Haematopoietic stem cell transplantation: a comparison between the accreditation process performed by competent authorities and JACIE in Italy.

There have been great advances over the last decades in haematopoietic stem cell (HSC) transplantation, using either bone marrow, peripheral blood or cord blood-derived stem cells. The coming into force of the European legislation on tissues and cells and the consequent transposition of Directives into national laws have required the health authorities in the Member States (MS) and the scientific societies to review the transplantation activities to ensure the circulation of safe HSC products. Here, the regulatory inspection process performed by the Competent Authorities and the professional voluntary accreditation process of the Transplant Programmes active in Italy is compared.

Transportation of cellular therapy products: report of a survey by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) collaborative.

BACKGROUND AND OBJECTIVES: Most cell therapy products (CTP) are infused or processed shortly after collection but in some cases this may be delayed for up to 48 h. A number of variables such as temperature and cell concentration are of critical importance for the integrity of CTP during this time. MATERIALS AND METHODS: We conducted a survey of cellular therapy laboratories to ascertain current practices for CTP transportation. RESULTS: There were 194 respondents of whom 90% shipped or received CTP--84% allogeneic, 71% autologous and 62% therapeutic cells. Processing facilities shipped or received the following products--hematopoietic progenitor cells (HPC), Marrow 73%; HPC, Apheresis 90%; HPC, Cord Blood 54% and others 14%. Other CTP included donor lymphocytes, mesenchymal stem cells (MSC), natural killer cells, buffy coat neutrophils and virus-specific cytotoxic T lymphocytes (CTL). More than 70% of respondents believed that it was acceptable for CTP to be held for up to 2 h without checking the temperature or cell density and a similar proportion agreed that putting products in containers to control parameters such as temperature within this time period was unnecessary. The majority of centres shipped or received between 1 and 10 CTP annually and 66% received products taking more than 2 h to ship. Of these, 82% specified the conditions for temperature in transit whilst 57% monitored temperature in transit and 74% of these used a data logger. The temperature range most commonly specified was 18-24 degrees C. The majority of processing facilities did not request an adjustment to the cell density even for products taking more than 2 h to reach their facility. More than 90% of respondents tested HPC for CD34(+) cells, viability and sterility; 40-48% performed colony-forming unit-granulocyte macrophage (CFU-GM) analysis. Only viability was thought by > 50% of respondents to be impacted by temperature, cell density and other parameters. CONCLUSION: Understanding current practice will help in the design of future studies for CTP storage and transportation.

Absence of damaging effects of stem cell donation in unrelated donors assessed by FISH and gene variance screening.

ASTRACT: Granulocyte-Colony-Stimulating factor (G-CSF) is currently the standard mobilising agent for peripheral blood stem cell (PBSC) donation. Concerns that it may trigger chromosome aberrations similar to those observed in leukaemia patients were refuted but long-term effects of G-CSF mobilisation on genome integrity remains unclear. In the setting of a multi-centre clinical trial we screened blood samples from 50 PBSC donors at cellular and gene level for aberrations common in haematological malignancies using fluorescence in situ hybridisation (FISH) and next generation sequencing (NGS) assays. Analysis of samples collected before, on the day of donation, 90 and 180 days after G-CSF admission confirmed the absence of short-term effects in PBSC donors on both quiescent and dividing cells. This data did not differ from the results of 50 individuals tested 3-5 years after bone marrow donation and 50 healthy persons. NGS using a panel targeting 54 genes recurrently affected in myeloid disorders (TruSight Myeloid panel, Illumina) showed that the gene profiles of samples from 48 PBSC donors remained stable throughout the study period. These data strongly indicate absence of detrimental effects on the genome integrity caused by PBSC donation.

Practical guidelines for applying statistical process control to blood component production.

Legislation, guidelines and recommendations for blood components related to statistical process control (SPC) and the selection of a quality monitoring (QM) sampling regimen are subject to misinterpretation and lack practical guidance on implementation. The aim of this article is: to review and interpret applicable European legislation and guidelines and to develop an SPC strategy that meets these requirements; and to provide practical guidance on the selection and application of appropriate techniques and the interpretation of resultant blood component QM data. A methodology is presented which utilizes: an algorithm to select an appropriate quality-monitoring strategy for the blood component parameter under consideration; a range of straightforward, validated SPC techniques for variable data and an assessment of process capability (Cpk) and blood component parameter 'criticality' to determine the sampling regimen. The methodology was applied to routine National Health Service Blood and Transplant (NHSBT) blood component data for 2005-2006. Cpk values ranged from 0.22 to >3 and their predicted non-conformance rates were close to those observed (23 to <0.001%). Required sample size ranged from 0.01 to 10%. Chosen techniques identified significant deviation from 'as validated' performance within an appropriate time-scale. Thus the methodology was straightforward to apply and prompted the choice of a clinically and operationally appropriate sampling regimen and analysis for each blood component parameter. This evidence-based, targeted use of SPC for blood component monitoring provides an essential focus on processes with a low capability in achieving their specifications.

Multicenter study on in vitro characterization of dendritic cells.

BACKGROUND: There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability. METHODS: CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol. RESULTS: Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion. DISCUSSION: In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.

Current status of JACIE accreditation in Europe: a special report from the Joint Accreditation Committee of the ISCT and the EBMT (JACIE).

JACIE (Joint Accreditation Committee of the ISCT and the EBMT) launched its first official inspection programme in January 2004. Since then, 35 centres in Europe have been inspected. Almost all were found to be functioning at a high level of excellence, with the majority having only minor deficiencies in compliance with the standards. In one-third of centres there were more significant deficiencies. The most common deficiencies were in quality management, and a survey of the applicant centres confirmed this was the area where centres experienced most difficulty in preparation for accreditation. Following correction of deficiencies, 28 centres have at the time of writing achieved full accreditation. Implementation of JACIE required a significant investment of time and resources by applicant centres. The majority required at least 18 months to prepare for accreditation and 85% needed to employ a quality manager and/or data manager on an ongoing basis. However, all centres felt their programme had benefited from the implementation of JACIE. In addition to the inspection and accreditation of individual centres, JACIE maintains an educational programme including training courses for inspectors and for centre preparation. JACIE is also working closely with other international organisations working in cellular therapy to develop international standards for all aspects of stem cell transplant. The recent implementation of Directive 2004/23/EC has provided an impetus for the implementation of JACIE in EU member states and in particular the requirements for safety of imported tissues and cells have emphasised the need for global harmonisation.

Terminology and labeling of cellular products-2: Implementation plan.

The publication of new standards for terminology and labeling marks an important step in ensuring consistency and traceability of cellular therapies at the global level. However, it is only with the widespread implementation of the standard that the benefits can be truly realized. This paper provides guidance on the practical aspects of adopting these new standards for organizations with differing current levels of computerization. It discusses project management, equipment, licensing, and validation topics.

Terminology and labeling of cellular products: 1. Standards.

The International Cellular Therapy Coding and Labeling Advisory Group was established to address the growing need for standardization of terminology and labeling for cellular therapy products as a result of increasing international transfer of these products. This paper presents new standards for terminology and labeling. These standards have been developed through a consultative process and are supported by key professional and accreditation bodies. By using these standards, together with the unique donation identification numbers and international product reference tables provided by the International Society of Blood Transfusion (ISBT) 128 Standard, consistency and traceability can be assured at the global level. A companion paper provides guidance on the implementation of the ISBT 128 system.

Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation.

The COBE Spectra is used to volume/red blood cell (RBC) deplete BM before transplantation or cryopreservation. We have audited our results to identify the effect of transit time, refrigerated storage, age and cellular composition on mononuclear cells (MNC) and CD34+ cell recoveries, volume/RBC depletion and neutrophil engraftment. In total, 88 consecutive collections from autologous (n = 25) and allogeneic (n = 63) donors were included. The mean collection volume was 1250 +/- 398 ml with RBC content of 341 +/- 113 ml. The MNC and CD34+ cell recoveries were 83.3 +/- 18.5 and 88.1 +/- 18.9%, respectively, volume depletion was 88.2+/-4.4% and RBC depletion 98.3 +/- 1.8%. Neutrophil engraftment was achieved in 20.1 +/- 6.4 days. Factors affecting MNC and CD34+ cell recoveries were transit time (P = 0.0060), overall age (P < 0.0210) and MNC/CD34+ cell concentrations (P < 0.0313). The presence of crenated RBC also reduced CD34+ cell recovery (P = 0.0028). Refrigerated storage did not adversely affect cell recovery (P > 0.8161) or neutrophil engraftment (P = 0.8959). This study demonstrates that time in transit, overall age, MNC and CD34+ cell concentrations and RBC condition were important factors affecting processing. RBCs show artefacts soon after collection at ambient temperatures and these may interfere with the separation and collection of MNC/CD34+ cells. Refrigeration at 4-6 degrees C during transit and storage may reduce formation of RBC artefacts and maximize MNC and CD34+ cell recoveries.

Topical issues in unrelated donor haematopoietic stem cell transplants: a report from a workshop convened by the Anthony Nolan Trust in London - 2005.

Over more than three decades, The Anthony Nolan Trust (ANT) has provided an unrelated donor (UD) for over 4000 children and adults lacking a suitable family member donor, and has remained at the forefront of developments in haematopoietic stem cell transplantation (HSCT) and bone marrow register management. These three decades have seen major changes in clinical practice of UD-HSCT, including new indications, increased use of alternative haematopoietic cell sources, significant improvement of the outcome as a result of better support care, less-toxic conditioning regimens, and better donor selection, and expansion to older patients with higher comorbidities. In order to foster our goal of improving UD-HSCT availability and outcome in a progressively more complex clinical scenario, a new initiative from ANT was launched in 2005 to convene an experts workshop to address the topical issues in this field. Four consecutive panels addressed factors influencing donor selection and transplant outcome, the use of cord blood, regulatory and accreditation issues, and future developments in this field. This report summarizes the discussions held in this workshop, which will likely develop into a periodic event where transplant clinicians, scientists and registry members will meet to share their experience and vision in the field of UD-HSCT.

Unrelated donor bone marrow transplantation for children and adolescents with Philadelphia-positive acute lymphoblastic leukemia.

PURPOSE: Few patients with Philadelphia-positive acute lymphoblastic leukemia (Ph-positive ALL) have been cured by chemotherapy alone. Registry figures show that 38% of patients who have a matched-sibling bone marrow transplant (BMT) are disease-free 2 years after transplant, but the majority of patients lack a sibling donor. Most modern ALL protocols recommend unrelated donor (UD) BMT for patients with Ph-positive ALL in first complete remission (CR1), but the outcome of this is unknown. PATIENTS AND METHODS: We report the results of 15 children and adolescents who had a T-cell depleted UD-BMT for Ph-positive ALL. Thirteen of 15 had been previously treated on United Kingdom ALL protocols. Nine were in CR1 and six had more advanced disease. Eleven donor recipient pairs were matched at HLA-A, HLA-B, HLA-DR, and HLA-DQ, and four were mismatched at one or two HLA loci. RESULTS: The incidence of greater than grade I acute and chronic graft-versus-host disease (GVHD) was low (13% and 8%, respectively). Six patients have relapsed and seven patients survive at a median of 21 months post-BMT; six of seven are disease free. All seven survivors are in full-time education or work. The 2-year overall and disease-free survivals are 44% +/- 13% and 37% +/- 13% (+/- SE). None of four patients who had mismatched donors survived, but seven of 11 matched recipients survive (P < .05). CONCLUSION: UD-BMT can produce prolonged disease-free survival in young patients with Ph-positive ALL who otherwise would have an extremely poor outlook.

Early emergence of PNH-like T cells after allogeneic stem cell transplants utilising CAMPATH-1H for T cell depletion.

CAMPATH-1H (C-1H) is widely used in vivo and / or in vitro for T cell depletion in hematopoietic SCT. This humanised monoclonal antibody is specific for CD52, a marker coexpressed on the majority of human lymphocytes with CD48 and other glycosylphosphatidyl-inositol (GPI) anchored proteins. We detected CD52 / CD48 dual expression on >99% of CD3(+) lymphocytes from normal individuals and all 15 post-SCT patients whose transplants did not utilise C-1H. By contrast, 23 / 26 patients with transplants involving C-1H (in vivo, in vitro or both) exhibited populations lacking CD52 expression that accounted for 49.7% (4.2-86.2%) of the CD3+ lymphocytes (median and range) in samples evaluated at a median of 2 months post-SCT. Most CD52- cells also lacked CD48 expression. These GPI- T cells were of either donor or mixed donor / recipient origin. They were predominant in the early months after SCT at times of profound lymphopenia and inversely correlated with the recovery of the absolute lymphocyte count (r= - 0.663, P<0.0001). The presence of CD52- cells has been correlated previously with clinical outcome after CAMPATH therapy for both malignant and nonmalignant diseases.

An optimised biphasic culture system for the generation of functional dendritic cells from patients with acute lymphoblastic leukaemia at presentation and in clinical remission.

We have tested the hypothesis that functional dendritic cells (DC) may be generated from patients with acute lymphoblastic leukaemia (ALL). We evaluated the production of DC from blast cells taken at presentation from nine children with ALL. Blast cells were expanded in serum-free medium supplemented with Flt3L, G-CSF, GM-CSF, IL-3, IL-6 and SCF for 7 days and subsequently stimulated with Flt3L, GM-CSF and TGF-beta for a further 14 days, with the addition of TNF-alpha for the final 48 h of culture. Cultured cells had the morphological appearance of DC and expressed the DC-associated antigens CD1A (range 2-87%) and CD83 (15-44%). Expression of the co-stimulatory molecules CD80 and CD86 was increased and the majority of these cells retained their expression of CD34 (73+/-4%) and HLA-DR (79+/-5%). Seven of the nine ALL had a leukaemia-specific abnormality and DC generated from five of these seven cases were derived from the leukaemic clone. Leukaemic DC derived from four HLA-A*02-positive ALL pulsed with CMV-associated peptides could induce significant proliferation of peptide-specific CD8+ T cells. This specificity was verified using tetrameric complexes of HLA class l/antigenic peptide. DC could also be generated from cells taken at times of complete remission of ALL and from normal controls using these culture conditions. These findings show that functional DC can be generated both from ALL blasts and from patients in remission; these might be utilised in future for immunotherapeutic strategies in the treatment of ALL.

Effective emetic control during conditioning of children for bone marrow transplantation using ondansetron, a 5-HT3 antagonist.

Preparation for bone marrow transplantation (BMT) uses the extremely emetogenic combination of chemotherapy and total body irradiation (TBI). Ondansetron is a selective 5-HT3 antagonist and has clear anti-emetic capabilities. The efficacy of the drug was assessed in 15 children (aged 2-17 years) who received high dose cyclophosphamide (on days -6 and -5) and TBI (days -3 to 0 inclusive). During days -6 to -4 when the emetic effect of cyclophosphamide would be most pronounced, 12 of the 15 patients (80%) had fewer than five emetic episodes during their worst 24-h period, 11 (73%) had fewer than three vomits whilst nine (60%) experienced no vomiting or retching. Eleven patients progressed to TBI and 10 (91%) had fewer than five emetic events in the worst 24-h period (days -3 to +2), six (55%) had no vomiting at all. Of 100 evaluable 'patient-days' 83 (83%) were without any vomiting or retching and a further 10 'patient-days' had only one or two emetic episodes. There were no significant side-effects noted and in particular no extrapyramidal reactions. Headaches and constipation, which have been seen in adult studies, were not reported by patient or parent on any of the study days and transient elevation of liver enzymes were noted in only two patients. Ondansetron has a major role in preparing patients for BMT.

Ultraviolet irradiation for the prevention of graft-versus-host disease and graft rejection in bone marrow transplantation.

This review describes an approach to the prevention of graft-versus-host disease (GVHD) and graft rejection following allogeneic BMT that differs from conventional methods. Ultraviolet (UV) irradiation inhibits the proliferative responses of lymphoid cells to mitogens and alloantigens by inactivation of T lymphocytes and dendritic cells, and in animal models this can prevent both GVHD and graft rejection. It is important that the marrow repopulating capacity of haemopoietic stem cells is not damaged by the irradiation process. We have found that polymorphic microsatellite markers are a sensitive way of assessing the impact of UV irradiation on chimerism after BMT in rodents.

Graft-versus-host disease in children receiving HLA-identical allogeneic bone marrow transplants with a low adjusted T lymphocyte dose.

Fourteen children with high risk leukaemia received allogeneic bone marrow transplants from HLA-identical MLC-compatible sibling donors. All bone marrows were T cell depleted and a T cell addback was prepared from the donor's peripheral blood so that the mean total number of CD3+ cells given was 2.6 (1.0-4.1) x 10(5)/kg recipient body weight. This was administered as a short infusion prior to the bone marrow. The children were conditioned with 1440 cGy fractionated total body irradiation and cyclophosphamide 120 mg/kg and were not given cyclosporin A or methotrexate. All patients engrafted and none showed late graft rejection. Acute graft-versus-host disease (GVHD) developed in nine of 14 children and required treatment with steroids. Two children with grade IV GVHD and one with grade I acute GVHD who subsequently developed severe chronic GVHD died. There have been two relapses (both fatal) and one death from cytomegalovirus pneumonitis. Survival is currently 57% (8/14) with a mean follow-up of 548 days (range 384-810). A high incidence of GVHD which was fatal in three patients can occur despite infusion of low T cell numbers in the absence of post-graft immunosuppression.

Early lymphocyte recovery is an important determinant of outcome following allogeneic transplantation with CD34+ selected graft and limited T-cell addback.

We evaluated the outcome of 29 patients (age 22-60 years), who received a CD34+selected related (n=16) or unrelated graft (n=13) with limited T-cell addback (TCAB) (median 5.9 x 10(4)/kg) following full-intensity conditioning for haematological malignancies. In all, 16 patients (55%) had either advanced disease or previous transplants. The cumulative incidences of grade 2-4 acute GVHD were 15.4 and 19.2% and that for chronic extensive GVHD were 35 and 37% in related and unrelated graft recipients, respectively. The strongest predictor of nonrelapse mortality and overall survival was the absolute lymphocyte count (ALC) at 30 days; patients with ALC<0.35 x 10(9)/l having an NRM and OS of 59.2 and 24.7%, compared to 10 and 90% in those with a higher ALC. Patients with acute leukaemia had poorer survival and this was associated with a lower ALC as well. Thus, TCAB with a CD34+ selected graft resulted in a comparable outcome in both older and younger patients, but the survival was strongly influenced by early lymphocyte recovery.

A laboratory comparison of T cell depletion by CD34+ cell immunoaffinity selection and in vitro Campath-1M treatment: clinical implications for bone marrow transplantation and donor leukocyte therapy.

Donor leukocyte infusions (DLI) have been used effectively to induce remission in patients who relapse after BMT. Using CD34+ cell immunoaffinity enrichment, donor T cells may be captured in the unadsorbed (residual) fraction and we assessed this as a potential source of functional T cells for post-BMT immunotherapy. We extended our study to compare CD34+ cell selection and antibody-mediated cell lysis using Campath-1M and measured T cell-depletion, CD34+ cell recovery and relative progenitor proliferative potential. The recovery of CD3+ cells (responsive to IL-2 or PHA) in the unadsorbed fraction was 84+/-12% (mean+/-s.d.) using a laboratory scale CD34+ cell selection process (CEPRATE LC). The immunoselected (CD34+ cell enriched) product contained 55+/-12% of the starting CD34+ cells (purity, 75+/-6%) with recoveries of 44+/-12% and 42+/-13% for CFU-GM and BFU-E respectively. T cell depletion was 99.8+/-0.2% (FACS) and the frequency of clonable T cells estimated at 1:640 (limiting dilution assay). In comparison, Campath-1M-treated marrow samples gave recoveries of CD34+ cells, CFU-GM and BFU-E of 50+/-7%, 78+/-20% and 79+/-18%, respectively. The frequency of clonable T cells was 1:2700 despite an estimated T cell depletion of 98.4+/-1.9%. Data obtained from four BM harvests processed on the clinical grade CEPRATE SC system was comparable in every respect to the laboratory scale system. The yield of 1259 +/- 222 x 10(6) CD3+ cells in the unadsorbed fraction would allow for multiple graded incremental T cell aliquots for DLI for patients with acute leukaemia.

Pediatric marrow transplantation for acute leukemia using unrelated donors and T-replete or -depleted grafts: a case-matched analysis.

A retrospective, case-matched analysis of the short-term toxicity, risk of GVHD and relapse as well as outcome in pediatric unrelated marrow transplantation was conducted by comparing recipients of T-replete and -depleted grafts in a two-center setting. Both groups contained 30 patients with acute leukemia matched by age at transplant, gender, primary diagnosis and disease status. Acute (90% vs 53%) and chronic (48% vs 0%) GVHD were more common among recipients of T-replete grafts. No significant differences in graft rejection/failure or viral infections were encountered between the two groups. Relapses were more prevalent (37% vs 15%) among recipients of T-depleted grafts. Outcome (EFS) was similar in the two groups. Consequently, in the analysis of transplant outcome, the higher risk of procedure-related, toxic complications among pediatric recipients of T-replete marrow grafts appears to be balanced by an increased risk of relapse among the recipients of T-depleted grafts.

Immune reconstitution after BMT in children.

Serial assessment of peripheral blood T and B cell recovery and serum immunoglobulins was performed in 19 children for the first year following BMT and compared with normal values established from healthy children. Immunophenotypic analysis on bone marrow was performed in selected cases by Southern blotting of the immunoglobulin heavy chain (IgH) gene. We found no significant differences between T cell-replete or depleted allogeneic bone marrow transplants. Lymphocyte numbers were low until 9 months post-BMT. T cell numbers (CD2, CD3, CD5) were also low until 12 months but B cell numbers (CD19) became normal at 3 months. Both CD4+ and CD8+ T cell subsets were low post-BMT with depression of CD4+ greater and more prolonged than that of CD8+. No overshoot of CD8+ was seen. The principal effect of GVHD or its treatment was further depression of CD4+ cells but with no increase in CD8+; recovery of B cells was also delayed. Recovery of IgG was slow with only six of 11 children reaching an age-adjusted normal level by 1 year, whereas there was more rapid recovery of IgM and IgA. Several children had an increase in lymphocytes of immature appearance in their bone marrow at varying times post-BMT with increased cells of phenotype CD19+, CD10+, HLA-DR+ and TdT+. In each case Southern blotting showed a germline pattern of the IgH indicating a polyclonal early B cell regenerative population.