The Graft Processing subcommittee of the Worldwide Network for Blood and Marrow Transplantation wrote this guideline to assist physicians and laboratory technologists with the setting up of a cell ...processing laboratory (CPL) to support a hematopoietic stem cell transplant program, thereby facilitating the start-up of a transplant program in a new location and improving patient access to transplantation worldwide. This guideline describes the minimal essential features of designing such a laboratory and provides a list of equipment and supply needs and staffing recommendations. It describes the typical scope of services that a CPL is expected to perform, including product testing services, and discusses the basic principles behind the most frequent procedures. Quality management (QM) principles specific to a CPL are also discussed. References to additional guidance documents that are available worldwide to assist with QM and regulatory compliance are also provided.
Virus-Specific T cells (VSTs) have emerged as a safe and promising treatment for cytomegalovirus (CMV) reactivation in patients after solid organ or hematopoietic stem cell transplant. The potency of ...allogeneic VSTs has recently been called into question in phase 3 clinical trials. The production capacity of CMV-specific interferon-γ (IFN-γ) is used as a biomarker of VST product potency and a tool for the selection of off-the-shelf VST products. However, IFN-γ production capacity upon CMV peptide stimulation is variable and unpredictable. Sole reliance on this post-production marker may result in production inefficiencies and suboptimal protocols for VST product generation.
Aim: to identify the biological parameters that predict VST IFN-γ production, aiming to guide the upstream selection of VST material donors and further enhance the potency of VST products.
This was a single-center retrospective study of VSTs manufactured using either hematopoietic stem cell donor-derived (DD) or third-party (TP) derived peripheral blood mononuclear cells (MNCs) from 2017 to 2023. Following Pepmix (pp65 and IE-1) stimulation, MNCs were cultured with IL-4 and IL-7, then harvested as VST products. Both MNCs' and VSTs' characterization and IFN-γ production were analyzed by flow cytometry.
A total of 274 VSTs (116 DD and 158 TP) were generated during the study period from 240 individuals. Among them, 64.1% (n=153) were CMV seropositive. VSTs derived from CMV seropositive individuals exhibited significantly higher CMV-specific IFN-γ production (mean: 4.1±4.5, range: 0-26.1%) compared to those derived from CMV seronegative individuals (mean: 0.1±0.1, range: 0-0.8%). Comparing VSTs and MNCs in CMV positive versus negative status revealed that seropositive individuals contained a higher fraction of CD3+CD56+NKT cells (1.6±1.7% vs 1.0±1.3% in VSTs and 3.0±2.7% vs 1.8±1.6% in MNCs) and CD8+T cells (38.7±17.2% vs 25.6±16.5% in VSTs and 20.4±7.6% vs 14.6±5.6% in MNCs). A positive correlation was observed between IFN-γ production and the product content of CD3+CD56+NKT and CD8+T cells.
Our data supports the selection of CMV seropositive individuals as MNC donors for manufacturing process of CMV-peptide stimulated VST products. The in vitro expansion of IFN-γ expressing CD3+CD56+NKT and CD8+T cells is superior in CMV-seropositive donor derived MNCs and VST products. CMV seropositivity should be used as a primary donor selection criteria for the generation of CMV-specific VST products.
Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, ...fluorescence-activated cell sorting (FACS)-sorted CD34+Thy1+ Lin− peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin−progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of ≥7.2 × 105 cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count ANC >0.5 × 109/L, 16 days) and platelet recovery (platelets > 50 × 109/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.
SUMMARY
The functional and phenotypic properties of normal human CD3+ CD5‐ T cells which have a higher frequency of cytotoxic cells than CD3+CD5+ T lymphocytes have been described. Using three‐ and ...four‐colour immunofluorescence flow cytometric cell sorting, the CD3+CD5‐ and CD3+CD5+ populations were subdivided into αβ or γδ T cell receptor positive cells. The four subsets were examined for the in vitro cytotoxic activity and were also stimulated with mitogens in limiting‐dilution assays to measure the frequencies of proliferating and interleukin‐2 (IL‐2) producing cells. CD3+CD5‐αβ, CD3+CD5‐γδ+ and CD3+CD5+γδ+ cells had lower frequencies of proliferating and IL‐2‐producing cells than did CD3+CD5+αβ+ cells. However, the cytotoxic activity of the different phenotypes was higher in the CD3+CD5‐ subsets, especially when these cells were γδ+ Expression of γδ or lack of expression of CD5 appeared to be associated with the acquisition of cytolytic potentials. CD8 was expressed on 20% of fresh CD3+γδ+ cells. Cultured γδ cells retained the expression of γδ, but quickly lost that of CD8 and with time modulated the expression orCD5, The expression of CD5 was found to be higher on sorted CD3+CD5+γδ‐ than on CD3+CD5+γδ+ cells. These observations indicate that γδ is preferentially expressed on CD5‐negative or weakly positive T lymphocytes and that CD3+CD5‐γδ cells appear to constitute a discrete small subset of mature T lymphocytes which are cytotoxic in nature. However, the exact immunological function of these cells and their place in T cell ontogeny are yet to be elucidated.
Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, ...fluorescence-activated cell sorting (FACS)-sorted CD34+Thy1+ Lin− peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin−progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of ≥7.2 × 105 cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count ANC >0.5 × 109/L, 16 days) and platelet recovery (platelets > 50 × 109/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.
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