56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr.

Vorsitz

Autoimmune neutropenia of infancy is almost exclusively caused by IgG antibodies

Background The relevance of IgM antibodies in autoimmune mediated haemocytopenias remains a matter of debate. For autoimmune neutropenia, conflicting results were published. This study was performed as part of the Giessen Neutropenia Registry which propectively included children with suspected autoimmune neutropenia.Methods Children with suspected autoimmune neutropenia were prospectively enroled and data central to their suspected diagnosis were collected over 3 years. Sera were tested for the presence of autoantibodies against neutrophils by granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT). The presence of IgG and IgM antibodies was assesed in a subgroup of patients with positive initial GIFT in whom spontaneous remission of their neutropenia was documented.Results A total of 406 children were included in the study. A remission was reported for 114 patients during the study period. Of those, 96 had an initial positive GIFT. Their median age at diagnosis was 11 months (7-17.5), and their mean neutrophil count was 293 (±267)/µL, ranging from 0 to 975. In total 75/96 (78%) of children had at least one documented infection at diagnosis. In 29/96 (30%), a bone marrow aspirate was available. At the documented time point of remission, children were 27 months (18-41) old. In the initial sample send for diagnosis, IgG antibodies against neutrophils were detected in 94/96 children (98%), and IgM antibodies were detected in 2/96 (2%). We did not observe children with autoantibodies of both Ig classes. Conclusion To our knowledge, this is the first study to demonstrate the contribution of IgG or IgM antibodies in a well-defined clinical cohort with a typical course including, spontaneous disease remission. IgG antibodies were present in 98%. Only very rarely, IgM antibodies can be detected. We conclude that in general, screening for IgG autoantibodies is appropriate as a first diagnostic step. In cases with strong clinical suspicion but negative GIFT with anti-IgG, anti-IgM testing may be considered. Offenlegung Interessenkonflikt: Keine.

Immunohematology

Freier Vortrag (Reinhold-Eckstein-Investigator-Award)

The impact of antibody subclasses on anti-HPA-1a mediated platelet phagocytosis

Background Accelarated clerance of maternal IgG antibody (anti-HPA-1a)-opsonized fetal platelets by phagocytes represents the major mechanism in cases of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Until today, limited studies examining the impact of IgG antibody subclasses have been conducted. Such study is mainly hampered by the absence of good standards for anti-HPA-1a antibody subclasses and reliable platelet phagocytosis assay. In this study, both limitiations are carefully addressed.Methods Different IgG subclasses (IgG1, -IgG2, -IgG3 and -IgG4) of recombinant monoclonal antibody anti-HPA-1a (moAb 26.4) and effector-silent moAb 26.4 (IgG1-LALAP) were produced. Binding of antibody subclasses onto platelets was tested by flow cyometry using fluorescence labelled subclass-specific and Fcγ-specific secondary antibodies. The clearance of anti-HPA-1a sensitized platelets was analyzed by Whole Blood Platelet Phagocytosis Assay (WHOPPA). Platelets were labelled with pH sensitive dye (pHrodo-SE), opsonized with different moAb 26.4 subclasses and subjected to WHOPPA. The rate of pHrodo entgulfed platelets by monocytes was measured by flow cytometry.Results All 26.4 IgG subclasses did not interact with HPA-1bb, but bound equally to HPA-1aa typed platelets when tested with Fcγ-specific secondary antibodies by flow cytometry. In contrast, IgG subclass bound moAb 26.4 could only be detected by subclass specific secondary antibodies; bound moAb 26.4 IgG1 only reacted with anti-IgG1, but not anti-IgG2, -3 or -4. This specific reaction was also found with IgG2, -3 and -4. Analysis by WHOPPA showed that except effector-silent 25.4-IgG1 LALAP, all 26.4 IgG sublasses induced significant platelet phagocytosis by monocytes, but not by neutrophils. Highest phagocytosis rate was observed with 26.4-IgG3 (˜78.7%), followed by IgG1 (˜51.1%), IgG4 (˜35.4%), and IgG2 (˜19.1%).Conclusion Here, we enable reliable analysis of platelet phagocytosis caused by anti-HPA-1a IgG subclass using different subclasses of moAb 26.4 as standard. Our observation that moAb 26.4 IgG3 subclass induced platelet phagocytosis most strongly is interesting and is in line with the high capability of IgG3 to induce effector functions, most probably via high affinity FcγRI receptor on monocytes. The question wheather anti-HPA-1a IgG3 abs are dominated in maternal sera with severe FNAIT is intriguing.Offenlegung Interessenkonflikt: Alle Autoren haben kein Interessenkonflikt

Freier Vortrag

NAIT due to a presumably new alloantibody against Gp IIb

Background Neonatal alloimmune thrombocytopenia (NAIT) is a disorder of the newborn due to fetomaternal incompatibility of platelet antigens. Polymorphisms associated with platelet alloimmunisation are primarily located in ITGB3 &amp; ITGA2B. The latter one encodes for the α chain of Gp IIb/IIIa and to date, nine variants with corresponding alloantigens are known. The rare variant rs547581737 of ITGA2B (estimated frequency &lt;10-4) leads to p.Arg774Trp. So far, no alloimmunisation is described for this variant.Methods Here, we present a case of a male newborn with a suspected NAIT and a presumably new alloantibody. Blood samples of all direct family members were analysed. Antibody detection was performed by indirect MAIPA (in-house assay). Serological analysis included a crossmatch between paternal platelets and maternal serum. Initial genotyping was performed by commercial SSP-PCR (BAGene HPA-TYPE, BAG Diagnostics). For sequencing of ITGA2B and ITGB3 a direct taq-cycle method was chosen.Results No maternal platelet specific alloantibody could be detected by indirect MAIPA using a routine platelet panel. For HPA-1, -2, -3, -4, -5, -6, -9, -15 systems, no incompatibility between mother and newborn was found by SSP genotyping. However, a crossmatch between maternal serum and paternal platelets was strongly positive (Gp IIb/IIIa). Thus, the existence of an antibody against a low-frequency antigen was assumed and sequencing of both eligible genes (ITGA2B and ITGB3) was initiated. The rare variant rs547581737 with a nucleotide exchange in exon 23 (c.C2320T) was found in the father and the newborn, but not in the mother. At GnomAD, an allele frequency of 4.6x10E-5 is given for this variant and no homozygote sample was found so far.Conclusion In Europeans, NAIT is caused by anti-HPA-1a in the majority of cases, though several cases with antibodies to low-frequency antigens have been described. Thus, the rare variant of ITGA2B in our case is probably also responsible for maternal alloimmunisation and neonatal thrombocytopenia. Further serological investigations are needed to prove this antibody specificity. However, our case demonstrates once again that a serological crossmatch is essential for detecting antibodies to novel antigens.Offenlegung Interessenkonflikt: All authors declare no conflicts of interest.

Prof.

Dose-dependent elimination of HPA-1a platelets by subcutaneous RLYB212, a monoclonal antibody to prevent fetal and neonatal alloimmune thrombocytopenia

Background Fetal and neonatal alloimmune thrombocytopenia (FNAIT), based on a rare development of maternal alloantibodies against fetal platelets, may result in potentially catastrophic bleeding. No preventative FNAIT treatments are currently available. Prophylactic treatment with a monoclonal human platelet antigen (HPA)-1a antibody (RLYB212) is intended to drive rapid phagocytosis of fetal-derived HPA-1a antigen in maternal circulation to prevent alloimmunization to HPA-1a.Methods Following approval by the University Hospital Ethics Committee, participant informed consent was obtained. We assessed the dose-effect relationship of subcutaneous monoclonal anti-HPA-1a antibody (RLYB212) to eliminate HPA-1ab platelets transfused to HPA-1bb participants. Participants (HPA-1bb/human leukocyte antigen [HLA]-A2–negative healthy males) received a single dose of RLYB212 (0.09 mg or 0.29 mg) or placebo on day 1, followed by transfusion with 10 X109 HPA-1ab/HLA-A2–positive platelets on day 8. The proportion of HPA-1ab platelets in circulation over 7 days was determined by flow cytometry using HLA-A2 antibodies.Results RLYB212 rapidly eliminated HPA-1ab platelets, with a mean reduction in platelet elimination half-life of ≥90%, thus meeting proof-of-concept criteria. Platelet elimination kinetics of RLYB212 were dose-dependent and biphasic, with a mean lag phase of 4.8 h (0.09 mg) and 2.0 h (0.29 mg) vs 15.0 h for placebo; mean terminal half-life was 5.8 h (0.09 mg) and 1.6 h (0.29 mg) for RLYB212 vs 71.7 h for placebo. Conclusion Subcutaneous RLYB212 treatment resulted in dose-dependent and rapid elimination of HPA-1ab platelets in HPA-1bb participants. Platelet elimination kinetics were consistent with those of RhD(+) erythrocytes transfused to RhD-negative individuals after intramuscular administration of anti-RhD, which is known to prevent RhD alloimmunization when administered within 72 h of a suspected fetal-maternal hemorrhage. Our data support the potential use of subcutaneous RLYB212 as a prophylactic for FNAIT.Offenlegung Interessenkonflikt: Study-Funding: Rallybio, IPA, LLCInvited talks abstract/summary

Prod. Dr.

Evaluation of knowledge about fetal and neonatal alloimmune thrombocytopenia among gynaecologists and midwives in Germany: A survey-based study

Background Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) is a potentially life-threatening condition characterized by maternal alloantibodies against fetal platelets, which can lead to severe fetal and newborn thrombocytopenia. Recognizing clinically relevant FNAIT is important for the management of subsequent pregnancies, but little is known about the knowledge of midwives and gynaecologists on this subject. This study aimed to assess the knowledge of these professions regarding FNAIT.Methods We designed a questionnaire with qualitative and quantitative questions for use among midwives and gynaecologists working in clinical and ambulatory pregnancy, childbirth and postpartum care. The survey consisted of questions regarding the etiology, clinical manifestations, diagnosis, and management of FNAIT. Participants' responses were collected online via SurveyMonkey and analyzed using descriptive statistics.Results Preliminary results show, that 40% (12/32) of midwives and 19% (11/57) of gynecologists questioned, were aware of the most common signs and symptoms of FNAIT, while the majority of midwives and gynecologists would refer children with symptoms to further diagnostics based on typical symptoms. Clinically less significant symptoms relating to extracranial bleeding were more often ignored than more severe symptoms like intracranial hemorrhage or severe thrombocytopenia in laboratory testing. The survey is ongoing.Conclusion We designed a questionnaire with qualitative and quantitative questions for use among midwives and gynaecologists working in clinical and ambulatory pregnancy, childbirth and postpartum care. The survey consisted of questions regarding the etiology, clinical manifestations, diagnosis, and management of FNAIT. Participants' responses were collected online and analyzed using descriptive statistics.Offenlegung Interessenkonflikt: The authors report no conflicts of interest.

Frequency of antiplatelet antibodies in patients with thrombocytopenia after SARS-CoV-2 vaccination

Background Thrombocytopenia has been reported as a complication following vaccination against SARS-CoV-2. We analyzed clinical data and antiplatelet antibodies of patients who developed de-novo or recurrent thrombocytopenia in temporal relationship to SARS-CoV-2 vaccination.Methods Sera of patients with thrombocytopenia (platelet count &lt; 150 Gpt/L) sent to the Greifswald laboratory after SARS-CoV-2 vaccination between March 1st 2021 and March 31st 2022 were tested for antiplatelet antibodies against glycoprotein (GP) IIb/IIIa, GPIb/IX/V and GPIa/IIa by the indirect monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. Anti-platelet factor 4 (PF4) antibodies were tested by IgG specific ELISA. Clinical features (age, sex, vaccine type and date of vaccination, bleeding symptoms) of patients were extracted from the laboratory submission form.Results We identified 156 patients (68 females, median age: 57 years) with thrombocytopenia who tested negative for anti-PF4 antibodies: after vector-based (n=74), mRNA-based (n=72), or a heterologous, first vector-based + second mRNA-based vaccination (n=10). Median platelet count was 33 Gpt/L and symptom onset was 5 days post-vaccination. Antiplatelet antibodies were found after different vaccination schemes to a similar extend: vector-based vaccines 25.7%; mRNA-based vaccines 22.2%; and heterologous vaccinations 30%. Patients testing positive for antiplatelet antibodies had lower platelet counts (Fig.1) and reported more often bleeding symptoms.Conclusion In general, vaccination against SARS-CoV-2 is a safe measure to control the COVID-19 pandemic, but patients who experience new bleeding symptoms a few days after vaccination with low platelet counts might be screened for antiplatelet antibodies. In this study, we found antiplatelet antibodies in about 20-30% of patients, which suggests a similar pathogenesis but independent of the type of vaccine.Offenlegung Interessenkonflikt: Andreas Greinacher reports grants and non-financial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc., Rovi, Sagent, Biomarin/Prosensa, personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, Werfen (Instrumentation Laboratory), and nonfinancial support from Boehringer Ingelheim, Portola, Ergomed, GTH e.V., Universitätsmedizin Greifswald is one of the owners of a patent for a solid phase assay to detect HIT and VITT antibodies.

Immunohematology 2 - Non Red Cells

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