viability and clinical toxicity of thawed and washed haematopoietic progenitor cells
Top of pageAbstractCryopreservation and thawing of haematopoietic stem cells are associated with cell loss and infusion related toxicities. We analysed viability, total nucleated cell (TNC) and CD34+ cell recovery, and infusion related toxicities of 952 thawed and washed pro ugg sale ducts. Mean TNC and CD34+ viable cells recoveries were 55.918.6 and 98.036.5%, respectively. Mean cell viability was 68.2518.9%. TNC recovery was correlated with viability but independent of the initial nucleated cell concentration. No difference in TNC recovery or viability was observed according to underlying diseases, except for myeloma, for which these variables were significantly lower (PTop of pageIntroductionOver the last decade, the clinical applications of autologous haematopoietic stem cell transplantation have considerably increased. Haematopoietic stem cell transplantation is still mainly used for the treatment of haematological malignancies, but also in other disorders such as solid cancers.1Autologous haematopoietic progenitor cells (HPC) can be collected from either peripheral blood after mobilization by growth factors following or not chemotherapy, or from bone marrow, the majority of patients receiving cryopreserved peripheral blood HPC. The major concern for cell preservation resides in the freezing step that can damage cells and thus requires the addition of cryoprotectants in order to optimize cell viability and function at the time of thawing.2 HPC are usually frozen in autologous plasma or macromolecules in solution with a final concentration of 10% dimethylsulphoxide (DMSO) and then stored in liquid or gas phase nitrogen until reinfusion.3, 4 In spite of these precautions, cryopreservation is associated with variable loss of cell viability and a reduction in the absolute number of viable CD34+ cells available for reinfusion.5, 6, 7 It has been related to the direct effect of low temperature and the formation of ice crystals,8 or to DMSO only.9Infusion of thawed products may induce a wide variety of adverse events usually mild to moderate. The most frequent adverse events reported in the literature include nausea, vomiting, hypo or hypertension and bradycardia. Headache, abdominal cramps, diarrhoea, flushing, chills, fever and chest tightne ugg sale ss have also been described.10, 11, 12 Severe reactions, such as life threatening cardiologic, neurologic, pulmonary, anaphylactic and renal events13, 14, 15, 16, 17, 18 are considered to be rare events. The collection process started on the fifth day of recombinant human granulocyte colony stimulating factor administration or during the haematologic recovery after chemotherapy and recombinant human granulocyte colony stimulating factor, if at least 10 CD34+ cells per microlitre were present in the peripheral blood sample. Haematopoietic stem cells were collected on a continuous flow blood cell separator (COBE spectra cell separator, Gambro BCT, Lakewood, CO, USA). A median number of three times the patient’s blood volume was processed each time. A minimum of 3 106 CD34+ cells per kilogram of patient weight and per graft was collected. Consent was obtained prior to the procedure from all patients following institutional guidelines. For microbial culture, 1 ml of graft sample was inoculated into a blood culture flask from a commercial source in both aerobic and anaerobic conditions (Bact/Alert SA and SN, Biom Marcy l’Etoile, France). Samples were incubated for 14 days at 37 The cryoprotectant solution was composed of voluven (Fresenius Kabi, Sevres, France) and DMSO (B Braun, Boulogne, France). The volume of the cryoprotectant solution was calculated to obtain a final volume of 140 ml per bag and the quantity of DMSO was calculated to achieve a DMSO final concentration of 10% (vol/vol). The cryoprotectant solution was chilled to 4 and added to the cells in freezing bags. The bags were transferred to a controlled freezer (NICOOL, Airliquide, France), cooled at 1.6 per minute to 40 and at 9 per minute to 120 and then stored in gas phase nitrogen until reinfusion.Although it has been shown that haematopoietic stem cells may retain engraftment capacity after extended cryostorage,21 the effect of storage duration on cell recovery and viability was analysed in two groups as follows: one transplanted with cells cryopreserved for less than 4 months and another for more than 4 months.Thawing, washing and infusionImmediately after removal from nitrogen tanks, frozen bags were thawed in a 37 water bath. The bag was removed from the water bath when only an ice cube remained visible and the cell suspension obtained was immediately diluted with a saline solution with 10% acid citrate dextrose anticoagulant (ACD; MacoPharma, Tourcoing, France) to a final volume of 600 ml. for 6 min. At the end of this first centrifugation step, supernatant waste was removed from the cell washer by fixing the flow at 100 ml min 1. The remaining cell suspension was automatically agitated for 80 s. Saline/acid citrate dextrose solution was added again and the washing process was repeated twice when only one bag had to be thawed, or three times if two bags were thawed. The cell product was then gently diluted in a glucose 5% solution, filtered and adjusted to a final infused volume of about 200 ml. Samples were taken for cell count, viability and bacteriological control assessment. The graft infusion was performed through a standard 170 m blood filter in order to remove macroscopic clumps before infusion. The presence of macroscopic clumps in the graft was recorded both by the technicians at the end of the thawing and washing procedures, and the physicians before transplantation. Cell suspensions were infused into the patient throug ugg sale h a central venous catheter in 45 min to 1 h.Cell countNucleated cells from leucapheresis products and thawed bags were counted on an automated cell counter (Sysmex, Roche Diagnostics, Meylan, France).The proportions of mononuclear cells and CD34+ cells were identified by flow cytometry.Briefly, 1 106 cells were incubated with anti CD34 phycoerythrin and CD45 fluorescein isothiocyanate conjugated monoclonal antibodies (all purchased from Becton Dickinson, San Jose, CA, USA). After a washing step in phosphate buffered saline, the samples were incubated for 15 min at room temperature with 1 ml of ammonium chloride solution (Ortho mune lysing reagent, Ortho, Raritan, NJ, USA), washed again and then acquired on a flow cytometer (FACS calibur with Cell Quest software, Becton Dickinson). A unique gate based on CD34 staining of low side scatter events was performed to identify the CD34+ cell population. The mononuclear cells were identified by flow cytometry as low and intermediate side scatter events, which include lymphocytes, monocytes and early progenitors. Cell viability was assessed by propidium iodide using a flow cytometric procedure. Cell recoveries, expressed as percentages, were calculated according to cell count obtained before freezing and after the thawing procedure as follows: (cell number after thawing/cell number before freezing) 100. Differences between the results of comparative tests were considered significant if the two sided P value was less than 0.05. All statistical analyses were performed using the SAS 9.1 software (SAS Institute, Cary, NC, USA).Top of pageResultsCell recovery and viabilityTotal nucleated cell recovery and the cell viabi ugg sale lity were 55.918.6 % (range 11 and 68.2518.9% (range 15 respectively. As expected, total NC recovery (TNCR) strongly correlated with viability (r2=0.66, P 4; Figure 1a) and inversely correlated, as cell viability, with the granulocytic cell concentration before cryopreservation (r2=0.43, P 4; Figure 1b). Conversely, TNCR and the viability after thawing were neither linked with the TNC count before cryopreservation, nor to the concentration of TNCs frozen per millilitre (not shown). We next addressed the question of whether or not the initial diagnosis influenced the TNCR at the time of graft thawing. Whereas no differences for TNCR and viability were observed for patients with solid cancers, for Hodgkin lymphoma, non Hodgkin lymphomas or various haematological diseases (P>0.05 for each group comparison), a slight but significant (PTable 1). These results were independent of the initial TNC count or TNC concentration per frozen bag, thus suggesting that TNCs from myeloma patients were less resistant to the thawing procedure.Figure 1.(a) Comparison between TNCR and viability, and (b) comparison between TNCR and granulocytic cells concentration. TNCR, total nucleated cell recovery.Full figure and legend (116K)We next analysed the relationship between storage duration, TNCR and viability in all grafts. The majority of graft products were thawed during the first 4 months following apheresis (n=826, 82.1%). One hundred and eighty products (17.9%) were thawed more than 4 months after cryopreservation, with a maximum period of 111 months. TNCR and viability were found to be similar in these two statistically comparable groups (55.9 versus 54.6%, and 68.3 versus 67.6%, respectively), thus confirming that long term storage did not seem to profoundly affect cell recovery (not shown).We next analysed similar parameters regarding the CD34+ haematopoietic stem cells. Mean CD34+ cell recovery was found to be 98.036.5% (range 12 We could neither find correlation between CD34+ cell recovery and viability, nor between haematopoietic progenitor cell recovery and initial CD34+ cell count or concentration at the time of freezing (not shown). Moreover, CD34+ cell recovery was not correlated with granulocytic cell concentration. In addition, CD34+ cell recovery was independent of the initial diagnosis, with mean recoveries of 100.4, 94.5, 95.2, 99.0 and 100.8% for solid cancers, Hodgkin lymphoma, non Hodgkin lymphomas, various haematological diseases and myeloma, respectively. Lastly, as observed for TNCR, CD34+ cell recovery was not modified by the duration of storage, since mean recovery for products thawed before or after 4 months was 98 and 98.3%, respectively (not shown).