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iv. PROCESSING, CRYOPRESERVATION AND INFUSION Page 2
- The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft. Liseth K, Bjorsvik S, Grottebo K, Bruserud O. Cytotherapy. 2005;7:328-33.
(Also see Citations #11, 14, 15, 20 and 21.)
DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when peripheral blood progenitor cells (PBPC) used for autologous transplantations were cryopreserved with four different DMSO concentrations.
Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples.
Results indicated that PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO.
The authors concluded that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.
[Although this study was done using PBPC, the results should be of interest to cord blood bankers.]
- Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood. Meyer T, Hofmann B, Zaisserer J, Jacobs V, Fuchs B, Rapp S, Weinauer F, Burkhart J. Cytotherapy. 2006;8:265-76.
(Also see Citations #3, 6, 7, 9, 20 and 21 )
The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs.
The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5x107 MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed.
The authors emphasized that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.
- Cryopreservation of Hematopoietic Cells. Rowley SD. In: Blume KG, Forman SJ, Appelbaum FR (eds) Thomas' Hematopoietic Cell Transplantation. 3rd edition. Malden (MA), Blackwell Publishing, 2004, pp 599-612.
Although this comprehensive review is not related exclusively to cryopreservation of cord blood (CB) units, it contains much information that is of critical importance for CB banking and transplantation.
The author reviews the scientific basis for cryopreservation including the physics of cooling and warming of cell products. The only "assay" for the cryosurvival of hematopoietic cells is engraftment after transplantation. Progenitor cell assays such as culture for CFU-GM after thawing can be predictive of engraftment, but the validity of these assays must be determined for each group of patients and the assay used. The different cooling properties of vials compared to bags diminishes the reliability of these small aliquots for determining cryosurvival. If an attempt is made to use small aliquots for clinical decisions, a system of cooling, thawing, washing and culture that correlates with engraftment kinetics must first be developed.
Cooling rates for hematopoietic cells are reviewed. Different cells have different optimal cooling rates, and the optimal cooling rate is also dependent on the type and concentration of cryoprotectant used. Cooling at slow rates limits intracellular ice nucleation, and mechanical disruption of the cell from ice recrystallization during warming is less likely. Rapid warming is appropriate.
Some laboratories perceive storage in the liquid phase of liquid nitrogen to be safer because of temperature gradients in vapor-phase refrigerators and because of the larger quantity of nitrogen present. However, liquid nitrogen can serve as a reservoir for viruses. This problem was dramatically illustrated by the transmission of hepatitis B infection to at least three patients whose cells were immersed in the same liquid nitrogen refrigerator as those of the index case.
The duration of storage may be indefinite if adequate temperatures are maintained and appropriate cryopreservation techniques are used. DMSO and HES need not be removed before infusion if consideration is given to the potential toxicities of these agents. Most centers performing autologous or allogeneic marrow hematopoietic cell transplantation infuse the cells within a few minutes after thawing and without any post-thaw processing.
A high incidence of generally mild, infusion-related morbidity has been reported with the infusion of either marrow of peripheral blood and this is generally attributed to the effects of DMSO compounded by the presence of lysed blood cells. More significant adverse reactions include the rare anaphylactic reaction and profound hypotension. Skin flushing, dyspnea, abdominal cramping, nausea and diarrhea have been attributed to DMSO-induced histamine release. These complaints resolve over a few hours and are treated symptomatically. DMSO may also cause increased blood pressure and bradycardia, which may be maximal about 1-3 hours after completion of the hematopoietic cell infusion. Cardiac rhythm abnormalities generally resolve spontaneously within 24 hours of infusion. CNS complications are rare and generally related to the amount of DMSO infused. Encephalopathy has been reported and may promptly resolve following plasmapheresis.
The authors summarize by stating that current cryopreservation techniques are satisfactory for the treatment of many patients, but there has been no comprehensive study of hematopoietic cell cryobiology to quantify the cell losses resulting from cell freezing. There is considerable, but generally minor, toxicity from the currently used cryoprotectants.
- Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples. Laroche V, McKenna DH, Moroff G, Schierman T, Kadidlo D, McCullough J. Transfusion 2005; 45:1909-1916.
This study examines the effect of the wash step as well as that of postthaw storage on various quality control variables of UCB units. Ten units were thawed and washed. Samples were removed from each unit at six time points: prefreeze, immediately postthaw, immediately postwash, and 1, 2 and 5 hours postwash. On each sample, total nucleated cell (TNC) count, CD34+ cell enumeration, colony-forming unit (CFU)-granulocyte-macrophage, and viability assays were performed.
Results indicated that TNC counts decreased postthaw and at subsequent time points; the postthaw TNC recovery was 89% compared to 82% postwash. TNC recovery decreased to 90% of postwash (82% of postthaw) values and 83% of postwash (76% of postthaw) values at 2 and 5 hours postwash, respectively. CD34+ cell loss postthaw was not significant. Viability decreased postthaw and plateaued over time. CFUs were significantly lower postthaw, recovering postwash.
Some results are particularly noteworthy. The mean TNC count decreased significantly at each step of processing. Of the 18% total loss of TNCs associated with standard postcryopreservation processing, the washing step accounted for 7 percent. The recovery of CD34+ cells was 97% postthaw and a remarkable 148.9% postwash (95% Confidence Interval (CI), 112.8-185%) (This was not statistically significant.) The majority of the increase in CD34+ cells occurred after washing. CFUs decreased to 17% of their prefreeze level but increased postwash returning to 64% of prefreeze values.
The authors conclude that thawing and washing result in a substantial loss of cells, with TNC loss approaching 20% when compared with prefreeze counts; the wash step was responsible for nearly half of the cell loss. Elapse of time postwash resulted in further loss of nucleated cells but no detectable significant changes in CD34+cell content and viability and/or CFU.
The authors comment that the rationale behind RBC depletion has been to reduce the total volume of the product being frozen to maximize storing capacity. However, a number of publications presenting data on post-RBC depletion processing have shown total cell losses falling in the range of 20-30%. Washing postthaw has been justified by the in vitro inhibitory effect of DMSO on progenitor cell viability and clonogenicity, better control over the thawing conditions, and reduction in infusional toxicity related to DMSO.
The authors indicate that their data suggest that cell loss associated with thawing and washing significantly impacts the number of UCB units suitable for transplantation in patients weighing 40 kg or more. Patients weighing between 40 and 60 kg would seem to gain more if the washing step was removed.
The authors suggest that their results support the need for an assessment or reevaluation of the rationale for washing in cord blood processing. The potential benefits of removing debris and DMSO should be weighed against the impact of cell loss in the infused product.
- Cryopreserved human haematopoietic stem cells retain engraftment potential after extended (5-14 years) cryostorage. Spurr EE, Wiggins NE, Marsden KA, Lowenthal RM, Ragg SJ. Cryobiology. 2002;44:210-7.
The effect of long-term cryostorage (5-14 years) on the viability and functional capacity of haematopoietic stem cells (HSCs) was investigated in 40 bone marrow and peripheral blood harvests using standard in vitro methods, the colony forming unit-granulocyte/macrophage (CFU-GM) assay and a single platform viable CD34(+) cell absolute count by flow cytometry. Forty percent of harvests had CD34(+) HSC counts of at least 0.7 x 106/kg bodyweight and 85% had CFU-GM counts of at least 1.0 x 105/kg bodyweight. These values represent minimum requirements for safe transplantation at the authors' institution. Based on these results, it appears that HSC collections can remain adequate for safe transplantation after up to 14 years of cryostorage. However, as deterioration of HSC quality and viability may occur, some precautions may be warranted, namely harvesting higher than normal numbers of HSCs in collections intended for long-term storage and repeating in vitro assays on harvests after long-term storage prior to transplantation.
- Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres. Windrum P, Morris TC, Drake MB, Niederwieser D, Ruutu T; EBMT Chronic Leukaemia Working Party Complications Subcommittee. Bone Marrow Transplant. 2005;36:601-3.
The cryoprotectant dimethyl sulfoxide (DMSO) is known to have toxic side effects, yet guidelines for its use in stem cell transplantation do not exist. To assess current practice in the use of DMSO and the incidence of DMSO-related complications, a single page questionnaire was mailed to 444 EBMT centers involved in autologous transplantation. The responses from 97 centers showed a wide variation in practice between transplant units regarding the concentration of DMSO used, daily DMSO dose restriction and the use of cell washing. There was an upper limit in the amount of DMSO given per day by 57 centers as follows: 80 g (n=5), 60 6 (n=9), 40 g (n=12), 20 g (n=2) and others (n=29). Among "others", the most frequently cited limit was 1 g/kg. Five centers washed cells before return while some did so "sometimes".
The overall incidence of DMSO toxicity was approximately one in 70 transplants and most cases were cardiovascular and respiratory in nature. Other effects such as CNS, renal, hepatic and anaphylaxis are less common. Only once case of mortality attributed to DMSO was cited, and that was in a patient with cardiac amyloidosis and end-stage renal failure. There was a trend to reduced complication rates in centers using lower concentrations of DMSO or washing cells prior to return.
A large-scale prospective study of the strategies for reduction in exposure to DMSO and reduction in toxic effects is required before guidelines in the use of DMSO in stem cell cryopreservation can be promulgated.
- Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood. Yang H, Zhao H, Acker JP, Liu JZ, Akabutu J, McGann LE. Cryobiology. 2005;51:165-75.
This study was designed to evaluate the pre- and post-cryopreservation effect of Me2SO and Me2SO removal on the enumeration of CD45+ and CD34+ cells by flow cytometry. While a logical predictor of hematologic recovery would be the establishment of a minimum post-cryopreservation dose of viable CD34+ cells, the lack of a validated or standardized test for post-thaw enumeration of CD34+ cells has hindered this development.
In most studies, Me2SO is removed prior to the enumeration of CD34+ cells from post-thaw samples, although some investigators perform the enumeration of CD34+ cells in the presence of different concentrations of Me2SO. In practice, thawed mobilized peripheral hematopoietic progenitor cells containing Me2SO are infused into patients, whereas thawed cord blood hematopoietic cells are [usually] washed to remove Me2SO before infusion. Some studies suggest that the toxicity of Me2SO to progenitor cells warrants its removal. However, Me2SO toxicity to human progenitor cells has not been found at a concentration of 10% (v/v) at either 4 or 37°C for incubation of up to one hour.
Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22°C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD.
The results indicated that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw progenitor cells mobilized from peripheral blood and umbilical cord blood were a function of the concentration of Me2SO. Without cryopreservation, when 10% Me2SO is present, recovery and viability of mobilized peripheral blood CD34+ cells but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both mobilized peripheral blood and umbilical cord blood before and after cryopreservation.
The authors concluded that their results indicate that to reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.
- Cord Blood Transplantation Study (COBLT): cord blood bank standard operating procedures. Fraser JK, Cairo MS, Wagner EL, McCurdy PR, Baxter-Lowe LA, Carter SL et al. J Hematother 1998; 7:521-561.
The Cord Blood Transplantation Study (COBLT) Cord Blood Bank Standard Operating Procedures were published in 1998. The medical coordinating center publishes updates on the EMMES website (www.EMMES.com).
