Peripheral blood stem cells Number of viable CD34 + cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation

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1 (22) 29, Nature Publishing Group All rights reserved /2 $25. Peripheral blood stem cells Number of viable CD34 + cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation DS Allan 1, M Keeney 1, K Howson-Jan 1, J Popma 1, K Weir 1, M Bhatia 2, DR Sutherland 3 and IH Chin-Yee 1 1 Division of Hematology, University of Western Ontario, London, Ontario, Canada; 2 Robarts Research Institute, London, Ontario, Canada; and 3 The Toronto Hospital, Toronto, Canada Summary: Reduced CD34 + cell viability due to cryopreservation has unknown effects on subsequent hematopoietic engraftment in autologous transplantation. Thirty-six consecutive autologous peripheral stem cell collections were analyzed for absolute viable CD34 + cell numbers at the time of stem cell collection and prior to reinfusion. Viable CD34 + cells were enumerated using single platform flow cytometry and the molecular exclusion dye 7-amino actinomycin D. The median number of viable CD34 + cells was /kg at the time of harvest and /kg after thawing. When viable CD34 + cells enumerated after thawing were 2., 2. 5., or /kg, the median time to platelet engraftment was 17, 12 and 1 days, respectively (P.5 for comparison of the group with /kg and the other two groups), and the median time to neutrophil engraftment was 13, 14 and 12 days, respectively (P = NS). A minimum of CD34 + cells/kg was harvested in 33 of 36 patients (92%) but only 19 of 36 (52%) patients met this threshold at the time of reinfusion. The reduced numbers of viable CD34 + cells measured prior to re-infusion is associated with time to platelet engraftment and may be useful in monitoring stem cell loss during processing and identifying patients at risk of graft failure. (22) 29, DOI: 1.138/sj/bmt/ Keywords: CD34; viability; engraftment; autologous; transplantation Peripheral blood stem cells mobilized by chemotherapy and/or cytokines are the preferred source of hematopoietic stem cells in the setting of autologous transplantation. 1 Assessing graft adequacy prior to myeloablative chemotherapy typically involves determining the yield of stem Correspondence: Dr IH Chin-Yee, Division of Hematology, London Health Sciences Centre, 8 Commissioner s Road East, London, Ontario, N6A 4G5 Canada Received 12 June 21; accepted 14 March 22 cells at the time of leukapheresis by enumeration of stem/progenitor cells expressing the cell surface antigen CD34. 2 The CD34 antigen is expressed on rare mononuclear cells in the peripheral blood and on 2 4% of mononuclear cells in the marrow. These putative stem cells have multilineage progenitor cell activity in vitro 3,4 and are capable of reconstituting long-term hematopoiesis in nonhuman primates 5 and in human subjects following myeloablative chemotherapy. 6 These cells can be mobilized into the peripheral blood in significantly higher numbers using recombinant cytokines alone 7 or in combination with chemotherapy. 8,9 The adequacy of leukapheresed autologous peripheral stem cell harvests is typically described by the yield of CD34 + cells collected. Numerous groups have established that rapid hematopoietic reconstitution following myeloablative chemotherapy depends upon the number of CD34 + cells collected. 2,1 12 A minimum threshold of CD34 + cells/kg in the PBSC collection has been associated with prompt engraftment 1,2 and CD34 + cell doses greater than /kg 11 and greater than /kg 12 may further accelerate hematopoietic recovery. Processing and cryopreservation undoubtedly render some cells nonviable and diminish the absolute number of CD34 + cells available for reinfusion. The impact of reduced cell viability on time to hematopoietic reconstitution in autologous peripheral stem cell transplantation has not been systematically studied. A single platform flow cytometric method is available for rapid determination of absolute CD34 + cells, based on guidelines developed for the International Society of Hematotherapy and Graft Engineering (ISHAGE) The single step method was adapted from a dual platform strategy that required a hematology analyzer for determination of total white cell concentration. 13 The dual platform method has been well validated in several clinical studies that included rates of hematopoietic reconstitution. 12,16,17 Guidelines for CD34 + cell enumeration by flow cytometry have been prepared by ISHAGE, 15 the European Bone Marrow Transplant Group 18 and the British Committee for Standards in Haematology 19 supporting flow cytometry as the reference standard for CD34 + cell enumeration. The simpler single platform method was evaluated in 72 patients at a single

2 968 institution and displayed excellent correlation with the dual platform method. 13 Single platform CD34 + cell enumeration by flow cytometry allows real-time assessments of graft adequacy at the time of collection to ensure minimum thresholds are reached. As well, this method can be applied after thawing cryopreserved stem cells to assess the effect of processing. Viability of mononuclear cells is determined using the exclusion dye 7-amino actinomycin D and can be easily combined with absolute CD34 + cell counting to enumerate viable CD34 + cells. 13,2 The accuracy of CD34 + cell enumeration and viability testing was extremely high in a recent study of thawed cord blood samples, 21 providing important validation of the single platform methodology. The aim of this study was to determine the cumulative effects of cryopreservation, thawing and washing of peripheral blood stem cell collections on CD34 + cell viability and to evaluate the association between the number of thawed CD34 + cells and rates of hematopoietic engraftment following autologous peripheral blood stem cell transplantation. Patients and methods Patients and transplantation protocol Thirty-seven consecutive patients who underwent autologous stem cell transplantation between February 1999 and January 21 at the London Health Sciences Centre were identified. One patient was excluded from analysis due to lack of availability of CD34 + cell viability testing. Following informed consent, patients underwent autologous peripheral blood stem cell transplantation for multiple myeloma (16 patients), relapsed Hodgkin s lymphoma (14 patients), relapsed non-hodgkin s lymphoma (three patients), and as consolidation therapy for germ cell tumors (three patients). To mobilize CD34 + cells, patients received one of three regimens: (1) recombinant human granulocyte colony-stimulating factor (G-CSF) 1 g/kg/day for 5 days (3 patients); (2) cyclophosphamide 2.5 g/m 2 followed by G-CSF for 11 days (one patient); or (3) cyclophosphamide, G-CSF and recombinant human stem cell factor (SCF) 2 g/kg/day for 11 days (five patients). Leukapheresis was performed using a COBE Spectra machine (Sorin Biomedica Canada, Ontario, Canada) after the mobilization treatment to obtain a target yield of CD34 + cells/kg. Disease-specific myeloablative chemotherapy was administered 3 days prior to receiving the stem cells. Patients with multiple myeloma received melphalan 2 mg/m 2 or melphalan 14 mg/m 2 with total body irradiation; patients with Hodgkin s disease and non-hodgkin s lymphoma received etoposide 6 mg/kg and melphalan 16 mg/m 2 ; and patients with germ cell tumors received etoposide 19 mg/m 2 and carboplatin 2 g/m 2. Patients did not receive recombinant cytokines during the period of hematopoietic reconstitution. Blood products were transfused using the following general thresholds or at the discretion of the treating physician. Two units of packed red cells were transfused if the hemoglobin was less than 8 g/l and 5 units of random donor platelets if platelets were less than /l or less than /l and the patient was febrile. Cryopreservation and processing Peripheral stem cell products were either processed immediately after collection or stored overnight at 4ºC following the addition of 5 ml of autologous plasma. All PBSC packs were processed by centrifugation at 42 r.p.m. for 9 min and excess plasma was removed while maintaining the white blood cell count below /l. Concentrated stem cell packs were then diluted with an equal volume of a mixture composed of 2 parts M199 tissue culture media (Canadian Life Technologies, Toronto, Canada), 2 parts patient plasma, and 1 part dimethylsulfoxide (DMSO, final concentration 1%). Ratecontrolled freezing was performed using a liquid nitrogen rate-controlled freezer (Planar Biomed, Mississauga, Canada) beginning at a rate of 1ºC/min to 5ºC, quick freezing from 5ºC to 1ºC, followed by rate-controlled freezing at a rate of 1ºC/min to 6ºC and then 5ºC/min to 1ºC. The PBSC product was then stored in liquid nitrogen at 1ºC until the day of reinfusion. The concentration of protein in the final product was not determined routinely. Prior to reinfusion, PBSC packs were thawed in a waterbath at 4ºC followed by the addition of acid citrate dextrose (ACD; MedSep Corporation, Ottawa, Canada; the volume added was 2% of stored PBSC volume) and 5 units deoxyribonuclease (DNAse, Sigma, Oakville, Canada). The thawed product was then added to a unit of compatible allogeneic donor red blood cells and washed on a COBE 2991 machine to reduce the risks associated with infusing unwashed cells. Washing was performed similarly to previously reported methodology 22 by diluting PBSC packs with saline to a final volume of 6 ml followed by centrifugation at 3 r.p.m. for 1 min. Supernatant waste was removed at 1 ml/min using the COBE 2991 machine and the remaining product was agitated for 3 min. Saline was added again to yield a final volume of 6 ml and the washing process was repeated until the supernatant was clear or until 9 min had elapsed. Absolute CD34 counts Absolute numbers of CD34 + cells were enumerated immediately following harvest and an aliquot of the washed donor red cells containing the CD34 + cells for reinfusion was sent for flow cytometric analysis. All samples were analyzed using the three-color single platform variant of the ISHAGE guidelines and included the addition of the viability dye 7-amino actinomycin D (7-AAD) as previously described. 13,14 This method allows the measurement of viable CD34 + cells directly by a flow cytometer without the need for a hematology analyzer, and a direct assessment of total leukocyte and CD34 + cell viability in a single tube. The details of this method have been extensively described elsewhere 13,14 and only the basics are described here. StemKIT (Beckman-Coulter/Immunotech, Hialeah, FL, USA) which utilizes CD45FITC/CD34PE and Stem- COUNT fluorescent immunospheres was used for determining absolute CD34 + counts. One hundred l of diluted blood was added to 2 l of CD45FITC (clone J33)/CD34PE (clone 581) and 2 l 7-AAD (final concentration 1 g/ml). All samples were prepared in duplicate.

3 Tubes were then incubated for 15 min at 4ºC and the red blood cells lysed with ammonium chloride for 1 min. Samples were stored on ice until analysis (within 1 min). There were no wash steps used in the procedure. Immediately before analysis 1 l of StemCOUNT fluorospheres were added and tubes analyzed on a Beckman-Coulter XL- MCL flow cytometer. A total of 75 CD45 events were collected per tube (see Figure 1). CD34 PE FLOW COUNT A 1 3 E E G Lymphs CD45 FITC Clustered CD events C CD45 FITC 5. E2 CD E CD45 FITC H Flow count TIME D CD34 + low CD34 PE Viable CD34 + =88/m l FS D 6. Lymphs gated from 1. B FS 8. 7-AAD + (dead) AAD Figure 1 Flow cytometric analysis of a post-thaw, washed, apheresis sample. Histogram 1 shows all events with region A drawn to include all viable CD45 + leukocytes (ie excluding the dead 7-AAD+ events in region I, histogram 8). Histogram 2 is gated on viable CD45 + events (region A, histogram 1) and CD34 + events are identified in region B. In histogram 3, events from gates I, A and B are displayed and clustered events identified as CD in region C. In histogram 4, events sequentially gated from I, A, B and C are viable CD34 + cells, identified as region D, a generic lymph-blast region. Histogram 5 shows CD45 vs CD34. Histogram 6 is a discriminator check on lymphocytes from region F, histogram 1. Histogram 7 shows counting beads, selected by their bright fluorescence, which allows calculation of the volume counted. Region H is placed on singlet beads. The apheresis sample contained 88 viable CD34 + cells/ l. Engraftment data Peripheral blood cell counts were determined daily. Patient charts were reviewed to verify the time to platelet recovery, defined as the first of 3 consecutive days with a platelet count greater than /l and without platelet transfusion in the 48 h preceding the first of these 3 consecutive days. Time to neutrophil recovery was defined as the first of 3 days with a neutrophil count greater than /l. The transfusion record was reviewed for each patient and information regarding fevers, use of antibiotics, and number of days in hospital was recorded. Statistical analysis Statistical analysis was performed using PS statistical software. Mann Whitney rank sum test was used to assess differences in the number of CD34 + cells between patients who engrafted before or after the median. Fisher s exact test was performed to compare differences in the proportion of patients meeting specific thresholds of CD34 + cell numbers in patient groups engrafting before or after the median. The Kruskal Wallis one-way analysis of variance on ranks was followed by pairwise multiple comparisons according to Dunn s method to detect differences in rates of engraftment between the three groups of patients stratified on the basis of CD34 + cell numbers. Results Cohort patient characteristics The 36 patients studied had a median age of 41 (range 16 68). Diagnosis, total days admitted to hospital, use of antibiotics, and transfusion requirements are presented in Table 1. Cohort stem cell characteristics The median number of CD34 + cells harvested during leukapheresis was /kg (range ) with 99% mononuclear cell viability (range 99 1%). Following cryopreservation, stem cell products contained a median of viable CD34 + cells/kg (range ) with 76% overall mononuclear cell viability (range 46 91%). At the time of stem cell harvesting, the number of patients with fewer than 2 1 6, , or greater than CD34 + cells/kg was three, 2, and 13, respectively, while the number of patients in each stratification after thawing of cryopreserved stem cells was 17, 13 and six, respectively. Specifically, six of 13 patients (46%) who initially had greater than CD34 + cells/kg harvested had only /kg following cryopreservation and one patient had fewer than /kg remaining. In addition, 13 of 2 patients (65%) with CD34 + cells/kg collected had fewer than /kg prior to reinfusion. Although 33 of 36 patients (92%) exceeded the minimum threshold of CD34 + cells/kg at the time of collection, only 19 patients (53%) achieved this threshold at the time of reinfusion. 969

4 97 Table 1 Baseline characteristics Patient characteristics No. patients 36 Age, median (range) 41 (16 68) Diagnosis Multiple myeloma 16 Hodgkin s lymphoma 14 Non-Hodgkin s lymphoma 3 Germ cell tumour 3 Stem cell characteristics median (range) Absolute CD34 + /kg harvested 3.6 ( ) 1 6 /kg Absolute CD34 + /kg post thaw 2. (.7 11) 1 6 /kg Mononuclear cell viability harvested 99% (99 1) Mononuclear cell viability post thaw 76% (46 91) Engraftment characteristics median (range) Days to platelet /l 14 (9 42) Days to neutrophils /l 13 (9 22) Days of i.v. antibiotics 8 (2 19) Units of transfused red cells 2 ( 2) Units of transfused platelets 12 ( 9) Days admitted to hospital 15 (5 44) Table 2 Comparing early and slower platelet engraftment Platelet engraftment median (14 d) median (14 d) No. patients Days to engraftment, median (range) 12 (9 14) 18 (15 42) CD34 + cells/kg harvested, median (range) 5. ( ) 1 6 a 2.9 ( ) 1 6 CD34 + cells/kg post thaw, median (range) 3. (.8 11) x1 6 b 1.7 (.7 2.7) 1 6 No. patients with viable CD34 + cells/kg At time of harvest 2 (91%) 12 (86%) P NS Post thaw 15 (68%) 2 (14%) P.2 No. of patients with viable CD34 + cells/kg At time of harves 11 (5%) 2 (14%) P.4 Post thaw 6 (27%) ( %) P.6 a P.2; b P.3 (see Methods). Time to engraftment The median time to platelet and neutrophil engraftment was 14 days (range 9 42) and 13 days (range 9 22), respectively. When viable CD34 + cells enumerated after thawing were /kg (group I), /kg (group II), or /kg (group III), the median time to platelet engraftment was 17 (range 12 42), 12 (range 11 16) and 1 days (range 9 14), respectively (P.5 for pairwise comparison between groups I and II and between groups I and III; see Methods). The median time to neutrophil engraftment for groups I, II and III was 13 (range 1 22), 14 (range 1 19) and 12 days (range 9 13), respectively (P values not significant in pairwise comparison between groups). Patients with recovery of platelets after the median of 14 days were compared with those engrafting platelets on or before the median (see Table 2). For the 14 patients engrafting beyond the median, the number of viable CD34 + cells measured after thawing was /kg (range ) compared with /kg (range )forthe 22 patients engrafting on or before the median (P.3). Although 86% of patients who engrafted beyond 14 days exceeded the minimum threshold of CD34 + cells/kg at the time of harvesting, only 14% had this critical number after thawing whereas 68% of those who engrafted earlier still had cells/kg after thawing (P.2). Of those engrafting on or before 14 days, 27% had CD34 + cells/kg after thawing compared with % of patients who engrafted later (P.6). A similar comparison was made concerning neutrophil engraftment (see Table 3). The 15 patients engrafting after the median of 13 days were compared with the 21 patients engrafting on or before the median. The number of viable CD34 + cells after thawing was /kg (range ) for patients engrafting after the median compared with CD34 + cells/kg (range ) for patients engrafting on or before 13 days (P.12).

5 Table 3 Comparing early and delayed neutrophil engraftment Neutrophil engraftment 971 median (13 d) median (13 d) No. patients Days to engraftment, median (range) 12 (9 13) 15 (14 22) CD34 + cells/kg harvested, median (range) 5.2 ( ) 1 6 a 3.2 ( ) 1 6 CD34 + cells/kg post-thaw, median (range) 2.5 (.8 11) 1 6 b 1.8 (.7 3.2) 1 6 No. patients with viable CD34 + cells/kg At time of harvest 19 (9%) 13 (87%) P NS Post thaw 12 (57%) 5 (33%) P NS No. patients with viable CD34 + cells/kg At time of harvest 12 (57%) 1 (7%) P.4 Post thaw 6 (29%) (%) P.3 a P.14; b P.12 (see Methods). Of patients engrafting after the median, only 33% met the threshold of CD34 + cells/kg measured after thawing and none had /kg. Of those engrafting on or before the median, 57% had CD34 + cells/kg (P.19) and 29% had /kg after thawing (P.3). Discussion Single platform flow cytometry is a simple one-step method that allows determination of viable CD34 + cell numbers prior to reinfusion of stem cell products. This method allows accurate assessment of viability loss due to stem cell processing. Previous studies have assessed the effect of various storage conditions on loss of CD34 + viability 23,24 although, to our knowledge, no studies have reported a correlation with clinical engraftment data. A quantitative relationship between the number of viable thawed CD34 + cells and time to platelet engraftment but not neutrophil recovery is suggested by our data. Interestingly, loss of CD34 + cell viability causes some patients to fall below the threshold of /kg and these patients, on average, experienced slower hematopoietic reconstitution. The effect of reduced CD34 + cell viability seems more important for platelet engraftment than for neutrophils although the small numbers in our study limit direct comparisons. Moreover, all patients who have high yields of CD34 + cells, greater than viable CD34 + cells/kg, engrafted on or before the median. Despite adequate harvests of greater than CD34 + cells/kg in 92% of patients in this study, almost half of patients (46%) had fewer than viable CD34 + cells/kg reinfused. Although /kg has been identified as a minimum target of CD34 + cells at the time of harvest to ensure rapid engraftment, it is unclear what minimum threshold is required at the time of reinfusion. Although our data do not identify a lower threshold of viable CD34 + cells needed to ensure successful engraftment, a 4% reduction in CD34 + cell viability due to stem cell processing and cryopreservation observed in our patients suggests that fewer than /kg may be sufficient. Methods of handling stem cells which reduce losses associated with cryopreservation, thawing and washing may lower the threshold of CD34 + cells required at the time of harvesting to ensure rapid engraftment. Identifying patients at risk of delays in hematopoietic reconstitution may alter some aspects of patient care following PBSC transplantation. Cytokines could prove beneficial in this population. Faster recovery of neutrophils was reported for subgroups of patients that received cytokines following stem cell infusion in two retrospective studies. 11,16 Slower platelet recovery, however, is often more affected by reduced CD34 + cell numbers and some studies have suggested post-infusion cytokines may further delay platelet engraftment. 16,25 Prophylactic antibiotics might be appropriate if increased rates of infection were detected in this group and more aggressive nutritional interventions such as total parenteral nutrition may be considered earlier in these patients. CD34 + cell numbers probably combine with other factors in determining time to hematopoietic reconstitution. Different myeloablative conditioning regimens have variable stem cell toxicity and previous chemotherapy regimens can affect the efficacy of stem cell harvesting and regrowth of the autograft. Infection and antibiotics may also suppress engraftment in some patients. Larger studies looking at more homogenous patient populations may better define the contribution of viable CD34 + cell dose and time to engraftment. Assessing CD34 + cell viability at different stages of processing allows for accurate quality control. Alterations in the cryopreservation or collection technique can be measured in their effect on CD34 + cell viability. Also, products that are cryopreserved for lengthy periods, such as cord blood samples, can be assessed for quality using the single platform flow cytometric methodology. 26 In summary, cryopreservation and processing of autologous stem cell collections significantly reduces the number of viable CD34 + cells available for reinfusion. The number of viable CD34 + cells reinfused is associated with time to hematopoietic engraftment in autologous transplantation, particularly recovery of platelet production. Loss of CD34 + cell viability causes many patients to fall below 2 1 6

6 972 CD34 + cells/kg. Measuring CD34 + cell viability may be useful in quality control assessments of stem cell processing and further studies may reveal ways of identifying patients at risk of delayed engraftment. Acknowledgements We wish to recognize the statistical support of Mr Larry Stitt and Ms Leslie Gray-Statchuk and the administrative support of Ms Elizabeth Wood. References 1 To LB, Haylock DN, Simmons PJ, Juttner CA. The biology and clinical uses of blood stem cells. Blood 1997; 89: Bender JG, To LB, Williams S, Schwartzberg LS. Defining a therapeutic dose of peripheral blood stem cells. J Hematother 1992; 1: Katz F, Tindle RW, Sutherland DR, Greaves MF. Identification of a membrane glycoprotein associated with hematopoietic progenitor cells. Leuk Res 1985; 9: Sutherland DR, Keating A. The CD34 antigen: structure, biology and potential clinical applications. J Hematother 1992; 1: Berenson RJ, Andrewes RG, Bensinger WI et al. Antigen CD34-positive marrow cells engraft lethally irradiated baboons. J Clin Invest 1988; 81: Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991; 77: Sheridan WP, Begley CG, Juttner CA et al. Effect of peripheral blood progenitor cells mobilized by filgastrim (G-CSF) on platelet recovery after high dose chemotherapy. Lancet 1992; 339: Elias AD, Ayash L, Anderson KC et al. Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte macrophage colony stimulating factor for hematologic support after high dose intensification for breast cancer. Blood 1992; 79: Schwartzberg LS, Birch R, Hazelton B et al. Peripheral blood stem cell mobilization by chemotherapy with and without recombinant human granulocyte colony-stimulating factor. J Hematother 1992; 1: Reiffers L, Leverger G, Marit G et al. Hematopoietic reconstitution after autologous blood stem cell transplantation. In: Gale RP, Champlin RE (eds). : Current Controversies. Proceedings of Sandoz-UCLA Symposium. Liss: New York, 1988, p Weaver CH, Hazelton B, Birch R et al. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995; 86: Ketterer N, Salles G, Raba M et al. High CD34 cell counts decrease hematologic toxicity of autologous peripheral blood progenitor cell transplantation. Blood 1998; 91: Keeney M, Chin-Yee I, Weir K et al. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. Cytometry 1998; 34: Gratama JW, Keeney M, Sutherland DR. Enumeration of CD34+ hematopoietic stem and progenitor cells. Curr Prot Cytometry 1999; 6: Sutherland DR, Anderson L, Keeney M et al. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. J Hematother 1996; 5: Bensinger W, Appelbaum F, Rowley S et al. Factors that influence collection and engraftment of autologous peripheralblood stem cells. J Clin Oncol 1995; 13: Tricot G, Jagannath S, Vesole D et al. Peripheral blood stem cell transplants for multiple myeloma: identification of favorable variables for rapid engraftment in 225 patients. Blood 1995; 85: Serke S, Johnsen HE. A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts. Bone Marrow Transplant 21; 27: Barnett D, Janossy G, Lubenko A et al. Guideline for the flow cytometric enumeration of CD34+ haematopoietic stem cells. Clin Lab Haematol 1999; 21: Loken MP. Peripheral blood stem cell quantitation. In: Owens MA, Loken MP (eds). Flow Cytometry Principles for Clinical Laboratory Practice. Wiley-Liss: New York, 1995, pp Yang H, Acker JP, Hannon J et al. Damage and protection of UC blood cells during cryopreservation. Cytotherapy 21; 3: Beaujean F, Hartmann O, Kuentz M et al. A simple, efficient washing procedure for cryopreserved human hematopoietic stem cells prior to reinfusion. Bone Marrow Transplant 1991; 8: Ruiz-Arguelles GJ, Ruiz-Arguelles A, Perez-Romano B et al. Filgastrim-mobilized peripheral-blood stem cells can be stored at 4 degrees and used in autografts to rescue high-dose chemotherapy. Am J Hematol 1995; 48: 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: De Magalhaes-Silverman M, Donnenberg AD, Lister J et al. Factors influencing mobilization and engraftment in patients with metastatic breast cancer undergoing PBSC transplantation. J Hematother 1999; 8: Brocklebank AM, Sparrow RL. Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy. Cytometry 21; 46: