BackgroundThe best way to monitor Chimeric Antigen Receptor (CAR)-T cells persistence and expansion in vivo after infusion, and the significance of observed fluctuations over time remains controversial. As living drugs, these therapies do not follow classical pharmacokinetic patterns. Therefore, reliable quantification of circulating CAR-T cells is essential to explore the relations between in vivo expansion and persistence on one hand, tumor response and occurrence of side-effects on the other hand; this work will prepare integration of this information in harmonized post-CAR-T Cells intervention algorithms. Conventional flow cytometry protocols rely on multi-step wash procedures that increase processing time and may reduce sensitivity. We here developed and validated a one-step no-wash flow cytometry assay for routine CAR-T monitoring in patients treated with approved autologous CAR-T Cells.MethodsCAR-T cell monitoring was implemented between 2021 and 2024 in patients treated with autologous CD19-directed CAR-T therapies at our institution with a classical two steps and wash flow cytometry method. Analytical validation included determination of detection and quantification limits, linearity, precision, and inter-laboratory reproducibility. In 2024, the classical method was considerably optimized to a one-step no-wash format to reduce manual handling and improve sensitivity. Clinical relevance was assessed in a cohort of 29 patients treated with axicabtagene ciloleucel, correlating CAR-T expansion metrics with clinical endpoints.ResultsThe optimized one-step no-wash assay markedly improved analytical sensitivity, achieving a limit of detection of 0.3 cells/µL and a lower limit of quantification of 1.0 cells/µL, versus 2.0 and 5.0 cells/µL, respectively, when using the previous two steps and wash protocol, while demonstrating a strong concordance (r² = 0.984). Inter-assay coefficients of variation remained below 9%, confirming maintained precision despite workflow simplification. In 29 patients treated with axicabtagene ciloleucel (axicel), peak CAR-T expansion significantly correlated with objective responses (p< 0.01) and occurrences of immune effector cell–associated neurotoxicity syndrome (ICANS) (p = 0.02), supporting the clinical relevance of flow cytometry–based CAR-T monitoring in routine practice.ConclusionThis one-step no-wash flow cytometry assay combines enhanced sensitivity with improved operational efficiency and provides clinically informative CAR-T cell monitoring in standard-of-care settings.

Cell therapy units are facing persistent saturation of their liquid nitrogen storage capacity for cell therapy products (CTPs). This situation compromises the safety of CTPs due to the ageing and increasing number of storage containers, leading to a higher risk of equipment failure. It also results in a significant rise in costs, mainly related to liquid nitrogen consumption. It is therefore essential to control overstocking for both economic and environmental reasons. This work represents an update of the 2014 recommendations, notably introducing a recommended maximum storage duration of 5 years for CTPs, which account for 64% of the stock despite a very low probability of clinical use. In addition, recommendations concerning the criteria for destruction of intrafamilial cord blood units and associated cell banks have been added.

Allogeneic stem-cell transplantation (allo-SCT) is met with increased toxicity and relapse in elderly patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The nonmyeloablative conditioning (NMAC) regimen combining 2GyTBI, fludarabine, and cyclophosphamide (FluCyTBI) with post-transplant cyclophosphamide (PTCy) has been described as well adapted to AML/MDS patients aged ≥70yo in the haplo-identical setting. PTCy has then been implemented as basis for GVHD prophylaxis in allo-SCT with unrelated donors (UD). We here report our experience with a single FluCyTBI+PTCy platform for both haplo and UD allo-SCT in older AML or high-risk MDS patients. We retrospectively analyzed 203 patients transplanted at our center between 2015 and 2024. Median age at transplant was 69. Donors were haplo, mismatched UD, and matched UD in 64%, 18% and 18% of patients, respectively. Day+100 grade III-IV acute GVHD (aGVHD) and 3-y moderate/severe chronic GVHD (cGVHD) rates were 8% and 18%. Donor age ≥35yo was predictive of increased aGVHD III-IV and female-to-male sex-mismatch was associated with increased moderate/severe cGVHD. 3-y NRM rate was 15%. 3-y OS was 62%, with only monosomal karyotype predicting worse survival. FluCyTBI+PTCy is safe and produces promising outcomes in both haplo and UD settings. Beyond HLA-matching, non-HLA donor-related factors constitute critical determinants of patient outcome.

TCRαβ/CD19 depletion enables mismatched and haploidentical hematopoietic stem cell transplantation (HSCT) without the use of a post-transplant alkylating agent and is particularly relevant for pediatric nonmalignant disorders. However, the complexity of graft engineering limits its availability to specialized centers. Indeed, implementing such a process and obtaining regulatory approval are resource-intensive endeavors that require sufficient patient volume. To address these limitations, we established a centralized platform for the real-time nationwide distribution of fresh TCRα/β/CD19-depleted grafts in France, prioritizing bringing the cells to the patients, rather than the patients to the cells. This study aimed to evaluate the feasibility and challenges of this unique model of centralized, nationwide manufacturing of fresh TCRαβ/CD19-depleted grafts. In this retrospective cohort study (2014 to 2025), after on-site or off-site collections, all haploidentical or mismatched unrelated donors that underwent TCRα/β/CD19 depletion were manufactured at a single academic facility and delivered to nationwide transplant centers. Manufacturing performance, product compliance, logistics (vein-to-vein and delivery times), and early hematologic recovery were analyzed and compared for 10 transplant centers. A total of 55 products were distributed after undergoing TCRα/β/CD19 depletion for Inborn Errors of Immunity (n = 36), DNA repair/bone marrow failure (n = 15), and malignancies (n = 4). Despite significant heterogeneity in starting apheresis materials across different collection centers, manufacturing performance was robust and reliable, with comparable CD34+ recovery, TCRαβ, and CD19 depletion efficacy between on-site and off-site collections. Infused CD34 dose was similar between on-site and off-site apheresis (9.81 × 106/kg versus 9.91 × 106/kg), and TCRα/β doses were <1 × 105 in 96% of products. Vein-to-vein time met the <36-h target in 52/55 cases (95%), including the majority of off-site collection and infusion procedures. Delivery time remained within the <6-h specification for all on-site infusions and 14/16 off-site infusions. Early neutrophil and platelet recovery were comparable between groups, with identical 50% recovery times of 15 days post-HSCT. Centralized fresh manufacturing and nationwide distribution of TCRαβ/CD19-depleted grafts are feasible, safe, and logistically robust. This approach represents a valuable intermediate model between local fresh production and centralized cryopreserved manufacturing, particularly in light of the reported limitations of cryopreservation on graft quality and immune effector cell integrity. It may provide a scalable organizational framework for other academic cellular therapy and ATMP platforms.

The discontinuation of the CS2 version of the semi-automated CliniMACS Plus device in 2024 prompted validation of a CD34+ immunoselection process using the fully automated CliniMACS Prodigy (Miltenyi Biotec). This transition was leveraged to evaluate Prodigy's performance on thawed apheresis products. Results were compared with fresh donor apheresis previously processed on the CliniMACS Plus and, within this validation, with fresh products processed on Prodigy. Fresh apheresis products from rhG-CSF-mobilized related donors were processed using either device. Cryopreserved apheresis products intended for destruction were thawed (Viathaw, Cytiva), washed on Sepax-II and resuspended in MACS GMP PBS/MgCl₂ with HSA and rh-DNase prior to CD34+ selection on Prodigy. Flow cytometry (Stem Kit-Beckman Coulter) was used for CD34+ and CD45+ cell quantification. From fresh products, immunoselections yielded similar CD34+ purities (95% versus 97%) and viabilities (95% versus 99%) for CliniMACS Plus (n = 10) and Prodigy (n = 10), respectively. T-cell depletion was slightly better with the CliniMACS Plus (0.1 × 10⁴ versus 0.4 × 10⁴ CD3⁺/kg), while CD34+ recovery was more efficient with Prodigy (66% versus 53%). Processing thawed apheresis with the Prodigy archieved 98% CD34+ purity, 51% recovery, and 0.3 × 10⁴ CD3⁺/kg, all within our center's acceptance criteria. The fully automated workflow significantly reduced hands-on time (2 h versus 5 h) and operator intervention. Prodigy platform enables reproducible CD34+ immunoselection from both fresh and thawed products, improving standardization, efficiency, and flexibility in graft engineering and ATMP manufacturing. The ability to process thawed products also broadens clinical options whenever cryopreserved products are available.

Les unités de thérapie cellulaire sont confrontées à une saturation persistante de leurs capacités de stockage en azote des préparations de thérapie cellulaire (PTC). Cette situation compromet la sécurité des PTC en raison du vieillissement et de l’augmentation du nombre de containers, avec un risque accru de défaillance des équipements. Elle engendre également une hausse importante des coûts, liée principalement à la consommation d’azote liquide. Il apparaît donc essentiel de mieux maîtriser le stockage, pour des raisons à la fois économiques et environnementales. Cet atelier constitue une actualisation des précédentes recommandations de 2014, en introduisant notamment une durée maximale de conservation de cinq ans, dans un contexte où les PTC conservées depuis plus de cinq ans représentent 64 % du stock, alors que leur probabilité d’utilisation clinique reste très faible. Par ailleurs, des recommandations relatives aux critères de destruction des unités de sang placentaire intrafamiliales et à la cellulothèque ont été ajoutées.

Cet article a pour but d’aborder les alternatives à la COBE2991® (Terumo BCT©) pour la préparation de produits de thérapie cellulaire, en raison de son arrêt de commercialisation prévu en 2025. Les unités de thérapie cellulaire (UTC) doivent trouver des méthodes alternatives face à des restrictions réglementaires et à l’obsolescence des équipements. Actuellement, la COBE2991® (Terumo BCT©) est largement utilisée en France, mais son remplacement nécessitera des validations multiples et un ajustement des pratiques, car il n’existe pas d’équipement unique équivalent. Les recommandations pour harmoniser ces pratiques ont été discutées lors d’un atelier spécifique en septembre 2024 après une enquête menée auprès de 25 centres de thérapie cellulaire francophones.

The aim of this article is to discuss alternatives to COBE2991® (Terumo BCT©) for the preparation of cell therapy products, in view of its planned cessation of commercialization in 2025. Cell therapy units need to find alternative methods in the face of regulatory restrictions and equipment obsolescence. Currently, COBE2991® (Terumo BCT©) is widely used in France, but its replacement will require multiple validations and adjustment of practices, as there is no single equivalent equipment. Recommendations for harmonizing these practices were discussed at a specific workshop in September 2024, following a survey of 25 French-speaking cell therapy centers.

Allogeneic stem cell transplantation (Allo-SCT) is the only rapidly available curative treatment modality in patients with severe sickle cell disease (SCD). The development of reduced-toxicity myeloablative conditioning (RT-MAC) regimen and the use of partially matched family donors with post-transplantation cyclophosphamide (PT-Cy) have widened the access to Allo-SCT. Antibodies against donor-specific HLA (DSA) increase the risk of engraftment failure in HLA mismatched Allo-SCT. We report the results of five patients with SCD, whereas three with DSA, who underwent an unmanipulated haploidentical stem cell transplantation (Haplo-SCT) after a busulfan-based RT-MAC regimen with PT-Cy. To reduce the risk of engraftment failure, a sequential two courses pharmacological pre-transplant immune suppression (PTIS) phase was added prior to the conditioning regimen. All patients engrafted successfully. The procedure was well tolerated. None of the patients developed acute GVHD, whereas one developed moderate chronic GVHD. After a median follow-up of 5 years (range, 2.2–9), all patients are free of pain with excellent quality of life. Our report shows that Haplo-SCT after a RT-MAC regimen is feasible and safe with stable long-term engraftment and excellent disease control. The risk of graft failure can be abrogated by adding a PTIS phase prior to initiating the conditioning regimen.

Haploidentical stem cell transplantation (Haplo-SCT) using non-myeloablative conditioning regimen (NMAC) has extended the feasibility of allogeneic transplantation, notably in older patients. However, there is few data specifically focusing on patients aged 70 years and over with AML and MDS. Thus the benefit of transplantation in this population is still debated. Here we report our single center experience of peripheral blood Haplo-SCT with NMAC and post-transplantation cyclophosphamide in AML and MDS patients aged 70 years and over. We analyzed 50 patients (27 AML, 23 MDS) with a median age of 72 years (70–77), 12/50 (24%) with active disease at Haplo-SCT. Cumulative incidence of grade 3–4 acute and moderate or severe chronic GVHD were 6% and 25%, respectively. Non-relapse mortality (NRM) at day +100 was 0%. NRM, relapse, PFS and OS at 3 years were 16%, 18%, 66%, and 69%, respectively. Among patients who were disease free at 2 years post Haplo-SCT, 88% are living without immunosuppressive treatment. Peripheral blood Haplo-SCT is feasible in selected AML/MDS patients over 70 years, without any early NRM. It produces long-term disease control and survival. Thus, age by itself should not be considered as a formal barrier to Haplo-SCT.

La pandémie COVID-19 a bouleversé les activités de greffe et a nécessité le recours à la cryopréservation des greffons de cellules souches hématopoïétiques allogéniques pour sécuriser la procédure, tant au niveau du patient que du donneur. La cryopréservation en situation allogénique, habituellement anecdotique, a été utilisée par tous les centres francophones. Les données collectées auprès de 24 centres ont été analysées afin d’évaluer l’impact de la cryopréservation sur la qualité du greffon. Cette analyse démontre le rôle délétère du transit prolongé (plus de 48heures) sur la récupération des progéniteurs CD34+, l’importance de l’augmentation de la dose de progéniteurs CD34+ demandée, justifiant la nécessité d’un contrôle de qualité après décongélation.

The COVID-19 pandemic disorganized the allogeneic stem cell transplantation activities all over the world, with the necessity to cryopreserve allografts to secure the procedure for both the recipient and the donor. Cryopreservation, usually anecdotal, has been used by all the French speaking centers; data collected from 24 centers were assessed in order to determine the impact of cryopreservation on the quality of allografts. Our analysis clearly demonstrates that increasing transit time (more than 48hours) is deleterious for CD34+ recovery, legitimates the slight increase of the requested CD34+ cell dose with respect to the average recovery rate as well as the importance of the quality control on the infused product.

Abstract After T-cell replete haploidentical stem cell transplantation, GVHD prophylaxis with post-transplant cyclophosphamide (PT-Cy) is now evaluated in unrelated donor (UD) transplants, where antithymocyte globulin (ATG) remains standard. We report the outcome of patients transplanted from HLA-10/10 matched unrelated donor (MUD) treated with PT-Cy (n=30), in comparison with a historical cohort treated with ATG (n=64). In the PT-Cy group, we observed lower 2-4 acute GVHD (23% vs. 45%, p=0.014), lower chronic GVHD (all grades: 13% vs 33%, p=0.029; moderate to severe: 10% vs. 27%, p=0.039) but no difference in the relapse (20% vs. 11%, p=0.628), non-relapse mortality (3% vs 11%, p=0.169), progression free survival (77% vs 78%, p=0.638) and overall survival (87% vs 83%, p=0.602). Neutrophil (19 vs 17 days, p=0.049) and platelet (26 vs 10 days, p<0.001) recovery was significantly delayed in the PT-Cy group. Then, we followed the GVHD and the immunosuppressive treatments (IST) prevalence in disease free patients as marker of quality of life. At 6 months, 5% and 36% in the PT-Cy group were living with GVHD and IST, versus 26% (p=0.030) and 64% (p=0.049). We conclude that PT-Cy is an effective GVHD prophylaxis in 10/10-HLA MUD allo-SCT, representing a valuable alternative to ATG.

Introduction: Donor recruitment in allogeneic hematopoietic stem cell transplantation (HSCT) is a multi-step process involving several stakeholders: patients, donors, coordinators, clinicians, HLA typing lab and blood collection center. With few, if any software existing on the market to perform these steps securely and efficiently, we aimed at developing an in-house academic software to streamline this process. Our objectives were: to reduce delays, facilitate interactions between stakeholders through secure real-time sharing of information and documents and limit the workload at each step. Methods: We set off by listing the steps involved in donor recruitment, analyzing their outputs and time required to accomplish them, documenting the delays between them and establishing key performance indicators. We then created the Prep@gref software that includes an interactive list of patients awaiting an allogenic HSCT, with work lists specific to each of the previously-listed steps (updated in real time), documents and information necessary for proceeding (or not) with donor recruitment and automated alerts for deadlines to accomplishing the individual tasks by each stakeholder. Training stakeholders and optimizing the software preceded its introduction into routine practices. Measurable outcomes were: median time to donor identification (related and unrelated donors), non-quantifiable variables include: stakeholder feedback and ease-of-use of the software. Results: Tracking key performance indicators shows that the time for matched related donor (MRD) identification has reduced by a median of 29% over the past 5 years: 34 days pre-Prep@gref to 24 days since the introduction of Prep@gref into routine use. Time for mismatch related donor (MMRD) has reduced by a median of 32% (36 days pre-Prep@gref versus 25 days post). Unrelated donor search reduced by 21% (67 days versus 53 days, respectively). Time for adoption of the software by stakeholders could explain the slow amelioration of indicators over time. Increase in activity of the transplant program (21% since the introduction of Prep@gref into routine use) and the COVID-19 pandemic did not significantly affect the time for donor identification. Positive feedback from clinicians and coordinators favor its positive impact on practices. Conclusion: Prep@gref software represents a prototype of complex care process modeling, evolving from the traditional organizational set up in many hospitals for donor recruitment. Saving times and being able to delegate tasks is key in addressing shortage of human resource that periodically happen in our activities, while absorbing the growing workload and increasing complexities of these tasks.