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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.

Hemopoietic stem-cell transplant patients may require intensive blood component support. Complications of transfusions include transmission of viral and bacterial infections, transfusion-associated graft-versus-host disease and transfusion-related acute lung injury. Alloimmunization to red cell antigens may cause difficulties in selecting compatible blood, while alloimmunization to HLA expressed on platelets may cause subsequent platelet transfusion refractoriness. It is essential to define robust transfusion policies and procedures and these should be regularly audited. This article reviews blood component transfusion in the setting of hemopoietic stem-cell transplant and specifically discusses the management of ABO-mismatched transplants, the prevention of cytomegalovirus transmission, the prevention of transfusion-associated graft-versus-host disease and the use of granulocyte transfusions.

Unrelated donor SCT activity is increasing, and in 5-10% of cases a subsequent donation of stem cells or donor lymphocytes may be requested. Second donations of stem cells are not associated with an increased chance of donor complications, but the yield of CD34+ cells may be lower in some donors. It is acceptable practice for any registry to request subsequent donations and it is recommended that donors should be counselled about this possibility before their first donation. Guidance is provided on the requirements for further medical assessment, the procedures used to agree requests, frequency and timing of donation and timing and duration of donor follow up. Bone Marrow Transplantation (2011) 46, 1409-1412; doi:10.1038/bmt.2010.323; published online 7 February 2011

BACKGROUND: Most hematopoietic progenitor cell (HPC) products are infused or processed shortly after collection, but in some cases this may be delayed for up to 48 hours. A number of variables such as temperature and cell concentration are of critical importance for the integrity of HPCs during this time. STUDY DESIGN AND METHODS: We evaluated critical variables using cord blood HPC units that were divided equally and stored at 4°C versus room temperature (RT) for up to 96 hours. Total nucleated cell (TNC) and mononuclear cell (MNC) counts, viable CD34+ cell counts, and CD45+ cell viability as well as colony-forming unit–granulocyte-macrophage (CFU-GM) present over time at each temperature were determined. RESULTS: Overall, the data indicate that with the exception of viable CD34+ cells, there was a significant decrease in each variable measured for 72 to 96 hours and, with the exception of viable CD34+ cells and CFU-GM, the reductions were significantly greater in RT units than 4°C units. There was an increase in viable CD34+ count for units where TNC count was greater than 8.5 × 109/L, compared with units where TNC count was less than 8.5 × 109/L, that was different for each storage temperature. CONCLUSIONS: Cord blood HPC collections maintained at 4°C retained higher TNC counts, MNC counts, and CD45+ cell viability over a 72- to 96-hour storage period.

Haematopoietic stem cell transplantation (HSCT) is widely used to treat patients with a range of haematological and non-haematological disorders. Both bone marrow and peripheral blood stem cell collection are associated with morbidity and, very rarely, mortality. We investigated the information that exists to adequately inform donors about the relative merits of each procedure. We carried out a systematic review analysing data from six prospective randomised controlled trials of related donors and discuss here the merits and drawbacks of this approach. Registry data mostly describes patient outcome but stem cell donor registries collect and report information on unrelated donors which could easily be extended to related donors. Further well-designed, randomised studies are required.

JACIE was initiated as a small pilot project in Spain in 2000 and launched as a formal Europe-wide inspection program in January 2004. Since 2000, over 150 applications for accreditation have been received by the JACIE Office and more than 130 inspections have been completed in European centers and facilities. Almost all of these 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 centers there were more significant deficiencies. The most common deficiencies were in quality management. Following correction of deficiencies 86 centers have to date achieved full accreditation and many more are nearing the completion of the process. Implementation of JACIE involves a significant investment of time and resources by applicant centers. The majority require at least 18 months to prepare for accreditation and 85% have needed to employ a quality manager and/or data manager on an ongoing basis. However, all centers felt their program had benefited from the implementation of JACIE. JACIE is also working closely with other international organisations related to cellular therapy as part of the Alliance for the Harmonisation of Cell Therapy Accreditation (AHCTA), which is examining the differences in existing standards and aiming to develop international standards for all aspects of stem cell transplantation. In particular the requirements for safety of imported tissues and cells has emphasised the need for global harmonisation. The recent implementation of Directive 2004/23/EC and the associated Commission Directives 2006/1 7/EC and 2006/86/EC has provided an impetus for the implementation of JACIE in European Union (EU) member states. It will be important in the future to examine how JACIE can co-operate with the EU Competent Authorities (CA) to ease the burden of the inspection process for haemopoietic stem cell (HSC) transplant programs.

BACKGROUND: Clinical transplant outcome with umbilical cord blood (UCB) as source of hematopoietic progenitor cells (HPCs) is, among other factors, determined by the total number of viable nucleated cells and/or CD34+ cells in the unit. Quantitative and qualitative losses by processing and cryopreservation and by thawing and washing before transfusion may occur, however. Another reason for a discrepancy between the number of cells in the unit released by the cord blood bank and found in the transplant center may be technical differences in cell counting methods between the two sites. STUDY DESIGN AND METHODS: With the collaborative group for Biomedical Excellence for Safer Transfusion (BEST), an interlaboratory exercise was conducted among nine sites for thawed UCB variables: total nucleated cells, CD34+ cells, viability, and HPC cultures. Three frozen UCB samples were shipped, with instructions for thawing, counting, and HPC plating. RESULTS: Unexpectedly samples arrived at all nine receiving centers without detectable hematopoietic progenitor colony-forming cells. Nevertheless, wide interlaboratory ranges for viability were obtained. The proportion of viable cells was found higher with manual methods, but all viability assays used in the study overestimated functional progenitor cells. CONCLUSIONS: The results underscore the complexity of evaluation of frozen-thawed cord blood cells and the need for standardization of assessment.

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.

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. J. Clin. Apheresis, 2007. © 2007 Internal Cellular Therapy Coding and Labeling Advisory Group.

Summary Robin Coombs was the last survivor of the distinguished group of immunologists that included Philip Gell, John Humphrey, John Marrack, Peter Medawar and Robert White and who were responsible for the renaissance of British Immunology after the Second World War. He is best remembered for describing the antiglobulin test that bears his name. The antiglobulin test revolutionised the diagnosis of haemolytic diseases and the compatibility testing of blood for transfusion. In all, Coombs authored over 200 scientific papers. Haemagglutination reactions became widely used in the diagnosis of a range of infectious agents. Together with Philip Gell, he devised the classification of allergic reactions; these were published in the textbook ‘Clinical Aspects of Immunology’, which he and Gell first edited in 1963 and which became the leading textbook on medical immunology. Robin Coombs was also one of the founders of the British Society for Immunology.

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.

BACKGROUND: Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation. STUDY DESIGN AND METHODS: Four exercises were conducted by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements. Exercise 1 was an intralaboratory exercise in which the reproducibility of cell measurements was determined. Exercises 2 and 3 involved the shipment of identical processed cord blood samples. In Exercise 2, laboratory-specific methods were utilized. In Exercise 3, two commercial CD34+ cell methods (Stem-Kit and TruCOUNT) were used. In Exercise 4, CD34+ cell levels were determined on repetitive regating of identical list-mode files. RESULTS: Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. In Exercise 2, all laboratories except one utilized HA with an impedance technology and determined comparable results for NC and MNC levels, whereas the other laboratory utilized a HA with an optical counting method. Substantial variation was observed on measuring CD34+ cells with ranges of 32 to 141, 32 to 66, and 25 to 116 CD34+ cells per µL for the three identical samples. In Exercise 3, on the use of one specific commercial assay, the ranges of CD34+ levels were 214 to 411 and 62 to 178 cells per µL for the two identical samples. Nearly all participating laboratories determined comparable CD34+ levels on the use of identical list-mode files. CONCLUSION: These studies indicate that substantial variability in CD34+ cell levels were determined with flow cytometry. The variability in NC and MNC levels was minimal with HA methodology.

Acute lymphoblastic leukaemia (ALL) responds well to chemotherapy and the majority of children and a significant proportion of adults are cured of their disease after primary therapy. However, a number of patients relapse and allogeneic transplantation following conditioning with chemotherapy and radiotherapy offers the possibility of long-term survival in a proportion of these patients. A significant number of patients with ALL develop disease that is refractory to further therapy. The infusion of unmodified donor lymphocytes (DLI) following relapse after allogeneic transplantation has been shown to be curative in patients with chronic myeloid leukaemia (CML). However, in ALL the success rate is much lower. The results of in vitro and limited in vivo studies suggest that it may be possible to manipulate lymphocytes from the transplant donor to produce cytotoxic T-lymphocytes (CTL) with increased effectiveness in killing patients' ALL cells. This may be done in a number of ways. For example, some strategies utilise the patients dendritic cells (DC) to present tumour antigens to donor lymphocytes and convert them into CTL either by pulsing DC taken in remission with ALL cells or lysate, fusing such 'normal' DC with ALL cells or using DC cultured from the patient's ALL cells. Other approaches include exploiting the expression of leukaemia-specific antigens such as the proteinase PR-3 or the zinc finger transcription factor Wilms tumour-1 protein (WT-1) to stimulate CTL responses. Alternatively, immunotherapeutic strategies might exploit differences in minor histocompatibility antigens such as HA-1 and HA-2 between donor and recipient. These are expressed solely on haemopoietic cells making them suitable targets for donor derived anti-leukaemic cells. In vivo studies to date suggest that educated T-cells may have a role to play in the treatment of relapsed and refractory ALL in the future.

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

Summary. Despite the introduction of platelet additive solutions for the preparation of pooled platelet components, only a few studies of limited scope have evaluated the clinical efficacy of platelets stored in these solutions. The current report presents an analysis of data to evaluate the response to the transfusion of pooled buffy-coat components suspended in storage solution with reduced (35%) plasma content in comparison with 100% plasma products. During the euroSPRITE clinical trial of platelet components treated with a pathogen inactivation process, control treatment group platelet components were prepared in 100% allogeneic donor plasma (plasma control) or in platelet additive solution (T-Sol™) mixed with plasma (T-Sol control). Control group thrombocytopenic patients received either plasma control or T-Sol control platelet components. One-hour and 24-h platelet count increments (CIs) and corrected count increments (CCIs) were analysed for these two types of preparation. In addition, haemostatic assessments were conducted for each transfusion. One-hour and 24-h mean platelet CIs and post-transfusion haemostatic scores were not significantly different for patients receiving platelet components suspended in 100% plasma and T-Sol plasma mixtures. Pooled buffy-coat platelet components prepared in reduced plasma content mixtures provided therapeutic platelet CIs with effective haemostasis.

Summary. All haemopoietic cell lineages arise from multipotential self-renewing stem cells that give rise to committed progenitor cells. These progenitor cells subsequently differentiate into more lineage-committed cells with a restricted range of plasticity. A hierarchical order is considered to exist, where lineage commitment and differentiation are thought to be irreversible. As cells differentiate, they gradually lose the ability to self-renew. The most primitive haemopoietic progenitor cells have the ability to reconstitute long-term haemopoiesis in myeloablated recipients. However, as cells differentiate, there is an orchestrated silencing of some genes and activation of others, resulting in lineage commitment and generally a reduction in proliferative ability. Here, we discuss potential differences between normal and leukaemic stem cells, some of which may have therapeutic implications.

Summary. The predictable neutropenia that follows allogeneic stem cell transplantation (ASCT) may be associated with recurrence of previous life-threatening infection. We describe nine patients with either previous invasive aspergillosis (IA) or considered to be at high risk of developing IA who underwent ASCT with prophylactic granulocyte transfusions. The study group, when compared with a control group, had a significant reduction in the incidence and duration of fevers (P

A number of haematological and non-haematological malignancies can be successfully treated using high-dose chemotherapy +/- irradiation followed by haematopoietic progenitor cell transplantation. Post transplant, thrombocytopenia and neutropenia always occur and patients require platelet transfusions. It may be possible to reduce the period of thrombocytopenia by re-infusion of ex vivo expanded megakaryocyte progenitors (MP), derived from the progenitor cell graft. We have investigated the expansion of MP from CD34+ enriched cells from normal bone marrow (NBM) and peripheral blood (PB) and remission BM or PB samples from patients with haematological malignancies. CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin (TPO), interleukin 1 (IL-1), IL-6 and stem cell factor (SCF) for 7 d, then cell proliferation was assessed by flow cytometry using lineage-specific markers. It was possible to significantly expand the number of MP cells from all sources. There were no major differences in yields of MP from normal BM or PB, or BM from multiple myeloma and non-Hodgkin's lymphoma patients. However, expansion of MP in acute myeloid leukaemia samples was lower than all other samples and the number of megakaryocyte colony-forming units was reduced. Several cytokine combinations were evaluated to optimize MP expansion from NBM. Equivalent yields of MP were obtained using TPO and one of IL-1, IL-3, granulocyte-macrophage colony-stimulating factor or SCF, suggesting that large cytokine combinations are not necessary for this procedure. It should be possible to scale up the culture conditions described to produce effective MP doses for clinical transplantation.

The increasing success of human leucocyte antigen (HLA)-matched sibling donor (MSD) transplants and combination immunosuppressive treatments have dramatically improved the prognosis of severe aplastic anaemia (SAA) in children and young adults. For patients who lack a MSD there is a significant minority who fail immunosuppressive therapy or suffer from a severe constitutional aplastic anaemia in which immunosuppression would be ineffective. Alternative donor bone marrow transplantation (AD-BMT) has only had limited success in this context. We report the successful outcome of AD-BMT in eight consecutive patients aged 7 months to 15 years, six of whom had acquired aplastic anaemia who had previously failed to respond to immunosuppression, and two of whom had a severe (non-Fanconi) constitutional aplastic anaemia. All eight patients had received multiple red cell and platelet transfusions. We used a new combination of agents for pretransplant conditioning aiming to maximize immunosuppression and minimize toxicity, consisting of Campath-1G or -1H, cyclophosphamide and low-dose total body irradiation (LD TBI) or fludarabine. Toxicity was minimal and all eight children are alive, well and free of disease at a median follow-up of 32 months. We suggest that this approach could facilitate the successful treatment of children with SAA in whom immunosuppressive therapy has failed or is not appropriate.

Disparity for HLA in unrelated donor bone marrow transplantation (BMT) increases the risk of graft rejection and graft-versus-host disease (GVHD) and may compromise transplant outcome. We have compared the outcome of matched and mismatched transplants from unrelated donors in 137 children with acute lymphoblastic leukemia (ALL). Their disease status was complete remission (CR)-1, 24 patients; CR-2, 88 patients; CR-3, 18 patients; CR-4, 2 patients; and relapse, 5 patients. CAMPATH monoclonal antibodies were used for T-cell depletion and cyclosporin A was given to 134 children together with short-course methotrexate in 43, mainly when there was HLA disparity. Fifty-two donor/recipient pairs were HLA-mismatched, 41 at HLA-A and -B and 11 at HLA-DR and -DQ loci. Overall graft failure was increased in recipients of marrow mismatched at either HLA-A, -B, -DR, or -DQ (15.7% v 4.8%; P = .057) mainly because there was a higher proportion of children with primary graft failure (11.8% v 1.2%; P = .012). The presence of an HLA-C locus mismatch did not independently increase the likelihood of graft failure. There was no significant difference in the incidence of acute GVHD ≥ grade 2 between the matched and mismatched groups (P = .849). For patients in CR-2, the risk of relapse post-BMT was significantly lower if leukemic relapse occurred off-treatment (P = .005). The Kaplan-Meier overall and leukemia-free survival (LFS) estimates for recipients of matched and mismatched BMT, respectively, at 36 months were 49% versus 42% (P = .380) and 45% versus 40% (P = .654). Although HLA mismatching results in an increased occurrence of primary graft failure with T-cell–depleted allografts, it allows more donors to be identified rapidly for children with ALL without compromising overall transplant outcome.

This study retrospectively reviews infections over a 7-year period in 60 consecutive adults (median age 25 years) undergoing their first unrelated donor bone marrow transplant (UD-BMT). T-cell depletion was employed in 93%. More than half the patients had one or more severe, potentially life-threatening, infections. There was a high incidence of invasive fungal infections (Aspergillus 17, Candida four), despite the use of itraconazole or amphotericin prophylaxis. Ten Aspergillus infections occurred beyond 100 d. Two patients (11%) with invasive aspergillosis survived. Clustering of infections was noted, with invasive fungal infections significantly associated with bacteraemias (OR 3.73, P = 0.06) and multiple viral infections (OR 4.25, P = 0.05). There were 21 severe viral infections in 16 patients, with CMV disease occurring in four patients only; viral pneumonitis was predominantly due to ‘community respiratory’ viruses. Most early bacteraemias (68%) were due to Gram-positive organisms. The majority of episodes of Gram-negative sepsis were caused by non-fastidious non-fermentative bacteria, such as Pseudomonas spp. and Acinetobacterspp., historically regarded as organisms of low pathogenicity. In patients with successful engraftment and minimal graft-versus-host disease, late infections suggestive of continued immune dysfunction (shingles, recurrent lower respiratory infections, Salmonella enteritis and extensive warts) were common.

The efficacy of allografting in acute lymphoblastic leukemia (ALL) is heavily influenced by remission status at the time of transplant. Using polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis, we have investigated retrospectively the impact of submicroscopic leukemia on outcome in 64 patients receiving allogeneic bone marrow transplantation (BMT) for childhood ALL. Remission BM specimens were taken 6 to 81 days (median, 23) before transplant. All patients received similar conditioning therapy; 50 received grafts from unrelated donors and 14 from related donors. Nineteen patients were transplanted in first complete remission (CR1) and 45 in second or subsequent CR. MRD was analyzed by PCR of Ig or T-cell receptor δ or γ rearrangements, electrophoresis, and allele-specific oligoprobing. Samples were rated high-level positive (clonal band evident after electrophoresis; sensitivity 10−2 to 10−3), low-level positive (MRD detected only after oligoprobing; sensitivity 10−3 to 10−5), or negative. Excluding 8 patients transplanted in CR2 for isolated extramedullary relapse (all MRD−), MRD was detected at high level in 12 patients, low level in 11, and was undetectable in 33. Two-year event-free survival for these groups was 0%, 36%, and 73%, respectively (P < .001). Follow-up in patients remaining in continuing remission is 20 to 96 months (median, 35). These results suggest that MRD analysis could be used routinely in this setting. This would allow identification of patients with resistant leukemia (who may benefit from innovative BMT protocols) and of those with more responsive disease (who may be candidates for randomized trials of BMT versus modern intensive relapse chemotherapy).

We report the results of a retrospective study of the role of intensive care unit (ICU) admission in the management of 367 children who underwent bone marrow transplantation (BMT) at a tertiary referral institution. 39 patients (11%) required 44 ICU admissions for a median of 6 d. 70% received marrow from unrelated donors, half of which were mismatched; 80% had leukaemia and two-thirds were considered high-risk transplants. Respiratory failure was the major reason for admission to ICU. 75% of admissions required mechanical ventilation (for a median of 5 d) and 20 patients had lung injury as defined by the criteria of the Seattle group. None of 11 patients with proven viral pneumonitis survived (P = 0.06) and only one of 20 patients with lung injury survived (P 6 months. ICU admission can be beneficial to selected children post-BMT but it may be less useful in proven viral pneumonitis. Where mechanical ventilation is required, the duration of this support should be limited unless there is rapid improvement.

Recent reports suggest an increased incidence of cytomegalovirus (CMV) infection in recipients of unrelated donor (UD) bone marrow transplantation (BMT). In this study we have collated the incidence of CMV infection and disease in sequential UD (n = 119) and related donor (RD; n = 79) BMT performed in a single institution over a 7-year period. Low-risk patients (CMV seronegative recipient and donor) accounted for 51% of UD BMT (n = 61) and 62% of RD BMT (n = 49), with CMV excretion documented in one RD BMT only. The remaining high-risk patients received identical prophylaxis regimens with aciclovir and intravenous immunoglobulin (IVIG). Two groups consisting of 58 UD BMT (median age 9.0 years, range 0.7–45.3 years) and 30 RD BMT (median age 13.6 years, range 1.6–47.6 years) were analysed. CMV reactivation/re-infection was documented in 15 UD BMT (26%) and 10 RD BMT (33%) (P = 0.72), and CMV disease in four UD BMT (8%) and four RD BMT (13%) (P = 0.533). In this series the risk of CMV excretion and disease following UD BMT was similar to that following RD BMT.

Granulocyte-colony-stimulating factor (G-CSF) is used for the mobilization of hemopoietic stem cells in healthy donors. It has a number of common side effects such as bone pain, which resolve rapidly after administration is discontinued. Recent publications have raised concern that it might act as a trigger for the development of hematologic malignancy in susceptible individuals, possibly by causing genomic instability, but to date there is no evidence that healthy volunteer donors who receive G-CSF are at any increased risk. Ongoing studies aim to confirm whether or not G-CSF can cause chromosomal abnormalities in healthy donors. In the UK, the British Bone Marrow Registry and Anthony Nolan Trust give G-CSF to donors who have agreed to donate peripheral blood stem cells. It is recommended by the UK Registries at present that all stem cell donors are given updated information explaining the current uncertainties with regard to the use of G-CSF before they give informed consent to its administration. This information is based on a statement agreed by the World Marrow Donor Association for use by individual donor registries. Further, it is our current practice that all donors who have received G-CSF, as well as marrow donors who do not, should be under regular review for at least 10 years to allow the occurrence of any long-term adverse events to be documented.

Advances in stem cell research over the past few decades have coincided with large increases in haemopoietic stem cell transplantation (HSCT) using either bone marrow, peripheral blood or cord blood-derived stem cells. Alongside this growth has come an increase in the role played by regulatory bodies, both governmental and professional, to ensure that those undertaking such procedures do so in a manner so as to minimize the risk to patients. Interestingly, government legislation encompasses not only cellular therapies, but also the use of tissues and organs, as many of the processes and procurement procedures involved are similar. In this review, we analyse the trends in HSCT, describe the development and impact of legislation within Europe on this practice and outline the vital role played by the UK blood services in providing robust and high-quality HSCT services.

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.

T hese standards have been developed through the co-operative endeavor of the following organizations: AABB, American Society for Blood and Marrow Transplantation (ASBMT), American Society for Apheresis (ASFA), European Group for Blood and Marrow Transplantation (EBMT), Foundation for the Accreditation of Cellular Therapy (FACT), ICCBBA, International Society of Blood Transfusion (ISBT), International Society for Cellular Therapy (ISCT), ISCT Europe, Joint Accreditation Committee of ISCT and EBMT (JACIE), National Marrow Donor Program (NMDP), and the World Marrow Donor Association (WMDA). The use of standard terminology will help to ensure a common understanding of product definitions. The labeling standard, supported by the ISBT 128 information system, will ensure unique global identification of cellular therapy products, an international reference table for product descriptions, and label design that is consistent worldwide. The organizations supporting these standards believe that their adoption will significantly improve the quality, safety, and traceability of cellular therapy products. In developing the terminology standard the Advisory Group recognized that the frequent movement of cellular therapy products between countries (and continents) required a consistent use of terminology between ISBT 128 and other published standards (e.g., Circular of Information, AABB, FACT, JACIE, Netcord). The Advisory Group has attempted to achieve these aims by removing complexity and redundancy from the coding system wherever possible and, by wide consultation, agreeing to terminology acceptable to all for inclusion in future publication of their standards and guidance. TERMINOLOGY STANDARD

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.

Only some acute lymphoblastic leukemia (ALL) cells are thought to be capable of proliferating to maintain the leukemic clone, and these cells may be the most relevant to target with treatment regimens. We have developed a serum-free suspension culture (SC) system that supported growth of B-ALL cells from 33 patients for up to 6 weeks. ALL cells from 28 cases (85%) were expanded in this system, and growth was superior in SC than in long-term bone marrow culture. To characterize ALL progenitors, cells were sorted for expression of CD34 and CD10 or CD19 and the subfractions assayed in SC and in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Cells capable of long-term proliferation in vitro and NOD/SCID repopulation were derived only from the CD34+/CD10- and CD34+/CD19- subfractions, and these cells could engraft secondary recipients. The engrafted cells had the same immunophenotype and karyotype as was seen at diagnosis, suggesting they had differentiated in vivo. These results demonstrate that ALL cells capable of long-term proliferation in vitro and in vivo are CD34+/CD10-/CD19-. This suggests that cells with a more immature phenotype, rather than committed B-lymphoid cells, may be the targets for transformation in B-ALL.

We compared the incidence and outcome of preemptively treated cytomegalovirus (CMV) infection, lymphocyte recovery and non-CMV infections between two different TCD modalities, one employing CD34+ selection and T-cell add-back (TCAB), preceded by Campath-1H in vivo (CD34+/TCAB group, n=29), and the other using grafts incubated with Campath-1H in vitro (Campath-1H in vitro group, n=32). The probabilities of CMV reactivation and recurrence were 67 and 83.6% in the CD34+/TCAB group and 42.9 and 20% in the Campath-1H group (P=0.07 and 0.02). Donor sero-positivity reduced CMV reactivation in the Campath-1H group, but not in the CD34+/TCAB group. The durations of positive PCR/antigenemia positivity and antiviral therapy were also significantly longer in the CD34+/TCAB group. However, only two patients developed CMV disease in each group. The mean absolute lymphocyte counts (x 10(9)/l) at 30 days (0.27 vs 0.4, P=0.03) and 100 days (0.77 vs 1.4, P=0.01) were significantly lower in the CD34+/TCAB group along with a higher incidence of non-CMV infections in CMV at-risk patients, but not in the CMV low-risk group. These findings suggest that the modality of TCD should be tailored according to the CMV risk status, and CMV sero-positive patients should receive a less extensively T-cell-depleted graft and a CMV sero-positive graft if possible.

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

A nucleic acid-targeted photochemical treatment (PCT) using amotosalen HCl (S-59) and ultraviolet A (UVA) light was developed to inactivate viruses, bacteria, protozoa, and leukocytes in platelet components. We conducted a controlled, randomized, double-blinded trial in thrombocytopenic patients requiring repeated platelet transfusions for up to 56 days of support to evaluate the therapeutic efficacy and safety of platelet components prepared with the buffy coat method using this pathogen inactivation process. A total of 103 patients received one or more transfusions of either PCT test (311 transfusions) or conventional reference (256 transfusions) pooled, leukoreduced platelet components stored for up to 5 days before transfusion. More than 50% of the PCT platelet components were stored for 4 to 5 days prior to transfusion. The mean 1-hour corrected count increment for up to the first 8 test and reference transfusions was not statistically significantly different between treatment groups (13,100 +/- 5400 vs 14,900 +/- 6200, P =.11). By longitudinal regression analysis for all transfusions, equal doses of test and reference components did not differ significantly with respect to the 1-hour (95% confidence interval [CI], -3.1 to 6.1 x 10(9)/L, P =.53) and 24-hour (95% CI, -1.3 to 6.5 x 10(9)/L, P =.19) posttransfusion platelet count. Platelet transfusion dose, pretransfusion storage duration, and patient size were significant covariates (P

Summary. The impact of transfusion of leucodepleted platelet concentrates (PCs) on cytomegalovirus (CMV) disease was assessed in 215 allogeneic (145 unrelated and 70 related donor) transplants over 3 years. In 43%, both donor and patient were CMV seronegative (CMV–/–). All received CMV-seronegative red cells and random leucodepleted PCs. No CMV disease occurred in any CMV–/– (low risk) transplant. CMV infection occurred in 31 seropositive patients (26%); 13 died and five deaths were attributable to CMV disease. When compared with historical controls, who received CMV-seronegative PCs, we found no difference in transfusion-acquired CMV in the current cohort.

We conducted a retrospective review of the clinical features and outcome of adenovirus infection in 572 consecutive patients transplanted in a single centre over a 10 year period. One hundred patients (17%) had a total of 105 episodes of adenovirus infection diagnosed at a median of 18 days post transplant (range 2-150 days). The incidence was higher in children than adults (21% vs 9%, P < 0.001) and in unrelated donor vs matched sibling donor transplants (26% vs 9%, P < 0.001). Diarrhoea and fever were the most common presenting features. Reflecting these symptoms, the most common site of isolation was the stool. Serotypes 1, 2 and 7 were the most frequently seen (total of 41/68 or 60% of evaluable cases). In six patients (6%) adenovirus infection was the direct cause of death occurring at a median of 72 days post transplant (range 18-365 days). Five of these six patients had pulmonary involvement and four had associated graft-versus-host disease (GVHD). Three further patients were considered to have severe adenoviral disease (total incidence 9%). Isolation of virus from multiple sites correlated with a poor outcome (P < 0.001). Comorbid viral infection was common in this group with 50% of all patients having other viruses isolated (predominantly polyoma virus and cytomegalovirus). We conclude that adenovirus is commonly isolated after bone marrow transplant and is a cause of significant morbidity but was a rare cause of mortality (6/572 = 1%) in our patient group as a whole. The relative infrequency of severe infection will make it difficult for the transplant physician to decide which patients should receive experimental antiviral drugs such as ribavirin and cidofovir or immunomodulatory therapy with donor white cell infusions.

The outcome for 39 patients with acute myeloid leukemia (AML) in remission who had CAMPATH 1M T cell-depleted unrelated donor bone marrow transplantations (BMTs) is described. Conditioning was mainly with cyclophosphamide (120 mg/kg) and total body irradiation (TBI) (14.4 Gy), but 5 patients received busulfan in place of TBI and 200 mg/kg cyclophosphamide. All patients received cyclosporin, and short-course methotrexate was given to recipients of mismatched grafts. The patient population was predominantly pediatric (median age, 10 years), but one third of the patients was aged 15 years or above. Twenty-five patients were in second complete remission (CR2), and 14 had high-risk CR1 disease (primarily failed remission induction or antecedent myelodysplastic syndrome, often with complex cytogenetic abnormalities). Both recipient and donor were cytomegalovirus seronegative in 15 of 37 cases (38%); 51% of patients were matched for HLA class I and II. Grade II to IV acute graft-versus-host disease (GVHD) occurred in 24% of patients; chronic GVHD occurred in 5 of 31 evaluable patients (16%), 4 extensive and 1 limited. Relapse occurred in 5 cases (13%); 1 of these 5 patients survives, 24 months after a second unrelated donor transplantation. Two of these relapses were associated with secondary graft failure (incidence rate, 5%). All patients engrafted primarily. Severe viral infection was the major transplant-associated complication, with 12 episodes in 9 patients, 5 of them lethal. Twenty-five patients survive at a median follow-up of 44 months (range, 2-102 months), with estimated actuarial overall and disease-free survival rates at 44 months of 61% (SE 8%) and 57% (SE 8%), respectively. Nineteen patients are more than 2 years post-BMT and may be cured. The functional status of long-term survivors is excellent, with 19 of 21 patients who survive 6 months or more in full-time employment or full-time students. These encouraging results suggest that in patients lacking a sibling donor, unrelated donor BMT for AML in remission achieves survival figures as good as or better than those reported on patients with autologous stem cell transplantation, and that T-cell depletion of grafts is associated with a low relapse rate and an excellent functional status. However, only a randomized study comparing unrelated donor BMT and auto-grafting will resolve which of these treatment strategies is better for patients with AML.Biol Blood Marrow Transplant 2000;6(6):646-53.

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.

Respiratory syncytial virus (RSV) is known to cause acute lung injury in the immunocompromised host, especially recipients of bone marrow allografts. Specific prognostic factors for the development of severe life-threatening disease remain to be identified as does the optimum treatment of established disease. Over a 5-year period the incidence and outcome of RSV in BMT recipients was analysed retrospectively. Prognostic factors assessed included type of transplant, engraftment status at the time of infection, the presence of lower respiratory tract disease, viral genotype and treatment received. During the study period, 26 of 336 (6.3%) allogeneic stem-cell recipients were identified as having RSV. Five patients (19.2%) died as a direct result of RSV. One patient died secondary to an intracranial bleed with concomitant RSV. There were four patients with graft failure (two primary and two secondary) attributable to the presence of RSV, two of whom subsequently died of infections related to prolonged myelosuppression. The presence of lower respiratory tract infection and a poor overall outcome was the only statistically significant association. Unrelated donor transplants and AML as the underlying disease appeared to be associated with a poorer outcome. Engraftment status, viral genotype and RSV treatment received did not correlate with outcome. We conclude that future studies are required to identify early sensitive and reproducible prognostic factors of RSV in the immunocompromised host. The roles of intravenous and nebulised ribavirin need to be clarified by prospective controlled trials.

We have analysed the behaviour of minimal residual disease (MRD) after allogeneic bone marrow transplantation (allo-BMT) in 71 children with acute lymphoblastic leukaemia (ALL). The method relied on PCR of IgH, TCRdelta and/or TCRgamma gene rearrangements followed by electrophoretic size resolution and allele-specific oligoprobing. Patients were similarly conditioned; 55 received marrow from unrelated donors and 16 from related donors. MRD was assessed at various time-points up to 24 months after BMT. Three children were not evaluable due to transplant-related mortality. MRD was detected in 28/32 patients (88%) who relapsed post-BMT; 16 were positive at all times and 12 were initially negative but became positive at a median of 3 months (range 1.5-11) prior to relapse. In contrast, only eight of 36 (22%) patients who remained in continuing complete remission (CCR) (median follow-up 43 months, range 20-94) showed MRD at any time after BMT (P

An advantage of CD34+ cell selection over antibody purging is that a component allograft is produced comprising a stem cell enriched and an unadsorbed fraction, the latter containing T cells which may be used for post-transplant immunotherapy. Initial reports with PBSC allografts suggested that T cell depletion (TCD) by CD34+ cell selection and post-graft cyclosporin A +/- methotrexate was insufficient prophylaxis against acute GVHD. We compared sequential TCD (of a CD34+ cell-selected fraction) using a second (CD2) immunoaffinity step or Campath-1M monoclonal antibody and complement. Since a high stem cell 'dose' enhances engraftment across HLA barriers and improves overall post-transplant outcome, the recovery of CD34+ cells and progenitors were assessed. Sequential positive (CD34+) and negative (CD2+) immunoaffinity selection resulted in a 3.4 log depletion of T cells as compared to a 4.05 log depletion when CD34+ cell selection was followed by Campath-1M treatment. Recoveries of CD34+ cells, CFU-GM and BFU-E following double depletion using CD34+ cell selection plus CD2+ cell depletion were 28, 25 and 17% as compared to 20, 18 and 16% when CD34+ cells were treated with Campath-1M. The unadsorbed fraction contained 85% of the original T cells, from which donor leukocyte infusions in the range of 10(5) to 10(7) CD3+ cells per kg body weight of the recipient were harvested. Despite the advantages of component allografts, the loss of stem/progenitor cells may restrict sequential TCD steps unless single BM harvests are supplemented and/or replaced with mobilised PBSCs.

Graft failure is a common and severe complication of unrelated donor bone marrow transplantation (UD-BMT). However, there are few reports of a second UD-BMT in this setting. We describe 12 patients with graft failure (five primary, seven secondary) who had a second transplant, five from their original donor and seven from a different donor. Their median age was 9 years. Two patients died before day 10 of regimen-related toxicity. Nine of 10 evaluable patients engrafted in a median of 17 days. Secondary graft failure was seen in one patient. Transplant-related morbidity was significant. Six of nine developed acute GHVD, there were five severe infections and five patients developed Bearman grade 3 or 4 extramedullary toxicity. Overall, five patients survive at a median of 38 months after the second BMT and two are in continuous complete remission. Second transplants from unrelated donors for graft failure can result in prolonged survival.

Umbilical cord blood (UCB) is an important source of stem cells for transplantation and may cause less GvHD than non-T-depleted bone marrow (BM). Only 25% of paediatric patients have an HLA matched sibling and for the remainder an unrelated BM or UCB donation may also not be available. A directed UCB (DCB) collection from a newborn sibling may be the only opportunity for a transplant until the sibling can donate BM. Matched DCB donations have been shown to give better long-term results than matched unrelated UCB or BM but DCB collection is not as easy to control as unrelated banking in hospitals established as collection sites. In the context of mandatory licensing under the European Union Tissues and Cells Directive introduced in April 2006, we have reviewed NBS DCB banking. Over ten years, 268 DCB collections were made, 233 (87%) for an existing sibling with a disease treatable by transplantation, and the remainder for families with a history of an inherited high risk disease. Diagnoses included 114 haematological malignancies, 68 hereditary anaemias and 53 immune or metabolic deficiencies. Collections were cryopreserved within 24 h without volume reduction. Volume mL Total TNC ×108 Total CD34 ×106 TNC Viability % Median 73 8.8 2.3 99.0 Mean ± S.D. 76 ± 32 9.9 ± 5.5 3.3 ± 3.4 98.2 +3.7 Range 14–173 1.0–31.0 0.13–26.7 73.0–100.0 88% of collections exceeded 40 ml UCB, the cut-off used by some banks including NHS-CBB. Despite the logistical difficulties in organising collections from many hospitals the cell counts and viabilities of DCB collections compare favourably with collections for UCB banks. The decision to continue storage was made once the cell counts and tissue typing results were also known. Of units collected for existing siblings, 67 (28%) were a 10:10 match for HLA -A, -B, -Cw, -DRB1 and -DQB1. Of these, 13 units were transplanted, with 10 for non-malignant disorders, including one collected following pre-implantation tissue typing for a sibling affected by DBA. Median time to neutrophil engraftment was 18 days (range 14–28) and 30 days (range 11–59) for platelets. DCB collection provides the option of immediate transplant with the potential of future sibling donation of stem cells or lymphocytes. DCB banking for high-risk families is therefore a useful service to provide and complements the work of unrelated UCB banks.

The use of granulocyte colony-stimulating factor in volunteer blood and marrow registry donors

The accurate and rapid determination of the origin of haemopoietic cells may provide valuable information as to the aetiology of, and most appropriate therapy for, leucopenia following allogeneic bone marrow or peripheral blood stem cell transplantation. We describe an approach to the analysis of chimaerism post bone marrow transplantation (BMT) based on the immunomagnetic capture of white cells combined with microsatellite polymerase chain reaction (PCR) and resolution of products by polyacrylamide gel electrophoresis (PAGE). This non-isotopic method enables the chimaeric status to be determined from as little as 1.0 ml of profoundly leucopenic peripheral blood (WBC < or =0.1 x 10(9)/l) and has been applicable to all donor/recipient pairs tested so far. Results are available within 6 h of blood sampling and lineage-specific chimaerism is possible. Furthermore, blood transfusions do not interfere with the analysis.

The role of unrelated donor bone marrow transplantation (UD-BMT) in the management of patients with acute myeloid leukaemia (AML) is uncertain. We describe 18 patients with a median age of 13 years (range 4-31) who received an ex vivo T-cell-depleted UD-BMT for AML (13 in second complete remission (CR2) and five in first complete remission (CR1) with high-risk features). Nine donor recipient pairs were fully matched; eight of these donor-recipient pairs had a single class I HLA mismatch; one patient had both single class I and class II HLA mismatches. Grade II GVHD of the skin occurred in four patients (22%) and limited chronic GVHD in two patients (11%). There have been four deaths: one from relapse and three from infection. With a median follow-up of 27 months, 14 patients survive and the actuarial event-free survival at 2 years is 70 +/- 20% (95% confidence interval). We conclude that unrelated donor BMT can result in prolonged disease-free survival in children and young adults with AML.