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57. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Univ.-Prof. Dr.

Collection efficiency of mononuclear cells in offline extracorporeal photopheresis: Can processing time be shortened?

Stem Cell Transplantation

Sammeleffizienz mononukleärer Zellen bei der extrakorporalen Offline-Photopherese: Kann die Prozessdauer verkürzt werden?

Extracorporeal photopheresis (ECP) is a well-known but lengthy cell therapy used for various conditions like cutaneous T-cell lymphoma and graft-versus-host disease. The number of mononuclear cells (MNCs) required for a clinical response to ECP is still uncertain. The purpose of this retrospective study was to determine the number of lymphocytes, monocytes and neutrophils in mononuclear cell products (MCP) by flow cytometry and the collection efficiency in the offline ECP setting.We collected data from 10 different patients who underwent 162 ECP procedures using the Spectra Optia device for MNC collection. The white blood cell (WBC) count of MCP was measured using a hematology analyzer. MNCs were analyzed for CD45 and CD14 expression by flow cytometry to exactly determine the collected lymphocyte and monocyte fractions.Collected MCP showed significant cell yields, with 55.3×10⁶/kg MNCs and 41.1×10⁶/kg lymphocytes. MCP were characterized by a high MNC percentage (81.3%) and a low percentage of neutrophils (18.7%). Mean collection efficiency for WBCs and for MNCs was 23.9% and 62.0%, respectively. The MNC fraction showed a moderate to high correlation between peripheral blood cell count of patients and MCP count.This study is among the few reports that demonstrate the monocyte-to-lymphocyte relation in MCP for ECP as determined by flow cytometry. Compared to historical data from inline ECP, offline ECP processing of one total blood volume results in significantly higher cell yields. Hence, to reduce the burden on patients, we propose that the offline ECP processing time can be significantly shortened.The authors declare no conflict of interest.

Poster

Dr. med.

Prof., Dr.

PD, Dr.

Management of severely HLA-alloimmunized patients who receive allogeneic hematopoietic stem cell transplantation or autologous gene therapy

Management von schwer HLA-immunisierten Patienten im Rahmen einer allogenen hämatopoetischen Stammzelltransplantation oder autologen Gentherapie

Transfusion plays a critical role in supportive care for patients who receive allogeneic hematopoietic stem cell transplantation (HSCT) or autologous gene therapy. Platelet (PLT) transfusions are needed for prolonged aplasia periods and blood banks must ensure adequate transfusion management for every patient, even if complicated by severely alloimmunization against human leukocyte antigens (HLA). Today, there still is no consensus on the best strategy to treat severe HLA-alloimmunization or on the mean fluorescence intensity (MFI) threshold to be considered clinically significant in this setting.1We report on 5 patients (age 17 to 23 years) who received HSCT or autologous gene therapy for different diseases (beta-thalassemia and myelodysplastic syndrome). All patients showed a long transfusion history and severe HLA-alloimunization: HLA-antibody-screen using Luminex technology revealed virtual panel reactive antibodies (vPRA) of 98-100%. Donors were selected, whose platelets lack the HLA antigens against which the patient´s alloantibodies are directed, focusing on HLA antibodies with MFI&gt;5000. PLT transfusion effectivity was frequently controlled by 1-hour posttransfusion increment. When HSCT was scheduled clinicians got into contact with the transfusion medicine team. The blood bank established a list of possible donors in the donor data base, where around 4600 HLA-typed donors are listed and coordinated donor acquisition with the blood donation center.In aplasia periods regularly 3-4 matched donors are needed weekly. One of the reported patients only needed 13 days until PLT independency after HSCT and received just 8 PLT transfusions, likely based on splenectomy done in childhood. The other patients needed 20 to 28 days to PLT recovery and received 17 to 30 PLT transfusions. There were no major bleeding events.Platelet transfusion management of severely HLA-alloimmunized patients who receive allogeneic HSCT or autologous gene therapy is resource intensive as it requires proper pre-planning, close communication between hematologists, blood bank and blood donation center and a large HLA-typed donor pool. Without secured access to HLA-selected apheresis platelets innovative and curative therapies of hematologic diseases wouldn´t be possible.1 Blandin et al.,Platelet transfusion refractoriness and anti-HLA immunization. Transfusion. 2021 Jun;61(6):1700-1704.doi:10.1111/trf.16358Es besteht kein Interessenkonflikt

PD Dr.

Prof. Dr.

Luminex-based HLA antibody testing: a comparison of two commercially available assays

Luminex-basierte HLA-Antikörpertests: ein Vergleich von zwei kommerziell erhältlichen Assays

Luminex HLA antibody (AB) screening and identification has established itself as the preferred platform for determination of HLA-immunization in transplantation immunology and transfusion medicine. Due to the high sensitivity of the One Lambda (OL) kit we used previously, false-positive results were often obtained. For this reason, we performed comparative HLA-AB testing with the luminex-based assays from One Lambda and Immucor (IM).Assays from OL LABScreen Mixed Class I &amp; II and IM LIFECODES® LifeScreen XP were used according to the manufacturer's instructions. In case of different results, LABScreen Single Antigen (SA) HLA Class I/II from OL were applied as a reference. Assays were performed on Luminex LX200. We measured and compared HLA AB of 102 patients. 91 were included in this analysis. 2 were excluded due to bead failures, 2 due to missing SAs measurements and 7 due to undetermined results. Accuracy, sensitivity and specificity were calculated.As can be seen in figure 1, OL and IM have an almost similar accuracy (87.91% vs. 85.71%), but OL has a higher sensitivity (92.31% vs. 53.85%) for class I HLA ABs. IM shows a better specificity (98.46% vs. 86.15%) for class I. For class II HLA ABs, IM shows more accuracy (94.51% vs. 84.62%) and specificity (98.72% vs. 87.18%). The sensitivity is equal for both assays (69.23%).Based on the data, OL provides better results for class I and IM for class II HLA ABs. However, it should be noted that the assays use different antigen panels and therefore detect ABs differently. SAs assays from OL were used to check for discrepancy results, but the ongoing controversy over setting appropriate cut-offs for positivity must also be considered. In order to verify this more precisely, it is planned to check the discrepant results with the IM SA kits.Hereby we declare that there is no conflict of interest.

Dip.Ing. (FH)

Bone Marrow Collection with the BioAccess system– a field report

Knochenmarksammlung mit dem BioAccess System – Ein Anwenderbericht

According to DKMS Donor Center gGmbH, bone marrow collection (BMC) for leukaemia and other hemoglobinopathies is still done in ~10% of the cases. As the collection set of Fresenius Kabi AG Germany wasn`t available anymore, it was necessary to establish an alternative. We started collecting stem cells with the system of BioAccess Inc., USA (temporary licensed by BfARM) as from September 2022. This study was done to verify the suitability of the set concerning handling and for validation purpose.Bone marrow collection was done with the BioAccess Marrow Collection System (BioAccess Inc., USA; MH-2150) by an accompanying risk based validation (n=11). Volume was calculated by weighing the collection bag and compared to the bag´s scaling. This is important for the operator during bone marrow collection. Quality parameter as hematocrit (hct), dose of nucleated cells (NC) with cell viability, visual inspection and microbiological contamination of the cell suspension were analyzed. Additionally, microbiological contamination due to luer-lock connections and a potential cell loss by filtration were considered. Data are presented as median values.The collected volume was 1378 mL. One product didn`t meet the specification of ≤ 1650 mL without any critical consequence for donor and patient. Comparing weight to scaling, there is a deviation below 1000 mL of up to 100 mL, whereas at higher volumes weight and scaling accord to each other. Due to weight of patient and donor 1.75 x 108 NC/kg body weight (1.3-4.92 x108) were collected. Dose of 0.10 x108 NC, 100% viability and hct of 29.9% met the specifications of ≤ 3 x 108 NC, ≥ 80% viability and hct ≤ 60%, respectively. There was no microbiological contamination. One BMC showed coagulation despite second filtration and extra anticoagulant. Set integrity was given in all cases. Cell loss due to filtration was 0% (n=10) and 13% (n=1).The operators attested an easy handling of BioAccess Marrow Collection System and the analytical data fulfilled our specifications. Deviations were based on medical decisions or donation phenomena. As there is only one set of filters included, a completely new collection set has to be opened when filter blockage makes it necessary. The BioAccess Marrow Collection System is an adequate alternative for the former collecting set of Fresenius Kabi.There is no conflict of interests.

Accelerated CD34-Quantification in Peripheral Blood Stem Cell Donations

Beschleunigte CD34 Messung bei der peripheren Stammzellspende

DKMS Collection Center (CC) gGmbH, established in 2023, completes the value chain within DKMS by offering apheresis of stem cells. We aim to minimize apheresis time for best donor safety and maximize capacities for more second life chances of blood cancer patients. The bottleneck is the CD34+ stem cell quantification time due to transportation to an external laboratory. Process insourcing is considered to be a potential alternative. Comparative testing for reliability has been conducted.Peripheral blood samples from donors were collected at day of apheresis. Viable CD34+ stem cells were quantified using Fluorescence CD34 Counter ADAMII™ by NanoEntek according to manual. Briefly, 20 µl of blood was incubated with 5 µl staining solution for 20 min. Then, 35 µl cell lysis buffer was added and incubated for 10 min. Finally, 35 µl sample was applied to specimen slide and incubated for 3 min while device calibrated. Single sample per run measurements were conducted. Results were validated against traditional FACS analysis, conducted at external, qualified testing laboratory via Navios EX by Beckman Coulter. Statistical analysis was performed in GraphPad Prism using linear regression testing.Viable CD34+ cells/µl were measured in n=43 blood samples of stem cell donors. Results from ADAMII™ and traditional FACS were plotted against each other (figure 1). Linear regression (y= 0.8596x + 11.69) was conducted. A correlation of r²=0.9065 between the two methodologies was demonstrated. Measuring several samples at a time revealed to be time-consuming.The strong correlation of 90.65 % demonstrates reliability of ADAMII™ to evaluate donors&quot; mobilized stem cells for apheresis time extrapolation. Tests with apheresis products for indication of drug dose and official authorization are required before implementation of the technology. We consider it to be a potential in-house alternative that maintains high product quality. In general, the system is well suited for low-throughput analysis without extensive training and experience needed.Nothing to disclose

Dr. rer. nat.

Comparison of an NGS and ddPCR based assay for the quantification of mixed chimerism post hematopoetic stem cell transplantation

Vergleich eines NGS- und ddPCR-basierten Tests zur Chimärismusbestimmung nach hämatopoetischer Stammzelltransplantation

Chimerism analysis is essential in the clinical follow-up of patients after allogeneic stem cell transplantation to monitor engraftment, graft failure, and predict relapse. Detecting low levels of mixed chimerism (&lt;0.1%) is clinically relevant, allowing early adjustment of treatment strategies. Various molecular methods with different sensitivities for assessing the relative proportions of donor and recipient cells are commonly employed, such as STR, qPCR, dPCR, and NGS-based assays.Until 2023, our laboratory used the Mentype® DIPscreen Kit (Biotype) to identify deletion/insertion polymorphisms (DIPs) in a 33-plex PCR, which occur individually in either the donor or recipient. Two recipient-specific markers were selected for digital droplet PCR (ddPCR) to detect chimerism with a high sensitivity of 0.06%. To reduce workflow complexity, we implemented the Devyser Chimerism NGS Kit (One Lambda). Informative markers are determined by performing a multiplex PCR on 24 biallelic Indels. In a second PCR, individual barcodes and sequencing adapters are introduced, and the amplicons are sequenced on the NovaSeq platform (Illumina). Data analysis is conducted using the Advyser software (One Lambda).One hundred patients and EQA samples were analyzed with the Devyser Chimerism NGS Kit and compared to ddPCR results. For all samples, at least two informative markers (on average 8) were identified. With the defined limit of at least 4,000 reads per marker, a sensitivity of 0.025% is theoretically achievable. Out of 31 samples defined as complete chimerism by ddPCR, 29 had values below 0.02%, and two were below 0.06%. The remaining samples had mixed chimerism between 0.06% and 90%, in concordance with ddPCR results, with a mean coefficient of variation (CV) of 7%. Two samples showed a background of &gt;0.06%, calculated from all homozygous non-informative markers, indicating sample contamination.The Devyser Chimerism NGS Kit detects mixed chimerism with high sensitivity, comparable to ddPCR (0.06%). Below this threshold, distinguishing between background and real chimerism is challenging. The results demonstrate high inter-assay accuracy (CV &lt;5%) and concordance with ddPCR. Evaluating 24 markers per sample, rather than two, allows for more reliable control of contamination. Among 189 patient/donor pairs analyzed so far, informative markers were unavailable in only two cases (1%).no conflict of interest

Image-based fluorescence cell counting for mobilized blood samples can support decision making for hematopoietic stem/progenitor apheresis readiness

Bildbasierte Fluoreszenz-Zellzählung in mobilisierten Blutproben zur Unterstützung der Entscheidungsfindung bezüglich des Stammzell-Apheresebeginns

The method for enumeration of hematopoietic stem/progenitor cells (HPC) described in the European Pharmacopoeia is based on flow cytometry with sequential gating according to the ISHAGE guidelines. The high precision and accuracy of this method have been consistently demonstrated. We identify a space for point-of-care (POC) technology for enumeration of circulating HPCs to determine apheresis readiness of mobilized patients or enumeration of HPC dose during apheresis.We posit that these uses could potentially be assisted by the non-compendial cell counter ADAMII (NanoEntek, Seoul, Korea). ADAMII is a compact table-top fluorescent imager facilitating acquisition of 75 individual fields of vision with four image sets each (bright field and fluorescence channels for CD34, CD45 and a viability dye) which are digitally overlaid to detect marker co-expression. ADAMII thus provides direct assessment of target cell concentration without the need for counting beads. We assessed the performance of ADAMII for HPC enumeration in 42 random mobilized blood (MPB) and 39 mobilized apheresis (HPC-A) samples relative to conventional compendial single-platform flow cytometry (SCE, Becton-Dickinson, Heidelberg, Germany).Viable HPC enumeration was mostly congruent between the methods for MPB (range 36-380/µL) and HPC-A (range: 331-8695/µL), with R²=0.9046, slope 0.9447 for MPB and R²=0.9724, slope 1.0758 for HPC-A, although differences between the methods exceeding 10% were noted in almost half, exceeding 20% in a quarter of the samples. A quite good correlation was equally observed for viable leukocyte count (R²=0.9154 and R²=0.8295 for MPB and HPC-A, respectively) and HPC frequency (R²=0.9347 and R²=0.9755 for MPB and HPC-A, respectively).Because MPB samples in the relevant decision range for patient apheresis readiness were insufficiently represented, analysis of a second MPB cohort with low HPC concentration samples (0-30/µL) was initiated and will be presented at the DGTI meeting. Our preliminary data indicate suitability of ADAMII as POC analysis tool to determine apheresis readiness of patients and HPC content in apheresis products which remains, however, to be corroborated in low-mobilization samples.JL and CYP are employees of NanoEntekt, manufacturers of the device evaluated here. The other authors have no declarations with respect to this work.

PD Dr. med.

Professor Dr. med.

Prof. Dr. med.

Real-world data of autologous peripheral blood stem cell collection

Real-world Daten zur peripheren Sammlung von autologen Blut-Stammzellen

Autologous stem cell collection (ASCC) from the peripheral blood after mobilization with granulocyte-colony stimulating factor (G-CSF) remains an important option for patients with hemato-oncological diseases as part of initial therapy or at relapsed/refractory stage. We aimed to identify factors predictive of successful CD34+ ASCC.We retrospectively evaluated 255 patients who underwent an ASCC (median age 60.5 years, range 7.5-74 years) at our institution from 2017 to 2023. The predicted minimal number of CD34+ cells to be collected was calculated as follows: [(peripheral CD34+ cells/µL) × adjusted collection efficiency of 30%] × total liters processed per kg body weight (bw).229 patients had hematological diseases (multiple myeloma (MM), n=115; non-Hodgkin"s lymphoma (NHL), n=102; Hodgkin's lymphoma (HL), n=11; amyloidosis, n=1), 15 patients had germ-line tumors, 8 had Ewing"s sarcoma, and 3 had multiple sclerosis. 159 patients were of male gender.Overall, 333 ASCC were performed. In 78 patients (31%) more than 1 apheresis session was necessary to reach the collection goal (range 2-4). In addition to G-CSF, the CXCR4-antagonist plerixafor was given in 24 patients (9%) (MM and NHL, n=9, each; germ-line tumor, n=3; HL, n=2; Ewing"s sarcoma, n=1).Total CD34+ collection was in median 6.6 × 106 CD34+ cells/kg bw (range, 0.2-62.7), 3.5 × 106 CD34+ cells/kg bw (range, 0.6-14.4), 3.2 × 106 CD34+ cells/kg bw (range, 0.3-6.7) and 5.2 × 106 CD34+ cells/kg bw (range, 2.1-8.4) on the 1st, 2nd, 3rd and 4th apheresis session, respectively.Predictive factors for a higher CD34+ cell collection at first apheresis were male sex (p = 0.003) as well as CD34+ cell count/µl in peripheral blood (p &lt; 0.0001). White blood cell count, platelets, hemoglobin, and age had no significant predictive value. Predicted versus observed number of CD34+ cells/kg bw collected demonstrated a very strong linear correlation (r = 0.93, p &lt; 0.0001).Of the routinely monitored indicators in ASCC, male sex and CD34+ cell count in peripheral blood were predictive factors of the collection yield. The CD34+ cell count was used to prospectively calculate the minimal number of collected CD34+ cells in order to determine the necessary duration of the individual apheresis session.Nothing to disclose.

Dr.-Dr.

Banking of the World's First Allogeneic Undirected Cryopreserved Hematopoietic Stem Cell Transplant "Allogeneic cryoPBSC, DKMS-1": An overview on the first year

Banking der weltweit ersten allogenen ungerichteten kryokonservierten hämatopoetischen Stammzelltransplantation &quot;Allogene cryoPBSC, DKMS-1&quot;: Ein Überblick über das erste Jahr

Peripheral blood stem cells (PBSC) as main source for transplantation are mobilized by G-CSF-administration and collected by apheresis. In many cases, cell amounts exceed the requested dose. After cryopreservation these extra cells are an undirected stem cell source with fast availability and clear quality specifications. DKMS Stem Cell Bank offers a cellular product based on highest standards and holds both manufacturing authorization and the federal license for "Allogeneic cryoPBSC, DKMS-1".Only persons who are requested for a directed donation, might donate cells for cryopreservation. The DKMS Donor Center (DKMS DC) identifies suitable candidates with the highest probability for a successful collection by the help of an algorithm. Cooperating apheresis centers collect the prioritized directed product and the product intended for cryopreservation considering donor safety and health. Subsequently, PBSC transported to, processed and tested at DKMS Stem Cell Bank (DKMS SCB). Quality of PBSC products is verified before freezing and after thawing. The corresponding data sets are listed by DKMS Registry and German National Bone Marrow Donor Registry (ZKRD).During the first year of stem cell bank activity, routine processes were successfully implemented at DKMS DC, apheresis centers and DKMS SCB. Staff of apheresis centers were trained and number of cooperating apheresis centers was increased to three. The apheresis centers obtained manufacturing authorization and federal license for the product intended for cryopreservation. Storage of cryopreserved products were increased and quality of stored products is verified regularly. We observed a high willingness of donors to donate for an additional undirected stem cell product. Shortly after listing of the first ADCUs (Adult Donor Cryopreserved Unit), transplantation centers were interested in our products.DKMS SCB has evolved from a pure cord blood bank to a stem cell bank and provides the world's first allogeneic undirected cryopreserved PBSC product. For safe and efficient banking processes, starting challenges were coped on all involved sites. Subsequently, up-scaling of manufacturing is in preparation. We are looking forward to further cooperating apheresis centers, the further increase of our inventory as well as numerous requested ADCUs for shipment.The authors have no conflict of interest to disclose.

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