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The role of depletion of dimethyl sulfoxide before autografting: on hematologic recovery, side effects, and toxicity
Biology of Blood and Marrow Transplantation 10:135-141 (2004) 䊚 2004 American Society for Blood and Marrow Transplantation 1083-8791/04/1002-0006$30.00/0 doi:10.1016/j.bbmt.2003.09.016
The Role of Depletion of Dimethyl Sulfoxide before Autografting: On Hematologic Recovery, Side Effects, and Toxicity R. Syme,1 M. Bewick,2 D. Stewart,3 K. Porter,2 T. Chadderton,4 S. Glu¨ck1,5,6 1
Oncology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; 2Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario, Canada; 3Tom Baker Cancer Centre, Calgary, Alberta, Canada; 4 GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania; 5 Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; 6Pharmacology & Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Correspondence and reprint requests: Stefan Glu¨ck, MD, PhD, FRCPC, Department of Oncology, Faculty of Medicine, University of Calgary, 1331 29th St. N.W., Calgary, Alberta T2N 4N2, Canada (e-mail: [email protected]). Received August 15, 2003; accepted September 25, 2003
ABSTRACT Cryopreservation of stem cells after collection from peripheral blood or bone marrow for autologous transplantation necessitates protection with dimethyl sulfoxide (DMSO). Unfortunately, DMSO, when infused with the thawed cell suspension, may induce serious complications and side effects. To assess whether depletion of DMSO before autografting affects safety and efficacy, 56 consenting consecutive patients treated with highdose chemotherapy and autologous blood stem cell transplantation were assigned to obtain either an untreated or DMSO-depleted autograft. On the day of transplantation, the cryopreserved cells were thawed and infused to the patient either immediately or after washing 3 times in normal saline supplemented with 6% anticoagulant citrate dextrose solution. Cell count with viability, clonogenic assay, and phenotyping were performed before and after thawing and after washing. Hematologic recovery, side effects, and complications were recorded. The in vitro and clinical data on 56 patients show that the depletion of DMSO in vitro before autografting does not induce a significant loss of cell number, viability, colony-forming unit– granulocytemacrophage activity, or number of CD34ⴙ cells. Furthermore, it leads to a safe and sustained engraftment. The complications and side effects, as recorded by continuous monitoring, were substantially less; however, the procedure takes 3 to 4 hours of laboratory work per patient. © 2004 American Society for Blood and Marrow Transplantation
KEY WORDS High-dose chemotherapy ● Autologous blood stem cell transplantation sulfoxide ● Cryopreservation
INTRODUCTION High-dose chemotherapy (HDCT) and autologous blood stem cell (ABSC) transplantation are increasingly used for the treatment of hematologic malignancies [1]. As part of bone marrow (BM) or ABSC transplantation, collected cells are frozen and stored for prolonged periods. Because cells can be injured by the direct effect of low temperatures and by the formation of ice crystals, which then leads to cell dehydration, a cryoprotectant is used to maintain cell integrity. Ideally, a cryoprotectant should maintain the quality of the collection product without causing any
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adverse effects to the recipient of the transplantation or to the cells being preserved. Dimethyl sulfoxide (DMSO) is the cryopreservation agent most often used by transplantation centers. Although it is usually relatively nontoxic to patients in small amounts, reinfusion of solutions containing substantial volumes of DMSO has been associated with adverse effects (up to 20%) [2]. Sudden and severe hypotension can result from the intravenous (IV) infusion of DMSO, presumably from histamine-induced vasodilation [2]. Skin flushing, dyspnea, abdominal cramping, nausea, and diarrhea can also be attributed to DMSO-induced 135
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histamine release [3]. Other adverse effects associated with DMSO include headache, fever, hemolysis, encephalopathy, and cardiovascular symptoms [4-8]. These may result in increased morbidity, prolonged hospitalization, and increased treatment-related costs. So far, only a few reports [9-12] have investigated the in vitro toxicity of DMSO on hematopoietic stem cells. It has been postulated that DMSO can be toxic to hematopoietic cells and BM stromal cells [13]. In view of the potential toxicities associated with the administration of DMSO to patients, it may be advantageous to eliminate it from the autograft before reinfusion. This study compared cell quality and quantity, engraftment kinetics, and adverse reactions related to the reinfusion of DMSO-depleted versus DMSO-containing autografts. This study was performed in patients who had received HDCT with ABSC transplantation for metastatic breast cancer in an outpatient setting previously described by our group [14].
METHODS Patients
Fifty-six patients receiving HDCT and ABSC transplantation for metastatic breast cancer were evaluated. All patients participated in a single-center study in Sudbury. The institutional review board approved the study. All patients signed informed consent. Included in our study were patients between 18 and 55 years old (median, 42 years) who had not had previous chemotherapy for metastatic breast cancer. They had evaluable or measurable disease, and the disease either progressed with hormonal treatment or was hormonereceptor negative. No patients with central nervous system metastasis were allowed on study. The first 21 patients received an untreated autograft, whereas the following 35 had DMSO depletion before reinfusion of their autograft. These were consecutive, consenting patients. Chemotherapy Regimens and ABSC Collection
All patients underwent 3 to 4 cycles of induction chemotherapy (IDC) with cyclophosphamide, doxorubicin, and 5-fluorouracil. A growth factor, recombinant human granulocyte colony-stimulating factor (rhG-CSF, Neupogen; Amgen Canada Inc., Mississauga, ONT), was administered subcutaneously (SC) from the day after IDC until the apheresis procedures were completed. When the white blood cell count (WBC) reached 2.5/nL and the platelet count was 50/nL, apheresis was performed daily on 4 consecutive days as described previously [15]. Apheresis was performed with the Fenwal CS 3000 Plus blood separator (Baxter Healthcare Corporation, Deerfield, IL). A total blood volume of 10 L per apheresis was pro136
cessed at a flow rate of 60 to 70 mL/min [15-17]. Collections were continued until a value of at least 2 ⫻ 106 CD34⫹ cells per kilogram body weight was reached. Each apheresis product (AP) obtained was cryopreserved in 10% (vol/vol) DMSO and stored in liquid nitrogen until the time of reinfusion [15]. The HDCT consisted of cyclophosphamide 6 g/m2 bodysurface area (BSA), mitoxantrone 70 mg/m2 BSA, and either carboplatin 800 mg/m2 BSA or vinblastine 12 mg/m2 BSA, all administered IV. Then, 24 to 48 hours later, the autograft was reinfused, and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) or rhG-CSF at a dose of 5 g/kg body weight SC was administered until hematologic recovery. High-Dose Chemotherapy
Only patients whose disease did not progress on IDC and who did not experience severe toxicity or major organ damage proceeded to HDCT. HDCT consisted of cyclophosphamide 2 g/m2 IV, mitoxantrone 23.3 mg/m2 IV, and mesna 300 mg/m2 IV every 8 hours. This was delivered on 3 consecutive days for a cumulative dose of cyclophosphamide 6 g/m2 and mitoxantrone 70 mg/m2. Appropriate hydration and premedications were administered. On day 4, the paclitaxel dose starting at 250 mg/m2 BSA was delivered as a 3-hour IV infusion. Premedication, including dexamethasone at 20 mg by mouth, was given 12 and 6 hours before infusion, as was histamine receptor1 and histamine receptor2 antagonist therapy. The 4 days of chemotherapy were administered in the hospital. The patients were usually discharged from the hospital for outpatient observation on the day after HDCT. After 48 hours of rest, the ABSC were reinfused, and rhG-CSF was administered at 5 g/kg BW SC daily until hematologic recovery (absolute neutrophil count, 1.5 ⫻ 109/L). Handling of the AP before Reinfusion
With aseptic technique, the cryocyte freezing bags (Baxter Healthcare Corporation), each containing 94 mL of cryopreserved AP, were removed from liquid nitrogen storage and rapidly thawed to just above freezing in a 40°C water bath. They were then placed on ice to maintain the temperature at 4°C. In the first 21 consecutive patients, the AP was resuspended in 60-mL syringes and infused into a central line. In the next 35 patients, the following manipulations were performed aseptically at 4°C in a laminar flow hood. The following sterile wash solution was prepared in advance and was used for all washes: to a 1000-mL 0.9% sodium chloride injection USP bag (Baxter Healthcare Corporation), 60 mL of anticoagulant citrate dextrose solution USP Formula A (Baxter Healthcare Corporation) was injected. A Fenwal
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plasma transfer set with a spike and needle adapter (Baxter Healthcare Corporation) was inserted into the bag to facilitate washes. This solution was then placed on ice to maintain the temperature at approximately 4°C. Each thawed AP was transferred from the cryocyte freezing bag into a 300-mL Fenwal transfer pack container with a sample site coupler inserted (Baxter Healthcare Corporation). The transfer line was then clamped, sealed with a hemostat, and cut. Each pack was then filled with 150 to 200 mL of wash solution, gently mixed, and centrifuged at 1200 rpm for 10 minutes at 4°C. The supernatants were then pressed off by using a Fenwal plasma extractor into a 2000-mL transfer pack container (Baxter Healthcare Corporation), into which a Fenwal plasma transfer set (Baxter Healthcare Corporation) with a spike and needle adapter had been inserted; the original line with spike had been sealed and cut off. The remaining cells were gently resuspended and combined into one of the transfer packs by using a 60-mL syringe. Two additional washes, each consisting of 150 to 200 mL of the wash solution, followed by centrifugation at 1200 rpm for 10 minutes at 4°C, were performed. The cells were then resuspended in a volume of 40 to 60 mL of the wash solution. A small sample was reserved for cell counts, clonogenic assay, and CD34⫹ phenotyping. The remainder was transferred into a 60-mL syringe and reinfused into the patient.
and 1% fetal bovine serum. Analysis regions were gated on the total population according to the published International Society of Hematotherapy and Graft Engineering guidelines [18]. A modified but standard clonogenic assay according to the method described by Fauser and Messner [19] was used. In brief, 2 ⫻ 105 cells per milliliter were plated into 30-mm suspension dishes (Life Technologies, Grand Island, NY), which contained a semisolid medium consisting of 2% methylcellulose (Shin-Etsu Chemical Company Ltd., Tokyo, Japan), Iscove-modified Dulbeccas` medium supplemented with 26% fetal bovine serum containing 2-mercapto-ethanol (4 ⫻ 10⫺5 mol/L; Sigma Chemicals), recombinant human interleukin-3 (5 ng/mL), erythropoietin (2.5 U/mL), and recombinant human GM-CSF (10 ng/mL). The cells were then incubated for 2 weeks in a humidified atmosphere supplemented with 5% CO2. The resulting colony number was evaluated with an inverted light microscope. A colony was considered as containing ⬎50 cells. Colony-forming unit– culture (CFU-C), burst-forming unit, erythroid (BFU-E), and colony-forming unit– granulocyte, erythrocyte, megakaryocyte, macrophage (CFU-GEMM) were scored separately. Clonogenic assays were performed on 20 of the 21 AP with DMSO and 20 new AP from new metastatic breast cancer patients whose product was washed to remove the DMSO.
Quality Assessment of the Autografts
Statistical Analysis
The cell number was obtained through a manual method by using a 0.2% Tu¨erks solution (Fluka Chemicals, Buchs, Switzerland) in a modified Neubauer chamber (Fluka Chemicals). Viability was determined with the trypan blue exclusion method (0.4% solution; Sigma Chemicals, St. Louis, MO). The following antibodies were used for immunophenotyping of the cells with a Coulter Epics Elite cellsorter analyzer (Fullerton, CA): anti-CD33 (Becton Dickinson, San Jose, CA), anti-CD34 (Becton Dickinson), and anti-CD38 (Becton Dickinson). Briefly, 100 L of the cell suspension at a concentration of 106 cells per milliliter was incubated with 20 L of the appropriate monoclonal antibody for 20 minutes on ice. Cells were washed in phosphate-buffered saline containing 0.02% sodium azide (Sigma Chemicals),
Statistical analysis was performed with the computer software SPSS (SPSS Inc., Chicago, IL). Statistical significance was reached if a P value of ⬍.05 was obtained. Continuous variables were analyzed with the Student t test. No adjustments were made for multiple comparisons. Data related to toxicity were analyzed by the Mann-Whitney U test. This study could detect a difference in the time to obtain a WBC ⬎0.5/nL of ⬎2.5 days between the groups with a probability of 90% ( error of .10), detecting an ␣ error set at .05 [20]. RESULTS To determine whether cell quality was similar between the groups, we first examined cell number
Table 1. In Vitro Results for Cell Quantity of the Reinfused Autograft*
Variable
DMSO-Containing (n ⴝ 21)
DMSO-Depleted before Washing (n ⴝ 35)
DMSO-Depleted after Washing (n ⴝ 35)
% Loss
MNC (ⴛ 108/kg BW) CD34ⴙ (ⴛ106/kg BW)
3.47 (2.1-5.89) 4.2 (1.22-21.1)
3.45 (1.3-12.9) 4.15 (1.9-16.3)
2.91 (0.8-9.1) 2.70 (0.4-19.5)
15.23 ⴞ 18.47 (0.1-54.5) 29.04 ⴞ 31.12 (0-79.2)
*The first column represents the 21 autografts reinfused after without further processing. The second and third columns represent the 35 autografts that were evaluated before and after the washing procedure. Data are median or mean ⫾ SD (range).
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Table 2. Clonogenic Cells of the Washed AP and DMSO Containing AP: Comparison of Populations* Colony Type
DMSO-Containing (n ⴝ 20)
CFU-C BFU-E CFU-GEMM
DMSO-Washed (n ⴝ 20)
63.95 ⴞ 76.3 (4-334) 25.6 ⴞ 29.9 (0-100) 46.05 ⴞ 78.7 (0-356) 95.4 ⴞ 106.7 (2-382) 0.65 ⴞ 0.99 (0-4) 1.5 ⴞ 2.7 (0-11)
P Value Side Effect .043 .104 .20
*Data are mean ⫾ SD (range). Values represent the number of colonies per 2 ⫻ 105 plated cells.
and viability. Table 1 summarizes the data for the cell number, viability, clonogenic cells, and CD34⫹ cells in both groups. The initial cell number was similar in the 2 groups. After washing, there was a decrease in the total mononuclear cell (MNC) count, but this was not significant. Viability was ⬎80% in all groups. The washing procedures showed a trend for total cell loss, but the differences between groups did not reach statistical difference. The number of CD34⫹ cells remained equal in both the DMSO-containing and the DMSO-depleted prewash groups. There was a decrease in the number of CD34⫹ cells after washing; however, this loss does not correlate with changes in the time to engraftment. When colonies were assessed, no difference was found in the number of BFU-E or CFU-GEMM; however, a small but significant difference was found in the number of CFU-C (Table 2). Despite this, the median hematologic recovery, defined as a WBC count of ⬎0.5/nL, occurred after 12 days (range, 8-15 days) in the DMSO group, as compared with 11.0 days (range, 9-18 days) in the DMSO-depleted group (Table 3). A platelet count ⬎20/nL was reached in 11 days (range, 7-16 days) in the DMSO group as compared with 12 days (range, 7-24 days) in the DMSO-depleted group. The numbers of packed red blood cells (PRBC) and platelet transfusions were also comparable between groups. Patients in the DMSO group Table 3. Data for the Comparison of Patients Who Had DMSO in Their Autograft with Those Whose Product Was Washed before Transplantation*
Variable Days to WBC 0.5 ⴛ 109/L Days to Plts 20 ⴛ 109/L Days to ANC 0.5 ⴛ 106/L Units of platelet transfusions Units of packed red blood cell transfusions
DMSOContaining (n ⴝ 21)
DMSOWashed (n ⴝ 35)
P Value
12 (8-15) 11 (7-16) 13 (9-17) 1.7 (0-6)
11 (9-18) 12 (7-24) 12 (9-19) 1.9 (0-6)
.0455 .2599 .0242 .653
2.7 (0-6)
2.0 (0-12)
.397
WBC indicates white blood count; Plts, platelets; ANC, absolute neutrophil count. *Mann-Whitney analysis was conducted for patients who had DMSO in the reinfused autograft and those who had the washing procedure before transplantation. 138
Table 4. Side Effects and Complications within the First 12 Hours after Reinfusion of the Autograft* DMSOContaining (n ⴝ 21)
Rash Abdominal cramps Back/bone pain Dysgeusia
5 11 6 19
Dyspnea/cough Macrohematuria/proteinuria Cardiovascular
13 7 10
DMSOWashed (n ⴝ 35)
P Value
0 .0037 0 <.0001 5 .160 None, some <.0001 occasionally 3 <.0001 2 .0062 7 .0297
WHO indicates World Health Organization. *Numbers represent patients who experienced WHO grade 2 toxicities or more in each category (Fisher exact test).
needed, on average, 1.7 platelet transfusions and 2.7 PRBC transfusions, whereas the DMSO-depleted group needed 1.9 and 2.0 transfusions for platelets and PRBC, respectively. These differences were not statistically significant. Patients in the DMSO group stayed, on average, 21.5 days in the hospital, compared with 19.3 days in the DMSO-depleted group. This difference was not statistically significant (P ⫽ .065). It is interesting to note that certain adverse effects tended to be less frequently observed in the DMSO-depleted group. Nausea, vomiting, and diarrhea occurred significantly less often (P ⬍ .05) in the DMSO-depleted group (Table 4), whereas constipation tended to be more common in the DMSO-depleted group (P ⫽ .051). Abdominal cramping, mucositis, fatigue, facial flushing, and discomfort (likely due to cyclophosphamide) were observed at similar frequencies in each group. Febrile neutropenia was not observed more frequently in the DMSO-depleted group. One patient in the DMSO group experienced a grade 4 hemorrhage. No episodes of sudden and severe hypotension, hemolysis, or encephalopathy were observed in either group. One patient experienced a grade 2 cardiac adverse event in the DMSO group, whereas 1 patient experienced a grade 1 cardiac adverse event in the DMSOdepleted group. The patients with DMSO-depleted autografts rarely complained of dry cough or temporary dysgeusia, in contrast to patients who received with unwashed autografts. DISCUSSION The use of a cryoprotectant is essential to maintain the viability and function of AP when it is stored at very low temperatures. DMSO most often serves this purpose in AP cryopreservation procedures. This agent is selected for its relatively low toxicity in most patients. Nevertheless, adverse effects as major as sudden and severe hypotension or encephalopathy, ob-
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served in recipients of ABSC transplants, have been associated with DMSO. Further, the severity of adverse reactions has been related to the amount of DMSO in the graft [21,22]. In the laboratory, the quality of the autograft product is measured by the cell viability, the total number of MNC, and the number of CD34⫹ cells. Clonogenic assay and immunophenotyping reflect committed and early progenitor cells. Clinically, the quality of the autograft is reflected by the time to hematopoietic engraftment. Some investigators have assessed the in vitro effect of DMSO washing on the quality of autografts. Beaujean et al. [23] have described a DMSO-washing procedure used on cryopreserved, thawed BM and AP grafts before autologous transplantation. Their approach consisted of a stepwise dilution with 2% human serum albumin followed by centrifugation. In the 12 instances in which APs were involved, the mean recovery of MNC and colony-forming unit– granulocyte-macrophages was 95.6% and 93.9%, respectively, with a mean cell viability of ⬎90%. BM results were similar but slightly lower. The differences between BM and AP recovery were not significant. Menichella et al. [24] compared 2 different procedures for BM processing. The first one involved hydroxyethyl starch as a sedimentary agent, whereas the second one involved a semiautomated procedure with a blood cell processor (SteriCell; DuPont, Wilmington, DE). With both procedures, MNC and nucleated cell recovery, as well as red blood cell removal, exceeded 80%. These investigators have demonstrated that, by following their methods, DMSO can be washed from autografts without affecting the quality of the leukapheresis products in vitro. The next step is to compare the efficacy and safety of washed versus unwashed grafts in the clinical setting. Other components besides DMSO may be removed by washing, some of which could be involved in the engraftment of the ABSC. In a previous, smaller study, we showed sustained MNC numbers, viability, and CD34⫹ subsets, as well as clonogenicity, after washing [25]. The cell numbers, viability, and composition did not change significantly with the DMSOwashing procedure. More recently, one group has described a novel washing/enzymatic digestion protocol to remove DMSO from AP [26]. This study demonstrated that the protocol was feasible, with no complications related to the specific toxicity of reinfusion; however, they did not compare reinfusion of washed with unwashed AP, and, further, the use of deoxyribonuclease in a clinical setting may not be practical. Our study was designed to evaluate whether DMSO could be depleted in vitro from AP collection products without affecting its ability to restore the hematopoietic system after HDCT in breast cancer patients. As seen in Table 3, hematopoietic recovery was similar between the group that received an ABSC collection product containing DMSO and the group
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in which DMSO had been depleted. The number of days to reach a WBC count greater than 0.5/nL and a platelet count greater than 20/nL was not statistically different between groups and was comparable with previously published results. Transfusions were generally performed when the platelet count was less than 10/nL and the hemoglobin was less than 80 g/L. Here again, no difference in hematologic support was observed between groups. Some small differences were encountered when clonogenic assays were performed. The washed group produced significantly less CFU-C than the group with DMSO. This was a small difference and likely not clinically relevant, because there were no differences in time to engraftment between groups. No differences in BFU-E or CFU-GEMM were found between groups. The second aspect of our evaluation concentrated on the adverse effects experienced by our patients (Table 4). The main finding relates to nausea, vomiting, and diarrhea, which were less intense and less frequent in patients in DMSO-depleted group. Certain adverse reactions were more difficult to evaluate. Some aspects of the GM-CSF toxicity profile are shared with DMSO, namely, fever and abdominal cramping [27]. Abdominal cramps can occur early in the course of GM-CSF administration, whereas fever generally occurs on hematopoietic recovery and has a recognizable pattern [27]. Fever related to GM-CSF would tend not to occur at the same time as the reinfusion of ABSC, whereas abdominal cramps are more difficult to assign to a specific cause because the first dose of GM-CSF would occur within 24 hours of ABSC transplantation. Therefore, in view of the small population size, the results are not sufficient to eliminate the possibility of a difference in the incidence of abdominal cramps between groups. Fever associated with neutropenia, mucositis, fatigue, facial flushing, and discomfort (cyclophosphamide) are common after a course of intense chemotherapy. All were equally distributed between groups. Because chemotherapy regimens and the distribution of patients who received either G-CSF or GM-CSF during HDCT were similar between groups (data not shown), no link to those variables can be made. Febrile neutropenic episodes had no direct link to ABSC reinfusion. They occurred later after several days of neutropenia and could be attributed to bacterial infection or GM-CSF administration [27]. Before using CD34⫹ selection methods, life-threatening adverse events due to DMSO toxicity were observed. In a recently presented study in which CD34⫹-selected (and volume-reduced) autografting was randomized compared with reinfusion of unmanipulated autografts, no differences in neutropenic recovery were observed, but the differences due to DMSO toxicity were statistically significant [8]. This exactly confirms our observation. As we gained expe139
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rience with the ABSC procedure, patients stayed in the hospital for shorter periods of time. However, this transition occurred over a prolonged period of time. Presently, the transplantation procedure is performed on an outpatient basis [14]. Therefore, in our center, hospital stay is not a good indicator of the severity of morbidity. Patients in the DMSO group had undergone transplantation before the patients in the DMSO-depleted group. Therefore, eventually the patients who received a DMSO-depleted AP would be hospitalized for a shorter period [28]. Of importance is that nausea, vomiting, and diarrhea can be very inconvenient to patients, and decreased morbidity associated with the reinfusion of ABSC can facilitate the transition of this procedure to the outpatient setting. One possible downside is that depleting AP of DMSO takes between 3 and 4 hours of laboratory work per patient. This can be a limiting factor, depending on the laboratory facilities, staff support, and number of transplantations performed. HDCT followed by peripheral blood stem cell transplantation was one of the promising approaches to high-risk and metastatic breast cancer in the 1990s. In 1999, several studies were presented that did not show any benefit in survival of these patients, and toxicity became unacceptably high [29]. In conclusion, the depletion of DMSO in vitro, before autografting, did not prolong the time to WBC and platelet recovery, nor did it increase the necessary hematologic support. Most importantly, morbidity related to the procedure was reduced. If laboratory support permits, DMSO depletion of APs can be performed in vitro to decrease procedure-related morbidity.
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ACKNOWLEDGMENTS We thank Terry Koski of the Clinical Trials Department for the data collection.
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The Role of Depletion of Dimethyl Sulfoxide before Autografting: On Hematologic Recovery, Side Effects, and Toxicity R. Syme,1 M. Bewick,2 D. Stewart,3 K. Porter,2 T. Chadderton,4 S. Glu¨ck1,5,6 1
Oncology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; 2Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario, Canada; 3Tom Baker Cancer Centre, Calgary, Alberta, Canada; 4 GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania; 5 Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; 6Pharmacology & Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Correspondence and reprint requests: Stefan Glu¨ck, MD, PhD, FRCPC, Department of Oncology, Faculty of Medicine, University of Calgary, 1331 29th St. N.W., Calgary, Alberta T2N 4N2, Canada (e-mail: [email protected]). Received August 15, 2003; accepted September 25, 2003
ABSTRACT Cryopreservation of stem cells after collection from peripheral blood or bone marrow for autologous transplantation necessitates protection with dimethyl sulfoxide (DMSO). Unfortunately, DMSO, when infused with the thawed cell suspension, may induce serious complications and side effects. To assess whether depletion of DMSO before autografting affects safety and efficacy, 56 consenting consecutive patients treated with highdose chemotherapy and autologous blood stem cell transplantation were assigned to obtain either an untreated or DMSO-depleted autograft. On the day of transplantation, the cryopreserved cells were thawed and infused to the patient either immediately or after washing 3 times in normal saline supplemented with 6% anticoagulant citrate dextrose solution. Cell count with viability, clonogenic assay, and phenotyping were performed before and after thawing and after washing. Hematologic recovery, side effects, and complications were recorded. The in vitro and clinical data on 56 patients show that the depletion of DMSO in vitro before autografting does not induce a significant loss of cell number, viability, colony-forming unit– granulocytemacrophage activity, or number of CD34ⴙ cells. Furthermore, it leads to a safe and sustained engraftment. The complications and side effects, as recorded by continuous monitoring, were substantially less; however, the procedure takes 3 to 4 hours of laboratory work per patient. © 2004 American Society for Blood and Marrow Transplantation
KEY WORDS High-dose chemotherapy ● Autologous blood stem cell transplantation sulfoxide ● Cryopreservation
INTRODUCTION High-dose chemotherapy (HDCT) and autologous blood stem cell (ABSC) transplantation are increasingly used for the treatment of hematologic malignancies [1]. As part of bone marrow (BM) or ABSC transplantation, collected cells are frozen and stored for prolonged periods. Because cells can be injured by the direct effect of low temperatures and by the formation of ice crystals, which then leads to cell dehydration, a cryoprotectant is used to maintain cell integrity. Ideally, a cryoprotectant should maintain the quality of the collection product without causing any
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adverse effects to the recipient of the transplantation or to the cells being preserved. Dimethyl sulfoxide (DMSO) is the cryopreservation agent most often used by transplantation centers. Although it is usually relatively nontoxic to patients in small amounts, reinfusion of solutions containing substantial volumes of DMSO has been associated with adverse effects (up to 20%) [2]. Sudden and severe hypotension can result from the intravenous (IV) infusion of DMSO, presumably from histamine-induced vasodilation [2]. Skin flushing, dyspnea, abdominal cramping, nausea, and diarrhea can also be attributed to DMSO-induced 135
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histamine release [3]. Other adverse effects associated with DMSO include headache, fever, hemolysis, encephalopathy, and cardiovascular symptoms [4-8]. These may result in increased morbidity, prolonged hospitalization, and increased treatment-related costs. So far, only a few reports [9-12] have investigated the in vitro toxicity of DMSO on hematopoietic stem cells. It has been postulated that DMSO can be toxic to hematopoietic cells and BM stromal cells [13]. In view of the potential toxicities associated with the administration of DMSO to patients, it may be advantageous to eliminate it from the autograft before reinfusion. This study compared cell quality and quantity, engraftment kinetics, and adverse reactions related to the reinfusion of DMSO-depleted versus DMSO-containing autografts. This study was performed in patients who had received HDCT with ABSC transplantation for metastatic breast cancer in an outpatient setting previously described by our group [14].
METHODS Patients
Fifty-six patients receiving HDCT and ABSC transplantation for metastatic breast cancer were evaluated. All patients participated in a single-center study in Sudbury. The institutional review board approved the study. All patients signed informed consent. Included in our study were patients between 18 and 55 years old (median, 42 years) who had not had previous chemotherapy for metastatic breast cancer. They had evaluable or measurable disease, and the disease either progressed with hormonal treatment or was hormonereceptor negative. No patients with central nervous system metastasis were allowed on study. The first 21 patients received an untreated autograft, whereas the following 35 had DMSO depletion before reinfusion of their autograft. These were consecutive, consenting patients. Chemotherapy Regimens and ABSC Collection
All patients underwent 3 to 4 cycles of induction chemotherapy (IDC) with cyclophosphamide, doxorubicin, and 5-fluorouracil. A growth factor, recombinant human granulocyte colony-stimulating factor (rhG-CSF, Neupogen; Amgen Canada Inc., Mississauga, ONT), was administered subcutaneously (SC) from the day after IDC until the apheresis procedures were completed. When the white blood cell count (WBC) reached 2.5/nL and the platelet count was 50/nL, apheresis was performed daily on 4 consecutive days as described previously [15]. Apheresis was performed with the Fenwal CS 3000 Plus blood separator (Baxter Healthcare Corporation, Deerfield, IL). A total blood volume of 10 L per apheresis was pro136
cessed at a flow rate of 60 to 70 mL/min [15-17]. Collections were continued until a value of at least 2 ⫻ 106 CD34⫹ cells per kilogram body weight was reached. Each apheresis product (AP) obtained was cryopreserved in 10% (vol/vol) DMSO and stored in liquid nitrogen until the time of reinfusion [15]. The HDCT consisted of cyclophosphamide 6 g/m2 bodysurface area (BSA), mitoxantrone 70 mg/m2 BSA, and either carboplatin 800 mg/m2 BSA or vinblastine 12 mg/m2 BSA, all administered IV. Then, 24 to 48 hours later, the autograft was reinfused, and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) or rhG-CSF at a dose of 5 g/kg body weight SC was administered until hematologic recovery. High-Dose Chemotherapy
Only patients whose disease did not progress on IDC and who did not experience severe toxicity or major organ damage proceeded to HDCT. HDCT consisted of cyclophosphamide 2 g/m2 IV, mitoxantrone 23.3 mg/m2 IV, and mesna 300 mg/m2 IV every 8 hours. This was delivered on 3 consecutive days for a cumulative dose of cyclophosphamide 6 g/m2 and mitoxantrone 70 mg/m2. Appropriate hydration and premedications were administered. On day 4, the paclitaxel dose starting at 250 mg/m2 BSA was delivered as a 3-hour IV infusion. Premedication, including dexamethasone at 20 mg by mouth, was given 12 and 6 hours before infusion, as was histamine receptor1 and histamine receptor2 antagonist therapy. The 4 days of chemotherapy were administered in the hospital. The patients were usually discharged from the hospital for outpatient observation on the day after HDCT. After 48 hours of rest, the ABSC were reinfused, and rhG-CSF was administered at 5 g/kg BW SC daily until hematologic recovery (absolute neutrophil count, 1.5 ⫻ 109/L). Handling of the AP before Reinfusion
With aseptic technique, the cryocyte freezing bags (Baxter Healthcare Corporation), each containing 94 mL of cryopreserved AP, were removed from liquid nitrogen storage and rapidly thawed to just above freezing in a 40°C water bath. They were then placed on ice to maintain the temperature at 4°C. In the first 21 consecutive patients, the AP was resuspended in 60-mL syringes and infused into a central line. In the next 35 patients, the following manipulations were performed aseptically at 4°C in a laminar flow hood. The following sterile wash solution was prepared in advance and was used for all washes: to a 1000-mL 0.9% sodium chloride injection USP bag (Baxter Healthcare Corporation), 60 mL of anticoagulant citrate dextrose solution USP Formula A (Baxter Healthcare Corporation) was injected. A Fenwal
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plasma transfer set with a spike and needle adapter (Baxter Healthcare Corporation) was inserted into the bag to facilitate washes. This solution was then placed on ice to maintain the temperature at approximately 4°C. Each thawed AP was transferred from the cryocyte freezing bag into a 300-mL Fenwal transfer pack container with a sample site coupler inserted (Baxter Healthcare Corporation). The transfer line was then clamped, sealed with a hemostat, and cut. Each pack was then filled with 150 to 200 mL of wash solution, gently mixed, and centrifuged at 1200 rpm for 10 minutes at 4°C. The supernatants were then pressed off by using a Fenwal plasma extractor into a 2000-mL transfer pack container (Baxter Healthcare Corporation), into which a Fenwal plasma transfer set (Baxter Healthcare Corporation) with a spike and needle adapter had been inserted; the original line with spike had been sealed and cut off. The remaining cells were gently resuspended and combined into one of the transfer packs by using a 60-mL syringe. Two additional washes, each consisting of 150 to 200 mL of the wash solution, followed by centrifugation at 1200 rpm for 10 minutes at 4°C, were performed. The cells were then resuspended in a volume of 40 to 60 mL of the wash solution. A small sample was reserved for cell counts, clonogenic assay, and CD34⫹ phenotyping. The remainder was transferred into a 60-mL syringe and reinfused into the patient.
and 1% fetal bovine serum. Analysis regions were gated on the total population according to the published International Society of Hematotherapy and Graft Engineering guidelines [18]. A modified but standard clonogenic assay according to the method described by Fauser and Messner [19] was used. In brief, 2 ⫻ 105 cells per milliliter were plated into 30-mm suspension dishes (Life Technologies, Grand Island, NY), which contained a semisolid medium consisting of 2% methylcellulose (Shin-Etsu Chemical Company Ltd., Tokyo, Japan), Iscove-modified Dulbeccas` medium supplemented with 26% fetal bovine serum containing 2-mercapto-ethanol (4 ⫻ 10⫺5 mol/L; Sigma Chemicals), recombinant human interleukin-3 (5 ng/mL), erythropoietin (2.5 U/mL), and recombinant human GM-CSF (10 ng/mL). The cells were then incubated for 2 weeks in a humidified atmosphere supplemented with 5% CO2. The resulting colony number was evaluated with an inverted light microscope. A colony was considered as containing ⬎50 cells. Colony-forming unit– culture (CFU-C), burst-forming unit, erythroid (BFU-E), and colony-forming unit– granulocyte, erythrocyte, megakaryocyte, macrophage (CFU-GEMM) were scored separately. Clonogenic assays were performed on 20 of the 21 AP with DMSO and 20 new AP from new metastatic breast cancer patients whose product was washed to remove the DMSO.
Quality Assessment of the Autografts
Statistical Analysis
The cell number was obtained through a manual method by using a 0.2% Tu¨erks solution (Fluka Chemicals, Buchs, Switzerland) in a modified Neubauer chamber (Fluka Chemicals). Viability was determined with the trypan blue exclusion method (0.4% solution; Sigma Chemicals, St. Louis, MO). The following antibodies were used for immunophenotyping of the cells with a Coulter Epics Elite cellsorter analyzer (Fullerton, CA): anti-CD33 (Becton Dickinson, San Jose, CA), anti-CD34 (Becton Dickinson), and anti-CD38 (Becton Dickinson). Briefly, 100 L of the cell suspension at a concentration of 106 cells per milliliter was incubated with 20 L of the appropriate monoclonal antibody for 20 minutes on ice. Cells were washed in phosphate-buffered saline containing 0.02% sodium azide (Sigma Chemicals),
Statistical analysis was performed with the computer software SPSS (SPSS Inc., Chicago, IL). Statistical significance was reached if a P value of ⬍.05 was obtained. Continuous variables were analyzed with the Student t test. No adjustments were made for multiple comparisons. Data related to toxicity were analyzed by the Mann-Whitney U test. This study could detect a difference in the time to obtain a WBC ⬎0.5/nL of ⬎2.5 days between the groups with a probability of 90% ( error of .10), detecting an ␣ error set at .05 [20]. RESULTS To determine whether cell quality was similar between the groups, we first examined cell number
Table 1. In Vitro Results for Cell Quantity of the Reinfused Autograft*
Variable
DMSO-Containing (n ⴝ 21)
DMSO-Depleted before Washing (n ⴝ 35)
DMSO-Depleted after Washing (n ⴝ 35)
% Loss
MNC (ⴛ 108/kg BW) CD34ⴙ (ⴛ106/kg BW)
3.47 (2.1-5.89) 4.2 (1.22-21.1)
3.45 (1.3-12.9) 4.15 (1.9-16.3)
2.91 (0.8-9.1) 2.70 (0.4-19.5)
15.23 ⴞ 18.47 (0.1-54.5) 29.04 ⴞ 31.12 (0-79.2)
*The first column represents the 21 autografts reinfused after without further processing. The second and third columns represent the 35 autografts that were evaluated before and after the washing procedure. Data are median or mean ⫾ SD (range).
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Table 2. Clonogenic Cells of the Washed AP and DMSO Containing AP: Comparison of Populations* Colony Type
DMSO-Containing (n ⴝ 20)
CFU-C BFU-E CFU-GEMM
DMSO-Washed (n ⴝ 20)
63.95 ⴞ 76.3 (4-334) 25.6 ⴞ 29.9 (0-100) 46.05 ⴞ 78.7 (0-356) 95.4 ⴞ 106.7 (2-382) 0.65 ⴞ 0.99 (0-4) 1.5 ⴞ 2.7 (0-11)
P Value Side Effect .043 .104 .20
*Data are mean ⫾ SD (range). Values represent the number of colonies per 2 ⫻ 105 plated cells.
and viability. Table 1 summarizes the data for the cell number, viability, clonogenic cells, and CD34⫹ cells in both groups. The initial cell number was similar in the 2 groups. After washing, there was a decrease in the total mononuclear cell (MNC) count, but this was not significant. Viability was ⬎80% in all groups. The washing procedures showed a trend for total cell loss, but the differences between groups did not reach statistical difference. The number of CD34⫹ cells remained equal in both the DMSO-containing and the DMSO-depleted prewash groups. There was a decrease in the number of CD34⫹ cells after washing; however, this loss does not correlate with changes in the time to engraftment. When colonies were assessed, no difference was found in the number of BFU-E or CFU-GEMM; however, a small but significant difference was found in the number of CFU-C (Table 2). Despite this, the median hematologic recovery, defined as a WBC count of ⬎0.5/nL, occurred after 12 days (range, 8-15 days) in the DMSO group, as compared with 11.0 days (range, 9-18 days) in the DMSO-depleted group (Table 3). A platelet count ⬎20/nL was reached in 11 days (range, 7-16 days) in the DMSO group as compared with 12 days (range, 7-24 days) in the DMSO-depleted group. The numbers of packed red blood cells (PRBC) and platelet transfusions were also comparable between groups. Patients in the DMSO group Table 3. Data for the Comparison of Patients Who Had DMSO in Their Autograft with Those Whose Product Was Washed before Transplantation*
Variable Days to WBC 0.5 ⴛ 109/L Days to Plts 20 ⴛ 109/L Days to ANC 0.5 ⴛ 106/L Units of platelet transfusions Units of packed red blood cell transfusions
DMSOContaining (n ⴝ 21)
DMSOWashed (n ⴝ 35)
P Value
12 (8-15) 11 (7-16) 13 (9-17) 1.7 (0-6)
11 (9-18) 12 (7-24) 12 (9-19) 1.9 (0-6)
.0455 .2599 .0242 .653
2.7 (0-6)
2.0 (0-12)
.397
WBC indicates white blood count; Plts, platelets; ANC, absolute neutrophil count. *Mann-Whitney analysis was conducted for patients who had DMSO in the reinfused autograft and those who had the washing procedure before transplantation. 138
Table 4. Side Effects and Complications within the First 12 Hours after Reinfusion of the Autograft* DMSOContaining (n ⴝ 21)
Rash Abdominal cramps Back/bone pain Dysgeusia
5 11 6 19
Dyspnea/cough Macrohematuria/proteinuria Cardiovascular
13 7 10
DMSOWashed (n ⴝ 35)
P Value
0 .0037 0 <.0001 5 .160 None, some <.0001 occasionally 3 <.0001 2 .0062 7 .0297
WHO indicates World Health Organization. *Numbers represent patients who experienced WHO grade 2 toxicities or more in each category (Fisher exact test).
needed, on average, 1.7 platelet transfusions and 2.7 PRBC transfusions, whereas the DMSO-depleted group needed 1.9 and 2.0 transfusions for platelets and PRBC, respectively. These differences were not statistically significant. Patients in the DMSO group stayed, on average, 21.5 days in the hospital, compared with 19.3 days in the DMSO-depleted group. This difference was not statistically significant (P ⫽ .065). It is interesting to note that certain adverse effects tended to be less frequently observed in the DMSO-depleted group. Nausea, vomiting, and diarrhea occurred significantly less often (P ⬍ .05) in the DMSO-depleted group (Table 4), whereas constipation tended to be more common in the DMSO-depleted group (P ⫽ .051). Abdominal cramping, mucositis, fatigue, facial flushing, and discomfort (likely due to cyclophosphamide) were observed at similar frequencies in each group. Febrile neutropenia was not observed more frequently in the DMSO-depleted group. One patient in the DMSO group experienced a grade 4 hemorrhage. No episodes of sudden and severe hypotension, hemolysis, or encephalopathy were observed in either group. One patient experienced a grade 2 cardiac adverse event in the DMSO group, whereas 1 patient experienced a grade 1 cardiac adverse event in the DMSOdepleted group. The patients with DMSO-depleted autografts rarely complained of dry cough or temporary dysgeusia, in contrast to patients who received with unwashed autografts. DISCUSSION The use of a cryoprotectant is essential to maintain the viability and function of AP when it is stored at very low temperatures. DMSO most often serves this purpose in AP cryopreservation procedures. This agent is selected for its relatively low toxicity in most patients. Nevertheless, adverse effects as major as sudden and severe hypotension or encephalopathy, ob-
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served in recipients of ABSC transplants, have been associated with DMSO. Further, the severity of adverse reactions has been related to the amount of DMSO in the graft [21,22]. In the laboratory, the quality of the autograft product is measured by the cell viability, the total number of MNC, and the number of CD34⫹ cells. Clonogenic assay and immunophenotyping reflect committed and early progenitor cells. Clinically, the quality of the autograft is reflected by the time to hematopoietic engraftment. Some investigators have assessed the in vitro effect of DMSO washing on the quality of autografts. Beaujean et al. [23] have described a DMSO-washing procedure used on cryopreserved, thawed BM and AP grafts before autologous transplantation. Their approach consisted of a stepwise dilution with 2% human serum albumin followed by centrifugation. In the 12 instances in which APs were involved, the mean recovery of MNC and colony-forming unit– granulocyte-macrophages was 95.6% and 93.9%, respectively, with a mean cell viability of ⬎90%. BM results were similar but slightly lower. The differences between BM and AP recovery were not significant. Menichella et al. [24] compared 2 different procedures for BM processing. The first one involved hydroxyethyl starch as a sedimentary agent, whereas the second one involved a semiautomated procedure with a blood cell processor (SteriCell; DuPont, Wilmington, DE). With both procedures, MNC and nucleated cell recovery, as well as red blood cell removal, exceeded 80%. These investigators have demonstrated that, by following their methods, DMSO can be washed from autografts without affecting the quality of the leukapheresis products in vitro. The next step is to compare the efficacy and safety of washed versus unwashed grafts in the clinical setting. Other components besides DMSO may be removed by washing, some of which could be involved in the engraftment of the ABSC. In a previous, smaller study, we showed sustained MNC numbers, viability, and CD34⫹ subsets, as well as clonogenicity, after washing [25]. The cell numbers, viability, and composition did not change significantly with the DMSOwashing procedure. More recently, one group has described a novel washing/enzymatic digestion protocol to remove DMSO from AP [26]. This study demonstrated that the protocol was feasible, with no complications related to the specific toxicity of reinfusion; however, they did not compare reinfusion of washed with unwashed AP, and, further, the use of deoxyribonuclease in a clinical setting may not be practical. Our study was designed to evaluate whether DMSO could be depleted in vitro from AP collection products without affecting its ability to restore the hematopoietic system after HDCT in breast cancer patients. As seen in Table 3, hematopoietic recovery was similar between the group that received an ABSC collection product containing DMSO and the group
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in which DMSO had been depleted. The number of days to reach a WBC count greater than 0.5/nL and a platelet count greater than 20/nL was not statistically different between groups and was comparable with previously published results. Transfusions were generally performed when the platelet count was less than 10/nL and the hemoglobin was less than 80 g/L. Here again, no difference in hematologic support was observed between groups. Some small differences were encountered when clonogenic assays were performed. The washed group produced significantly less CFU-C than the group with DMSO. This was a small difference and likely not clinically relevant, because there were no differences in time to engraftment between groups. No differences in BFU-E or CFU-GEMM were found between groups. The second aspect of our evaluation concentrated on the adverse effects experienced by our patients (Table 4). The main finding relates to nausea, vomiting, and diarrhea, which were less intense and less frequent in patients in DMSO-depleted group. Certain adverse reactions were more difficult to evaluate. Some aspects of the GM-CSF toxicity profile are shared with DMSO, namely, fever and abdominal cramping [27]. Abdominal cramps can occur early in the course of GM-CSF administration, whereas fever generally occurs on hematopoietic recovery and has a recognizable pattern [27]. Fever related to GM-CSF would tend not to occur at the same time as the reinfusion of ABSC, whereas abdominal cramps are more difficult to assign to a specific cause because the first dose of GM-CSF would occur within 24 hours of ABSC transplantation. Therefore, in view of the small population size, the results are not sufficient to eliminate the possibility of a difference in the incidence of abdominal cramps between groups. Fever associated with neutropenia, mucositis, fatigue, facial flushing, and discomfort (cyclophosphamide) are common after a course of intense chemotherapy. All were equally distributed between groups. Because chemotherapy regimens and the distribution of patients who received either G-CSF or GM-CSF during HDCT were similar between groups (data not shown), no link to those variables can be made. Febrile neutropenic episodes had no direct link to ABSC reinfusion. They occurred later after several days of neutropenia and could be attributed to bacterial infection or GM-CSF administration [27]. Before using CD34⫹ selection methods, life-threatening adverse events due to DMSO toxicity were observed. In a recently presented study in which CD34⫹-selected (and volume-reduced) autografting was randomized compared with reinfusion of unmanipulated autografts, no differences in neutropenic recovery were observed, but the differences due to DMSO toxicity were statistically significant [8]. This exactly confirms our observation. As we gained expe139
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rience with the ABSC procedure, patients stayed in the hospital for shorter periods of time. However, this transition occurred over a prolonged period of time. Presently, the transplantation procedure is performed on an outpatient basis [14]. Therefore, in our center, hospital stay is not a good indicator of the severity of morbidity. Patients in the DMSO group had undergone transplantation before the patients in the DMSO-depleted group. Therefore, eventually the patients who received a DMSO-depleted AP would be hospitalized for a shorter period [28]. Of importance is that nausea, vomiting, and diarrhea can be very inconvenient to patients, and decreased morbidity associated with the reinfusion of ABSC can facilitate the transition of this procedure to the outpatient setting. One possible downside is that depleting AP of DMSO takes between 3 and 4 hours of laboratory work per patient. This can be a limiting factor, depending on the laboratory facilities, staff support, and number of transplantations performed. HDCT followed by peripheral blood stem cell transplantation was one of the promising approaches to high-risk and metastatic breast cancer in the 1990s. In 1999, several studies were presented that did not show any benefit in survival of these patients, and toxicity became unacceptably high [29]. In conclusion, the depletion of DMSO in vitro, before autografting, did not prolong the time to WBC and platelet recovery, nor did it increase the necessary hematologic support. Most importantly, morbidity related to the procedure was reduced. If laboratory support permits, DMSO depletion of APs can be performed in vitro to decrease procedure-related morbidity.
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ACKNOWLEDGMENTS We thank Terry Koski of the Clinical Trials Department for the data collection.
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