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Vaccine-induced immune thrombotic thrombocytopenia (VITT; synonym, thrombosis with thrombocytopenia syndrome, is associated with high-titer immunoglobulin G antibodies directed against platelet factor 4 (PF4). These antibodies activate platelets via platelet FcγIIa receptors, with platelet activation greatly enhanced by PF4. Here we summarize the current concepts in the pathogenesis of VITT. We first address parallels between heparin-induced thrombocytopenia and VITT, and provide recent findings on binding of PF4 to adenovirus particles and non-assembled adenovirus proteins in the 2 adenovirus vector-based COVID-19 vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S. Further, we discuss the potential role of vaccine constituents such as glycosaminoglycans, EDTA, polysorbate 80, human cell-line proteins and nucleotides as potential binding partners of PF4. The immune response towards PF4 in VITT is likely triggered by a proinflammatory milieu. Human cell-line proteins, non-assembled virus proteins, and potentially EDTA may contribute to the proinflammatory state. The transient nature of the immune response towards PF4 in VITT makes it likely that-as in heparin-induced thrombocytopenia -marginal zone B cells are key for antibody production. Once high-titer anti-PF4 antibodies have been formed 5 to 20 days after vaccination, they activate platelets and granulocytes. Activated granulocytes undergo NETosis and the released DNA also forms complexes with PF4, which fuels the Fcγ receptor-dependent cell activation process, ultimately leading to massive thrombin generation. Finally, we summarize our initial observations indicating that VITT-like antibodies might also be present in rare patients with recurrent venous and arterial thrombotic complications, independent of vaccination.

Background: Plasma transfusion is one of the basic treatments in patients with major blood loss. The anti-A and anti-B antibodies contained in the plasma demand ABO blood group compatibility. This is limiting the use of plasma in emergency situations and can cause a shortage in the supply of plasma of certain blood groups. We developed a method for anti-A and anti-B depletion by adsorbing plasma isoagglutinins using red blood cells. Materials and Methods: Three units of fresh frozen plasma were thawed after quarantine storage, pooled, and an aliquot of red cell concentrate was added. After 2 h of incubation at room temperature antibody-red-cell complexes were removed by centrifugation, the isoagglutinin-depleted plasma was split into three units and deep frozen. Isoagglutinin titers, free hemoglobin, residual red cells, clotting factor activity, and sterility of plasma units were determined after isoagglutinin depletion and a double freeze-thawing procedure. Results: Anti-B titers in group A plasma were reduced from values of 1:64 to 1:1 or lower, anti-A titers in group B plasma decreased from values of 1:128 to at least 1:16. Postprocedure clotting factor activities were preserved with 88.0 ± 7.3% (factor V), 106.9 ± 11.4% (factor VIII), and 84.0 ± 7.5% (factor XI) fulfilling the quality control requirements. No residual red cells were found, but free hemoglobin slightly increased to 53.7 ± 5.2 μmol/L. All units were sterile. Discussion: We described a method for the production of anti-A- and anti-B-depleted plasma in a closed system that uses standard equipment. The resulting isoagglutinin-depleted plasma may allow for blood group independent plasma transfusion.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Vaccination using the adenoviral vector COVID-19 vaccine ChAdOx1 nCoV-19 (AstraZeneca) has been associated with rare vaccine-induced immune thrombotic thrombocytopenia (VITT). Affected patients test strongly positive in platelet factor 4 (PF4)/polyanion enzyme immunoassays (EIAs), and serum-induced platelet activation is maximal in the presence of PF4. We determined the frequency of anti-PF4/polyanion antibodies in healthy vaccinees and assessed whether PF4/polyanion EIA+ sera exhibit platelet-activating properties after vaccination with ChAdOx1 nCoV-19 (n = 138) or BNT162b2 (BioNTech/Pfizer; n = 143). In total, 19 of 281 participants tested positive for anti-PF4/polyanion antibodies postvaccination (All: 6.8% [95% confidence interval (CI), 4.4-10.3]; BNT162b2: 5.6% [95% CI, 2.9-10.7]; ChAdOx1 nCoV-19: 8.0% [95% CI, 4.5% to 13.7%]). Optical densities were mostly low (between 0.5 and 1.0 units; reference range

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures 2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ addition on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4°C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4°C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4°C vs. 4°C + 10mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p 2+ ≥ 4mM prevents 4°C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was reduced in cold-stored platelets (45.65 ± 11.59% vs. RT stored platelets 56.38 ± 29.36; p = 0.042) but normal at 4°C + 10 mM Mg2+ (55.22 ± 11.16%, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4°C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4°C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.

Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intraindividual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by using differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolor flow cytometry was used to characterize resting and stimulated platelet subpopulations, and platelet age was determined based on RNA and HLA-I labeling. Signal transduction was analyzed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Compared with small platelets, large platelets adhered faster to collagen under flow and formed larger thrombi. Among the large platelets, a highly reactive juvenile platelet subpopulation was identified with high glycoprotein VI (GPVI) expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content, and increased platelet reactivity. There was a stronger difference in Akt phosphorylation and activation upon collagen stimulation between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population, explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage, and immune thrombocytopenia.

Background: SARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that-like autoimmune heparin-induced thrombocytopenia-is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies. Aims: To understand how the SARS-CoV-2 vaccine ChAdOx1 nCov-19 can induce anti-PF4 antibodies and how these antibodies induce thrombosis Methods: We investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using 3D-super-resolution microscopy, dynamic light scattering, and transmission electron microscopy. Vaccine composition was analyzed by mass spectrometry. Experimental vascular leakage models assessed early post-vaccine reactions. We evaluated VITT antibody-mediated platelet activation and formation of procoagulant DNA-containing neutrophil extracellular traps (NETs), including within VITT patient cerebral venous thrombi. Results: Biophysical analyses showed HEK cell line proteins and free virus proteins in the vaccine, formation of complexes between PF4 and vaccine constituents (including viral proteins) that were recognized by VITT antibodies. In a murine model, EDTA (vaccine constituent) increased microvascular leakage with dissemination of virus-and cell culture-derived human proteins. Free viral proteins and preexisting antibodies in normal human sera reacting with vaccine-containing constituents, likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. PF4-binding polyanions (polyphosphates, DNA) enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven NETs formation;further evidence for NETosis included elevated NETs biomarkers and low DNase activity in VITT sera, and NETs/ neutrophil-rich cerebral vein thrombi extracted from VITT patients. Conclusions: ChAdOx1 nCoV-19 vaccine constituents form antigenic complexes with PF4, and EDTA increases microvascular permeability, enhancing risk of early post-vaccination acute inflammatory reactions;PF4/polyanion antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. Resulting high-titer anti-PF4 antibodies activate platelets and induce neutrophil activation with NETosis, further fueling the VITT prothrombotic response.

INTRODUCTION: In the light of the ongoing SARS-CoV-2 pandemic, convalescent plasma is a treatment option for CO­VID-19. In contrast to usual therapeutic plasma, the therapeutic agents of convalescent plasma do not represent clotting factor activities, but immunoglobulins. Quarantine storage of convalescent plasma as a measure to reduce the risk of pathogen transmission is not feasible. Therefore, pathogen inactivation (e.g., Theraflex®-MB, Macopharma, Mouvaux, France) is an attractive option. Data on the impact of pathogen inactivation by methylene blue (MB) treatment on antibody integrity are sparse. METHODS: Antigen-specific binding capacity was tested before and after MB treatment of plasma (n = 10). IgG and IgM isoagglutinin titers were tested by agglutination in increasing dilutions. Furthermore, the binding of anti-EBV and anti-tetanus toxin IgG to their specific antigens was assessed by ELISA, and IgG binding to Fc receptors was assessed by flow cytometry using THP-1 cells expressing FcRI and FcRII. RESULTS: There was no significant difference in the isoagglutinin titers, the antigen binding capacity of anti-EBV and anti-tetanus toxin IgG, as well as the Fc receptor binding capacity before and after MB treatment of plasma. CONCLUSION: MB treatment of plasma does not inhibit the binding capacity of IgM and IgG to their epitopes, or the Fc receptor interaction of IgG. Based on these results, MB treatment of convalescent plasma is appropriate to reduce the risk of pathogen transmission if quarantine storage is omitted.

In life sciences, the material properties of suspended cells have attained significance close to that of fluorescent markers but with the advantage of label-free and unbiased sample characterization. Until recently, cell rheological measurements were either limited by acquisition throughput, excessive post processing, or low-throughput real-time analysis. Real-time deformability cytometry expanded the application of mechanical cell assays to fast on-the-fly phenotyping of large sample sizes, but has been restricted to single material parameters as the Young’s modulus. Here, we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition, our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties., Real-time deformability cytometry (RT-DC) is used for mechanical cell phenotyping but is limited to a single snapshot per cell and can only measure elastic modulus. Here the authors introduce dynamic RT-DC which can measure elasticity and viscosity in single cells by following dynamic cell shape changes.

Platelet transfusion has become essential therapy in modern medicine. Although the clinical advantage of platelet transfusion has been well established, adverse reactions upon transfusion, especially transmission of bacterial infection, still represent a major challenge. While bacterial contamination is favored by the storage of platelets at room temperature, cold storage may represent a solution for this important clinical issue. In this study, we aimed to clarify whether plasma has protective or detrimental effects on cold-stored platelets. We investigated the impact of different residual plasma contents in apheresis-derived platelet concentrates, stored at 4°C or room temperature, on platelet function and survival. We found that platelets stored at 4°C have higher expression of apoptosis marker compared to platelets stored at room temperature, leading to accelerated clearance from the circulation in a humanized animal model. While cold-induced apoptosis was independent of the residual plasma concentration, cold storage was associated with better adhesive properties and higher response to activators. Interestingly, delta (δ) granule-related functions, such as ADP-mediated aggregation and CD63 release, were better preserved at 4°C, especially in 100% plasma. An extended study to assess cold-stored platelet concentrates produced under standard care Good Manufacturing Practice conditions showed that platelet function, metabolism and integrity were better compared to those stored at room temperature. Taken together, our results show that residual plasma concentration does not have a cardinal impact on the cold storage lesions of apheresis-derived platelet concentrates and indicate that the current generation of additive solutions represent suitable substitutes for plasma to store platelets at 4°C.

Measurements of magneto-optical relaxation signals of magnetic nanoparticles functionalized with biomolecules are a novel biosensing tool. Upon transmission of a laser beam through a nanoparticle suspension in a pulsed magnetic field, the properties of the laser beam change. This can be detected by optical methods. Biomolecular binding events leading to aggregation of nanoparticles are ascertainable by calculating the relaxation time and from this, the hydrodynamic diameters of the involved particles from the optical signal. Interaction between insulin-like growth factor 1 (IGF-1) and its antibody was utilized for demonstration of the measurement setup applicability as an immunoassay. Furthermore, a formerly developed kinetic model was utilized in order to determine kinetic parameters of the interaction. Beside utilization of the method as an immunoassay it can be applied for the characterization of diverse magnetic nanoparticles regarding their size and size distribution.