Rev. Bras. Hematol. Hemoter. vol.37 número3

w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

Mobilization

and

collection

of

CD34

+

cells

for

autologous

transplantation

of

peripheral

blood

hematopoietic

progenitor

cells

in

children:

analysis

of

two

different

granulocyte-colony

stimulating

factor

doses

Kátia

Aparecida

de

Brito

Eid

_,

_Eliana

_Cristina

_Martins

_Miranda

_,

Simone

dos

Santos

Aguiar

a_{UniversidadeEstadualdeCampinas(UNICAMP),Campinas,SP,Brazil}

b_{CentroInfantilBoldrini,Campinas,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received29July2014 Accepted15September2014 Availableonline17February2015

Keywords:

Hematopoieticstemcell mobilization

Leukapheresis

Granulocytecolony-stimulating factor

Autologoustransplantation

a

b

s

t

r

a

c

t

Introduction:Theuseofperipheralhematopoieticprogenitorcells(HPCs)isthecellchoice inautologoustransplantation.Theclassicdoseofgranulocyte-colonystimulatingfactor (G-CSF)formobilizationisasingledailydoseof10␮g/kgofpatientbodyweight.Thereisa theorythathigherdosesofgranulocyte-colonystimulatingfactorappliedtwicedailycould increasethenumberofCD34+_{cellscollectedinfewerleukapheresisprocedures.}

Objective:Theaimofthisstudywastocompareafractionateddoseof15␮gG-CSF/kgof bodyweightandtheconventionaldoseofgranulocyte-colonystimulatingfactorinrespect tothenumberofleukapheresisproceduresrequiredtoachieveaminimumcollectionof 3×106_CD34+_{cells/kgbodyweight.}

Methods:Patientsweredividedintotwogroups:Group10–patientswhoreceivedasingle dailydoseof10␮gG-CSF/kgbodyweightandGroup15–patientswhoreceivedafractioned doseof15␮gG-CSF/kgbodyweightdaily.Theleukapheresisprocedurewascarriedoutinan automatedcellseparator.Theautologoustransplantationwascarriedoutwhenaminimum numberof3×106_CD34+_{cells/kgbodyweightwasachieved.}

Results:Group10comprised39patientsandGroup15comprised26patients.Atotalof146 apheresisprocedureswereperformed:110(75.3%)forGroup10and36(24.7%)forGroup 15.For Group10,a medianofthree(range:1–7)leukapheresis proceduresanda mean of8.89×106 _CD34+_{cells/kgbodyweight(±9.59)werecollectedwhereasforGroup15the}

correspondingvalueswereone(range:1–3)and5.29×106_{cells/kgbodyweight(±4.95).A}

sta-tisticallysignificantdifferencewasfoundinrelationtothenumberofapheresisprocedures (p-value<0.0001).

∗ _{Correspondingauthorat:RuaEngArturCanguc¸u,275,apt21,VilaAndradeNeves,130070-293Campinas,SP,Brazil.}

E-mailaddress:[email protected](K.A.d.B.Eid). http://dx.doi.org/10.1016/j.bjhh.2015.02.006

Conclusions: Tocollectaminimumtargetof3×106_CD34+_{cells/kgbodyweight,the}

admin-istrationofafractionateddoseof15␮gG-CSF/kgbodyweightsignificantlydecreasedthe numberofleukapheresisproceduresperformed.

©2015Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.Allrightsreserved.

Introduction

Theautologoustransplantationofperipheralhematopoietic progenitorcells (HPCs)iswidelyusedtotreatsolidtumors andlymphomasinchildren,adolescentsandyoungadults.1–3 Since the end of the 1980s the use of leukapheresis has beenthemostusedmethodtoharvestcellsforautologous transplantationasitisconsideredtobeeffectiveandsafe, pro-motingafasterhematopoieticrecoveryandposinglessriskto thepatient(nogeneralanesthesiaandnoanemia).Asa con-sequence,thismethodisassociatedwithlowercostandthere islessriskofcontaminationofthegraftbyneoplasticcells comparedtobonemarrow.4_{AccordingtotheCenterfor} Inter-nationalBloodandMarrowTransplant Research(CIBMTR),5 around32,000transplantswerecarriedoutinthelastdecade with,inmostcases,thecellsourcebeingperipheralHPCs.5_In 91%ofcaseswhereabonemarrowtransplantisneededinthe childpopulationoftheUS,peripheralHPCsareharvestedwith themostcommonreasonsfortreatmentbeinglymphomas andsolidtumors.5

Myelosuppressive chemotherapy or high doses of chemotherapy in association with granulocyte-colony stimulating factor (G-CSF) has been successfully used in the mobilization of peripheral HPCs with a reduction of contamination byneoplastic cells.6,7 _{These treatments are} safeandwelltoleratedwithalargecapacityformobilization andactionagainstneoplasticcells.Cyclophosphamide(CY), aloneorincombinationwithotheragents,isthemost com-monlyused chemotherapydrug8 _{although other regimens} suchastheifosfamide,carboplatinandetoposide(ICE)and dexamethasone,adriamycinandcisplatin regimens (DHAP) arealsoemployed.9–11_{However,themosteffectiveregimen,} withthemostsuitableintensityofmobilization,remainsto bedefined.4,12

G-CSFisthemostpotentcytokineavailable4_andtheone mostcommonlyusedforthemobilizationofperipheralHPCs.5 G-CSFhaslowtoxicityandiswelltolerated.Themost com-mon side effect ismild bone pain, beginning aftertwo or threeapplications,13 _{however, few patients needto reduce} thedoseordiscontinuetreatment.4_Inautologous transplan-tation,theclassicG-CSFdoseformobilizationis10␮g/kgof patient body weight (bw) via subcutaneous administration onceaday.12,14 _{Somestudieshaveshownthatlargerdoses} of G-CSF and fractionated doses given in two daily appli-cationsincrease the number ofCD34+ _{cells collected with} alowernumber ofleukapheresisprocedures.15,16 _Moreover, inpatientswhosemobilizationfailedwiththeconventional dose of G-CSF, an increase in the dose to 12.5–50␮g/kg bw/daycanbesuccessful.6,17 _{SubcutaneousG-CSF} adminis-trationachievesamaximumserum levelwithin2–8hafter

applicationwithahalf-lifeof3–4h.18_{Thus,asingledaily} appli-cationmaynotbeoptimal.18

Therecognizedmethodofharvesting peripheralHPCsis large-volumeleukapheresis(LVL).5 _{Withcurrenttechnology,} leukapheresiscanbecarriedoutinveryyoungchildrenwith lowweights(<10kg),allowingasufficientnumberofCD34+ cellstobecollectedforcompletebonemarrowrecoveryafter highdosesofsafelyappliedchemotherapy.18–20

Thedeterminationofthetime atwhich tobegin leuka-pheresis isbasedonseveralfactors,suchasthe kineticsof leukocyterecoveryaftermyelosuppressivechemotherapy,the peripheralplateletcount,theabsolutenumberofleukocytes intheperipheralbloodandtheconcentrationofcirculating peripheralCD34+_cells.21–23_{Themostcommonlyapplied} crite-riaare anabsoluteleukocyte count≥1×103 _{cells/␮Land a} CD34+_{cellconcentration≥10cells/␮L.}22,23

TheincreasedcirculationofperipheralCD34+ _cellsafter mobilizationlastsashorttimeandthusitisfundamentalthat apheresisiscarriedoutduringthisperiodinordertocollect asufficientnumberofCD34+_{cells.ThepeakinCD34}+_cellsis reachedaftertheleukocytenadirintherecoveryfromaplasia causedbymyelosuppressivechemotherapy.24_Themomentat which there isa sufficientquantity ofCD34+ _{cells for} col-lectionbyapheresisiswhentheperipheralleukocytecount reaches≥1×103_{cells/␮Lafterrecoveryfromthenadir.}25 Con-sequently,it isproposed thattheperipheralHSCcollection shouldbeginwhenthetotalleukocytecountreaches≥1×103 cells/␮LafterthenadirandtheconcentrationofCD34+_cellsin peripheralbloodis≥10cells/␮L.22,23_{Oneofthebestindicators} ofhematopoieticrecoveryinautologoustransplantationisthe amountofinfusedCD34+_cells/kgbw.16_{Theminimumvalues} fortheacceptablenumberofCD34+_{cellstoachieveafast,safe} andeffectiveengraftmentafterautologoustransplantationis between2and5×106_/kgbw.4,26,27

InordertoobtaintheidealnumberofCD34+_{cellscollected} forautologoustransplantationinchildren,thisstudy inves-tigates whether the useofG-CSF ata fractionateddoseof

15␮g/kgbwwouldreducethenumberofleukapheresis

proce-duresrequiredtoachieveaminimumnumberofCD34+_cells of3×106_{/kgbwandcomparestheresultswiththoseobtained} usingtheconventionalsingledoseof10␮g/kgbwG-CSF.

Methods

Table1–Characteristicsofpatientsineachgroup.

Variable Group10

n=39

Group15

n=26

Gender–n(%)

Male 23(59) 13(50)

Female 16(41) 13(50)

Race–n(%)

White 32(82) 20(77)

Non-White–n(%) 7(18) 6(23) Age–median(range) 12(1–20) 6(1–22)

Diagnosis–n(%)

HL 19 2

NHL 6 2

Solidtumor 14 22

PriorCT–median(range) 2(1–3) 2(1–4)

HL: Hodgkin lymphoma; NHL: Non-Hodgkin lymphoma; Solid tumor: Germ cell tumor, Neuroblastoma, Ewing sarcoma and Medulloblastoma;CT:Chemotherapytreatment

andmobilizedwithchemotherapyandG-CSF.Thestudywas approvedbytheappropriateethicscommitteeandinformed consentformsweresignedbythepatientsortheirguardians. Thepatientsweredividedintotwogroups:Group10was comprisedofpatientswhoreceivedasingledailydoseof10␮g G-CSF/kgbwbetweenDecember1998andDecember2008and Group15wascomprisedofpatientswhoreceiveda fraction-ateddailydose(definedbelowinthesectiononG-CSF)of15␮g G-CSF/kgbwbetweenAugust2010andApril2013.Thepatient characteristicsaredetailedinTable1.

Mobilization

chemotherapy

The mobilization chemotherapy regimens (MCR) adminis-tered using cyclophosphamide (Regimen A) included CY (cyclophosphamide 4g/m2_{/day×1 day), Topo/CY} (topote-can 0.75mg/m2_{/day×5 days and cyclophosphamide} 250mg/m2_{/day×5 days), Cy/Vp-16 (cyclophosphamide} 4–7g/m2_{/day×1 day and etoposide 4g/m}2_{/day×1 day),} Cy/MTX/VP-16 (cyclophosphamide 4–7g/m2_{/day×1 day,} methotrexate8g/m2_{/day×1dayandetoposide4g/m}2_/day×1 day) and Cy/Adria (cyclophosphamide 2.4g/m2_/day×2 days and adriamycin 20mg/m2_{/day×3 days) and those} without cyclophosphamide (Regimen B) were ICE (ifos-famide 3g/m2_{/day×3 days, carboplatin 500mg/m}2_/day×2 days and etoposide 150mg/m2_{/day×3 days), TIP (taxol} 175mg/m2_{/day×1 day, ifosfamide 1.2g/m}2_{/day×5 days} and cisplatin 20mg/m2_{/day×5 days), IFO/Vp-16} (ifos-famide 2.5g/m2_{/day×5 days, cisplatin 40mg/m}2_/day×4 days, doxorubicin 10mg/m2_{/day×4 days and} etopo-side 125mg/m2_{/day×4 days), DHAP (dexamethasone} 40mg/day×4 days, cytarabine: 400mg/m2_{/day×1 day and} cisplatin100mg/m2_/day×1day).

Granulocyte-colonystimulatingfactor

G-CSF(Granulokine®_{;Roche,SP,Brazil/Leucin}®_;Bergamo,SP, Brazil/Filgrastine®_{;Blausiegel,SP,Brazil)isthemostpotent} cytokineavailableandisthemostusedinthemobilization

ofperipheralHPCs5,6_{asithassynergisticactionswithother} growthfactorswhichinducemobilization.Inthisstudythe administrationofG-CSF wasstarted onedayafterthe end oftheMCR.TheG-CSFwasadministeredsubcutaneouslyat 6:00a.m.asasingledoseforGroup10(10␮g/kgbw)andas twodosesforGroup15,10␮g/kgbwat6:00a.m.and5␮g/kg bwat6:00p.m.Thiswascontinueddailyforbothgroupsuntil successfulcollectionoftheminimumamountof3×106_CD34+ cells/kgbworuntilthecharacterizationoflackofmobilization wasdeterminedbyanunsuccessfulcollection.

Vascularaccess

A non-completely implantable two-way central venous catheterwasintroducedintoallpatientsinordertocarryout theLVL.

Large-volumeleukapheresis

Leukapheresis was carriedout inan automaticcontinuous flow cell separator with the anticoagulant citratedextrose solution (ACD-A– CS3000plus®_{; Cobe-spectra}®_{; Com.tec}®_). ThefirstLVLprocedureforGroup15wascarriedoutafterthe MCRwith≥1×103_{cells/␮Lofleukocytesand≥10cells/␮Lof} CD34+_{intheperipheralbloodandforGroup10with≥1×10}3 cells/␮Lofleukocytes.Patientswhoweighed<20kgreceived priming withirradiatedand filteredpackedred blood cells (PRBCs) (20mL/kgbw).Duringthe LVLall patientsreceived intravenous(IV)replacementofcalcium,sodium,potassium andmagnesiumandwereundercontinuousmonitoring.Four blood volumeswere processedperleukapheresisprocedure and if symptoms and/or signs of hypocalcemia related to the anticoagulant used in the cell separator system were observed, adose of0.2mL/kgbwof10%calciumgluconate was administered intravenously. All patients who did not reachtheminimumnumberofCD34+_cells(3×106_/kgbw)in thefirstleukapheresisprocedureweresubmittedtoanother procedureonthefollowingdayregardlessofthenumberof leukocytesandCD34+_{cellsintheperipheralblood.Alimitof} twomoreleukapheresisprocedureswassetforGroup15but withnolimitinthenumberofproceduresforGroup10.

Completebloodcountafterlarge-volumeleukapheresis

A complete blood count was obtained aftereach LVL pro-cedure.Aprophylactic transfusionwascarried outwithan irradiatedandfilteredplateletconcentrate(PC)ifthenumber ofplateletsintheperipheralbloodwas<10×103_{cells/␮Land} irradiatedandfilteredPRBCtransfusionifthehemoglobin(Hb) was<8.0g/dL.

Peripheralhematopoieticprogenitorcellanalysis

Table2–Mobilizationchemotherapyregimens(MCR).

MCR Group10 Group15 p-value

A 23 22 0.03

B 16 4

Statisticalanalysis

Adescriptiveanalysisofallvariablesinvolvedwascarriedout andthentheChisquaredorFisherexacttestswereapplied asappropriatetothecategoricalvariableswhiletheStudent

t-testwasappliedtothecontinuousvariablestocomparethe meansofGroups10and15.Statisticalsignificancewassetfor

p-values<0.05.TheStatisticalPackagefortheSocialSciences (SPSS)version21.0wasuseinallanalyses.

Results

Fifty-three patients received 10␮g G-CSF/kg bw between December 1998 and December 2008 (Group 10). Fourteen patientswereexcludedfromtheanalysis:eightmobilizedonly withG-CSF,onewas29yearsofageandmobilizationfailed infivecases.BetweenAugust2010andApril2013,35patients receivedafractionateddoseof15␮gG-CSF/kgbwdaily(Group 15)withninebeingexcludedfromtheanalysisdueto mobi-lizationfailure.

Ofthe65patientsanalyzed,55.4%weremale,themedian age was ten years (1–22), 80% were white and 55.4% were diagnosed with solid tumors. Themedian number of pre-mobilizationchemotherapycyclesreceivedwastwoforboth groups;varyingfromonetothreeforGroup10andonetofour forGroup15,withnostatisticallysignificantdifference,and withnoinfluenceonthenumberofCD34+_{cellscollected.}

Withregard tothe MCR, Regimen A was used in23/39 (59%) of cases of Group 10 and 22/26 (84%) of cases of Group15resultinginastatisticallysignificantdifference(p -value=0.03)(Table2).However,nodifferenceintheleukocyte recoverytime(≥1×103 _{cells/␮L) wasobservedbetweenthe} tworegimensandtheMCRdidnotinfluencethenumberof leukapheresisprocedurespergroupandnopatientshad MCR-relatedcomplications.

Regimen A: CY (cyclophosphamide 4g/m2_{/day×1 day),}

Topo/CY(topotecan0.75mg/m2_{/day×5daysand} cyclophos-phamide 250mg/m2_{/day×5 days), Cy/Vp-16} (cyclophos-phamide 4–7g/m2_{/day×1 dayand etoposide 4g/m}2_/day×1 day),Cy/MTX/Vp-16(cyclophosphamide4–7g/m2_/day×1day, methotrexate8g/m2_{/day×1dayandetoposide4g/m}2_/day×1 day)andCy/Adria(cyclophosphamide 2.4g/m2_/day×2days andadriamycin20mg/m2_{/day×3days).}

RegimenB:ICE(ifosfamide3g/m2_{/day×3days,carboplatin}

500mg/m2_{/day×2 days and etoposide 150mg/m}2_/day×3 days), TIP (taxol 175mg/m2_{/day×1 day, ifosfamide} 1.2g/m2_{/day×5 days and cisplatin 20mg/m}2_/day×5 days/_{), IFO/Vp-16 (ifosfamide 2.5g/m}2_{/day×5 days,} cis-platin40mg/m2_{/day×4days,doxorubicin 10mg/m}2_/day×4 days and etoposide 125mg/m2_{/day×4 days), DHAP} (dexa-methasone40mg/day×4days,cytarabine:400mg/m2_/day×1 dayandcisplatin100mg/m2_/day×1day).

AllpatientsshowedgoodtolerancetoG-CSF administra-tion; no applications were suspendedor reduced ineither groupandonlyfive(13%)patientsofGroup10andtwo(7%) patientsofGroup15reportedmildbonepain.

Themediannumberofpre-leukapheresisleukocyteswas 14.4×103_{cells/␮L(range:0.9–50.3)forGroup10and22.3×10}3 cells/␮L(range:3.0–73.9)forGroup15.Themedian concen-tration of peripheral CD34+ _{cells was 27.9 per ␮L (range:} 1.10–135.0)forGroup10and29.5cellsper␮L(range:8.0–90.0) for Group 15, with no statistically significant difference between values. Nopatients had complicationsduring the leukapheresisprocedureineithergroup;nohypovolemiawas observedinchildrenwhoweighed<20kgandonlytwo(5%) patientsinGroup10reportedmildparesthesiarelatedtothe useoftheanticoagulant.Thirty-twopatients,15(38%)from Group 10 and 17(65%) from Group15,had platelet counts <50×103_{cells/␮Lduringthepre-leukapheresisperiod.} How-ever,notypeofbleedingoccurredandonlyone(3%)patientof Group15receivedpost-leukapheresisirradiatedandfiltered PC.Noneofthepatientsfromeithergroupreceiveda trans-fusion ofirradiatedand filtered PRBCs afterleukapheresis. Three(7%)casesinGroup10and11(42%)casesinGroup15 receivedprimingofirradiatedandfilteredPRBCsduringthe leukapheresisprocedure.

Atotalof146leukapheresisprocedureswerecarriedout, 110inGroup10and36 inGroup15,withanaverage dura-tion of4h(±2);thesecond leukapheresisprocedure ofone patient in Group 15 was interrupted half-way through the procedureduetomalfunctioningofthecellseparator;there was malfunctioningofthe catheterinfour patients. More-over, one patient of Group 10 and one of Group 15 had prolonged LVLtimes and it wasnotpossible tocollect the minimum number of CD34+ _{cells in two cases of Group} 15.Amedianofthree(range:1–7)leukapheresisprocedures were performedforGroup10withmediansof7.22×108_/kg bw (range: 1.28–20.70) and 8.89×106_{/kg bw (range: 0.3–45)} ofnucleatedcells(NCs) andCD34+ _{cells,respectivelybeing} collected. In Group 15 there was a median of one leuka-pheresisprocedure(range:1–3)andmediansof10.17×108_/kg bw NCs (range:1.22–37.0) and 5.29×106 _CD34+ _{cells/kg bw} (range:0.6–27.8)werecollected(Figures1and2).Themedian

10µg/kg/w of G-CSF 15µg/kg/w of G-CSF

P=0.06 25

17.84 15.28

13.24 9.94 8.2

7.36 6.1

5.1 4.4 4.12

3.7

2.46 2.07 1.22

T

otal of n

ucleated cells (

x 108)

P=0.02 30.0

24.0

18.0

12.0

6.0

0.0

T

o

tal of collected CD34+ (x 106)

10µg/kg/w of G-CSF 15µg/kg/w of G-CSF

°

Figure2–TotalnumberofcollectedCD34+_cells.

°

5

4

3

2

1

T

o

tal of aphereses

P<0.001

10µg/kg/w of G-CSF 15µg/kg/w of G-CSF

Figure3–Numbersofleukapheresis.

numberofdaysofleukocyterecoverywaseight(range:0–18) forGroup10andten(range:2–13)forGroup15(p-value=0.15). Thus,statisticallysignificantdifferencesbetweengroupswere observedforthenumberofleukapheresisprocedures(p-value <0.0001)(Figure3)andthenumberofCD34+_{cellscollected} (p-value=0.02),whilethetotalNCcountresultedinafavorable tendencytowardGroup15(p-value=0.06).

A statistically significant distribution was observed on comparing groups in respect to pre-apheresis peripheral

Table3–CollectedCD34+cellsversuspre-leukapheresis

peripheralCD34+cellcount.

Collectedcells (×106_{cells/␮L/kgbw)}

Pre-leukapheresisCD34+_count (×106_{cells/␮L/kgbw)}

p-value

<10 ≥10

<3.0×106 _{9 22 0.04}

≥3.0×106 _{3 31}

Bw:bodyweight.

CD34+ _{cells (≥10 or<10 cells/␮L)and thenumber ofCD34}+ cellscollected(≥3.0or<3.0×106_{cells/␮L)(Table3).}

Discussion

Some authors23,29 _{suggest that the number of pre-MCR} receivednegativelyinfluencesthenumberofCD34+_cells col-lected,whichwasnotobservedinthisstudy.TheMCRusedin thisstudydidnotcausecomplications,eveninpatients tak-ing highdosesofcyclophosphamide(4–7g/m2_).Itisknown thateachpatientrespondsinadistinctwaytomobilization andthatotherparametersmayhaveanegativeeffect,suchas thetimebetweenthediagnosisandharvest,previous irradia-tion,thrombocytopeniaatthetimeofmobilizationandmany otherfactorscitedintheliterature.5,23,28,29 _{Consequently,in} thisstudyitwasnotpossibletodefinethebestmobilization chemotherapyregimenasisreportedintheliterature.4,12

The administration of G-CSF was well tolerated as the onlysideeffectreportedbythepatientswasmildbonepain in 10% of the cases which is the side effect most com-monlyreported.13_{PrimingwithirradiatedandfilteredPRBCs} ofchildrenweighingbelow20kgallowedtheLVLprocedure to be safely carried out without complications. In the two casesofmildanticoagulant-relatedparesthesia,thecondition wasrevertedusingintravenouscalciumgluconate.Problems related tothe non-completely implantedtwo-way catheter werefewwithonlyaround6%ofthecathetersmalfunctioning, alowerratethanreportedintheliterature.22,29

Withtheexceptionofoneleukapheresisprocedure(1/146), whichwasinterruptedhalfwaythrough,thecellseparators functioned acceptablyand therewasnoneedfortechnical adjustmentintheotherleukapheresisprocedures.Thetime ofleukapheresiswaswelltoleratedincludingbyunder 5-year-oldchildrenastherewerenocomplaintsfrompatientswho experiencedprolongedprocessingtimes;thesepatientsdid notshowsignsorsymptomsofanticoagulant-related hypocal-cemia.

Theabsoluteperipheralleukocytecount(≥1×cells/␮L)and CD34+_{cellconcentration(≥10cells/␮L)werefoundtobegood} parameters tostart leukapheresis,inparticularthe periph-eralCD34+_{cellconcentration(≥10cells/␮L),eventhoughthis} parametercannotbeusedforallpatients.

Conclusions

Thisstudydemonstratedthatthemobilizationandcollection ofperipheralHPCsinchildrenisviableandsafe.Theabsolute peripheralleukocytecount(≥1×103_{cells/␮L)andCD34}+_cell concentration(≥10cells/␮L),whenusedtogether,aregood parameterstoindicatethestartofleukapheresis.The fraction-atedapplication of15␮gG-CSF/kgbwsignificantlyreduced thenumberofleukapheresisproceduresneededtocollecta minimumof3×106_{cells/kgbwCD34}+_cells.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest

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1. LadensteinR,PötschgerU,HartmanO,PearsonAD,Klingebiel T,CastelV,etal.28yearsofhigh-dosetherapyandSCTfor neuroblastomainEurope:lessonsfrommorethan4000 procedures.BoneMarrowTransplant.2008;41Suppl. 2:S118–27.

2. MatthayKK,ReynoldsCP,SeegerRC,ShimadaH,AdkinsES, Haas-KoganD,etal.Long-termresultsforchildrenwith high-riskneuroblastomatreatedonarandomizedtrialof myeloablativetherapyfollowedby13-cis-retinoicacid:a children’soncologygroupstudy.JClinOncol.

2009;27(7):1007–13.

3. BurkhardtB,ReiterA,LandmannE,LangP,LassayL,

DickerhoffR,etal.Pooroutcomeforchildrenandadolescents withprogressivediseaseorrelapseoflymphoblastic

lymphoma:areportfromtheBerlin-Frankfurt-Munster Group.JClinOncol.2009;27(20):3363–9.

4. CastroCGJr,BrunettoA,etal.Transplanteautogênicode células-troncohematopoiéticasemlinfomasdeHodgkinna infânciaeadolescência.Experiênciadoservic¸odeoncologia pediátricadohospitaldeClínicasdePortoAlegre.RBHH. 2011;33Suppl.1:109.

5. NerviB,LinkDC,DiPersioJF.Cytokinesandhematopoietic stemcellmobilization.JCellBiochem.2006;99(3):690–705. 6. PasquiniMC,WangZ,HorowitzMM,GaleRP.2010reportfrom

theCenterforInternationalBloodandMarrowTransplant Research(CIBMTR):currentusesandoutcomesof hematopoieticcelltransplantsforbloodandbonemarrow disorders.ClinTransplant.2010;8:7–105.

7. RavagnaniF,SienaS,BregniM,SciorelliG,GianniAM, PellegrisG.Large-scalecollectionofcirculating

haematopoieticprogenitorsincancerpatientstreatedwith high-dosecyclophosphamideandrecombinanthuman GM-CSF.EurJCancer.2014;26(5):562–4.

8. BensingerWI,WeaverCH,AppelbaumFR,RowleyS,Demirer T,SandersJ,etal.Transplantationofallogeneicperipheral bloodstemcellsmobilizedbyrecombinanthuman granulocytecolony-stimulatingfactor.Blood. 2014;85(6):1655–8.

9. WatanabeT,KawanoY,KanamaruS,OnishiT,KanekoS, WakataY,etal.Endogenousinterleukin-8(IL-8)surgein granulocytecolony-stimulatingfactor-inducedperipheral bloodstemcellmobilization.Blood.1999;93(4):1157–63. 10.BurtnessBA,PsyrriA,RoseM,D’AndreaE,Staugaard-HahnC,

Henderson-BakasM,etal.AphaseIstudyofpaclitaxelfor mobilizationofperipheralbloodprogenitorcells.Bone MarrowTransplant.1999;23(4):311–5.

11.SuredaA,PetitJ,BrunetS,BoquéC,AventínA,MartinoR, etal.Mini-ICEregimenasmobilizationtherapyforchronic myelogenousleukaemiapatientsatdiagnosis.BoneMarrow Transplant.1999;24(12):1285–90.

12.VelasquezWS,CabanillasF,SalvadorP,McLaughlinP,Fridrik M,TuckerS,etal.Effectivesalvagetherapyforlymphoma withcisplatinincombinationwithhigh-doseAra-Cand dexamethasone(DHAP).Blood.1998;71(1):117–22. 13.CaraccioloD,GavarottiP,AgliettaM,BondesanP,FaldaM,

GalloE,etal.High-dosesequential(HDS)chemotherapywith bloodandmarrowcellautograftassalvagetreatmentinvery poorprognosis,relapsednon-Hodgkin’slymphoma.Bone MarrowTransplant.1993;12(6):621–5.

14.WatanabeH,WatanabeT,SuzuyaH,WakataY,KanekoM, OnishiT,etal.Peripheralbloodstemcellmobilizationby granulocytecolony-stimulatingfactoraloneandengraftment kineticsfollowingautologoustransplantationinchildrenand adolescentswithsolidtumor.BoneMarrowTransplant. 2006;37(7):661–8.

15.AnderliniP,PrzepiorkaD,SeongD,MillerP,SundbergJ, LichtigerB,etal.Clinicaltoxicityandlaboratoryeffectsof granulocyte-colony-stimulatingfactor(filgrastim) mobilizationandbloodstemcellapheresisfromnormal donors,andanalysisofchargesfortheprocedures. Transfusion.1996;36(7):590–5.

16.AkizukiS,MizorogiF,InoueT,SudoK,OhnishiA. Pharmacokineticsandadverseeventsfollowing5-day repeatedadministrationoflenograstim,arecombinant humangranulocytecolony-stimulatingfactor,inhealthy subjects.BoneMarrowTransplant.2000;26(9):939–46. 17.AnderliniP,ChamplinR.Useoffilgrastimforstemcell

mobilisationandtransplantationinhigh-dosecancer chemotherapy.Drugs.2014;62Suppl.1:79–88. 18.EngelhardtM,BertzH,AftingM,WallerCF,FinkeJ.

High-versusstandard-dosefilgrastim(rhG-CSF)for mobilizationofperipheral-bloodprogenitorcellsfrom allogeneicdonorsandCD34(+)immunoselection.JClinOncol. 2014;17(7):2160–72.

19.KrögerN,ZellerW,HassanHT,KrügerW,GutensohnK, LöligerC,etal.StemcellmobilizationwithG-CSFalonein breastcancerpatients:higherprogenitorcellyieldby deliveringdivideddoses(2×5microg/kg)comparedtoa singledose(1×10microg/kg).BoneMarrowTransplant. 1999;23(2):125–9.

20.KobbeG,SöhngenD,BauserU,SchneiderP,GermingU,Thiele KP,etal.FactorsinfluencingG-CSF-mediatedmobilizationof hematopoieticprogenitorcellsduringsteady-state

hematopoiesisinpatientswithmalignantlymphomaand multiplemyeloma.AnnHematol.1999;78(10):456–62. 21.CecynKZ,SeberA,GinaniVC,Gonc¸alvesAV,CaramEM,

OguroT,etal.Large-volumeleukapheresisforperipheral bloodprogenitorcellcollectioninlowbodyweightpediatric patients:asinglecenterexperience.TransfusApherSci. 2005;32(3):269–74.

22.FontanaS,GroebliR,LeibundgutK,PabstT,ZwickyC, TaleghaniBM.Progenitorcellrecruitmentduring

individualizedhigh-flow,very-large-volumeaphaeresisfor autologoustransplantationimprovescollectionefficiency. Transfusion.2006;46(8):1408–16.

23.RavagnaniF,ColucciaP,NottiP,ArientiF,BompadreA,Avella M,etal.Peripheralbloodstemcellcollectioninpediatric patients:feasibilityofleukapheresisunderanesthesiain uncompliantsmallchildrenwithsolidtumors.JClinApher. 2006;21(2):85–91.

24.GinaniV,SeberA,etal.Leucaféresesdegrandevolumepara coletadecélulas-troncohematopoiéticasperiféricas

25.ToLB,RobertsMM,HaylockDN,DysonPG,BranfordAL,Thorp D,etal.Comparisonofhaematologicalrecoverytimesand supportivecarerequirementsofautologousrecoveryphase peripheralbloodstemcelltransplants,autologousbone marrowtransplantsandallogeneicbonemarrowtransplants. BoneMarrowTransplant.1992;9(4):277–84.

26.CagnoniPJ,ShpallEJ.Mobilizationandselectionof CD34-positivehematopoieticprogenitors.BloodRev. 1996;10(1):1–7.

27.MarquesJF.Mobilizac¸ãoecoletadascélulasprogenitoras periféricashemopoiéticasperiféricasparatransplante

autólogoempacientesonco-hematológicos.RevBras HematolHemoter.2000;22(2):135–6.

28.DelamainM.Correlac¸ãoentreaquantidadedecélulasCD34+ circulanteseacoletaporaféresedeCPPempacientes onco-hematológicos.Campinas,SãoPaulo,Brasil:

UniversidadeEstadualdeCampinas;2004.Tesedemestrado. 29.DregerP,KlössM,PetersenB,HaferlachT,LöfflerH,Loeffler