Human leukocyte antigen (HLA) loss-mediated relapse is an important mechanism of relapse after haploidentical or mismatched unrelated donor (MMUD) allogeneic hematopoietic stem cell transplantation (allo-HCT), impacting the efficacy of donor lymphocyte infusion (DLI) and second transplants. Real-world use of HLA loss testing is poorly characterized. We conducted an international survey of 62 transplant physicians from 11 countries across North America, Europe, Asia, and Central and South America to assess practices surrounding HLA loss testing after haploidentical or MMUD allo-HCT. We also applied a next-generation sequencing (NGS)-based indel chimerism HLA loss assay to 58 samples from 36 patients who relapsed post-transplant, of whom 29 samples with recipient chimerism greater than 5% were evaluable for HLA loss assessment. Most respondents practiced in academic centers (76%). For molecular relapse, 74.5% used DLI alone; for hematologic relapse, 72.5% favored a second allo-HCT. Most (85.7%) preferred a different donor for second transplants, but HLA loss testing was infrequent prior to second allo-HCT (21%) or DLI (17.7%), with no differences by center type or geography. Testing barriers included cost (88.1%), limited assay availability (87.8%), and long turnaround times (44.5%). At our center, application of an NGS-based indel chimerism assay to 29 evaluable samples (recipient chimerism >5%) identified HLA loss in 3 patients (10.3%), including both complete and partial haplotype loss. Confirmatory NGS HLA typing in one patient validated the indel chimerism findings. The assay was rapid, sensitive, did not require patient-specific markers, and could reduce both cost and turnaround time compared to conventional methods, directly addressing the major barriers to HLA loss testing identified in the international survey. HLA loss testing remains underutilized worldwide. Increased awareness and broader access to rapid, cost-effective assays are needed, alongside prospective studies to establish optimal testing strategies.

IntroductionDonor-derived cell-free DNA (dd-cfDNA) has emerged as a promising non-invasive marker for assessing allograft status and guiding clinical management in transplant recipients. Its utility in kidney transplantation has repeatedly been demonstrated in large studies showing a strong association between elevated dd-cfDNA levels and allograft injury or rejection. This study evaluated the performance of a centralized next-generation sequencing (NGS)-based assay for measurement of dd-cfDNA in patients post-kidney transplantation.MethodsThe TransplantTrace cfDNA Kidney assay utilizes 50 insertion–deletion (indel) markers to discriminate dd-cfDNA. Evaluation of analytical performance included determination of input requirements, analytical sensitivity and specificity, as well as accuracy and precision parameters. Diagnostic performance was evaluated in a retrospective cohort of 104 post-transplantation samples by comparing dd-cfDNA results with biopsy-confirmed rejection.ResultsThe assay required low DNA input (2 ng) and demonstrated high analytical sensitivity, with a verified limit of detection of 0.2% and limit of quantification of 0.3% dd-cfDNA. Analytical accuracy was excellent (R2 = 1.00), with high repeatability and reproducibility across the reportable range of 0.2–30% dd-cfDNA. In the clinical validation, the assay showed high concordance with biopsy-confirmed rejection and excellent discriminatory performance for differentiating active from non-active rejection (AUC of 0.980). At a 1% cut-off, the assay exhibited a positive predictive value of 100%, supporting confident identification of patients likely to have treatable graft injury, while a high negative predictive value (95.6% at 15% prevalence) supports its reliability in ruling out active rejection.ConclusionThe TransplantTrace cfDNA Kidney assay demonstrated robust analytical performance and strong clinical concordance with biopsy-confirmed rejection status. Its high diagnostic accuracy supports reliable identification and exclusion of active rejection, with the potential to reduce reliance on invasive biopsy procedures in patients with elevated serum creatinine but low dd-cfDNA levels. In summary, the findings of this study support the implementation and use of this centralized assay for measurement of dd-cfDNA in patients post-kidney transplantation.

Thalassemia and sickle cell disease are inherited hemoglobinopathies caused by pathogenic variants in the globin genes and represent a major global health burden. Despite major advances in screening and diagnostics, challenges persist due to extensive genetic heterogeneity and complex genotype-phenotype relationships. Conventional workflows typically combine hematologic and biochemical analyses with targeted DNA-based testing. However, traditional molecular approaches are often sequential and labor-intensive, with limited capacity to detect the full spectrum of pathogenic variation. Advances in next-generation sequencing (NGS) now enables integrated and comprehensive strategies to support hemoglobinopathy diagnostics and screening follow-up. Currently available NGS-based platforms allow simultaneous detection of diverse variant classes, including sequence variants and copy number alterations, across multiple disease-relevant genes, including genetic modifiers that may influence disease severity. This review summarizes the genetic basis of thalassemia and sickle cell disease and compiles traditional and emerging molecular testing methodologies. It further discusses the strengths, limitations and utility of NGS-based platforms, and considers their role in shaping future screening and diagnostic workflows for hemoglobinopathies.

Donor-derived cell-free DNA (dd-cfDNA) has emerged as a minimally invasive biomarker of allograft injury following solid organ transplantation. However, its clinical performance and interpretability depend strongly on how dd-cfDNA is measured, reported, and integrated into existing care pathways. This narrative review outlines the biological rationale for dd-cfDNA monitoring and explores the key preanalytical and analytical factors affecting test performance, with an emphasis on comparing measurement technologies and commonly used diagnostic systems. We examine major assay strategies, including next-generation sequencing approaches and PCR-based methods, including digital PCR, and discuss how assay design influences the need for donor/recipient genotyping, analytical sensitivity, susceptibility to clinical confounders (e.g., early post-operative injury, infection, leukopenia, and multi-organ DNA sources), turnaround time, batching, and quality control requirements in centralized versus decentralized testing models. We synthesize evidence for clinical validity and utility across transplanted organs, focusing on use cases such as early detection of injury, risk stratification, and supporting biopsy decisions, while highlighting ongoing challenges like threshold harmonization, inter-platform comparability, and imperfect specificity for distinguishing rejection from non-rejection injury. Overall, dd-cfDNA serves as a valuable adjunct for graft surveillance, but broader clinical applications will require standardized guidance for assay performance and reporting, platform-aware interpretation frameworks, and prospective outcome-focused studies to define optimal testing intervals and decision thresholds.

BACKGROUND:Allogeneic hematopoietic stem cell transplantation (HSCT) is a treatment primarily for hematological malignancies. Infections, relapse, and graft versus host disease (GvHD) are some of the most common adverse events following HSCT. We aimed to evaluate whether the lymphocyte function of the stem cell graft was associated with these events. STUDY DESIGN AND METHODS:We included peripheral blood stem cell (PBSC) grafts from 53 donors to HSCT recipients. The grafts' lymphocyte function was assessed by in vitro blast formation, expression of activation markers, and cytokine production upon stimulation. The cellular composition of the grafts was determined with flow cytometry. Results were compared with the incidence of infections, GvHD, relapse, and survival in the HSCT recipients. RESULTS:We found an association between lower graft T cell response upon mitogen or superantigen stimulation and cytomegalovirus reactivation (p = .013 and p = .0028). Recipients of grafts with an above-median programmed death-1 (PD-1) expression on CD8+ T cell blasts had a higher incidence of relapse and lower overall survival (p = .02). Both findings remained significant in the multivariable analysis. DISCUSSION:Measurement of the lymphocyte response upon mitogen stimulation of PBSC grafts might provide beneficial knowledge of graft-related factors that contribute to the incidence of complications after HSCT.

Donor-specific immunomodulatory cells (DSIMC) have successfully induced donor-specific tolerance in clinical living donor liver transplantation. These cells are generated from recipient peripheral blood mononuclear cells (PBMCs) stimulated with irradiated donor PBMC in the presence of costimulatory blockade, yielding a heterogeneous population of recipient-derived immune cells including T cells, B cells, and NK cells, in which CD4 + regulatory T cells (Tregs) are thought to play a central role. Here, we examined the cell populations and functions crucial for donor-specific immunomodulation within the DSIMC product. DSIMC were generated from PBMC of healthy volunteers using belatacept to achieve costimulatory blockade. Tregs were enriched by CD4 + CD25 + magnetic-cell activated cell sorting (MACS) or CD25 + CD127 -/low fluorescence-activated cell sorting (FACS). CD4 + , CD8 + , CD19 + , and CD25 + CD127 -/low fractions were also individually depleted from DSIMC. Immunomodulatory function of each resulting population was assessed by tritium-labelled thymidine incorporation in mixed lymphocyte cultures (MLC), and cytokine-producing capacity was evaluated by ELISA/ELISpot assays. Unsorted DSIMC exhibited donor-specific suppression at lower cell numbers than CD4 + CD25 + enriched cells, while CD4 + CD25 - T cells showed no immunosuppressive effect. Depletion of CD4 + , CD8 + , or CD19 + cells did not substantially impair immunomodulatory function. In contrast, depletion of the CD25 + CD127 -/low fraction reduced inhibitory function and abolished donor specificity. Depletion of CD4 + or CD25 + CD127 -/low reduced IFN-γ and IL-10 production in response to donor stimulation compared with unsorted DSIMC, implicating CD4 + Tregs as important contributors to cytokine production under allogeneic stimulation. Furthermore, Treg expansion following donor antigen restimulation was observed in unsorted DSIMC but not in CD4 + CD25 + -enriched cells, suggesting that Tregs can be induced from non-Treg CD4 + populations within the DSIMC. In conclusion, CD4 + CD25 + CD127 -/low FoxP3 + -associated cells appear to contribute to donor-specific immunomodulation; however, the enrichment of CD4 + CD25 + T cell diminished DSIMC efficacy. Since the unsorted DSIMC showed stronger activity, we suggest that multiple cell populations may act together to mediate the DSIMC effect.

Allogeneic haematopoietic stem cell transplantation (HSCT) is the only curative therapy for patients diagnosed with acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). γδ+ T-cells, a rare subpopulation of T-cells with both innate and adaptive anticancer functions, have been understudied and the role of different γδ+ T-cell subsets after HSCT is unclear. Objectives:We aimed to characterise the γδ+ T-cells reconstitution post-HSCT, investigating their association with graft-versus-host disease (GvHD) treatment and occurrence, and cytomegalovirus (CMV) reactivation. Methods:Peripheral blood samples from AML and MDS patients and their respective donors were immunophenotyped by flow cytometry before and at several time points after HSCT. Additionally, next-generation sequencing of the TRG locus was performed to assess clonal dynamics and repertoire diversity. Results:Early after HSCT, γδ+ T-cells showed a similar phenotype compared to pre-HSCT, and stable repertoires were observed up to 6 months post-HSCT. Patients with acute GVHD presented a higher frequency of CD8 in γδ+ T-cells, and γδ+ T-cell and subsets frequencies were not affected by anti-thymocyte globulin prophylaxis. Donor-derived Vδ2+ T-cell central memory phenotype was associated with a reduced risk of CMV reactivation in the recipient and with repertoire disturbance. Effector memory cells displaying a specific HLA-DR+ CD86+ phenotype were associated with CMV reactivation and a higher proportion of hyperexpanded clonotypes prior to transplantation. Conclusion:Overall, Vδ1+ and Vδ2+ T-cell phenotypic profiles and γδ TCR repertoire remain stable post-HSCT. This stability is disrupted by CMV reactivation, which is potentially associated with γδ+ T-cells of donor origin and γδ TCR repertoire.

Due to their ability to differentiate into the full diversity of blood cells, CD34+ hematopoietic stem and progenitor cells (HSPC) can be used to treat a variety of diseases. In autologous transplantation CD34+HSPC are harvested when patients are in remission, cryopreserved and later reinfused after therapy. In many cases the harvested cells are cryopreserved for several years. It is unclear how extended, long-term cryostorage affects the quality of CD34+HSPC. This study assessed quality markers in a unique library of 30 CD34+HSPC grafts, cryopreserved for up to 34 years. The samples were divided into 3 groups (<10y, 10-19y and ≥20y) based on their time in cryostorage. Viability indicators, phenotypic as well as functional markers were evaluated. Our results concluded that CD34+HSPC cryostored grafts were resilient to time. No quality marker, except production of selected cytokines, differed between the first and second decade of preservation. After more than two decades of preservation the viability of total leukocytes (CD45+7-AAD-) (P = 0.041), HSPC (CD34+7-AAD-) (P = 0.015), the functionality measured by CFU (P = 0.005) and Th1 and Th2 cytokine production of the grafts were significantly decreased. Despite this, grafts preserved more than twenty years, retained some viability and some ability to form colonies. In addition, most of the live cells retained enzymatic function and capacity to produce cytokines. In conclusion, although more cells die with time, the cells surviving cryopreservation retain some functional capacity after more than two decades of storage. Based on this study, it is hard to identify a time-limit for cryostorage.

Objectives:The clinical outcome after allogeneic haematopoietic stem cell transplantation (aHCT) relies greatly on the efficient recovery of T cells. Several studies have investigated the short-term γδ T cell reconstitution and their role in clinical outcomes following haematopoietic stem cell transplantation. Nevertheless, their long-term characteristics and impact have remained largely unknown. Methods:We analysed γδ T cells from 20 recipient/donor pairs at phenotypic, clonotypic and functional levels to assess their reconstitution ≥ 8 years (median 18 years) post-transplantation using high-parameter flow cytometry and next-generation sequencing of the TCR γ-chain. Results:γδ T cells displayed comparable phenotypic characteristics between recipients and matching donors. The Vδ2+ subset showed a more activated phenotype and cytokine production, while the Vδ1+ and non-Vδ2 T cells maintained long-term CMV control. TCR γ-chain composition in long-term survivors was largely restored, with no significant differences in gene segment usage or diversity. A small cohort of recipients with severe chronic graft-versus-host disease (GVHD) showed overrepresented donor-derived private clonotypes. Furthermore, we also found elevated HLA-DR+Vδ1+ T cells in recipients with severe chronic GVHD. Conclusion:Overall, γδ T cells reconstitute with a normalised repertoire, high functional capacity and sustained CMV control ability. An increased proportion of activated Vδ1+ T cells correlates with chronic GVHD severity, indicating a potential therapeutic target.

The rs2204985 single-nucleotide genetic polymorphism is a common genetic variant located within the T-cell receptor locus that influences thymopoiesis, with the GG genotype presenting higher thymic function than AA counterparts. Recent reports have presented discordant results regarding its effect on the clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT); therefore, evaluation of independent cohorts is warranted. Here, we further investigate the donor polymorphism impact on patient outcome after allo-HSCT in a single-center observation case report from Karolinska University Hospital, Sweden. We retrospectively evaluated 521 adult patients and investigated the impact of the donor rs2204985 genotype on their clinical outcomes post-transplantation. We observed no significant effect of donor genotype on mortality or relapse. The GG genotype was associated with a higher incidence of severe (IIb-IV) acute graft-versus-host disease (GVHD) as compared with AG/AA (P = 0.008), but with no impact on chronic GVHD incidence. The same effect is observed in a subgroup of patients treated with unrelated donors (P = 0.02) but not with human leukocyte antigen-matched sibling donors. A multivariate analysis confirmed the impact of the rs2204985 genotype (P = 0.009) independently of other parameters, including recipient age or sex. Our data suggest that, in this single-center cohort, the donor rs2204985 AA/AG genotype, despite not significantly affecting relapse or survival, is associated with lesser risk of acute GVHD development. ©2025 International Society for Cell & Gene Therapy. Published by Elsevier Inc.

Cryopreservation is a well-established method for extending platelet shelf-life and addressing supply shortages. Traditionally, this involves dimethyl sulfoxide (DMSO) as a cryoprotective agent (CPA), but recent studies suggest that using controlled rate freezing (CRF) with only NaCl may offer a less toxic alternative. To explore further optimization, this study assessed whether adding 10% choline chloride–glycerol, a deep eutectic solvent (DES), could enhance platelet quality in CRF/NaCl cryopreservation. Ten double-dose buffy coat platelet units were divided into test (DES-treated) and control (NaCl-only) groups. After DES exposure (10% for 20 min), all units were prepared using the NaCl protocol and frozen at −80 °C with CRF equipment, then stored for over 90 days. Upon thawing and reconstitution in AB plasma, no significant differences were observed in platelet content post-thaw between control and test units (255 ± 43 vs. 257 ± 41 × 109/unit), post-thaw recovery (>85%): respectively, Δψ (JC-1% pos 63 ± 15 vs. 68 ± 17), LDH (% of total 10 ± 6 vs. 9 ± 6), (CD63% 77 ± 9 vs. 82 ± 7), (CD62P % 72 ± 15 vs. 76 ± 11), (CD42b % 78 ± 9 vs. 80 ± 9), (CD61% 79 ± 9 vs. 78 ± 9), (CD41% 81 ± 11 vs. 83 ± 7), (PAC-1% 33 ± 10 vs. 32 ± 8), (Pecam-1% 78 ± 11 vs. 80 ± 8), (GPIV % 72 ± 10 vs. 74 ± 11), (LAMP-1% 26 ± 14 vs. 11 ± 9), (MPCD61+ % 41 ± 11 vs. 46 ± 10), (ROTEM CT 56 ± 7 vs. 55 ± 6), (ROTEM CFT 110 ± 70 vs. 106 ± 67) and (ROTEM MCF 35 ± 6 vs. 36 ± 6). These findings support the feasibility of CPA-free CRF-based platelet cryopreservation while maintaining functional integrity.

This study presents the analytical performance of a new Next-Generation Sequencing (NGS) assay designed to detect Human Leukocyte Antigen (HLA) loss. Unlike existing methods, this assay offers increased sensitivity, broader applicability, and does not require prior knowledge of specific HLA mismatches, making it a more versatile tool for post-transplant monitoring. The main goal was to determine whether this assay can reliably identify HLA loss in post-transplant patients and provide clinically actionable information for relapse management. Furthermore, the clinical utility of the assay was assessed in patients undergoing Hematopoietic Stem Cell Transplantation (HSCT) with haploidentical or HLA-mismatched unrelated donors (MMUD). The study included both artificial and clinical samples, which were analyzed using the present assay to examine insertion-deletion (indel) markers located within and adjacent to the HLA region. The results demonstrated that the new assay exhibits excellent correlation with the One Lambda Devyser Chimerism assay in samples without HLA loss, achieving a detection limit of 0.25%. Furthermore, the study showed that the markers employed in the assay can effectively identify the occurrence and location of HLA loss. These findings could potentially influence clinical decision-making, when the donor source of retransplants or Donor Lymphocyte Infusions (DLI) need to be re-considered.

Despite the considerable progress in acute myeloid leukemia (AML) treatment, relapse after allogeneic hematopoietic stem cell transplantation (HSCT) is still frequent and associated with a poor prognosis. Relapse has been shown to be correlated with an incomplete eradication of CD34+ leukemic stem cells prior to HSCT. Previously, we have shown that a novel CD34-directed, bispecific T-cell engager (BTE) can efficiently redirect the T-cell effector function toward cancer cells, thus eliminating leukemic cells in vitro and in vivo. However, its impact on γδ T-cells is still unclear. In this study, we tested the efficacy of the CD34-specific BTE using in vitro expanded γδ T-cells as effectors. We showed that the BTEs bind to γδ T-cells and CD34+ leukemic cell lines and induce target cell killing in a dose-dependent manner. Additionally, γδ T-cell mediated killing was found to be superior to αβ T-cell mediated cytotoxicity. Furthermore, we observed that only in the presence of BTE the γδ T-cells induced primary AML blast killing in vitro. Importantly, our results show that γδ T-cells did not target the healthy CD34intermediate endothelial blood–brain barrier cell line (hCMEC/D3) nor lysed CD34+ HSCs from healthy bone marrow samples.

Human Vγ9Vδ2 T lymphocytes are regarded as promising effector cells for cancer immunotherapy since they have the ability to eliminate several tumor cells through non-peptide antigen recognition. However, the cytotoxic function and the mechanism of Vγ9Vδ2 T cells leading to specific killing of cholangiocarcinoma cells are yet to be confirmed. In this study, we established a protocol for ex vivo expansion of Vγ9Vδ2 T cells from healthy donors’ peripheral blood mononuclear cells by culture with zoledronate and addition of IL-2, and IL-15 or IL-18 or neither. Testing the cytotoxic capacity of cultured Vγ9Vδ2 T cells against cholangiocarcinoma cell lines showed higher reactivity than against control cells. Surface expression of CD107 was detected on the Vγ9Vδ2 T cells, suggesting that these cells limit in vitro growth of cholangiocarcinoma cells via degranulation of the perforin and granzyme pathway. Analysis of molecular signaling was used to demonstrate expression of pro- and anti-survival genes and a panel of cytokine genes in Vγ9Vδ2 T cells. We found that in the presence of either IL-15 or IL-18, levels of caspase 3 were significantly reduced. Also, IL-15 and IL-18 stimulated cells contained cytotoxicity against cholangiocarcinoma cells, suggesting that stimulated Vγ9Vδ2 T cells may provide a feasible therapy for cholangiocarcinoma.