Rev. Bras. Hematol. Hemoter. vol.39 número3

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

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Review

article

Thrombin

generation

assays

for

global

evaluation

of

the

hemostatic

system:

perspectives

and

limitations

Rita

Carolina

Figueiredo

Duarte

a

_,

_Cláudia

_Natália

_Ferreira

a

_,

Danyelle

Romana

Alves

Rios

b

_,

_Helton

_José

_dos

_Reis

a

_,

_Maria

_das

_Grac¸as

_Carvalho

a,∗ a_Universidade_Federal_de_Minas_Gerais_(UFMG),_Belo_Horizonte,_MG,_Brazil

b_Universidade_Federal_de_São_João_del-Rei_(UFSJ),_{Divinópolis,}_MG,_Brazil

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Articlehistory:

Received15April2016 Accepted30March2017 Availableonline9May2017

Keywords:

Thrombingeneration CATmethod Hemostasis

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b

s

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t

Theexistingtechniquestoevaluatehemostasisinclinicallaboratoriesarenotsensitive enoughtodetecthypercoagulableandmildhypocoagulablestates.Underdifferent exper-imental conditions, the thrombin generation test maymeet these requirements. This techniqueevaluatestheoverallbalancebetweenprocoagulantandanticoagulantforcesand hasprovidednewinsightsinourunderstandingofthecoagulationcascade,aswellasof thediagnosisofhypocoagulabilityandhypercoagulabilityconditions.Thrombingenerated inthethrombingenerationtestcanbequantifiedasplatelet-richorplatelet-poorplasma usingthecalibratedautomatedthrombogrammethod,whichmonitorsthecleavageofa fluorogenicsubstratethatissimultaneouslycomparedtotheknownthrombinactivityin anon-clottingplasmasample.Thecalibratedautomatedthrombogrammethodisanopen system,inwhichdifferentantibodies,proteins,enzymesandpeptidescanbeintroducedto answerspecificquestionsregardinghemostaticprocesses.Thethrombingenerationtesthas greatclinicalpotential,suchasinmonitoringpatientstakinganticoagulantsandantiplatelet drugs,screeningforgeneticoracquiredthromboticdisorders,andevaluatingbleedingrisk controlinpatientswithhemophiliausingbypassagentsorreplacementtherapy.Different toconventionalcoagulationtests,thethrombingenerationtestcanbeusedforanoverall evaluationofhemostasis,theresultsofwhichcanthenbeusedtoevaluatespecific char-acteristicsofhemostasis,suchasprothrombintime,activatedpartialthromboplastintime, andlevelsoffibrinogenandothercoagulationfactors.Theintroductionofthismethod willcontributetoabetterunderstandingandevaluationofoverallhemostaticprocesses; however,thismethodstillrequiresstandardizationandclinicalvalidation.

©2017Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author_at_:_Clinical_and_{Toxicological}_Department,_Universidade_Federal_de_Minas_Gerais_(UFMG),_Av._Antônio_Carlos,_6627,

31270-910Pampulha,BeloHorizonte,Brazil.

E-mailaddress:mgcarvalho@farmacia.ufmg.br(M.G.Carvalho). http://dx.doi.org/10.1016/j.bjhh.2017.03.009

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Introduction

Different aspects of hemostasis can be studied using the methods that are currently available to researchers and clinicallaboratories.These includecoagulometryand chro-mogenic methods, which assess separate aspects of the hemostaticprocess.However,thesemethodsdonotprovide anoverall evaluationof hemostasis.1 _The _method _of

eval-uation is chosen depending on the patient’s clinical data, andmayseparatelyprovideimportantinformationabout pri-mary,secondaryandtertiaryphasesofhemostasis,inaddition tonatural inhibitors such asprotein C (PC)protein S (PS), antithrombin(AT)andtissuefactorpathwayinhibitor(TFPI). Molecular methods also contribute to the investigation of hemostaticdisorders,inparticular,incasesofgenetic resis-tancetoactivatedPC(aPC),whichisprimarilycausedbyfactor VLeidenand other uncommonmutations,and incasesof hyperprothrombinemiacausedbytheG20210Aprothrombin mutation.2_Despite_the_diversity_of_laboratory_methods_used

toevaluatehemostaticprocesses,noneofthecurrently avail-ablemethodsisabletoassessallthephasesofhemostasis. Thus,theresultsobtainedusingtheseconventionalmethods arenotalwaysassociatedwithclinicalmanifestations.2

Traditionallyusedmethodshavesatisfactorysensitivityfor moderateandseverehypocoagulability,butnotfor hyperco-agulableormildhypocoagulablestates.Suchmethodsonly provideinformationaboutthebeginning ofthecoagulation process, and therefore, the resultof the test is not repre-sentativeoftheentireclotformationprocess,asmeasured using the total thrombin generation capacity.2–4 _The

typi-calcoagulometricmeasurements,suchasprothrombintime (PT)andactivatedpartialthromboplastintime(aPTT), mea-sure onlythe clottingtime corresponding to the initiation phaseofthecoagulationprocess.Furthermore,theend-point ofthesetestsoccursaftertheformationofonly5%oftotal thrombin.2,4 _Therefore, _PT_and _aPTT_reflect_only_the _initial

coagulation process while the formation of thrombin and fibrin isstill occurring.2,5 _A _greater _amount _of_thrombin _is

generatedduringtheamplificationandpropagationphases, resulting in an exponential increase in thrombin, which becomesinactivatedbyphysiologicalanticoagulantssuchas alpha-2-macroglobulin,AT,PCandPS.1,2 _Therefore,

conven-tionaltestsdonotprovideinformationabouttheamplification andpropagationphasesofthehemostaticsystem.1,2

Theintroductionofamethodabletoevaluatetheentire coagulation process is highly desirable, as this could bet-ter reflect bleeding and thrombotic risks. Many attempts havebeen madeto achieve this goal,which would ideally accuratelyreflectallcomponentsandconditionsofthe hemo-staticprocess,includingplatelets,coagulationfactors,natural inhibitors, theendotheliumand its interactions,aswell as fibrinolysisandbloodflow.Severalauthorshavesuggestedthe needtointroduceinvitromethodsrepresentativeofthemain physiologicalaspectsofhemostasisasapossiblesolution.3,6–8

Attemptstodevelopamethodtocomprehensivelyevaluate hemostasisbeganseveraldecadesago.In1953,MacFarlane and Biggs9 _were _the _first _to_report _thrombin _generation _in

thebloodusingalaboriousandtime-consumingtechnique, whichrendereditinapplicableforuseintheclinicalpractice.

Inthesameyear,PitneyandDacie10_reported_the

measure-ment of thrombin generation inplasma. Many years later, convincedoftheneedforacomprehensivetestforthebroader assessmentofhemostasis,theillustriousProfessorCoenraad Hemkeretal.attheUniversityofMaastricht(Netherlands)1,11

improvedandsemi-automatedthethrombingeneration tech-nique,initiallyemployingachromogenicmethod,andlatera fluorogenicmethod.12_This_improvement_contributed_greatly

tothesuccessfuluseofthistechniqueinnumerousstudies. Duetothelackofinformationaboutglobaltestsof throm-bingeneration,wepresentashortdiscussionofthistechnique withemphasisonthecalibratedautomatedthrombogram® (CAT) method. Inthis concise review,we present method-ological aspectsofthe thrombin generation test(TGT), the evaluationofhemostaticcomponentsundersomeanalytical conditions,theuseofthetestinexperimentalstudies, poten-tialclinicalapplicationsasaglobalcoagulationtest,aswellas itslimitationsandfutureperspectives.

Thrombin

generation

assays

and

the

calibrated

automated

thrombogram

method

Thrombinisakeyproteininvolvedintheregulationof hemo-staticprocesses;ithasbothprocoagulantandanticoagulant properties.13_While_in_vivo_thrombin_generation_can_be

evalu-atedbymeasuringthethrombin-antithrombincomplex(TAT) andprothrombinfragments1+2(F1+2),exvivoTGTaimsto evaluatetheendogenouscapacityoftheoverallhemostatic potential.Therefore,whilehighlevelsofTATandF1+2 rep-resent thepathological activation ofin vivocoagulation,ex vivo thrombin generation reflects the endogenous capacity ofthe hemostaticsystem, and canbeindicative of throm-botic or hemorrhagic risk. TGTcontinuouslymeasures the proteolyticactivityofthrombinformedinplasmausing chro-mogenic or fluorogenic substrates following the activation ofclottingusingatriggeringagent,aswascomprehensively shownbyLecutetal.1_The_synthetic_substrate,_which_is

cou-pledtoachromogenorfluorophore,isselectivelycleavedby thrombin,releasingthechromogenorfluorophore.The out-putsignaliscontinuouslymeasured,andisproportionalto theamountofthrombinpresentinthereaction,thekinetics ofwhichcomprisetwostages.Thefirst,theinitiationstage, correspondingtothecoagulationtimemeasuredusingtests suchasPTandaPTT,canbeinhibitedbyTFPI.Thesecond,the amplification/propagation stage,isfollowedbyaresolution phaseresultingfromtheactionofvariousinhibitorspresent inplasma,suchasaPC,ATandalpha-2-macroglobulin.

TheCAT method,developed byHemkeret al.12 _enables

thequantificationofthrombinconcentrationsinplatelet-rich (PRP) or platelet-poor plasma (PPP) bymonitoring the sep-aration ofafluorogenic substrate,whichis simultaneously compared to known thrombin activity in a non-clotting plasmasample.12_This_thrombin_calibrator_contains_a_known

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Plasma (PPP/PRP)

Thrombin

Cleavage of the fluorescent substrate (Z-Gly-Gly-Arg-7-amino-4methylcoumarin)

Reading of the fluorescence intensity Releasing of

fluorophore

TF + phospholipids + calcium

Figure1–Schematicofthethrombinformationreaction andcleavageofthefluorescentsubstrate.

(TF),phospholipids(amplifytheeffectofTF)andcalciumin theplasma,resultsincoagulationactivationandsubsequent generation of thrombin. Thrombin cleaves the fluorescent substrate(Z-Gly-Gly-Arg7-amino-4-methylcoumarin)thatis addedtothereactioninalaterstep,releasingafluorophore whosefluorescenceintensityovertimeisproportionaltothe concentrationofthrombinformed(Figure1).12,14

Aftermeasurementsaretakenina96-wellplateusinga fluorimeter(Fluoroscan,ThermoScientific),Thrombinoscope BVsoftwareisusedtoconvertfluorescenceunits(RFU)into thrombin concentrations(nM).Thesoftwarealsocalculates thrombogramparameterssuchaspeakandendogen throm-bin potential(ETP) from the area under the curve and the peakthrombinconcentration,respectively.Thefluorescence intensityisconvertedtothrombinconcentration(nM)usinga referencecurvepreparedbymeasuringtheconversionrateof thesubstratewithaknownconcentrationofthrombin.12,14,15

Comparisonofthetwosignals(testandcalibrationsamples) allows a calculation of the thrombin concentration in the plasma(Figure2).

Well (sample)

Fluca

PPP HNBSA

Low/High TF

10 min

37ºC 10 min

37ºC Plasma

Plasma

PPP HNBSA

Well (calibrator)

Calibrator

400 700

600 500 400 300 200

Fluorescence (RFU) Fluorescence (RFU) 100

0 300

200

100

0

0 10

Time (min)

Thrombin (nM)

Thrombin generation curve

Time (min)

0 5 10 15 20 25

20

250

200

Peak time

Peak

Leg time

ETP

150

100

50

0

30 40 0 10

Time (min)

20 30 40

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Peak

ETP Lag time

0 100

Thrombin (nM)

200 300

10 20

Time (min)

30

Figure3–Parametersofthethrombingenerationcurve usingthecalibratedautomatedthrombogram(CAT) method.

Thethrombingenerationcurve(Figure3)ischaracterized byaninitiationphase (lag-time) followedbythe formation oflargeamountsofthrombin(propagation),culminatingina peakthrombinconcentration,andfinallyinhibitionof throm-bin generation by natural anticoagulants.15 _The _ETP _(area

underthecurve),representstheamountofthrombinformed over60min.11_Clot_formation_is_known_to_occur_at_the_end_of

thelag-time,therefore,thedurationofthisparameter corre-spondstotheclottingtime.14 _The_coagulation_state_can_be

inferredfromanalysisoftheseparametersfromthe throm-bingeneration curve.A prolongedlag-time and reductions inbothETP andpeak valuesindicate astate of hypocoag-ulability,characterizedbylessthrombin generation.Onthe otherhand,higherthrombingenerationischaracterizedby reducedlag-time,andincreasedETPandpeakvalues, indicat-ingahypercoagulablestate.1

Asmentionedpreviously,theCATmethodisbasedonthe useofasyntheticfluorogenicsubstrate that issensitive to theactionofthrombin.16 _However,_this _synthetic_substrate

canbecleavednotonlybyfreethrombin,butalsoby throm-binboundtoalpha-2-macroglobulin,potentiallyresultingin highervaluesforthrombingeneration.Toaccountforthis, theCATmethodusesanalgorithmthatnullifiestheactivity ofthrombinlinkedtoalpha-2-macroglobulin.12_Similar_to_the

CATmethod,thereagentsforthisareavailablefrom Diagnos-ticaStago(France).Severalversionsofthistestarecurrently commerciallyavailableforchromogenic(Innovance Endoge-nousThrombinPotential assay,Siemens; Pefakit Thrombin Dynamics Test,Pentapharm, Switzerland) and fluorimetric (TechnothrombinTGA,Germany)techniques.Theadvantages anddisadvantagesofthesemethods,aswellascomparisons, includingthereagentconcentrationsanduseoffluorogenic orchromogenicsubstrates,havebeenreportedelsewhere.1,17

AnadvantageofTGTisthattheoperationmodesarevery flexible, and the experimentalconditions are designed for specificpurposes.Theflexibilityofthismethodallows numer-ous investigations using various types and concentrations ofreagents thatactivatecoagulation,withandwithoutthe additionofdifferentsubstances(e.g.,aPCand thrombomodu-lin),cells(e.g.,platelets)ordifferentbuffers.Anadvantageof thefluorogenicmethodoverthechromogenicmethodisthat

fibrinogendoesnotinfluencetheresults.Asinvivoactivation ofcoagulationoccursinthepresenceofbloodcells,therehas beenmuchresearchintothedevelopmentofthistechnique forusewithwholeblood.18

As mentioned by Lipets and Ataullakhanov,5 _although

otherstimulicanbeused,thethrombingenerationreaction isgenerallytriggeredbytheadditionofloworhigh picomo-lar (pM)concentrations ofTF, inaddition to phospholipids andCa2+_ions,_in_order_to_measure_the_bleeding_or_thrombotic

potential.Dependingontheintendedpurposeofthetest,the amountofTFaddedmaybetterreflectparticularaspectsofthe hemostaticmechanism.Forexample,whenhighamountsof TF(≥10pM)areadded,thereactionisveryfastwithreduced

sensitivityforfactorsoftheintrinsicpathway.However,atTF concentrationsbetween2and5pM,thereactionismore sen-sitivetodeficienciesoffactor(F)VIII,FIXandFXI.19_Therefore,

inPPPsamples,the CATmethodissufficientlysensitiveto detectalldeficienciesofcoagulationfactors(withthe excep-tion ofFXIII)andthe effectofallanticoagulants, including vitaminKantagonists,heparinoids,and directinhibitorsof FXa and thrombin, as previously reported by Brinkman.20

WithPRPsamples,thismethodissensitiveforcasesofvon Willebranddisease,andisabletoshowtheeffectofplatelet inhibitorssuchasaspirinandabciximab.Furthermore, throm-bin generation may beinhibited bythe addition ofaPC or thrombomodulin,reflectingcongenitaloracquireddisorders oftheaPC/PSpathway.12,17,21

Application

of

the

thrombin

generation

test

as

a

potential

tool

for

experimental

and

clinical

studies

Manythrombingenerationassayshavebeencreatedto eval-uatetheclinicalpotentialofthistechnique,particularlythe correlationbetweenTGTparametersandriskofbleeding,and venous andarterialthrombosis.1,18 _Of_all _the_thrombogram

parameters, ETPappearstobe themostwidely used,as it is bettercorrelated tothe clinical phenotype, as described byLipetsandAtaullakhanov.5_A_number_of_potential_clinical

applicationsofTGThavebeen identified,suchastoassess treatmentfailureofpatientswhohavethromboticdisorders and are receiving oralanticoagulants, heparin, antiplatelet drugsoracombinationofsuchdrugsandtocomprehensively evaluatethethromboticorhemorrhagicstatusofat-risk indi-viduals.Someofthemostimportantreportsarepresented inTable1.Inthe researchfield,TGThasbeenwidelyused in various parts of the world. Briefly, TGT has been used to help elucidate new hemostatic mechanisms by explor-ing variousplasmacomponents, suchasthosereportedby Peraramelli et al.,22 _Spronk _et _al.,23 _Omarova _et _al.24 _and

Zamolodchikovetal.25_These_studies_have_explored_the_role

ofPSinTFPIactivity,22_provided_new_insights_into_thrombin

formation23 _and _reported_a _novel_mechanism _for_the

inhi-bitionofthrombin-mediatedFVactivationbythefibrinogen

␥′ _peptide.24 _Furthermore_studies_have_investigated_the_role

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Table1–Potentialclinicalapplicationsofthethrombingenerationtest.

Author Mainobjectivesofthestudy

Kesselsetal.,26_{Arachchillage}_et_al.27_and

Zabczyketal.28

Tomonitortreatmentwithoralanticoagulantsandantiplateletdrugs.

AlDierietal.29 _To_assess_bleeding_risk_in_patients_with_von_Willebrand_disease.

Simionietal.,30_Castoldi_et_al.,31_Alhenc-Gelas

etal.32_and_Castoldi_at_al.33

ToevaluatehypercoagulablestatesinpatientswiththeprothrombinG20210A mutation,factorVLeiden,andantithrombinorproteinSdeficiency. Tripodietal.34_and_Hron_et_al.35 _To_use_the_thrombin_generation_test_to_predict_the_recurrence_of_venous

thromboembolism.

Lewisetal.36_and_Matsumoto_et_al.37 _To_estimate_bleeding_risk_in_patients_with_hemophilia_using_factor_VIII_or_factor_VIIa.

vanHylckamaetal.38 _To_assess_risk_of_deep_venous_thrombosis.

Brummel-Ziedinsetal.39 _To_discriminate_between_acute_and_stable_coronary_artery_disease.

Segersetal.40 _To_use_the_thrombin_generation_test_to_identify_novel_genetic_risk_factors_for_venous

thromboembolism.

Carcaillonetal.41 _To_correlate_thrombin_generation_with_coronary_heart_disease_and_acute_ischemic

strokeintheelderly.

Tchaikovskietal.42 _To_investigate_changes_and_determinants_of_thrombin_generation_and_activated_protein

Cresistanceinthefirst16weeksofgestationinwomenwithahistoryofpreeclampsia. Boschetal.43 _To_evaluate_patients_undergoing_cardiac_surgery.

Orsietal.44 _To_evaluate_possible_{pathophysiological}_mechanisms_that_may_contribute_to_the

bleedingtendencyobservedinpatientswithdenguefever. Kamphuisenetal.45 _To_evaluate_{cardiovascular}_risk_in_patients_with_hemophilia.

Gouldetal.46 _To_assess_procoagulant_potential_of_intact_neutrophil_{extracellular}_traps_released_from

activatedneutrophils.

Picoli-Quainoetal.47 _To_investigate_{hypercoagulability}_during_the_very_early_phases_of_the_host_response_to

aninfectionovertheclinicalcourseofsepsisandsepticshock.

Loeffenetal.48 _To_evaluate_the_{hypercoagulable}_profile_of_patients_with_stent_thrombosis.

Ziaetal.49_and_Glintborg_et_al.50 _To_evaluate_{hypercoagulability}_in_women_using_oral_{contraceptives.}

Dargaudetal.51 _To_identify_an_autosomal_dominant_bleeding_disorder_in_a_family,_caused_by_a

thrombomodulinmutation.

Barcoetal.52_and_Honickel_et_al.53 _To_evaluate_the_effect_of_prothrombin_complex_concentrate_in_reversing_the

anticoagulanteffectofrivaroxabananddabigatran.

contributedtoourunderstandingofhemostaticmechanisms, howeverthesearebeyondthescopeofthisreport.

Limitations

and

perspectives

AlthoughTGThasmany potentialclinicalapplicationsand considerableadvantagesoveralmostallothertechniques,it alsohassomelimitationsthatneedtobeovercomebeforeits useinclinicallaboratories.Althoughthis techniquecanbe performedusingPPPorPRP,thisinvolvesatime-consuming process,whichpreventsitsuseforrapiddiagnosis.Anideal technique would be performed using whole blood sam-plescontaining all blood cells,whichwould reproduce the

invivoconditionsbetter.18_It_should_be_noted_that_there_are

other teststhat can beused forthe overall assessmentof hemostasis, such as thromboelastography (TEG® systems, Haemonetics Corporation,Braintree, MA, USA) and throm-boelastometry (ROTEM®-Analyser, Tem Innovations GmbH, Munich,Germany),bothofwhichareperformedusingwhole blood. Thromboelastography, a global test widely used in the management ofacute hemorrhages, is alsocapable of detecting hypercoagulable states.54 _However, _according _to

Lancé,13_this_method_does_not_fully_reflect_the_effects_of_using

low-molecular-weightheparinorthedirectuseoforal antico-agulants,northeeffectsofinheritedordrug-inducedplatelet dysfunction.TGTpresents awider rangeofpotential clini-calapplicationscomparedtothromboelastography.Thus,the applicationofTGTonwholeblood(containingallbloodcells)

mayoutweightheadvantagesofTGTonplasma,asthisassay maybetterreflecttheinvivoconditions.

ThelackofreferencevaluesforspecificTGTconditions, suchasthetypeandconcentrationofthetriggeringagent, orwhether ornotacontactinhibitorfactor isused,makes theuseofTGTdifficultinclinicallaboratories,asitrequires appropriateinterpretationoftheresults.AsstatedbySpronk etal.,55_there_is_no_consensus_as_to_whether_a_contact

path-wayactivationinhibitorshouldbeused,forexample,whether corn trypsin inhibitor is required. These reference values shouldideallybeestablishedforeachcenteraccordingtothe adoptedprotocol,andwouldrequireidenticalconditionsfor patientandcontrolsamples,includingbloodcollection, sam-plepreparationandstorage.Anotherimportantlimitationof TGTisthelackofsensitivitytochangesintheendothelium. Althoughitisincreasinglybeingrecognizedasamoreprecise testthatreplicatestheinvivohemostaticconditions,TGTstill lacksofficialstandardization,despiteseveralstudieshaving beenconducted.55–57_Therefore,_it_is_mandatory_to

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characteristics andconditions toensurethereproducibility and accuracyofTGTare required.54 _In_line _with_this,

Dar-gaudetal.8_evaluated_a_standardized_protocol_for_measuring

thrombin generation usingthe CATmethod inan interna-tionalmulti-centerstudy.Theseauthorsdemonstratedthat theuseofstandardconditions,suchasidenticalequipment, standardizedreagents,referenceplasmaforthe normaliza-tionofresults,andthesametestprocedure,showedagreat reductionin assay variability comparedto previously pub-lisheddata.Their datademonstrated thatthestandardized TGTmethodologyevaluatedinthisreporteffectivelyreduces thevariabilityofthisassaytoacceptablelimits.

Inshort,weagreewithOthman58_on_the_need_for_a

stan-dardized global test that can reliably detect, predict and monitorhemostaticstatusforcliniciansandresearchers,in bothclinicalandexperimentalstudies.

Conclusions

Basedonthecurrent literatureTGTaimstogloballyassess hemostasis,providinginformationabouttheinitiation, ampli-fication/propagationandresolutionphases.Theresultsfrom thistestare morerepresentativeofthephysiological state, whichmaybetterreflectthehemostaticphenotypecompared toroutinetests,andhasgreatpotentialfortheevaluationof therisksofbleedingandthrombosis.However,TGTisnotyet availableforclinicaluse,asitstillrequiresstandardizationand validation.Theuseofastandardizedmethodmayreducethe variabilityofthisassaytoacceptablelimits.Finally,anideal coagulationtestdoes notexistyet. However,new develop-mentsarecontinuouslyemergingaimedatimprovingexisting testsinordertogloballyassesshemostasis,particularlyTGT usingtheCATmethod.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethanktheBrazilianagenciesFAPEMIG/SESandMS/CNPq forfundingthisstudy.

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