Managing severe warm autoimmune haemolytic anaemia after the SARS-Cov- 2 vaccine with emergency splenectomy and eculizumab

Autoimmune haemolytic anaemia (AIHA) is rare but described after the SARS-CoV- 2 Pfizer-BioNTech vaccine. We present a case of severe refractory warm AIHA after this vaccine, managed with emergency splenectomy and complement inhibition with eculizumab. A male in his teens with a history of liver transplant for biliary atresia (aged 2 years) and AIHA (aged 6 years) presented to his district general hospital with jaundice, dark urine, fatigue and chest discomfort 48 h after the first dose of SARS-CoV- 2 Pfizer-BioNTech vaccine (BNT162b2 mRNA). Investigations revealed haemoglobin (Hb) of 70 g/L and bilirubin of 98 mumol/L, which was treated as AIHA. The patient initially responded to prednisolone (1 mg/kg, 60 mg) but subsequently deteriorated and failed to respond to second-line rituximab (375 mg/m2) and two units of packed red blood cells (PRBC). By day 29 the patient had developed life-threatening anaemia culminating in a Hb of 35 g/L (after transfusion), lactate dehydrogenase (LD) of 1293 units/L and bilirubin of 228 mumol/L. This necessitated an immediate transfer to our tertiary centre for specialist support. Further investigations revealed a haptoglobin <0.1 g/L and direct antiglobulin test (DAT) strongly positive for IgG (4+) and negative for C3d. The peripheral blood film showed severe anaemia, nucleated red cells, anisocytosis and spherocytes with no autoagglutination, schistocytes or platelet clumps. Thrombocytopaenia (platelets 49 +/- 109/L) was present. Differentials were ruled out, such as paroxysmal nocturnal haemoglobinuria and heparin-induced thrombocytopaenia. HIV and hepatitis serology were negative, as were adenovirus, cytomegalovirus and Epstein-Barr virus PCR assays. A CT showed splenomegaly of 15.5 cm. Urinalysis found urobilinogen and bilirubin at high concentrations and negative urinary haemosiderin. Together, the investigations were consistent with warm AIHA. On day 29, four units of PRBC were transfused alongside 100 mg methylprednisolone and 1 g/kg IVIG. On day 30 the patient deteriorated despite the escalated treatment: Hb had only increased to 54 g/L, bilirubin was 200 mumol/L and LD was rising. Considering this life-threatening fulminant haemolysis, an emergency splenectomy was performed. This slowed haemolysis but did not completely ameliorate it: by day 33 the patient had received 15 units of PRBC. Thus, eculizumab, a terminal complement pathway inhibitor, was trialled to arrest intravascular haemolysis, alongside rituximab, repeat IVIG 1 g/kg, prednisolone 40 mg and tacrolimus 2 mg. This showed a favourable response, requiring less frequent transfusions and settling haemolysis. This case highlights the rare complication of warm AIHA with the SARS-CoV- 2 Pfizer-BioNTech vaccine, the use of emergency splenectomy for disease control, and the potential of eculizumab for refractory cases.

Membrane dysfunction in patients with severe COVID-19 treated with venovenous extracorporeal membrane oxygenation - analysis from a pooled data from a single centre

Objectives: It is well known that severe COVID-19 is associated with complex immunological and inflammatory dysregulation. Both these physiopathological events translate to a high risk of major thrombotic or hemorrhagic events. In patients treated with venovenous extracorporeal membrane oxygenation (VVECMO), membrane dysfunction might affect systemic oxygenation and limit its duration-expectancy. This study aimed to assess the possible causes of extracorporeal membrane failure in COVID-19 patients and its impact on outcome. Method(s): Retrospective, single-center, observational case-control study involving adult COVID-19 patients admitted to an ECMO referral centre in a tertiary university hospital. All patients required VVECMO for acute respiratory failure, including 48 cases who needed one or more extracorporeal membrane exchanges and 45 controls (no membrane exchange). These two groups were compared for demographic characteristics, severity of the disease using validated scores (SAPS II and SOFA), duration of ECMO run, coagulation assessment, cumulative anticoagulation dose, associated complications, and outcomes (ICU and hospital mortality). Result(s): Most patients were males (71.0%) and younger than 50 years (79.5%). Median ECMO run duration was significantly longer in the case group (35.0 vs 14.0 days, p <0.001), as well as ICU length-of-stay (45.5 vs 28 days, p <0.001). Membrane exchange tended to be associated with sepsis (56% vs 33%, p=0.037), major hemorrhage (58% vs 43%, p=0.022), heparin-induced thrombocytopenia (25% vs 9%, p=0.054), higher D-dimer title (17.36 ng/dL vs 7.5 ng/dL, p=0.07) and lower platelet counts (133.000/muL vs 154.000/muL). Median SAPS II (32.0 vs 33.0, p=0.20) and the mortality (27% vs 24%, p >0.99) were similar between these groups. Conclusion(s): In patients with SARS-CoV-2 pneumonia and severe hypoxemia treated with VVECMO support the emergence of infection, coagulopathy and inflammation were associated with high risk of membrane dysfunction. No impact on mortality could be confirmed from these data. Anticoagulation monitoring and dosing strategies should be reinforced to promote membrane protection.

Significance of heparin induced thrombocytopenia (HIT) in COVID-19: a systematic review and meta-analysis.

Heparin-induced thrombocytopenia (HIT) occurs in approximately 3% of patients receiving heparinoids. About 30-75% of patients with type 2 of HIT develop thrombosis as a result of platelet activation. The most important clinical symptom is thrombocytopenia. Patients with severe COVID-19 are among those receiving heparinoids. This meta-analysis performed to picture the current knowledge and results of published studies in this field. Three search engines were searched and 575 papers were found. After evaluation, 37 articles were finally selected of which 13 studies were quantitatively analyzed. The pooled frequency rate of suspected cases with HIT in 13 studies with 11,241 patients was 1.7%. The frequency of HIT was 8.2% in the extracorporeal membrane oxygenation subgroup with 268 patients and 0.8% in the hospitalization subgroup with 10,887 patients. The coincidence of these two conditions may increase the risk of thrombosis. Of the 37 patients with COVID-19 and confirmed HIT, 30 patients (81%) were treated in the intensive care unit or had severe COVID-19. The most commonly used anticoagulants were UFH in 22 cases (59.4%). The median platelet count before treatment was 237 (176-290) x 103/µl and the median nadir platelet count was 52 (31-90.5) x 103/µl.

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.

The Complexities of Managing Hypertriglyceridemia-Induced Pancreatitis in a Critically-Ill COVID-19 Patient

Introduction: Hypertriglyceridemia-induced pancreatitis (HTP) is a variant of pancreatitis requiring unique management. The complications of COVID-19 and its treatments can make HTP therapy more nuanced. This case describes a patient who presented in diabetic ketoacidosis (DKA) with HTP, and COVID-19. The patient developed renal and respiratory failure, necessitating hemodialysis (HD) and extracorporeal membrane oxygenation (ECMO), complicating an otherwise straightforward medical management plan. Case Description: A morbidly obese (BMI 38.9 kg/m2) 43-year-old male presented to an outside hospital with abdominal pain, and vomiting, and was found to have HTP with triglycerides (TG) >2000 mg/dL (<149 mg/dL) and presumed new-onset type 2 Diabetes (HbA1c 10.9%) with DKA. Treatment with fluids, intravenous (IV) insulin infusion and plasmapheresis were initiated. He developed hypoxia after receiving over 17 liters of fluids and was intubated, subsequently developing renal failure and was transferred to our tertiary center for HD and ECMO. On admission, he tested positive for COVID-19, rhabdomyolysis [creatinine kinase 5600 U/L (30-200 U/L)], HTP [TG 783 mg/dL (<149 mg/dL), lipase 461 U/l (7-60 U/L)], glucose 269 mg/dL (not in DKA), transaminitis [AST 184 U/L (4-40 U/L), ALT 61 U/L (4-41 U/L)] and renal failure (GFR 10 ml/min/1.73m2). IV insulin infusion was initiated for hyperglycemia worsened by COVID-19 dexamethasone treatment. Plasmapheresis was performed twice with minimal effect at maintaining a low TG. Fenofibrate was not initiated due to renal failure;Lovaza could not be given via oral gastric tube;Atorvastatin was attempted once rhabdomyolysis resolved, with subsequent worsening of liver function tests. Heparin infusion was initiated for deep vein thrombosis treatment and HTP but was stopped after development of heparin induced thrombocytopenia. The patient developed worsening hypoglycemia requiring cessation of IV insulin, hypotension requiring maximum pressor support, and worsening sepsis leading to his death. Discussion(s): This case illustrates the challenges of managing a patient with HTP and COVID-19. It demonstrates how a normally straightforward treatment algorithm can become increasingly complex when factoring the patient's comorbid conditions. The case highlights the importance of knowing both treatment indications and contraindications for HTP. In this case, HTP may have been the initial diagnosis, straightforward for most endocrinologists, but its treatments and comorbid conditions ultimately made the landscape more challenging, limiting effective management and ultimately leading to this patient's demise.Copyright © 2023

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.

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).

An Unusual Case of Combined Thrombosis and Amegakaryocytopenia Resembling Thrombosis With Thrombocytopenia Syndrome Following COVID-19 Infection in an Unvaccinated Patient.

As a global community, we have learned that the manifestations of severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2), infection, or coronavirus disease 2019 (COVID-19), extends far beyond respiratory compromise. Thrombocytopenia is thought to occur secondary to increased platelet consumption. Platelet activation and platelet-mediated immune inflammation contribute towards the thromboembolic complications seen in COVID-19 patients. In this report, the authors present the unusual case of a 75-year-old female with a history of COVID-19 infection who presented with a transient ischemic attack, thrombocytopenia, and amegakaryocytopenia.

Extracorporeal membrane oxygenation in COVID-19: Results of the Croatian Extracorporeal Membrane Oxygenation Referral Center.

At the beginning of the COVID-19 pandemic, the role of extracorporeal membrane oxygenation (ECMO) was uncertain and the outcomes of ECMO-treated patients were unfavorable. During the pandemic, medical community realized that carefully selected patients may benefit from ECMO support. The goal of the study was to present the outcomes of ECMO-treated patients with severe COVID-19 ARDS referred to the respiratory ECMO hub in Croatia and to determine variables that influenced the outcome. Our study included all adult patients with confirmed COVID-19 ARDS that required ECMO treatment, in the period between February 2020 and April 2022. All ECMO circuits were veno-venous with femoro-jugular configuration, with drainage at the femoral site. A total of 112 adult patients with COVID-19 induced ARDS were included in the study. All patients had veno-venous ECMO treatment and 34 survived. Surviving patients were discharged home either from the hospital or from a designated rehabilitation facility. The mortality was associated with the incidence of nosocomial bacteremia, occurrence of heparin induced thrombocytopenia and acute renal failure. In order to reduce the mortality in COVID-19 ECMO patients, the treatment should be started as soon as criteria for ECMO are met. Furthermore, complications of the procedure should be detected as soon as possible. However, despite even the optimal approach, the mortality in COVID-19 ECMO patients will surpass that of non-COVID-19 ARDS ECMO patients, mostly due to poor resolving and long lasting ARDS with longer ECMO runs and ensuing infectious complications.

Use of fondaparinux in patients with heparin-induced thrombocytopenia on veno-venous extracorporeal membrane oxygenation: A three-patient case series report.

Heparin-induced thrombocytopenia is a life-threatening immune-mediated complication of unfractionated heparin therapy. Fondaparinux is a therapeutic alternative, but it has limited evidence for its use in patients on extracorporeal membrane oxygenation (ECMO). We present a series of three adult patients with COVID-19 on ECMO who were diagnosed with heparin-induced thrombocytopenia after 7-12 days of unfractionated heparin treatment and were switched to fondaparinux. Fondaparinux was initiated with an intravenous loading dose of 5 mg, followed by a dose of 2.5 mg subcutaneously every 8-12 h. Dosage was adjusted according to daily measured anti-Xa concentration with a target range of 0.4-0.7 mg/L. The total duration of treatment with fondaparinux and ECMO ranged from 13 to 26 days. One major bleeding episode unrelated to fondaparinux therapy was observed, and the transfusions requirement was also low in all patients. The ECMO circuit was changed once in each patient. This series provides a deep insight into the use of fondaparinux over an extended period of time in patients on ECMO. Based on the presented data, fondaparinux can be considered a reasonable and affordable anticoagulant in patients without a high risk of bleeding.

Use of nafamostat mesilate for anticoagulation during extracorporeal membrane oxygenation: A systematic review.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) represents an advanced option for supporting refractory respiratory and/or cardiac failure. Systemic anticoagulation with unfractionated heparin (UFH) is routinely used. However, patients with bleeding risk and/or heparin-related side effects may necessitate alternative strategies: among these, nafamostat mesilate (NM) has been reported. METHODS: We conducted a systematic literature search (PubMed and EMBASE, updated 12/08/2021), including all studies reporting NM anticoagulation for ECMO. We focused on reasons for starting NM, its dose and the anticoagulation monitoring approach, the incidence of bleeding/thrombosis complications, the NM-related side effects, ECMO weaning, and mortality. RESULTS: The search revealed 11 relevant findings, all with retrospective design. Of these, three large studies reported a control group receiving UFH, the other were case series (n = 3) or case reports (n = 5). The main reason reported for NM use was an ongoing or high risk of bleeding. The NM dose varied largely as did the anticoagulation monitoring approach. The average NM dose ranged from 0.46 to 0.67 mg/kg/h, but two groups of authors reported larger doses when monitoring anticoagulation with ACT. Conflicting findings were found on bleeding and thrombosis. The only NM-related side effect was hyperkalemia (n = 2 studies) with an incidence of 15%-18% in patients anticoagulated with NM. Weaning and survival varied across studies. CONCLUSION: Anticoagulation with NM in ECMO has not been prospectively studied. While several centers have experience with this approach in high-risk patients, prospective studies are warranted to establish the optimal space of this approach in ECMO.

New perspectives on the induction and acceleration of immune-associated thrombosis by PF4 and VWF.

Platelet factor 4 (PF4), also known as chemokine (C-X-C motif) ligand 4 (CXCL4), is a specific protein synthesized from platelet α particles. The combination of PF4 and heparin to form antigenic complexes is an important mechanism in the pathogenesis of heparin-induced thrombocytopenia (HIT), but vaccine-induced immune thrombotic thrombocytopenia (VITT) related to the COVID-19 vaccine makes PF4 a research hotspot again. Similar to HIT, vaccines, bacteria, and other non-heparin exposure, PF4 can interact with negatively charged polyanions to form immune complexes and participate in thrombosis. These anions include cell surface mucopolysaccharides, platelet polyphosphates, DNA from endothelial cells, or von Willebrand factor (VWF). Among them, PF4-VWF, as a new immune complex, may induce and promote the formation of immune-associated thrombosis and is expected to become a new target and therapeutic direction. For both HIT and VITT, there is no effective and targeted treatment except discontinuation of suspected drugs. The research and development of targeted drugs based on the mechanism of action have become an unmet clinical need. Here, this study systematically reviewed the characteristics and pathophysiological mechanisms of PF4 and VWF, elaborated the potential mechanism of action of PF4-VWF complex in immune-associated thrombosis, summarized the current status of new drug research and development for PF4 and VWF, and discussed the possibility of this complex as a potential biomarker for early immune-associated thrombosis events. Moreover, the key points of basic research and clinical evaluation are put forward in the study.

Cerebral venous thrombosis after ChAdOx1 nCoV-19 vaccination: a systematic review

OBJECTIVE: To perform a systematic review of case reports or case series regarding thrombosis with thrombocytopenia syndrome (TTS) and cerebral venous thrombosis (CVT) related to ChAdOx1 nCoV-19 vaccination to address the clinical features, laboratory findings, treatment modalities, and prognosis related with CVT. SUBJECTS AND METHODS: We included 64 TTS patients from 19 articles, 6 case series and 13 case reports, in which thrombosis occurred after the first dose of ChAdOx1 nCoV-19 vaccination published up to 30 June 2021 in Embase, ePubs, Medline/PubMed, Scopus, and Web of Science databases. RESULTS: Of the 64 TTS patients, 38 (59.3%) had CVT. Patients with CVT were younger (median 36.5 vs. 52.5 years, p<0.001), had lower fibrinogen levels (130 vs. 245 mg/dL, p=0.008), had more frequent history of intracerebral hemorrhage (ICH), and had higher mortality rate (48.6% vs. 19.2%, p=0.020) than that of patients without CVT. In multivariable analysis, the possibility of presence of CVT was higher in younger age groups [odd ratio (OR): 0.91, 95% confidence interval (CI): (0.86-0.97, p<0.001)] and those with accompanying intracerebral hemorrhage (ICH) (OR: 13.60, 95% CI (1.28-144.12, p=0.045). CONCLUSIONS: Our study demonstrated that CVT related to ChAdOx1 nCoV-19 vaccination was associated with younger age, low levels of fibrinogen, presence of ICH and more frequent mortality compared to those of non-CVT. If TTS occurs after ChAdOx1 nCoV-19 vaccination, the presence of CVT in patients with young age or ICH should be considered.

High-Risk Pulmonary Embolism During Labor: JACC Patient Care Pathways.

While in labor, a 37-year-old woman developed acute dyspnea, hypoxemia, and tachycardia. Transthoracic echocardiography demonstrated severe right ventricular dilation and dysfunction, raising the suspicion of acute pulmonary embolism. The patient indeed had bilateral pulmonary embolism, necessitating percutaneous thrombectomy. Her course was complicated by another saddle pulmonary embolus, heparin-induced thrombocytopenia, and COVID-19 infection. This clinical case illustrates the importance of prompt diagnosis of acute pulmonary embolism in a peripartum female patient, the multidisciplinary approach of management, and how to approach clinical complications such as heparin-induced thrombocytopenia. Furthermore, long-term management in acute pulmonary embolism is presented.

Anti-Platelet4 Antibodes Testing in Pandemic: Which Test to Use

Introduction: Heparin-induced thrombocytopenia (HIT) is a severe adverse reaction to heparin caused by heparin-dependent, platelet-activating anti-plateletfactor 4 (PF4)/heparin antibodies. Vaccine-induced thrombocytopenia and thrombosis (VITT) following Astrazeneca vaccine has been described, associated with IgG anti-PF4 antibodies. Only ELISA immunoassays have been shown to detect anti-PF4 antibodies in these patients. Diagnosis of both HIT and VITT requires confirmation of heparin-dependent, platelets activating antibodies to avoid overdiagnosis and overtreatment Anti-PF4 laboratory assay requests during pandemic are in most of the cases of COVID positive vaccinated subjects, vaccinated healed from COVID19 subjects and COVID negative vaccinated subjects. Aim of our study was to investigate which laboratory test to use in patients with suspected HIT in this particular historical period Methods: Thirty patients with suspected HIT/ VITT were tested with three anti-PF4 immunoassays (HIT Ab Latex Immunoassay-Werfen, Acustar HITIgG CliA-Werfen, HPIA ELISA-Stago): 8 Astrazenca suspected VITT;1 Moderna and 1 Pfizer suspected VITT, 20 suspected HIT (9/20 COVID positive and 11/20 COVID negative patients). In order to confirm immunoassay positivity Platelet Aggregation Test (PAT) functional test was performed in all patients found to be positive for at least one assay. Result(s): 3/8 suspected Astrazenca VITT tested positive only by ELISA assay 2/3 tested positive by PAT (confirmed VITT). Both patients with suspected Pfizer and Moderna VITT tested negative for all immunoassays. 4/9 COVID patients with suspected HIT tested positive only by ELISA assay, only 1 of them tested positive by PAT. 4/11 COVID negative suspected HIT tested positive by all immunoassays performed and 1/11 tested positive only by ELISA and CliA immunoassays, among these 5 subjects only 2 were confirmed HIT by PAT functional assay Conclusion(s): With the only exception of suspected Astrazeneca VITT no superiority of IgG-ELISA over CliA or Latex immunoassay in sensitivity to HIT was observed. No single immunoassay method detected all probable HIT cases;if a single test is negative, a second immunoassay or a platelet activation assay should be considered where there is strong clinical suspicion Interestingly by using Bayesan diagnosis of HIT and CliA conservative negative cut-off < 0.13 U/ml suggested in the literature (Marchetti et al. 2020) all our CliA negative (results < manufacturer's cut-off of 1.0 U/mL) and ELISA positive suspected HIT/VITT were correctly classified as positive by CliA assay.

Comparison between unfractionated heparin (UFH) and fondaparinux on platelets and D-dimer level in COVID-19 patients with hypercoagulation

Introduction: This study aimed to compare the clinical effects between UFH and fondaparinux in COVID-19 patients with hypercoagulation. Material(s) and Method(s): This was a prospective cohort study. Samples were taken consecutively from hospitalized COVID-19 patients with hypercoagulation who received UFH or fondaparinux based on the standardized guidelines. A total of 71 patients met the inclusion criteria. Patients were evaluated for platelet and D-dimer values before and after administration of UFH or fondaparinux. Result(s): Although there was no difference in D-dimer reduction between the two groups (p = 0.44), fondaparinux showed a greater reduction, 26% against 22% for UFH. While on platelets, there was a significant difference (p = 0.04) between fondaparinux and UFH. Fondaparinux showed a reduced thrombocytopenia impact, as seen by an increase in pre-and post-therapy platelets of up to 50%, compared to 16% in UFH. In regard to the incidence of Heparin-Induced Thrombocytopenia (HIT), there was no significant difference between post-UFH therapy and post-fondaparinux therapy (p = 0.361). Conclusion(s): Fondaparinux did not reduce platelet levels as much as UFH, but there was no difference between the fondaparinux group compared to the UFH group in the effect of decreasing D-dimer levels and the sign of HIT. Copyright © 2022 Via Medica.

Virus-cell interactions of the ChAdOx1 viral vector give insights into vaccine-induced immune thrombotic thrombocytopenia

The ChAdOx1 nCoV-19 vaccine (AZD1222/Vaxzervia) adapted from the chimpanzee adenovirus Y25 (ChAd-Y25) has been critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. However, as part of the largest vaccination campaign in history, a potentially lifethreatening clotting disorder, thrombosis with thrombocytopenia, resembling heparin-induced thrombocytopenia (HIT), has been observed in a minority of AZD1222 patients following the first but not the second dose. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterised by development of thromboses at uncommon sites such as the cerebral venous sinuses and the splanchnic veins, with concomitant thrombocytopaenia. Therefore, to determine how ChAdOx1 may contribute to this novel disorder, it is critical to investigate the vector-host interactions of ChAdOx1. Structural and in vitro analysis of the fiber knob responsible for the primary virus-cell interaction suggests that coxsackie and adenovirus receptor (CAR) is the primary ChAdOx1 receptor. However, ChAdOx1 infection of CAR(-) human vascular endothelial cells has been demonstrated in vitro, suggesting ChAdOx1 may be using additional receptors. Dual tropism has been demonstrated in other human adenoviruses, with HAdV-D26 and HAdV-D37 both using sialic acid and CAR for transduction. Furthermore, coagulation factor X (FX), a factor demonstrated to bind to the hexon and facilitate human adenovirus type 5 (HAdV-C5) transduction via a CARindependent pathway does not increase ChAdOx1 infection, with amino acid alignment between the hexon proteins suggesting ChAdOx1 is unable to bind FX. Taken together, these findings suggest ChAdOx1 uses additional as yet unknown mechanisms for transduction, which may further contribute to the pathogenesis of VITT.

Implications of novel Adenovirus-Host interactions in Thrombosis with Thrombocytopenia Syndrome

In this study we investigated a link between adenovirus-based vaccines, deployed to fight the SARS-CoV-2 pandemic, and lifethreatening thromboembolisms after vaccination. Post-marketing surveillance showed that, following vaccination, Vaxzevria (ChAdOx1 based, AstraZeneca) and Jcovden (Adenovirus type 26 based, Johnson & Johnson) are associated with reduced platelet counts (thrombocytopenia) and blood clots (thrombosis) in some individuals. This extremely rare condition, with a rate between 1:50,000 - 1:350,000 cases per vaccinated individual, is above background rates of thrombosis in the population and can lead to fatal ischemic events including cerebral venous thrombosis, intracranial haemorrhage, and pulmonary embolism. It has been termed vaccine induced thrombotic thrombocytopenia (VITT) or thrombosis with thrombocytopenia syndrome (TTS). Heparin induced thrombocytopenia (HIT) is another condition with a similar clinical presentation to TTS. In HIT, immunoaggregates are formed due to the presence of strong anti-selfantibodies directed against Platelet Factor 4 (PF4). When similar anti-PF4 antibodies were detected in TTS patients, we investigated whether there could be a link between the adenovirus vectors used in the vaccines and PF4. This study demonstrates a direct interaction between adenovirus capsids and PF4 using surface plasmon resonance. We then utilized an integrative structural biology workflow including cryo-electron microscopy and molecular dynamics to characterize and demonstrate the mechanism of this interaction. These results demonstrate a previously unknown adenovirushost interaction and provide critical clues as to the underlying mechanism which causes TTS, including how these pathogenic anti-PF4 antibodies may be induced. We are therefore able to present a hypothesis as to the route of pathogenesis in TTS.