Whole Blood Procoagulant Platelet Flow Cytometry Protocol for Heparin-Induced Thrombocytopenia (HIT) and Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) Testing.

Heparin-induced thrombocytopenia (HIT) is a well-characterized, iatrogenic complication of heparin anticoagulation with significant morbidity. In contrast, vaccine-induced immune thrombotic thrombocytopenia (VITT) is a recently recognized severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria, AstraZeneca) and Ad26.COV2.S (Janssen, Johnson & Johnson) vaccines against COVID-19. The diagnosis of HIT and VITT involve laboratory testing for antiplatelet antibodies by immunoassays followed by confirmation by functional assays to detect platelet-activating antibodies. Functional assays are critical to detect pathological antibodies due to the varying sensitivity and specificity of immunoassays. This chapter presents a protocol for a novel whole blood flow cytometry-based assay to detect procoagulant platelets in healthy donor blood in response to plasma from patients suspected of HIT or VITT. A method to identify suitable healthy donors for HIT and VITT testing is also described.

Heparin-Induced Thrombotic Thrombocytopenia (HITT) and Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Similar but Different.

Heparin-induced thrombocytopenia (HIT) represents an autoimmune process whereby antibodies are formed against heparin in complex with platelet factor 4 (PF4) after heparin administration. These antibodies can be detected by a variety of immunological assays, including ELISA (enzyme-linked immunosorbent assay) and by chemiluminescence on the AcuStar instrument. However, pathological HIT antibodies are those that activate platelets in a platelet activation assay and cause thrombosis in vivo. We would tend to call this condition heparin-induced thrombotic thrombocytopenia (HITT), although some workers instead use the truncated abbreviation HIT. Vaccine-induced (immune) thrombotic thrombocytopenia (VITT) instead reflects an autoimmune process whereby antibodies are formed against PF4 after administration of a vaccine, most notably adenovirus-based vaccines directed against COVID-19 (coronavirus disease 2019). Although both VITT and HITT reflect similar pathological processes, they have different origins and are detected in different ways. Most notable is that anti-PF4 antibodies in VITT can only be detected immunologically by ELISA assays, tending to be negative in rapid assays such as that using the AcuStar. Moreover, functional platelet activation assays otherwise used for HITT may need to be modified to detect platelet activation in VITT.

Covax-19/Spikogen: The first exclusively Australian-developed human vaccine in four decades

Introduction/Aim: The development of safe and effective vaccines is crucial to conquering the COVID-19 pandemic. Recombinant proteins represent the best understood and reliable approach to pandemic vaccine delivery with well-established safety;however, they face challenges in design, structural characterisation, manufacture, potency testing and ensuring adequate immunogenicity. Method(s): Our team used in silico structural modelling to design a vaccine based on a stabilised spike protein extracellular domain (ECD). The insect cell expressed recombinant spike ECD was formulated with Vaxine's proprietary Advax-CpG55.2 adjuvant. Result(s): The vaccine known as Covax-19 or SpikoGen induced high titers of antibody and memory T-cells which translated to protection against SARS-CoV-2 infection in hamsters, ferrets, and aged monkeys. Despite numerous challenges along the journey, clinical trials in Iran during a major wave of delta variant infection confirmed SpikoGen vaccine was 78% effective in reducing risk of severe disease and with no evidence of vaccine-associated thrombosis, myocarditis, or sudden death, receiving marketing approval under emergency use authorisation in Iran on 6 October 2021. This made it the first recombinant spike-protein vaccine in the world to be approved, and the first Australian-developed human vaccine to receive marketing approval in four decades. Since approval millions of doses have been administered and additional trials in Australia and Iran have confirmed its effectiveness as a booster to prevent waning immunity, as well as its safety and effectiveness in children from the age of 5 years. The ongoing Australian and overseas clinical trial program is focussed on gaining better understanding the effect of dosing intervals on vaccine immunogenicity, gathering additional data on use as a booster, and development of new variant formulations. Conclusion(s): Covax-19/Spikogen is safe and effective adjuvanted recombinant protein vaccine.

Adverse reactions to COVID-19 vaccines: Differences and similarities

Background: COVID-19 virus vaccines are associated with adverse events. We aim to characterize and compare adverse reactions to different COVID-19 vaccines in a Portuguese centre. Method(s): Retrospective analysis of patients with adverse reactions to COVID-19 vaccines referred to our Immunoallergology Department between January and October 2021. The patients were divided according to the vaccine used: Pfizer/BioNTech (Pf), Moderna (M), or AstraZeneca (AZ). Result(s): 123 patients were included. 64 patients (52%) reacted to the Pf vaccine (77% women, mean age 49 years old);15 (12%) to the M vaccine (87% females, mean age 50 years old);and 44 (36%) to the AZ vaccine (75% women, mean age 64.8 years old). All groups showed a higher number of non-immediate reactions (>6h after inoculation): 59% for Pf, 60% for M, and 91% for AZ. Reactions to Pf and M were more frequently allergic-like (63% and 60%, respectively). Reactions to AZ were predominantly non-allergic (64%). The most frequently reported reactions for Pf and M were: sensation of throat tightness (Pf 31%, M 20%), urticaria (Pf 30%, M 27%), angioedema (Pf 17%, M 33%), constitutional non-specific symptoms (Pf 25%, M 27%), and local reactions on the inoculation site (Pf 20%, M 33%). There were 8 (13%) patients with suspected anaphylaxis with Pf, 3 (20%) with M, and none with AZ. The most frequently reported reactions for AZ were cardiovascular events (30%): myocardial, cerebral or pulmonary thromboembolic events (n = 6), phlebitis (n = 5), myocarditis (n = 1), and vaccine-induced immune thrombotic thrombocytopenia (n = 1). Other common reactions were constitutional non-specific symptoms (32%), local reactions on the inoculation site (18%), urticaria (23%), angioedema (14%), and non-urticaria rash (14%). Conclusion(s): Adverse reactions were more common in women. The mRNA vaccines were more frequently associated with allergic-like reactions, including anaphylaxis. In contrast, AZ vaccine was associated with nonallergic cardiovascular reactions. Up to 1/3 of patients in each group reported constitutional non-specific symptoms and local reactions on the inoculation site.

Coagulopathies after vaccination against SARS-CoV-2: The sole solution might lead to another problem

The common reported adverse impacts of COVID-19 vaccination include the injection site's local reaction followed by various non-specific flu-like symptoms. Nevertheless, uncommon cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) and cerebral venous sinus thrombosis (CVST) following viral vector vaccines (ChAdOx1 nCoV-19 vaccine, Ad26.COV2 vaccine) have been reported. This literature review was performed using PubMed and Google Scholar databases using appropriate keywords and their combinations: SARS-CoV-2, adenovirus, spike protein, thrombosis, thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia (VITT), NF-kappaB, adenoviral vector, platelet factor 4 (PF4), COVID-19 Vaccine, AstraZeneca COVID vaccine, ChAdOx1 nCoV-19 COVID vaccine, AZD1222 COVID vaccine, coagulopathy. The s and titles of each article were assessed by authors for screening and inclusion English reports about post-vaccine CVST and VITT in humans were also collected. Some SARS-CoV-2 vaccines based on viral vector, mRNA, or inactivated SARS-CoV-2 virus have been accepted and are being pragmatic global. Nevertheless, the recent augmented statistics of normally very infrequent types of thrombosis associated with thrombocytopenia have been stated, predominantly in the context of the adenoviral vector vaccine ChAdOx1 nCoV-19 from Astra Zeneca. The numerical prevalence of these side effects seems to associate with this particular vaccine type, i.e., adenoviral vector-based vaccines, but the meticulous molecular mechanisms are still not clear. The present review summarizes the latest data and hypotheses for molecular and cellular mechanisms into one integrated hypothesis demonstrating that coagulopathies, including thromboses, thrombocytopenia, and other associated side effects, are correlated to an interaction of the two components in the COVID-19 vaccine.Copyright © 2022, Iranian Pediatric Hematology and Oncology Society. All rights reserved.

Platelet-activating functional assay resolution in vaccine-induced immune thrombotic thrombocytopenia: differential alignment to PF4 ELISA platforms.

Background: Anti-platelet factor 4 (PF4) antibodies in vaccine-induced immune thrombotic thrombocytopenia (VITT) appear to be transient, with discrepant persistence depending on the platform used for detection. Objectives: We aimed to report a longitudinal study of antibody persistence using 2 ELISA platforms and 2 platelet-activating functional assays in a clinical cohort of patients with VITT referred for follow-up testing. Methods: In total, 32 Australian patients with VITT or pre-VITT, confirmed by expert adjudication, with samples referred for clinical follow-up were included. Clinical follow-up assays, including Stago and Hyphen ELISAs, procoagulant platelet flow cytometry, and modified PF4-serotonin-release assay, were performed according to the pattern of reactivity for that patient at diagnosis. Results: The median follow-up was 24 weeks after diagnosis. A general decline in anti-PF4 antibody levels and platelet-activating capacity over time was observed with a more rapid median time to resolution of 16 weeks by functional assay vs 24 weeks by Stago ELISA. Decline in platelet-activating antibody levels detected by functional assays mirrored Stago ELISA titer but not Hyphen. However, 87% of patients received a documented second vaccination and 74% received an mRNA booster with no reported adverse events. Conclusion: Anti-PF4 antibodies persist longer than functional platelet-activating antibodies in VITT but do not warrant avoidance of subsequent vaccinations. Persistence detection is assay-dependent. Stago ELISA may be a surrogate where functional assays are unavailable for follow-up testing of confirmed patients with VITT.

Autoimmune Diseases Affecting Hemostasis: A Narrative Review.

Hemostasis reflects a homeostatic mechanism that aims to balance out pro-coagulant and anti-coagulant forces to maintain blood flow within the circulation. Simplistically, a relative excess of procoagulant forces can lead to thrombosis, and a relative excess of anticoagulant forces can lead to bleeding. There are a wide variety of congenital disorders associated with bleeding or thrombosis. In addition, there exist a vast array of autoimmune diseases that can also lead to either bleeding or thrombosis. For example, autoantibodies generated against clotting factors can lead to bleeding, of which acquired hemophilia A is the most common. As another example, autoimmune-mediated antibodies against phospholipids can generate a prothrombotic milieu in a condition known as antiphospholipid (antibody) syndrome (APS). Moreover, there exist various autoimmunity promoting environments that can lead to a variety of antibodies that affect hemostasis. Coronavirus disease 2019 (COVID-19) represents perhaps the contemporary example of such a state, with potential development of a kaleidoscope of such antibodies that primarily drive thrombosis, but may also lead to bleeding on rarer occasions. We provide here a narrative review to discuss the interaction between various autoimmune diseases and hemostasis.

Vaccine-induced immune thrombotic thrombocytopenia post dose 2 ChAdOx1 nCoV19 vaccination: Less severe but remains a problem.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but established complication of 1st dose ChAdOx1 nCoV19 vaccination (AZD1222), however this complication after dose 2 remains controversial. OBJECTIVES: To describe the clinicopathological features of confirmed cases of VITT post dose 2 AZD1222 vaccination in Australia, and to compare this cohort to confirmed cases of VITT post 1st dose. METHODS: Sequential cases of clinically suspected VITT (thrombocytopenia, D-Dimer > 5x upper limit normal and thrombosis) within 4-42 days of dose 2 AZD1222 referred to Australia's centralised testing centre underwent platelet activation confirmatory testing in keeping with the national diagnostic algorithm. Final classification was assigned after adjudication by an expert advisory committee. Descriptive statistics were performed on this cohort and comparative analyses carried out on confirmed cases of VITT after 1st and 2nd dose AZD1222. RESULTS: Of 62 patients referred, 15 demonstrated presence of antibody mediated platelet activation consistent with VITT after dose 2 AZD1222. Four were immunoassay positive. Median time to presentation was 13 days (range 1-53) platelet count 116x10^9/L (range 63-139) and D-dimer elevation 14.5xULN (IQR 11, 26). Two fatalities occurred. In each, the dosing interval was less than 30 days. In comparison to 1st dose, dose 2 cases were more likely to be male (OR 4.6, 95% CI 1.3-15.8, p = 0.03), present with higher platelet counts (p = 0.05), lower D-Dimer (p = 01) and less likely to have unusual site thromboses (OR 0.14, 95% CI 0.04-0.28, p = 0.02). CONCLUSIONS: VITT is a complication of dose 2 AZD1222 vaccination. Whilst clinicopathological features are less severe, fatalities occurred in patients with concomitant factors.

Long-term outcome of patients with vaccine-induced immune thrombotic thrombocytopenia and cerebral venous sinus thrombosis

Introduction In early 2021, unanticipated thromboses, including cerebral venous sinus thrombosis (CVST) with thrombocytopenia, emerged as an adverse reaction (ADR) in patients who had been vaccinated with the AstraZeneca ChAdOx1 nCoV-19 vaccine. This ADR was termed vaccine-induced immune thrombotic thrombocytopenia (VITT) or thrombosis with thrombocytopenia syndrome (TTS). Although sporadic in nature, VITT can result in severe disease in the individual vaccinee. We followed up on the outcomes and status of neurological recovery of 49 cases of VITT with CVST that were reported to PEI. Method Assessment of the Extended Glasgow Outcome Scale (GOS-E) was performed within 3-6 months after the initial hospital admissions. Individual Glasgow Coma Scale (GCS) scores were reported by phone or electronically via a questionnaire or medical report by the treating physician of the hospital to which the patient was initially admitted. If a GCS score was not reported, an expert determined a score based on the patient's medical report. For most patients, follow-up was pursued about 3-6 months after hospital admission. The reported outcomes describe the patients' neurological status at 5-38 weeks (mean 20 weeks) after hospital admission. Outcomes were identified in 44 of the original 49 cases. Results Patient outcomes ranged from good recovery (13 patients, 29.6 %) to moderate disability (11 patients, 25.0 %) and severe disability or vegetative state (6 patients, 13.6 %). Fatal outcomes were reported in 14 patients (31.8 %). As anticipated, initial low GCS scores were associated with poor outcomes. By contrast, GCS scores > 10 were typically associated with improved neurological outcomes. Moreover, platelet count nadirs were correlated with patient outcomes. Low platelet counts were observed in fatal cases (GOS-E 1) with a mean count of 17,000 platelets/muL). Likewise, patients with better neurological outcomes (GOS-E scores of 5-6 and 7-8) presented with mean counts of 61,000 thrombocytes/muL. However, the course of the disease was not always predictable and showed significant individual variability. Conclusion We provide data on the outcome of VITT cases with CVST upon vaccination with the AstraZeneca adenoviral vector ChAdOx1 nCoV-19 COVID- 19 vaccine and found that the recovery of patients from CVST was very heterogeneous. While some patients exhibited good recoveries, others developed severe disabilities and major long-term complications. Collectively, our findings highlight the importance of paying attention to early signs of increased intracranial pressure and the onset of thrombocytopenia in patients with a recent history of vaccination with the AstraZeneca adenoviral vector ChAdOx1 nCoV-19 COVID-19 vaccine.

Acute limb ischemia secondary to vaccine-induced inmune thrombotic thrombocytopenia (VITT) after ChAdOx1 nCoV-19

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a syndrome that resembles to heparin-induced thrombocytopenia (HIT). Platelet factor 4 (PF-4) reacts to a vaccine component resulting formation of immune complex that stimulates an autoimmune reaction triggering platelet consumption causing thrombus formation and producing thrombotic events. When suspected is important to confirm for make a correct anticoagulation management to avoid complications related to unfractioned and low weight heparins use. In this report we describe a case of acute limb ischemia secondary to ChAdOx1 nCoV-19 vaccine (Astrazeneca, Cambridge, UK)Copyright © 2022

Anti-PF4 antibodies after SARS-CoV-2 vaccines: Long term follow-up

Introduction A subgroup of anti-platelet factor 4 (PF4) antibodies can activate platelets via Fcgamma RIIA and cause thrombotic and thrombocytopenic diseases such as heparin-induced thrombocytopenia and vaccine-induced immune thrombotic thrombocytopenia (VITT). Nonpathological anti-PF4 antibodies are detected in 1-7 % of healthy blood donors and in 2-8 % of SARS-CoV-2 vaccinated individuals. In this study, we investigated the long-term course of anti- PF4 antibodies detected after the first SARS-CoV-2 vaccination in healthy subjects and in patients with VITT. Method Five healthy subjects (all female, median age (range): 40 years (29-62) ) who had anti-PF4 antibodies after the first vaccination with ChadOx1 nCov19 (Vaxzevria, AstraZeneca-Oxford) were included. None of the subjects developed VITT. Blood samples were collected as part of a longitudinal study (TuSeRe:exact) evaluating the immune response to SARS-CoV-2 vaccines among employees of an University Hospital. In addition, data from 4 patients with VITT (3 female, median age (range): 44 years (22 -62 years)) were included for long-term follow-up of anti-PF4 antibodies. Anti-PF4/heparin antibodies were measured using a commercially available ELISA assay (Zymutest HIA IgG, Hyphen BioMed, France). Platelet activation was tested with a modified heparin- induced platelet aggregation assay (HIPA). Results In the non-VITT group, the median (range) OD for IgG anti-PF4/heparin antibodies was 0.69 (0.60-1.83) after the first vaccination. Blood samples were available up to 16 months after the first vaccination (range: 5-16 months). Anti-PF4 antibody levels decreased in all subjects despite further vaccination. However, antibody levels returned to pre-vaccination levels in only one subject. In one subject who had received two doses of ChadOx1 nCov19, anti-PF4 antibodies remained above OD 1.0 at the last follow-up. All samples were negative in the modified HIPA assay. Patients with VITT received mRNA-based vaccine as second vaccination against SARS CoV2. No significant drop in platelet count or new thromboembolic complication was observed. Conclusion Nonpathological anti-PF4 antibodies can be detected even several months after the first vaccination. The clinical significance of these antibodies in case of subsequent exposure to a vector vaccine or heparin is not yet clear. Furthermore, subsequent vaccination seems safe in VITT patietns.

Subsequent vaccinations in VITT patients other than SARS-CoV2 vaccination

Introduction Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare, but severe side effect after vaccination with adenovirus vector-based vaccines (ChAdOx1 nCoV-19, AstraZeneca and Ad26.COV2.S, Johnson & Johnson/ Janssen) in which platelet activating anti-platelet factor 4 (PF4) antibodies cause thrombocytopenia and thrombosis at unusual sites. Patients and treating physicians are concerned about whether other vaccinations can also trigger thrombosis in patients with a history of VITT. We showed that VITT patients can safely receive their second and third vaccination against Covid-19 with an mRNA-based vaccine. [1] However, there is limited information on whether other vaccines than against Covid-19 could booster platelet activating anti-PF4 antibodies. Uncertainty increased after a report of VITT caused by human papilloma vaccination. [2] Method In our follow-up study of patients with laboratory confirmed VITT (EUPAS45098), an anti-PF4/heparin IgG enzyme immune assay (EIA) and a PF4-dependent platelet activation assay (PIPA) were performed at regular intervals and after each vaccination reported to us. Results Seventy-one VITT patients (43 female, median age at VITT diagnosis 48, range 18-80) were followed for a median of 56 weeks (range: 13-77 weeks). During the follow-up period, eight vaccinations other than against Covid-19 were reported: Six vaccinations against influenza (three Influvac, two Vaxigrip Tetra, one Influsplit Tetra) and two consecutive vaccinations against tick-borne encephalitis (TBE) in one patient. In six patients who received vaccination against influenza, all patients showed decreasing or stable EIA optical density (OD) levels. None of them showed a reactivation of platelet-activating anti- PF4-antibodies in the PIPA. The patient who was vaccinated against TBE twice showed stable EIA OD levels and remained negative in the PIPA throughout. No new thrombosis or recurrent thrombocytopenia were observed after any vac- cination. Five out of six patients still received therapeutic anticoagulation, one patient did not receive any anticoagulative drug (Fig. 1). Conclusion Similar to observations after consecutive mRNA-vaccinations against Covid-19 in VITT patients, vaccinations against influenza and TBE very unlikely reactivate platelet-activating anti-PF4-antibodies. Further follow up of the VITT patient cohort is performed to detect any new safety signal related to recurrence of VITT. (Table Presented).

Developing an assay to distinguish between HIT and VITT antibodies

Introduction Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare, but severe side effect after Covid-19 and other vaccinations. First cases of VITT-mimicking antibodies in unvaccinated patients with recurrent thrombosis have been described. Differentiation between heparin-induced thrombocytopenia (HIT) and VITT is difficult in some patients. Widely used enzyme-linked immunoassays (EIA) cannot differentiate between the two, some of them even fail to detect VITT antibodies. So far, differentiation between HIT-like and VITT-like anti-PF4 antibodies can only be performed in specialized laboratories by functional tests using the heparin-induced platelet activation (HIPA) or PF4-induced platelet activation (PIPA) test. We have developed an assay, which can distinguish between HIT and VITT antibodies and can be used in any hospital laboratory. Method Confirming platelet-activation assays (HIPA and PIPA) were performed as described.[1] We defined 3 cohorts: 1) Negative controls (n = 112, including 35 healthy donors from before 2020, 46 clinical patients suspected for HIT but with negative EIA and HIPA and 31 non-thrombotic patients);2) classical HIT-patients with positive EIA and HIPA (n = 121);3) typical VITT patients (n = 63;presenting after vaccination with adenoviral vector-based Covid-19 vaccine and positive EIA and PIPA). Samples were analyzed by an automated coagulation analyzer ACL AcuStar (Werfen / IL Inc., Bedford, MA, USA) using HemosIL AcuStar HIT-IgG(PF4-H) and a prototype of VITT-IgG(PF4) assay according to the manufacturer's protocol. For both assays, raw data was analyzed as relative light units (RLU). Results All VITT samples were positive in the prototype VITT-assay (Fig. 1);only a few (n = 9;14.3 %) also showed weakly positive results in the HIT-assay. On the other hand, most of the HIT samples showed positive results in the HIT-assay (113;93.4 %), 34 of them (30.1 %) also reacted positive in the prototype VITT-assay (12 of them strongly;10.6 %), and three demonstrated an antibody pattern like autoimmune VITT. Negative control samples where all non-reactive in the HITassay and served to adjust the cutoff for the prototype VITT-assay. Conclusion The different reaction pattern of samples of HIT and VITT patients using HemosIL AcuStar HIT-IgG(PF4-H) and a VITT prototype assay was able to distinguish between the two antibody entities for the first time. The combination of assays can facilitate a rapid decision whether heparin may be used for treatment and also identify patients with autoimmune-VITT as a cause of recurrent thrombosis. (Table Presented).

Analysis of hematologic adverse events reported to a national surveillance system following COVID-19 bivalent booster vaccination.

Hematologic complications, including vaccine-induced immune thrombotic thrombocytopenia (VITT), immune thrombocytopenia (ITP), and autoimmune hemolytic anemia (AIHA), have been associated with the original severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines. However, on August 31, 2022, new formulations of the Pfizer-BioNTech and Moderna vaccines were approved for use without clinical trial testing. Thus, any potential adverse hematologic effects with these new vaccines remain unknown. We queried the US Centers for Disease Control Vaccine Adverse Event Reporting System (VAERS), a national surveillance database, through February 3, 2023, all reported hematologic adverse events that occurred within 42 days of administration of either the Pfizer-BioNTech or Moderna Bivalent COVID-19 Booster vaccine. We included all patient ages and geographic locations and utilized 71 unique VAERS diagnostic codes pertaining to a hematologic condition as defined in the VAERS database. Fifty-five reports of hematologic events were identified (60.0% Pfizer-BioNTech, 27.3% Moderna, 7.3% Pfizer-BioNTech bivalent booster plus influenza, 5.5% Moderna bivalent booster plus influenza). The median age of patients was 66 years, and 90.9% (50/55) of reports involved a description of cytopenias or thrombosis. Notably, 3 potential cases of ITP and 1 case of VITT were identified. In one of the first safety analyses of the new SARS-CoV-2 booster vaccines, we identified few adverse hematologic events (1.05 per 1,000,000 doses), most of which could not be definitively attributed to vaccination. However, three reports of possible ITP and one report of possible VITT highlight the need for continued safety monitoring of these vaccines as their use expands and new formulations are authorized.

Pituitary apoplexy following adenoviral vector-based COVID-19 vaccination

Pituitary apoplexy (PA) may complicate the course of coronavirus disease 2019 (COVID-19), posing a potential threat to life. Among vaccines designed to prevent COVID-19, there are those adenoviral vector-based, such as Vaxzevria® (formerly COVID-19 Vaccine AstraZeneca). The product insert states that it can cause very rare coagulation disorders, in particular thrombosis with thrombocytopenia syndrome in some cases accompanied by bleeding, cerebrovascular venous or sinus thrombosis, and thrombocytopenia, including immune thrombocytopenia, also associated with bleeding. Here, we report the onset of PA after Vaxzevria® in a 28-year-old healthy Caucasian female, who experienced long-lasting tension-type headache, hyperprolactinemia and menstrual changes, without thrombocytopenia or thrombosis.

Acute pulmonary thromboembolism after messenger RNA vaccination against coronavirus disease 2019: A case report.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is defined as thrombosis after inoculation of adenovirus vector vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VITT rarely occurs with messenger RNA vaccines, and the use of heparin for VITT is also controversial. A 74-year-old female patient with no risk factors for thrombosis was brought to our hospital after loss of consciousness. Nine days before admission, she had received the third vaccine against SARS-CoV-2 (mRNA1273, Moderna). Immediately after transport, cardiopulmonary arrest occurred, prompting extracorporeal membrane oxygenation (ECMO). Pulmonary angiography showed translucent images of both pulmonary arteries, resulting in the diagnosis of acute pulmonary thromboembolism. Unfractionated heparin was administered, but D-dimer subsequently became negative. Pulmonary thrombosis remained in large volume, indicating that heparin was ineffective. Treatment was shifted to anticoagulant therapy using argatroban, which increased D-dimer level and improved respiratory status. The patient was successfully weaned from ECMO and ventilator. Anti-platelet factor 4 antibody examined after treatment initiation showed negative results; however, VITT was considered as an underlying condition because of the time of onset after vaccination, the ineffectiveness of heparin, and the absence of other causes of thrombosis. In case heparin is not effective, argatroban can be an alternative therapy against thrombosis. Learning objective: During the coronavirus disease 2019 pandemic, treatment with vaccine against severe acute respiratory syndrome coronavirus 2 has been widely performed. Vaccine-induced immune thrombotic thrombocytopenia is the most common thrombosis after adenovirus vector vaccines. However, thrombosis can also occur after messenger RNA vaccination. Though commonly used for thrombosis, heparin may be ineffective. Non-heparin anticoagulants should be considered.

Long VITT: A case report.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) has been described following adenovirus vector-based COVID-19 vaccines. This condition is associated with important morbidity and mortality following thrombosis related complications. Diagnosis is confirmed based on results of platelet factor 4 ELISA detecting anti-PF4 antibodies and of platelet-activation assay. Initial treatment strategy has been established but long-term management and follow up remain unclear. Most platelet-activation tests become negative after 12 weeks. We describe a case of VITT which can now be characterized as long VITT. The patient initially had a lower limb ischemia, pulmonary embolism and cerebral vein thrombosis. He was treated with prednisone, intravenous immunoglobulin, argatroban and had a lower limb revascularization surgery. Rivaroxaban was then initiated for the acute treatment and continued for the secondary prevention of recurrent events. The patient still demonstrates positive platelet-activation tests and thrombocytopenia after more than 18 months of follow-up. No recurrent thrombosis or bleeding event have occurred. He is not known for any relevant past medical history other than alcohol consumption and slight thrombocytopenia (130 × 109/L since 2015). It is unclear if the ongoing and more important thrombocytopenia could be explained by the persistent platelet-activating anti-PF4 antibodies or the patient's habits. Managing long VITT is challenging considering uncertainty regarding risks and benefits of long-term anticoagulation and potential needs of additional treatment. Additional data is needed to offer optimal long-term management for this patient population. We suggest that long VITT diagnosis definition might include the persistence within patient serum/plasma of anti-PF4 platelet-activating antibodies with clinical manifestations (e.g., thrombocytopenia) for more than 3 months.

Characterization of adenovirus interactions with PF4 using a novel ELISA-qPCR technology

The COVID vaccines Janssen and AstraZeneca, based respectively on adenovirus (AdV) serotypes AdV26 and ChAdOx1, have been associated with rare cases of vaccine-induced thrombotic thrombocytopenia (VITT). It was recently demonstrated that the AdVs of the vaccines can bind to the blood protein platelet factor 4 (PF4), an interaction very likely to be involved in VITT. Since there are hundreds of known AdV serotypes, we hypothesized that certain serotypes have a lower affinity for PF4. We therefore aimed to screen a library comprising dozens of serotypes from different AdV species. For this purpose, we established the ELISA-qPCR technology. Like in standard ELISA, AdV viral particles are allowed to specifically interact with PF4 proteins coated on a plate. However, the revelation is not performed by antibody staining, but by qPCR after the genomes of bound AdVs are released through alkaline heat lysis. This technology enables fast, accurate and unbiased assessment of virus molecular interactions. Unlike most tested serotypes, the species D AdV37, AdV69 and AdV70 did not bind to PF4. Even though the ELISA-qPCR technique is not sensitive enough to detect potential low-affinity interactions, these serotypes may avoid or decrease the risk of VITT and represent safer candidates for vaccine or gene therapy vector development. In order to gain deeper insights into the mechanism of virion binding to PF4, we tested how AdV5 affinity for PF4 was affected by genetic removal or PEGylation of different hypervariable regions (HVR) of the hexon protein of the capsid.

Incidence of anti-platelet factor4/polyanionic antibodies, thrombocytopenia, and thrombosis after COVID-19 vaccination with ChAdOx1 nCoV-19 in Thais

Background: Prevalence of antiplatelet-factor 4 (PF4)/polyanionic antibodies occurring after vaccination with ChAdOx1 nCoV-19 was low. Most of these antibodies are weak and not associated with vaccine-induced thrombotic thrombocytopenia. It remains unknown whether these antibodies are preexisting or occur after the vaccination. Aim(s): In this study, we demonstrated the incidence of anti-PF4/ polyanionic antibodies, thrombocytopenia, and thrombosis after vaccination with ChAdOx1 nCoV-19 in Thais. Method(s): We conducted a prospective study in health care workers and the general population who received COVID-19 vaccination with ChAdOx1 nCoV-19. Blood collection for complete blood count, D-dimer, and anti-PF4/ polyanionic antibodies was performed before vaccination (day 0), day 10, and day 28 after vaccination. Anti-PF4/ polyanionic antibodies were detected using enzyme-link immunosorbent assay (ELISA). Functional assay with platelet aggregation was performed for all positive anti-PF4/ polyanionic antibody ELISA tests. Result(s): A total of 720 participants receiving the first, second, or third booster dose of ChAdOx1 nCoV-19 were included in the study. Baseline characteristics are presented in Table 1. Three participants developed seroconversion. Therefore, the incidence of anti-PF4/ polyanionic antibodies was 0.42% (95% confidence interval 0.08, 1.23). However, these antibodies were low titer. Fourteen (1.9%) participants had preexisting anti-PF4/ polyanionic antibodies before the vaccination but the optical density of anti-PF4/ polyanionic antibodies did not significantly increase over time (Figure 1). None of the anti-PF4/ polyanionic positive sera induced platelet aggregation. Abnormal D-dimer levels following the vaccination were not different among the positive and negative anti-PF4/ polyanionic groups (11.8% vs. 13.2%, p = 0.86). Thrombocytopenia occurred in one person with negative anti-PF4/ polyanionic antibodies. No clinical thrombosis occurred. Conclusion(s): We found a low incidence of seroconversion of anti-PF4/ polyanionic antibodies after vaccination with ChAdOx1 nCoV-19 in Thais. Most of the anti-PF4/ polyanionic antibodies are preexisting and did not significantly increase after vaccination with ChAdOx1 nCoV-19. Some participants with anti-PF4/ polyanionic antibodies had elevated D-dimer levels. However, no thrombocytopenia and thrombosis were observed. (Table Presented).