Overnight Storage of Autologous Stem Cell Apheresis Products Before

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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 engraftment 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 109/l and platelets X50 Â 109/l. gender, tumor type, disease status, prior therapy and Leukapheresis for all patients was performed using a Cobe- high-dose regimen. Multiple regression analysis was then Spectra, cell separator (Cobe Canada, Scarborough, used to study the effect of a predictor with other variables Ontario, Canada) using a continuous collection procedure being simultaneously controlled. according to the manufacturer’s protocols for mononuclear harvesting. Venous access was through a double or triple lumen dialysis catheter inserted into the internal jugular Results vein. Products were targeted to achieve a minimum of X 6 X 6 2.0 Â 10 CD34 þ cells/kg and a preferred 5.0 Â 10 Patient characteristics CD34 þ cells/kg. The clinical characteristics of the two patient groups are summarized in Table 1. All adult patients (age X18 years) Overnight storage and processing PBSC who underwent ASCT for either NHL or Hodgkin’s The autologous PBSC leukapheresis products of patients disease were included in this analysis. One hundred who underwent mobilization before September 2000 were twenty-eight patients (56%) enrolled had aggressive

Bone Marrow Transplantation Next-day PBSC cryopreservation does not impair engraftment MD Parkins et al 611 Table 1 Patient characteristics NHL, 55 (24%) indolent NHL and 46 (20%) with Hodgkin’s disease. The two groups of patients were similar Variable Overnight storage Immediate (n ¼ 98) processing with respect to age, sex, underlying diagnosis and CD34 þ (n ¼ 131) stem cell dose. The disease status and associated number of prior chemotherapy regimens, and the high-dose Median age (range) in years 47 (18–69) 46 (19–69) chemotherapy regimens use for ASCT were statistically Male gender 58 (59%) 77 (59%) different between groups. Diagnosis Hodgkin’s disease 18 (18%) 28 (21%) Aggressive NHL 57 (58%) 71 (54%) Viability testing and bacterial culture of apheresis products Indolent NHL 23 (23%) 32 (24%) Viability testing of CD34 þ cells using ﬂow cytometry and 7-AAD dye for a subset of autografts that underwent Disease status* next-day cryopreservation demonstrated 91–100% viability Refractory 14 (14%) 22 (17%) First remission 44 (45%) 29 (22%) (median 99%), with absolutely identical results before and Relapsed 40(41%) 80(61%) after overnight storage. In the immediate cryopreservation group before September 2000, none of the bacterial cultures Prior chemotherapy treatments* were positive post-apheresis and only two (2.0%) were 1 82 (84%) 87 (66%) 2 12 (12%) 30(23%) positive post-thaw (Enterobacter agglomerans and Gram- X3 4 (4%) 14 (11%) positive bacilli and cocci). In the overnight storage group after September 2000, one (0.8%) of the bacterial cultures Prior radiation treatment 12 (12%) 41 (31%) was positive post-apheresis (Gram-positive bacilli) and three (2.3%) were positive post-thaw (one with Bacillus sp Mobilization chemotherapy DICEP+G-CSF 72 (73%) 96 (73%) and two Gram-positive cocci). Other+G-CSF 26 (27%) 35 (27%)

Single apheresis 90(92%) 121 (92%) Early hematopoietic engraftment post-ASCT Hematopoietic engraftment by timing of autograft cryo- High-dose regimens* 2 preservation is summarized in Table 2. Median and mean Melphalan 200 mg/m 19 (19%) 78 (60%) 9 Melphalan 180mg/m 2 3 (3%) 26 (20%) times to platelet recovery 420 Â 10 /l occurred 1 and 2.5 TBI 500 cGy days earlier, respectively in the overnight storage/next-day BEAM 52 (53%) 20(15%) cryopreservation group (95% confidence interval (CI) of FluBu 14 (14%) 1 (1%) difference in means 1.1–3.9, P ¼ 0.0007). When organized Other 10(10%) 6 (5%) into arbitrarily defined categories, the frequency of early CD34+ cells infused: median 15.9 (1.7–56.9) 13.6 (1.1–89.2) (p9 day), intermediate (10–13 days) and delayed (X14 (range) Â 106/kg Â 106/kg days), platelet engraftment was 35, 62 and 3%, respectively for patients who received next-day cryopreserved auto- Abbreviations: BEAM ¼ carmustine 300 mg/m2, etoposide 800 mg/m2, grafts, compared to 14, 68 and 18%, respectively for ara-C 1600 mg/m2, melphalan 140mg/m 2; DICEP ¼ dose-intensive cyclo- patients who received immediately cryopreserved auto- phosphamide, etoposide and cisplatin; G-CSF ¼ granulocyte colony- stimulating factor; NHL non-Hodgkin’s lymphoma; FluBu fludarabine grafts (Po0.0001). Median and mean times to ANC ¼ ¼ 9 250mg/m 2 IV, busulfan 12.8 mg/kg IV; TBI ¼ total body irradiation. recovery 40.5 Â 10 /l occurred 1 and 0.8 days earlier, *Po0.05. respectively in the overnight storage/next-day cryopreser-

Table 2 Initial and long-term hematopoietic engraftment post-ASCT

End point Cell line Overnight storage Immediate processing (n ¼ 98) (n ¼ 131)

Median days to initial engraftment (range) Platelets 420 Â 109/l 10days (7–17) 11 days (8–89) ANC 40.5 Â 109/l 10days (7–14) 11 days (7–15)

Median blood counts 6 months post-ASCTa (range) WBC count Â 109/l 4.8 (0.6–12.6) 5.0 (1.3–14.3) Platelet count Â 109/l 199 (33–447) 188 (26–365) Hemoglobin g/l 130(72–158) 127 (87–168)

% Patients with low blood counts 6 months post-ASCTa WBC o4 Â 109/l 28.4% 21.1% Platelets o150 Â 109/l 19.3% 26.3% Hemoglobin o120g/l for 43.2% 48.3% women or o137 g/l for men

Abbreviations: ANC ¼ absolute neutrophil count; ASCT ¼ autologous stem cell transplantation; WBC ¼ white blood cell count. aExcludes 17 and 30patients in overnight storage and immediate cryopreservation groups, respectively, who had relapsed, died or had missing data by 6 months post-ASCT.

Bone Marrow Transplantation Next-day PBSC cryopreservation does not impair engraftment MD Parkins et al 612 vation group (95%CI of difference in means 0.5–1.1, 18 months. At 18 months post-ASCT, 92 (71%) patients Po0.0001). When organized into arbitrarily defined transplanted for aggressive NHL, 36 (65%) patients categories, the frequency of early (p9 day), intermediate transplanted for indolent NHL and 36 patients (78%) (10–12 days) and delayed (X13 days) ANC engraftment transplanted for Hodgkin’s disease remain in remission. was 15, 82 and 3%, respectively for patients who received Single covariate Cox proportional hazard modeling de- next-day cryopreserved autografts, compared to 5, 86 and monstrated a nonsignificant HR of 0.68 for the next-day 9%, respectively for patients who received immediately cryopreserved group relative to immediately cryopreserved cryopreserved autografts (P ¼ 0.006). group (95%CI 0.38–1.22, P ¼ 0.198) indicating no statis- Time to event analysis using single covariate Cox tical difference in the rate of relapse between the two study proportional hazard modeling to compare the two groups groups. By multivariate analysis, factors independently of cryopreservation timing for rates of platelet and ANC associated with higher relapse risk 18 months post-ASCT recovery revealed hazard ratios (HRs) of 1.94 (95%CI were aggressive NHL relative to Hodgkin’s disease (HR 1.47–2.56, Po0.0001) and 1.86 (95%CI 1.42–2.43, 3.96; 95%CI 1.40–11.24, P ¼ 0.01), relapsed (HR 3.66; Po0.0001), respectively, suggesting that engraftment was 95%CI 1.55–8.61, P ¼ 0.003) and refractory (HR 5.64; achieved more rapidly when autografts were stored over- 95%CI 2.19–14.54, Po0.001) disease status relative to first night before cryopreservation. Multivariate analysis using remission, and male gender (HR 1.98; 95%CI 1.04–3.79, multiple covariate Cox proportional hazard modeling P ¼ 0.04). revealed that only overnight autograft storage before Regarding OS, a single covariate Cox proportional cryopreservation (HR 1.88; 95%CI 1.39–2.55, Po0.0001) hazard model revealed a nonsignificant HR of 0.68 for and the infused CD34 þ cell dose/kg (HR 1.01; 95%CI overnight autograft storage (95%CI 0.39–1.17, P ¼ 0.16) 1.01–1.02, Po0.0001) were significantly associated with indicating that timing of autograft cryopreservation was time to platelet recovery. Factors significantly associated not a predictor of death. By multivariate analysis, factors with more rapid ANC recovery included overnight auto- independently associated with higher risk of death post- graft storage before cryopreservation (HR 1.96; 95%CI ASCT were aggressive NHL (HR 2.60; 95%CI 1.01–6.65, 1.47–2.62, Po0.0001), infused CD34 þ cell dose/kg (HR P ¼ 0.047) relative to Hodgkin’s disease, relapsed (HR 2.68; 1.01; 95%CI 1.00–1.01, P ¼ 0.019) and female gender 95%CI 1.33–5.37, P ¼ 0.006) and refractory (HR 3.76; (HR 1.41; 95%CI 1.07–1.87, P ¼ 0.015), whereas relapsed 95%CI 1.71–8.26, P ¼ 0.001) disease status relative to first (HR 0.69; 95%CI 0.47–1.00, P ¼ 0.047) or refractory remission, use of total body irradiation (TBI) (HR 2.30; disease status (HR 0.56; 95%CI 0.36–0.87, P ¼ 0.01) were 95%CI 1.12–4.73, P ¼ 0.02) and age (HR 1.05; 95%CI associated with delayed ANC recovery compared to first 1.02–1.08, Po0.0001). The HR of 1.049 for age indicates remission status. that a 1-year increase in age was associated with a 4.9% increase in the relative risk of dying post-ASCT. Hematopoietic reconstitution at 6 months post transplantation Discussion Table 2 summarizes the proportion of patients who had blood counts below the lower limit of normal 6 months Myeloablative chemotherapy and ASCT is increasingly post-ASCT according to timing of autograft cryopreserva- used to improve outcomes of patients with relapsed or poor tion. By multivariate, logistic regression analysis, there was prognosis lymphoma. However, given the frequent require- no statistically significant association between immediate or ment for multiple apheresis procedures in order to attain next-day autograft cryopreservation and the proportion of the minimally acceptable CD34 þ cell dose for ASCT, cost individuals who had a normal platelet count 4150 Â 109/l 6 and inconvenience of autograft collection and cryopreser- months post-ASCT (P ¼ 0.22), hemoglobin levels 4120g/l vation are of concern.10 Immediate autograft processing (P ¼ 0.15) or WBC 44.0 Â 109/l (P ¼ 0.18). This logistic and cryopreservation has been the standard policy at many regression analysis detected an association between older centers with the assumption that this maximizes autograft age (odds ratio (OR) 1.05, P 0.001) and male gender (OR o viability.11 In Calgary, because of the increasing use of 2.43, P ¼ 0.006) and low hemoglobin levels, as well as ASCT, restraints on the healthcare system and the study between lower CD34 þ cell dose/kg (OR 1.06, P ¼ 0.005) published by Lazarus et al.9 demonstrating feasibility of and low platelet counts 6 months post-ASCT. Linear next-day cryopreservation, we changed policy in September regression analysis was also performed using actual 2000 and began storing autografts overnight before observed blood counts 6 months post-ASCT (results not cryopreservation. This change in policy allowed single but shown) and gave results consistent with those of the logistic more prolonged, high volume leukapheresis and efficient regression. Although long-term transfusion dependence time utilization by laboratory personnel. was not assessed directly, the number of individuals with The results of our study suggest that storage of significantly reduced hemoglobin 80g/l (1/89 (1%) vs o autologous PBSC grafts at 2–61C overnight before proces- 0/106 (0%), P ¼ 0.456) or platelets 50 Â 109/l (1/89 (1%) o sing and cryopreservation does not have a deleterious vs 4/106 (4%), P ¼ 0.378) did not differ between groups. impact on early engraftment, blood counts 6 months post- ASCT, relapse rates or OS. In fact, the rate of early RFS and OS engraftment was significantly faster in patients who Of the 65 patients who relapsed throughout the entire received next-day cryopreserved autografts compared length of this study, 51 (78.5%). did so within the first to the immediately processed autografts. Although this

Bone Marrow Transplantation Next-day PBSC cryopreservation does not impair engraftment MD Parkins et al 613 finding was likely due to the use of historical controls in this of overnight storage on positively selected autografts. In retrospective study, the association between overnight this study, there was no significant difference in days to autograft storage and more rapid early engraftment neutrophil recovery (10.5 vs 11) or platelet recovery (13.5 vs persisted despite controlling for differences in patient 13). Of note, CD34 þ selection is a technically laborious characteristics and treatment protocols between study process, associated with increased cost, and has not been groups in multivariate analysis. Another, albeit less likely, proven to improve clinical outcome of ASCT.19 and unsubstantiated explanation for this finding is possible RFS and OS were evaluated in this study assessing ex vivo expansion of the autograft through overnight overnight autograft storage before cryopreservation be- storage before cryopreservation.12,13 cause we have reported that CD34 þ dose influences both The retrospective, observational nature of this study relapse and death,14 and because there is a theoretical introduces several inherent limitations. The patient popula- potential for decrease in tumor contamination with over- tions were heterogeneous with respect to diagnosis and night storage as cancerous cells have decreased ex vivo disease status. Newer technologies and supportive mechan- survival.20 As far as we have been able to determine, this is isms have been developed that may have positively the first study which has demonstrated that there is not a influenced outcome of the more recently treated group correlation between overnight autograft storage before receiving next-day cryopreserved autografts. The condi- cryopreservation and RFS or OS. As expected, factors we tioning regimens were significantly different between the found to be most strongly associated with inferior RFS and two groups, with a change from melphalan/TBI during the OS were diagnosis of aggressive NHL, as well as relapsed time of immediate autograft cryopreservation, to BEAM or refractory disease status. (carmustine, etoposide, ara-C, melphalan) during the more In conclusion, this study demonstrates that PBSC recent next-day autograft cryopreservation. Although this autografts containing 45 Â 106 CD34 þ cells/kg can be change in conditioning regimen may influence long-term safely stored overnight at 2–61C, without adversely outcomes such as relapse and death, it is unlikely to affect effecting the time to early engraftment, blood counts 6 engraftment rates. months post-ASCT, RFS or OS. It is important to establish Predictors of engraftment recognized in the literature are whether the same is true for products of between 2 and predominantly the number of CD34 þ cells infused and 5 Â 106 CD34 þ cells/kg. The use of next-day autograft prior alkylator therapy.14–17 In the present study, CD34 þ cryopreservation allows large volume apheresis to proceed dose correlated with time to platelet and neutrophil later in the day, gives time for assessment of tumor recovery. Other independent predictors for neutrophil but contamination before cryopreservation if one desires, and not platelet recovery identified in this study include female permits staff to process and freeze the autograft in a gender and absence of either refractory or relapsed disease. convenient, cost-effective manner. We suggest that centers This study confirms a previous report that overnight considering a change in policy to overnight autograft storage of stem cells does not adversely impact engraftment storage before cryopreservation execute a validation pro- potential.9 It needs to be emphasized, however, that both tocol comparing fresh and overnight stored apheresis groups received fairly large CD34 þ cell doses, and that we products for CD34 þ counts, tests for viability and cannot exclude an adverse effect of overnight autograft possibly clonogenic capacity. This is particularly important storage before cryopreservation in the setting of a small for centers that have a significant proportion of autografts CD34 þ cell dose. Only two patients in our overnight containing o5 Â 106 CD34 þ cells/kg. autograft storage group received ASCT with o3 Â 106 CD34 þ cells/kg and only five received 3–5 Â 106 CD34 þ cells/kg. This seven-patient sample size is too small to Acknowledgements analyze and draw statistical conclusions regarding engraftment kinetics. Of interest, in multivariate analyses evaluat- We thank Maggie Yang and Jan McLaughlin for database ing the association between CD34 þ cell dose/kg and management, Susan Berrigan from blood bank who supplied engraftment, the HRs were close to 1.0, probably because data, policies and procedures and Dr Karen Kopciuk and Thomas Speidel for statistical analysis. the vast majority of patients in the study received ASCT with 45 Â 106 CD34 þ cells/kg, and also because all patients received G-CSF post-ASCT to hasten ANC References recovery. Two previously reported studies have assessed the impact 1 Stewart DA, Guo D, Luider J, Auer I, Klassen J, Ching E et al. of overnight storage of autografts that had been subjected Factors predicting engraftment of autologous blood stem cells: to purging with CD34 þ selection. Koc et al.18 using CD34+ subsets inferior to the total CD34+ cell dose. Bone immuno-affinity columns directed against CD34 þ assessed Marrow Transplant 1999; 23: 1237–1243. the effect of overnight storage of positively selected PBSC 2 Siena S, Schiavo R, Pedrazzoli P, Carlo-Stella C. Therapeutic of 11 patients against historical controls and found no relevance of CD34 cell dose in blood cell transplantation for J Clin Oncol difference in time to neutrophil recovery (11 days), cancer therapy. 2000; 18: 1360–1377. 3 Jestice HK, Scott MA, Ager S, Tolliday BH, Marcus RE. however, there was significant delay in platelet recovery Liquid storage of peripheral blood progenitor cells for (13 vs 28.5 days). Samples stored overnight had a mean of transplantation. Bone Marrow Transplant 1994; 14: 991–994. 2.4-fold less CD34 þ cells infused suggesting potential 4 Hechler G, Weide R, Heymanns J, Koppler H, Havemann K. loss or differentiation. In 2000, Lazarus et al.9 reported a Storage of noncryopreserved periphered blood stem cells for randomized control trial of 53 patients to assess the impact transplantation. Ann Hematol 1996; 72: 303–306.

Bone Marrow Transplantation Next-day PBSC cryopreservation does not impair engraftment MD Parkins et al 614 5 Pettengell R, Woll PJ, O’Connor DA, Dexter TM, Testa NG. 13 McNiece I, Jones R, Bearman SI, Cagnoni P, Nieto Y, Viability of haemopoietic progenitors from whole blood, bone Franklin W et al. Ex vivo expanded peripheral blood marrow and leukapheresis product: effects of storage media, progenitor cells provide rapid neutrophil recovery after high- temperature and time. Bone Marrow Transplant 1994; 14: dose chemotherapy in patients with breast cancer. Blood 2000; 703–706. 96: 3001–3007. 6 Beaujean F, Pico J, Norol F, Divine M, Le Forestier C, 14 Stewart DA, Guo D, Luider J, Auer I, Klassen J, Morris D Duedari N. Characteristics of peripheral blood progenitor cells et al. A low CD34+ cell dose predicts relapse and death early frozen after 24 h of liquid storage. J Hematother 1996; 5: following autologous blood stem cell transplantation. Hematol 681–686. 2001; 6: 19–27. 7 de Kreuk AM, Jonkhoff AR, Zevenbergen A, Hendriks EC, 15 Weaver CH, Hazelton B, Birch R, Palmer P, Allen C, Schuurhuis GJ, Ossenkoppele GJ et al. Storage of unprocessed Schwartzberg L et al. An analysis of engraftment kinetics as G-CSF-mobilized whole blood in a modiﬁed Leibovitz’s L15 a function of the CD34 content of peripheral blood progenitor medium preserves clonogenic capacity for at least 7 days. Bone cell collections in 692 patients after the administration of Marrow Transplant 2001; 28: 145–155. myeloablative chemotherapy. Blood 1995; 86: 3961–3969. 8 Sugrue MW, Hutcheson CE, Fisk DD, Roberts CG, Mageed 16 Tricot G, Jagannath S, Vesole D, Nelson J, Tindle S, Miller L A, Wingard JR et al. The effect of overnight storage of et al. Peripheral blood stem cell transplants for multiple leukapheresis stem cell products on cell viability, recovery, and myeloma: identiﬁcation of favourable variables for rapid cost. J Hematother 1998; 7: 431–436. engraftment in 225 patients. Blood 1995; 85: 588–596. 9 Lazarus HM, Pecora AL, Shea TC, Koc ON, White JM, 17 Gandhi MK, Jestice K, Scott MA, Bloxham D, Bass G, Gabriel DA et al. CD34+ selection of hematopoietic blood Marcus RE. The minimum CD34 threshold depends on prior cell collections and autotransplantation in lymphoma: over- chemotherapy in autologous peripheral blood stem cell night storage of cells at 4 degrees C does not affect outcome. recipients. Bone Marrow Transplant 1999; 23: 9–13. Bone Marrow Transplant 2000; 25: 559–566. 18 Koc ON, Gerson SL, Phillips GL, Cooper BW, Kutteh L, 10Meehan KR, Areman EM, Ericson SG, Matias C, Seifeldin R, Van Zant G et al. Autologous CD34+ cell transplantation for Schulman K. Mobilization, collection, and processing of patients with advanced lymphoma: effects of overnight storage autologous peripheral blood stem cells: development of a on peripheral blood progenitor cell enrichment and engraft- clinical process with associated costs. J Hematother Stem Cell ment. Bone Marrow Transplant 1998; 21: 337–343. Res 2000; 9: 767–771. 19 Friedman J, Lazarus HM, Koc ON. Autologous CD34+ 11 Magrin S, Gentile S, Santoro A, Indovina A, Scime R, Hauser enriched peripheral blood progenitor cell (PBPC) transplant- D et al. Collection, processing and storage of peripheral ation is associated with higher morbidity in patients with blood stem cells (PBSC). Haematologica 1991; 76 (Suppl 1): lymphoma when compared to unmanipulated PBPC trans- 55–57. plantation. Bone Marrow Transplant 2000; 26: 831–836. 12 Reiffers J, Cailliot C, Dazey B, Attal M, Caraux J, Boiron JM. 20Widmer L, Pichert G, Jost LM, Stahel RA. Fate of Abrogation of post-myeloablative chemotherapy neutropenia contaminating t(14; 18)+ lymphoma cells during ex vivo by ex vivo expanded autologous CD34-positive cells. Lancet expansion of CD34-selected hematopoietic progenitor cells. 1999; 354: 1092–1093. Blood 1996; 88: 3166–3175.

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