at unusual sites, low fibrinogen, and elevated plasma D-dimer, generally manifesting between 4 and 28 days following vaccination. Detection of anti-platelet factor antibodies using an enzyme-linked immunosorbent assay (ELISA) is often confirmatory. Although several similar principles subside in most diagnostic criteria for VITT, the presentation of a positive ELISA assay, use of expert hematology and neurology opinion, and exclusion of possible VITT cases outside the "standard" 4 to 28-day timeframe have contributed a lack of global standardization for defining VITT. Accordingly, the global and regional incidence of VITT differs according to the diagnostic pathway and case definition used. This has influenced the public perception of VITT's severity and the decision to use adenovirus vector-based vaccines for limiting severe acute respiratory syndrome coronavirus 2 infection. We hereby delineate the recognized pathogenic mechanisms, global incidence, discrepancies in diagnostic criteria, recommended treatments, and global implications to vaccine hesitancy from this coagulopathy.

Vaccine-associated thrombocytopenia.

Vaccination is the most cost-effective means of preventing and even eliminating infectious diseases. However, adverse reactions after vaccination are inevitable. In addition to common vaccine-related adverse reactions, some rare but serious adverse reactions have been reported, including secondary immune thrombocytopenia (ITP). The measles-mumps-rubella (MMR) vaccine is currently the only vaccine for which a cause-effect relationship with immune thrombocytopenia has been demonstrated with an incidence of approximately 0.087-4 per 100,000 doses, and the complication is mostly observed in children. In addition, thrombocytopenia can be induced by coronavirus disease 2019 (COVID-19) vaccines following COVID-19 vaccination primarily occurs within a few weeks post-vaccination. The condition mostly occurs in elderly individuals with no sex differences. Its incidence is approximately 0.80 to 11.3 per million doses. Some patients have previously suffered from chronic ITP likely to develop exacerbation of ITP after COVID-19 vaccines, especially those who have undergone splenectomy or are being treated with >5 medications. Based on clinical practice, first-line treatments for vaccine-associated thrombocytopenia are essentially limited to those used for primary ITP, including glucocorticoids and intravenous immunoglobulin (IVIg).

Potential mechanisms of vaccine-induced thrombosis.

Vaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare syndrome characterized by high-titer anti-platelet factor 4 (PF4) antibodies, thrombocytopenia and arterial and venous thrombosis in unusual sites, as cerebral venous sinuses and splanchnic veins. VITT has been described to occur almost exclusively after administration of ChAdOx1 nCoV-19 and Ad26.COV2.S adenovirus vector- based COVID-19 vaccines. Clinical and laboratory features of VITT resemble those of heparin-induced thrombocytopenia (HIT). It has been hypothesized that negatively charged polyadenylated hexone proteins of the AdV vectors could act as heparin to induce the conformational changes of PF4 molecule that lead to the formation of anti-PF4/polyanion antibodies. The anti-PF4 immune response in VITT is fostered by the presence of a proinflammatory milieu, elicited by some impurities found in ChAdOx1 nCoV-19 vaccine, as well as by soluble spike protein resulting from alternative splice events. Anti-PF4 antibodies bind PF4, forming immune complexes which activate platelets, monocytes and granulocytes, resulting in the VITT's immunothrombosis. The reason why only a tiny minority of patents receiving AdV-based COVID-19 vaccines develop VITT is still unknown. It has been hypothesized that individual intrinsic factors, either acquired (i.e., pre-priming of B cells to produce anti-PF4 antibodies by previous contacts with bacteria or viruses) or inherited (i.e., differences in platelet T-cell ubiquitin ligand-2 [TULA-2] expression) can predispose a few subjects to develop VITT. A better knowledge of the mechanistic basis of VITT is essential to improve the safety and the effectiveness of future vaccines and gene therapies using adenovirus vectors.

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.

NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of FcγRIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and FcγRIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively, leading to better patient outcomes.

Second-dose ChAdOx1 and BNT162b2 COVID-19 vaccines and thrombocytopenic, thromboembolic and hemorrhagic events in Scotland.

We investigated thrombocytopenic, thromboembolic and hemorrhagic events following a second dose of ChAdOx1 and BNT162b2 using a self-controlled case series analysis. We used a national prospective cohort with 2.0 million(m) adults vaccinated with two doses of ChAdOx or 1.6 m with BNT162b2. The incidence rate ratio (IRR) for idiopathic thrombocytopenic purpura (ITP) 14-20 days post-ChAdOx1 second dose was 2.14, 95% confidence interval (CI) 0.90-5.08. The incidence of ITP post-second dose ChAdOx1 was 0.59 (0.37-0.89) per 100,000 doses. No evidence of an increased risk of CVST was found for the 0-27 day risk period (IRR 0.83, 95% CI 0.16 to 4.26). However, few (≤5) events arose within this risk period. It is perhaps noteworthy that these events all clustered in the 7-13 day period (IRR 4.06, 95% CI 0.94 to 17.51). No other associations were found for second dose ChAdOx1, or any association for second dose BNT162b2 vaccination. Second dose ChAdOx1 vaccination was associated with increased borderline risks of ITP and CVST events. However, these events were rare thus providing reassurance about the safety of these vaccines. Further analyses including more cases are required to determine more precisely the risk profile for ITP and CVST after a second dose of ChAdOx1 vaccine.

Relapse of immune thrombotic thrombocytopenic purpura (iTTP) possibly triggered by COVID-19 vaccination and/or concurrent COVID-19 infection.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease that may be triggered by inflammation, including infection or vaccination. Since the start of the COVID-19 pandemic, several case reports were published on de novo or relapsed immune TTP (iTTP) in COVID-19-infected patients. Case reports of iTTP episodes following vaccination against COVID-19 are also emerging. We report a case of relapsed iTTP in a patient who received Moderna mRNA-1273 SARS-CoV-2 vaccine and developed concurrent severe COVID-19 infection. The patient's iTTP was successfully managed with caplacizumab, therapeutic plasma exchange and high-dose steroids. We summarise published cases of iTTP associated with COVID-19 infection or vaccination.

Successful Treatment of Immune Thrombocytopenic Purpura with Intracranial Hemorrhaging and Duodenal Bleeding Following SARS-CoV-2 Vaccination.

Several vaccines have been developed for coronavirus disease 2019 - caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - in record time. A few cases of immune thrombocytopenic purpura (ITP) following SARS-CoV-2 vaccination have been reported. We herein report a 90-year-old man who received the Pfizer-BioNTech SARS-CoV-2 vaccine (BNT162b2) and developed severe thrombocytopenia with intracranial hemorrhaging and duodenal bleeding, consistent with vaccine-related ITP. He was successfully treated with intravenous immunoglobulin, prednisolone, and eltrombopag and discharged without cytopenia. Vaccine-related ITP should be suspected in patients presenting with abnormal bleeding or purpura after vaccination.

Severe case of refractory immune thrombocytopenic purpura requiring splenectomy after the COVID-19 vaccine.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder caused by autoantibodies against platelet antigens resulting in platelet destruction and inhibition of platelet production. Occasionally, an inciting event such as a virus or vaccination can precipitate ITP. Several cases of ITP have been reported after the BTN162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) SARS-CoV-2 (COVID-19) vaccines. All reported cases of post-vaccination ITP have resolved with medical therapy until this case.A man in his mid-20s developed bleeding from ITP 2 weeks after receiving the second dose of the Pfizer SARS-CoV-2 vaccine. All inpatient medical treatment attempts failed. On hospital day 40, a splenectomy was performed and he ultimately recovered and was discharged.Awareness of potential vaccination side effects is a fundamental element of refining and improving patient safety. This case illustrates that given the right circumstances, serious refractory ITP can develop in response to the second dose of the Pfizer SARS-CoV-2 vaccine.

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.

Laboratory testing for VITT antibodies.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a highly prothrombotic disorder that like heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4). However, unlike HIT-where heparin at low concentrations (0.1-0.5 U/mL) typically enhances antibody-induced platelet activation, platelet activation by VITT sera is usually inhibited by heparin. Further, conventional platelet activation assays for HIT, such as the serotonin-release assay (SRA) and heparin-induced platelet activation (HIPA) test, often yield negative or atypical results when testing VITT sera. Nevertheless, VITT (like HIT) is a "clinical-pathological" disorder whereby laboratory detectability of platelet-activating anti-PF4 antibodies is crucial for diagnosis. VITT antibodies follow 2 fundamental principles of HIT laboratory testing: (1) high probability of a positive PF4-dependent enzyme-immunoassay (EIA), and (2) high probability of a positive platelet activation assay. However, optimal detection of VITT in platelet activation assays requires the addition of PF4, for example, PF4-enhanced SRA (PF4-SRA) and PF4-enhanced HIPA (PIPA). A novel whole blood assay, called the PF4-induced flow cytometry-based platelet activation (PIFPA) assay, exhibits high sensitivity and specificity for VITT. HIT and VITT sera/plasmas differ in their reactivity in rapid HIT immunoassays (90-97% sensitivity for HIT, <25% sensitivity for VITT), consistent with distinct antigen sites on PF4 recognized by HIT and VITT antibodies.

The clinical and laboratory diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious adverse syndrome occurring 5 to 30 days after adenoviral vector COVID-19 vaccination. Therefore, a practical evaluation of clinical assessments and laboratory testing for VITT is needed to prevent significant adverse outcomes as the global use of adenoviral vector vaccines continues. We received the clinical information and blood samples of 156 patients in Canada with a suspected diagnosis of VITT between April and July 2021. The performance characteristics of various diagnostic laboratory tests were evaluated against the platelet factor 4 (PF4)-14C-serotonin release assay (SRA) including a commercial anti-PF4/heparin immunoglobulin G (IgG)/IgA/IgM enzyme immunoassay (EIA, PF4 Enhanced; Immucor), in-house IgG-specific anti-PF4 and anti-PF4/heparin-EIAs, the standard SRA, and the PF4/heparin-SRA. Of those, 43 (27.6%) had serologically confirmed VITT-positive based on a positive PF4-SRA result and 113 (72.4%) were VITT-negative. The commercial anti-PF4/heparin EIA, the in-house anti-PF4-EIA, and anti-PF4/heparin-EIA were positive for all 43 VITT-confirmed samples (100% sensitivity) with a few false-positive results (mean specificity, 95.6%). These immunoassays had specificities of 95.6% (95% confidence interval [CI], 90.0-98.6), 96.5% (95% CI, 91.2-99.0), and 97.4% (95% CI, 92.4-99.5), respectively. Functional tests, including the standard SRA and PF4/heparin-SRA, had high specificities (100%), but poor sensitivities for VITT (16.7% [95% CI, 7.0-31.4]; and 46.2% [95% CI, 26.6-66.6], respectively). These findings suggest EIA assays that can directly detect antibodies to PF4 or PF4/heparin have excellent performance characteristics and may be useful as a diagnostic test if the F4-SRA is unavailable.

Vaccine-induced immune thrombocytopaenia and thrombosis (VITT) after COVID-19 vaccination.

COVID-19 represents a global health emergency, causing significant morbidity and mortality. Multiple vaccines have been distributed worldwide to control the spread of this pandemic. Several reports of thrombosis and thrombocytopaenia have been described after vaccination. These have been termed vaccine-induced immune thrombocytopaenia and thrombosis (VITT). We report a fatal case of VITT after receiving the first dose of Ad26.COV2.S vaccine. A man in his 30s developed thrombocytopaenia, massive haemoperitoneum due to spleen rupture and extensive portal and femoral vein thrombosis. The patient rapidly developed multiple organ failure and died. We attributed this condition to the vaccine due to the temporal relationship, presence of thrombosis and thrombocytopaenia, high levels of platelet factor 4 antibodies and exclusion of other diagnoses. Healthcare providers should be aware of such rare but fatal complications of COVID-19 immunisation, as early diagnosis of VITT may improve prognosis by allowing timely appropriate treatment.

Vaccine-induced immune thrombotic thrombocytopenia in a male after Ad26.COV2.S vaccination presenting as cerebral venous sinus thrombosis.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare and life-threatening complication that can occur after COVID-19 vaccination. After the first reports of VITT and CVST in 2021 after Ad26.COV2.S vaccination, the FDA and CDC recommended an emergency pause on 13 April 2021, and after extensive safety reviews, on 23 April 2021, the CDC's Advisory Committee on Immunization Practices (ACIP) reaffirmed its original recommendation for use of the Ad26.COV2.S vaccination. As of 31 August 2021, in the United States, 54 cases of VITT following Ad26.COV2.S vaccination (37 female, 17 male) have been reported out of 14.1 million total shots given, 29 of which had cerebral venous sinus thrombosis (CVST). With more data, on 16 December 2021, the CDC endorsed the ACIP recommendations for individuals to receive an mRNA COVID-19 vaccine in preference over the Ad26.COV2.S vaccination. We report a rare case of a male with confirmed VITT and CVST following Ad26.COV2.S vaccination.