56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Prof. Dr.

Vorsitz

PD Dr.

Prolonged thrombocytopenia and severe transfusion reaction after ABO-incompatible allogeneic hematopoietic stem cell transplantation in a patient with chronic myelomonocytic leukemia

Background Major ABO-incompatible allogeneic hematopoietic stem cell transplantation (allo-HCT), is a common practice and represents a challenging transfusion scenario. Prolonged thrombocytopenia with increased platelet transfusion needs is one of its reported adverse effects, and this has been linked to the persistence of recipient anti-donor isoagglutinins.Methods A 55-year-old male patient, O, Rh(D)-positive, with chronic myelomonocytic leukemia (CMML) underwent major incompatible allo-HCT from a A, Rh(D)-negative donor. He suffered prolonged thrombocytopenia and multiple transfusion reactions after A, Rh(D)-negative platelet transfusions.Results A transfusion reaction work-up was initiated. The minor compatibility test was negative. No eluate was performed due to the absence of detectable IgG on the posttransfusion direct antiglobulin test (DAT) sample. Microbiological evaluation of the remaining platelets revealed no bacterial contamination. HLA-work-up revealed HLA-antibodies class I versus HLA B76 in weak reaction strength. Due to the rarity of the HLA trait, HLA-matched platelet transfusions were not possible. An additional platelet work-up was performed. No platelet-specific antibodies were detected. Finally, anti-A1 IgM titers in the patient were assessed showing a titer of &gt;1:2048. Based on this result, platelet transfusions were restricted to O Rh(D)-negative donors.Conclusion In case of transfusion reactions against platelet products in major ABO-incompatible allo-HCT patients, isoagglutinin monitoring should be considered and a change in the platelet transfusion protocol maybe beneficial in patients presenting high isotiters against recipient"s blood type.Offenlegung Interessenkonflikt: All authors declare no competing conflict of interest

Immunotherapy

ePoster

Phenotype characterization of monocyte-derived dendritic cells from human umbilical cord blood

Background Umbilical cord blood (UCB) describes a valuable resource of stem cells for pediatric and ethnic minority stem cell transplantations. Monocytes, as part of the innate immune system, are able to generate dendritic cells (MoDC), and thus play an important role in T-cell activation and cellular therapies. However, UCB-MoDC have not been extensively characterized in previous studies. This study aims to characterize the phenotype of UCB-MoDC to assess possible applications in cellular therapies.Methods In order to characterize UCB-derived monocytes and MoDC, CD14+ monocytes were magnetically isolated from healthy adult donors and UCB-derived PBMC (purity &gt;95%), differentiated with IL-4 and GM-CSF for 5 days followed by their maturation (IL-1β, IL-6, TNFα, IFNγ, PGE2) for additional 2 days. Characterization and survey of monocyte and MoDC viability and surface marker expression for activation, migration and differentiation were analyzed on d0, d5 and d7 by flow cytometry.Results Freshly isolated UCB CD14+ monocytes displayed significantly lower frequencies in CD11c, HLA-DP,DQ,DR, and Fas-L expression in comparison to adult CD14+ monocytes. Comparison between adult and UCB-MoDC cultures revealed no significant differences in vitality and cell recovery. Expression of key MoDC markers such as CD209 and CD40 were similar between adult and UCB-MoDC. However, UCB-MoDC displayed significantly lower frequencies for CD11c and CD80 expression while expression levels were significantly reduced for CD11c, CD83, CD274 (PD-L1) and CCR7 in comparison to adult MoDC cultures. Simultaneously, frequencies of CD14 and CD16 expressing UCB-MoDC were significantly greater than in adult MoDC cultures.Conclusion UCB provide a viable source of monocytes for the generation of MoDC. Further investigations will clarify if the observed differences in co-stimulator and chemokine receptor expression translates to functional differences in comparison to adult MoDC in order to evaluate their potential for potential future clinical applications.Offenlegung Interessenkonflikt: The authors have no conflict of interests to declare.

Antibodies inhibiting cell-to-cell spread of herpes simplex virus type 1 protect against viral reactivations: a retrospective study

Background Herpes simplex viruses (HSV) cause ubiquitous human infections. For vaccine development, knowledge concerning correlates of protection is essential. Therefore, we investigated (I) if humans are in principle capable producing cell-to-cell spread inhibiting antibodies against HSV and (II) whether this capacity is associated with a reduced HSV-1 reactivation risks.Methods We established a high-throughput HSV-1-ΔgE-GFP reporter virus-based assay and evaluated 2,496 human plasma samples for HSV-1 glycoprotein E (gE) independent cell-to-cell spread inhibiting antibodies. Subsequently, we conducted a retrospective survey among the blood donors to analyze the correlation between the presence of cell-to-cell spread inhibiting antibodies in plasma and the frequency of HSV reactivations.Results In total, 128 of the 2,496 blood donors (5.1%) exhibited high levels of HSV-1 gE independent cell-to-cell spread inhibiting antibodies in the plasma. None of the 147 HSV-1 seronegative plasmas exhibited partial or complete cell-to-cell spread inhibition, demonstrating the specificity of our assay. Individuals with cell-to-cell spread inhibiting antibodies showed a significantly lower frequency of HSV reactivations compared to subjects without sufficient levels of such antibodies.Conclusion This study contains two important findings: (I) upon natural HSV infection, some humans produce cell-to-cell spread inhibiting antibodies and (II) such antibodies correlate with protection against recurrent HSV-1. Moreover, these elite neutralizers may provide promising material for immunoglobulin therapy and information for the design of a protective vaccine against HSV-1.Offenlegung Interessenkonflikt: No conflicts of interest

Follow-up of serotype-specific antibodies after sequential vaccination with two pneumococcal vaccines in kidney transplant recipients

Background To reduce the morbidity and mortality from invasive pneumococcal disease, vaccination against Streptococcus pneumoniae is recommended in kidney transplant recipients, using sequential vaccination with the 13-valent pneumococcal conjugate vaccine Prevenar 13 (PCV13) and the polysaccharide vaccine Pneumovax 23 (PPSV23). However, there are currently no data on serological responses after sequential vaccination in this cohort.Methods In the current study, we sequentially vaccinated 46 kidney transplant recipients with PCV13 and PPSV23 and determined serotype-specific and global anti-pneumococcal antibody responses in the year following vaccination.Results After vaccination, serotype-specific and global antibody concentrations were significantly higher compared to baseline. We observed that serotype-specific antibody responses varied by serotype (2.2- to 2.9-fold increase after 12 months). The strongest responses after 12 months were detected against the serotypes 9N and 14. Global IgG2 antibodies against pneumococci revealed the highest increase, IgM antibodies the lowest. Sequential vaccination with both vaccines achieved higher antibody levels in comparison with a historical cohort studied at our institute, that was vaccinated with PCV13 alone. During the 12-months follow-up period, none of the patients developed pneumococcal-associated pneumonia or vaccination-related allograft rejection.Conclusion In conclusion, we strongly recommend sequential vaccination over single immunization in kidney transplant recipients. * These authors contributed equally to the work and share first authorship.Offenlegung Interessenkonflikt: None.

Prof.

Unstimulated autologous and allogenic mononuclear cell apheresis

Background Autologous lymphocytes can be used therapeutically for the production of CAR-T cells or other cellular therapeutics. The dose of lymphocytes could be important in this context. We present data on allogeneic and autologous mononuclear cell apheresis (MNC-A) to obtain starting material for CAR-T cells, donor lymphocyte infusions (DLI) or allogeneic lymphocytes for research (RL). We hypothesise that there are differences in cell dynamics between allogeneic and autologous MNC-A.Methods All MNC-A procedures between 10/2009 and 04/2023 were extracted from our electronic records (n=1312). MNC-A was performed with the Cobe Spectra or the Spectra Optia. We started with MNC-A for CAR-T-cells in 2019 using exclusively the Spectra Optia cell separator. The absolute lymphocyte count (ALC) was obtained before and after apheresis. Collection efficiency 2 was calculated as follows: CE2=separated total blood volume * CD3+ cell count in peripheral blood /collected CD3+ cells. Data is presented as median with interquartile ranges. A Mann-Whitney test or a Wilcoxon matched pairs test was used to test for significance.Results For CAR-T-cell-MNC-A (n=48), ALC was 690/µl (60; 1480/µl) and CD3+ cell count was 432/µl (268; 915/µl) before MNC-A. The total body blood volume was processed (PTBV) 2.0-fold (1.8; 2.1 fold). CE2 for CD3+ cells was 76.9% (67.3; 87.6%). After CAR-T-Cell-MNC-A, ALC decreased to 520/µl (370; 940/µl) (75% (59; 98%)). For all allogeneic MNC-A (DLI and RL, n=1260), ALC was significantly higher than in CAR-T-cell-MNC-A: 1550/µl (1310; 1860/µl) (p= 0.0086) and decreased to 87% (78; 96%). Comparable DLI-MNC-A (n=66) with respect to PTBV (1.9 (1.85; 1.9) fold) were extracted from the database. These matched DLI-MNC-A again showed significantly less reduction in post-MNC-A lymphocytes to 90% (81; 102%) than observed in CAR-T-cell-MNC-A (p= 0.0079).Conclusion Lymphodepleted patients had a significantly lower pre apheresis lymphocyte count than healthy donors and showed a significantly reduced capacity to mobilize lymphocytes into blood indicating a severe impairment of the body lymphocyte pool.Offenlegung Interessenkonflikt: none

Hyperglycaemia and dyslipidaemia synergistically amplify TLR-mediated inflammatory macrophage responses

Background Hyperglycemia (HG) is a critical factor in the initiation of diabetic complications. Macrophages are key innate immune cells that regulate inflammatory responses which are responsible for the development of micro- and macrovascular complications. Increased expression of toll-like receptor 4 (TLR4) has been inked to type 2 diabetes (T2D). Here for the first time, we systemically addressed the role of hyperglycemia in the regulation of TLR system in primary human macrophages.Methods Expression of TLR 1-9 was examined in primary human monocyte-derived homeostatic M(NS), inflammatory M1(IFNgamma) and healing M(IL4) macrophages in normoglycemic and hyperglycemic (HG) conditions by RT-PCR and flow cytometry. Cytokine secretion was quantified by ELISA.Results HG induced upregulation in expression of TLR1 and TLR8 in M0 macrophages, TLR1, TLR2 and TLR6 in M1, and TLR4 and TLR5 in M2. HG potentiated TLR4-mediated response of M2 to LPS and significantly enhanced production of IL1beta. In M (IL4), HG in combination with PAM3CSK4 (PAM3), synthetic triacylated lipopeptide, ligand for TLR1/TLR2 amplified expression of TLR4, enhanced production of IL1beta, and supressed production of IL10.Conclusion We found that hyperglycaemia alone enhances inflammatory potential of homeostatic, inflammatory, and healing macrophages by increasing specific profiles of TLRs. In combination with dyslipidaemic ligands, hyperglycaemia can switch the inflammatory program in healing (M2) macrophages towards supporting vascular inflammatory complications in diabetic patients.Offenlegung Interessenkonflikt: No

The HLA specific IgG panel from recipient CD38+/138+ B cells represents a resilient surrogate marker for safe solid organ transplantation

Background HLA specific antibodies in peripheral blood leads to the exclusion of donors carrying the corresponding HLAs while the absence of DSAs leads to the acceptance of donors, even in the presence of repeated HLA mismatches. As a secondary immune response might remain unseen the need to provide a robust method for immune monitoring before SOT becomes obvious. The existence of circulating antibodies cannot be translated into reliable information on the memory B cell compartment.Methods We generated an artificial secondary immune response by re-expose recipient B cells to repeated HLA mismatches in vitro. Utilizing soluble HLA technology, recombinant HLA class I or II molecules were expressed and affinity purified. From whole blood of recipients polyclonal B cell activation was performed using the TLR7/8 agonist Resiquimod and IL-2 to generate memory B cell-derived plasma cells. A sophisticated ELISPOT assay has been developed using recombinant HLA molecules as targets for secreted IgGs from CD19+/27+/138+/38+ B cells. Autologous recombinant HLA antigens served as negative controls.Results This artificial secondary immune response allowed unequivocal detection of memory B cells against a specific HLA antigen. We could detect HLA specific memory B cells in recipients where no DSAs following a documented immunization record have been specified. In other cases, the emerge of a secondary immune response following acceptance of a repeated mismatch could be virtually excluded, when no HLA specific memory B cells could be detected.Conclusion The ELISPOT enables the quantitative and qualitative detection of HLA-specific memory B cells and to dissect donor/recipient incompatibilities that would remain unseen with accepted screening methods. In living kidney Tx, this method has been applied to adjust donor selection to prevent humoral immune responses. This detection of a secondary immune response has the potential to be used in routine diagnostics, as it provides robust and incontrovertible data on the individual immunization status.Offenlegung Interessenkonflikt: Es besteht kein Interessenkonflikt.

HLA-B*57:01/small molecule interactions induce inflammatory processes and trigger the outcome of Steven Johnson Syndrome post CBZ administration

Background Extensive clinical trials prior to license of a drug could not prevent certain cellular drug-mediated severe immune reactions. Dependent on the genotype, Carbamazepine (CBZ) hypersensitivities cause diverse clinical pictures as MPE, DRESS (HLA-A*31:01) or more fatal diseases SJS or TEN (HLA-B*15:02). Recently, the association between HLA-B*57:01 and CBZ administration could be demonstrated to result in SJS/TEN.Methods Genetically engineered human B-LCLs expressing sHLA-B*57:01 molecules were treated with CBZ or its metabolite carbamazepine-10,11-epoxide (EPX). The incapability of these B-LCLs to metabolize CBZ makes them an ideal system for analyzing the influence of CBZ and EPX orthogonally. HLA/drug complexes were purified and the availability of CBZ or EPX was monitored by mass spectrometry. The cellular alteration through HLA/drug interdependence was measured by full proteome analysis.Results The data reveal significant stronger engagement of B*57:01 to the metabolite EPX than to CBZ. Subsequent full proteome analysis of engineered B*57:01+ cells following CBZ or EPX treatment uncovered an unknown mechanism; HLA-B*57:01/EPX complexes introduced drastic proteomic alterations as the induction of inflammatory processes through the upstream kinase ERBB2 and the upregulation of NFκB and JAK/STAT pathway implying a pro-apoptotic, pro-necrotic shift in the cellular response. Simultaneously, anti-inflammatory pathways and associated effector proteins were downregulated.Conclusion Fundamental knowledge of drug-susceptible HLA molecules, their drug antagonist and the disequilibrium of the proteomic content will certainly facilitate personalized and safe medication. Offenlegung Interessenkonflikt: Es besteht kein Interessenkonflikt.

Sulfated glycans prevent platelet-related cancer progression

Background Cancer growth requires physical contact between platelets and tumour cells. This extracellular supply of growth factors is essential for cell growth. All dividing cells have receptors for platelets. Blocking them inhibits cell growth and prevents metastasis and drug resistance. Heparins and even non-anticoagulant highly sulfated relatives can effectively block these receptors, offering a well-tolerated and cost-effective treatment that may reduce cancer drug doses and minimise side effects.Methods Heparins can prevent cancer-associated thrombosis in tumour patients and slow down tumour progression. High platelet counts cause more rapid tumour growth. These observations are related. All primary cell cultures from tumours and normal tissues require a supplement, usually foetal bovine serum, to thrive. This supplement contains growth factors necessary for cell division. These are not synthesised in cancer cells but in megakaryocytes in the bone marrow and are transported in our circulation in platelets and released from platelets during serum production. Receptors for growth factors enable cells to take up these. A mechanism must therefore exist in vivo which allows cells to acquire these factors. Results All cells in the cell cycle, malignant and benign, possess platelet receptors. After docking to these receptors, platelets fuse with the cell membrane and release their contents into the cytoplasm. Proliferating cells now receive growth factors and the molecular machinery for making the same. The anticoagulant effect of heparins and their blockade of the platelet receptor are independent of each other. Blocking the physical contact between cells and platelets inhibits cell growth. This is the mode of action of the heparins on tumour growth. Remarkably, not only is cell proliferation thereby blocked competitively but it also prevents metastasis. Such adjuvant treatment also prevents resistance to anticancer drugs.Conclusion Modified non-anticoagulant heparins and highly sulfated heparans, devoid of anticoagulatory activities, also effectively block these receptors. In contrast, fondaparinux does not affect the platelet binding to cells. Platelets thus assume a new, hitherto little-appreciated role in regulating cell growth. The dosage of anticancer drugs can be reduced through such adjuvant treatment. Adverse side effects will become rarer, and metastases and chemoresistance will be a thing of the past.Offenlegung Interessenkonflikt: Der Autor verneint Interessenkonflikte. Er ist CVO (Chief Visionary Officer) des kleinen Unternehmens Cell Receptor GmbH, und dessen Muttergesellschaft, welche gegründet wurden, um eine klinische Studie zum vorstehenden Thema zu ermöglichen und wundert sich darüber, dass einfache und effektive Verfahren zur Verbesserung onkologischer Ergebnisse nicht die ihnen gebührende Beachtung finden.

Ten years of manufacturing MSC at the IKT Ulm â€“ perspectives for autologous MSC therapies?

Background Application of mesenchymal stromal cells (MSCs) as advanced therapy medicinal product (ATMP) is very versatile due to their regenerative and immunomodulatory properties. Approximately 90 therapeutic doses have been produced for 9 phase 1 - 3 non-commercial clinical trials at IKT Ulm. Autologous non-cryopreserved MSC were used within 24 after production for bone augmentation, treatment of fractures, osteonecrosis, osteoarthritis and multiple sclerosis.Methods MSC used in 9 clinical trials are manufactured in a xenogenic free system established at 5 different manufacturing centers in Europe. Heparinized bone marrow (BM) is received within 24 hours and seeded in alphaMEM supplemented with platelet lysate (50.000/cm2). Medium exchange is performed 2x per week for 10 or 14 days and harvested MSC (MSCP0) are reseeded at a density of 4000 cells/cm2 for 5 or 7 days. The investigational medicinal product (IMP) is packaged in syringes in physiological saline supplemented with or without 5% human serum albumin and immediately shipped after release to be applied to the patient within 24 hours or 48 hours using an accompanied transport service of a qualified courier company.Results All trials have proven the feasibility and safety of MSC so far. There were no adverse events. Consolidation after one year was &gt;90% in ORTHO1 trial (non-union bone fractures), &gt;70% in ORTHO2 trial (osteonecrosis of femoral head) and 100% in MAXILLO1 trial (bone augmentation prior to dental implant) The use of autologous, non-cryopreserved MSC may be critical. Due to yet unknown factors, failure of manufacturing clinical doses occurred for some patients. Factors that may influence the manufacturing process are e.g. transport time, experience of the physician performing the small volume BM collection, but also patient variation and costs for manufacturing autologous MSC for individual therapy are very high.Conclusion The use of autologous MSC is safe, effective but expensive. Failure of MSC manufacturing cannot be excluded. Cost reduction could be performed by using automated, large scale bioreactor systems for manufacturing of huge amounts of cells from one or few donors for allogeneic applications. A product from the shelf could be transported frozen, without the request for an accompanied transport. We have tested different bioreactor systems for their suitability to isolate and/or expand MSC.Offenlegung Interessenkonflikt: The authors declare no commercial, proprietary or financial interest. The manufacturing of MSC and the clinical trials were financed by projects within the European Union"s Seventh Framework Programme and the European Union"s Network HORIZON2020. The materials presented and views expressed here are the responsibility of the authors only. The European Union Commission takes no responsibility for any use made of the information set out.

Stem Cells

Qualitiy of cord blood derived stem cells after 15 years of storage in the vapour phase over liquid nitrogen

Background Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells. So far, no end of shelf life has been defined for such stem cell products stored cryopreserved in the vapour phase over liquid nitrogen. We present cell content and viability measurements of UCB derived stem cell concentrates and attached sampe tubes thawed after 15 years of storage.Methods A total of 13 cord blood derived stem cell concentrates and 25 pilot tubes were thawed for further measurements and investigations. For each individual sample, the relative content of CD34+ cells, CD45+ cells, mononuclear cells (MNC) and viability of CD34+ cells and CD45+ cells were determined by flow cytometry. In addition, CFU tests were performed with each sample. Based on these values the absolute cell counts and recovery rates (CD34+, CD45+, MNC, CFU) were calculated.Results The recovery rate of CD34+ cells of the UCB concentrates was 78.6 +/- 20.1 % (mean +/- standard deviation). Viability of CD34+ cells was 88.4 +/- 5.3 %. The CFU recovery rate of UCB concentrates was 78.6 +/- 39.6 %. Cell viability, relative and absolute cell content, and the recovery rates of the different cell types and CFU were significantly lower in the corresponding pilot tubes compared to the UCB concentrate bags. However, there were significant correlations of the absolute number of different cell types in the PRB concentrates compared to the corresponding pilot tubes.Conclusion The quality of UCB stem cell concentrates after 15 years of storage is good. A high recovery rate of CD34+ cells and good viability were found. CFU testing showed very good clonogenic potential of the UCB products. These results are in line with published research. Cord blood can be used for transplantation after 15 years of storage. Pilot tubes are representative for the respective cord blood unit. However, quality of the pilot tubes after long-term storage is worse than the PRB concentrates.Offenlegung Interessenkonflikt: No conflicts of interest to declare

Effect of expansion media on functional characteristics of bone marrow-derived mesenchymal stromal cells

Background Therapeutic application of mesenchymal stromal cells (MSC) implies their ex vivo expansion in growth media that can affect MSC characteristics. Human platelet lysate (PL) is a xenogeneic-free (XF) cell culture supplement but its mostly unknown composition is a big drawback. Hence, defined serum- and xenogeneic-free (SF/XF) media were developed, though, most of them led to poor cell growth. We expanded MSC in either XF or SF/XF media or in mixtures of both and analyzed functional characteristics.Methods MSC were expanded in aMEM+8%PL (XF, media 1), StemMACS (Miltenyi Biotec; SF/XF, media 13), or in mixtures of both media (media 4, 7, 10; for composition see Table 1). Proteome of media and cells was only analysed for XF and SF/XF approaches while further analyses were performed for all media. Specific growth factors, cytokines, chemokines, hormones and other factors were determined in media and MSC conditioned media. Consumption of growth factors or secretion of functionally relevant factors was analysed. Expression of surface antigens related to metabolism, adhesion, differentiation and other cellular processes was investigated. Differentiation potential and migratory capacity of MSC were determined as functional cell characteristics.Results Comparison of proteome of XF and SF/XF media identified various proteins exclusive for each media. Respective cells showed significant alterations in protein expression involved in several cellular pathways. Analysis of additional factors showed higher concentrations in XF media except for fibroblast growth factor-2 and insulin. Significantly different consumption of growth factors like platelet derived growth factor was observed and confirmed by significant differences in receptor expression. Secretion of functionally relevant factors could be identified and varied significantly for cells of different media (see Table 1). Cells of all media retained their differentiation potential but migration of cells was impaired by SF/XF conditions.Conclusion Metabolism of cells varied for XF and SF/XF media as shown by proteome analysis. This may rely on availability of nutrients as indicated by divergent consumption and secretion pattern of cells. Impaired migratory potential of cells grown in SF/XF media may be unfavorable for MSC homing towards sites of injury. Since therapeutic applications require different MSC characteristics, growth media may be utilized as priming approach for specific purposes.Offenlegung Interessenkonflikt: The authors declare no commercial, proprietary or financial interest. MR and HS work for IKT Ulm, manufacturing PL. VJ, MR and HS received funding from the Bundesministerium für Verteidigung, the European Union"s Seventh Framework Programme and the European Union"s Network HORIZON2020. The materials presented and views expressed here are the responsibility of the authors only. The European Union Commission takes no responsibility for any use made of the information set out.

Dr. Dr.

Approval process for allogeneic undirected cryopreserved HSC transplants - regulatory and procedural challenges

Background Hematopoietic stem cell transplants are able to give patients a second chance of life. A significantly reduced time-to-transplant, an improved possibility of transplantation coordination and previously known product specifications of the selected transplant - these are the advantages of an inventory of undirected cryopreserved PBSC. "Allogeneic cryoPBSC, DKMS-1" was approved in April 2023 by the Paul Ehrlich Institute (PEI)as the world's first allogeneic undirected cryopreserved PBSC product .Methods For approval project, structural and procedural precautions were carried out. A GMP clean room unit has been built and qualified. Appropriate production and quality control processes had to be validated. DKMS SCB applied for extension of the manufacturing authorization. . A registration dossier was created for the PEI including relevant details on production, quality control methods, end product specifications, quality and shelf life data as well as a clinical and an analytical expert report. Premises and processes were inspected by the Saxony State Directorate. Moreover, collection centers that provide the undirected apheresis product as starting material also require a manufacturing license and an approval.Results In May 2019, the first scientific advice with the PEI for the approval of "Allogeneic cryoPBSC, DKMS-1" took place. Regulatory, structural and procedural preparations were required in preparation of the acceptance inspection by the Saxony State Directorate in November 2020. 19 months have passed between inspection to manufacturing authorization.,PEI requested rectification and additional information, in a majority focused on donor ethics and end product specification. 14 months have passed from submission of the approval dossier to the final approval. In April 2023, manufacturing authorization was given to the DKMS Collection Center. Donor selection criteria has to be documented and assessed for 4 years after approval using real life data.Conclusion An approval process is time-consuming - for the applicant and also for the responsible authorities. Active communication between both parties and the early involvement of the authorities minimizes effort, misunderstanding, and simplifies the approval process. In case of "Allogeneic cryoPBSC, DKMS-1", the world's first allogeneic undirected cryopreserved PBSC product, process from scientific advice to the final approval took 4 years.Offenlegung Interessenkonflikt: Keine.

miRNA profiling to assess protective effects of MSC-conditioned medium on cisplatin-induced injury in proximal tubule epithelial cells

Background Aberrant expression of microRNAs (miRNAs) appears associated with the progression of renal disease. Mesenchymal stromal cells (MSCs) exert a protective effect in damaged renal cells through the release of cytokines, growth factors and extracellular vesicles. We speculate that MSC-conditioned medium or EVs contained within it exert a protective effect, by modulating miRNA expression in injured renal cells and by this post-transcriptional gene regulation.Methods To mimic in vitro injury and MSC-mediated protection, conditionally- immortalized proximal tubule epithelial cells (ciPTECs) were treated with cisplatin for 1h and then treated for a further 23h with cisplatin plus adipose-derived Stromal Cells (ASC)-derived conditioned medium (CM-ASC). Viability, metabolic activity and migratory capacity in a scratch wound healing assay were then assessed. Further, mRNAs were prepared from ciPTECs treated with/without cisplatin and with/without MSC-CM and assessed by small-RNASeq.Results Cisplatin-treated ciPTECs showed reduced viability and metabolic activity, ameliorated by ASC-CM. Further, cisplatin significantly reduced the migration in the scratch wound assay, CM levelled it up even exceeding control levels. EVs failed to reproduce the results obtained by the CM. Small-RNASeq analysis identified differentially expressed miRNAs in all groups, particularly in the cisplatin-ASC-CM group compared to the ASC-CM group. Some of these miRNAs are known to regulate apoptosis and cellular fitness, while others have not been described in this context yet and are subject to further investigation now. Conclusion Our results demonstrate that MSC-CM per se exerts a protective effect reducing cisplatin cytotoxicity in renal proximal tubule cells. miRNA profiles are changed, suggesting that MSC-CM affects post-transcriptional gene regulation.Offenlegung Interessenkonflikt: No conflicts of Interest.

Dynamics in Apheresis of human hematopoietic Progenitor Cells

Background The yield of CD34+ cells obtained by apheresis (HPC-A) crucially depends on the success of CD34+ cell mobilization in combination with the efficiency of CD34+ cell collection. The collection efficiency 2 (CE2) describes the collection process under the assumption of a steady state of CD34+ cell count in peripheral blood throughout the whole procedure. We state the hypothesis that the capacity to maintain the steady state varies between different donors.Methods All HPC-A procedures between 27AUG2009 and 14APR2023 were extracted from our electronic records (n=5780, autologous: n=1922, allogeneic: n=3858). HPC-A was performed with the Cobe Spectra or the Spectra Optia. Patients were usually mobilized with G-CSF and chemotherapy. Plerixafor could be used in addition for poor mobilizers. Allogeneic donors were mobilized with G-CSF alone. As the collection efficiency 1 cannot always be calculated from routine data, in addition to CE2 we introduced a process efficiency (PE) calculated by (Number of collected CD34+ cells/ (total blood volume[l] *CD34+ in peripheral blood [cells/l])*100%. Data is given as mean ± S.D.. A Mann-Whitney test was used to test for significance.Results CE2 was higher in allogenic than autologous HPC-As (62±34% vs. 58±25%). Using the Spectra Optia the difference was 69±38% (allogenic, n=2727) vs. 58±24% (autologous, n = 1434). Males had higher CE2 in allogeneic (63±38% vs. 60±25%) and autologous HPC-As (60±27% vs. 56±19%). CE2 was slightly higher in multiple myeloma (MM) patients than in other lymphomas (60±20% vs. 57±34). PE was higher in allogeneic than in autologous HPC-As (142±60% vs. 136±70%). There was no difference between MM and lymphoma patients (134±62% vs. 135±81%). For all differences p&lt; 0.001. Linear regressions showed a slight decrease of CE2 on the processed blood volume (PBV) (-2.8%/PBV). PE increased with PBV (58.4%/PBV) or time (0.55%/min).Conclusion Different parameters contribute to the success of HPC-A: gender, allogeneic and autologous collection as well as the type of disease influence the effiency of CD34+ cell collection. Multivariate analysis will be provided to better characterize the contributing properties to HPC-A.Offenlegung Interessenkonflikt: none

Improvements in unrelated donor search for hematopoietic stem cell transplantation over the last 10 years –Experience of the search unit Ulm

Background Hematopoietic stem cell transplantation is a curative treatment for various high-risk diseases of the hematopoietic system. Transplantation from unrelated donors is the most widely used transplant source as many patients lack a matched sibling donor. The search unit of the Institute for Clinical Transfusion Medicine and Immunogenetics Ulm conducts unrelated donor searches for 23 clinics across Germany. In this analysis, we summarize the trends and improvements over the last 10 years.Methods The search database of the search unit in Ulm was queried for all searches between 01.01.2013 and 31.12.2022. We generated descriptive statistics with the aim to identify trends and changes over time using RStudio and R for Windows Version 4.1.3.Results A total of 11035 searches were performed. Search numbers increased from 1085 (2013) to 1196 (2022), which leads to an average increase of 1.4% per year (determined per linear regression). The proportion of highly urgent searches decreased from 25.5 to 17.1% per year. Match rates showed almost constant improvement from 2013 (10/10: 73.1%, 9/10 24.8%, 8/10 2.1%) to 2022 (10/10: 82.3%, 9/10: 16.8%, 8/10: 0.9%). In all years search success rates were greater than 95% (defined as at least one donor found that met minimum requirements of the transplant center). Search duration defined as time from initiation of the search until first donor is identified improved significantly between 2013 (mean 34d, median 15d) and 2022 (mean 17d, median 13d).Conclusion Increasing numbers of upfront high-resolution HLA-typed donors are available worldwide. The larger donor pool and the high quality of HLA-typing in registry donors led to substantial improvements in donor search, manifesting in better match rates and shorter search duration. This benefits patients in urgent need of an allogenic stem cell transplantation in Germany.Offenlegung Interessenkonflikt: No conflicts of interest are present

Postersession 5
Immunotherapy | Stem Cells

Appointment

Vorsitz

Topics

Programme

Postersession 5Immunotherapy | Stem Cells

Appointment

Vorsitz

Topics

Programme

Postersession 5
Immunotherapy | Stem Cells