Transfusion and Apheresis Science 44 (2011) 139–147

Contents lists available at ScienceDirect

Transfusion and Apheresis Science

journal homepage: www.elsevier .com/ locate/ t ransc i

Large volume leukapheresis: Efficacy and safety of processing patient’stotal blood volume six times

Ines Bojanic a,⇑, Klara Dubravcic b, Drago Batinic b, Branka Golubic Cepulic a, Sanja Mazic a,Darko Hren c, Damir Nemet d, Boris Labar d

a Department of Transfusion Medicine and Cellular Therapy, University Hospital Centre Zagreb, Kispaticeva 12, Croatiab Department of Immunology, University Hospital Centre Zagreb, Kispaticeva 12, Croatiac University of Split, Faculty of Philosophy, Zajceva bb, Split, Croatiad Department of Internal Medicine, University Hospital Centre Zagreb, Kispaticeva 12, Croatia

a r t i c l e i n f o

Article history:Received 16 December 2010Accepted 20 January 2011

1473-0502/$ - see front matter � 2011 Elsevier Ltddoi:10.1016/j.transci.2011.01.005

⇑ Corresponding author. Address: Department of Tand Cellular Therapy, University Hospital Centre Za10000 Zagreb, Croatia. Tel.: +385 1 2388708; fax: +

E-mail addresses: ibojanic@kbc-zagreb.hr (I. Boj@zg.htnet.hr (K. Dubravcic), drago.batinic@zg.t-cbgolubic@kbc-zagreb.hr (B.G. Cepulic), smazic@kbcdhren@ffst.hr (D. Hren), damnemet@inet.hr (D. Ninet.hr (B. Labar).

a b s t r a c t

Large-volume leukapheresis (LVL) differs from standard leukapheresis by increased bloodflow and an altered anticoagulation regimen. An open issue is to what degree a furtherincrease in processed blood volume is reasonable in terms of higher yields and safety. In30 LVL performed in patients with hematologic malignancies, 6 total blood volumes wereprocessed. LVL resulted in a higher CD34+ cell yield without a change in graft quality.Although a marked platelet decrease can be expected, LVL is safe and can be recommendedas the standard procedure for patients who mobilize low numbers of CD34+ cells and whenhigh number of CD34+ cells are required.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Peripheral blood has become a widely accepted sourceof hematopoietic stem cells because of easier accessibilityand faster engraftment [1]. Since rapid and sustainedengraftment following high-dose therapy depends on thenumbers of stem cells reinfused, efforts are directed to-wards harvesting sufficient numbers of CD34+ cells. Thequality of the peripheral blood stem cell (PBSC) graft is af-fected by the effectiveness of mobilization of stem cells,proper timing of collection, and the efficiency of collectiontechnique [2]. Although sufficient PBSCs may be obtained

. All rights reserved.

ransfusion Medicinegreb, Kispaticeva 12,385 1 2388699.anic), klara.dubravcicom.hr (D. Batinic),

-zagreb.hr (S. Mazic),emet), boris.labar@

in a single leukapheresis, the majority of patients requirerepeated procedures. An alternative to repeated apheresescould be processing larger volumes of blood during oneprocedure. Large-volume leukapheresis (LVL) involves pro-cessing the patient’s total blood volume (TBV) at leastthree times during a single procedure, and differs fromstandard leukapheresis by processing a larger blood vol-ume, increasing the blood flow rate, use of additional hep-arin anticoagulant as a longer duration of the procedure[1–5]. Unfortunately, there is no standardized protocolfor LVL and between centers there are differences regard-ing the blood volume processed and the duration of theprocedure. An open issue is to what degree a further in-crease in the processed blood volume is reasonable interms of higher cell yields and safety.

The purpose of this study was to investigate the effi-ciency and safety of processing 6 TBVs. We prospectivelyanalyzed the kinetics of peripheral blood cells and theCD34+ cell yield during LVL, and intraapheresis recruit-ment of CD34+ cells. Finally, we evaluated whether theprolonged procedure and processing 6, instead of 4, TBVsaltered the quality of the leukapheresis product.

140 I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147

2. Patients and methods

2.1. Patients

The study was performed on a group of 30 patients trea-ted at the University Hospital Centre, Zagreb (Table 1). Allpatients were candidates for high-dose chemotherapy fol-lowed by autologous PBSC transplantation. PBSCs weremobilized by a combination of disease specific chemother-apy protocols and 10 lg/kg/day s.c. granulocyte colony-stimulating factor (G-CSF) filgrastim (Neupogen, Roche,Switzerland). The study was approved by the local ethicscommittee, and written informed consent for PBSC collec-tion by LVL and additional testing was obtained from allpatients.

2.2. Methods

2.2.1. PBSC collectionPBSCs were collected using a Cobe-Spectra cell separa-

tor (MNC program, software version 6.0) (Gambro BCT,Lakewood, CO, USA). Venous access was established usinga dual lumen central venous catheter (Dualise-Cath, Vygon,France), placed in subclavian (27 patients), jugular (2 pa-tients) or femoral vein (1 patient).

Leukapheresis started when the peripheral blood CD34+cell count reached 10 � 106/L. A minimum of 30 � 109/Lplatelets was required before leukapheresis. Patients withpreapheresis counts of P20 � 106/L CD34+ cells were con-sidered good mobilizers, while those with a CD34+ count<20 � 106/L were considered poor mobilizers. Twelve pa-tients were poor mobilizers, and 18 were good mobilizers.

The target yield of CD34+ cells was 3.5 � 106/kg of bodyweight (BW), except in patients with multiple myelomawho were scheduled for double transplantation with targetyield of 7 � 106/kg BW.

During LVL the total volume of processed blood equaled 6patients’ TBVs, calculated by the instrument, based onweight, height and gender. The inlet flow rate was setaccording to instrument calculations. The collection ratewas 1.0 ml/min while the collected fraction was maintainedunder manual control at a hematocrit of approximately 1%.

Table 1Patients’ characteristics.

Gender (male/female) 16/14Age (years)* 50.1 ± 10.9 (19–61)Body weight (kg)* 78.3 ± 15.4 (57–108)Total blood volume (ml)* 4618.6 ± 140.5 (3463–6761)

Diagnosis (N)Multiple myeloma 20Non-Hodgkin’s lymphoma 8Hodgkin’s disease 2

Mobilization (N)Cyclophosphamide (4 g/m2) 20HDIM 7ICE 1Mini BEAM 1DHAP 1

* Mean ± SD (range); HDIM = high-dose ifosfamide and mitoxantrone;ICE = ifosfamide, carboplatin and etoposide; mini BEAM = carmustine,etoposide, cytarabine and melphalan; DHAP = dexamethasone, cytarabineand cisplatin.

A combination of solution citrate dextrose formula A(ACD-A, Baxter, Deerfield, IL, USA) and heparin (Heparin,Belupo, Croatia) was used as anticoagulant. The additionof 6 IU of heparin per 1 ml of ACD-A allowed us to enhancethe ACD-A to whole blood ratio to 1:24. Therefore the inletflow rate was doubled and, in the same time, twice theblood volume was processed.

In order to investigate the volume-dependent kinetics ofthe CD34+ cell yield after each TBV processed, the WBC col-lection set was modified. The collection bag was replacedwith a six collection bag set which was connected to theWBC set collection line by a sterile connection device. Themodified set allowed removal of the collected cells everytime one TBV had been processed therefore the product col-lected during processing each TBV could be analyzed.

When the optimal interface for MNC collection wasestablished, the first collection bag was opened; the bagswere changed after processing 1�, 2�, 3�, 4�, 5� and6� the patients’ TBV. All aphereses were performed bythe same operator and parameters were held constantthroughout the procedure.

Patients were monitored for adverse reactions and re-corded reactions were classified according to the NationalCancer Institute Common Toxicity Criteria (CTC-NCI) Clas-sification, Version 2 [4]. In order to prevent symptoms ofhypocalcemia, during LVL all patients received 0.5 g cal-cium chloride/10 kg BW in 100 ml of saline solution.

2.2.2. Laboratory evaluation of peripheral blood andleukapheresis products

Peripheral blood samples taken preapheresis and afterprocessing each TBV, and samples from each leukapheresisbag were analyzed for WBC, MNC, platelet and CD34+ cellcounts. Afterwards, products collected in the first four bagswere pooled into one bag, while products collected in thefifth and sixth bag were pooled into another bag. Sampleswere taken from each pooled bag and the quality of theproducts was compared with respect to CD34+ cell count.An intraindividual comparison of the collected CD34+ cellswas done with respect to their differentiation- (CD38,CD90 and HLA-DR) and lineage-associated markers(CD117, CD33 and CD41). The numbers of colony-formingunits: granulocyte macrophage (CFU-GM), burst formingunits-erythroid (BFU-E) and mixed units (CFU-MIX) weredetermined in two pooled bags as well. Finally, productsfrom both bags were pooled into one final bag and ana-lyzed for WBC, MNC, platelet and CD34+ cell count.

Cell yields were expressed as both the content of cells ina product and the cell yield per kg of patient’s BW. Totalyield was defined as the sum of the yields of 6 bags. Cumu-lative yield was defined as the sum of the yields collectedduring processing each TBV. CD34+ cell collection effi-ciency [6,7] and recruitment factor [8–10] for the variouscell populations were calculated as previously described.

Complete blood counts on the peripheral blood samplesand leukapheresis products were obtained on an auto-mated cell counter the ADVIA 120 (Bayer, Leverkusen,Germany). WBC differential counts were performed manu-ally using Wright-Giemsa-stained specimens. Mononu-clear cells (MNC) were defined as the sum of monocytesand lymphocytes.

I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147 141

CD34+ cells were analyzed by flow cytometry usingFACSCalibur (BD Biosciences, Heidelberg, Germany) fol-lowing the standard ISHAGE procedure for cell stainingwith anti-CD34-PE (clone 8G12) and anti-CD45-FITC (clone2D1) monoclonal antibodies (BD Biosciences) [11]. CD34+cell subsets were analyzed using monoclonal antibodiesto CD38, HLA-DR, CD90, CD117, CD41 and CD33 (BDBiosciences).

Short-term culture assay for (CFU-GM, BFU-E and CFU-MIX) was performed using MethoCult H4433 medium(StemCell Technologies, Vancouver, Canada) [12]. Thenumber of colonies was evaluated after 14 days of incuba-tion at 37 �C in a humidified atmosphere with 5% CO2.

Electrolytes calcium, potassium, phosphorus, and mag-nesium were tested in peripheral blood samples obtainedpre- and postapheresis using an Olympus AU 400 analyzer(Olympus Diagnostica, Tokyo, Japan).

2.2.3. Statistical analysisData were tested for normality using the Kolmogorov

Smirnov test. All distributions were normal, thus themeans and standard deviations as descriptors and para-metric procedures were used for all analyses.

Repeated measures ANOVA was used to test changes inthe peripheral blood cells during LVL. If repeated measuresANOVA resulted in a statistically significant F-ratio, weused paired samples t-tests to test the differences betweenneighboring points of measurement. In these cases weused Bonferonni’s correction for multiple comparisons: topreserve the overall p < 0.05 level of statistical significance,p < 0.008 was considered statistically significant whenthere were 6 comparisons, and p < 0.007 when there were7 comparisons. Furthermore we used within-between sub-jects ANOVA to test the possible effects of diagnosis (multi-ple myeloma vs. lymphoma) or the level of CD34+ cellmobilization (good vs. poor mobilizers) on changes inperipheral blood cells during LVL. A 2 � 2 between subjectsANOVA was used to test the effect of the diagnosis and le-vel of CD34+ cell mobilization on recruitment factor. Inde-pendent samples t-test was used to test the differences inCD34+ cell yield between patients with multiple myelomaand those with lymphoma, as well as between good andpoor mobilizers. We used Spearman’s rho coefficient totest the association between CD34+ cell total yield withthe preapheresis CD34+ cell count. The influence of pro-cessing 6 TBVs as opposed to standard processing of 4 TBVson the success of collecting the necessary number ofCD34+ cells in patients with lymphoma was tested usingMcNemar’s test. Differences in CD34+ cell subpopulationsin the product collected during processing of the first 4TBVs and the product collected during processing of thefifth and sixth TBVs were tested using a paired samplest-test. The level of statistical significance was set at 0.05for all analyses and they were performed using SPSS 13.0for Windows (SPSS Inc., Chicago, IL).

3. Results

Thirty aphereses performed in 30 patients were ana-lyzed. A mean volume of 26.97 ± 5.48 l (range 17.83–

37.29 l) was processed during 305.8 ± 7.6 min (range290–324 min) with a mean flow rate of 91.7 ± 18.8 ml/min (60.2–131.9 ml/min). The patients received a meanvolume of 1120.8 ± 230.1 ml ACD-A (range 740–1510 ml)and 6725.2 ± 1380.6 U of heparin (range 4440–9060 U).The mean volume of total apheresis products was291.9 ± 13.2 ml (range 255–335 ml). After processing 6TBVs, the mean total CD34+ cell yield was 4.71 ± 3.79(range 0.91–16.90) � 108/kg BW, and was significantlyhigher in good mobilizers than in poor mobilizers,(6.54 ± 3.93 � 106/kg vs. 1.98 ± 0.79 � 106/kg, p < 0.001).There were no significant differences in CD34+ yield be-tween the myeloma and lymphoma patients.

There was a strong correlation between the preaphere-sis peripheral blood CD34+ cell count and the total CD34+cell yield (q = 0.860 and p < 0.001). In both good and poormobilizers, the total CD34+ yield correlated significantlywith the preapheresis CD34 count (q = 0.83 and q = 0.89,respectively; p < 0.0001).

3.1. Kinetics of peripheral blood cells during LVL

The baseline hematological parameters and theirchanges during apheresis after processing each of the 6TBVs are summarized in Table 2 and Fig. 1. LVL significantlydecreased the circulating WBC, MNC, and CD34+ cell counts(p < 0.001). Post-hoc analyses revealed that this effect wasdue to the significant difference in number of WBC, MNCand CD34+ cells between the baseline values and valuesafter processing the first TBV (p < 0.001). We found no sig-nificant interaction between the change in peripheral bloodCD34+ cell count and diagnosis (p = 0.212) or the level ofmobilization of CD34+ cells (p = 0.62). The platelet countdecreased significantly during LVL through each of the se-ven points of measurement (p < 0001).

3.2. Kinetics of PBSC collection

PBSCs harvested during processing of each TBV volumewere collected in a separate bag, with a mean volume of48.6 ± 5.1 ml. The yields of cells and the CD34+ cell collec-tion efficiency during LVL are summarized in Table 3 andFig. 2. A statistically significant change in yield of WBC,MNC, and CD34+ cells during LVL was found (p < 0.001).Post-hoc analyses revealed that this effect was due to thesignificantly higher yield of all cells while processing thesecond TBV compared to the first TBV (p < 0.001). After-wards, the mean yield of WBC, MNC and CD34+ cells re-mained stable while processing the remaining 5 TBVs. Nosignificant interaction between the kinetics of CD34+ cellyield and diagnosis (p = 0.380) or level of CD34+ cellsmobilization were found (p = 0.886). Although the totalnumber of harvested CD34+ cells was higher for goodmobilizers, the kinetics of CD34+ cell collection during dif-ferent stages of LVL were the same for all patients.

The cumulative yield of CD34+ cells increased continu-ously during LVL with each processed TBV (p < 0.001), whichconfirmed the fact that the number of CD34+ cells collectedduring apheresis is related to the volume of blood processed.

The average total CD34+ cell collection efficiency duringLVL was 70.9 ± 33.2%. CD34+ cell collection efficiency was

0

10

20

30

40

50

60

70

80

90

100

baseline 1x 2x 3x 4x 5x 6xTotal blood volume processed

Perc

ent o

f bas

elin

e va

lues

(%)

platelets

WBC

MNC

CD34+ cells

Fig. 1. Decrease of WBC, MNC, CD34+ cells and platelets during LVL expressed as a mean percentage from baseline values.

Table 2Kinetics of WBC, MNC, CD34+ cells and platelets in the peripheral blood during LVL (mean ± SD). Levels of statistical significance were calculated comparing theresults after each processed total blood volume (TBV) with the following.

ProcessedTBV

Peripheral blood cells

WBC �109/L

WBC decreasefrom baseline(%)

MNC �109/L

MNC decreasefrom baseline(%)

CD34+ �106/L

CD34+ celldecrease frombaseline (%)

Platelet �109/L

Plateletsdecrease frombaseline (%)

Baseline 14.39 ± 7.68 100 2.02 ± 0.81 100 48.35 ± 44.80 100 99.75 ± 40.17 100p* <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.0011� 12.10 ± 6.91 83.20 ± 10.74 1.38 ± 0.57 69.27 ± 12.69 27.86 ± 23.19 63.46 ± 20.38 79.06 ± 33.55 79.26 ± 9.06p* NS NS p = 0.004 NS NS NS <0.001 <0.0012� 11.83 ± 6.69 82.48 ± 12.64 1.26 ± 0.51 64.23 ± 14.71 26.71 ± 25.29 58.17 ± 18.01 71.75 ± 32.69 72.24 ± 12.73p* NS NS 0.005 0.007 NS NS <0.001 <0.0013� 11.64 ± 6.57 80.07 ± 10.20 1.15 ± 0.41 58.58 ± 10.37 23.42 ± 23.00 51.20 ± 17.02 63.41 ± 25.46 64.55 ± 11.23p* NS NS 0.006 0.003 NS NS <0.001 <0.0014� 11.41 ± 6.59 78.01 ± 12.39 1.05 ± 0.38 53.98 ± 11.91 21.02 ± 19.73 47.72 ± 21.27 58.75 ± 25.41 59.88 ± 13.61p* NS NS NS NS NS NS <0.001 <0.0015� 11.16 ± 6.47 76.67 ± 14.50 1.01 ± 0.35 52.54 ± 13.01 19.63 ± 16.13 46.47 ± 19.08 55.20 ± 25.14 56.65 ± 16.21p* NS NS NS NS NS NS <0.001 <0.0016� 11.09 ± 6.53 76.01 ± 14.56 12.14 ± 8.46 50.35 ± 16.21 17.61 ± 14.05 45.95 ± 18.64 49.44 ± 21.46 51.65 ± 15.89F6,156 19.168 46.828 42.029 138.430 15.368 61.054 62.312 130.902p** <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

* p Paired samples t-tests.** p Repeated measures ANOVA.

142 I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147

lowest during the first TBV processing, but afterward re-mained stable. No significant interaction between thechange in CD34+ cell collection efficiency and diagnosis(p = 0.309) or the level of CD34+ cell mobilization wasfound (p = 0.173).

3.3. Comparison of the total CD34+ cell yield collected duringprocessing 4 TBVs vs. 6 TBVs

Processing 6 TBVs versus 4 TBVs resulted in signifi-cantly higher CD34+ cell yields; 4.71 ± 3.79 � 106/kg vs.

3.28 ± 2.89 � 106/kg, respectively (p < 0.001). If leukaphe-resis was stopped after processing 4 TBVs, the targetCD34+ cell yield would have been achieved after onlyone procedure in 9 (30%) patients. However, when the pro-cedure was prolonged to processing 6 TBVs, the target dosewas successfully achieved in 20 (66.7%) patients (p =0.001).

The results for the group of 20 myeloma patients wereanalyzed separately because their target dose was doubled.With processing 6 TBVs, satisfactory CD34+ counts for tan-dem transplantation was achieved in 6 (30%) patients, for

Table 3Yields of WBC, MNC and CD34+ cells collected in separate bags and CD34+ cell collection efficiencies (CE) during each total blood volume processed(mean ± SD). Levels of statistical significance were calculated comparing the results after each processed total blood volume (TBV) with the following.

Processed TBV Yield

WBC � 109/L WBC � 108/kg MNC � 109/L MNC � 108/kg CD34+ � 106/L CD34+ � 106/kg CD34+ cell CE (%)

1� 149.7 ± 61.4 0.87 ± 0.43 80.3 ± 39.5 0.45 ± 0.23 12.6 ± 10.7 0.71 ± 0.61 40.6 ± 18.7p* NS p < 0.001 p < 0.001 p < 0.001 NS p = 0.004 p < 0.0012� 162.1 ± 57.8 1.02 ± 0.41 88.4 ± 36.6 0.56 ± 0.22 14.3 ± 14.2 0.87 ± 0.73 68.6 ± 25.3p* NS NS NS NS NS NS NS3� 150.9 ± 68.3 0.97 ± 0.36 82.2 ± 33.5 0.53 ± 0.19 14.5 ± 16.2 0.87 ± 0.84 74.4 ± 32.6p* NS NS NS NS NS NS NS4� 152.6 ± 48.6 0.96 ± 0.32 80.5 ± 29.8 0.50 ± 0.19 13.4 ± 14.1 0.82 ± 0.82 73.8 ± 29.2p* NS NS NS NS NS NS NS5� 156.7 ± 46.3 0.98 ± 0.28 81.6 ± 32.1 0.48 ± 0.18 13.6 ± 10.1 0.80 ± 0.60 74.4 ± 24.2p* NS NS NS NS NS NS NS6 159.1 ± 46.0 1.01 ± 0.34 79.6 ± 27.5 0.49 ± 0.19 12.9 ± 9.1 0.82 ± 0.51 79.5 ± 28.3F5,125 1.489 3.187 3.979 4.808 0.242 3.082 9.927p** 0.198 0.041 0.002 <0.001 0.943 0.012 <0.001

Total product 149.7 ± 61.4 5.71 ± 1.97 80.3 ± 39.5 3.32 ± 3.86 126.2 ± 107.2 4.71 ± 3.79 70.9 ± 33.2

* p Paired samples t-test.** p Repeated measures ANOVA.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1x 2x 3x 4x 5x 6xTotal blood volume processed

Yiel

d

CD34+ cells x10E6/kg BW

MNC x10E8/kg BW

WBC x10E8/kg BW

Fig. 2. Yields of WBC, MNC and CD34+ cells/kg BW collected after each blood volume processed during large volume leukapheresis expressed as a mean.

I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147 143

single transplantation in 9 (45%) patients, while productswith counts <3.5 � 106 CD34+/kg were collected in 5(25%) patients. If leukapheresis was stopped after process-ing 4 TBVs, only one patient (5%) would have collected en-ough CD34+ cells for tandem transplantation and 6 (30%)for single transplantation.

3.4. Quality of products collected during different stages of LVL

The intraindividual comparison of the quality of cellscollected while processing 4 TBVs and cells collected whileprocessing the fifth and sixth TBVs are presented in Table4. The CD34+ cell subset analysis revealed no significantdifference in the composition of products collected during

different stages of LVL. Likewise, the number of CFU-GM,BFU-E and CFU-MIX collected from processing 4 TBVswas not significantly different from the number of CFUscollected from processing the fifth and sixth TBVs.

3.5. Recruitment of cells during LVL

The total number of cells in the peripheral blood pre-apheresis and postapheresis, the total number of cells inthe leukapheresis products, and the recruitment factorfor WBC, MNC, granulocytes, CD34+ cells and plateletsare presented in Table 5. The recruitment factor was calcu-lated for each of these cells in order to find out whethercell-specific recruitment occurred during LVL. The highest

Table 4Comparison of the quality of leukapheresis products collected during different stages of LVL: subset analysis of CD34+ cells and numbers of CFU-GM, BFU-E andCFU-MIX colonies /105 cells after short-term culture.

Subsets of CD34+ cells TBV processed 1–4 TBV TBV processed 5th and 6th times p*

CD38+ (% CD34+ cells) 98.90 ± 2.33 99.07 ± 1.67 0.669CD34+ CD38+ (x108/L) 12.74 ± 10.47 12.13 ± 11.09 0.238HLA-DR+ (% CD34+ cells) 98.67 ± 1.84 99.02 ± 1.30 0.100CD34+ HLA-DR+ (�108/L) 11.90 ± 10.29 11.30 ± 10.45 0.147CD90+(% CD34+ cells) 46.69 ± 19.77 47.72 ± 20.82 0.320CD34+ CD90+ (x108/L) 6.80 ± 6.7 6.76 ± 6.3 0.884CD117+ (% CD34+ cells) 94.15 ± 4.75 94.48 ± 4.64 0.529CD34+ CD117+ (�108/L) 11.39 ± 9.12 11.12 ± 9.19 0.349CD41+ (% CD34+ cells) 21.97 ± 19.28 19.74 ± 20.09 0.080CD34+ CD41+(�108/L) 2.34 ± 2.20 2.21 ± 2.16 0.508CD33+ (% CD34+ cells) 89.10 ± 13.49 89.52 ± 12.33 0.416CD34+ CD33+ (�108/L) 10.91 ± 8.44 10.68 ± 8.55 0.483

Number of colonies/105 cellsCFU-GM 77.63 ± 31.88 73.65 ± 24.27 0.344BFU-E 47.19 ± 27.72 48.94 ± 25.23 0.558CFU-MIX 16.28 ± 7.57 15 ± 5.31 0.301

* p Paired samples t-tests.

Table 5Preapheresis and postapheresis total number of cells in peripheral blood, total number of collected cells in the leukapheresis products and recruitment factor.

Parameters Preapheresis Postapheresis Total number of harvested cells Recruitment factor*

WBC (�108) 676.70 ± 416.73 527.36 ± 368.78 439.69 ± 156.81 1.64 ± 0.54MNC (�108) 96.32 ± 45.07 46.56 ± 20.49 205.47 ± 103.26 2.72 ± 0.69Granulocyte (�108) 580.37 ± 385.53 480.79 ± 355.94 234.21 ± 137.95 1.42 ± 0.51CD34+ cells (�106) 226.80 ± 204.88 83.37 ± 68.77 540.12 ± 562.95 3.23 ± 1.61Platelets (�109) 469.47 ± 210.93 227.70 ± 87.77 313.72 ± 183.12 1.17 ± 0.20

* Recruitment factor = absolute number of cells in blood postapheresis + absolute number of cells collected/absolute number of cells in bloodpreapheresis.

144 I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147

recruitment factor (3.2 ± 1.6) was found for CD34+ cells. Astatistically significant difference in the CD34+ cell recruit-ment factor was found between good (2.8 ± 0.4) and poormobilizers (4.1 ± 0.5), respectively (p = 0.044). There wasno difference in recruitment factors related to the patients’diagnoses (p = 0.112).

3.6. Side effects and electrolyte changes during LVL

All LVLs were well tolerated, and none of procedureshad to be discontinued. Apheresis related adverse reac-tions were restricted to mild symptoms of citrate toxicity.Only one patient experienced mild perioral paresthesias,classified as grade 1. Although significant decreases inplatelet count were observed after LVL, no bleeding epi-sodes occurred, and there was no need for transfusionsupport.

All analyzed electrolytes decreased significantlywith respect to the basal values: calcium 5.18 ± 4.10%,(p = 0.005), potassium 11.75 ± 7.46% (p < 0.001), magne-sium 11.98 ± 5.44% (p < 0.001), and phosphorus 17.22 ±16.06% (p = 0.024). Although calcium concentration de-creased significantly with respect to basal values, it stillremained within reference values (2.38 ± 0.40 mmol/L vs.2.24 ± 0.22 mmol/L) [13]. Other analyzed electrolytes de-creased markedly below the reference interval: potassium3.92 ± 0.35 mmol/L vs. 3.47 ± 0.38 mmol/L, magnesium0.71 ± 0.08 vs. 0.65 ± 0.09, and phosphorus 0.87 ± 0.28

mmol/L vs. 0.73 ± 0.2 mmol/L [13]. Nevertheless, none ofour patients experienced clinically relevant side effects re-lated to changes in these electrolytes.

4. Discussion

Leukapheresis has been successfully used in PBSC col-lection for over 20 years, however several topics still re-main unresolved regarding the optimal method ofcollection and the volume of processed blood. Processingof larger blood volumes in a single LVL may improveCD34+ cell yield, consequently reducing the number of re-quired procedures and diminishing the total cost of collec-tions [2,4]. Another rationale for use of LVL is the narrowpeak of the CD34+ cells in the peripheral blood, presentonly for a short period after mobilization, and thereforethe optimal time for successful collection would not likelybe missed [14].

Results of previous studies which compared standardvs. LVL processing varied considerably [2,4,10,14–18]. Itis difficult to establish a control group with comparable pa-tients due to high interindividual variability of the pa-tients’ characteristics, such as age, gender, TBV, stage ofdisease, previous treatment, and baseline blood count val-ues. Even if the same patient is analyzed the following day,the peripheral blood CD34+ cell count could halve or dou-ble overnight. The optimal study design would be to com-

I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147 145

pare products collected during the same procedure at dif-ferent times, which was done in this study, so differencescaused by interindividual variability were excluded. Stud-ies of kinetics of the PBSC enrichment during LVL showedvariable results [3,8–10,19–31], which could be the conse-quence of the differences in volumes of processed blood.Therefore, the processed blood volume in our study wasstandardized and strictly related to the patient’s TBV. Pro-cessing 6 TBVs was accomplished by doubling the inletflow rate and the additional use of heparin, along with pro-longation of procedure to 5 hours. The duration of LVLcould have been prolonged even more, but was limited to5 hours because of the patients’ comfort and tolerance,and in view of the working hours of the apheresis unit,quality control and cell processing laboratories. Time-con-suming LVL raises an issue of the patient’s compliance, butprevious studies showed that cooperation for a prolongedapheresis procedure could be achieved even in pediatricpatients [17,25,26].

A strong correlation between the preapheresis CD34+cell count and the total CD34+ cell yield was found, bothin good and poor mobilizers. Accordingly to our results aswell as other reports [5,22,27,29], even in the LVL setting,the preapheresis CD34+ cell count is still the best predictorof the outcome of PBSC collection, although the volume ofblood processed during LVL also affects the total yield.

LVL is feasible only if the level of CD34+ cells in theblood is maintained throughout the procedure. Althougha decline in CD34+ cell level was documented in all pa-tients, our study showed that even after processing 6 TBVsthere was no exhaustion of the peripheral blood CD34+cells. The kinetic study showed that a decrease in CD34+count is related to the number of the TBVs processed.The drop in CD34+ cell count was most evident at thebeginning of leukapheresis as cells were packed into theapheresis device set [3,26]. At the same time, the separatordraws patient’s blood while returning saline to fill the setlines, which can cause hemodilution and contribute to aCD34+ cell decrease.

Our results confirm that CD34+ cells were collected at asteady rate throughout the LVL [9,19,26,30]. The lowestCD34+ cell yield and collection efficiency were observedat the very beginning of LVL, which can be explained bythe time required to establish an interface, and by a dilu-tion effect in the first collection bag, caused by automatedfilling with saline [19,29,31]. In the remaining collectionperiod, the CD34+ cell yield and collection efficiency werestable, without any time-dependent changes, as observedby others [9,10]. Furthermore, the MNC purity remainedconstant during LVL, confirming that enhanced blood vol-ume processing does not affect the product quality interms of higher granulocyte contamination during the finalstages of apheresis [19]. It is important to emphasize that,in spite of a decrease in the peripheral blood CD34+ cellcount during LVL, the total cumulative CD34+ cell yield in-creased steadily after each blood volume processed. Thekinetics of CD34+ cell collection were similar and stableregardless of diagnosis or level of CD34+ cell mobilization,therefore all patients could benefit from LVL [32].

The use of modified sets enabled us to compare howmany cells would be collected after processing standard

4 TBVs vs. 6 TBVs. CD34+ cell yield was higher when leuk-apheresis was extended, which could be particularly bene-ficial in poor mobilizers. Our results confirmed that thelikelihood for collecting target number of CD34+ cellswas greater with use of LVL than with standard leukaphe-resis [4,28].

Current protocols for myeloma patients require tandemtransplantation [33,34], and it is common practice to col-lect sufficient cells for two grafts. That is sometimes chal-lenging in heavily pretreated patients, particularly thosewho previously received lenalidomide [4,35]. Moreover,some data demonstrated different mobilization of PBSCsand myeloma cells, with peak levels of the myeloma cellsoccurring a few days after CD34+ cells [36–38]. Since LVLallows collection of more CD34+ cells, fewer days are re-quired to obtain the target cell dose, and use of LVL couldresult in lower tumor contamination of the graft [36–38].Zubair et al. [33] estimated that by using LVL in myelomapatients, the cost savings could be greater than $7597per patient.

A leukapheresis product consists of hematopoietic stemand progenitor cells in different stages of differentiation[39–41]. Previous studies, [39,41,42] as well as ours, re-ported that the majority of collected CD34+ cell coexpres-sed CD38, HLA-DR or CD33 associated with a commitmentto different hematopoietic lineages. Our study first ana-lyzed CD34+ cell subpopulations in products collectedwhile processing as many as 6 TBVs, and showed no differ-ence in the composition of products collected during dif-ferent stages of LVL. This finding argues against apreferential release of particular CD34+ cell subsets duringLVL [27]. Our data confirm that LVL results in a higherCD34+ cell yield without change in graft quality.

Higher CD34+ cell yields harvested by processing a lar-ger volume of blood was explained by a steady recruitmentof PBSCs during leukapheresis [8,9,18,19]. In our study,threefold more CD34+ cells were collected than were pres-ent in the blood before leukapheresis. The recruitment waslimited to MNC and CD34+ cells, which were predominantlyremoved during the procedure. This was also observed byother authors [8,23,24,43]. Interestingly, the recruitmentfactor for CD34+ cells in our study was significantly higherin poor mobilizers than in good mobilizers, which points tothe importance of LVL in patients who mobilize low numberof CD34+ cells [17]. The underlying mechanisms of PBSCsrecruitment during LVL are not clear, and few hypotheseshave been proposed. Some authors explained recruitmentof PBSCs merely by the stimulating effect of G-CSF adminis-tration [44], while others suggested that it was mediated bya negative feedback mechanism caused by the decline of theperipheral blood CD34+ cell count during LVL [8,10]. An-other possible mechanism is modification of PBSCs homingand transmigration caused by changes in their microenvi-ronment, induced by use of heparin [15,45,46] and cit-rate-induced hypocalcemia [27,47].

Results of our study are in favor of LVL but some draw-backs should also be mentioned. LVL is definitely time-consuming because processing 6 TBVs requires 5 hours.Implementation of LVL has implications on the workinghours of apheresis department as well as the quality con-trol and cell processing laboratory. Timely completion of

146 I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147

LVL requires high blood flow rates. In some studies itwasn’t possible to establish the required inlet flow rate,and the procedures were shortened because of catheter re-lated problems [10,18]. We didn’t observe any problem re-lated to high inlet flow rate, but all our patients had anapheresis catheter which we recommend for LVL. LVLmay result in an excess of collected CD34+ cells, thereforeit should not be used in patients who mobilized high num-ber of CD34+ cells.

A limiting factor for widespread use of LVL may be thepatients’ tolerance of the procedure [2,3]. Larger volumesof infused anticoagulant causes electrolyte imbalance suchas hypocalcemia, metabolic alkalosis, hypokalemia andhypomagnesemia [48]. In agreement with previous reports[33,48], we did not observe an increase in the number ofadverse events during LVL collection. Buchta et al. [49]showed that prophylactic calcium infusion during LVL re-duced the incidence of citrate-related symptoms withoutaffecting the technical performance or the number ofCD34+ cells collected, as was confirmed by our results.Our study also confirmed a marked platelet decrease afterprocessing each TBV with a halved platelet count after LVL[3,48]. The additional heparin administration enables thereduction of the infused volume of citrate anticoagulant,but might represent an additional risk factor for bleedingcomplications in thrombocytopenic patients with centralvenous catheters [50]. Heparin-induced thrombocytopenia(HIT) and the associated thrombotic complications coulddevelop in a newly exposed patient, or rapid-onset compli-cations might occur in a patient with a recent prior historyof HIT [51]. Processing up to 6 TBVs according to our re-sults could be performed safely because no severe adversereactions occurred, including bleeding complications.Although it lasts longer, patients will probably toleratean extra hour of collection easier than another procedureon consecutive days which would increase the total costof treatment and expose them to the risks of central ve-nous line complications and additional leukaphereses [24].

LVL could be strongly recommend for patients whomobilized a low number of CD34+ cells, and patientswho need a high dose of CD34+ cells, including doubletransplantation or in vitro processing of the leukapheresisproduct where a significant loss of cells is expected. Ifthe role of LVL is considered in the setting of new mobiliz-ing agents, such as plerixafor used in case of previouslyunsuccessful mobilization [52,53], there is no doubt thatLVL should be performed to collect the maximum numberof PBSCs in one procedure. From the data presented, weconclude that processing of 6 TBVs during LVL is an effi-cient and safe technique, which significantly reduces thenumber of aphereses needed to obtain target number ofCD34+ cells. Whether or not a further increase of the pro-cessed blood volume could improve the quality of the graftmust be evaluated in future studies.

References

[1] Moog R. Management strategies for poor peripheral blood stem cellmobilization. Transfus Apher Sci 2008;38:229–36.

[2] Gasová Z, Marinov I, Vodvárková S, Böhmová M, Bhuyian-LudvíkováZ. PBPC collection techniques: standard versus large volume

leukapheresis (LVL) in donors and in patients. Transfus Apher Sci2005;32:167–76.

[3] Rowley S, Yu J, Gooley T, et al. Trafficking of CD34+ cells into theperipheral circulation during collection of peripheral blood stemcells by apheresis. Bone Marrow Transplant 2001;28:649–56.

[4] Abrahamsen J, Stamnesfet S, Liseth K, Hervig T, Bruserud O. Large-volume leukapheresis yields more viable CD34+ cells and colony-forming units than normal-volume leukapheresis, especially inpatients who mobilize low numbers of CD34+ cells. Transfusion2005;45:248–53.

[5] Monacada V, Bolan C, Yau Y, Leitman S. Analysis of PBPC cell yieldsduring large-volume leukapheresis of subjects with a poormobilization response to filgrastim. Transfusion 2003;43:495–501.

[6] Rowley S, Prather K, Bui K. Collection of peripheral blood progenitorcells with an automated leukapheresis system. Transfusion1999;39:1200–6.

[7] Schreiner T, Wiesneth M, Krug E, et al. Collection of allogeneicperipheral blood progenitor cells by two protocols on an apheresissystem. Transfusion 1998;38:1051–5.

[8] Knudsen L, Nikolaisen K, Gaarsdal E, Johnsen H. Kinetic studiesduring peripheral blood stem cell collection show CD34+ cellrecruitment intra-apheresis. J Clin Apher 2001;16:114–9.

[9] Cull G, Ivey J, Chase P, Picciuto R, Herrmann R, Cannell P. Collectionand recruitment of CD34+ cells during large-volume leukapheresis. JHematother 1997;6:309–14.

[10] Moller A, Dickmeiss E, Geisler C, Christensen L. Recruitment ofCD34+ cells during large-volume leukapheresis. J Hematother StemCell Res 2001;10:837–53.

[11] Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland D. Singleplatform flow cytometric absolute CD34+ cell counts based on theISHAGE gudielines. Int Soc Hematother Graft Eng Cytometry1998;34:61–70.

[12] Smalcelj R, Kusec V, Thune S, Petrovecki M. Circulatinghematopoietic progenitors are not altered in patients with post-transplant erythrocytosis. Haematologica 1998;83:948–9.

[13] Painter P, Cope J, Smith J. Reference information for the clinicallaboratory. In: Burtis C, Ashwood E, Bruns D, editors. Tietz textbookof clinical chemistry and molecular diagnostics. St. Louis: ElsevierSaunders; 2006. p. 2251–318.

[14] Abe T, Makimoto A, Kawano Y, et al. Intra-apheresis recruitment ofblood progenitor cells in children. Transfusion 1998;38:944–50.

[15] Humpe A, Riggert J, Munzel U, et al. A prospective, randomized,sequential, crossover trial of large-volume versus normal-volumeleukapheresis procedures: effect on progenitor cells andengraftment. Transfusion 1999;39:1120–7.

[16] Smolowicz A, Villman K, Tidefelt U. Large-volume apheresis for theharvest of peripheral blood progenitor cells for autologoustransplantation. Transfusion 1997;37:188–92.

[17] Torrabadella M, Olivé T, Ortega JJ, Massuet L. Enhanced HPCrecruitment in children using LVL and a new automated apheresissystem. Transfusion 2000;40:404–10.

[18] Passos-Coelho J, Machado M, Lucio P, Leal-Da-Costa F, Silva M,Parraiera A. Large-volume leukaphereses may be more efficient thanstandard-volume leukaphereses for collection of peripheral bloodprogenitor cells. J Hematother 1997;6:465–74.

[19] Cassens U, Ostkamp-Ostermann P, van der Werf N, Garritsen H,Ostermann H, Sibrowski W. Volume-dependent collection ofperipheral blood progenitor cells during large-volumeleukapheresis for patients with solid tumours and haematologicalmalignancies. Transfus Med 1999;9:311–20.

[20] Hillyer C, Lackey Dr, Hart K, et al. CD34+ progenitors and colony-forming units-granulocyte macrophage are recruited during large-volume leukapheresis and concentrated by counterflow centrifugalelutriation. Transfusion 1993;33:316–21.

[21] Hillyer C, Tiegerman K, Berkman E. Increase in circulating colony-forming units-granulocyte-macrophage during large-volumeleukapheresis: evaluation of a new cell separator. Transfusion1991;31:327–32.

[22] Passos-Coelho J, Braine H, Davis J, Huelskamp A. Predictive factorsfor peripheral- blood -progenitor-cell collections using a singlelarge-volume leukapheresis after cyclophosphamide andgranulocyte–macrophage colony stimulating factor mobilization. JClin Oncol 1995;13:705–14.

[23] Smolowicz A, Villman K, Berlin G, Tidefelt U. Kinetics of peripheralblood stem cell harvests during a single apheresis. Transfusion1999;39:403–9.

[24] Bojko P, Stellberg W, Küdde C, et al. Kinetic study of CD34+ cellsduring peripheral blood stem cell collections. J Clin Apher1999;14:18–25.

I. Bojanic et al. / Transfusion and Apheresis Science 44 (2011) 139–147 147

[25] Gorlin J, Vamvakas E, Cooke E, et al. Large-volume leukapheresis inpediatric patients: processing more blood diminishes the apparentmagnitude of intra-apheresis recruitment. Transfusion1996;36:879–85.

[26] Yamaguchi E, Yamato K, Miyata Y. Kinetics of peripheral blood stemcell collection in large-volume leukapheresis for pediatric patientsundergoing chemotherapy and adult patients before chemotherapy.J Hematother Stem Cell Res 2000;9:565–72.

[27] Murea S, Goldschmidt H, Hahn U, Pförsich M, Moos M, Haas R.Successful collection and transplantation of peripheral blood stemcells in cancer patients using large-volume leukaphereses. J ClinApher 1996;11:185–94.

[28] Bojko P, Scharifi M, Stössel K, Seeber S. Comparison of processingfour and five times the patients’ blood volume during peripheralblood stem cell collection and analysis of CD34+38-and CD34+49d+subsets during apheresis. J Cancer Res Clin Oncol 2002;128:19–28.

[29] Fabritiis P, Gonzalez M, Meloni G, et al. Monitoring of CD34+ cellsduring leukapheresis allows a single, successful collection ofhemopoietic progenitors in patients with low numbers ofcirculating stem cells. Bone Marrow Transplant 1999;23:1229–36.

[30] Humpe A, Riggert J, Meineke I, et al. A cell-kinetic model of CD34+cell mobilization and harvest: development of a predictive algorithmfor CD34+ cell yield in PBPC collections. Transfusion 2000;40:1363–70.

[31] Cassens U, Momkvist P, Zuehlsdorf M, et al. Kinetics of standardizedlarge volume leukapheresis in patients do not show a recruitment ofperipheral blood progenitor cells. Bone Marrow Transplant2001;28:13–20.

[32] Majado M, Minguela A, Gonzalez-Garcia C, et al. Large-volume-apheresis facilitates autologous transplantation of hematopoieticprogenitors in poor mobilizer patients. J Clin Apher 2009;24:12–7.

[33] Zubair A, Rymer R, Young J, et al. Multiple myeloma patientsreceiving large volume leukapheresis efficiently yield enough CD34+cells to allow double transplants. J Clin Apher 2009;24:6–11.

[34] Barlogie B, Attal M, Crowley J, et al. Long-term follow-up ofautotransplantation trials for multiple myeloma: update ofprotocols conducted by the Intergroupe Francophone du Myelome,Southwest Oncology Group, and University of Arkansas for MedicalSciences. J Clin Oncol 2010;28:1209–14.

[35] Kumar A, Dispenzieri A, Lacy M, et al. Impact of lenalidomidetherapy on stem cell mobilization and engraftment post-peripheralblood stem cell transplantation in patients with newly diagnosedmyeloma. Leukemia 2007;21:2035–42.

[36] Gazitt Y, Tian E, Barlogie B, et al. Different mobilization of myelomacells and normal hematopoietic stem cells in multiple myeloma aftertreatment with cyclophosphamide and granulocyte–macrophagecolony-stimulating factor. Blood 1996;87:805–11.

[37] Desikan K, Jagannath S, Siegel D, et al. Collection of morehematopoietic progenitor cells with large volume leukapheresis inpatients with multiple myeloma. Leuk Lymphoma 1997;28:501–8.

[38] Gazitt Y, Reading C, Hoffman R, et al. Purified CD34+Lin-Thy+ stemcells do not contain clonal myeloma cells. Blood 1995;86:381–9.

[39] Stewart A, Imrie K, Keating A, Anania S, Nayar R, Sutherland D.Optimizing the CD34+ and CD34+Thy-1+ stem cell content ofperipheral blood collections. Exp Hematol 1995;23:1619–27.

[40] Haas R, Mohle R, Murea S, et al. Characterization of peripheral bloodprogenitor cells mobilized by cytotoxic chemotherapy andrecombinant human granulocyte colony-stimulating factor. JHematother 1994;3:323–30.

[41] Humpe A, Riggert J, Koch S, Legler T, Munzel U, Kohler M.Prospective, randomized, sequential, crossover trial of large-volume vs. normal volume leukapheresis procedures: effects onsubpopulations of CD34+ cells. J Clin Apher 2001;16:109–13.

[42] Haas R, Mohle R, Pforsich M, et al. Blood-derived autografts collectedduring granulocyte-colony-stimulating factor-enhanced recoveryare enriched with early Thy-1+ hematopoietic progenitor cells.Blood 1995;85:1936–43.

[43] Benjamin R, Linsley L, Fountain D, et al. Preapheresis peripheralblood CD34+ mononuclear cell counts as predictors of progenitorcell yield. Transfusion 1997;37:79–85.

[44] Cassens U, Barth I, Baumann C, et al. Factors affecting the efficacy ofperipheral blood progenitor cells collections by large-volumeleukaphereses with standardized processing volumes. Transfusion2004;44:1593–602.

[45] Watt S, Williamson J, Genevier H, et al. The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursorcells with early myeloid and B-lymphoid cell phenotypes. Blood1993;82:2649–63.

[46] Yong K, Watts M, Shaun Thomas N, Sullivan A, Ings S, Linch D.Transmigration of CD34+ cells across specialized and nonspecializedendothelium requires prior activation by growth factors and ismediated by PECM-1 (CD31). Blood 1998;91:1196–205.

[47] Mohle R, Haas R, Hunstein W. Expression of adhesion molecules andc-kit on CD34+ hematopoietic progenitor cells: comparison ofcytokine mobilized blood stem cells with normal bone marrowand peripheral blood. J Hematother 1993;2:483–9.

[48] Humpe A, Riggert J, Munzel U, Köhler M. A prospective, randomized,sequential crossover trial of large-volume versus normal-volumeleukapheresis procedures: effects on serum electrolytes, plateletcounts, and other coagulation measures. Transfusion 2000;40:368–74.

[49] Buchta C, Macher M, Bieglmayer C, Hocker P, Dettke M. Reduction ofadverse reactions during autologous large-volume PBSC apheresisby continuous infusion of calcium-gluconate. Transfusion2003;43:1615–21.

[50] Malachowski M, Comenzo R, Hillyer C, Tiegerman K, Berkman E.Large-volume leukapheresis for peripheral blood stem cell collectionin patients with hematologic malignancies. Transfusion 1992;32:732–5.

[51] Linenberger M. Collection of cellular therapy products by apheresis.In: Areman E, Loper K, editors. Cellular therapy: principles, methods,and regulations. Bethesda: AABB; 2009. p. 251–64.

[52] Choi H, Yong C, Yoo B. Plerixafor for stem cell mobilization inpatients with noon-Hodgkin’s lymphoma and multiple myeloma.Ann Pharmacother 2010;44:117–26.

[53] Fruehauf S, Veldwijk M, Seeger T, et al. A combination ofgranulocyte-colony-stimulating factor (G-CSF) and plerixaformobilizes more primitive peripheral blood progenitor cells thanG-CSF alone: results of a European phase II study. Cytotherapy2009;11:992–1101.