Fresh PBSC harvests, but not BM, show temperature-related loss of CD34 viability during storage and transport

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1 Cytotherapy (2006) Vol. 8, No. 2, 158/165 Fresh PBSC harvests, but not BM, show temperature-related loss of CD34 viability during storage and transport V Antonenas 1, F Garvin 1, M Webb 1, M Sartor 2, KF Bradstock 1,2,3 and D Gottlieb 1,3,4 1 Sydney Cellular Therapies Laboratory, 2 Flow Cytometry Unit and 3 Blood and Marrow Transplant Service, Westmead Hospital, Sydney, New South Wales, Australia, and 4 University of Sydney, New South Wales, Australia Background The optimum conditions for storage and transport of freshly harvested HPC in the liquid state are uncertain. It is not specified in commonly applied standards for stem cell transplantation. We used a viable CD34 assay to determine the optimum temperature for maintaining progenitor cell viability in freshly harvested BM and PBSC. Our aim was to identify standardized conditions for storage and transport of marrow or peripheral blood products that would optimize CD34 recovery, leading to better transplant outcomes. Methods Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC and 12 BM) and stored at refrigerated temperature (2 /88C), room temperature (18/248C) and 378C for up to 72 h. Samples were analyzed for viable CD34 cells/ml at 0, 24, 48 and 72 h. Results The mean viable CD34 yield prior to storage was 7.7/10 6 /kg (range 0.7/30.3). The mean loss of viable CD34 cells in HPC products at refrigerated temperature was 9.4%, 19.4% and 28% at 24, 48 and 72 h, respectively. In contrast, the mean loss of viable CD34 cells at room temperature was 21.9%, 30.7% and 43.3% at 24, 48 and 72 h, respectively. No viable CD34 cells remained after storage at 378C for 24 h. Only PBSC products and not BM showed temperaturerelated loss of CD34 viability. Greater loss of viable CD34 cells was observed for allogeneic PBSC compared with autologous PBSC. Discussion These results demonstrate that the optimum temperature for maintaining the viability of CD34 cells, during overnight storage and transport of freshly harvested HPC, is 2/88C. These findings will allow the development of standard guidelines for HPC storage and transport. Keywords hemopoietic progenitor cells, storage and temperature, viable CD34 cells. Introduction High-dose chemotherapy with autologous stem cell support is recognized as the standard of care for a variety of diseases including multiple myeloma, lymphoma and some types of solid tumor. Myeloablative and nonmyeloablative allogeneic stem cell transplants are a curative option for many patients with hematologic malignancy. The number of both autologous and allogeneic transplants has been steadily rising over the last two decades. This increasing use of hemopoietic stem cells poses important issues related to transport and storage of stem cell products. Although BM has been conventionally used for hemopoietic transplantation following high-dose therapy, PBSC are now the preferred choice of stem cell product in most instances of transplantation [1,2]. Autologous PBSC are usually cryopreserved immediately after harvest, while allogeneic PBSC are typically infused within 24 h of apheresis collection. However, more prolonged storage and transport of HPC is becoming common practice for various reasons. Increasingly, apheresis products from unrelated donors have to be transported from distant collection centers to the point of transplant. In Correspondence to: Vicki Antonenas, Sydney Cellular Therapies Laboratory, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Darcy Rd, Westmead, NSW 2145, Australia ISCT DOI: /

2 Optimum temperature for storage and transport of fresh HPC 159 some cases up to 48 h transit time is involved. Some unrelated PBSC products are collected on consecutive days and, in an effort to be more efficient and costeffective, collection centers may store the first product overnight and then ship with the second collection. Finally, overnight storage of apheresis products may be necessary when specialized processing is required, or to fit in with laboratory workflow. In the last 10 years there have been reports on various factors affecting the storage of freshly harvested HPC products. Most analyzes have been based on trypan blue for viability, measurement of total nucleated cells (TNC) and colony-forming unit (CFU) assays [3/5]. Most information exists for autologous HPC stored overnight (16 /18 h) at 48C. Less data are available for room temperature and extended storage conditions. There is incomplete information on allogeneic HPC samples, in particular regarding the storage of unrelated PBSC. Few studies have evaluated the survival of viable CD34 cell numbers, which are known to be critical to the speed of engraftment and survival of the recipient. There are no consensus guidelines for the storage and transport of HPC products. National Marrow Donor Program (NMDP) standards recommend that marrow be infused within 24 h of collection and that transport temperature be determined by the transplant center [6]. The current FACT and JACIE standards used for the shipping of products from collection centers to transplant facilities do not specify transport temperatures, and only recommend a range of ideal temperatures from 18C to 248C [7,8]. We have undertaken a study examining the effect of different temperatures on storage of both autologous and allogeneic stem cell products for time periods similar to those that elapse in the normal transport of stem cells used in transplantation. We have studied the survival of viable CD34 cells using a novel single Figure 1. Viable CD34 flow histograms on a day 3 PBSCH product stored at room temperature. (a) All CD45 leukocytes in region 1 (R1). (b) Viable and non-viable leukocytes gated on R1. (c) Viable CD45 events (i.e. R1 and not R2) and CD34 events identified in R3. (d and e) Sequentially gated from R3, R4, and R5, respectively. (f) Ungated events and the bead count obtained from R7.

3 160 V Antonenas et al. platform assay recently developed in our laboratory. Our results will enable standardization of guidelines for storage, transport and handling of fresh HPC. autologous and 14 were from allogeneic donors; of the BM harvests, four were autologous and eight were allogeneic. Methods Fresh HPC samples Forty-six fresh HPC samples (34 PBSC and 12 BM) were taken from adult and pediatric patients and donors undergoing HPC transplantation for hematologic malignancies, with informed consent. There were 24 autologous, 17 matched sibling allogeneic and five matched unrelated transplants. Of the 34 PBSC collections, 20 were Storage conditions PBSC and BM products were received and analyzed within 2 h of collection, before processing/cryopreservation or for infusion. The mean viable CD34 yield collected was 7.7/10 6 /kg (n/46, range 0.7/30.3). A small aliquot (1 /2 ml) was aseptically removed from the HPC product for routine analysis, including white cell counts (WCC), viable CD34 analysis, blood group Figure 2. Viable CD34 cells/ml from HPC products stored at 0, 24, 48 and 72 h at (A) a refrigerated temperature of 4 o C, (B) room temperature (18/24 o C) and (C) 37 o C.

4 Optimum temperature for storage and transport of fresh HPC 161 confirmation and sterility testing. The remainder of the aliquot was then transferred into sterile tubes, which were stored either at refrigerated temperature (2 /88C), room temperature (18 /248C) or 378C (in incubator). Small samples were then removed from these aliquots at 24 h (day 1), 48 h (day 2) and 72 h (day 3) to determine viable CD34 counts. The cell concentration of samples used for this study was adjusted if necessary to maintain a WCC of B/300/10 9 /L. The total WCC was obtained using an Advia 120 (Bayer) hematology analyzer. Viable CD34 assay and flow cytometry Viable CD34 analysis was performed according to the method described by Sartor et al. [9]. Briefly, 10 ml ofcell suspension was labeled with appropriately titered CD34 / PE and CD45 /FITC Ab and with 7AAD in TRU- COUNT tubes (BD Bioscience, San Jose, CA, USA) and incubated for 15 min at room temperature. PBS, 500 ml containing 0.5% human albumin, was added to the tube immediately prior to analysis by flow cytometry. List mode data was acquired on a FACSCalibur and analyzed using Cell Quest 3.1 software (BD Bioscience). Effect of different storage temperatures on viable CD34 cells in HPC products We examined a total of 46 HPC products, stored at refrigerated, room and 378C temperatures for up to 72 h (Figure 2). The viable CD34 results for all stem cell harvests, PBSC and BM, stored at 48C, are shown in Figure 2A. Overall, we found stability of CD34 viability in HPC products at 48C for up to 72 h, although some cases demonstrated a slight decrease in numbers of viable CD34/mL, most obvious after 48 h. In contrast, there was a greater decline in the number of viable CD34/mL stored at room temperature, (Figure 2B), while we found no viable CD34 cells remaining in HPC products at 378C after 24 h (Figure 2C). At all storage temperatures, the greatest decline in viable CD34 numbers was observed in samples with an initial count/1000/ml. Figure 3 shows the mean percentage loss of viable CD34 cells after storage of HPC products at refrigerated and room temperatures over 3 days. There was a progressive loss of Statistical methods All statistical analysis was performed using Prism Software. Results in column graphs represent mean values9/sem error bars. Significance was determined using unpaired t- tests (two-tailed). Results Viable CD34 cells in freshly harvested HPC products We used a no-lyse single-platform viable CD34 flow cytometry assay, recently described by our group [9], to determine the optimum storage and transport temperature for maintaining the viability of CD34 cells in freshly harvested HPC. Figure 1 shows an example of the gating strategy for CD34 analysis on a day 3 PBSC product stored at room temperature. Initially, the viability of CD34 cells from HPC collection bags was compared with the tube analysis used for this study. Ten HPC samples (six allogeneic and four autologous PBSC) were tested after 24 h storage at 48C. We found a significant correlation between the mean viable CD34 counts/ml obtained from HPC bags compared with those stored in tubes (r 2 /0.993, P B/0.0001). Figure 3. PBSC and BM HPC products stored at refrigerated (2/ 88C) and room temperature (18/248C) for up to 3 days. The mean percentage loss of viable CD34 cells is higher for HPC products stored at room temperature at all time points compared with HPC products stored at 48C.

5 162 V Antonenas et al. viable CD34 cells, which was more pronounced at room temperature at all time points. Effect of different temperatures on CD34 viability in stored PBSC and BM We next investigated whether there was a difference in the loss of CD34 viability at different temperatures based on HPC type, i.e. PBSC compared with BM. We found significantly greater loss of viable CD34 cells for PBSC stored at room temperature compared with 48C at all time points (Figure 4A). The median loss of viable CD34 cells in PBSC at 48C was 5.5%, 14% and 35% at 24, 48 and 72 h, respectively. In contrast, the median loss of viable CD34 cells in PBSC at room temperature was 22.5%, 26% and 53% at 24, 48 and 72 h, respectively (P-values 0.003, and 0.008, respectively). In contrast, there was no temperature-based difference in the loss of viable CD34 cells from BM at any time point (Figure 4B). CD34 viability in HPC products from adults or children To investigate whether the age of the donor had an effect on the CD34 viability during storage, we compared HPC products from adults with those from pediatric patients (B/15 years of age). As shown in Figure 5, the difference in the loss of viable CD34 cells between the two age groups was not statistically significant over time. Effect of different storage temperatures on CD34 viability in autologous and allogeneic HPC products Allogeneic peripheral blood HPC are collected from normal donors who have been mobilized with G-CSF alone, whereas autologous patients have been mobilized with chemotherapy and G-CSF. We examined whether there was a difference in CD34 viability between stored autologous and allogeneic peripheral blood HPC collections. Surprisingly, as shown in Figure 6, there was significantly greater loss of viable CD34 cells for allogeneic PBSC compared with autologous PBSC. We also evaluated autologous and allogeneic BM samples (n / 5), but found no significant difference between refrigerated and room temperature (data not shown). The mean loss of viable CD34 cells in allogeneic PBSC at 48C was 16%, 28% and 37% at 24, 48 and 72 h, while at room temperature the corresponding values were 35%, 37% and 46.5% (P/ 0.003, and 0.026, respectively). To investigate further the difference in the Figure 4. Median percentage loss of viable CD34 at refrigerated and room temperature storage on (A) PBSC and (B) BM products for 24, 48 and 72 h.

6 Optimum temperature for storage and transport of fresh HPC 163 loss of viable CD34 cells between autologous and allogeneic PBSC, we compared the white cell count, percentage of neutrophils and platelet count in these two different types of samples. As shown in Figure 7, there was no difference between the autologous and allogeneic HPC harvests for the median values of WCC and the percentage of neutrophils. However, we found a significantly increased platelet content for the allogeneic PBSC (median count 2208/10 9 /L) compared with autologous PBSC (median count 469/10 9 /L), P/ Figure 5. Autologous refrigerated HPC products; comparing pediatric with adult samples. Discussion This study analyzed the effects of different storage temperatures on the viability of cells in PBSC and BM HPC products. Our results have particular significance because they have been obtained using assays assessing the viability of CD34 stem cells whose number determines the success and speed of engraftment after transplantation. To our knowledge this is the first rigorous analysis of CD34 viability in human HPC products and has direct implications for how these products should be handled. Traditionally, evaluation of HPC harvests after collection has been performed by trypan blue dye exclusion assay, Figure 6. Mean percentage loss of viable CD34 at refrigerated and room temperature storage on autologous PBSC and allogeneic PBSC products at 24, 48 and 72 h. Mean values quoted in text.

7 164 V Antonenas et al. Figure 7. Comparison of WCC, percentage neutrophils and platelet counts in autologous and allogeneic HPC products. with viabilities typically 90% or higher for the total population. However, this method fails to identify the viability of rare cells, particularly CD34 cells. Another frequently used tool to evaluate stem cell harvests, colony assays performed on short-term liquid storage products, is labor intensive, has poor reproducibility and assesses only one type of myeloid progenitor cell [10,11]. Not surprisingly, the use of these assays to assess progenitor cell viability after harvest has produced widely divergent results [12,13], including some that differ from those we report here. A number of previous studies have indicated that shortterm storage of autologous PBSC at refrigerated temperature does not result in deterioration in total nucleated cell numbers, with retention of trypan blue viability counts of /90% and high percentage recoveries of CFU [3,5,13 / 15]. Refrigerated temperatures were found to be optimal for recovery of long-term culture initiating cells for short periods [16]. Two studies suggested little alteration following storage even at room temperature for up to 24 h [17,18]. In contrast, using the viable CD34 assay, we show that the mean percentage loss of viable CD34 cells after 24 h is significantly higher for cells stored at room temperature (21.9%) than for products stored at 48C (9.4%). Using the same traditional assays, other reports have indicated that autologous PBSC stored for up to 72 h at 48C can be used successfully for transplantation without cryopreservation. The trypan blue viability was more than 90% in all samples studied [4,19]. However, our data demonstrate that even at 48C there is an incremental loss of viable CD34 cells in autologous BM and PBSC stored for 24, 48 and 72 h and that this loss is worsened by storage at room temperature. These data are supported by those of Pettengell et al. [20], who reported a decline in CFU recoveries for autologous PBSC stored at 48C for up to 72 h. In contrast to other data [21], we found no viable CD34 cells remaining in HPC products at 378C after 24 h. In this study, PBSC products, but not BM, showed temperature-related loss of CD34 viability. We found no difference in CD34 viability between BM cells stored at either 48C or room temperature for 72 h. Few studies have addressed the question of whether BM and PBSC have different storage condition requirements. Delforge et al. [22] reported a 3% loss of colony-forming cells in BM stored for 96 h at 48C, but a significant loss of 95% of PBSC stored at the same temperature. In another study of five allogeneic PBSC products stored at 1/68C for up to 3 days, CFU, LTC-IC, and CD34 cell numbers were maintained without alteration [23]. In contrast, we found a greater percentage loss of viable CD34 cells in allogeneic PBSC compared with BM stored at 48C for the same time periods. We cannot identify why allogeneic PBSC demonstrate greater loss of CD34 viability. One possibility is toxicity as a result of the greater WCC or granulocyte content typically present in these collections. We also observed a higher number of platelets in allogeneic PBSC collections that may result in leucocyte entrapment in platelet fibrin aggregates. These issues require further investigation. Our data quantify for the first time the CD34 cell loss with storage at different temperatures. Even more importantly, they indicate that maintenance of CD34 cell viability is not identical in PBSC and BM products at different temperatures. To date, most guidelines have adopted the same transport temperatures for PBSC as for BM, and although these have been validated

8 Optimum temperature for storage and transport of fresh HPC 165 historically for marrow products no validation for PBSC has taken place. While the current practice of storing and transporting products at either refrigerated or room temperatures is reasonable for BM, PBSC products should only be stored and transported at refrigerated temperatures. For the sake of simplicity, we suggest that all HPC products be stored and transported at this temperature. This will improve consistency of handling, allow for standardization of methodology between laboratories and permit incorporation of recommendations into various national guidelines for stem cell storage and transportation. Most importantly, reducing the loss of viable CD34 cells in stem cell products is likely to result in better transplant outcomes for patients undergoing various forms of blood and marrow transplantation. References 1 Korbling M, Juttner C, Henon P et al. Autologous blood stem cell versus bone marrow transplantation. Bone Marrow Transplant 1992;10 Suppl 1:144 /8. 2 Pettengell R, Morgenstern GR, Woll PJ et al. Peripheral blood progenitor cell transplantation in lymphoma and leukemia using a single apheresis. Blood 1993;82:3770 /7. 3 Lasky LC, McCullough J, Zanjani ED. Liquid storage of unseparated human bone marrow. Evaluation of hematopoietic progenitors by clonal assay. Transfusion 1986;26:331 /4. 4 Jestice HK, Scott MA, Ager S et al. Liquid storage of peripheral blood progenitor cells for transplantation. Bone Marrow Transplant 1994;14:991 /4. 5 Koc ON, Gerson SL, Phillips GL et al. Autologous CD34 cell transplantation for patients with advanced lymphoma: effects of overnight storage on peripheral blood progenitor cell enrichment and engraftment. Bone Marrow Transplant 1998;21:337 /43. 6 NMDP Standards, 18th edn. St Paul, MN: National Donor Program, Available at complete_03.pdf. 7 FACT Standards, 2nd edn Available at standards/pdf. 8 JACIE Standards, 2nd edn June Available at jacie standards/pdf. 9 Sartor M, Antonenas V, Garvin F et al. Recovery of viable CD34 cells from cryopreserved hemopoietic progenitor cell products. Bone Marrow Transplant 2005;36:199 / To LB, Haylock DN, Simmons PJ, Juttner CA. The biology and clinical uses of blood stem cells. Blood 1997;89:2233 / Parker JW, Metcalf D. Production of colony-stimulating factor in mixed leucocyte cultures. Immunology 1974;26:1039 / Preti RA, Razis E, Ciavarella D et al. Clinical and laboratory comparison study of refrigerated and cryopreserved bone marrow for transplantation. Bone Marrow Transplant 1994;13:253 / Burnett AK, Tansey P, Hills C et al. Haematological reconstitution following high dose and supralethal chemo-radiotherapy using stored, non-cryopreserved autologous bone marrow. Br J Haematol 1983;54:309 / Ager S, Scott MA, Mahendra P et al. The use of noncryopreserved PBSC in autologous transplantation. Bone Marrow Transplant 1995;16:633 /4. 15 Sugrue MW, Hutcheson CE, Fisk DD et al. The effect of overnight storage of leukapheresis stem cell products on cell viability, recovery, and cost. J Hematother 1998;7:431 /6. 16 Petzer AL, Gunsilius E, Zech N et al. Evaluation of optimal survival of primitive progenitor cells (LTC-IC) from PBPC apheresis products after overnight storage. Bone Marrow Transplant 2000;25:197 / Beaujean F, Pico J, Norol F et al. Characteristics of peripheral blood progenitor cells frozen after 24 hours of liquid storage. J Hematother 1996;5:681 /6. 18 Lazarus HM, Pecora AL, Shea TC et al. CD34 selection of hematopoietic blood cell collections and autotransplantation in lymphoma: overnight storage of cells at 48 C does not affect outcome. Bone Marrow Transplant 2000;25:559 / Ruiz-Arguelles GJ, Ruiz-Arguelles A, Perez-Romano B et al. Filgrastim-mobilized peripheral-blood stem cells can be stored at 48 and used in autografts to rescue high-dose chemotherapy. Am J Hematol 1995;48:100 /3. 20 Pettengell R, Woll PJ, O Connor DA et al. Viability of haemopoietic progenitors from whole blood, bone marrow and leukapheresis product: effects of storage media, temperature and time. Bone Marrow Transplant 1994;14:703 /9. 21 Niskanen E. Preservation of human granulopoietic precursors following storage in the nonfrozen state. Transplantation 1983;36:341 /3. 22 Delforge A, Ronge-Collard E, Stryckmans P et al. Granulocytemacrophage progenitor cell preservation at 48 C. Br J Haematol 1983;53:49 / Moroff G, Seetharaman S, Kurtz JW et al. Retention of cellular properties of PBPCs following liquid storage and cryopreservation. Transfusion 2004;44:245 /52.

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