Experimental Hematology 27 (1999) 526–532

0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(98)00045-9

Granulocyte colony–stimulating factor–mobilizedperipheral blood stem cells in

b

-thalassemia patients:kinetics of mobilization and composition of apheresis product

Karen Li

a

, Annie Wong

a

, Chi Kong Li

a

, Matthew Ming Kong Shing

a

,Ki Wai Chik

a

, Kam Sze Tsang

b

, Howard Lai

a

, Ting Fan Leung

b

, and Patrick Man Pan Yuen

a

,

a

Departments of Paediatrics,

b

Anatomical and Cellular Pathology,The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, Republic of China

(Received 11 March 1998; revised 24 June 1998; accepted 12 August 1998)

b

-Thalassemias are often associated with bone marrow expan-sion and immunomodulation in terms of lymphocyte subsetsand cytokine levels in the peripheral blood. The mobilization ofperipheral blood stem cells (PBSC) by cytokines in such abackground has not been reported. If achieved, the apheresisproduct could be used as a stem cell back-up for

b

-thalassemiapatients prior to bone marrow transplant. PBSC collectionmay also become a means for providing stem and progenitorcells for gene manipulation and therapy of this disorder. Theaim of the study was to assess the administration of G-CSF inmobilizing stem and progenitor cells in these patients and tocompare the kinetics of CD34

1

cells and lymphocyte subsetswith those of healthy PBSC donors. Results showed that theCD34

1

cells were effectively mobilized by G-CSF (10–16

m

g/day per kg) in 20 thalassemia patients and 11 healthy donors.Although no significant difference was observed in levels ofdaily stem cell counts between the two groups of subjects, a 1day delay in achieving peak levels of CD34

1

cells was observedin the majority of thalassemia patients. The peak increase ofCD34

1

cells was 21.5-

6

6.1-fold and 30.8-

6

7.6-fold of thebasal steady-state levels in thalassemia patients and healthydonors, respectively. Similar to the situation of healthy donors,G-CSF stimulated essentially the CD34

1

cells and the myeloidlineage (granulocytes, monocytes) in thalassemia patients andhad a slight effect on lymphocyte subsets (T-helper, T-suppres-sor, NK, and B cells) and activation (CD25, HLA-DR, andCD45RO). Compositions of the apheresis products, includingCD34

1

CD38

2

, CD34

1

CD33

1

and CD34

1

HLA-DR

2

cells, weresimilar in the two groups of subjects. Correlation studiesshowed that the level of CD34

1

cells in the PB is a good indica-tor of that in the apheresis product (

r

5

0.88,

p

,

0.001). Thestudy has demonstrated that under close monitoring of CD34

1

cell levels in PB, the mobilization by G-CSF and collection ofPBSC in

b

-thalassemia patients are feasible. © 1999 Interna-

tional Society for Experimental Hematology. Published byElsevier Science Inc.

Keywords:

Peripheral blood stem cell—Granulocyte-colony

stimulating factor—

b

-Thalassemia

Introduction

Since the first successful transplantation for

b

-thalassemiamajor in 1982 [1], bone marrow transplant (BMT) remainsthe only curative treatment for this disorder. However, thereare risks of severe, transplant-related complications, partic-ularly in the older

b

-thalassemia patients with hepatomeg-aly and portal fibrosis [2]. Graft rejection may occur in up to30% of high risk patients and some patients may developmarrow aplasia [3]. Some centers have adopted the policyof saving autologous BM as back-up stem cells for reinfu-sion if marrow aplasia develops. An alternative way of ob-taining back-up stem cells is the collection of peripheralblood stem cells (PBSC) after mobilization by granulocyte-colony stimulation factor (G-CSF). Mobilized PBSC fromthalassemia patients could also be used for gene manipula-tion as a potential therapy of the disorder [4–6]. However,impaired mobilization of PBSC was reported in some pa-tients with inherited hematopoietic disorders such aschronic granulomatous disease and adenosine deaminasedeficient severe combined immunodeficiency disease [7].Thalassemia patients suffer from marrow expansion and im-munomodulation, including the alterations of lymphocytesubsets, abnormal levels of cytokines, and presence of allo-antibodies [8–14]. We have previously shown that somethalassemia children have decreased NK levels and in-creased activation status in terms of the expression of inter-leukin-2 receptor (CD25), HLA-DR and CD45RO on lym-phocytes [15]. An increase in serum levels of cytokines andsoluble antigens such as sCD25, tumor necrosis factor

Offprint requests to: Chi Kong Li, M.B.B.S., Department of Paediatrics,Room G15, Lady Pao Children’s Cancer Centre, Prince of Wales Hospital,Shatin, N.T., Hong Kong, China; Email: ckli@cuhk.edu.hk

K. Li et al./Experimental Hematology 27 (1999) 526–532

527

(TNF), and IL-8 has also been noted in thalassemia patients[11,12]. The mobilization of PBSC against this immunolog-ical background has not been reported. This project aimedto study the efficiency of using G-CSF in the mobilizationof CD34

1

stem and progenitor cells in thalassemia patientsand to compare the kinetics of mobilization with that ofhealthy PBSC donors.

Materials and methods

Patients, donors, and G-CSF treatment

Recombinant human G-CSF (Amgen, Inc, Thousand Oaks, CA)was administered (in the morning) subcutaneously to 20 consecu-tive patients who had

b

-thalassemia major and 11 consecutivehealthy donors of whom 3 were for the thalassemia patients (Table1). There were no significant differences between the two groupsin terms of age, G-CSF treatment and the number of apheresis. Themajority of subjects were under 17 years of age except three fromthe thalassemia group (ages 18, 18, and 21 years) and three fromthe donor group (ages 19, 31, and 36 years). The female-to-maleratio was higher in the thalssemia group. G-CSF was initially givenat 16

m

g/kg per day (single dose) but later in the study, the dosagewas reduced to 10

m

g/kg per day as reports have shown that thislower dosage is also effective in mobilizing stem cells. After 4days of G-CSF treatment, stem cell harvest was performed on day5 with the Fenwal CS3000 (Baxter, Deerfield, IL) or Cobe Spectra(COBE Laboratory Inc., Lakewood, CO) cell separators. The pro-cessing blood volume for each apheresis was targeted at 2 timesthe blood volume. The minimal target CD34

1

cell dose was 1

3

10

6

/kg from thalassemia patients as backup stem cells and 3

3

10

6

/kg recipient body weight from healthy donors for allogeneic PBSCtransplant. The daily G-CSF treatment continued until stem cellharvests were completed. Informed consent was obtained from par-ents and from donors if they were over 16 years old. The study wasapproved by the Ethical Committee of The Chinese University ofHong Kong.

Flow cytometry analysis

PB was collected before G-CSF administration (day 1) and dailyafterwards until one day after the completion of stem cell harvest-ing. Nucleated cells, CD34

1

stem and progenitor cells, lymphocytesubsets were measured by flow cytometry. This was performed ona FACScan instrument (Becton Dickinson, San Jose, CA [BD]) us-

ing three-color combination of fluorochrome-conjugated antibod-ies. Whole blood and PBSC samples (1

3

10

6

cells) were lysedwith the FACS lysing solution (BD) according to the manufac-turer’s protocol. A total of 78,000 and 10,000 cells were acquiredfor enumerating CD34

1

cells and lymphocyte subsets, respec-tively, using the Lysis II software. All antibodies were products ofBD unless described otherwise. The following antibodies were used:isotypic controls, CD34-FITC, CD34-PE, CD34-PerCP, CD38-PE,HLA-DR-PerCP, CD33-PE, CD19-FITC (Dako, Denmark), CD3-PerCP, CD4-FITC, CD8-PE, CD3-FITC/CD16CD56-PE, CD122-PE, CD45RA-FITC, CD45RO-PE, CD28-PE, CD16-FITC (Dako),CD3-PerCP and CD8-PerCP. Mononucleated cells (MNC) weregated by using either CD45-FITC/CD14-PE or CD45-FITC/SSC.At least two CD34 tubes were acquired and the results averaged,after subtracting the background cells of the isotypic control tubes.The T-helper, T-suppressor, and NK cells were identified asCD3

1

CD4

1

, CD3

1

CD8

1

, and CD3

2

CD16

1

CD56

1

respectively.CD34

1

subsets are expressed as the percentage of cells in theMNC population and lymphocyte subsets as percentage of cells inthe lymphocyte population. Complete white cell counts were deter-mined by the Coulter counter.

Statistical analysis

We used the Mann-Whitney Rank Sum Test to compare immu-nophenotypes of white cell and CD34

1

cell subsets in the thalas-semia patients and normal donors. The distribution of peak valuesof CD34

1

cells were compared between the two groups, using theTest of Proportion. The correlation between CD34

1

cells in the pe-ripheral blood (PB) and in the PBSC was analyzed using the Spear-man Correlation Coefficient. A

p

value of

,

0.05 was consideredstatistically significant. Results were expressed as mean

6

stan-dard error of the mean (SEM).

Results

Kinetics of stem cell mobilization

No serious side effects were observed, other than mild bonepain and fever in some subjects in both groups. The increaseof CD34

1

cells in the PB in terms of percentage of MNC,absolute cell count per milliliter, and the number of fold ofthe baseline concentrations are depicted in Figure 1. Therewas no significant difference in levels of CD34

1

cells ateach day after G-CSF administration in thalassemia patientswhen compared with those in donors. The peak increase ofCD34

1

cells reached 21.5-

6

6.1-fold (range 4.2- to 82.4-fold) and 30.8-

6

7.6-fold (range 10.6- to 71.3-fold) ofbaseline levels in thalassemia patients and normal donors,respectively. However, the majority of thalassemia patientsexperienced a peak level of CD34

1

cells at day 6 (range,days 4–8) while most of normal donors had peak CD34

1

cell levels at day 5 (range, days 4–6) (Table 2). A signifi-cantly higher proportion of thalassemia patients had peakpercentages of CD34

1

cells at day 6 when compared withthe donors (

p

5

0.008). We did not encounter nonre-sponders to G-CSF in either group, although one thalas-semic patient had peak CD34

1

cell count at day 8.

Table 1.

Clinical data of thalassemia patients and normal donors

Thalassemia patients Normal donors

Mean

6

SE Range Mean

6

SE Range

Number of subjects 20 11Female:male ratio 10:10 3:8Age (years) 11.9

6

1.1 3.0–21.0 15.4

6

3.2 0.80–36.0Dose of G-CSF

(

m

g/kg/day) 12.5

6

0.6 10.0–16.0 12.4

6

0.8 10.0–16.0Duration of G-CSF

(days) 4.7

6

0.2 4.0–6.0 4.9

6

0.1 4.0–5.0Number of apheresis 1.7

6

0.2 1.0–3.0 1.9

6

0.2 1.0–3.0

528

K. Li et al./Experimental Hematology 27 (1999) 526–532

Kinetics of white cell mobilization

There were no significant differences in the proportion andnumber of white cell populations in thalassemia patientsand donors at each day after G-CSF treatment. The whitecell counts increased by 3.6-

6

0.8-fold and 4.1-

6

1.5-foldafter 24 hours of G-CSF treatment (day 2) and reached4.9-

6

2.3-fold and 5.4-

6

1.3-fold by day 5 in thalassemiapatients and donors, respectively. The percentages of granu-locytes in the white cell population increased by 1.3-

6

0.1-fold and 1.4-

6

0.1-fold at day 2 when compared to baselinelevels (day 1), and stayed at high levels (maximum levels of1.5-

6

0.2-fold; 1.6-

6

0.3-fold) until day 7. The absolutegranulocyte counts were also increased in the two groups ofsubjects (maximum levels of 6.9-

6

3.7-fold; 7.9-

6

2.5-fold). The percentages of monocytes decreased at day 2(0.6-

6

0.2-fold; 0.5-

6

0.1-fold) and gradually returned tobaseline percentages at day 6. The absolute monocytecounts, however, were increased up to 4.0-

6

2.2-fold and5.3-

6

1.7-fold in the thalassemia patients and normal do-nors, respectively. The percentages of lymphocytes weredecreased (0.4-

6

0.1-fold; 0.3-

6

0.2-fold) after 24 hoursof G-CSF treatment and stayed at similar levels throughoutthe period of study. The absolute cell counts of lymphocyteswere slightly increased (2.0-

6

0.8-fold; 1.8-

6

0.4-fold).Effects of G-CSF on the proportions of lymphocyte sub-

sets are depicted in Figure 2. There was no significant dif-ference between the daily level of all subsets in the thalas-semia patients and donors. The percentages of lymphocytesubsets did not display significant changes during G-CSFtreatment.

Apheresis products

Table 3 summarizes the CD34

1

cell subsets in the first aph-eresis collection from patients and donors. There were nosignificant differences in the CD34

1

cell subsets and lym-phocyte subsets (CD33, HLA-DR, CD38, CD3, CD4, CD8,

counts over baseline levels. There was no significant difference in the dailyCD34

1

cell levels between the thalassemia patients and healthy donors.

Figure 1.

Mobilization of CD34

1

cells by G-CSF. G-CSF at 10–16

m

g/kgbody weight were administered to 20 thalassemia patients (

j

) and 11healthy donors (

h

). Peripheral blood CD34

1

cells were analyzed daily,starting at baseline levels (day 1) before the first G-CSF treatment. Results(mean

6

SE) are expressed as the percentages of CD34

1

cell in MNC,absolute cell counts per milliliter of PB and fold increase in absolute cell

Table 2.

Distribution of peak CD34

1

cells in peripheral blood

Percentage of subjects in each group

% Thalassemias % Normal donors

Day 5 Day 6 Day 5 Day 6

% MNC 35 55* 73 18*Absolute counts 42 47 64 18Fold increase 31 46 67 33

The figures represent percentages of subjects in each group who experi-enced peak CD34

1

cell levels at day 5 and day 6 of mobilization.*There was a significantly high proportion (

p

5

0.008) of thalassemiapatients who had peak percentages of CD341 cells at day 6 when comparedwith the donors.

K. Li et al./Experimental Hematology 27 (1999) 526–532 529

Figure 2. Mobilization of lymphocyte subsets by G-CSF. Results (mean 6 SE) are expressed as percentages of subsets in the lymphocytes. There was no sig-nificant difference in the daily levels of subsets between the thalassemia patients (j) and healthy donors (u).

530 K. Li et al./Experimental Hematology 27 (1999) 526–532

CD19, CD16.CD56, CD122, CD25, CD45RA, CD45RO,CD28) as expressed in percentages or absolute cell countsin the two groups. There was a strong correlation betweenthe percentage of CD341 cells in the PBSC harvest and inthe PB collected at the same day (regression coefficient r 50.83, p , 0.001 for thalassemia patients; r 5 0.90, p , 0.001for donors; r 5 0.88, p , 0.001 for all samples [Fig. 3]).

DiscussionAllogeneic bone marrow and more recently, cytokine-mobi-lized peripheral blood stem cells have been applied success-fully for the transplant of b-thalassemia patients [2,3,16].However, these patients are iron-overloaded and possiblyimmunomodulated as a complication of frequent bloodtransfusion [8–14]. Our previous study has demonstrateddecreased NK cells and high levels of activated lympho-cytes in some Chinese, thalassemia children in terms ofCD25, HLA-DR, and CD45RO expression [15]. Levels ofthese subsets were correlated with the number of transfu-sions given to the patients. The response to G-CSF and thecollection of PBSC in b-thalassemias have not been re-ported. Our present results have shown that CD341 cellswere effectively increased in terms of percentages of MNC,folds of basal levels, and absolute cell counts in both groupsof subjects. Our data on healthy donors were in broad agree-ment with those reported on healthy donors of older ages[17–25]. Dreger et al. [17] reported a 31-fold enrichment ofCD341 cells from nine healthy donors with peak valuesconstantly occurring 4 days after G-CSF treatment at 10 mgper day per kg of body weight. Using the same dose, Grigget al. [24] observed a CD341 peak between day 4 and 6 in15 donors. Although there was no significant difference inpeak daily stem cell counts between the two groups of sub-jects, there was a trend towards better mobilization in the

normal donors. This was again demonstrated in the delay of1 day in achieving peak levels of CD341 cells in the major-ity of thalassaemia patients. The proportions of stem andprogenitor subsets in the apheresis products from thalas-semia patients, including CD341CD382, CD341CD332,and CD341HLA-DR2 cells, were comparable with thoseobtained from our healthy donors or from other studies[18,20–22]. In our center, the apheresis product was cryo-preserved as back-up stem cells. Since we did not encounterany need for the re-infusion of PBSC to the patients, wehave no engraftment data for comparing with autologous or

Table 3. CD341 cell subsets in the apheresis product

Thalassemia patients Normal donors

Mean 6 SE Range Mean 6 SE Range

WCC (3106/mL) 242 6 42.0 39.9–623 316 6 53.8 64.4–532MNC (%WC) 84.3 6 3.2 50.8–100 75.2 6 6.4 32.0–97.4

(3106/mL) 208 6 34.7 35.7–476 267 6 50.9 47.1–480CD341 (% MNC) 0.50 6 0.07 0.10–1.27 0.66 6 0.12 0.29–1.54

(3106/mL) 1.05 6 0.23 0.09–2.81 1.49 6 0.35 0.17–3.78CD341 CD382 (% MNC) 0.01 6 0.01 0–0.02 0.01 6 0.01 0–0.04

(3106/mL) 0.02 6 0.01 0–0.07 0.05 6 0.02 0–0.14CD341DR1 (% MNC) 0.47 6 0.07 0.18–0.97 0.44 6 0.10 0.09–0.95

(3106/mL) 0.90 6 0.20 0.02–1.99 1.02 6 0.27 0.38–2.52CD341DR2 (% MNC) 0.12 6 0.02 0.04–0.30 0.11 6 0.04 0–0.33

(3106/mL) 0.29 6 0.07 0.08–0.72 0.35 6 0.17 0–0.97CD341CD331 (% MNC) 0.19 6 0.04 0.02–0.59 0.22 6 0.07 0.01–0.72

(3106/mL) 0.33 6 0.09 0.03–1.16 0.43 6 0.16 0.01–1.58

Results are expressed as percentages of mononucleated cells (MNC) and absolute cell counts in the first apheresis product. No significant difference wasfound between thalassemias and donors in these parameters.

Figure 3. Correlation of CD341 cells in PB and PBSC. Results areexpressed as percentages of CD341 cells in MNC of the PB and PBSCobtained on the same day of mobilization. There was a strong correlationbetween the percentages of CD341 cells in the PBSC harvest and in the PBcollected at the same day. Regression coefficients were r 5 0.83, p ,

0.001 for thalassemia patients (j); r 5 0.90, p , 0.001 for donors (u);and r 5 0.88, p , 0.001 for all samples. The linear correlation line repre-sents all samples.

K. Li et al./Experimental Hematology 27 (1999) 526–532 531

allogeneic transplants of other diseases. To our knowledge,the re-infusion of autologous PBSC to b-thalassemia pa-tients has not been documented. However, the infusion ofback-up autologous marrow and blood cells have been usedsuccessfully for the rescue of primary allogeneic graft fail-ure in leukemia patients [26].

The kinetics of mobilizing CD341 and white cell subsetshas confirmed that the effects of G-CSF are essentially onthe increase of circulating CD341 cells and the myeloid lin-eage [27]. The proportion and concentration of granulocyteswere greatly affected. The concentrations of monocytes andlymphocytes were increased to lesser extents. The percent-ages of lymphocyte subsets, including the T (helper andsuppressor), B, and NK cells were not altered during thecourse of treatment. The activation status (CD25, HLA-DRand CD45RO) of these subsets were also not changed.While we have previously shown that NK levels were lowerin thalassemia patients, particularly in the chronic trans-fused group [15], the expansion of NK cells was at levelssimilar to those in healthy donors. The slight increase of ab-solute lymphocyte and subset counts could possibly be initi-ated at the level of stem and progenitor cells rather than aspecific effect of G-CSF on the lymphocyte lineage.

The reason for the slight delay in the mobilization ofstem cells in the thalassemia patients is unclear. One possi-ble explanation is the presence of high levels of erythroidprogenitors in the bone marrow of thalassemia patients. Ku-wabara et al. [28] suggested that G-CSF receptor-mediatedendocytosis in bone marrow might be a major clearancesystem of G-CSF. However, the expression of G-CSF re-ceptors on the erythroid lineage has not been shown [29–31], in spite of the high levels of burst-forming unit-erythro-cytes present in the apheresis product mobilized by G-CSF[20,21,24]. Other mechanism could involve the interactionof G-CSF with endogeneous cytokines on stem cell mobili-zation. Lombardi et al. [12] reported a transfusion-corre-lated increase of TNF antigen in the serum of thalassemiapatients. TNF-a is a bidirectional regulator of hematopoie-tic progenitor cells [32–34] and may interact with G-CSFactivity by down-regulating the expression G-CSF receptors[32]. The delay of mobilization observed in this study, how-ever, has not significantly affected the overall yield andcomposition of PBSC harvests. This delay could be over-come by the close monitoring of CD341 cell levels in thePB before collection. The correlation of the percentages ofCD341 cells in the PB and in PBSC has indicated the use-fulness of regular monitoring of PB during mobilization.Similar observations were reported in the collection of au-tologous PBSC from cancer patients [23] and allogeneicPBSC from healthy donors [22,24]. In our thalassemiagroup, 9 of 20 patients required only one apheresis toachieve the collection of 1 3 106 CD341 cells/kg of bodyweight.

Our results have shown that under close monitoring ofCD341 cell levels on PB, the procurement of stem and pro-

genitor cells in b-thalassemia patients by a relatively stan-dard dose of G-CSF is feasible. The procedure of PBSC mo-bilization does not require surgery and avoids the morbidityrelated to general anesthesia and the aspiration of a largevolume of bone marrow. The collection of autologousPBSC could, thus, provide an alternative choice of obtain-ing back-up stem cells for thalassemia patients before theyundergo transplantation. With the advancing technology ofgene manipulation and therapy, G-CSF mobilized PBSCmay become a means of providing target cells for correctingthe defective b-globin gene. Further work is needed to ver-ify the potential use of the mobilized stem and progenitorcells for gene transfer and therapy of this disorder.

AcknowledgmentsWe thank Ms. Suparna Gangopadhyay of the Department of Paedi-atrics for data entry; Ms. Grace Lai of the Centre for Clinical Trials& Epidemiological Research for statistical analysis; BMT nursesand laboratory staff for patient care and technical assistance; andthe Hong Kong Paediatric Bone Marrow Transplant Fund for fi-nancial support.

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