57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Univ.-Prof. Dr. med.

Vorsitz

Prof. Dr.

PD Dr. med.

Prof.

Dr. med.

Low but not absent risk of alloimmunization against RHD in patients receiving RHD-incompatible red blood cells during liver transplantation

Hemotherapy and Patient Blood Management

Geringes Risiko der Anti-D-Bildung in RHD-negativen Patienten nach Lebertransplantation, die RHD-positive Erythrozytenkonzentrate erhalten haben

Transfusion demand is high in liver transplantation (LT) and thus, RHD-positive red blood cell concentrates (RBCs) are frequently given to RHD-negative patients during LT. It has been suggested that formation of anti-D antibodies is then rare, probably due to immunosuppression, in these patients. We assessed the rate of anti-D formation in RHD-negative patients undergoing LT who were transfused with RHD-positive RBCs.RHD-type and transfusion history of patients who underwent LT were reviewed retrospectively. In RHD-negative patients, who received RHD-positive RBCs, results of antibody screening test (indirect antiglobuline test and with papain treated RBCs) and direct antiglobulin test as well as medical records were retrospectively evaluated.527 patients underwent 576 LT within the observation period between 2010 und 2023. 87 patients were RHD-negative, of whom 42 were transfused with RHD-positive RBCs. In 34 of them, an antibody screening test result was available more than 2 after weeks after the last RHD-positive RBC was administered. In two of them, a transient, weak anti-D antibody was detectable. In one of these patients, anti-D was also detectable after elution, without overt signs of hemolysis. In both patients, anti-D disappeared rapidly in the further course.Transient anti-D occurred in a minority of RHD-negative patients undergoing LT after transfusion of RHD-positive RBCs. The evidence of a primary immune response was low. To save stocks of RHD-negative RBCs, transfusion of RHD-positive RBCs to RHD-negative patients during LT can be considered as relatively safe probably due to immunosuppression but should be limited to men and women beyond the childbearing age as precautionary measure.none

Abstract lecture

Dr. rer. nat.

Long read RHD-RHCE sequencing of samples with inconsistent fetal RHD real-time PCR results

Immunohematology

Long read RHD-RHCE Sequenzierung zur Abklärung der nicht eindeutigen real-time PCR Ergebnisse zur fetalen RHD Bestimmung

According to German guidelines, non-invasive fetal RHD analysis should be offered to every RhD-negative pregnant woman to avoid unnecessary anti-D prophylaxis for women with an RHD negative fetus. The fetal RHD genotype can reliably be determined in maternal blood by real-time PCR (rtPCR) in the vast majority of cases. Long-read RHD-RHCE sequencing was used to resolve discrepancies between RhD phenotype and genotyping.We use two different rtPCR test systems in parallel for fetal RHD detection in maternal blood: the commercial Free DNA Fetal Kit RhD Duplex Kit (Institut de Biotechnologies Jacques Boy) and an in-house rtPCR method. Both methods utilize amplification of exon 5, 7, and 10 of the RHD gene on the CFX Opus Real-Time PCR System (Biorad). Early amplification (Ct&lt;34.5) of at least one of the RHD exons indicates the presence of maternal RHD sequences. For sequencing, RHD and RHCE are enriched either by long-range PCR, covering the whole genes of RHD and RHCE, or by a customized hybridization-based panel (Twist Bioscience). Long read sequencing is performed on a Sequel IIe (PacBio), and data are analyzed using SeqNext software (JSI).In 33 out of 2008 samples for fetal RHD genotyping, the presence of maternal RHD sequences was suggested despite negative RhD serology. Nine samples showed early amplification of all exons, ten samples showed amplification of exons 7 and 10, and 14 samples of exon 10 only. Long-read RHD and RHCE sequencing has been completed for 11 samples so far. The presence of maternal RHD sequences was confirmed in all samples. In samples with early exon 5, 7, and 10 amplification, RHD11, RHD15, two RHD01EL.08 alleles, and a novel allele with a splice site mutation were detected. All 3 samples with early exon 7 and 10 amplification were identified as RHD08N.01. Three samples with early exon 10 amplification can be explained by hybrid RHD-CE-D alleles.In 1.6% of serologically RhD-negative samples, the presence of maternal RHD sequences was detected by rtPCR. Complete long read sequencing of RHD and RHCE genes enabled the determination of the underlying genetic basis for missing RhD expression. Rare genetic variants were detected in our patient population from Germany. Long read sequencing is a powerful tool to assign rare RHD and RHCE alleles, even with complex genetic structures like RHD-RHCE hybrid alleles.No conflicts to declare

PD Dr.

Use of a simplified maldi-TOF workflow without DNA purification for mass-scale blood donor typing

Benutzung eines vereinfachten maldi-TOF Arbeitsprozesses ohne DNA Aufreinigung zur Hochdurchsatz-Spendertypisierung

In our blood service, we use simplified limited quality molecular typing (SLQMT) to identify blood donors with useful antigen pattern including rare donors. SLQMT is devised for an optimal information to effort ratio, simplicity, non-calls and rare miscalls are accepted, because relevant results are confirmed by serology before unit release. We evaluated the impact of a switch from pooled capillary electrophoresis (PCE) to MALDI-TOF (mTOF) in 2019.A single well mTOF multiplex PCR was devised predicting 24 SNV including those determined previously. DNA was prepared using Sigma Extract-N-Amp reagents, amplifed in plates ( 2 µl DNA + 3 µl primer/Taq mix + 2 µl of extension primer mix) and analyzed on a mass array Dx analyzer 4 with chip prep module (Agena Bioscience). Initial results were cross-validated with PCE-typed samples; "new" SNV were validated by sequencing. Percentage of no-calls, number of donors tested, rare donors detected, donated units with predicted antigen typing results (including PCE) and discrepancies to PCE or serology results were monitored.The number of SNV investigated by mTOF (n=24) increased (PCE: n=9). Donors tested per year were similar (mTOF: 14817 (mean, range: 14474 to 15377), PCE: 20861). No call rates were JKa 2.2% (PCE: 4.6%), Fya 2.9% (13.6%), M 10.2% (4.9%), S 9.9% (8.4%), Coa 2.2% (2.7%), Lub 8.3% (1.7%), Yta 2.1% (3.3%), Kpb 8.1% (6.7%). We detected 558 rare donors, most frequenly Yt(a-) (n=192) and Co(a-) (n=166). "New" types of rare donors included Lu:-8 (n=48), Sc:-1 (n=4) and LW(a-) (n=3). Neither Js(a+b-) nor In(a+b-) donors were found consistent with heterozygote prevalences of 0.051% (n=33) for Js(a+b+) and 0.003% (n=2) for In(a+b+). In 2023, the 31% of donated units had typing results for Jka (Fya : 31%, M 30%, S 30%). Rare units numbered 1163, including 474 Yt(a-) and 370 Co(a-). Among 135 discrepancies detected since 29-Aug-2023, 67 were due to incorrect "old" PCE-typing, 41 to incorrect serologic results and 27 to incorrect mTOF predictions, most frequently antigen N (n=10). Only one misprediction was caused by a silent allele (JK*02N[c.1000G&gt;T]).Our simplified mTOF workflow extends the SLQMT concept and allows long-term sustained molecular donor typing. Most relevant antigens are predicted with sufficient accuracy at limited cost. Adding more SNV is unlikely to give more information without disproportionately high increases in workload.no COI to declare

Rapid full-length haplotyping method of RHD/RHCE by nonspecific long-range PCR-based sequencing

Schnelle Methode zur Haplotypisierung von RHD/RHCE in voller Länge durch unspezifische long-range PCR Sequenzierung

Resolving allele-specific sequences (haplotypes) for the RHD and RHCE gene can be highly challenging. Molecular techniques like cloning or cDNA sequencing have been utilized for this purpose, but they are not suitable in time-sensitive situations. Here, we introduce a rapid long-read sequencing method for haplotype reconstruction at the RH locus, exemplified by three distinct cases for which both serological and genotypic tests failed to offer unambiguous RHCE allele assignment.Standard serological methods showed RH:1,2,3,4,5 phenotype in two donor cases. Additionally, PCR-SSP kits revealed heterozygous c.733C&gt;G resulting in V+VS+ antigen expression. The third case was a patient urgently requiring transfusion; serotyped RH:1,2,-3,4,5 and molecular analyses showed presence of c.667G&gt;T. To fully cover exons 1 to 10 from RHD and RHCE, we designed five generic primer pairs co-amplifying both homologous genes. PCR amplicon lengths ranged from 12.3 to 15.2 kb for RHD and 13.3 to 15.2 kb for RHCE. Overlaps of PCR amplicons comprised &gt;1 kb and were chosen to lie within polymorphic regions to facilitate variant phasing. Pooled PCR products were barcoded allowing multi-sample Nanopore sequencing on MinION flow cells.Long-range PCRs and library preparation were completed within half a day. Sequencing produced &gt;20,000 reads in ~4 hours. Variant calls enabled full-length haplotype reconstruction in all three samples by identifying variants in overlapping regions. The c.733C&gt;G variant was phased to different RHCE backgrounds: one donor exhibited a RHCE*ce allele (RHCE*01.20.01 | RHCE*04), while the other displayed the V+VS+ causing variant on a RHCE*Ce background (RHCE*02.30 | RHCE*03). Contrary to frequency-based expectations, the c.667G&gt;T variant found in the patient was linked to RHCE*Ce (RHCE*02.22) associated with weak C rather than lying on RHCE*ceMO (RHCE*01.07, partial c), resulting in transfusion recommendation from a RH:1,-2,-3,4,5 donor.Here, we present an amplicon-based Nanopore sequencing approach capable of rapidly and reliably obtaining complete RHD and RHCE haplotypes, at least in the absence of complex structural variation. This method is particularly well-suited for urgent scenarios where precise genetic allele data is critical for ensuring optimal blood provision. To the best of our knowledge, no other technique offers the ability to accurately infer RHD/CE haplotype sequences within a time frame of one working day.No conflict of interest

Abstract lecture (Abstract winner)

Mechanism of low birth weight in fetal-neonatal alloimmune thrombocytopenia (FNAIT): antibody-dependent impaired trophoblast function

Mechanismus des niedrigen Geburtsgewichts bei fetal-neonataler Alloimmunthrombozytopenie (FNAIT): Antikörper-abhängige Beeinträchtigung der Trophoblastenfunktion

In addition to thrombocytopenia, a reduction in birth weight has been reported as a clinical feature of fetal-neonatal alloimmune thrombocytopenia (FNAIT), potentially indicating an impaired placental function. Previous analyses have shown that the most common antibodies in FNAIT, anti-HPA-1a, react not only with aIIbb3 on platelets but also with avb3, which is expressed on the trophoblast. Using a human first-trimester trophoblast cell line (HTR-8), we investigated the effects of antibodies on trophoblast cell function.Trophoblast network formation and remodeling were investigated in HTR-8 cells using time-lapse video microscopy. VEGF pre-stimulated cells were cultured on Matrigel and the effects of antibodies against b3, av, or avb3 on network formation were studied. In addition, using a silicone insert, a standardized wound area was generated, and the migratory capacity of HTR-8 cells was evaluated. Cellular effects of antibody binding were investigated using a flow-cytometry-based assay for the detection of reactive oxygen species (ROS).A significant inhibition of the trophoblast network formation was observed following incubation with complex-specific antibodies, but not with anti-b3 or anti-av. Similarly, in contrast to single-chain specific antibodies, the presence of complex-specific antibodies significantly slowed down the migration of HTR-8 cells into the wound area and induced a delayed wound closure. Finally, a 1.5-fold increase in cellular ROS production was observed following incubation with complex-specific antibodies.Our results suggest that upon binding of complex-specific antibodies recognizing avb3, a signaling pathway is initiated, which promotes the release of ROS. In vitro, this signal impairs the migratory capacity of trophoblast cells. We consider this mechanism to be one potential cause of impaired placental growth, which affects the birth weight of FNAIT babies.NA

MSc.

Safety and efficacy of Nipocalimab in pregnant individuals at high risk for early-onset severe Hemolytic Disease of the Fetus and Newborn (HDFN): results from the phase 2 UNITY study

Sicherheit und Wirksamkeit von Nipocalimab bei Schwangeren mit hohem Risiko für eine früh einsetzende schwere hämolytische Erkrankung des Feten und des Neugeborenen (HDFN): Ergebnisse der Phase-2-Studie UNITY

The UNITY phase 2 study evaluates the efficacy and safety of nipocalimab, a neonatal Fc receptor (FcRn) blocking monoclonal antibody, to reduce the risk of fetal anemia, intrauterine transfusions (IUT), and poor outcomes for pregnancies at high risk of early-onset severe hemolytic disease of the fetus and newborn (EOS-HDFN). Nipocalimab aims to inhibit placental transfer of HDFN-associated alloantibodies from mother to fetus and to lower circulating maternal alloantibody levels.This open-label, single-arm study (NCT03842189) enrolled RhD (D) or Kell (K) alloimmunized individuals (fetal anti-D ≥32 and anti-K ≥4, 8–14 weeks [wks] gestational age [GA] at screening) with singleton pregnancies at high risk for EOS-HDFN. Weekly 30 or 45 mg/kg intravenous nipocalimab was administered between 14-35 wks GA, with delivery targeted for 37 wks, and follow-ups at 24 wks postpartum (mothers) and 96 wks postnatal (infants). Primary efficacy endpoint is proportion of pregnancies with a live birth at ≥32 wks GA without an IUT. Secondary endpoints will report antenatal and postnatal management.Of 14 pregnancies enrolled with high risk EOS-HDFN, 1 was not included and 12/13 (92.3%) pregnancies were live births (median GA at delivery =36 5/7 wks [29 2/7–37 3/7]), of which 7 were at &gt;32 wks GA without IUTs (median GA=37 1/7 wks [35 6/7–37 3/7]) and 5 were with IUT (IUT initiation ≥28 wks). One pregnancy resulted in fetal demise (IUT complications). Of 12 live births, 11 received phototherapy (median 87.0 hours [12–301]) and 6 received ≥1 simple transfusion (median number 2 [1–6], in first 12 weeks of life; 5 IUTs, 1 exchange transfusion). No maternal/neonatal deaths/serious infections; 4 fetal SAEs occurred: fetal growth restriction, subchorionic hematoma, fetal heart rate deceleration, and placental abruption at delivery.This first prospective interventional study demonstrates the potential for nipocalimab, an FcRn blocking antibody, to alter the underlying disease mechanism impacting the antenatal and postnatal management of fetal anemia in pregnancies at high risk for EOS-HDFN. These findings support a favorable benefit–risk profile of nipocalimab and warrant further evaluation of this drug in a phase 3 trial in pregnant individuals at risk for severe HDFN.KJM serves as the overall principal investigator for the phase 2 trial of nipocalimab (UNITY); has received funding from Momenta Pharmaceuticals, Inc., paid on his behalf to the McGovern Medical School–UT Health and from Janssen Pharmaceuticals, Inc., paid on his behalf to Dell Medical School at The University of Texas at Austin for a clinical trial on a monoclonal antibody for the treatment of HDFN; has served on the steering committees and advisory boards for clinical studies for Momenta Pharmaceuticals, Inc., and Janssen Pharmaceuticals but has not received funding for these activities; receives royalty funding from UpToDate, Inc., for authorship on various chapters, consulting fees from Health Management Associates, Inc., for consultation on the formation of fetal centers, consulting fees from BillionToOne, Inc., paid on his behalf to Dell Medical School at The University of Texas at Austin, and honoraria from GLC Healthcare, Inc., for podcast content on HDFN; and serves as a nonpaid consultant for immunology for Janssen Pharmaceuticals, Inc.LEL, JHL, AM, VS, LBS, MLT, SS-K, and YK are employees of Janssen Research &amp; Development, LLC, and hold stock/stock options from Johnson &amp; Johnson.DO, EL, and ET have received consulting fees for membership of steering committees and advisory boards for clinical studies from Momenta Pharmaceuticals, Inc., and Janssen Pharmaceuticals, Inc.EV serves as the principal investigator of UNITY and CLARITY studies in The Netherlands.JS, UJS, RSM, and RD have no conflicts of interest.MDK serves as the chair of the RCOG Genomic Taskforce and as a senior principal clinical scientist in the Medical Genomics Research Group, UK.JBB has receivedconsulting fees for membership of advisory boards for Janssen Pharmaceuticals, Inc., UCB, Argenx, and Rallybio.

Raum 13

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Immunohematology

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