Increased apoptosis in cryopreserved autologous hematopoietic progenitor cells collected by apheresis and delayed neutrophil recovery after transplantation: a nested case-control study

Cytotherapy, 2012; 14: 205–214

Increased apoptosis in cryopreserved autologous hematopoietic progenitor cells collected by apheresis and delayed neutrophil recovery after transplantation: a nested case-control study

LUKE WU1,2, AYMAN AL-HEJAZI3,5, LIONEL FILION2, ROBERT BEN2,4, MICHAEL HALPENNY6, LIN YANG6, ANTONIO GIULIVI6 & DAVID S. ALLAN1,2,3,5 1Regenerative

Medicine Program, Ottawa Hospital Research Program, Ottawa, Canada, Departments of and Immunology, 3Medicine and 4Chemistry, University of Ottawa, Ottawa, Canada, 5Blood and Marrow Transplant Program, The Ottawa Hospital, Ottawa, Canada, and 6Stem Cell Processing Laboratory, Canadian Blood Services, Ottawa, Canada 2Biochemistry, Microbiology

Abstract Background aims. Delayed neutrophil recovery following autologous hematopoietic stem cell transplantation (aHSCT) increases transplant-related morbidity. Apoptosis induced by cryopreservation and thawing of hematopoietic progenitor cells collected by apheresis (HPC-A) was investigated in this nested case-control study as a factor associated with delayed neutrophil recovery following aHSCT. Methods. Among patients with lymphoma who underwent aHSCT between 2000 and 2007 (n  326), 13 cases of primary delayed neutrophil recovery and 22 age- and sex-matched controls were identified. Apoptosis and viability were measured using multiparameter flow cytometry, and colony-forming capacity was determined using semi-solid methylcellulose assays. Results. HPC-A grafts from cases and controls had similar percentages of viable mononuclear cells (MNC) and CD34 progenitor cells, as determined by standard 7AAD dye exclusion methods measured before and after cryopreservation. Patients with delayed neutrophil recovery received increased numbers of apoptotic MNC (P  0.02) but similar numbers of apoptotic CD34 cells per kilogram measured after thawing. Apoptosis was more pronounced in MNC compared with CD34 cells after thawing, and apoptosis was negligible in freshly collected HPC-A products. Patients with delayed neutrophil recovery had fewer total colony-forming unites (CFU) and CFU-granulocyte–macrophages (GM) per 105 viable post-thaw MNC compared with controls (P  0.05). Conclusions. Increased numbers of apoptotic MNC in thawed HPC-A products are associated with delayed neutrophil recovery after aHSCT. Studies that address factors contributing to increased apoptosis are needed, and measuring apoptosis in thawed HPC-A may have a role in the assessment of graft adequacy. Key Words: apoptosis, autologous, cryopreserved, engraftment, neutrophil, peripheral blood stem cells

Introduction Optimal outcomes for patients undergoing highdose treatment and autologous hematopoietic stem cell transplantation (aHSCT) depend on the timely recovery of blood counts following reinfusion of hematopoietic progenitor cells collected by apheresis (HPC-A). The content of CD34 cells in HPC-A collections prior to cyropreservation is strongly associated with timely engraftment (1,2). Several studies have confirmed that patients reinfused with HPC-A containing  2.0  106 CD34 cells/kg are at increased risk of developing delayed neutrophil and platelet recovery, accompanied by a greater risk of bleeding and infectious complications,

prolonged length of hospital stay and increased mortality (1,3,4). Cellular processing and cryopreservation of autologous HPC-A products is known to reduce the number of viable CD34 available for reinfusion and can contribute to delayed engraftment (5,6). Despite infusing numbers of CD34 cells and total mononuclear cells (MNC) that exceed the minimum threshold associated with timely engraftment, some patients still experience delayed hematopoietic recovery (7,8). Although cryopreservation and thawing of stem cell grafts can reduce the number of live, or viable, MNC and CD34 cells (9,10), measured using simple dye exclusion methods, many of these viable

Correspondence: David Allan, MD, Blood and Marrow Transplant Program, Division of Hematology, The Ottawa Hospital Research Institute, 501 Smyth Road, Box 704, K1H 8L6, Ottawa, ON, Canada. E-mail: [email protected] (Received 20 November 2010; accepted 22 July 2011) ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare DOI: 10.3109/14653249.2011.610302

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cells may be undergoing apoptosis and have limited functional potential after reinfusion. Important levels of apoptosis have been reported within the populations of viable MNC and CD34 cells (11,12). In one study, apoptotic CD34 cells were identified by surface expression of Annexin V (AnnV) and were isolated from umbilical cord blood. The CD34 AnnV cells were significantly impaired in their capacity to engraft non-obese diabetic severe combined immunodeficient (NOD-SCID) mice compared with non-apoptotic CD34 AnnV– cells (11). Whether increased levels of apoptotic cells in HPC-A grafts influences hematopoietic reconstitution in patients having a sufficient number of viable CD34 cells prior to cryopreservation of HPC-A remains unknown. In this study, we investigated the association of apoptosis in thawed HPC-A grafts in cases of delayed neutrophil engraftment following aHSCT for non-Hodgkin’s lymphoma using a casecontrol design.

Methods Patient selection Patients who underwent autologous HPC-A transplantation for non-Hodgkin’s lymphoma at the Ottawa Hospital between January 2000 and December 2007, and who provided consent for the use of medical information for research purposes in accordance with our institutional research ethics board, were included in the study (n  326). Study cases were defined as patients with a persistent neutrophil count  1.0  109/L before day  30. Primary delayed neutrophil recovery was chosen as the main selection criterion, to avoid selection of patients who had initial neutrophil recovery while receiving granulocyte–colony-stimulating factor (G-CSF) and had a second period of neutropenia after G-CSF was stopped. In addition, persistent neutropenia until day  30 was considered to be a more robust measure of graft quality compared with days to neutrophil engraftment ( 0.5  109/L), as many factors can contribute to delays in the initial recovery of neutrophils to a level of 0.5  109/L. Fourteen cases were identified and an aliquot of HPC-A was stored and available for further analysis in 13 (93%). Controls were matched for age, gender, status of disease at transplant, number of CD34 cells/kg in the HPC-A graft measured before cryopreservation, and year of transplant. We identified a total of 22 patients for the control group within the same cohort of patients as the cases. All cases and controls were  18 years of age. All transplanted patients received G-CSF starting on day  7 after transplant until peripheral

blood neutrophils were  0.5  109/L for at least 2 consecutive days. Patients did not receive prophylactic antibiotics. Cases and controls received other similar supportive care measures and were all transplanted on the same ward at our hospital. Collection, storage and processing of peripheral blood HPC-A grafts were collected following multi-agent salvage chemotherapy or following single-agent cyclophosphamide and daily subcutaneous injections of G-CSF (5 μg/kg). Leukapheresis was performed using a standard technique with blood-processing volumes of 10–20 L, determined based on the peripheral blood CD34 level to allow the collection of  2.5  106 CD34 cells/kg. Leukapheresis products were volume reduced and cryopreserved in autologous plasma and an equal volume of freezing solution in a 500-mL Cryocyte™ freezing container (Baxter, Deerfield, IL, USA), with a final concentration of 5% dimethylsulfoxide (DMSO), 12% Plasma-Lyte A (Baxter), 4% human serum albumin and 2.2% (w/v) pentastarch (Bristol-Myers Squibb, Montreal, Canada). The Cryocyte container was placed directly into liquid nitrogen vapor overnight (i.e. using the dump freeze method), as described previously (13,14), and stored until the day of reinfusion. Cryopreserved aliquots separate from the HPC-A grafts were also stored in the vapor phase of liquid nitrogen in Nalgene 2-mL cryogenic vials (Thermo Fisher Scientific, Rochester, NY, USA). HPC-A aliquots were retrieved and thawed in a 37°C water bath and analyzed for this study, in accordance with our standard approach for thawing grafts prior to reinfusion in our clinical transplant program. Once thawed, the samples were diluted 6-fold, drop-wise over 5 min, with RPMI solution pre-warmed to 37°C containing 5.0  104 U/mL DNase I (Sigma-Aldrich, Oakvile, Canada, Germany) and 5 mM MgCl2 (Sigma-Aldrich). Subsequently, samples were centrifuged (Allegra X-15R centrifuge, Beckman Coulter) for 10 min at 250 g at 4°C and the cells resuspended in 2 mL RPMI media for further analysis by flow cytometry. Cell concentrations were determined using a hemocytometer. Colony-forming unit assays Clonogenic assays were performed on thawed aliquots using methylcellulose media (Methocult GF H4434; StemCell Technologies, Vancouver, Canada) in accordance with the manufacturer’s instructions. Briefly, cells were suspended in 1 mL Iscove’s modified Dulbecco’s media (IMDM) media with 2% fetal bovine serum (FBS; StemCell Technologies) at a concentration of 1  106 cells/mL. Cell

Apoptosis in thawed autologous HPC-A grafts and engraftment concentrations were determined based on the number of viable MNC after thawing, using trypan blue and a hemocytometer. A volume of 0.3 mL of the cell suspension was transferred into 2.7 mL of the methylcellulose media to obtain a final concentration of 1  105 cells/mL; 1.0 mL of the methylcellulose cell suspension was plated in duplicate into 35-mm diameter Petri dishes and incubated at 37°C and 5% CO2 for 14 days. Colony-forming units (CFU) were enumerated by morphology using an inverted microscope to discriminate erythroid (CFU-E), erythroblast-forming units (BFU-E), granulocyte– macrophage (CFU-GM) and granulocyte–erythroid– macrophage–monocyte (CFU-GEMM) colonies (more than 50 cells/colony). Flow cytometry Cells were analyzed by 5-color flow cytometry to measure viability and apoptosis in the specific cell populations shown in Figure 1. All fluorescent markers were incubated together in one tube. Following thawing of cryopreserved samples, cells were washed in five volumes of RPMI and centrifuged at 250 g for 10 min to remove DMSO. Cells were resuspended in 2 mL RPMI. To identify aldehyde dehydrogenase-expressing progenitor cells, 4  106 cells were suspended in 1 mL Aldecount assay buffer (StemCell Technologies). A volume of 0.5 mL of the cell suspension was diluted with 1.5 mL activated Biodipy–aminoacetaldehyde (BAA) (StemCell Technologies) into a tube labeled tube 2. A volume of 500 μL of this original sample was added immediately to 10 μL of diethylaminobenzaldehyde (DEAB; StemCell Technologies) in a separate tube, labeled tube 1, to inhibit (ALDH) activity and act as a negative control. Both tubes 1 and 2 were incubated at 37°C for 30 min, centrifuged at 250 g for 10 min and resuspended in 100 μL AnnV binding buffer (BD Pharmigen, Mississauga, Canada). AntiCD34–phycoerythrin-cyanine 7 (PCy7) (clone 581; Beckman Coulter, Mississauga, Canada), AnnV– phycoerythrin (PE) (BD Pharmigen), 7-actinomycin D (7AAD) (BD Pharmigen) and CD45–phycoerythrin-Texas-red (ECD–trademark) (clone J.33; Beckman Coulter) were added in combination into both tubes 1 and 2, and incubated at room temperature for 20 min. Both tubes were diluted with AnnV binding buffer and analyzed with a FC500 flow cytometer (Beckman Coulter). Cell number enumeration was performed with the use of CountBright™ counting beads (InVitrogen Corp., Toronto, Canada). Beads were added to the sample just prior to data acquisition, as recommended by the manufacturer. Hematopoietic CD34 progenitor cells were identified and enumerated using a single-platform method in accor-

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dance with guidelines published by the International Society for Hematotherapy and Graft Engineering (ISHAGE) (15), although the initial gating strategy employed side scatter plotted on a logarithm scale to enhance discrimination of CD45 events (Figure 1). Data analysis was performed using the Summit software (DAKO, Carpinteria, Mississauga, Canada, USA). The CD34 CD45low ALDHhigh progenitor region was set by the fluorescence of BAA in CD34 progenitors from the control tube where BAA catalysis was inhibited. Statistical analysis Mean values are reported  1 standard deviation and means were compared using the Mann–Whitney test using a 95% confidence level. Proportions were compared using Fisher’s exact test Results HPC-A grafts in study cases with delayed neutrophil recovery have similar levels of post-thaw viable MNC and CD34  cells compared with matched controls Study cases with delayed neutrophil recovery (n  13) were similar to controls (n  22) with regard to age, gender, diagnosis and the number of MNC and CD34 cells collected in HPC-A grafts (Table I). Study cases experienced longer delays to initial engraftment of neutrophils above 0.5  109/L (defined as the first of 3 consecutive days with neutrophils  0.5  109/L), had significantly longer delays to platelet recovery (first of 3 days with platelets  20  109/L and without platelet transfusion in the preceding 48 h) and longer hospital stays following transplantation, in comparison with controls. The number of patients exceeding the minimum threshold of 2.0  106 viable (i.e. live) CD34 cells/ kg at the time of collection was similar amongst study cases (11/13, 85%) and controls (20/22, 91%), and no significant difference was observed between study cases and controls with regard to the number of patients exceeding the optimal threshold of 5.0  106 viable CD34 cells/kg at the time of collection. Study cases with delayed neutrophil recovery and controls had similar percentages of live 7AAD– MNC and live CD34 cells following processing and thawing (Figure 2), as estimated using the standard exclusion dye 7AAD on thawed aliquots of HPC-A stored in liquid nitrogen. In general, HPC-A aliquots maintained high levels of cell viability in both the MNC population (91.0  1.3% for cases versus 91.6  0.7% for controls, P  0.7) and the CD34 cell population (84.6  2.7% for cases versus 85.5  1.4% for controls, P  0.8) after thawing.

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Figure 1. The gating strategy used to identify the various cell populations using flow cytometry. Cell concentrations were enumerated with fluorescent counting beads as identified in region 8 (A and B). The CD34 population was identified by plotting all events and gating for the CD45 population, defined as R1 (depicted in C). R1 was gated onto D and analyzed for CD34 events as defined by R2. R2 was gated onto E and analyzed for CD34 CD45low events as defined by R3. R3 was gated onto F and analyzed for FSClow and FSChigh events as defined by R5 and R4, respectively. CD34 progenitors are defined as the CD34 CD45low population identified in region 4 of F. To identify the CD34 progenitor population that was ALDHhigh, a portion of each original sample was incubated with DEAB in a separate tube to inhibit ALDH catalysis of the fluorescent marker BAA, which was identified in region 6 of G. The CD34 CD45low ALDHhigh progenitor region in the original tube were identified in region 7 of H. Both the original and DEAB control tubes were stained with anti-CD34–PCy7, AnnV–PE, 7AAD, CD45–ECD, activated BAA and fluorescent counting beads in combination.

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Table I. Characteristics of study cases with delayed neutrophil engraftment and matched controls. Characteristic n Age, mean years  SD Gender, male/female Hodgkin’s lymphoma/non-Hodgkin’s lymphoma CD34  cells  106/kg collected in HPC-A MNC  108/kg collected in HPC-A Neutrophil (%) collected in HPC-A Median number HPC-A collections (range) Days to ANC  0.5 (mean  SD) Days to PLT  20 (mean  SD) LOS, mean days  SD ICU admission, n (%) Non-relapse death before day  100, n (%)

Study cases

Controls

13 48  14 7/6 2/11 3.7  2.0 (7.9  3.5)  108 33.3  19.8 1 (1–3) 17.6  6.6 30.1  15.5 31.2  11.4 3 (23) 1 (8)

22 47  15 12/10 3/19 4.4  1.6 (8.1  4.4)  108 35.8  15.8 2 (1–3) 11.9  3.2 13.9  3.4 22.6  5.9 2 (9) 1 (5)

P

0.8 1.0 1.0 0.26 0.87 0.72 0.002  0.0001 0.006 0.3 1.0

ANC, absolute neutrophil count; PLT, platelets; LOS, length of hospital stay; ICU, intensive care unit.

In addition, the absolute number of viable MNC and CD34 cells available for reinfusion based on the patients’ weights was used to estimate the actual dose of cells for each patient and were similar for cases and controls (Figure 2). Further, the proportion of cases with delayed neutrophil recovery with a calculated dose of more than 2.0  106 live CD34 cells/kg at the time of reinfusion was similar to the controls (10/13, 77% versus 17/22, 77%), and likewise the proportion of patients receiving more than 5.0  106 live CD34 cells/kg at the time of reinfusion was the same in cases and controls (2/13 cases, 17% versus 6/22 controls, 27%). Study cases with delayed neutrophil recovery received more apoptotic MNC Although similarly high levels of viable MNC and viable CD34 cells, determined by measuring 7AAD exclusion alone, were maintained during cryopreservation and thawing in both cases and controls, we observed significant levels of apoptosis measured using multicolor flow cytometry, ranging from 16% to 88% of MNC and 3% to 30% of the CD34 fraction in the entire cohort of study cases and controls. In contrast, fresh HPC-A grafts sampled immediately following collection and processing but prior to freezing had low levels of apoptotic MNC (6.6  0.8%, n  3) and CD34 cells (1.0  0.2%, n  3) (Figure 3). Furthermore, in both fresh and cryopreserved samples, the CD34 ALDHhigh population identified using flow cytometry was found to be entirely viable and non-apoptotic ( 99.5% in all samples). In contrast to our findings with regard to the assessment of live versus dead cells using the exclusion dye 7AAD, aliquots of HPC-A from study cases with delayed neutrophil recovery had significantly more AnnV  MNC/kg compared with con-

trols (1.6  2.1 versus 1.0  1.8  108/kg, P  0.02) (Figure 2). The absolute number of non-apoptotic 7AAD- AnnV– cells, however, was similar between the cases and controls (1.6  2.4 versus 1.6  2.1  108/ kg). In contrast, the number of apoptotic AnnV CD34 cells was not different between cases and controls (2.3  0.4 versus 1.9  0.3  105/kg) and the number of non-apoptotic 7AAD– AnnV– CD34 cells was similar between cases and controls but markedly reduced compared with live 7AAD– measurements and pre-cryopreservation determinations (1.0  0.3 versus 1.4  0.2  106/kg for cases and controls, respectively, P  0.2) (Figure 4). Interestingly, there was a trend toward a lower dose of CD34 ALDHhigh cells/kg in study cases compared with controls (1.1  0.2  106 versus 1.7  0.2  106 cells/kg, P  0.06). We observed a trend toward a reduced proportion of study cases compared with matched controls that received more than 2.0  106 7AAD– AnnV– CD34 cells/kg (6/13 cases, 46% versus 17/22 controls, 77%, P  0.07) (Table II) but no significant difference between cases and controls with regard to the proportion of patients receiving more than 5.0  106 7AAD– AnnV– CD34 cells/kg, based on assessment of thawed HPC-A aliquots.

CFU levels are reduced in HPC-A grafts of patients with delayed neutrophil recovery CFU were enumerated after plating thawed MNC in methylcellulose assays. HPC-A grafts from study cases with delayed neutrophil recovery had reduced total CFU plating efficiencies compared with controls (20.4  3.4 colonies/2  105 cells versus 47.4  8.4/2  105 cells, P  0.022) and a reduced dose of total CFU/kg (1.2  0.2  105 colonies/kg versus 2.4  0.5  105 colonies/kg, P  0.05; Figure 5).

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Figure 3. Changes in cell viability and apoptosis after thawing cryopreserved HPC-A. Typical flow cytometry analysis of MNC (gated from R1 in Figure 1) (A), CD34 progenitors (gated from R4 in Figure 1) (B) and CD34 CD45low ALDHhigh progenitors (gated from R7 in Figure 1) (C) in freshly collected HPC-A and of MNC (D), CD34 progenitors (E) and CD34  CD45low ALDHhigh progenitors (F) after thawing cryopreserved HPC-A. The bottom quadrants of each plot represent viable non-necrotic cells that exclude 7AAD, while the quadrants on the right side of each plot represent apoptotic cells that bind AnnV–PE.

CFU-GM plating efficiencies were also reduced in cases of delayed engraftment (P  0.02; Figure 5) and there was a trend toward a lower dose of

CFU-GM/kg in study cases compared with controls (3.1  0.7  104 CFU-GM/kg versus 6.3  1.3  104 CFU-GM/kg, P  0.06).

Apoptosis in thawed autologous HPC-A grafts and engraftment

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Figure 2. MNC and delayed engraftment. Comparison between study cases with delayed neutrophil engraftment and matched controls regarding the absolute number of MNC infused/kg that were 7AAD– (A), 7AAD– AnnV (B) and 7AAD– AnnV– (C). ∗P  0.02.

Discussion The results of our case-control study suggest that reinfusion of HPC-A grafts containing increased levels of apoptotic cells is associated with prolonged primary delay of neutrophil recovery after aHSCT. Interestingly, study cases with delayed neutrophil recovery received equal numbers of ‘live’, or 7AAD–, MNC and CD34 cells, as measured after thawing cryopreserved HPC-A products using the dye exclusion method alone, suggesting that simply enumerating ‘live’ versus ‘dead’ cells may be insufficient in assessing the engraftment capacity of autologous HPC-A. Using a cell-surface marker for early apoptosis with multiparameter flow cytometry, we identified a greater dosage of apoptotic AnnV  MNC in HPC-A grafts of study cases compared with controls. Importantly, however, the number of

Figure 4. CD34 cells and delayed engraftment. Comparison between study cases with delayed neutrophil engraftment and matched controls regarding the absolute number of CD34 cells/kg that were 7AAD– (A), 7AAD– AnnV (B) and 7AAD– AnnV– (C). None of the populations of CD34 cells was significantly different between cases of delayed neutrophil engraftment and controls.

7AAD– AnnV– cells reinfused was similar between cases and controls. Taken together, our data suggest that the number of apoptotic cells reinfused may contribute to delayed engraftment or be a marker of reduced engraftment potential after aHSCT. The reduced number of CFU observed using cells from HPC-A of cases compared with controls was consistent with the observation that grafts containing increased numbers of apoptotic MNC had reduced engraftment potential. Previous studies have addressed the role of apoptotic cells in rates of hematopoietic engraftment, best illustrated by the marked reduction in engraftment capacity of selected human AnnV  umbilical cord blood MNC reinfused into NOD/SCID mice compared with more rapid engraftment of AnnV– cells (11). It remains unclear, however, what impact

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Table II. Proportion of study cases and controls reaching thresholds of viable CD34  cells at collection, after thawing, and after considering only viable and non-apoptotic cells after thawing. Graft characteristic  2.0  106

Study cases

Controls

P

11 (85) 10 (77) 6 (46)

20 (91) 17 (77) 17 (77)

0.6 1.0 0.07

2 (17) 2 (17) 2 (17)

8 (36) 6 (27) 3 (14)

0.3 0.7 1.0

CD34

viable cells/kg, n (%) At collection Post-thaw Post-thaw, non-apoptotic  5.0  106 viable CD34 cells/kg, n (%) At collection Post-thaw Post-thaw, non-apoptotic

increased numbers of apoptotic cells may have on the engrafting potential of similar numbers of CD34 cells in this model, although our data support a model whereby apoptotic cells may impede timely engraftment through unknown mechanisms. Apoptotic MNC may lose their accessory function to act as chaperones during the early phases of hematopoietic engraftment, or apoptotic cells may release factors that inhibit the homing capacity of hematopoietic progenitors. More study is required to gain mechanistic insight. The impact of cryopreservation on apoptosis in HPC-A grafts has also been investigated previously and our results are consistent with the observation that cryopreservation-associated apoptosis occurs to a larger extent in the MNC population compared with the CD34 fraction. Abrahamsen et al. (12) reported that mean levels of apoptosis in viable, or live, MNC recovered from cryopreserved HPC-A grafts was 32%, but only 7% in viable CD34 cells. Interestingly, more significant levels of apoptosis have been reported in HPC-A cryopreserved in 10% DMSO compared with 5% DMSO, suggesting that DMSO may contribute to the induction of apoptosis (10). Additional markers of apoptosis have been studied in HPC-A grafts. In a series of studies addressing the utility of the cell surface marker Syto16, significant levels of early apoptosis were detected in CD34 cells from HPC-A grafts frozen in 10% DMSO (16,17). The use of Syto16 enabled the establishment of lower thresholds of viable non-apoptotic cells associated with prompt hematopoietic engraftment after aHSCT (18). Of particular interest, very high yields of viable non-apoptotic cells, defined as Syto16high 7AAD– events, were observed in whole blood stem cell collections stored at 4°C for 72 h compared with frozen–thawed HPC-A collections, suggesting that freezing and thawing induced high levels of early apoptosis in hematopoietic stem and progenitor cells.

Our results also suggest a major effect of cryopreservation, as apoptosis in both the MNC and CD34 populations was minimal in freshly collected HPC-A samples immediately after leukapheresis and processing, whereas apoptosis was more extensive after thawing cryopreserved HPC-A. Other studies have also noted minimal apoptosis ( 5%) and viability loss ( 5%) in fresh HPC-A grafts (12). It appears likely that cryopreservation conditions and thawing procedures contribute significantly to the induction of apoptosis. In one study, cryopreservation of bone marrow MNC was shown to induce artificial cleavage of apoptosisrelated proteins, resulting in increased apoptosis of MNC (19). Although our data provide insight regarding the impact of apoptotic cells in autologous HPC-A grafts and their effect on delayed neutrophil engraftment following transplantation, different methods of freezing and many variables of the cryopreservation method may influence levels of cellular apoptosis. In addition to CD34 cell viability and the role of apoptotic cells in HPC-A grafts, other factors have been identified that may affect engraftment. A study by Oran et al. (20) has identified the extent of exposure to prior alkylator therapy and female gender as two factors that affect neutrophil engraftment. From the same study, preserved renal function and absence of neutropenic fever after transplantation were found to accelerate platelet engraftment specifically (20). In another study, Ergene et al. (21) found that a more prolonged interval between diagnosis and transplantation along with the Carmustine (BiCNU), etoposide, cytarabine arabinsoide and melphalan (BEAM) regimen of conditioning chemotherapy was associated with delayed neutrophil engraftment in univariate but not multivariate analysis. Both total colony and CFU-GM forming capacities have been correlated with hematopoietic stem cell (HSC) engraftment kinetics (22–24), although the methodology continues to have significant interlaboratory variability and the testing is time consuming and laborious. It is possible that some of these factors contributed to delayed neutrophil recovery in our study. Importantly, however, the cases and controls appeared well balanced in terms of disease severity and other clinical parameters, and our study was focused on patients undergoing transplantation for lymphoma, thereby providing a relatively homogeneous patient population. Further studies of delayed engraftment assessing the full spectrum of factors in a prospective manner may clarify more precisely the role of apoptotic cells in HPC-A grafts in relation to other factors. In conclusion, our study provides new insight regarding apoptosis as an important factor contributing

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Figure 5. CFU and delayed engraftment. The plating efficiency of total CFU (A) and CFU-GM (B) in HPC-A of study cases with delayed neutrophil engraftment and controls is presented along with the absolute number of total CFU (C) and CFU-GM (D)/kg recipient weight. ∗P 0.05; ∗∗P  0.06.

to delayed neutrophil recovery following aHSCT in patients with lymphoma. We did not address rates of platelet recovery specifically, as cases were identified based on criteria for neutrophil engraftment, and separate studies on platelet engraftment may be warranted. Further studies addressing thresholds of apoptosis that are associated with delayed engraftment and the role of apoptosis in combination with other factors may allow the development of more refined algorithms for the rapid assessment of graft adequacy using multiparameter flow cytometry in HPC-A transplantation. Future studies aimed at reducing cryopreservation-associated apoptosis are warranted and could be applicable to umbilical cord blood banking as well as HPC-A storage for autologous transplantation.

Acknowledgments This work was supported in part by an award from MITACS Accelerate (Canada). DSA is an Adjunct Scientist with Canadian Blood Services and a recipient of a Research Award from the Department of Medicine at the University of Ottawa and a New Investigator Award from Canadian Institutes of Health Research. RB holds a Canada Research Chair in Medicinal Chemistry. We are grateful for the patients and their families who entrusted their care to our team of physicians, nurses, pharmacists and health care providers at The Ottawa Hospital. We wish to gratefully acknowledge the assistance of Sheryl Mcdiarmid for expert assistance with the BMT database at The Ottawa Hospital, supported by The Ottawa Hospital Foundation.

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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Ketterer N, Salles G, Raba M, Espinouse D, Sonet A, Tremisi P, et al. High CD34() cell counts decrease hematologic toxicity of autologous peripheral blood progenitor cell transplantation. Blood. 1998;91:3148–55. 2. Weaver CH, Hazelton B, Birch R, Palmer P, Allen C, Schwartzberg L, 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;86:3961–9. 3. To LB, Haylock DN, Simmons PJ, Juttner CA. The biology and clinical uses of blood stem cells. Blood. 1997;89:2233–58. 4. Schiller G, Vescio R, Freytes C, Spitzer G, Sahebi F, Lee M, et al. Transplantation of CD34 peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood. 1995;86:390–7. 5. Allan DS, Keeney M, Howson-Jan K, Popma J, Weir K, Bhatia M, et al. Number of viable CD34() cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 2002;29:967–72. 6. Lee S, Kim S, Kim H, Baek EJ, Jin H, Kim J, et al. Post-thaw viable CD34() cell count is a valuable predictor of haematopoietic stem cell engraftment in autologous peripheral blood stem cell transplantation. Vox Sang. 2008;94:146–52. 7. Trebeden-Negre H, Rosenzwajg M, Tanguy ML, Lefrere F, Azar N, Heshmati F, et al. Delayed recovery after autologous peripheral hematopoietic cell transplantation: potential effect of a high number of total nucleated cells in the graft. Transfusion. 2010;50:2649–59. 8. Mineishi S, Saito T, Kanda Y, Tanosaki R, Tobinai K, Takaue Y. Delayed recovery of neutrophil counts after peripheral stem cell transplantation which improved with administration of a minimal dose of G-CSF: a case report. Jpn J Clin Oncol. 2001;31:43–5. 9. Reich-Slotky R, Colovai AI, Semidei-Pomales M, Patel N, Cairo M, Jhang J, et al. Determining post-thaw CD34 cell dose of cryopreserved haematopoietic progenitor cells demonstrates high recovery and confirms their integrity. Vox Sang. 2008;94:351–7. 10. Abrahamsen JF, Bakken AM, Bruserud O. Cryopreserving human peripheral blood progenitor cells with 5-percent rather than 10-percent DMSO results in less apoptosis and necrosis in CD34 cells. Transfusion. 2002;42:1573–80. 11. Shim JS, Cho B, Kim M, Park GS, Shin JC, Hwang HK, et al. Early apoptosis in CD34 cells as a potential heterogeneity in quality of cryopreserved umbilical cord blood. Br J Haematol. 2006;135:210–3. 12. Abrahamsen JF, Bakken AM, Bruserud O, Gjertsen BT. Flow cytometric measurement of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases. Bone Marrow Transplant. 2002;29:165–71.

13. Katayama Y, Yano T, Bessho A, Deguchi S, Sunami K, Mahmut N, et al. The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells. Bone Marrow Transplant. 1997;19:283–7. 14. Akkök CA, Liseth K, Nesthus I, Løkeland T, Tefre K, Bruserud O, et al. Autologous peripheral blood progenitor cells cryopreserved with 5 and 10 percent dimethyl sulfoxide alone give comparable hematopoietic reconstitution after transplantation. Transfusion. 2008;48:877–83. 15. Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. Single platform flow cytometric absolute CD34 cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry. 1998;34:61–70. 16. Schuurius GJ, Muijen MM, Oberink JW, de Boer F, Ossenkoppele GJ, Broxterman HJ. Large populations of nonclonogenic early apoptotic CD34-positive cells are present in frozen–thawed peripheral blood stem cell transplants. Bone Marrow Transplant. 2001;27:487–98. 17. de Boer F, Dräger AM, Pinedo HM, Kessler FL, Monnee-van Muijen M, Weijers G, et al. Early apoptosis largely accounts for functional impairment of CD34 cells in frozen–thawed stem cell grafts. J Hematother Stem Cell Res. 2002;11:951–63. 18. de Boer F, Dräger AM, Pinedo HM, Kessler FL, van der Wall E, Jonkhoff AR, et al. Extensive early apoptosis in frozenthawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation. Bone Marrow Transplant. 2002;29:249–55. 19. Schmidt-Mende J, Hellstrom-Lindberg E, Joseph B, Zhivotovsky B. Freezing induces artificial cleavage of apoptosis-related proteins in human bone marrow cells. J Immunol Methods. 2000;245:91–4. 20. Oran B, Malek K, Sanchorawala V, Wright DG, Quillen K, Finn KT, et al. Predictive factors for hematopoietic engraftment after autologous peripheral blood stem cell transplantation for AL amyloidosis. Bone Marrow Transplant. 2005;35:567–75. 21. Ergene U, Cagirgan S, Pehlivan M, Yilmaz M, Tombuloglu M. Factors influencing engraftment in autologous peripheral hematopoetic stem cell transplantation (HPC-AT). Transfus Apher Sci. 2007;36:23–9. 22. al-Fiar F, Prince HM, Imrie K, Stewart AK, Crump M, Keating A. Bone marrow mononuclear cell count does not predict neutrophil and platelet recovery following autologous bone marrow transplant: value of the colony-forming unit granulocyte-macrophage (CFU-GM) assay. Cell Transplant. 1997;6:491–5. 23. Yang H, Acker JP, Cabuhat M, Letcher B, Larratt L, McGann LE. Association of post-thaw viable CD34 cells and CFU-GM with time to hematopoietic engraftment. Bone Marrow Transplant. 2005;35:881–7. 24. Yoo KH, Lee SH, Kim HJ, Sung KW, Jung HL, Cho EJ, et al. The impact of post-thaw colony-forming units-granulocyte/ macrophage on engraftment following unrelated cord blood transplantation in pediatric recipients. Bone Marrow Transplant. 2007;39:515–21.