Rev. bras. ortop. vol.50 número6

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rev bras ortop.2015;50(6):729–738

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Original

Article

Quantification

of

platelets

obtained

by

different

centrifugation

protocols

in

SHR

rats

夽

João

Alberto

Yazigi

Junior

∗

_,

_João

_Baptista

_Gomes

_dos

_Santos,

_Bruno

_Rodrigues

_Xavier,

Marcela

Fernandes,

Sandra

Gomes

Valente,

Vilnei

Mattiolli

Leite

UniversidadeFederaldeSãoPaulo(Unifesp),SãoPaulo,SP,Brazil

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r

t

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Articlehistory:

Received28September2014 Accepted16December2014 Availableonline20October2015

Keywords: Plateletcount Centrifugation Platelet-richplasma

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Objective:ToquantifytheplateletconcentrationinthebloodofSHRrats,bymeansof differ-entcentrifugationprotocols,andtoevaluatewhatthemosteffectivemethodforobtaining plateletsis.

Methods:Weused40maleratsoftheisogenicSHRlineage.Theanimalsweredividedinto threegroups:control,usingwholebloodwithoutcentrifugation;singlecentrifugation,using wholebloodsubjectedtoasinglecentrifugationat200×gand400×g;anddouble cen-trifugation,usingwholebloodsubjectedonecentrifugationatdifferentrotations,followed bycollectionofwholeplasmasubjectedtoanothercentrifugationatdifferentrotations: 200×g+200×g;200×g+400×g;200×g+800×g;400×g+400×g;400×g+800×g.Samples of3mlofbloodweredrawnfromeachanimalbymeansofcardiacpuncture.Thebloodwas storedinVacutainercollectiontubescontaining3.2%sodiumcitrate.Thebloodfromthe controlgroupanimalswasanalyzedwithoutbeingsubjectedtocentrifugation.Afterthe bloodfromtheothergroupsofanimalshadbeensubjectedtocentrifugation,thewhole plasmawascollectedandsubjectedtoplateletcountinginthelowerthirdofthesample. Results:Weobtainedgreatestplateletenrichmentinthesubgroupwithtwocentrifugations comprising400×gfor10min+400×gfor10min,inwhichthemeanplateletconcentration was11.30timeshigherthanthatofthecontrolgroup.

Conclusion: Itwaspossibletoobtainahighplateletconcentrationusingviablesimple tech-niques,bymeansofcentrifugationofwholebloodanduseofcommonlyusedmaterials.The mosteffectivemethodforobtainingplateletconcentratewasfoundinsamplessubjected totwocentrifugations.

夽

StudycarriedoutattheLaboratoryofMicrosurgeryoftheDisciplineofHandandUpperLimbSurgery,DepartmentofOrthopedics andTraumatology,UniversidadeFederaldeSãoPaulo(Unifesp),SãoPaulo,SP,Brazil.

∗ _{Corresponding}_author_.

E-mail:junioryazigi73@yahoo.com.br(J.A.YazigiJunior). http://dx.doi.org/10.1016/j.rboe.2015.10.008

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Quantificac¸ão

do

número

de

plaquetas

a

partir

de

diferentes

métodos

de

centrifugac¸ão

em

ratos

da

linhagem

SHR

Palavras-chave:

Contagemdeplaquetas Centrifugac¸ão

Plasmaricoemplaquetas

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s

u

m

o

Objetivo: QuantificaraconcentraçãodeplaquetasdosanguederatosSHR,por meiode diferentesprotocolosdecentrifugação,eavaliarqualométodomaiseficazdeobtençãode plaquetas.

Métodos: Usamos40ratosmachosdalinhagemisogênicaSHR.Osanimaisforamdivididos emtrêsgrupos:Controle(GCT)-sanguetotalsemcentrifugação;ÚnicaCentrifugação(GUC) -sanguetotalsubmetidoaumaúnicacentrifugação:200ge400g;DuplaCentrifugação (GDC)-sanguetotalsubmetidoaumacentrifugação,seguidodecoletadoplasmatotal,e realizadoumacentrifugação,emdiferentesrotações:200g+200g;200g+400g;200g+800g; 400g+400g;400g+800g.Foramretirados3mldesanguedecadaanimalpormeiodepunção cardíaca.Osanguefoiacondicionadoemtubodecoletavacutainercomcitratodesódio3,2%. Osanguedosanimaisdogrupocontrolenãofoisubmetidoàcentrifugaçãoefoianalisado. Apósacentrifugaçãodosanguedosanimais,submetidoàcentrifugação,oplasmatotalfoi coletadoesubmetidoàcontagemdeplaquetasnoterçoinferiordaamostra.

Resultados: Obtivemos maior enriquecimento de plaquetas no subgrupo de duas centrifugações (400g por 10 minutos+400g por 10 minutos), no qual ocorreu uma concentraçãomédiadeplaquetas11,30vezessuperioremrelaçãoaogrupocontrole. Conclusão: Foipossívelobterumaaltaconcentraçãoplaquetária,comtécnicasimplese viável,pormeiodecentrifugaçãodosanguetotaleusodemateriaisdeusocorriqueiro;e métodomaiseficazdeobtençãodeconcentradodeplaquetasocorreunasamostras sub-metidasaduascentrifugações.

Introduction

Plateletsarecytoplasmicanucleatefragmentsfoundinblood andproducedbymegakaryocytesinthebonemarrow.1,2 _Of

the totalnumber of platelets present inthe body,70% are presentinthecirculationand30%inthespleen,remaining inthecirculationforanaverageof10 days,whentheyare removed bythe reticuloendothelial cells ofthe spleenand liver.1–3 _Platelets_are _directly_involved _in_several_major

dis-eases,asinseverethromboticsyndromesorconditionssuch asarterialthrombosis.4,5

Thehemostaticsystemisinherentlyresponsibleforthe maintenanceofbloodflowandvascularintegrity,asitisable toformaplugoveradamagedsurfaceofthevascular endothe-liumwhenitsuffersaninjury.Thus,hemostasisminimizes bloodlossandpromotestherestorationofthenormalvascular architecture.6–8

Theplatelet-rich plasma(PRP)istheautologousplatelet concentrationinasmallvolumeofplasma,obtainedby cen-trifugationofwholeblood,consideredanimportantsourceof growthfactors.Itsbasicconstitutionisgivenbythree com-ponents:plasma,leukocytesandplatelets.Theplatelet-rich fibrin (PRF) is a platelet concentrate, obtained from fibrin membrane,withhighpotentialfortissueregeneration.Thus, asinPRP,plateletconcentratescontainedinthePRFrelease growthfactors(GFs)thatenhancetheregenerationprocess. Additionally,thefibrinmatrixpromotesangiogenesis, facili-tatestheaccesstotheinjuredsite,withanimportantrolein tissuehealing.9

DescribedforthefirsttimeinFrancein2000,9_{platelet-rich}

fibrin(PRF),anewconceptandstilllittledescribedinthe treat-mentusingfibrin gel,isaplateletconcentrateoverafibrin membranewithahighpotentialforinjuryrepair.The mem-braneisobtainedfromautologousbloodwithouttheaddition ofexternalfactors.10

Itisknownthatplateletsactintheprocessofhemostasis, injuryhealingandreepithelialization,releaseseveralgrowth factors and thatatleastsevendifferent GFwere identified intheinitialphaseofinjuryhealing.Therefore,the release oftheseGFbyplatelet␣-granulesplaysanimportantrolein

the controlandproliferation ofmesenchymalcells, includ-ingfibroblasts.PRPisactivatedbyaddingcalciumions,with thereleaseofGFandtheexocytosisof␣-granulesand

trans-forms fibrinogeninto fibrin. Through this process,wealso obtain autologousfibringlue,alsocalledplateletgel orPRP gel. The PRP gel presentation facilitates its use and han-dlinginseveralsurgicalareasandpromotestheintegration offlapsandgrafts,suchasbone,skin,cartilageorfatcells. Theuse resultsinmorerapidand efficienthealing,dueto adhesioneffectofthegel,whichisproportionaltothepresent fibrinogen.11–13

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shouldbecollectedinadrytubetoobtainplatelet-rich fib-rin.

ThereareseveralavailablePRPpreparationsystemsusing abenchtop centrifuge, but inpreclinical and clinical stud-ies,theattainedresultsseemcontradictory.Thereisalackof moresystematicandconsistentstudiesonthesubject,which makesPRPpreparationremain anexperimentalprocedure, despiteitspromisingapplicationintissueregeneration.

Calixto,14 _when_studying_the _human_blood,_verified_that

thebestmethodofobtainingPRPistheoneinwhichasingle centrifugationismade,of200×gfor10min.Pereira,15_using

sevenprotocolstoobtainPRPinhorses,verifiedthatthehigher thegforceandthelongerthecentrifugationtime,thehigher wasthetotalplateletconcentration.

Asthe animalsmostoftenusedinresearchlaboratories areratsandduetothelackofstudiesinthisanimalmodel, wedecidedtoperformthisstudytoevaluatethebestmethod to obtain the best platelet concentration, in different pro-tocols, to quantify the concentration of blood platelets in SHRrats,usingdifferent centrifugationprotocolsand eval-uate what the most effective method is toobtain platelet concentrate.

Materials

and

methods

TheworkwasperformedintheLaboratoryofMicrosurgeryof theHandandUpperLimbSurgeryDisciplineofthe Depart-mentofOrthopedicsandTraumatologyandattheLaboratory ofHematologyoftheDepartmentofClinicaland Toxicologi-calAnalysesofourinstitution.Thestudywasapprovedbythe ResearchEthicsCommittee(RECn.0312/12).

Atotalof40malerats oftheisogenic SHRlineagewere usedinthestudy,weighing280–300g,providedbyCentrode Desenvolvimento de ModelosExperimentais para Medicina eBiologia.Theanimalswerekeptincollectivecages inthe vivarium,with5ratsineach,inacontrolledlight-darkcycle environment(12/12h),temperatureof21±2◦C_,_free_access_to

waterandfoodthroughouttheentirestudyperiod. Theanimalsweredividedintothreegroups:

(1)ControlGroup(CTG)–wholebloodwithoutcentrifugation (n=5).

(2)SingleCentrifugationGroup(SCG)–wholebloodsubjected toasinglecentrifugation.Thisgroupwasdivided intotwo subgroups:

(a) SCG-I:200×gcentrifugationfor10min(n=5) (b) SCG-II:400×gcentrifugationfor10min(n=5)

(3) Double Centrifugation Group(DCG) – wholeblood sub-jectedtocentrifugation,followedbywholeplasmacollection andsubjectedtoasecondcentrifugationatdifferentrotations. Thisgroupwasdividedintofivesubgroups:

(a) DCG-I:centrifugationat200×gfor10min,followedbya second200×gcentrifugationfor10min(n=5)

(b) DCG-II:200×gcentrifugationfor10min,followedbya sec-ondcentrifugationat400×gfor10min(n=5)

(c) DCG-III200×gcentrifugationfor10min,followedbya sec-ondcentrifugationfor10minat800×g(n=5)

Fig.1–Plasmacollectioninthe1.5mLEppendorftube.

(d) DCG-IV400×gcentrifugationfor10min,followedbya sec-ondcentrifugationfor10minat400×g(n=5)

(e) DCG-V:400×gcentrifugationfor10min,followedbya sec-ondcentrifugationfor10minat800×g(n=5)

Bloodandplasmasamplecollection

Theanimalswereanesthetizedwithanintraperitoneal injec-tionwithanestheticsolutionconsistingofxylazine1U/100g andketamine1U/100g.

After being anesthetized, 3ml of blood was withdrawn from each animalbycardiacpuncture.Thecollectedblood wasplacedinavacutainertubewithsodiumcitrateat3.2%.

Thebloodoftheanimalsfromthecontrolgroupwasnot submittedtocentrifugationandwasanalyzedaftershaking thetubefor2minat4◦_C_for_{homogenization.}

Samplesofwholeanimalbloodwerecentrifuged accord-ingtotheprotocoldescribedaboveforeachgroupinabench centrifuge(Eppendorf5810R)at4◦_C.

Aftercentrifugation,theanimals’bloodwassubjectedtoa singlecentrifugationandthewholeplasmawascollectedinto a1.5mLEppendorftubeandsubjectedtoplateletcountingin thelowerthirdofthesample.

For the animals’blood subjected totwocentrifugations, afterthefirstcentrifugationthewholeplasmawascollected ina1.5mLEppendorftubeandsubjectedtoanother centrifu-gation.Theplateletcountwasperformedinthelowerthirdof thesample(Fig.1).

Plateletcount

Theobtainedsampleswere subjectedtoplateletcountina hematologyanalyzer(AnimalBloodCounter–VetABC) cali-bratedforbloodparametersofrats(Fig.2).

Statisticalmethods

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Table1–Descriptionofresultinghematologicalparameters.

Variable Group Mean SD Median Minimum Maximum N p

Leukocytes CTRL 3060 219.1 3200 2700 3200 5 0.016

200×g-10min 420 465.8 200 100 1200 5

400×g-10min 420 549.5 200 100 1400 5

200×g-10min+200×g-10min 1160 2260.1 100 100 5200 5

200×g-10min+400×g-10min 1860 1105.9 1400 500 3100 5

200×g-10min+800×g-10min 2.700 3755.7 700 300 9200 5

400×g-10min+400×g-10min 3.020 2983.6 1500 400 7800 5

400×g-10min+800×g-10min 3160 3943.7 500 200 8700 5

Erythrocytes CTRL 7,120,000 358,956.8 7,020,000 6.640.000 7,530,000 5 <0.001

200×g-10min 22,000 16,431.7 20,000 10,000 50,000 5

400×g-10min 14,000 11,401.8 10,000 0 30,000 5

200×g-10min+200×g-10min 80,000 134,721.9 20,000 10,000 320,000 5

200×g-10min+400×g-10min 262,000 895,545 240,000 150,000 380,000 5

200×g-10min+800×g-10min 170,000 101,242.3 150,000 60,000 330,000 5

400×g-10min+400×g-10min 120,000 86,313.4 150,000 20,000 220,000 5

400×g-10min+800×g-10min 188,000 199,549.5 70,000 10,000 420,000 5

Platelets CTRL 648,800 30,094.9 648,000 614,000 695,000 5 0.013

200×g-10min 1,632,400 580,962.4 1650.000 682,000 2,122,000 5

400×g-10min 939,600 687,417.1 863,000 110,000 1,764,000 5

200×g-10min+200×g-10min 3,826,000 4920.808.4 456,000 29,000 970.0000 5

200×g-10min+400×g-10min 4,760,000 2254.550.953 530.0000 130.0000 720.0000 5

200×g-10min+800×g-10min 4,080,000 1685.823,241 310.0000 270.0000 6500.000 5

400×g-10min+400×g-10min 7,320,000 2819.929,077 7,300,000 2,800,000 9,700,000 5

400×g-10min+800×g-10min 5,500,000 1813.835,715 5200.000 3400.000 7600.000 5

Plasmavolume(mL) CTRL 0 0 0 0 0 5 <0.001

200×g-10min 0.72 0.11 0.8 0.6 0.8 5

400×g-10min 1.10 0.10 1.1 1 1.2 5

200×g-10min+200×g-10min 1.46 0.11 1.5 1.3 1.6 5

200×g-10min+400×g-10min 1.60 0.10 1.6 1.5 1.7 5

200×g-10min+800×g-10min 1.62 0.04 1.6 1.6 1.7 5

400×g-10min+400×g-10min 1.62 0.08 1.6 1.5 1.7 5

400×g-10min+800×g-10min 1.72 0.08 1.7 1.6 1.8 5

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Fig.2–Plateletcountinthehematologyanalyzer.

median,minimumandmaximum)werecomparedtothe sub-groupsusingKruskal–WallistestsfollowedbyDunn’smultiple non-parametriccomparisons,whennecessarytocomparethe subgroupstwobytwo.

Results

Theresultsweredepictedusingtablesandbarchartsthat rep-resentthemedianvaluesofeachparameteraccordingtothe subgroups;thetestswereperformedwithasignificancelevel of5%.

Table1showsthatallassessedparametersshowed statis-ticallysignificantdifferencebetweenthegroups(p<0.05).

Figs.3and 4suggesthigher amountsofleukocytes and erythrocytesinthecontrolgroup.

Fig.5showsthatthehighestplateletenrichmentoccurred inthesubgroupoftwo400×g+400×gcentrifugations.

TheplasmavolumevaluesshowninFig.6arehigherwith twocentrifugations(400×gfor10min+800×gfor10min).

Table2showsthattheresultingamountofleukocyteswas statisticallyhigherinthecontrolgroupwhencomparedtothe subgroupswithasinglecentrifugation(p=0.004andp=0.004) and inrelation tothe subgroupwith two 200×g centrifu-gations and10mineach (p=0.007).Onthe other hand,the subgroupwithtwo 400×g centrifugationsand10mineach showedstatisticallymoreleukocytesthanthesubgroupswith asinglecentrifugation(p<0.05)andthesubgroupwithtwo 200×gcentrifugationsand10mineach(p=0.031).

Table 3 shows that the amount of red blood cells was statistically higher in the control than in the other sub-groups(p<0.05),withtheexceptionofthesubgroupswithtwo centrifugationsof200×g-10min+400×g-10minand200×g -10min+800×g-10min (p=0.192 and p=0.056). These two subgroupsshowedstatisticallymoreredbloodcellsthanthe subgroups ofasinglecentrifugation and subgroupsoftwo 200×gcentrifugationsand10mineach(p<0.05).

Table4showsthattheamountofplateletswithtwo cen-trifugationswasstatisticallyhigherthanthecontrol,witha singlecentrifugation,andthesubgroupwithtwo centrifuga-tions,with200×gand10mineach(p<0.05).

Table5showsthattheobtainedplasmavolumewas statis-ticallysimilartothebehaviorofplatelets.Subgroupswithtwo centrifugationshavehighervolumethantheothersubgroups, exceptforthesubgroupwithtwocentrifugations,with200×g and10mineach.

Thetablesandfiguresshowthattheamountofplatelets with two centrifugations (400×g+400×g subgroup) was statisticallyhigherthanthecontrolwithonlyasingle centrifu-gation,whereasthesubgroupwithtwocentrifugations,with 200×gand10mineach(p<0.05)showedamean concentra-tionofplatelets (400×g+400×g)thatwas11.30-foldhigher comparedtothecontrolgroup.

Discussion

The literature shows techniques to obtain platelet-rich plasma, asthefirst oneused,onlyapplicableinahospital

0

CTRL

200g–10min 400g–10min

200g–10min+200g–10min200g–10min+400g–10min200g–10min+800g–10min400g–10min+400g–10min400g–10min+800g–10min 3500

3000

2500

2000

1500

Leukocytes

1000

500

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CTRL

200g–10min 400g–10min

200g–10min+200g –10min200g–10min+400g–10min200g–10min+800g–10min400g–10min+400g–10min400g–10min+800g–10min

8 000 000

7 000 000

6 000 000

5 000 000

4 000 000

3 000 000

2 000 000

1 000 000

0

Erythrocyte

Fig.4–Medianerythrocytevaluesaccordingtosubgroups.

structurewithplasmapheresismachinethatrequiresa tech-niciantomanipulateit.16_The_procedure_had_higher_morbidity

andrequiredsophisticatedtechnology.

ThesearchfortechniquestoobtainPRPwithlowercosts hasledtotheuseofsomesimplemethodswithtesttubes and a common centrifuge, which allowthe preparation of platelet-rich plasma at a much lower cost and in an out-patient setting.However, thesemethods are laborious and require learning from those who will perform the proce-dure.

The existing methods in the literature do not reach the same platelet concentrations. Someare insufficient to improvetissueregeneration.Thesefactorsmayexplainmuch ofthe criticismabout theeffectivenessand applicabilityof platelet-richplasma.

Another keyfactor isassociatedwiththe anticoagulant. Usually,sodiumcitrate3.2%isused.Thesodiumcitrate cap-turescalciumionsfrombloodandneutralizedthem,forming a compound named chelate that prevents clot formation. Additionally, thesodium citrate doesnot alter the platelet membranereceptorsand,consequently,thechelationprocess canbereversedbytheadditionofcalciumchloridetoform plateletgel.17

Therecenttechnologyallowsthe useofPRP withsmall bloodvolumes,thusminimizingtheneedforredbloodcell reinfusionandassociatedrisks.However,thereislittle infor-mationabouttheoptimalamountofplateletsforbettertissue repair.

TheliteratureshowsseveralprotocolsforobtainingPRP. Thenumberofcentrifugations(oneortwo)willdependonthe

CTRL

200g–10min 400g–10min

200g–10min+200g–10min200g–10min+400g–10min200g–10min+800g–10min400g–10min+400g–10min400g–10min+800g–10min

8 000 000

7 000 000

6 000 000

5 000 000

4 000 000

3 000 000

2 000 000

1 000 000

0

Platelets

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CTRL

200g–10min 400g–10min

200g–10min+200g–10min200g–10min+400g–10min200g–10min+800g–10min400g–10min+400g–10min400g–10min+800g–10min 0.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

Plasma volume (mL)

0.4

0.2

Fig.6–Medianplasmavolumevalues.

methodtobeused.Someauthorssuggestonlyone centrifuga-tion,whileotherreferencesindicateadoublecentrifugation protocol.

AnyprotocoltoobtainthePRPshouldconcentrateplatelets atitsmaximumtocorrespondtotheclinicalresultsreported

intheliterature.Butinadditiontoahighplatelet concentra-tionwithalargereleaseofgrowthfactors,itisveryimportant thattheintegrityofplateletsismaintained.Thegrowth fac-tors must bereleased from viable platelets, as during the actofgranularexocytosis,theplateletcompletesthetertiary

Table2–Resultsofmultipleleukocytecomparisons.

Leukocytes

Comparison Zvalue p

CTRLVS 200×g-10min 2.84 0.004

CTRLVS 400×g-10min 2.84 0.004

CTRLVS 200×g-10min+200×g-10min 2.70 0.007

CTRLVS 200×g-10min+400×g-10min 0.90 0.367

CTRLVS 200×g-10min+800×g-10min 1.04 0.298

CTRLVS 400×g-10min+400×g-10min 0.54 0.589

CTRLVS 400×g-10min+800×g-10min 1.11 0.267

200×g-10minVS 400×g-10min 0.00 >0.999

200×g-10minVS 200×g-10min+200×g-10min −0.14 0.890

200×g-10minVS 200×g-10min+400×g-10min −1.94 0.052

200×g-10minVS 200×g-10min+800×g-10min −1.80 0.071

200×g-10minVS 400×g-10min+400×g-10min −2.30 0.021

200×g-10minVS 400×g-10min+800×g-10min −1.73 0.083

400×g-10minVS 200×g-10min+200×g-10min −0.14 0.890

400×g-10minVS 200×g-10min+400×g-10min −1.94 0.052

400×g-10minVS 200×g-10min+800×g-10min −1.80 0.071

400×g-10minVS 400×g-10min+400×g-10min −2.30 0.021

400×g-10minVS 400×g-10min+800×g-10min −1.73 0.083

200×g-10min+200×g-10minVS 200×g-10min+400×g-10min −1.80 0.071

200×g-10min+400×g-10minVS 200×g-10min+800×g-10min 0.14 0.890

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Table3–Resultsofmultipleerythrocytecomparisons.

Erythrocytes

Comparison Zvalue p

CTRLVS 200×g-10min 3.90 <0.001

CTRLVS 400×g-10min 4.27 <0.001

CTRLVS 200×g-10min+200×g-10min 3.41 0.001

CTRLVS 200×g-10min+400×g-10min 1.30 0.192

CTRLVS 200×g-10min+800×g-10min 1.91 0.056

CTRLVS 400×g-10min+400×g-10min 2.44 0.015

CTRLVS 400×g-10min+800×g-10min 2.18 0.029

200×g-10minVS 400×g-10min 0.37 0.708

200×g-10minVS 200×g-10min+200×g-10min −0.49 0.627

200×g-10minVS 200×g-10min+400×g-10min −2.59 0.010

200×g-10minVS 200×g-10min+800×g-10min −1.98 0.047

200×g-10minVS 400×g-10min+400×g-10min −1.46 0.145

200×g-10minVS 400×g-10min+800×g-10min −1.72 0.086

400×g-10minVS 200×g-10min+200×g-10min −0.86 0.390

400×g-10minVS 200×g-10min+400×g-10min −2.97 0.003

400×g-10minVS 200×g-10min+800×g-10min −2.36 0.018

400×g-10minVS 400×g-10min+400×g-10min −1.83 0.067

400×g-10minVS 400×g-10min+800×g-10min −2.09 0.036

Dunn’smultiplecomparisontestresults.

Table4–Resultsofmultipleplateletcomparisons.

Platelets

Comparison Zvalue p

CTRLVS 200×g-10min −1.03 0.305

CTRLVS 400×g-10min −0.25 0.803

CTRLVS 200×g-10min+200×g-10min −1.19 0.233

CTRLVS 200×g-10min+400×g-10min −2.50 0.013

CTRLVS 200×g-10min+800×g-10min −2.29 0.022

CTRLVS 400×g-10min+400×g-10min −3.48 0.001

CTRLVS 400×g-10min+800×g-10min −2.91 0.004

200×g-10minVS 400×g-10min 0.78 0.437

200×g-10minVS 200×g-10min+200×g-10min −0.17 0.868

200×g-10minVS 200×g-10min+400×g-10min −1.47 0.142

200×g-10minVS 200×g-10min+800×g-10min −1.26 0.207

200×g-10minVS 400×g-10min+400×g-10min −2.45 0.014

200×g-10minVS 400×g-10min+800×g-10min −1.89 0.059

400×g-10minVS 200×g-10min+200×g-10min −0.94 0.346

400×g-10minVS 200×g-10min+400×g-10min −2.25 0.025

400×g-10minVS 200×g-10min+800×g-10min −2.04 0.041

400×g-10minVS 400×g-10min+400×g-10min −3.23 0.001

400×g-10minVS 400×g-10min+800×g-10min −2.66 0.008

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Table4–(Continued₎

Platelets

Comparison Zvalue p

Dunn’smultiplecomparisontestresults.

structureofthegrowthfactorproteins.Fragmentedplatelets candegranulate largeamounts ofgrowth factors,but with decreasedeffectiveness.18

In this study, we compared the number of platelets obtainedfromperipheralbloodandsubjectedtooneandtwo centrifugations;thehigherenrichmentoccurredinthegroup withtwocentrifugations.Theresultsaresimilar,when com-paredtotheresultsofsomepublishedstudies,suchastheone byCalixto.14

InthestudybyCalixto,14_carried_out_in_humans,_test_tubes

withtheanticoagulantsodiumcitrateat3.2%andEDTAwere used,which showed adifference inplatelet concentration, higherwhencitratewasused.Basedonthisfact,wechose thecitrateastheonlyanticoagulantinthecollectiontubes.

Vendraminetal.19_obtained_{platelet-rich}_plasma_of_better

qualityusingtwocentrifugationsat400×gfor10min+800×g for10min.Inourstudy,weobtainedahighplatelet concen-tration with 400×g+800×g; however, the highest amount of platelets was obtained with 400×g for 10min+400×g for10min.Thisresultshowsthatthe lysisofplatelets can occurabovethisforce,whichinfluencesitsfinal concentra-tion.

Several studies were performed inhumans, rabbits and horses;however,wedidnotfindcentrifugationprotocolsin rats.WiththeestablishmentofaprotocoltoobtainPRPinrats, whichreachedahighplatelet concentration,weencourage futurestudiestoevaluatetheclinicaleffectivenessof platelet-richplasma.Furtherstudiesmayemerge fromourprotocol

Table5–Resultsofplasmavolumecomparisons.

Plasmavolume

Comparison Zvalue p

CTRLVS 200×g-10min −0.69 0.488

CTRLVS 400×g-10min −1.39 0.166

CTRLVS 200×g-10min+200×g-10min −2.36 0.018

CTRLVS 200×g-10min+400×g-10min −3.38 0.001

CTRLVS 200×g-10min+800×g-10min −3.55 <0.001

CTRLVS 400×g-10min+400×g-10min −3.58 <0.001

CTRLVS 400×g-10min+800×g-10min −4.47 <0.001

200×g-10minVS 400×g-10min −0.69 0.488

200×g-10minVS 200×g-10min+200×g-10min −1.66 0.096

200×g-10minVS 200×g-10min+400×g-10min −2.69 0.007

200×g-10minVS 200×g-10min+800×g-10min −2.86 0.004

200×g-10minVS 400×g-10min+400×g-10min −2.88 0.004

200×g-10minVS 400×g-10min+800×g-10min −3.77 <0.001

400×g-10minVS 200×g-10min+200×g-10min −0.97 0.332

400×g-10minVS 200×g-10min+400×g-10min −2.00 0.046

400×g-10minVS 200×g-10min+800×g-10min −2.16 0.031

400×g-10minVS 400×g-10min+400×g-10min −2.19 0.028

400×g-10minVS 400×g-10min+800×g-10min −3.08 0.002

(10)

andexpanditsuseintheregenerationofnerves,tissue,bone andligamentgrafts.

Conclusion

Itwaspossibletoobtainahighplateletconcentration,usinga simpleandfeasibletechnique,throughwholeblood centrifu-gationandusingcommonmaterialssuchascollectiontubes, syringesandneedles.Themosteffectivemethodtoobtaina plateletconcentratewasachievedinthesamplessubjected totwocentrifugations(400×gfor10min+400×gfor10min), whichyieldedameanplateletconcentrationthatwas 11.30-foldhigherthanthatinthecontrolgroup.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ToDr.PrimaveraBorelliGarciaandtechnicianEdsonfromthe HematologyLaboratoryoftheDepartmentofClinicaland Tox-icologicalAnalysis,FaculdadedeCiênciasFarmacêuticasda USP,fortheirhelpandtechnicalsupportfortheresearch.

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