57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Mezagitamab interference in serological testing can be eliminated with anti-CD38 fab fragments

Late breaking abstract

Die Interferenz von Mezagitamab in serologischen Tests kann mit anti-CD38 Fab Fragmenten vermieden werden

- - -- - -- - -- - -C.S. is co-inventor of the patented AMIAT technology. C.S. and C.P.H. are both employees of imusyn GmbH &amp; Co. KG.INTRODUCTION: Administration of anti-CD38 antibodies is a state-of-the-art therapy for patients diagnosed with multiple myeloma. However, treatment with the currently approved anti-CD38 antibodies regularly results in widespread agglutination of red blood cells in indirect anti-human globulin tests (IAT), making the determination of irregular antibodies and timely transfusion of compatible blood a challenge.Mezagitamab, a novel monoclonal anti-CD38 antibody, is currently under clinical investigation, not only for the treatment of multiple myeloma but also for other indications such as generalized myasthenia gravis.METHODS: We tested mezagitamab against a range of test cells in the IAT and compared its interference to daratumumab. Additionally, we titrated mezagitamab to see if interference is likely to occur at the expected serum levels. Finally, we tested if DaraEx, a reagent containing Fab fragments of an anti-CD38 antibody, was able to eliminate the interference in the antigen masking indirect anti-human globulin test (AMIAT).RESULTS: Mezagitamab also interferes with the indirect anti-human globulin test, with comparable titers but weaker reaction strengths than daratumumab. DaraEx is able to completely overcome this interference in the AMIAT.CONCLUSIONS: Mezagitamab is likely to become a challenge for diagnostic laboratories once it will have been approved by the medical authorities. DaraEx treatment remains a simple and effective way to mitigate the interference of anti-CD38 antibodies.

Poster

Univ. Prof. Dr.

PD Dr.

CD34 positive selection of cryopreserved stem cell concentrates

CD34 Positiv Selektion von kryokonservierten Stammzellkonzentraten

- - -- - -- - -- - -no conflict of interestImmunomagnetic CD34 positive (CD34+) selection is a graft manipulation technique to lower the risk of graft versus-host-disease (GvHD) in patients receiving stem cell boosts for graft failure after allogeneic hematopoietic cell transplantation (HCT). We report about the successful administration of stem cell boosts with CD34+ selected cells from a portion of previously cryopreserved stem cell products in three children who initially underwent allogeneic HCT for malignant diseases.Cryopreserved stem cells were thawed carefully, supplemented with 10 Vol% citrate, 2500 IE DNase and 0.3 ml MgCl2 0.5 molar, and centrifuged to reduce DMSO and platelets to a minimum. The cells were incubated with immunomagnetic beads against CD34 for 30 minutes. Subsequently, two washing steps using PBS/EDTA buffer with 0.5% human albumin and a soft centrifugation program (300 g, 15 minutes) were performed. Before selection, the cells were adjusted to a volume of 150 ml and filtered through a 200 µm filter system to reduce cell aggregates and clots. CD34+ selection was performed using the CliniMacs plus device (Miltenyi Biotec). CD34+/CD45+ cells were analyzed by flow cytometry and sterility was tested by bacterial culture tests.Two cryopreserved haploidentical allogeneic peripheral stem cell grafts and one cryopreserved erythrocyte depleted bone marrow graft from an unrelated HLA-identical donor were thawed and CD34+ selected. Indications were secondary graft failure, cytopenia due to meta-iodobenzylguanidine (miBG) therapy and prolonged cytopenia after CAR-T-cell infusion, respectively. Basic values were 88, 129, and 39 x106 and after selection 44, 63, and 16 x106 viable CD34+ cells which results in a CD34 recovery rate of 51, 49, and 42%. Sterility testing was negative. The patients were transfused on day of selection with 1.2, 4.5, and 0.9 x106 CD34+ cells /kg body weight. The transfusions were well tolerated. All three patients engrafted and no GvHD occurred.CD34+ selection of cryopreserved stem cell grafts was safe and efficient in three cases of allogeneic stem cell boost. After thawing, the CD34 recovery rates were lower than in fresh grafts. Despite of extra-addition of anticoagulant one has to be aware of the risk, that cell aggregates and clots may occure and may hamper the processing. Thus, grafts should be filtered through a transfusion filter system before selection.

Miss

Analysis of production rate of blood components at northern zone blood transfusion centre (NZBTC) from January 2021 to December 2022

Analyse der Produktionsrate von Blutkomponenten im Bluttransfusionszentrum der nördlichen Zone von Januar 2021 bis Dezember 2022

- - -- - -- - -- - -Affiliation: The authors are afilliated with department of National Blood Transfusion Service in Tanzania. Author 1 is serves as medical laboratory scientist, author 2 holds a managerial position at National Blood Trnsfusion Service. The authors have no further conflict of interest to disclose.Background: Blood component preparation was developed in 1960 to separate blood products from one-unit whole blood by a specialized equipment called a Refrigerated Centrifuge. Blood components are separated from whole blood within 24 hours after collection. According to Africa Society of Blood Transfusion (AfSBT) Standards"; If a facility plans to use red cells only and to discard the plasma, the separation step can be performed at any point up to the expiry date unless an additive solution is being used, in which case separation must occur within a maximum of 7 days of collection, in conformance with manufacturer"s instructions. In the Northern Tanzania, it is only NZBTC which is capable of producing Packed Red Cells(PRCs), Fresh Frozen Plasma(FFP) and Platelets(PLT) by using all the two methods mentioned above.Methods: This is a descriptive cross-sectional study that analyzed blood components production data extracted from Blood Established Computer System (BECS) - eDelphynResults: A total of 17,754 units of blood were collected for the period of 2 years whereby 11,169 (63%) of blood components were produced from these units. In 2021 and 2022, 7,491 and 10,263 units were collected for each year whereby 5,771 (77.03%) and 5,398 (52.6%) of PRCs were produced respectively. For the year 2021, FFPs and PLTs produced were 2,261(30.2%), and 586 (7.8%) respectively while 1,720 (22.9%) were whole blood units. In 2022 components produced were 2,545 (24.8%) and 494 (4.8%) for FFPs and PLTs respectively and 4,865 (47.4%) remained as whole blood units.Conclusions: PRCs is the most common blood product produced followed by FFPs and PLTs respectively. Rate of blood components produced decreased in 2022 (PRC, FFP and PLT respectively) due to increased collection but not on components production capacity. The facility should ensure that all units collected are converted into blood and blood components as per AfSBT standard requirements. All necessary resources for blood component productions should be reliably available throughout the time including availability of the double/triple blood bags.

Prof. Dr. Dr.

Prof. Dr.

HLA-DOB: From the myth of low-polymorphism to the reality of high polymorphism

HLA-DOB: Vom Mythos Low-Polymorphismus zur Realität High-Polymorphismus

- - -- - -- - -- - -no conflict of interestCompared to the classical HLA genes known to be relevant for transplantation, there is limited data on HLA-DOB. HLA-DOB codes for the β-chain of the HLA-DO class II molecule, which influences the peptide loading of classical HLA class II molecules. Due to limited research on this locus, it was assumed earlier that HLA-DOB has a low degree of polymorphism.We disproved this hypothesis by developing a long-range PCR assay for HLA-DOB and typing of nearly 500 randomly selected Western European samples with next generation sequencing according to Illumina and the NGSengine. We extensively tested the assay&quot;s reliability and robustness, making it suitable for detailed research into this locus. It enables full-length sequencing of the HLA-DOB, including a large part of the untranslated regions, generating sequences with a length of approximately 9.2 kb. Any appearing unknown allele sequences were verified using nanopore sequencing by Oxford Nanopore Technologies.We found that slightly more than half of the alleles analyzed corresponded to HLA-DOB*01:01:01. In addition, the sequenced region included an indel, whose variations were observed in specific alleles. We also identified single nucleotide polymorphisms apparently linked to the 1066 bp deletion found in most HLA-DOB alleles. No significant Linkage Disequilibrium between HLA-DOB and the loci HLA-DOA, -DMB and -DQB1 were observed. However, we discovered a new HLA-DOB allele with an exon mismatch causing an amino acid substitution. In addition, we submitted 19 further HLA-DOB sequences to the IPD-IMGT/HLA database, which contained intron mismatches or served as extensions of already listed alleles.In summary, analyzing nearly 500 Western European samples uncovered new alleles, suggesting many more unknown sequences could be identified with a larger and more diverse sample cohort comprising different ethnicities. Our assay provides an excellent basis for further HLA-DOB research, including discovering new alleles and expanding statistical analyses and databases, making future HLA-DOB typing increasingly precise.

Professor

Disaster preparedness in blood supply and transfusion – Survey in Germany

Umfrage zur Entwicklung der Katastrophenvorsorge

- - -- - -- - -- - -Kein InteressenskonfliktBackgroundPreparedness for catastrophes and disaster is part of transfusion medicine since many years in Germany. In the past decades computer breakdowns, mass casualties, flood, and recently pandemia were the issues. Due to political developments in the past 2 years the ancient quotation &quot;si vis pacem para bellum&quot;, &quot;if you desire peace, prepare for war&quot; has become a benchmark discussed in media. Governments encourage experts to update preparedness for any issue.MethodsIn a pilot survey development of disaster preparedness within the last 2 years will be evaluated with respect blood donation, manufacturing, testing, issuing, transport and transfusion. Also, use of the &quot;Model preparedness plan of EDQM&quot;, issued in 2022 will be a topic. If the pilot survey is successful, all blood centers in Germany will be asked to participate.ResultsThe results of the pilot survey will be reported and discussed in the context of the international literature.ConclusionThis survey will provide additional support for setting scientific standards for disaster preparedness, which could then become part of the national guidelines in the context of the European SoHO regulations.

Shaken, not stirred – breaking up with traditional DNA fragmentation

Geschüttelt, nicht gerührt - Schluss mit traditioneller DNA-Fragmentierung

- - -- - -- - -- - -no conflict of interestsNext generation sequencing has become an integral part of modern molecular diagnostics. Regardless of the sequencing method employed fragmentation of DNA is an indispensable step within most library preparation protocols. To avoid the obstacles of enzymatic treatment, usually applied in high-throughput procedures, the objective was to establish a reliable, cost-efficient and safe method of mechanical fragmentation in a 96-well format.High molecular weight DNA isolated from either peripheral blood or saliva were utilised as starting materials. The method is based on fragmentation by shear forces generated during the implosion of cavitation bubbles. A vibrating mill with corresponding glass beads was deployed to generate these bubbles. The fragmentation degree was monitored using agarose gel electrophoresis.The results demonstrate that a high-throughput homogeneous fragmentation of human DNA across the entire 96-well plate is feasible using a vibrating mill. The digestion time, oscillation rate, material, geometry, size and quantity of the glass beads used as well as the plates employed can be optimised depending on the desired fragment size range. It is important to note that the sample itself also exert an influence on the fragmentation result, due to parameters such as volume, viscosity, pH value, presence of salts and temperature.The use of a vibrating mill for mechanical DNA fragmentation represents an innovative approach that is in direct competition with established conventional methods. The major advantage of this method is that no enzymes are employed, obviating the necessity for the creation of a corresponding milieu. Furthermore, no subsequent inactivation and any requisite buffer exchange is required. Therefore, DNA fragmentation utilising a vibrating mill is a safe, time- and cost-efficient method.

Prof.

Human serum-mediated proliferation of primary human cardiac cells is regulated via TGFβRI/II-SMAD2/3 axis

Die Proliferation primärer humaner kardialer Zellen wird induziert durch humanes Serum und reguliert über TGFβRI/II und SMAD2/3

- - -- - -- - -- - -Parts of this research were filed for a patent application entitled &quot;Method and medium for cultivating animal cells&quot;.Introduction: Adult human stem- and progenitor cells play a crucial role in tissue and organ repair, making them promising candidates for regenerative medicine. However, age-related diseases are often accompanied by a decline in endogenous stem cell-based regeneration. Blood serum has shown rejuvenating effects in animal studies, but its translation to human the system requires further elucidation of underlying molecular mechanisms. Further, a deeper understanding of stem cell proliferation at the molecular level is necessary to address age-related diseases and expand stem cell therapies. We here investigated the proliferative effects of human blood serum on adult human cardiac stem cells with a special focus on cell-type specific TFGβ signaling.Methods: Human cardiac stem cells (hCSCs) were treated with human blood serum and selective inhibitors for the TGFβ receptors TGFβRI and TGFβRII. Proliferation- and senescence assays served as functional outread. RNAseq was applied to gain a deeper insight into the molecular regulation of hCSC proliferation.Results: RNA-Seq of serum-treated hCSCs indicated the involvement of TGFβ signaling in regulating key mechanisms of cellular proliferation. Functional assays confirmed TGFβ as a major mediator of serum-induced effects in hCSCs. The application of human blood serum as well as of TGFβ1 resulted in increased proliferation and protection from senescence in hCSCs. Inhibition of this pathway showed that TGFβRI inhibition reduces serum-mediated proliferation, while the inhibition of both receptors, TGFβRI and TGFβRII, strongly increased hCSC proliferation.Conclusion: Our data indicate TGFβ signaling playing a dual role in the serum-mediated effects on hCSCs, crucial for their proliferation and senescence regulation. These findings enhance our understanding of the molecular basis of serum-mediated hCSC proliferation and senescence, contributing to potential advancements in regenerative medicine to treat age-related cardiovascular diseases. Further research into these pathways could improve therapeutic strategies for heart failure and other related conditions.

Applying explainable artificial intelligence on image flow cytometry data to improve granulocyte immunology testing

Die Anwendung von explainable artificial intelligence und image flow cytometry zur Verfeinerung der granulozytenimmunologischen Diagnostik

- - -- - -- - -- - -- Introduction: Granulocyte immunology is a small but relevant field, especially for the detection of diseases like the (often under-diagnosed) transfusion-related acute lung injury (TRALI). Guidelines for performing granulocyte immunology testing recommend screening using functional tests (GAT, granulocyte aggregation test), microscopy (GIFT, granulocyte immunofluorescence test) and flow cytometry (white blood cell/granulocyte immunofluorescence testing, Flow-WIFT/GIFT) as well as confirmatory testing using ELISA-based techniques.Methods: Mostly, granulocyte immunology testing is performed using separate tests for each assay, which is labour-intensive and time-consuming. In addition, microscopical assays are always prone to subjectivities. But with the recent advances in the field of image flow cytometry and computational image analysis using machine learning, multiple diagnostic steps could be joined together so that analysis steps could be automated. In this project we present a method for automated, high-throughput image flow cytometry analysis for combining GIFT and Flow-WIFT/GIFT. Comparisons were performed on specimen including allo- and auto-antibodies against HNA-1a, -1b, -1d, -2, -3a, -3b, -4a as well as HLA class I.Results: Outcomes between image flow cytometry and GIFT were greatly comparable (AUROC 0.984), although the resulting images had a lower quality when being compared to classical microscopy. The use of machine learning tools also allowed to automate the generation of a proposed classification from the high-throughput image generation. Explainable Artificial Intelligence (XAI) tools can also be used to give a human-understandable interpretation of the resulting images, which could later be evaluated by the technician (if needed). In addition, the results from image flow cytometry were identical to the Flow-WIFT/GIFT assay (AUROC 1). Furthermore, hands-on-time and turnaround-time could be greatly reduced.Conclusion: The use of image flow cytometry could be a major advancement in the field of granulocyte immunology. In addition, the use of machine learning and XAI techniques allows not only to automate image classification tasks but also to propose human-understandable interpretations of the images.

Prof. Dr. med.

Dr.med.

Increased occurence of Parvovirus B19V infection in pregnant women 2024 and intrauterine transfusion in the management of virus related fetal complications

Erhöhte Parvovirus B19 Infektion bei Schwangeren 2024 und intrauterine Transfusionen zur Behandlung der dadurch verursachten fetalen Komplikationen

- - -- - -- - -- - -keine InteressenskonflikteBackground: Between the end of 2023 and now, Germany has seen an outbreak of Parvovirus B19 (B19V) infection among pregnant women. B19V infection during pregnancy can lead to potentially life-threatening non-immune fetal hydrops in 1-5% of cases, which can be managed through intrauterine transfusion (IUT). Additionally, neurodevelopmental outcomes following fetal B19V infection are impacted by fetal anemia. IUT of red blood cells can alleviate anemia and reduce both the risk of hydrops and mortality associated with B19V. To minimize complications, it is recommended to perform intrauterine transfusions between 18 and 34 weeks of gestation. Study cohort/Results: Since 2024, 21 cases of B19V-associated fetal anemia have been admitted to our university hospital. All 21 pregnant women showed severe fetal anemia confirmed by Doppler ultrasonographic assessment of the peak velocity of systolic blood flow in the fetal middle cerebral artery. For 9 patients, fetal blood sampling was performed before and after IUT. Red cells from B19V-PCR negative blood donors were prepared in our blood bank to reach a hematocrit of 80%. IUT was performed within &lt; 12 hours after preparation. 21 cases of B19V-associated fetal anemia were treated with IUT of red cells. The gestational age of the patients ranged from 16 to 38 weeks, with an average of 21 weeks. Fetal hemoglobin levels ranged from 3.0 to 15.0 g/dL before intrauterine transfusion (n=15, Median 5.6 g/dL). Post-transfusion levels ranged from 11.2 to 15.8 g/dL (n=9, Median 14.4 g/dL). Most patients (20 out of 21) received a single IVT, while one patient additionally received IPT. Follow-up was conducted using MCA-PSV Doppler measurements: in 11 cases, a decrease from &gt;1.5 MoM to &lt;1.5 MoM was found; 2 cases showed an increase. Hydrops fetalis was observed in 7 cases. None of the pregnant women showed alterations in placental implantation or amniotic fluid. One case was complicated by a late miscarriage with fetal malformation. By the end of July, 7 deliveries between 38 and 38+6 weeks of gestation were recorded. Intrauterine transfusion appears to be a viable tool to treat severe B19V-associated fetal anemia. Further studies need to assess the long-term outcomes of IUT.

Dr. med.

Prolonged storage of purified granulocyte concentrates from pooled buffy coats

Verlängerte Lagerungsmöglichket von Granulozytenkonzentraten aus gepoolten Buffy Coats durch Aufreinigung

- - -- - -- - -- - -JA, FD, SD sind Angestellte der ARTCLINE GmbH und Co-Erfinder von Patenten.Die anderen Autoren haben keine Interessenkonflikte bezüglich dieser Studie.Background:In Germany, granulocyte concentrates are typically produced by apheresis and require donor pretreatment with G-CSF and dexamethasone, often resulting in time delays, limited availability and potential side effects for the donor.In contrast, producing GCs from pooled buffy coats (BCs) offers expanded availability and fewer donor risks. Recently, we published the production of purified GCs using BCs and hydroxyethyl starch or apheresis GCs (1, 2). Here, we present a manufacturing process involving the pooling of BCs and purification based on gelatine sedimentation to achieve the desired cell counts and extended storage. Viability, functionality, and metabolism of the cells were investigated during prolonged storage.Methods:Up to 20 ABO blood group-identical BCs were pooled. After adding gelatine (Gelafundin), the red blood cells sedimented. The remaining leukocyte-rich supernatant was washed three times with saline to reduce platelet content and was resuspended in donor plasma. The purified pooled GC (ppGC) was stored at 20–24°C without agitation for up to 96 hours. Cell count and viability, metabolic parameters, and functionality were monitored daily.Results:Pooling of 12 BCs resulted in ppGC with 8.5E9 granulocytes (N=12, range 5.3-12.2E9) corresponding to the EDQM specifications for pooled granulocyte concentrates (at least 5E9). Pooling of 20 BCs resulted in ppGC with 1.22E10 granulocytes (N=6, range 1.03-1.40E10) corresponding to the German Hemotherapy specifications for granulocyte concentrates (at least 1E10). During storage for 96h viability and phagocytosis rate were above 80%.Conclusion:Granulocyte concentrates (GCs) from pooled buffy coats meet required granulocyte cell counts and demonstrate an extended storability. Both hydroxyethyl starch and gelatine can be used. This may allow offering granulocyte concentrates as an of the shelf product in the future for immediate use and broad applicability for transfusion and extracorporeal immune therapy (3).Transfus Med Hemotherapy. 12. April 2024;1–10Transfusion (Paris). 2022;62:194–204Crit Care. 2011;15:R82

Donor vigilance – Severe adverse events of whole blood donation

Donorvigilanz – Schwerwiegende Nebenwirkungen der Vollblutspende

- - -- - -- - -- - -NoBackgroundThe new Transfusion Law opens the door for eligibility testing of whole blood donors (WBD) by a remote physician via telemedicine. Thus the incidence of severe adverse events (SAE) requiring immediate medical intervention is a matter of debate. In order to estimate to what extent a physician may be dispensible, we analyzed the incidences of AE and SAE according to the IHN classification among a total of 983.532 repeat (RD) and 78.192 first-time-donors (FTD) in 2023.MethodsWe analyzed all whole blood donations at the German Red Cross Blood Donation Services Baden-Württemberg - Hessia (BWH) and North-East (NE) in 2023 with regard to the occurance and severity of AE. The incidence of all AE and SAE were clustered according to the IHN classification for SAE requiring immediate medical treatment: 1. systemic reactions: vasovagal reaction (VVR) requiring medication, reaction with loss of consciousness (LOC) &lt;60s, LOC &gt;60s, hypertension, sickness, arrythmia, seizure, injury following VVR, injury related to WBD, cerebrovascular accident, allergic reaction, others; and 2. local reactions: infiltration, nerve injury, arterial puncture, local allergic reaction, cellulitis, thrombophlebitis and arteriovenous fistula.ResultsWe observed a total of 34.892 AE in RD (3.5% of all donations) and 7.583 AE in FTD (9.7%). Out of these, 2.477 (RD) and 1.435 (FTD) were classified as SAE as they required medical treatment by a physician. Thus the ratio of SAE was 1 in 397 donations and 1 in 54 for RD and FTD, respectively. Notably this ratio correlated inversely with age and was highest in FTD donors &lt; 24 yrs. (1 in 37) and RD &lt; 24 years (1 in 105). In contrast, donors at age 60-64yrs had 1 in 938 AE (RD) and 1 in 174 (FTD) and donors at age 65-69yrs. had 1 in 862 (RD) and 1 in 180 (FTD) SAE. Furthermore systemic SAE requring medical treatment were much more frequent versus local both in RD (1 in 434 vs. 1 in 4.684) and FTD (1 in 58 vs. 1 in 850).ConclusionWe observe a frequency of 1 in every 397 whole blood donation by RD and 1 in 54 FTD. With the introduction of telemedicine and the absence of a physician on-site, adapted strategies for an immediate medical intervention have to be introduced in case of SAE. Further investigations will be necessary in order not put WBD at risk carelessly. The incidence of SAE is not only correlated to the donor status but is also inversely correlated to age identifying young RD and FTD as the most vulnerable cohort.

Posteraustellung 15
Late-Breaking-Abstracts

Appointment

Programme

Posteraustellung 15Late-Breaking-Abstracts

Appointment

Programme

Posteraustellung 15
Late-Breaking-Abstracts