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.

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.

Ten years after the first inspection of a candidate European centre, an EBMT registry analysis suggests that clinical outcome is improved when hematopoietic SCT is performed in a JACIE accredited program.

In 2010, JACIE, the Joint Accreditation Committee of ISCT (International Society for Cell Therapy) Europe and EBMT (European group for Blood and Marrow Transplantation) celebrated the tenth anniversary of the first inspection of a European hematopoietic SCT program. JACIE standards establish the criteria for a comprehensive quality management program that covers all three major domains of activity that are necessary for the delivery of HSCT: clinical, collection and processing, as well as their interactions with ancillary and supportive activities. Although more than 200 European programs have applied for JACIE accreditation, and more than 100 have been granted accreditation, a recent retrospective analysis of the large-size EBMT registry of autologous and allogenic hematopoietic HSCT demonstrates that one of the factors affecting the overall survival of recipients of allogenic transplantation is the status of the transplant program regarding JACIE accreditation. This provides one of the first demonstrations that introduction of a quality management system contributes to the overall survival of patients treated with a highly specific medical procedure, and represents a milestone in the implementation of JACIE.

WMDA guidelines for subsequent donations following initial BM or PBSCs.

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.

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.

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.

The role of blood services and regulatory bodies in stem cell transplantation.

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.

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.

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.

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.

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.

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.

High peripheral blood progenitor cell counts enable autologous backup before stem cell transplantation for malignant infantile osteopetrosis.

Autosomal recessive osteopetrosis (OP) is a rare, lethal disorder in which osteoclasts are absent or nonfunctional, resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors, allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However, high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition, colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical.

Therapeutic efficacy of pooled buffy-coat platelet components prepared and stored with a platelet additive solution.

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.

Influence of cytomegalovirus (CMV) sero-positivity on CMV infection, lymphocyte recovery and non-CMV infections following T-cell-depleted allogeneic stem cell transplantation: a comparison between two T-cell depletion regimens.

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.