Mike Halpenny

Immunoablation and autologous haemopoietic stem-cell transplantation for aggressive multiple sclerosis: a multicentre single-group phase 2 trial.

BACKGROUND Strong immunosuppression, including chemotherapy and immune-depleting antibodies followed by autologous haemopoietic stem-cell transplantation (aHSCT), has been used to treat patients with multiple sclerosis, improving control of relapsing disease. We addressed whether near-complete immunoablation followed by immune cell depleted aHSCT would result in long-term control of multiple sclerosis. METHODS We did this phase 2 single-arm trial at three hospitals in Canada. We enrolled patients with multiple sclerosis, aged 18-50 years with poor prognosis, ongoing disease activity, and an Expanded Disability Status Scale of 3.0-6.0. Autologous CD34 selected haemopoietic stem-cell grafts were collected after mobilisation with cyclophosphamide and filgrastim. Immunoablation with busulfan, cyclophosphamide, and rabbit anti-thymocyte globulin was followed by aHSCT. The primary outcome was multiple sclerosis activity-free survival (events were clinical relapse, appearance of a new or Gd-enhancing lesion on MRI, and sustained progression of Expanded Disability Status Scale score). This study was registered at ClinicalTrials.gov, NCT01099930. FINDINGS Between diagnosis and aHSCT, 24 patients had 167 clinical relapses over 140 patient-years with 188 Gd-enhancing lesions on 48 pre-aHSCT MRI scans. Median follow-up was 6.7 years (range 3.9-12.7). The primary outcome, multiple sclerosis activity-free survival at 3 years after transplantation was 69.6% (95% CI 46.6-84.2). With up to 13 years of follow-up after aHSCT, no relapses occurred and no Gd enhancing lesions or new T2 lesions were seen on 314 MRI sequential scans. The rate of brain atrophy decreased to that expected for healthy controls. One of 24 patients died of transplantation-related complications. 35% of patients had a sustained improvement in their Expanded Disability Status Scale score. INTERPRETATION We describe the first treatment to fully halt all detectable CNS inflammatory activity in patients with multiple sclerosis for a prolonged period in the absence of any ongoing disease-modifying drugs. Furthermore, many of the patients had substantial recovery of neurological function despite their diseases aggressive nature. FUNDING Multiple Sclerosis Scientific Research Foundation.

Evaluation of the sterility testing process of hematopoietic stem cells at Canadian Blood Services

BACKGROUND: Sterility testing of hematopoietic stem cells (HSCs) at The Canadian Blood Services Stem Cell Laboratory is performed using BacT/ALERT aerobic (SA) culture bottles. This study was conducted to verify the efficacy of this method and to assess the use of the BacT/ALERT aerobic (BPA) and anaerobic (BPN) culture bottles for microbial testing of HSCs.

Quality controls of cryopreserved haematopoietic progenitor cells (peripheral blood, cord blood, bone marrow)

The final quality control of cryopreserved progenitor cells is a successful and persistent three lineage engraftment after transplantation. Of course, the stem cell providing institution is obliged to have a program for controlling and monitoring the manufacturing of cellular therapy products before the patients’ conditioning therapy is started. The FACT-JACIE Standards [1] and the Netcord ⁄ FACT Stan- dards prescribe that the director of the institute shall define tests and procedures for measuring and assaying cellular therapy products to ensure their safety, viability and integ- rity and shall also ensure that products meet predetermined release specifications. This requires specifications of assays and the definition of thresholds to allow release.

Validation of sterility testing of cord blood: challenges and results

Sterility testing for cord blood (CB) products is mandatory to prevent transplantation‐transmitted microbial infections. Here, the automated BacT/ALERT (bioMérieux) culture system was validated to detect microbial contamination in CB units processed at the Canadian National Public Cord Blood Bank.

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

BACKGROUND AIMS The ability of hematopoietic progenitor cells-apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented. METHODS In this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2-7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage). RESULTS The doses of nucleated and CD34(+) cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10(8) and 6.8 ± 4.3 × 10(6), respectively) and long- (4.0 ± 4.9 × 10(8) and 6.1 ± 3.4 × 10(6), respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10(9)/L (median 11 days for both short- and long-term storage) and platelets >20 × 10(9)/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10(6)/kg CD34(+) cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets). CONCLUSIONS Cryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.

Cell aggregation in thawed haematopoietic stem cell products visualised using micro-flow imaging.

Dear Sir, Cell aggregation is often observed after thawing peripheral blood progenitor cell products used in haematopoietic stem cell transplantation, and some groups have described methods to reduce cell clumping (Querol et al., 2000; Allan et al., 2002). Transplant centres typically follow standard procedures that guide the thawing conditions and rates of stem cell product infusion to limit cellular injury and prevent cell clumping. The impact on cells reinfused into recipients, however, is unknown and precise quantification of cell clumping has been difficult. Micro-flow imaging (MFI) is a novel method of imaging biologic fluids (Sharma et al., 2007; Huang et al., 2009) and provides a means for investigating cell aggregation following cryopreservation. MFI systems are specialised flow microscopes designed to measure the size distribution, concentration and morphological properties of multi-component populations of sub-visible particles in solution with high sensitivity and statistical accuracy. MFI is broadly used for evaluating populations of micro-particles encountered during bio-formulation development (Sharma et al., 2007; Huang et al., 2009). In this study, we used MFI as a new approach to analyse cell clumping in thawed haematopoietic progenitor cell products. Aliquots (1 mL) of autologous haematopoietic progenitor cells (HPC-A) products were utilised for our study in accordance with the protocol approved by our Research Ethics Board (REB). HPC-A products were volume reduced and cryopreserved in 5% dimethylsulfoxide (DMSO), 12% plasma-Lyte A (Baxter, Deerfield, IL, USA) and 2·2% (w/v) pentastarch by transferring directly to the vapour phase of liquid nitrogen in cryogenic vials. Haematopoietic progenitor products derived from umbilical cord blood (HPC-C) were collected using standard collection kits following informed consent and in accordance with the protocol approved by our institutional ethics review board. Mononuclear cells from HPC-C units were isolated by Ficoll gradient centrifugation and cryopreserved at a concentration of 1·5–2·5 × 107 cells mL−1 in cryogenic 2-mL vials in RPMI-1640 media (1 mL) with a final concentration of 5% DMSO, 12% Plasma-Lyte A and 2·2% (w/v) pentastarch by rate controlled freezing to −80◦C and then transferring the cryovials to the