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Journal of Clinical Oncology, Vol 17, Issue 7 (July), 1999: 2160
© 1999 American Society for Clinical Oncology

High- Versus Standard-Dose Filgrastim (rhG-CSF) for Mobilization of Peripheral-Blood Progenitor Cells From Allogeneic Donors and CD34+ Immunoselection

Monika Engelhardt, Hartmut Bertz, Matthias Afting, Cornelius F. Waller, Jürgen Finke

From the Department of Hematology/Oncology, University Medical Center, University of Freiburg, Freiburg, Germany.

Address reprint requests to Jürgen Finke, MD, University of Freiburg, Department of Hematology/Oncology, Hugstetterstr 55, 79106 Freiburg, Germany; email finke{at}mm11.ukl.uni-freiburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: The efficacy of a high- versus a standard-dose filgrastim (recombinant human granulocyte colony-stimulating factor, or rhG-CSF) regimen to mobilize peripheral-blood progenitor cells (PBPCs) for allogeneic transplantation was investigated in 75 healthy donors.

PATIENTS AND METHODS: From December 1994 to December 1997, 75 consecutive donors (median age, 38 years; range, 17 to 67 years) were assigned to two different schedules of rhG-CSF for PBPC mobilization. Fifty donors received 24 µg rhG-CSF/kg body weight (BW) divided into two daily subcutaneous injections (two doses of 12 µg, group A), whereas 25 were treated with 10 µg rhG-CSF once daily (group B). Apheresis was started on day 4 in group A and on day 5 in group B. Target CD34+ cell numbers in apheresis products were >= 4 x 106/kg recipient BW.

RESULTS: Cytokine priming and collection of PBPCs were equally well tolerated in both groups. Significantly higher CD34+ cell numbers in group A with 3.7 x 106/kg recipient BW/apheresis (0.47 x 106/L apheresis) compared with 2 x 106/kg recipient BW/apheresis (0.25 x 106/L apharesis) in group B were obtained (P < .05). Using standard aphereses (median, 9 L), two doses of 12 µg rhG-CSF/kg allowed collection of >= 4 x 106/kg CD34+ cells with two aphereses (range, one to three) in group A versus three aphereses (range, one to six) in group B (P < .015). Donor age, sex, and BW influenced the collection of CD34+ cell numbers: in particular, significantly higher apheresis results were obtained in donors younger than 40 years compared with donors older than 40 years of age (P < .05). In 65 CD34+ selection procedures using avidin-biotin immunoabsorption columns (Ceprate SC System, CellPro, Bothell, WA), a median CD34+ purity of 53%, CD34+ recovery of 40%, and the collection of 2 x 106/kg CD34+ cells/selection were achieved. In group A with higher CD34+ cells/kg/apheresis, CD34+ purity, recovery, and cell yields were 60%, 45%, and 2.3 x 106/kg/selection, respectively, as compared with 48%, 31%, and 0.7 x 106/kg in group B (P < .05).

CONCLUSION: Our results demonstrate that twice daily rhG-CSF (two doses of 12 µg/kg BM) compared with once daily rhG-CSF (10 µg/kg BW), in addition to being well tolerated, significantly improves PBPC mobilization, allows the collection of higher numbers of CD34+ cells with one or two standard aphereses, and facilitates subsequent selection procedures in healthy allogeneic donors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CYTOKINE-MOBILIZED peripheral-blood cells can be harvested after chemotherapy and/or cytokine priming and durably restore hematopoietic function when transplanted after myeloablative therapy.1-3 During the past 5 years, transplantation with mobilized peripheral-blood progenitor cells (PBPCs) has increasingly been used as an alternative approach to autologous bone marrow transplantation (ABMT).1-3 The relative ease of PBPC collection, the rapid engraftment with faster recovery of neutrophils and platelets, the reduced risk of serious infections, and the reduction of transfusion requirements after peripheral-blood progenitor-cell transplantation (PBPCT) compared with ABMT have been the main reasons for this development.2-5 Recently, several groups have advocated the use of PBPCs instead of bone marrow for allogeneic transplantation.6-10 PBPCs in allogeneic transplantation offer the advantage of eliminating the need for general anesthesia and result in rapid hematopoietic reconstitution.6-13 However, due to the approximately 1 log higher number of T cells, concern has been raised about a potentially higher incidence of graft-versus-host disease (GVHD) as compared with bone marrow transplantations.10 Early results suggest that PBPCs from HLA-matched related donors reliably result in rapid hematopoietic engraftment without increasing the incidence or severity of acute GVHD.7-9,12 The incidence of chronic GVHD after allogeneic PBPCT remains to be fully assessed by controlled randomized trials but has been suggested to occur more frequently in PBPC recipients than in marrow recipients.14-20 However, acute and chronic GVHD may be reduced by CD34+-selected stem-cell grafts that deplete T-cell numbers by 2 to 4 log.21,22

Mobilization of PBPCs from healthy donors is achieved with cytokines such as filgrastim (recombinant human granulocyte colony-stimulating factor, or rhG-CSF) or granulocyte-macrophage colony-stimulating factor. Both have demonstrated the ability to effectively mobilize PBPCs into the blood, although rhG-CSF is the preferred cytokine, because it has fewer side effects and higher mobilization efficacy.23-27 rhG-CSF has been applied in numerous clinical trials using different dose schedules.7-13 Currently, 10 µg rhG-CSF/kg is widely recommended; however, neither optimal schedules nor optimal dosages for PBPC mobilization have been established thus far. Because the application of 10 µg rhG-CSF/kg/d has been demonstrated to result in inconsistent amounts of CD34+ cells, we previously decided to increase the dosage to two daily doses of 12 µg rhG-CSF/kg. In our preceding analysis comparing high- versus low-dose rhG-CSF in 19 donors, we demonstrated that mobilization with two doses of 12 µg rhG-CSF compared with one dose of 10 µg rhG-CSF results in higher PBPC yields, although this did not reach statistical significance.28 In this study, 75 healthy allogeneic donors were included: 50 donors in group A received two daily doses of 12 µg rhG-CSF and 25 donors in group B received one daily dose of 10 µg rhG-CSF. Our findings in this large cohort of donors demonstrate that two doses of 12 ng rhG-CSF result in significantly higher CD34+ cell yields in blood and apheresis products, allow the collection of large CD34+ cell numbers with one or two standard aphereses, and facilitate selection procedures in allogeneic donors.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Donors
The initiation of the study was based on the observation that our first donors, who had insufficient CD34+ mobilization after one dose of 10 µg/kg rhG-CSF, showed marked improvement after two doses of 12 µg/kg rhG-CSF. Therefore, a single-center, prospective analysis was performed in 75 consecutive healthy allogeneic donors (48 males, 27 females) to determine the efficacy of 24 µg (two doses of 12 µg) versus 10 µg rhG-CSF (Neupogen, Amgen, Munich, Germany). Our analysis includes all donors mobilized in our center from December 1994 to December 1997. After our experience with two doses of 12 µg rhG-CSF, we pursued assignment to one dose of 10 µg versus two doses of 12 µg rhG-CSF, applying the following modus: donors of patients fulfilling the criteria and willing to participate in a randomized protocol comparing ABMT versus PBPCT received one dose of 10 µg rhG-CSF, whereas the other donors received high-dose (two doses of 12 µg) rhG-CSF. In group A, 50 donors received two doses of 12 µg rhG-CSF/kg body weight (BW) (2 x rhG-CSF) subcutaneously (SC), whereas in group B, 25 donors received 10 µg/kg (1 x rhG-CSF) SC once daily. The numbers of donors receiving 10 versus 24 µg rhG-CSF between 1994 and 1997 are listed in Table 1. Approximately two thirds of donors in the year 1994 were assigned to receive standard- compared with high-dose rhG-CSF. In 1995 to 1997, approximately two thirds of donors were assigned to receive high-dose rhG-CSF versus one third receiving standard-dose rhG-CSF. Although more donors in 1995 to 1997 received high-dose rhG-CSF, thus potentially affecting CD34+ mobilization and collection more in this group due to potential changes in the apheresis technique, CD34+ mobilization and collection were equally superior throughout the observation period of 1994 to 1997 in group A (2 x rhG-CSF) compared with group B (1 x rhG-CSF). Therefore, potential changes in the apheresis technique equally affected both groups between 1994 to 1997. Growth factor application dosing was rounded to standard-size ampoules of 300 or 480 µg rhG-CSF, resulting in median rhG-CSF doses of 23 (range, 18.4 to 26) and 10 (range, 7.4 to 12) µg/kg BW/d in groups A and B, respectively. All donors were offered oral prophylaxis with two doses of 500 mg of paracetamol per day for rhG-CSF–induced side effects. The study was carried out under the guidelines of the ethical committee of the University of Freiburg, and informed consent was obtained from each individual before start of mobilization.


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  		Table 1. Allogeneic Donor and Recipient Characteristics
 

Collection of PBPCs
Apheresis was started on day 4 after twice-daily rhG-CSF priming in group A and on day 5 after once-daily rhG-CSF priming in group B. Harvest was performed using a continuous flow cell separator (Baxter CS 3000, Baxter, Deerfield, IL, or Spectra, Cobe BCT, Inc, Lakewood, CO). In all donors, central venous access could be avoided and 16-gauge needles for venous access through cubital or antecubital veins were used. The target apheresis volume was 9 L of blood using a flow rate of 60 to 100 mL/min. rhG-CSF administration was continued twice daily in group A and once daily in group B until completion of apheresis, with a target cell dose of >= 4 x 106 CD34+ cells/kg recipient BW. Before and after apheresis, the total blood count was measured using an automated cell counter (Cell-Dyn 3500, Abbott, Wiesbaden, Germany). In peripheral-blood (PB) samples, the WBC count and CD34+ cells were determined. In apheresis products, WBC, mononuclear cells, CD34+, CD3+ cell numbers, and colony-forming units (CFU) were determined. A total of 36 PB samples and 191 apheresis products were analyzed. In donors who failed to collect >= 4 x 106 CD34+ cells with six aphereses, the PBPC collection was discontinued and a bone marrow harvest was performed 2 weeks after the PBPC collection.

Immunoaffinity Selection of CD34+ Cells
On 65 PBPC products, a CD34+ immunoselection (immunoaffinity column separation, Ceprate system, CellPro, Bothell, WA)21 for 30 patients was performed. The first PBPC apheresis product was stored at 4°C overnight, pooled with the second apheresis product before further processing, and washed with phosphate buffered saline (PBS). All PBS wash steps were performed at 400 g for 10 minutes. The CD34+ cell selections were performed according to the manufacturer's instructions. In brief, the washed cell pellet was resuspended into 330 mL of PBS. Three milliliters of biotinylated 12.8 IgM anti-CD34 monoclonal antibody (MoAb, CellPro) and 0.7 mL of 25% human serum albumin (HSA) were added to harvested mononuclear cells and passaged over a computer-driven avidin-immunoaffinity column device with a loading capacity of greater than 1011 mononuclear cells. The uncoupled MoAbs were washed out after an incubation at 22°C for 30 minutes. Adsorbed CD34+ cells were removed from the avidin column, washed in PBS, resuspended in a final volume of 10 to 15 mL (2 x 107 cells/mL), and frozen in the presence of 7.5% dimethylsulfoxide and 4% HSA in PBS supplemented with 10 U/mL heparin (Liquemin, Roche, Grenzach, Germany). Aliquots of the CD34+ fraction were analyzed for WBCs, CD34+ and CD3+ cell numbers, and colony-forming capacity. CD34+ cells were transferred into 50-mL freezing bags and stored in liquid nitrogen until transplantation. All cell processing and storage steps were performed according to Good Manufacturing Practice guidelines.

Immunofluorescence Staining
Samples from PB, apheresis products, and cell products after immunoseparation were analyzed by dual-color immunofluorescence as described previously.29 Briefly, 100 µL EDTA-PB, apheresis, and CD34+ selection products were incubated with anti-CD34 phycoerythrin (PE) and anti-CD45 (HLe-1 fluorescein isothiocyanate [FITC] conjugated; Becton Dickinson, Heidelberg, Germany), incubated for 20 minutes at 4°C, and washed once with PBS. Cells were analyzed using a FACScan analyzer (Becton Dickinson) equipped with a filter set for FITC-PE dual-color fluorescence. Isotype-identical antibodies (IgG1-FITC/IgG2a-PE; Becton Dickinson) were used as controls. The whole blood was lysed (FACS lysis solution, Becton Dickinson) at room temperature for 10 minutes, washed twice with PBS, and analyzed. We used forward-scatter versus right-angle light-scatter (FSC/SSC) dot plot to establish the blast/lymphocyte region (R1) and to exclude debris and granulocytes. The number of CD34+ cells was analyzed in a fluorescence-1 versus SSC dot plot. For PB samples, the percentage of CD34+ cells/mL was determined by multiplying the number of CD34+ cells by the WBC count. The analysis of CD34+ cell numbers in apheresis and positive selection products was performed by incubating with anti-CD34 PE-conjugated MoAB, anti-CD45 (HLe-1 FITC conjugated), and anti–CD3-PE (all from Becton Dickinson). The apheresis aliquot was lysed at room temperature for 10 minutes, washed twice with PBS, and analyzed. The number of CD34+ cells in each PBPC product was determined by multiplying the total number of WBCs in harvest products by the percentage of CD34+ cells. A FACScan analyzer was used, and data acquisition was performed with FACScan Lysis II software (Becton Dickinson). For analysis, a total of 50,000 to 100,000 cells were acquired.

Clonogenic Assay
MNCs (1 x 105) from apheresis samples and CD34+ cells (1 x 104) after immunoselection were cultured in 0.9% methylcellulose supplemented with Iscove's Modified Dulbecco's Medium and 30% fetal calf serum. Semisolid cultures were performed in duplicates, stimulated with recombinant human erythropoietin (1 U/mL), interleukin-3 (100 ng/mL), and granulocyte-macrophage colony-stimulating factor (100 ng/mL). Formation of more than 50 cells was scored as myeloid (CFU–granulocyte-macrophage [CFU-GM]), erythroid (burst-forming unit–erythrocyte [BFU-E]), and multilineage colonies (CFU-granulocyte, erythrocyte, monocyte, megakaryocyte [CFU-GEMM]) after 14 days with an inverted microscope. Colony numbers from apheresis and after CD34+ immunoselection were expressed as CFU-GM, BFU-E, CFU-GEMM, and total CFUs (CFU-GM + BFU-E + CFU-GEMM) per 100,000 cells.

Statistics
Comparisons among groups were made with standard statistical tests. Results are expressed as median and range except when stated otherwise. Statistical significance of the data obtained was analyzed by the Wilcoxon rank sum test and the Student's t test (Statworks, Cricket Software, Philadelphia, PA). The relationship between age and CD34+ apheresis results were estimated by linear regression and correlation analysis. A P value of less than .05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Donor Characteristics
Allogeneic donor and recipient characteristics are listed in Table 1. Group A and B donors were comparable regarding age and BW (Table 1).

Priming with rhG-CSF and harvesting of PBPCs were associated with tolerable toxicity (Table 2). In almost all donors, mild bone pain was observed. Approximately one half (20 of 50) of the donors who received 2 x rhG-CSF suffered from mild headaches (World Health Organization [WHO] grade 1). In group A, dosage of paracetamol had to be increased in four individuals (four of 50; 8%); in one donor, bone pain persisted for 15 days after completion of harvest (one of 50; 2%); and in one, dosage of rhG-CSF had to be reduced because of severe bone pain and headaches (WHO grade 3) (2%). No fatigue, nausea, or weight gain due to rhG-CSF priming were observed. Changes in PB counts, with increase of WBC counts and decrease of platelet numbers in donors of both groups during apheresis, are outlined in Table 2. In 52% versus 40% of donors, the WBC count during apheresis was less than 60,000/µL compared with 48% versus 60% of donors with WBC counts >= 60,000/µL in groups A and B, respectively. Platelet counts after completion of apheresis declined by <= 100,000/µL in 18% of donors in group A versus 24% of donors in group B (Table 2).


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  		Table 2. Number of Allogeneic Donors With rhG-CSF–Induced Effects
 

The median total rhG-CSF dose for PBPC mobilization (day 1 of rhG-CSF priming to the end of apheresis) was 8,640 µg versus 5,760 µg and 111 µg/kg BW versus 75 µg/kg BW in groups A and B, respectively. Thus, despite the more than two-fold higher rhG-CSF dose schedule in group A compared with group B, rhG-CSF could be discontinued earlier in group A due to fewer aphereses. This resulted in an only 1.5-fold increase of rhG-CSF applied to donors in group A compared with those in group B.

PB Results
In all donors, the WBC count increased to 30,000 to 60,000/µL on days 4 and 5 after rhG-CSF priming. Median WBC counts during the aphereses were 54.2 x 103/µL (range, 26.6 to 100 x 103/µL) in group A and 49.6 x 103/µL (range, 17.9 to 99.8 x 103/µL) in group B (P = .066), with higher WBC values on subsequent days of apheresis in group A compared with group B (Fig 1A). Platelet counts were comparable in groups A and B but were significantly reduced during apheresis (Fig 1B), although leukapheresis was not limited in any donor (threshold level <50,000/µL). PB values rapidly returned to normal after cessation of the rhG-CSF application and the end of apheresis.



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  		Fig 1. Analysis of PB results in both groups of healthy donors. (A) WBC counts increased on days 4 and 5 after rhG-CSF priming, reaching higher values in group A compared with group B, although this was not statistically significant. (B) Platelet counts were comparable in group A and B and were significantly reduced during apheresis. PB CD34+ cells (per mL [C] and in % [D]) were higher in group A compared with group B, especially on days 2, 3, and 4 of apheresis (* = P < .05).

 

Numbers of CD34+ cells in PB samples (for CD34+ cells/µL and percentage of CD34+ cells) were higher in group A, with 55.4/µL (range, 18.8 to 191/µL) and 0.1% (range, 0.03% to 0.23%) versus 38/µL (range, 6.4 to 66.2/µL) and 0.08% (range, 0.03% to 0.26%) in group B. On the first day of apheresis, blood CD34+ cells were comparable in donors in groups A and B (Fig 1C and 1D). On days 2, 3, and 4 of apheresis, however, group A donors showed significantly higher CD34+ cells (CD34+ cells/µL and percentage of CD34+ cells) compared with group B (Fig 1C and 1D).

Apheresis Results
The median apheresis volume was 9 L (range, 6 to 17 L) in both groups (P = .44). Apheresis volumes were comparable, with a median of 9 L in both groups for the total of aphereses performed in each group, as well as determined for each day of apheresis (days 1 to 4). In one donor of each group, large volume aphereses (17 L) were performed. This was due to inaccessibility of the donors on subsequent days and a CD34+ immunoselection being planned in both donors. WBC counts in apheresis products were higher in group A, with a median of 3.81 x 108/kg (range, 1.04 to 18.6 x 108/kg), compared with group B, with a median of 2.56 x 108/kg (range, 0.97 to 10.2 x 108/kg) (P = .092). Significantly lower WBC counts were observed at the end of the apheresis (day 4 of apheresis) in group B (Fig 2A).



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  		Fig 2. Comparison of apheresis results in groups A and B of donors. (A) WBC counts in apheresis products were higher in group A. (B) Significantly higher CD34+ cells (in %) were observed in group A than in group B. (C) Number of CD34+ cells calculated for the recipient's BW, and (D) number of CD34+ cells calculated for the donor's BW. On the first day of apheresis, comparable results in groups A and B were observed; however, on subsequent days of apheresis, CD34+ cell numbers in group B decreased. In the twice-daily rhG-CSF group, CD34+ cell numbers increased on the second day of apheresis and showed a continuous plateau, with CD34+ cell numbers of > 3 x 106 collected over a 4-day period. (E) Twice-daily compared with once-daily rhG-CSF increased the median cell content, with a higher total number of CD3+ T cells. However, the percentage of T cells in all apheresis products were identical (F) (*=P < .05).

 

In donors receiving 2 x rhG-CSF, the median number of CD34+ cells per single apheresis was 3.7 x 106/kg compared with 2 x 106/kg in the 1 x rhG-CSF group (Table 3). Whereas comparable results in groups A and B were observed on the first day of apheresis, on subsequent days of apheresis, CD34+ cell numbers in group B decreased considerably and harvest results deteriorated (Fig 2C and 2D). In the twice-daily rhG-CSF group, CD34+ cell numbers increased on day 2 of apheresis and showed a continuous plateau, with median CD34+ cell numbers of more than 3 x 106/kg collected over a 4-day period (Fig 2C and 2D). This was independent of whether this was calculated per recipient (patient) or donor BW (Fig 2C and 2D).


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  		Table 3. Collection of Allogeneic PBPCs in Healthy Donors
 

CD34+ cell numbers were also calculated per one liter apheresis to adjust for variations in apheresis volumes. This also revealed significantly higher CD34+ cell numbers of 0.47 x 106/L in group A compared with 0.25 x 106/L in group B (Table 3). This correlated with a higher percentage of CD34+ cells in apheresis products (1.02% in group A v 0.76% in group B) (Table 3, Fig 2B), with fewer aphereses in group A (two) than in group B (three), and with total number of CD34+ cells in apheresis products (Table 3). In 90% versus 64% of donors, target CD34+ numbers (>=4 x 106/kg BW) were obtained with one or two aphereses in group A and B, respectively, compared with 0% of donors in group A versus 24% of donors in group B needing >= four aphereses to obtain target cell numbers (Table 3).

Higher doses of rhG-CSF increased the total number of WBCs in apheresis products (Fig 2A) and CD3-positive T cells in group A (Table 3, Fig 2E). However, the percentage of T cells in all apheresis products was identical in groups A and B (Table 3). This was also observed when analyzed separately for each day of apheresis (Fig 2F).

In all donors in group A (50 of 50), and in 23 of 25 donors in group B, PBPC grafts contained >= 3 x 106/kg CD34+ cells. In group A, >= 90% of donors also obtained CD34+ numbers of >= 4 x 106/kg and >= 5 x 106/kg, whereas considerably fewer donors reached these target numbers in group B (>=4 x 106/kg, 20 of 25, 80%; >=5 x 106/kg, 15 of 25, 60%). Two donors in group B (two of 25, 8%; Table 3), one 43-year-old male and one 52-year-old female, failed to mobilize more than 2 x 106/kg CD34+ cells, and a bone marrow harvest was performed successfully for both.

Although more donors in 1995 to 1997 received high- compared with standard-dose rhG-CSF (Table 1), thus potentially effecting CD34+ mobilization and collection more in group A due to potential changes in apheresis technique, CD34+ mobilization and collection were equally superior throughout the observation period of 1994 to 1997 in group A (2 x 12 µg) (CD34+ cells/single apheresis [x106/kg BW]: 1994, 4.2; 1995, 5.1; 1996, 4.5; 1997, 3.6; and apheresis CD34+ cells [%]: 1994, 2.2; 1995, 1.7; 1996, 1.3; 1997, 1.3) compared with group B (1 x 10 µg rhG-CSF) (CD34+ cells/single apheresis [x106/kg BW]: 1994, 2.2; 1995, 2.1; 1996, 1.9; 1997, 2.0; and apheresis CD34+ cells [%]: 1994, 0.5; 1995, 0.9; 1996, 1.0; 1997, 1.0).

Influence of Sex, Age, and BW on CD34+ Cell Numbers
To assess whether sex, age, and BW of donors influence CD34+ cell numbers, apheresis results in both rhG-CSF groups were analyzed (Fig 3). Higher median CD34+ cell numbers were obtained in male versus female donors: in group A, 4.9 x 106/kg CD34+ cells/apheresis were obtained in male donors versus 4.5 x 106/kg in female donors, and in group B, 3.6 x 106/kg CD34+ cells/apharesis were obtained in male donors versus 1.3 x 106/kg in female donors (Fig 3A). However, donor BW differed in male versus female donors, with a median BW of 81.5 kg in male donors versus 70.4 kg in female donors in group A, and of 81 kg in male donors versus 63.5 kg in female donors in group B. When adjusting CD34+ cell numbers/kg BW to a constant BW of 81.5 kg in all donors, theoretically, a median harvest result of 4.9 x 106/kg CD34+ cells/apheresis in male versus 5.2 x 106/kg in female donors in group A was calculated, compared with 3.62 106/kg versus 1.7 106/kg in group B (Fig 3A). This suggests that in male and female donors of group A, sex may not affect apheresis results, but sex may affect results with lower doses of rhG-CSF. In younger (<40 years) versus older donors (> 40 years), CD34+ cell numbers in apheresis products were significantly higher: in group A with median CD34+ cells/apheresis of 5.4 x 106/kg in younger donors versus 4.1 x 106/kg in older donors (P = .013) and 3 x 106/kg in younger donors versus 2 x 106/kg in older donors in group B (P = .049) (Fig 3B). A correlation analysis of donor age and apheresis results demonstrated an age-related decline in CD34+ numbers in both groups of donors: an approximately two-fold difference in CD34+ cell numbers between group A versus group B was observed, and a decline in CD34+ cells of approximately 0.5 x 106/kg per decade of donor age (Fig 3C).



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  		Fig 3. To assess whether sex, age, and BW of donors influence CD34+ cell numbers, apheresis results in both rhG-CSF groups were analyzed. (A) Higher median CD34+ cell numbers were obtained in male versus female donors (group A, {block}; group B, shaded box). However, the BW differed in male versus female donors. If a constant BW of 81.5 kg was presumed in all donors, this theoretically allowed us to calculate a harvest result of 4.9 x 106/kg CD34+ cells/apheresis in male versus 5.2 in female donors in group A, compared with 3.62 and 1.7 x 106/kg in group B. (B) In young (< 40 years, {block}, shaded circle) versus older donors (> 40 years, {square}, {bigcirc}), CD34+ cell numbers in apheresis products were significantly higher. In group A ({block}), median CD34+ cells/apheresis were 5.4 x 106/kg in young versus 4.1 x 106/kg in older donors (P = .013) and 3 x 106/kg versus 2 x 106/kg in group B (shaded circle). (C) Correlation analysis of donor age and apheresis results demonstrates an age-related decline in CD34+ numbers in both groups of donors, with an approximately two-fold difference in CD34+ cell numbers between group A ({block}) versus group B (shaded circle) and a decline in CD34+ cells of approximately 0.5 x 106/kg per decade of donor age.

 

CD34+ Selection Results
For 30 patients, a CD34+ selection was performed, using a total of 65 immunoselection columns. Donors in both groups were comparable regarding sex distribution, age, and BW as outlined in Table 4. In order to reach >= 4 x 106/kg CD34+ cells, a median of two aphereses in group A and four in group B were required (P < .05). The median CD34+ purity in allogeneic blood products after positive selection was 53% (range, 11% to 96%). The CD34+ recovery was 40% (range, 14% to 98%), and the total CD34+ cell yield was 4.2 x 106/kg BW (range, 0.2 to 8.9 x 106/kg BW). The purity in selection products positively correlated with the percentage of CD34+ cells in apheresis products: with higher numbers of precolumn CD34+ cells in group A, yields of CD34+–selected products were higher compared with apheresis products with low numbers of CD34+ cells. As a result, improved CD34+ purity, improved CD34+ recovery, and increased CD34+ cell numbers in group A compared with group B were observed (Table 4). Consequently, in 16 of 19 donors (84.2%) in group A, >= 4 x 106 CD34+ cells after immunoselection were obtained, compared with only four of 11 donors (36%) in group B.


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  		Table 4. CD34+ Selection in Allogeneic Donors Using 2 x 12 µg or 1 x 10 µg rhG-CSF
 

Clonogenic Assays
Clonogenic assays were performed from apheresis products, CD34+–selected cell products, and induced myeloid (CFU-GM), erythroid (BFU-E), and multilineage colony formation (CFU-GEMM). In group A compared with group B, an increased clonogenic potential of apheresis and immunoselection products was observed: in apheresis products of groups A and B, a median of 45 and 33 CFU-GM (P = .4), 45 and 43 BFU-E (P = .9), 3.3 and 2.7 CFU-GEMM (P = .1), and 92 and 88 total CFUs (P = .3), respectively, were observed. In CD34+–selected cell products as compared with unselected apheresis products, colony numbers were increased approximately 10-fold, with CFU-GM of 310 versus 223 (P = .31), BFU-E of 497 versus 201 (P = .17), CFU-GEMM of 26.7 versus 46.7 (P = .17), and total colony numbers of 867 versus 463 (P = .136) in groups A and B, respectively. Higher CFU numbers in immunoselected cell products demonstrated that colony formation was significantly increased in immunoselected products compared with aphereses that correlated with an increase in positively selected cell products of at least 1 log CD34+ cells (~50% to 60% CD34+ cells after column selection) versus apheresis products (~0.5% to 5% CD34+ cells).

Number of Apheresis and CD34+–Selected Cells in Allogeneic Transplantation and Hematopoietic Engraftment
The grafts collected from both groups of donors were, with almost exact cell numbers, transplanted into patients, thus differences were not expected in the speed of hematopoietic recovery after allografting (Table 5). Whereas number of CD34+ cells were comparable, whether transplanted as unselected or CD34+–selected grafts, number of WBC and CD3+ cells—although similar between donor groups A and B—were approximately 100- and 250-fold decreased after CD34+ immunoselection (Table 5). Days of engraftment with WBCs greater than 1,000/µL and platelets greater than 20,000/µL were observed on days +11 and +13 in all patients: on days +11 and +13 in patients receiving group A grafts and on days +11 and +14 in patients receiving group B grafts.


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  		Table 5. Number of Cells Transplanted in Allogeneic Patients Using Group A and B Apheresis or CD34+–Selected Grafts
 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ease of collection and the rapid recovery after transplantation have made PBPCs an attractive source of hematopoietic progenitor cells for autologous and allogeneic transplantation.1-13 So far, a possible risk of more frequent and more severe acute GVHD after allogeneic PBPCT due to the high number of T cells in the graft has not been observed, and engraftment after allogeneic PBPC transplantation seems to be more rapid compared with bone marrow controls.6-9,13 A major advantage of allogeneic PB stem cells over bone marrow is their better suitability for graft engineering. This has dramatically increased the use of cytokine-mobilized allogeneic PBPCs over the last few years, and PBPCs may entirely replace bone marrow for allogeneic transplantation in the near future.

Donor safety is of major concern during cytokine treatment and harvest of PBPCs.23-28 We started apheresis on day 4 in group A, as previously reported by Körbling et al and others,8,28,30,31 and on day 5 in group B, because 10 µg rhG-CSF has shown to result in best PBPC mobilization on day 5 after rhG-CSF priming.9,13,23,32-35 For PBPC mobilization and treatment of neutropenia, rhG-CSF has been safely administered to a considerable number of healthy subjects, such as granulocyte donors, PBPC donors, and volunteers.23-28,31-33,36 Short-term effects of rhG-CSF for PBPC mobilization are predominantly minor, and toxicity is tolerable and transient.12,24-28 Although long-term rhG-CSF effects have not been reported to date and seem to be unlikely after more than 5 years of clinical experience, healthy donors will need to be observed for longer periods of time to determine the potential for eventual unwanted effects.37 In our study, typical side effects of rhG-CSF priming in both groups were bone pain, myalgia, and headaches. Generally, side effects such as bone pain, headache, and flu-like symptoms were comparable in both groups of donors, although more pronounced side effects were observed in 16% of patients in group A compared with patients in group B. Twenty donors (40%) receiving high-dose twice-daily rhG-CSF (2 x rhG-CSF) reported headache and four donors reported severe bone pain compared with seven donors (28%) reporting headache and no donor reporting severe bone pain in the standard-dose rhG-CSF group. In 48% versus 60% of donors in groups A and B, respectively, PB WBC counts persisted at less than 60,000/µL during apheresis, whereas in 6% of donors in group A but 16% of donors in group B, WBC counts were >= 80,000/µL during apheresis. Platelet counts of <= 100,000/µL were observed in 18% of donors in group A versus 24% of donors in group B. Therefore, due to the longer rhG-CSF exposure and greater apheresis numbers in group B, higher WBC counts and lower platelet counts were observed more frequently in this group of donors. Central catheters for venous access, which also pose a possible risk to healthy individuals, were not necessary for any donor in this study.

Recent data in donors have demonstrated that 3 to 5 µg rhG-CSF/kg/d results in less efficient blood progenitor-cell mobilization compared with higher rhG-CSF doses, such as 10 to 24 µg/kg BW/d.8,23,24,28,31 In pretreated cancer patients, higher doses (> 10 µg/kg BW/d) of rhG-CSF after chemotherapy for PBPC mobilization have been advocated, supporting the concept that CD34+ cell mobilization is facilitated and demonstrating that the efficacy of PBPC mobilization depends on the amount and schedule of rhG-CSF.34,38 The definite determination of the optimal dosage and schedule for stem-cell mobilization, especially in normal donors, however, remains to be defined. We have previously reported, in 19 allogeneic donors, that mobilization with two daily doses of 12 µg compared with one daily dose of 10 µg rhG-CSF results in higher PBPC yields in apheresis products.28 In line with our observation, Harada et al33 found a dose-escalating and time-dependent effect of 5, 10, and 15 µg rhG-CSF in nine allogeneic donors. However, because the number of patients was limited and differences of apheresis results in rhG-CSF groups did not reach statistical significance in both previous studies, we conducted this large prospective analysis. In this study, CD34+ cell numbers were determined in apheresis and blood products, because the blood CD34+ cell concentration has repetitively been shown to be predictive for CD34+ cell yields in apheresis products.6,26,39-41 Blood CD34+ cells were higher in patients who received twice-daily rhG-CSF, which correlated with better apheresis results in group A. In addition, the median number of aphereses to obtain target CD34+ cells (>= 4 x 106/kg BW) was two versus three for apheresis products and two versus four for CD34+–immunoselected cell products in group A versus group B, respectively. Because target CD34+ cells were obtained with fewer aphereses in group A, rhG-CSF could be discontinued earlier, resulting in an actual rhG-CSF dose increase of only 50% in group A compared with group B. Interestingly, CD34+ cell numbers decreased with subsequent apheresis days in group B, whereas with two doses of 12 µg rhG-CSF, CD34+ cell numbers increased on the second day of apheresis, reaching a continuous plateau with median CD34+ cell numbers of more than 3 x 106/kg BW collected over a 4-day period. This suggests a stable 4-day mobilization capacity of the twice-daily 12 µg regimen compared with the less efficient 10-µg regime, the latter of which results in poor harvest yields beyond day 7 and/or day 8 of the rhG-CSF application.23,24,42,43 Although we could successfully collect PBPCs in most healthy donors, almost all donors in group A obtained CD34+ numbers of >= 4 x 106/kg (98%) and >= 5 x 106/kg (90%), whereas considerably fewer donors in group B reached these target numbers (80% and 60%, respectively).

Previous analyses have indicated that donor sex, age, or BW may influence apheresis results.24,44-47 Our study confirms these results, finding a significant influence of these parameters on CD34+ cell numbers in harvest products, with particularly higher CD34+ cell numbers in male and young allogeneic donors (< 40 years). CD34+ cells declined with donor age, which theoretically allowed us to predict a decline in CD34+ cells/apheresis of 0.5 x 106/kg per decade of donor age, and which suggests that in young PBPC donors, higher CD34+ apheresis results can be anticipated.

We observed that with two daily doses of 12 µg rhG-CSF, absolute WBC and CD3-positive T-cell numbers in apheresis products were increased in group A compared with group B, although the percentage of T cells was identical in both groups. Similar numbers of T cells as observed in our analysis have been reported by others, with no increase of acute GVHD as compared with ABMT.7-9,27 This may be due to the fact that T-cell numbers in both groups are well above the safe threshold number of 1 x 105/kg BW, with no linear incidence of acute GVHD. Or it may be due to (1) an rhG-CSF–induced downregulation of the alloreactivity of infused T cells and increased number of suppressor cells,14,19 (2) an rhG-CSF–induced preferential differentiation of T-helper cells toward cytokine-secreting TH2 rather than TH1 cells,17 and/or (3) large numbers of rhG-CSF–modulated monocytes in PBPC grafts that suppress T-cell functions.18,19

Our analysis of CD34+ selection procedures revealed that the CD34+ purity and recovery rates were lower than those in patients undergoing autologous transplantation after chemotherapy plus cytokine mobilization,48 but these rates were comparable to results achieved in other studies of rhG-CSF–mobilized healthy donors.49 Higher platelet numbers, platelet aggregation, and platelet coating, which partly prevent the binding of stem cells to the anti-CD34+ antibody, have been suggested as factors that negatively affect CD34+ selection performance in allogeneic donors.49 We found that CD34+ purity, recovery, and total CD34+ cells were significantly higher in group A donors compared with group B donors. As a result, 84% of donors in group A obtained target CD34+ numbers after selection, compared with only 36% of donors in group B. This demonstrates that with higher CD34+ yields in apheresis products, better selection results are obtained. As a result, positive selection of CD34+ cells from rhG-CSF mobilized donors—although lower in purity and recovery than in autologous patients—is feasible and reproducible. However, improvement of purity and recovery rates in allogeneic PBPC products seems possible by using higher rhG-CSF doses, as observed with two daily doses of 12 µg rhG-CSF. Although higher rhG-CSF doses significantly improved CD34+ yields in apheresis products and significantly decreased apheresis numbers, PBPC grafts from group A and B donors that were transplanted in allogeneic patients did not differ in mononuclear cells, CD34+, or CD3+ numbers/kg BW, and the speed of hematopoietic recovery after allografting was comparable in patients transplanted with group A and B grafts. Our results of clonogenic assays were in line with increased cell yields in apheresis and CD34+ selection products in group A donors and demonstrated two doses of 12 µg as compared with one dose of 10 µg rhG-CSF increased the clonogenic potential of apheresis and immunoselection products and that colony formation after CD34+ selection was increased approximately 1 log compared with apheresis products.

In conclusion, our data illustrate that the number of CD34+ cells recovered by apheresis after different rhG-CSF doses may vary significantly. We found that high-dose twice-daily rhG-CSF was well tolerated, had few and manageable side effects, significantly increased CD34+ cells in harvest products, and significantly reduced the number of aphereses necessary to collect >= 4 x 106 CD34+ cells/kg. Our analysis is important because maximum allogeneic PBPC mobilization minimizes donor aphereses, reduces the rhG-CSF exposure, and increases the feasibility of allogeneic PBPC collection. Moreover, high CD34+ cell numbers obtained with two daily doses of 12 µg rhG-CSF more reliably allow graft engineering, such as CD34+ selection procedures. Therefore, reduced numbers of apheresis procedures and possibly reduction of costs can be achieved by short-term applications of higher doses of rhG-CSF. In addition, large-volume apheresis, in which three to four times the donor's total BW per aphereses is processed, has been shown to provide high CD34+ cell yields in adult and pediatric donors31,32 and may further increase stem-cell yields when combined with two daily doses of 12 µg rhG-CSF, preferentially with a single apheresis. Moreover, for donors with low baseline CD34+ cell counts, higher rhG-CSF doses could also prove beneficial.32 Finally, a higher probability of rapid hematopoietic engraftment with infusion of more than 5 x 106 CD34+ cells after allogeneic PBPCT has been observed, making maximum allogeneic PBPC mobilization even more desirable.7-10,12,13,50 This seems crucial also in relation to latest results in the field of telomeres, in which telomere shortening after autologous and allogeneic transplantation has been demonstrated.51-53 It has been suggested that telomere loss may be reduced by transplanting higher numbers of progenitor cells, which—by adding to the prompt neutrophil and platelet engraftment—might reduce or completely prevent a substantial telomeric decline in hematopoietic cells. Therefore, maximum PBPC mobilization for transplantation in allogeneic and autologous patients seems important and warrants further study.


   ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of D. Wider, E. Samek, B. Gross, and F. Lucquiaud, and the helpful logistic support of M. Schäfholz and B. Schuler. We thank Prof Dr R. Mertelsmann for his continuous support, for valuable suggestions, and for critically reading the manuscript.


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Submitted August 13, 1998; accepted February 26, 1999.




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