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Keywords:

  • healthy donors;
  • peripheral blood stem cells;
  • mobilisation;
  • aphaeresis;
  • G-CSF

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Mobilised peripheral blood is now the main source of stem cells collected from normal donors. We report our experience of mobilising and collecting 400 normal healthy donors using standardised procedures and techniques. Target recipient doses were reached with one aphaeresis in 63% of donors and with two aphaereses in 81% of donors. Approximately 2% of donors yielded such low progenitor values that they were termed ‘poor mobilisers’. There were minor effects of donor age, weight and sex and where possible, larger male donors under the age of 55 years should be selected. Two forms of granulocyte colony-stimulating factor (G-CSF) were used at the same dose and no significant difference was seen in the yield of CD34+ cells collected/l of blood processed. However, a greater number of granulocyte-macrophage colony-forming cells were harvested using lenograstim (glycosylated G-CSF) compared with filgrastim (non-glycosylated G-CSF; P = 0·002). CD34+ cell yields were also measured halfway through the aphaeresis procedure. This was found to be highly predictive of final yield and facilitated distribution of the stem cell product to other centres. The observation that CD34+ yields did not decline in the second half compared with the first half of aphaeresis suggests that the circulating cell numbers are not static.

Mobilised peripheral blood stem cells are routinely used as the source of stem cells in high-dose therapy and autologous haemopoietic stem cell transplantation (Gratwohl et al, 2005). A variety of mobilisation regimes are used but the combination of chemotherapy and granulocyte colony-stimulating factor (G-CSF) is most frequently employed (Gratwohl et al, 2005). The factors predicting for the magnitude of stem cell mobilisation have been well-documented (Haas et al, 1994; Bensinger et al, 1995; Watts et al, 1997a; Ketterer et al, 1998) and there is widespread agreement that mobilisation is relatively poor in certain disease states (e.g. myeloma and indolent lymphomas) and following certain chemotherapy regimens {e.g. chlorambucil, fludarabine and Mini-BEAM [BCNU(carmustine), etoposide, cytarabine, melphalan]} and after wide-field radiotherapy.

Mobilised peripheral blood stem cells are also widely used in allogeneic transplantation and the normal donors are mobilised with G-CSF alone. A large registry study suggested that the short-term safety profile of stem cell mobilisation and collection in normal donors was comparable to that of marrow harvesting (Anderlini et al, 2001). A recent review of similar large registry reports appears to support this observation (Pulsipher et al, 2006).

Relatively little information is available about the factors predicting for satisfactory stem cell collections from normal donors. Several groups have reported that older age is associated with poorer stem cell yields, above 55 years of age in one study (Anderlini et al, 1999) and 38 years in a Spanish registry analysis (de la Rubia et al, 2002) although age has not been found to be a significant predictive factor in other series (Grigg et al, 1995; Miflin et al, 1996; Arbona et al, 1998). Several groups have reported that progenitor cell collection yield is related to donor weight and that the level of the peripheral blood white cell count and the total number of nucleated cells collected is a predictor of the number of stem cells harvested (Engelhardt et al, 1999; Croop et al, 2000; Kroger et al, 2000). Studies in normal volunteers have suggested that higher doses of G-CSF result in better stem cell mobilisation (Dreger et al, 1994; Engelhardt et al, 1999; Martinez et al, 1999) and that a given dose split into twice daily subcutaneous injections may be preferable to a single injection (Kroger et al, 2004). These are important issues as they may assist in donor selection when more than one donor is available, allow optimisation of the mobilisation regimen and facilitate the logistics of carrying out repeated aphereses.

Registry data can provide useful information, but analysing data from multiple sites can be difficult to interpret. For this reason, we have analysed a consecutive series of stem cell collections from 400 normal healthy donors at a single institution where there are standardised mobilisation regimens, collection protocols and progenitor cell measurements, including the use of granulocyte-macrophage colony-forming cell (GM-CFC) assays.

In addition, a group of 42 normal donors was assessed to determine whether the CD34+ cell yield halfway through the aphaeresis procedure could predict accurately for the final yield at the end of the aphaeresis. This information is of particular importance where cells are to be shipped to a distant centre. If an adequate number of cells could be predicted for the first complete harvest, arrangements could be made to transport the cells that day in a timely manner. If the harvest is likely to be sub-optimal, arrangements can be made to store the cells overnight and then dispatch them the following day together with the second harvest.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Donors

Peripheral blood stem cells (PBSC) were collected from 400 normal healthy subjects between May 1996 and September 2005. The majority of donors (n = 215) were collected for related transplants within our own centre and the remaining 185 subjects were harvested in our centre as part of the Anthony Nolan Trust programme for unrelated allogeneic transplants. Donor characteristics are shown in Table I. All donors were medically examined and gave written, informed consent.

Table I.   Donor characteristics. Age and weight are shown as medians (range).
 Donor categoryAge (years)Weight (kg)
RelatedUnrelatedAllRelatedUnrelatedAllRelatedUnrelatedAll
Male11614726341 (12–74)37 (20–59)38 (12–74)80 (41–126)85 (57–126)83 (41–126)
Female993813741 (14–70)35 (20–56)39 (14–70)66 (45–107)64 (51–112)65 (45–112)
All21518540041 (12–74)37 (20–59)38 (12–74)74 (41–126)83 (51–126)78 (41–126)

Mobilisation and aphaeresis

All donors were mobilised with G-CSF at 10 μg/kg/d s.c. Related donors received filgrastim (non-glycosylated G-CSF; Amgen, Thousand Oaks, CA, USA) and unrelated donors were mobilised with lenograstim (glycosylated G-CSF; Chugai Pharma, London, UK). G-CSF was administered for four consecutive days and aphaeresis commenced on day 5. Peripheral blood CD34+ cell counts were not routinely measured prior to aphaeresis. If the harvest CD34+ cell target yield was not achieved a further injection of G-CSF was given on the same day. A maximum of two aphereses were performed. Aphaeresis was performed on either the Baxter CS3000 (Baxter Healthcare, Deerfield, IL, USA; n = 46), the COBE Spectra v4·0 (n = 53) or the COBE Spectra v6·0 AutoPBSC (Gambro BCT, Quedgeley, Gloucester, UK; n = 301). In all instances acid-citrate-dextrose formula A (ACD-A) was used as the anticoagulant. The median (range) total blood volume (TBV) processed was 2·6 l (1·5–3·2). The target recipient CD34+ cell dose for related donors was 4 × 106/kg. For unrelated donors the target recipient doses ranged from 4 to 10 × 106/kg according to the request from the recipient centre.

An aphaeresis kit with twin collection bags, manufactured by Gambro.BCT for the COBE Spectra v6·0 AutoPBSC, was used to harvest the cells of 42 of the donors. This allowed accurate assessment of the progenitor content from the first and the second half of the aphaeresis procedure without interfering with the integrity of the sealed kit. The intention of these measurements was to determine whether the final cell yield could be predicted reliably by the progenitor content at the midpoint of aphaeresis and therefore allow timely shipping of the harvest. A halfway recipient CD34+ cell dose of ≥2 × 106/kg was assumed to indicate that a final recipient CD34+ cell dose of at least 4 × 106/kg would be achieved on completion.

White blood cell (WBC) count and flow cytometry

A sample was removed from the pilot line of the harvests to assess progenitor cell content. The WBC of donor peripheral blood and harvest products was assessed using an automated cell counter (KX-21, Sysmex, Milton Keynes, Bucks, UK). All of the harvest samples were analysed fresh (within 2 h) of collection. Harvest CD34+ cell numbers were determined by flow cytometry using a Milan/Mulhouse protocol as described previously (Pollard et al, 1999) using phycoerythrin (PE)-conjugated HPCA-2 monoclonal antibody staining or appropriate matched control (Becton Dickenson. San Jose, CA, USA). This was followed by red cell lysis (Coulter Q-prep) and flow cytometry (Coulter Epics XL-MCL or Coulter FC500, Beckman Coulter, High Wycombe, Beds, UK). The CD34+ cell profile was taken from the total leucocyte population determined by forward/side scatter and a ‘cluster gate’ set around CD34+ expressing cells determined by the dual characteristics of low light scatter and PE staining properties. The absolute CD34+ cell count was derived from the CD34+ cell percentage and the electronic cell WBC count (dual platform). Solely to exclude the rare possibility of incomplete red cell lysis, a CD45-flurescein isothiocyanate (FITC) antibody (Becton Dickenson) was added in 2003 in accord with Nordic recommendations for the Milan/Mulhouse protocol (Johnsen et al, 1999).

Colony assays

To measure clonogenic function, cells were plated at 2·5 × 104/ml in a commercially available methylcellulose-based medium (Stem Cell Technologies, Vancouver, BC, Canada) with added recombinant growth factors (G-CSF 25 ng/ml, granulocyte-macrophage colony-stimulating factor (GM-CSF) 25 ng/ml, interleukin (IL)-3 30 ng/ml, stem cell factor (SCF) 10 ng/ml and Erythropoietin 2 U/ml). GM-CFC and erythroid burst-forming units (BFU-E) were scored after incubation for 14 d at 37°C with 4% CO2 as previously described (Watts et al, 2002).

Statistical analysis

Statistical analysis was performed using GB-STATTM v6·5 PPC from Dynamic Microsystems, Inc. Silver Spring, MD, USA.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Donor characteristics

In accordance with the Anthony Nolan Trust guidelines, most (80%) of the unrelated donors harvested were male. Among the related donors, male and female distribution was approximately equal. Unrelated donors tended to be younger than related donors with median (range) ages of 37 years (20–59) and 41 years (12–74), respectively (P < 0·001). There was also a significant difference in weight between the two donor groups, with unrelated donors weighing a median of 83 kg (range: 51–126 kg) and related donors weighing 74 kg (range: 41–126 kg; P < 0·0001), with the higher weight in the unrelated donors reflecting the larger proportion of male donors.

Recipient thresholds

A summary of recipient CD34+ cell doses achieved is shown in Fig 1. Recipient doses were available for 391 patients. Nine recipient weights were not known and an absolute number of cells only were requested. Based on a required recipient CD34+ cell dose of 4 × 106/kg, 248 donations (63%) achieved this after one aphaeresis procedure. Of the 143 donors achieving <4 × 106/kg recipient body weight in one harvest procedure, 121 were apheresed on the second day. After the second collection a further 76 donations reached the target CD34+ cell threshold. After a maximum of 2 d aphereses, 324 (81%) donations achieved the target recipient CD34+ cell dose of 4 × 106/kg, 37 (9%) gave recipient doses of between 2–4 × 106/kg and 8 (2%) attained <2 × 106/kg CD34+ cells. The cell doses, donor characteristics and outcomes of these 8 poor donations are summarised in Table II.

image

Figure 1.  Recipient CD34+ cell dose achieved at aphaeresis from 400 donors (CD34+ cell dose × 106/kg shown in brackets, see text for details).

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Table II.   Peripheral blood stem cell CD34+ cell doses × 106/kg, donor characteristics and action taken following eight poor donations.
No.Recipient CD34+ doseDonor CD34+ doseTotal CD34 × 106AgeWeight (kg)SexRelated/ unrelatedAction taken
  1. PBSC, peripheral blood stem cells, BM, bone marrow.

10·40·750·95279MRTransplanted with autologous back-up PBSC
20·90·744·62760MRNo further cell collection, ‘Top up’ for prior transplant
31·21·599·94567MRBM harvest, CD34 = 1·0 × 106/kg (7 d post-PBSC)
41·51·688·75254FUBM harvest, CD34 = 1·1 × 106/kg (3 d post-PBSC)
51·61·7136·02380MRNo further cell collection
61·61·8175·45895FRNo further cell collection
71·71·9136·04571MUAccepted by recipient transplant centre.
81·92·2133·45662FRNo further cell collection

Of the 22 donations that failed to reach the required recipient dose in one day, but were not re-harvested, 19 achieved ≥2 × 106/kg and were deemed adequate by the recipient transplant centre. The remaining three donors only achieved recipient cell doses of (a) 0·3, (b) 0·9 and (c) 1·2 × 106/kg CD34+ cells. Donor (a) had a peripheral blood CD34+ cell count of 0·006 × 109/l the following day and was deemed unsuitable for further aphaeresis and a bone marrow harvest was performed. This gave an additional recipient CD34+ cell dose of 0·7 × 106/kg. Donor (b) was only the fifth donor to be collected in the series and at that time optimal CD34+ doses were not well-defined for allogeneic transplantation. Donor (c) was a sibling donor who was re-harvested for a ‘top-up’ transplant procedure and was not subjected to further aphaeresis.

Factors correlating with total CD34+ cell yield

There was a wide range in the number of CD34+ cells collected at all ages and there was no significant correlation between the numbers of CD34+ cells collected when the data was viewed as a whole. Previous authors have identified 38 years (de la Rubia et al, 2002) and 55 years (Anderlini et al, 1999) as ages above which progenitor yields are, on average, reduced. In our data set, donors aged 55 years or more collected significantly fewer CD34+ cells than younger donors with a median (range) yield of 280 (43–624) and 342 (22–1491) × 106 CD34+ cells respectively (P < 0·001). At an age of 38 years, the difference in progenitor cell yield was much less marked than with a threshold of 55 years. In the 38–54 year age group, the median CD34+ cell number collected was very similar to those under 38 years of age. The median (range) yield was 380 (22–1355) below the age of 38 years and 309 (24–1491) CD34+ cells above this age (P = 0·3).

There was only a weak correlation between donor weight and the absolute CD34+ cell number collected (r = 0·43) but the difference in CD34+ cells collected from donors above and below the median weight of 78 kg was highly significant. The median (range) CD34+ cells collected from donors below 78 kg was 274 (22–1356) × 106 whereas donors that weighed >78 kg gave a median yield (range) of 438 (72–1491) × 106 (P < 0·0001).

Peripheral blood WBC counts were available for 397 donors. On the first day of aphaeresis the median WBC count of all donors was 41·4 × 109/l (range: 14·0–83·4). There was no correlation between the peripheral blood white cell count on the morning of the first aphaeresis and the absolute number of CD34+ cells collected, r = 0·18. However, an analysis of CD34+ cell yield from donors where the peripheral blood WBC count was above the median showed a significant increase compared with those donors below this threshold. Donors with a blood WBC count below 41·4 × 109/l collected a median (range) of 308(22–1491) × 106CD34+ cells compared with 380 (22–1380) × 106 where the WBC count exceeded this (P = 0·001).

A detailed comparison of cells collected from male and female donors is shown in Table III. Male donors achieved significantly greater numbers of both CD34+ cells and GM-CFC in absolute terms than female donors, median (range) 389 (21–1491) versus 270 (22–1355) × 106, for CD34+ cell counts (P < 0·0001) and 6769 (264–23 413) versus 4686 (586–19 124) × 104 for GM-CFC respectively (P < 0·0001). This difference could, however, be explained by the greater TBV and greater weight of male donors and so progenitor yields were also compared on the basis of cells collected/l of blood processed. Females harvested a median (range) CD34+ cell yield of 28·5 (2·1–91·3) × 106/l, whereas males harvested 29·6 (1·9–122·5), (P = 0·03). The number of GM-CFC/l of blood processed was not significantly different between male and female donors.

Table III.   A comparison of aphaeresis products from male and female donors.
 MaleFemaleP-value
  1. Values are given as median (range). Cell doses are based on the donor weight. P-values derived from Students t-test. NS, not significant; TBV, total blood volume.

PB WBC (×109/l)40·7 (14·0–83·4)43·6 (20·4–73·2)NS
Inlet volume (ml)13 464 (7530–18 995)10 000 (4656–17 735)<0·0001
TBV processed (l)2·6 (1·7–3·0)2·6 (1·5–3·2)NS
Harvest volume (ml)405 (56–871)266 (45–706)<0·0001
Harvest WBC (×109/l)112 (57–925)138 (26–820)NS
Harvest CD34 (%)0·90 (0·06–3·90)0·71 (0·09–2·00)<0·0001
CD34+ cells (×106)389 (21–1491)270 (22–1355)<0·0001
CD34+ dose (×106/kg)4·7 (0·4–18·0)4·1 (0·4–18·1)<0·001
CD34+ cell yield × 106/l blood processed29·6 (1·9–122·5)28·5 (2·1–91·3)0·03
GM-CFC × 1046769 (264–23 413)4686 (586–19 124)<0·0001
GM-CFC dose (×104/kg)82·1 (3·3–289·6)71·8 (10·3–251·6)<0·005
GM-CFC yield × 104/l blood processed531 (26–1817)525 (56–1886)NS

The peripheral blood white cell count on the morning of the first aphaeresis and the white cell count of the harvest product did not significantly differ between male and female donors. However, the percentage of CD34+ cells and the volume collected were significantly higher in males.

Type of G-CSF used

A statistically significant difference was seen in the number of CD34+ cells collected between related and unrelated donors, (P < 0·0001), probably due to the imbalanced ratio of male to female donors. The number of female-unrelated donors was considered too small for a statistical comparison with female-related donors, however a comparison of the effects of G-CSF type used between the male donors was feasible and is summarised in Table IV. The peripheral blood WBC count was marginally higher for male donors mobilised with filgrastim than with lenograstim on the morning of the first aphaeresis, median (range) 42·2 (14·0–83·4) versus 39·7 (16·9–80·5) × 109/l, (P = 0·02). There were significant increases in CD34+ cell yields in those males receiving lenograstim in terms of absolute numbers and as a dose based on the donor's weight but this difference was not apparent when the CD34+ cell yield/l of blood processed was compared. Significant increases were also seen in the number of colony-forming cells in absolute numbers and as a dose based on donor weight. In this instance, however, the increase was still significant when the GM-CFC yield/l of blood was compared. Lenograstim mobilised male donors achieved a median (range) yield of 622 (72–1817) × 104GM-CFC/l of blood processed compared with a median (range) of 467 (26–1700) × 104/l for filgrastim mobilised subjects (P = 0·002).

Table IV.   Comparison between male donors receiving filgrastim or lenograstim at 10 μg/kg/d.
 FilgrastimLenograstimP-value
  1. Values are given as median (range). Cell doses are based on the donor weight. P-values derived from Students t-test. NS, not significant; TBV, total blood volume.

n116147 
Age (years)41 (12–74)37 (20–59)0·01
Weight (kg)80 (41–126)85 (57–126)<0·001
PB WBC (×109/l)42·2 (14·0–83·4)39·7 (16·9–80·5)0·02
Inlet volume (ml)11 662 (7530–187100)14165 (9166–18 995)<0·0001
TBV processed (l)2·5 (1·7–2·8)2·6 (1·7–3·0)0·02
Harvest CD34%0·80 (0·06–2·30)0·93 (0·18–3·90)NS
CD34+ cells × 106313 (22–1404)438 (56–1491)<0·001
CD34+ dose × 106/kg4·0 (0·4–12·9)5·1 (0·8–18·0)0·002
CD34+ cell × 106/l blood processed27·5 (1·8–102·8)30·8 (4·12–122·5)NS
GM-CFC × 1045326 (264–23 350)8552 (984–23 413)<0·0001
GM-CFC dose × 104/kg67·1 (3·3–257·9)97·0 (13·9–289·6)<0·0001
GM-CFC × 104/l blood processed467 (26–1700)622 (72–1817)0·002

Multivariate analysis

The results of a multivariate analysis of factors influencing progenitor cell yields are summarised in Table V. The formulation of G-CSF used was not significant when applied to the yield of CD34+ cells/l of blood processed yet it was the major factor determining the yield of GM-CFC. Donor age and weight had little effect upon GM-CFC collection but both were significant with regard to CD34+ cell yield. Donor gender was not a factor influencing the mobilisation and collection of either CD34+ cells or GM-CFC/l of blood processed.

Table V.   Multivariate analysis of factors influencing progenitor cell yields.
 All donors (P-value)Male donors (P-value)
  1. Yields measured as cells collected/l of blood processed. NS, not significant; N/A, not applicable.

CD34+ cell yield
 SexNSN/A
 Age0·0340·037
 Weight (kg)<0·00010·015
 Type of G-CSFNSNS
GM-CFC yield
 SexNSN/A
 AgeNSNS
 Weight (kg)NSNS
 Type of G-CSF0·010·004

CD34+ cell yield and aphaeresis machine used

Three different aphaeresis machines were used for collecting PBSC from normal healthy donors. The CD34+ cell yields/l of blood processed were almost identical for the CS3000, Spectra v4·0 and Spectra v6·0 AutoPBSC. A comparison of the machine CD34+ cell yields showed no significant differences although numbers were small for both the CS3000 and COBE Spectra v4·0 (data not shown).

Mid-point aphaeresis CD34+ cell counts

Forty-two donors were apheresed using the Gambro.BCT twin-bag collection kit. Twenty-four donors achieved a recipient CD34+ cell dose of ≥2 × 106/kg midway through aphaeresis and, in every case, achieved the target dose of 4 × 106/kg when the entire procedure was completed. These harvests were all booked for shipping on the same day based on the mid-point measurement. Three aphaeresis procedures out of 18 that were deemed inadequate at the halfway stage also achieved the final desired recipient dose. Of the 15 patients not achieving the target dose after one day of aphaeresis, three were deemed acceptable by the transplant centre with recipient CD34+ cell doses of 3·2, 3·7 and 3·9 × 106/kg.

Eleven of the remaining 12 donors who did not reach the target recipient dose were apheresed for a second day. Six reached the target with five failing, but two were close to the threshold at 3·8 and 3·9 × 106/kg recipient body weight. One donor was not harvested again as his platelet count was below the guidelines issued by the Anthony Nolan Trust (<80 × 109/l).

No significant difference was seen between either the CD34+ cells or GM-CFC collected between the first and second half of the cell collection. The median (range) CD34+ cell and GM-CFC counts were 177 (34–584) × 106 and 3488 (320–9452) × 104 for the first half of the aphaeresis procedure and 176 (23–585) × 106 and 3488 (592–9284) × 104 for the second half respectively.

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The mobilisation with G-CSF and the harvesting of peripheral blood stem cells from normal donors appears to be both safe and efficient in many reports. Indeed, most allogeneic transplants now utilise peripheral blood rather than bone marrow as a source of stem cells (Gratwohl et al, 2005). This single institution study confirms the effectiveness of G-CSF mobilisation in a large group of normal healthy donors. A large single institution report from the MD. Anderson Center (Anderlini et al, 1999) showed that approximately 70% of their normal donors could achieve the required recipient dose of 4 × 106/kg in a single aphaeresis procedure. In our study 63% of donations from a single aphaeresis achieved this target threshold, although a further 19 collections with a CD34+ cell yield close to this target were deemed adequate by the recipient transplant centre, thus increasing the success rate to 68%. Eight donations (2%) failed to achieve a recipient CD34+ cell dose of 2 × 106/kg with 2 (0·5%) failing to achieve a dose of 1 × 106/kg after 2 d of collection. It is known that a proportion of patients with haematological malignancies fail to mobilise sufficient stem cells to provide support for high dose therapy (Watts et al, 2000). This study highlights the fact that some normal healthy individuals could also be referred to as ‘poor mobilisers’ although the reasons for this are not known.

Roberts et al (1995) were probably the first to report that the progenitor mobilisation response to G-CSF in healthy subjects is so broad that the lowest normal responses overlapped with some pretreated cancer patients. However, remobilisation studies in healthy subjects indicate that the interindividual variation is much less than that within individuals. In a cross-over remobilisation study comparing G-CSF preparations in 20 healthy subjects for example, we reported over a 10-fold variation between individuals but much less so in the same individuals. Notably, the two poorest responders were ‘poor mobilisers’ on repeat mobilisation regardless of the G-CSF type given (Watts et al, 1997b). These data are in accord with the similar progenitor yields on remobilisation of 13 donors (Anderlini et al, 1997), and confirmed in a similar study of 67 donors (Platzbecker et al, 2005) for male donors, although there was a significant reduction in CD34+ cell yields reported on remobilisation of female donors in this study (P = 0·008). The reason for this wide individual response to G-CSF in healthy subjects is not clear, although mobilisation studies in inbred mouse strains suggest that genetic factors play a part (Geiger et al, 2004).

The impact of donor age and weight was not major, but where there are two or more potential donors available, there is an advantage from the viewpoint of stem cell yield in using larger donors under the age of 55 years. It is noteworthy that there was no obvious impact of different ages under 55 years. The WBC count varied by almost six-fold amongst donors, with a range of 14·0–83·4 × 109/l after four doses of G-CSF but no prediction of CD34+ cell yield could be made from the white cell count on the day of the first aphaeresis.

Although the CD34+ cell and GM-CFC absolute number and dose collected were higher in male than female donors, this difference was markedly reduced when the yield/l of blood processed was considered. The CD34+ cell yield × 106/l of blood processed remained significant (P = 0·03) but not the GM-CFC yield × 104/l.

Two types of G-CSF were used in this study, the non-glycosylated form, filgrastim, for related donors and the glycosylated form, lenograstim, for unrelated donors. Both preparations were given at the same dose of 10 μg/kg/d. In two previous intra-individual ‘cross-over’ studies, male healthy subjects showed significantly increased progenitor numbers mobilised with glycosylated compared to non-glycosylated G-CSF given as a weight by weight dose of either 10 μg/kg/d (Hoglund et al, 1997) or 5 μg/kg/d (Watts et al, 1997b). It would be expected that this difference in potency would be evident between donors if the two groups receiving the two G-CSF formulations were large enough. Due to the small number of unrelated female donors in this study only a comparison of male donors has been reported here related to the G-CSF type. In accord with our findings, a recent large donor study of 501 patients (Fischer et al, 2005) reported significantly increased numbers of CD34+ cells in male donors mobilised with lenograstim (n = 173) compared with filgrastim (n = 166) in terms of absolute numbers and as a dose/kg donor weight, although the CD34+ cell yield/l of blood processed was not given and the study did not include colony assay data. Interestingly, no significant difference was seen between female donors receiving lenograstim (n = 88) or filgrastim (n = 74) in this report. Martino et al (2005) compared the mobilisation efficacy in 101 donors of filgrastim (n = 55) compared with lenograstim (n = 46) and reported no differences in either CD34+ cell or GM-CFC harvested. There were differences both in the way that G-CSF was given and in aphaeresis harvest timing in this study. G-CSF was administered as a divided dose each day rather than as a single daily injection, and aphaeresis commenced based on blood CD34+ cell counts. The G-CSF dose varied between 5 to 14 μg/kg/d although at comparable weight-by-weight doses for each G-CSF type. The majority of patients (over 60%) were harvested on day 4 and the remainder on days 5, 6 or 7 rather than commencing on a fixed day. There were also 22 female donors in each donor group receiving filgrastim or lenograstim in this study. In light of the Fischer study (2005), mixed gender donor groups may lessen differences in mobilisation between G-CSF type used.

Although the CD34+ cell yield/l blood processed was not significant between male donors in our comparison of G-CSF formulations, the median number of GM-CFC/l of blood processed was 25% greater with the glycosylated form (P = 0·002). GM-CFC are, on average, more primitive than the total CD34+ cell population and whether the increase in the clonogenic fraction with the different types of G-CSF is a biological potency issue (Mire-Sluis et al, 1995) or a qualitative functional difference between the two forms of G-CSF, is unknown. This also raises the issue of whether engraftment is more closely related to the CD34+ cell dose or the GM-CFC dose. Although we routinely make decisions based on CD34+ cell counts for logistic reasons, we have found GM-CFC levels to be more predictive of engraftment when progenitor cell numbers are limiting (Watts et al, 1997a).

Kroschinsky et al (2005) have recently reported the successful mobilisation of 25 healthy donors with a novel pegylated analog of G-CSF (Neulasta, Amgen, Thousand Oaks, CA, USA). The reduced clearance time of this form of G-CSF means that a single injection has a similar bioavailability to multiple daily injections of unconjugated G-CSF. In the Kroschinsky study, a single injection of 12 mg pegylated G-CSF was administered on day 1 and aphaeresis commenced on day 5 for all donors. The peak blood CD34+ cell levels were achieved earlier on days 4 and 5, but yields were not regarded as different from daily injections of G-CSF in the authors’ experience. They advised caution, however, in that the total cytokine dose was much higher than that normally given to healthy donors and that differences in the immune cell composition of the graft may give rise to different patterns of graft versus host/graft versus leukaemia effects (Kroschinsky et al, 2005).

Progenitor cell yields did not vary between the aphaeresis machines used. However, the COBE Spectra v6·0 AutoPBSC has the advantage of greater control of the WBC concentration (Watts et al, 2004). A WBC concentration below 0·2 × 109/l, may reduce the loss of clonogenic potential with cell products that require storage and transportation (Watts et al, 2003) and is in accord with UK guidelines (James, 2002).

Mid-point harvest CD34+ cell counts proved to be highly predictive of the final yield. All 24 of the 42 donors (57%) who had a halfway recipient CD34+ cell dose of 2 × 106/kg were successful in achieving a final recipient dose of at least 4 × 106/kg on completion that day. In practical terms, this meant that arrangements could be made for shipping the cells the same day as soon as the mid-point count was available. If the midpoint recipient CD34+ cell dose is close to 2 × 106/kg a modest prolongation of the aphaeresis procedure might obviate the need for a repeat procedure.

The observation that progenitor yields did not fall between the first and second half of the aphaeresis process is of particular interest. The efficiency of the aphaeresis process is thought to be about 50% (Rowley et al, 1999; Heuft et al, 2000). That is to say that for every blood volume processed, 50% of the CD34+ cells present in the donor circulation are harvested. The second half of the aphaeresis procedure should therefore yield considerably fewer progenitor cells than the first if circulating progenitor numbers are static. This was not the case, and although it is highly unlikely that stem cells are generated de novo from the marrow during the short period of aphaeresis, progenitor cells are adherent by nature and a significant proportion could be loosely marginating on vascular surfaces. The possibility that stem cells might be ‘recruited’ during aphaeresis has been suggested previously (Cull et al, 1997; Smolowicz et al, 1999; Knudsen et al, 2001) in patients with haematological malignancies. The present study showed that progenitor cells were collected pro rata in healthy donors depending on the duration of aphaeresis. It is tempting to speculate that one of the newer approaches to enhance mobilisation by reducing progenitor adhesion, such as AMD3100 (Fruehauf et al, 2005), might have the potential to rapidly increase the circulating pool of progenitors and thereby reduce the duration of aphaeresis required.

This study shows that the mobilisation of progenitor cells into the circulation of normal donors for use in allogeneic stem cell transplantation is highly efficient and that, in most cases, sufficient cells can be readily collected. It also demonstrates the large variation between normal healthy individuals in the extent of G-CSF mobilisation of haemopoietic stem cells and that careful choice of donor, and use of G-CSF type may help to optimise stem cell yields.

Acknowledgments

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors would like to acknowledge the staff of the haematology day care unit for the excellent aphaeresis service that they provide and for their active participation in this study.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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