57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr. med.

PD Dr. rer. nat.

Characterization of Anti-FVIII Antibodies in Acquired Hemophilia A Patients Using a bead-based multiplex immunoassay

Hemostaseology

Charakterisierung von Anti-FVIII-Antikörpern bei erworbenen Hämophilie-A-Patienten mit Hilfe eines Bead-basierten Multiplex-Immunoassays

Acquired hemophilia A (AHA) is an autoimmune disorder characterized by bleeding due to neutralizing antibodies against coagulation factor VIII (FVIII). The Nijmegen Bethesda assay (NBA) is the gold standard for detection of neutralizing antibodies (inhibitors). Whereas both, inhibitors and non-neutralizing antibodies can be detected by immunoassays.The in-house multiplex Luminex™ bead-based domain-epitope binding assay (LumiTope) comprised full-length (FL) and B-domain deleted (BDD) FVIII, along with nine purified FVIII single or multi-domains as previously described1. These proteins were coupled to magnetic beads to detect domain specific Immunoglobulin (IgG (IgG1-4), IgA, IgM and IgE) anti-FVIII antibodies in a large cohort of AHA patients. Data were acquired as Mean Fluorescence Intensity (MFI).The study included a total of 80 AHA patients. Demographic and baseline characteristics are detailed in Table 1. All patients were screened for anti-FVIII antibodies of IgG, IgM, IgA, and IgE isotypes, with no detection of anti-FVIII antibodies of the IgE subtype. Overall, the LumiTope assay demonstrated high sensitivity and specificity, with all ELISA-positive patients also testing positive in LumiTope. The BDD-FVIII bead exhibited higher MFI values compared to the FL-FVIII bead for anti-FVIII antibodies of the IgG isotype. MFI values for IgA and IgM antibodies were lower compared to IgG antibodies. AHA patients exhibited distinct differences in IgG subclasses and domain specificity of anti-FVIII antibodies compared to congenital hemophilia A (HA) patients. Analysis of IgG subtypes revealed that similar to HA patients IgG4 antibodies were predominant (96%). However, all other IgG subclasses were equally detected among AHA patients (IgG3 (60%), IgG1 (55%), and IgG2 (44%)). Interestingly, a comparison of the overall domain specificity of anti-FVIII antibodies in congenital and AHA patients showed that the majority of AHA patients had anti-FVIII antibodies directed towards the light-chain beads, specifically the C1C2 and C2 domain beads, whereas congenital H A patients primarily had antibodies directed towards the A2 and A2a2 bead.LumiTope assay provides a sensitive and specific method for detection but also domain specification of anti-FVIII-antibodies in AHA patients. Different domain specificities of the identified anti-FVIII antibodies may be associated with disease outcomes.The study was supported by Octapharma Germany. There is no other conflict of interest.

Poster

Dr. med. (Univ. Belgrad)

Dr. med.

Univ.-Prof. Dr. med.

Recombinant von Willebrand Factor in Bleeding Patients with Extracorporeal Circulation

Rekombinanter von Willebrand-Faktor bei blutenden Patienten mit extrakorporaler Zirkulation

Acquired von Willebrand Disease (aVWD) induced by extracorporeal circulation (ECC) is caused by loss of large multimers due to increased shear stress. Information is sparse regarding the contribution of ECC-aVWD to bleeding and wether or how to treat. As compared to plasma derived von Willebrand factor Vonicog alfa (rVWF) contains ultralarge multimers. We present pharmacodynamic data from patients with refractory bleeding who were treated with rVWF on an ultima ratio basis.Six patients with suspected ECC-aVWD were compared to a patient with congenital vWD (cVWD) type 2, all treated with rVWF (Veyvondi®, Takeda). VWF activity (VWF:Ac) as well as VWF antigen levels (VWF:Ag) (Innovance®, Siemens) were assessed before and immediately after substitution (trough and peak levels). VWF:Ac/VWF:Ag ratios (VWF:R) were used as a surrogate for high-molecular-weight multimers (HMWM). VWF:R &lt; 0.7 was considered indicative of loss of HMWM. Descriptive statistics were used for patient characteristics and therapeutic responses, including absolute and relative as well as body weight related values. n=10 measurements in six patients with ECC-aVWD and n=3 measurements in the patient with cVWD were analysed.Five out of six patients with suspected ECC-aVWD showed reduced VWF:R (range 0,23-0,64), while trough levels of VWF:Ac and VWF:Ag being normal. In contrast VWF:Ac in the patient with cVWD was reduced to 16%. Overall, increments of VWF:Ac and VWF:Ag after rVWF substitution ranged from +0,4 to +5,5%/1IU/kg b.w. and -0,9 to &gt;2,2%/1IU/kg b.w., respectively. The increase (mean value) of VWF:Ac per 1IU/kg b.w. rVWF was 1,5% and 1,3% in ECC-aVWD and cVWD, as compared to increases of vWF:Ag by 1,4% and 0,9%. Correspondingly, the mean difference between the VWF:R after and before substitution was 0,06 (ECC-aVWD) and 0,3 (cVWD) (0,002 and 0,007/1IU/kg b.w.) Trough levels of VWF:Ac and increment were inversely correlated (r = -0,64).ECC-aVWD shows reduced VWF:R with normal VWF:Ac. Thus loss of HMWM predominates and rVWF might specifically meet antihemorrhagic needs. Less effective correction of the VWF:R as compared to cVWD points to faster degradation. Overall the efficacy of rVWF substitution is highly variable, but is correlated inversely with trough levels of VWF:Ac. In future studies adressing the antihemorrhagic efficacy of VWF substitution in ECC-VWD, dosing should be targeted by monitoring the VWF:R.There is no conflict of interest.

PD Dr. med.

PD Dr.

Characterization of a novel SERPINC1 gene mutation (c.1247dupC) resulting in a secretion defect and type I antithrombin deficiency

Charakterisierung einer neuen SERPINC1-Genmutation (c.1247dupC), die zu einem Sekretionsdefekt und einem Antithrombinmangel Typ I führt

Antithrombin (AT), encoded by the SERPINC1 gene, acts as an important anticoagulant factor in hemostasis by inactivating coagulation factors IIa (thrombin), IXa, Xa, XIa and XIIa. AT deficiency poses a major risk for thromboembolic events. We identified a novel SERPINC1 frameshift mutation (c.1247dupC) in two sisters with confirmed AT deficiency and clinically relevant thrombosis and examined the effect of this mutation on cellular and molecular level [Ruf, M. et al. (2024): SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency. Thrombosis J. 22, 19. https://doi.org/10.1186/s12959-024-00589-5].The SERPINC1 mutation c.1247dupC, resulting in a frameshift in SERPINC1 coding sequence and subsequently altering the amino acid sequence of the AT C-Terminus (p.Ser417LysfsTer48), was identified by Next Generation Sequencing.Plasmids for the expression of wild-type and mutated SERPINC1 coding sequence fused to green fluorescent protein (GFP) or hemagglutinin tag (HA) were generated for transfection of HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot.The altered C-terminal sequence, caused by frameshift mutation p.Ser417LysfsTer48, affects the secretion of the mutated AT antigen. Since the GFP fusions of both wild-type and mutated AT are localized in the endoplasmic reticulum (ER) of the respective HEK293T cell lines, wild-type as well as mutated AT are able to enter the secretory pathway. In contrast to that, Western Blot analysis comparing cell lysates and cell culture supernatants of HEK293T cells transfected with constructs for GFP- or HA-tagged wild-type and mutated AT showed a strongly reduced secretion of the AT antigen with the p.Ser417LysfsTer48 mutation, indicating that the mutated AT is not able to pass the quality control of the ER.The p.Ser417LysfsTer48 mutation results in impaired secretion, which might be caused by polymerization induced by aberrant folding or by new cysteine residues introduced by the frameshift mutation, providing free thiol groups that are recognized by the thiol-dependent ER quality control. The here described mutation is causative for the type I AT deficiency observed in the two patients.There are no conflicts of interest.

Prof. Dr.

Prof. Dr.med.

Flow cytometry-based method to detect aggregates of non-muscle myosin IIA in erythrocytes

Nachweis von Aggregaten aus nicht-muskulärem-Myosin IIA in Erythrozyten mittels Durchflusszytometrie

Myeloproliferative neoplasms (MPN) are hematologic disorders characterized by clonal proliferation of one or more blood cell lines. Some MPN are associated with mutations in the JAK2 gene and affected patients have an increased thromboembolic risk. We could recently show, that patients with MPN show aggregates of the cytoskeletal protein non-muscle myosin IIA (NMMIIA) in erythrocytes in immunofluorescence microscopy (IF). we developed a flow cytometry-based method to detect NMMIIA aggregates in erythrocytes for early detection of MPN.Flow cytometry (FC) method was established using a mouse model and further validated with human samples. Whole blood was drawn from of Jak2+/VF mice (mutant) and corresponding Jak2+/+ wildtype mice (WT); and from patients with genetically confirmed MPN and healthy controls. Blood samples were fixed, permeabilized and stained with anti-NMMIIA-AF647 and anti-235a-PE. AF647-baseline fluorescence was set using samples of wild type mouse or healthy controls without NMMIIA-aggregates. We simultaneously prepared blood smears from human samples, fixed, and stained them with primary anti-NMMIIA- and secondary AF568-labelled antibody.Jak2-mutant mice showed a higher proportion of NMMIIA-aggregates compared to WT-mice in flow cytometry (8.6 % vs 1.3 %, p=0.0014). In patients with MPN and visible NMMIIA aggregates in IF, the mean percentage of NMMIIA-positive events in FC was 11.1 % vs 0.48 % in healthy controls (p&lt;0.0001). MPN-patients with no visible NMMIIA aggregates in IF displayed similar results in FC as healthy controls (0.66 %, p= 0.1422). Human blood samples were stored in glycine buffered saline for 7 days and analyzed again. Results were comparable to fresh samples. (healthy control 0.43%, MPN-patient with aggregates 14.6 %; MPN-patient without aggregates 0.73 %). None of the 50 healthy controls showed NMMIIA-aggregates in IF. 10 patients had aggregates in IF, of which only 1 was negative in FCAggregates of NMMIIA in erythrocytes can be detected by flow cytometry. The assay is fast and easy to apply to screen patients with possible MPN. We hypothesize that NMMIIA-aggregates in erythrocytes may also be found in patients with clonal hematopoiesis (CH), a stem cell aging related phenomenon, clinically ranging from healthy individuals to progression into MPN. This is currently investigated in an ongoing study.Disclosures available upon request

Dr.med.

Platelet function after orthopedic surgery – Increased risk due to hyperreactive platelets?

Thrombozytenfunktion nach orthopädischen Operationen – Erhöhtes Risiko durch hyperreaktive Thrombozyten?

Perioperative platelet consumption due to blood loss and coagulation stimulates de novo synthesis of juvenile platelets, which are characterized as RNA rich (reticulated) and HLA-I rich and more reactive. This study was performed to investigate, if prothrombotic juvenile platelets are synthesized in response to major orthopedic surgery, thereby orchestrating the common postoperative increased thrombotic risk.Twenty-seven patients undergoing total endoprosthesis or adjustment osteotomy were enrolled. Blood was drawn preoperative (day0) and postoperative (day1,3,5), incubated with 550µM Aspirin to equalize platelet function for the postoperative pain therapy and washed platelets were prepared. Juvenile platelets were assessed by measuring the immature platelet fraction (IPF), HLA-I and SYTO13 staining. Platelet activation was assessed via CD62p expression and procoagulant platelet formation (phosphatidyl serine+/CD62p+).Post surgery, none of the age markers tested, changed significantly (Fig. 1). Therefore, the overall proportion of juvenile platelets remained unaltered postoperatively. Congruently, platelet activation upon TRAP-6 stimulation showed only minor variation (Fig. 2A). Similar results were obtained after stimulation with ADP or CRP. However, postoperative platelet activation greatly differed between individuals (range 1.2-4-fold increase). Furthermore, the proportion of procoagulant platelets upon co-stimulation with TRAP-6 and convulxin remained stable at approximately 10%. Juvenile, HLA-rich platelets (top 20%) expressed 4 times more CD62p compared to HLA-poor platelets (lower 80%) upon TRAP-6 stimulation (p&lt;0.001, Figure 2B). Activation of both subfractions also did not change after surgery.Major orthopedic surgery does not seem to alter the proportion of juvenile platelets or global platelet function. Activation was higher in the juvenile compared to older platelet population but did not rise significantly postoperative. Consequently, the postoperative increased thrombotic risk is likely not caused by a rise of young hyperreactive platelets. However, a platelet associated risk of thrombosis may be present in individuals showing a more pronounced and persistent enhanced platelet activation pattern after surgery.Disclosures available upon request

Investigation of antibody-induced Fc-gamma-RIIA-mediated procoagulant platelet formation in real-time

Untersuchung der Antikörper-vermittelten Formation von prokoagulanten Plättchen in Echtzeit

Heparin-induced thrombocytopenia is a prothrombotic disease characterized by a rapid drop in platelet (PLT) count and high risk for the onset of thromboembolic events. Anti-platelet factor 4/heparin immunoglobulin G (IgG) immune complexes that activate PLTs and different immune cells are the causing factor for HIT. Although PLTs are well characterized for being pivotal in the complex pathophysiology of HIT, a deeper knowledge regarding the kinetics of antibody (Ab)-induced Fc-gamma-RIIA-mediated PLT alterations is missing. Utilizing a flow cytometry (FC)-based kinetic protocol and a simplified in vitro model of HIT, we aimed to investigate Ab-induced PLT alterations in real time.PLTs from healthy individuals were loaded with the calcium-indicator Fluo-5N which was followed by dual agonist stimulation with thrombin (THR; [0.125 U/mL]) and convulxin (CVX, agonist of collagen GPVI receptor; [500 ng/mL]) or with monoclonal antibody (moAb) anti-CD9 or isotype control (Iso; [both 5 µg/mL]) for 15 min under continuous measurement in FC. Changes in PLTs phenotype were assessed by co-staining with PE-labelled moAb PAC-1 and Annexin-V (AnxV)-Cy5, detecting the activated fibrinogen receptor and procoagulant PLT phosphatidylserine (PS), respectively.Compared to control, stimulation of PLTs with anti-CD9 resulted in the formation of procoagulant PLTs that were characterized by a high F5N signal, increased binding of AnxV and low binding of PAC-1. Although dual agonist stimulation with THR+CVX resulted in significant higher formation of F5N positive PLTs (mean %±SEM: 33.6±2.8 vs. 15.2±1.2), higher binding of AnxV was detected following stimulation with anti-CD9 (mean fold increase of AnxV binding±SEM: 25.23±1.4 vs. 3.7±0.5, p&lt;0.0001). Most importantly, while the presence of the direct thrombin inhibitor hirudin did not show the potential to prevent anti-CD9-induced procoagulant PLT formation, specific FcγRIIA blockade using moAb IV.3 resulted in a nearly complete abrogation of Ab-induced procoagulant PLT formation (mean %±SEM: 15.2±1.2 vs. 0.80±0.3).First findings from our kinetic studies indicate that anti-CD9 induces procoagulant PLTs in a FcγRIIA-dependent manner. Future studies with this time resolved model of Ab-induced PLT activation will allow the identification of the critical events that are essential for the generation of Ab-induced procoagulant PLTs in HIT.The authors have no conflicts of interest to disclose.

Posterausstellung 13

Wintergartenrestaurant + Foyer

Posterausstellung

Posterausstellung 13
Hemostaseology

Termin

Programm

Posterausstellung 13Hemostaseology

Termin

Programm

Posterausstellung 13
Hemostaseology