Bone Marrow Transplantation (2006) 38, 609–614 & 2006 Nature Publishing Group All rights reserved 0268-3369/06 $30.00 www.nature.com/bmt ORIGINAL ARTICLE Overnight storage of autologous stem cell apheresis products before cryopreservation does not adversely impact early or long-term engraftment following transplantation MD Parkins1, N Bahlis1,2, C Brown1,2, L Savoie1,2, A Chaudhry2, JA Russell1,2 and DA Stewart1,2 1Department of Medicine, University of Calgary and Tom Baker Cancer Centre, Calgary, Alberta, Canada and 2Department of Oncology, University of Calgary and Tom Baker Cancer Centre, Calgary, Alberta, Canada To reduce costs and avoid inconvenient overtime work, our Introduction institution changed policy in September 2000 so that autologous stem cell apheresis products were stored Numerous reports have demonstrated that the single most overnight before cryopreservation rather than immedi- important predictor of engraftment following autologous ately processed. This retrospective reviewwasconducted stem cell transplantation (ASCT) is the CD34 þ dose, to evaluate the possible impact of this policy change on with minimum and preferred targets of 2.0 Â 106 and hematopoietic engraftment following autologous stem cell 5.0 Â 106 cells/kg, respectively.1,2 In order to achieve these transplantation (ASCT). In total, 229 consecutive lym- targets, multiple apheresis procedures may be required. phoma patients who underwent a single, unpurged ASCT Cryopreservation of the autograft is usually carried out in Calgary between January 1995 and November 2003 within 2–4 h following the collection. Current cryopreser- were evaluated. Of these patients, 131 patients’ autografts vation practices are technically demanding and may require underwent immediate processing and cryopreservation experienced laboratory personnel to work extended hours before September 2000, and 98 patients’ autografts in order to process samples on the day of collection. As underwent next-day cryopreservation after overnight such, costs and inconvenience are concerning. storage following this date. Results of univariate and A number of in vitro experiments have shown that multivariate analyses demonstrated no adverse effect of storage of peripheral blood stem cells (PBSC) for short overnight storage before cryopreservation on the number periods of time does not adversely affect stem cell viability of days to initial engraftment of platelets or neutrophils, or engraftment potential.3–6 Storage beyond a period of on the proportion of patients with low blood counts 6 24–72 h has been associated with decreased nucleated cell months post-ASCT, or on lymphoma relapse rates or viability, decreased glucose concentration and increased overall survival post-ASCT. These data suggest that pH.7 It would seem that storage beyond this 24–72 h time overnight storage of the autograft before cryopreservation period risks suboptimal hematopoietic re-constitution. In does not adversely affect graft viability or influence long- 1998, Sugrue et al.8 demonstrated 100% cell viability and term disease status, and support the continued use of 99.5% cell recovery in samples stored at a median overnight storage of stem cells before cryopreservation as temperature of 151C overnight without an adverse effect a convenient, cost reduction measure. on engraftment in a sample of 12 patients whose overnight Bone Marrow Transplantation (2006) 38, 609–614. stored product was batched with next-day leukapheresis doi:10.1038/sj.bmt.1705501; published online 18 September product. This translated into a cost saving of 33%. 2006 In March 2000, Lazarus et al.9 published a small Keywords: hematopoietic stem cells; autologous trans- randomized trial demonstrating that overnight storage of plantation; lymphoma; engraftment purged autografts did not adversely impact early engraft- ment of lymphoma patients undergoing ASCT compared to traditionally processed purged autografts. Thereafter, our center adopted next-day autograft cryopreservation into routine clinical practice. The potential impact of next- day cryopreservation of unpurged autografts on short-term and long-term clinical outcome has not previously been reported. In this retrospective study of lymphoma patients Correspondence: Dr DA Stewart, Departments of Medicine and receiving a single, unpurged ASCT, we compare outcomes Oncology, University of Calgary and Tom Baker Cancer Centre, of immediate cryopreservation of autologous PBSC grafts 1331 – 29th Street NW, Calgary, Alberta, Canada T2N 4N4. E-mail: [email protected] with those of next-day autograft cryopreservation after Received 20 April 2006; revised and accepted 18 August 2006; published overnight storage at 2–61C in terms of time to early online 18 September 2006 engraftment post-ASCT, frequency of low blood counts Next-day PBSC cryopreservation does not impair engraftment MD Parkins et al 610 6 months post-ASCT, as well as relapse-free survival (RFS) immediately processed and cryopreserved as described and overall survival (OS). previously.1 Briefly, PBSC were volume adjusted to a final concentration of o3 Â 108 nucleated cells/ml. Dimethyl- sulfoxide (DMSO), 5% albumin or plasma, and either Patients and methods tissue culture medium TC 199 (before September 2004) or Plasma-Lyte A (after September 2004) were mixed with the Study population PBSC apheresis product to achieve a 10% DMSO final A retrospective analysis of all 229 (age 18–69 years) concentration. The processed suspension was transferred consecutive individuals treated with a single, unpurged into Baxter freezing bags and rate control frozen to À901C. ASCT for Hodgkin’s disease or non-Hodgkin’s lymphoma Following the change in laboratory practice in September (NHL) in Calgary between January 1995 and November 2000, the PBSC leukapheresis products were stored over- 2003 was performed. The November 2003 date was chosen night in the gas permeable Baxter collection bags at 2–61C to give a minimum follow-up of 18 months post-ASCT at and processed the following day using the same technique the time of initiating this retrospective study. Of these described above. Cell concentrations were not diluted or patients, 131 consecutive patients before September 2000 otherwise altered before overnight storage. Viability testing underwent ASCT with immediate cryopreservation of the was performed for a subset of 27 patients before and after leukapheresis product. From September 2000 to November overnight storage using flow cytometric evaluation of 2003, a further 98 consecutive patients underwent ASCT CD34 þ cells with 7-amino-actinomycin D (7-AAD) using a leukapheresis product that was stored overnight at staining (Immunotech, a Beckman Coulter Company, 41C before processing and cryopreservation. All patients Marseille, France). CD34 þ counts were not routinely gave written informed consent for autologous PBSC performed before and after overnight storage, and assays apheresis, autograft cryopreservation, ASCT and data for clonogenic capacity were not performed. Bacterial collection in the Calgary BMT database. Overnight cultures were performed on all apheresis products before autograft storage before cryopreservation was a standard cryopreservation and again post-thaw before infusion. The practice policy change in September 2000, not part of a apheresis nurse collected the pre-cyropreservation bacterial prospective clinical trial. As such, Research Ethics Board culture samples from the apheresis product. If the product approval was not obtained for next-day cryopreservation. was stored overnight, the culture samples were also stored This retrospective study was conducted as a quality overnight and processed the next day. assurance project to ensure that the policy change was not adversely impacting patient outcome. Statistical methodology Comparisons between the two groups, immediate vs Mobilization and collection of PBSC delayed processing, were performed using standard statis- Patients underwent combined chemotherapy plus granulo- tical means using Stata version 8.0. (Stata Corp., College cyte colony-stimulating factor (G-CSF) treatment for Station, TX, USA). Platelet and neutrophil engraftment PBSC mobilization. The most commonly used re-induction were defined as the first of three consecutive days that and PBSC mobilization regimen for lymphoma in Calgary platelets were 420 Â 109/l and absolute neutrophil count is DICEP (dose-intensive cyclophosphamide 5.25 g/m2, (ANC) 40.5 Â 109/l. We performed a w2 test of association etoposide 1.05 g/m2 and cisplatin 105 mg/m2) plus G-CSF to assess for statistical differences in platelet and neutrophil (300 mg/day if body weight o60kg, 480 mg/day if weight recovery between the two groups and hematologic recon- 60–90 kg or 600 mg/day if weight 490kg). Other lower stitution at 6 months post transplant. To test for impact of intensity regimens were used for patients who had a storage, Fisher’s exact test and t-tests were performed. minimal tumor burden, marrow biopsies free of contam- Univariate analyses were performed using a Cox inating lymphoma, significant co-morbid diseases or if they proportional hazard model of variance in conjunction with were enrolled on a clinical trial that mandated a particular simple regression, for the following potential predictors of chemotherapy regimen. Leukapheresis was initiated when engraftment and clinical outcome: immediate or next-day the blood CD34 þ count was at least X10 Â 106/l, white autograft cryopreservation, CD34 þ cell dose/kg, age, blood cells (WBC) X5 Â