rev bras hematol hemoter. 2017;39(3):199–201
w w w . r b h h . o r g
Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
Scientific
Comment
Scientific
comment
on:
“Quantitative
flow
cytometric
evaluation
of
CD200,
CD123,
CD43
and
CD52
as
a
tool
for
the
differential
diagnosis
of
mature
B-cell
neoplasms”
夽
Maura
Rosane
Valerio
Ikoma
∗Fundac¸ãoDrAmaralCarvalho,Jau,SP,Brazil
Sincethe 1980s,multiparametricflowcytometry(FCM)was broughttotheclinicallaboratoryfordiagnostic immunophe-notyping. The expression of antibodies bound to the membrane or intracellular receptors has been generally definedaspositiveornegativewithacutoffsetrelativetoa nonstainingcontrolpopulation. Itisknown thatthe inten-sityofthefluorescentsignalisproportional totheamount ofantibodyboundpercell,i.e.,itisproportionaltothe num-berofantigensitesexpressed.Thismeansthatthequantityof moleculespercellcanbemeasuredbytheintensityofantigen expression.
Oneofthemostimportantclinicalutilitiesoffluorescence intensity measurements is the diagnosis of hematological malignancies,bywhich aberrant phenotypesare identified accordingtotheover-orunder-expressionofvariouscellular proteinscomparedtothoseexpressedinnormalcells.
Inprinciple,antigendetectionbyFCMisdescribed qualita-tivelyashavingabsent,brightordimexpressions.Advances overthepasttwodecadeshaveresultedinthedevelopment ofFCM methodsand materialsthat permitmeasurements ofquantitativefluorescencewithimprovedlevelsofcontrol and interlaboratory precision. With these advances, inter-est in quantitativeflow cytometry (QFCM)has grown as a
DOIoforiginalarticle:http://dx.doi.org/10.1016/j.bjhh.2017.05.002.
夽
SeepaperbyArlindoetal.onpages252–8.
∗ Correspondingauthorat:FlowCytometryLaboratory.Fundac¸ãoDr AmaralCarvalho,R. DonáSilvéria,150,ChácaraBrazMiraglia,
17210-070Jaú,SP,Brazil.
E-mailaddress:maura.ikoma@amaralcarvalho.org.br
methodtoquantifytheexpressionandactivitiesofavariety ofproteinsandenzymesfordiagnostic,prognostic,and ther-apeuticpurposes.1Overtheyears,ithasbeenrecognizedthat
quantifyingthenumberofantigenspercellprovidesuseful informationthatshouldbeconsideredanintrinsicpartofthe cellularphenotypeprocess.2Acurrentexampleoftheutility
ofQFCMisthetherapeuticuseofhighdosesofthemonoclonal antibody(MoAb)anti-20(Rituximab)forchroniclymphocytic leukemia(CLL),whichhasadimexpressionofCD20onthe cellmembrane.
QFCMbasedonmeanfluorescentintensity(MFI)is rela-tivelynewandpendingstandardizationwhichrequiresstrict procedures comparedto those needed forqualitative FCM analysisduetotherequirementsofprecisequantitativeand absoluteresults.MFIisproportionaltothenumberof antibod-iesthatrecognizeandbindtothecellantigens,allowingexact quantification ofantigenexpression per cell.3 Quantitative
measurementisbasedonthecalibrationofthefluorescence axisofthenumberoffluorochromemoleculesattachedtothe cellordirectlyoftheantibodybindingcapacityusingstandard beads.2
The regulation and standardization of MFI-based tests are stillintheirearlystagealthoughmanyMFI-basedtests
http://dx.doi.org/10.1016/j.bjhh.2017.05.004
200
revbrashematolhemoter.2017;39(3):199–201arebeingperformedroutinelyaspartofacademicresearch studies.3
SomestudiesshowthatassaystoevaluateMFIwerenot reproducible,intra-andinter-laboratory.MFIvalueshavebeen reportedinavariablerangeofthecoefficientofvariation per-centage(CV%)between7and33% dependingontheMoAb tested,evenonusingthesameequipmentinthesame lab-oratory,and also using the same donorblood ondifferent days or at different times of the day.4,5 This means that
the whole process of FCM assays must be properly stan-dardized and validated to allow reliable and reproducible MFImeasurementsthatincludepre-analyticalandanalytical procedures.
The strict application of the samplepreparation proto-cols,theadequateMoAbtitration,controlsofreagentstability, theuseofbetterMoAb-fluorochromecombinationsand accu-ratedataanalysisare someofthemostimportanttoolsto guarantee accurate results inFCM. In this sense, previous reportsshowed the effectiveness ofstandardization guide-linestoachievereproducibleresults.6,7
Despite all the variables involved, calibration of instru-ments forquantification offluorescence measurements by FCMshouldbeconsideredaroutineandabsolutelynecessary procedure.Calibrationbeadsareusedtoassurecontrolofflow cytometerperformanceandlaserstability.Furthermore,some kindsofbeadscanbeusedtosetafixedoutputvalue(target MFI)foreachfluorescentchannel.Thesettingofthesetarget valuesintheinstrumentpriortoeachexperimentensurea moreaccurateandreproducibleassay.6
OthermethodsforstandardizationofMFIare:
(i) Converting MFI to known fluorescent units such as molecules of equivalent soluble fluorochrome (MESF). MESFbeads canbeusedtocreate astandardcurve of MFIversusMESFvaluesandcantranslateMFItomore universalMESFunits;
(ii) TransformingMFIinto antibodybindingcapacityunits (beadswithaknownnumberofantibodybindingsites arestainedtogetherwiththecellsamplessotheMFIof thebeadsshouldreflectthenumberofmoleculesbound toasinglecell);
(iii) By using quantitativefluorescentbeads labeledwith a knownnumberofphycoerythrin(PE)molecules,thecurve offluorescenceintensitycanbeextrapolatedtoevaluate thenumberofPEmoleculesboundpercell.2,3
Regardingthe article“Quantitativeflowcytometric eval-uation ofCD200, CD123, CD43and CD52as a tool forthe differentialdiagnosisofmatureB-cellneoplasms”byArlindo et al. inthis issue of the Revista Brasileira de Hematologia e Hemoterapia, theauthors shouldbeencouraged tocontinue withthisresearch.QFCMrepresentsadevelopingfieldwithin FCM,whichcanprovideanimportantunderstandingabout cellular mechanisms in hematological and immunological diseasesandconsequentlytheuseofsuchknowledgeintheir control.8
Theauthorsshowedtheirdiscernmentbyusingprotocols to maintain quality control, including instrument calibra-tion,theprocessofsamplepreparation,dataacquisitionand analysis. However, the process of validation and titration
ofthe MoAbused, whichled tothechoiceoftheseclones and fluorochromes for the research in question, was not shown.
Furthermore,it isimportanttoimprove themethodsof MFIevaluationtomakethe analysisless subjective.FCMis a semi-quantitative method for diagnostic immunopheno-typing,andsometimesit iscriticizedasbeingan‘intuitive’ methodofanalysis.Thus, therigorousmaintenanceofthe standard operational proceedings in all the phases of the immunophenotyping process isessentialtoensure reliable and reproducibleresults,minimizingthesubjectivityofthe method.
Regardingthesample,itisrecommendabletoincreaseall thestudygroups,excepttheCLLgroup,toallowabetter eval-uationofthevariabilityofexpressionofeachmarkerwithin eachgroupandcompareitwiththatofothergroups.Despite ofthis,theresultsobtainedwiththeexpressionsofthefour AbMochoseninthestudy weresimilartothosepreviously describedintheliterature.
ConcerningthefinaldiagnosisofthematureB-cell neo-plasms (MBCN), it is important to emphasize the current limitationsofFCMalonetoestablishthediagnosisofsomeof theseneoplasms,suchaslymphoplasmacyticlymphoma(LPL) andmarginalzonelymphoma(MZL);thedifferential diagno-sisbetweenthembyFCMisdifficultandcannotbeassumed without otherauxiliarydiagnosticmethods,untilnewFCM strategiesaredesigned.Oneattemptatcomparingcaseswith theprofileofsimilardiseasesistheMBCNcasedatabasethat isbeingdevelopedbytheEuroflowgroup.
Concluding,theinitiativetoimplementQFCMinthe diag-nosisofMBCNiscommendableandavant-gardeinBraziland shouldbestimulated.Congratulationstotheauthors!
Conflicts
of
interest
Theauthordeclaresnoconflictsofinterest
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