Rev. Bras. Hematol. Hemoter. vol.37 número5

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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

Original

article

Hemoglobin

A

2

values

in

sickle

cell

disease

patients

quantified

by

high

performance

liquid

chromatography

and

the

influence

of

alpha

thalassemia

Silvana

Fahel

da

Fonseca

a,∗

,

Tatiana

Amorim

b

,

Antônio

Purificac¸ão

b

,

Marilda

Gonc¸alves

c

_,

_Ney

_Boa-Sorte

b

a_Universidade_de_Brasília_(UnB),_Brasília,_DF,_Brazil

b_{Associac¸ão}_de_Pais_e_Amigos_dos_Excepcionais_(APAE),_Salvador,_BA,_Brazil c_Fundac¸ão_Oswaldo_Cruz_(FIOCRUZ),_Salvador,_BA,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10March2013

Accepted11May2015

Availableonline7June2015

Keywords:

HemoglobinA2

Alphathalassemia

Betathalassemia

Sicklecelldisease

Highperformanceliquid

chromatography

a

b

s

t

r

a

c

t

Background:Insicklecelldisease,thequantificationofHbA2isimportantforthedifferential

diagnosisbetweensicklecellanemia(HbSS)andHbS/␤0-thalassemia.

Objective:TodetermineHbA2levelsasquantifiedbyhighperformanceliquid

chromatogra-phyinpatientswithsicklecellanemia(HbSS)andwiththeSChemoglobinopathy,withor

withoutconcomitantalphathalassemia.

Methods:Thisisaretrospectivestudyof242childrenagedbetweentwoandsixyearswith

diagnosesofHbSSorHbSC.Thehemoglobinwasevaluatedusinghighperformanceliquid

chromatographyandalphathalassemia[3.7kbdeletion(−␣3.7)]wasdetectedbypolymerase

chainreaction.Patientswereclassifiedashomozygous(−␣3.7_/₋_␣3.7_),_heterozygous₍₋_␣3.7_/_␣_),

orhomozygouswild-type.AnalysisofvariancewasusedtocomparethemeanHbA2values

betweenthealphathalassemiagroups.

Results:Themean(±standarddeviation)HbA2concentrationsintheHbSSgroup(n=135)

was3.68±0.65%.ThemeanvaluesforindividualswithHbSSandheterozygous(n=28)or

homozygousforalphathalassemia(n=3)were3.98±0.64%and4.73±0.25%,respectively.

ThemeanHbA2ofalltheHbSCpatients(n=107)was4.01±0.507with4.29±0.41%and

4.91±0.22%inindividualsheterozygous(n=23)andhomozygous foralphathalassemia

(n=7),respectively.AllpatientshomozygousforalphathalassemiahadHbA2levelsabove

3.5%.However,Hb A2 values above5.2%wereseenin patientswithHbSS andHb SC,

independentlyofalphathalassemia.

Conclusion:HbA2levelsareelevatedinpatientswithHbSorHbC,andaredirectlyinfluenced

bythealphathalassemiagenotypes.

byElsevierEditoraLtda.Allrightsreserved.

∗ _{Corresponding}_author_at_:_SQN_214,_Bloco_C,_Apt_214,_AsaNorte,_70873-030_Brasília,_DF,_Brazil.

E-mailaddress:sfahel@unb.br(S.F.daFonseca).

http://dx.doi.org/10.1016/j.bjhh.2015.05.005

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Introduction

Sickle cell disease is one of the most common genetic

pathologiesintheworld.Itischaracterizedbyhomozygous

hemoglobinS(HbS)orHbSassociatedtootherHbvariants.1

Thereisgreatclinicalvariationintheclinicalmanifestations

betweensicklecelldiseasepatients;severalfactorsare

asso-ciatedwiththedifferentpresentations.Somedeterminants

are already well established, such as genetic, clinical and

laboratoryfactors,while others,suchaspsycho-social and

nutritionalfactors,havebeenlesswellstudied.2–5

Ofthe geneticfactors,the importanceofthephenotype

ofthe hemoglobinopathyiswellcharacterizedinthat

indi-vidualsdoublyheterozygousforsicklecellanemiaandthose

withHbS/␤0-thalassemiahaveamoresevereclinicalprofile.

Ontheotherhand,carriersofHbSCtogetherwithHbS/␤+

-thalassemiahavebetteroutcomes,whichmakesthecorrect

diagnosisofthesesyndromesanissueofgreatimportancefor

abetterunderstandingandadequateclinicalandtherapeutic

managementofpatients.4–6

Inthediagnosisofsicklecelldisease,quantificationofHb

A2, aswell asacomplete bloodcount(CBC),familyhistory

andclinicaldata,helptoestablishthedifferentialdiagnosis

betweensicklecellanemia(HbSS)andHbS/␤0-thalassemia.7,8

ThechoiceofthemethodologytoaccuratelymeasureHb

A2hasbeenthesubjectofmanydiscussionsinthemedical

lit-erature,andhighperformanceliquidchromatography(HPLC)

hasbeenregardedthemethodofchoiceformanyyears.The

referencevalueofHbA2inhealthyadultswhodonothave

thalassemiaisusuallybetween2.0and3.3%.5,9–12

In1996,Suhetal.13_reported_that_Hb_A

2valuesobtained

byHPLCincreasedsignificantlyinsamplescontainingHbS

andsuggestedthepossibilitythatHbSSindividualsmayhave

beenincorrectlydiagnosed withHbS/␤0-thalassemia.They

alsosuggestedthatincreasesinHbA2couldbeexplainedby

thepresenceofsmallercomponentsofHbSthatco-eluted

withHbA2.

In 2000, Shokrani et al.14 _proposed _that _blood _samples

withHb valuesofup to 5.9%and up to5.2% ofHb A2 as

analyzedbyHPLCfrom Hb SS or HbSC patients and

indi-vidualswiththesicklecelltrait(HbAS),respectivelycanbe

considerednormal.In2004,Headetal.7_confirmed_the

find-ingsofShokranietal.andexplainedthatthefalselyelevated

Hb A2 values were due to the presence of Hb S that had

sufferedpost-translationalmodifications,andconsequently

had the same retention time as Hb A2. They also

demon-stratedthatthedegreeofuncertaintydirectlycorrelated to

theconcentrationofHbS,whichishigherinHbSSpatient

samplesthaninsamplesfromindividualswithHbAS.

Addi-tionally,theyconcludedthatthepercentageofHbA2ishigher

in Hb AS individuals with alpha thalassemia (AT) than in

thosewithoutAT;andsuggestedthatthisoccursbecausethe

deltachainshavegreateraffinitytoalphachainsthanthe␤S

chains.7

Morerecently,studiesusingmassspectrometryhave

pro-posedthatelevatedHbA2valuesinsampleswithHbScanbea

consequenceoftheelutionofsmallerHbcomponentsformed

bya␤Schainassociatedwithanalpha-globinchainthatwas

modifiedbycarbamylationaftertranslation.15

TheinterferenceofanomalousHbinthequantificationof

HbA2isnotlimitedtothepresenceofHbSandHbC,butcan

alsoberelatedtothepresenceofother betachainvariants

suchasHbE,HbDandHbLepore.16,17

In2007,Ondeietal.,18 _in_an_effort_to_establish_reference

valuesfortheBrazilianpopulation,usedHPLCtoanalyze136

sampleswiththeHbASphenotype,106withATandHbAS,

18withHbSSandninewithHbSC,andconcludedthatHb

A2valuesof2.9–5.2%forHbAS,2.8–5.2%forATtogetherwith

HbAS,0.8–5.6%forHbSSand3.8–5.7%forHbSCshouldbe

considerednormal.

Considering the widespread use of HPLC inthe

labora-torypracticeandtheimportanceofanaccuratediagnosisto

definethetypeofsicklecelldiseaseinordertofacilitatebetter

clinical andlaboratoryfollow-upofthesepatients,the

cur-rentstudysoughttoverifytheHbA2valuesasquantifiedby

HPLC,insamplesofpatientswithHbSandHbC,inthe

pres-enceorabsenceofthe␣2-thalassemia3.7kbdeletionasboth

hemoglobinopathies maybeprevalent inthesame

popula-tion.

Methods

Studydesignandpopulation

Thisis aretrospective study conductedbetweenJune2008

andJune2009withaninitialsamplecomposedof287

chil-dren.Forty-fivewereexcluded:eightforpresentingadiagnosis

ofHb S/␤-thalassemia, elevenforhavingbeen analyzedby

HPLC after the transfusion of blood components, and 26

duetoirondeficiency.Thisleftatotalof242children

diag-nosedwithsicklecellanemiaorHbSCscreenedandfollowed

at the Neonatal Screening Referral Service (SRTN) of the

Associac¸ão de Pais eAmigos dos Excepcionaisin Salvador

(APAE/Salvador), Bahia, Brazil. The SRTN-APAE/Salvador is

registeredwiththeBrazilianMinistryofHealth(Ordinance822

–06/06/2001)asareferralserviceinthestateofBahia,Brazil.

Thisservicediagnosesandfollowsupallnewbornswithsickle

cellanemiainBahia,thestatewiththehighestincidenceof

thishemoglobinopathyinBrazil(onein601newborns/year).19

Theobjectivesandproceduresinvolvedinthestudywere

explained,andparentswhoagreedtoparticipatesignedan

informedconsentform.Allparentsofparticipatingchildren

wereasymptomatic,andweresubjectedtoaCBC,Hbprofiling,

andmeasurementofserumferritin.Allpresentedalaboratory

profilecompatiblewithheterozygosityforHbSorHbCand

normalironreserves.

Analysesandlaboratorytechniques

Venousbloodsampleswerecollectedin3.0-mLtubes

contain-ing 3.6mgK2-ethylenediaminetetraacetic acid (EDTA) as an

anticoagulationagentandinatubewithagelseparator.The

qualitativeandquantitativeHbprofileswereinvestigatedby

automatedHPLCusingtheVariantExpressapparatusandthe

␤-thalassemiaShortProgramkit(Bio-Rad,California,USA).

Cation-exchangeHPLCisaprocessinwhichamixtureof

molecules(suchasnormalandvariantHbs)withanet

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Table1–Distributionof␣3.7-thalassemiain242childrenwithsicklecelldisease.

Sicklecelldisease ␣3.7_-Thalassemia _p_-Value

␣␣/␣␣ −␣/␣␣ −␣/−␣

n % n % n %

HbSS 104 77.0 28 20.7 3 2.2

0.233

HbSC 77 72.0 23 21.5 7 6.5

Total 181 74.8 51 21.0 10 4.1

HbSS:sicklecellanemia;HbSC:SChemoglobinopathy.

ofmoleculesinanegatively chargedstationaryphasein a chromatographycolumn,followedbytheirelutioninamobile phase.Inthemobilephasealiquidwithanincreased con-centrationofcationsflowsthroughthecolumn;thecations inthemobilephasecompetewiththeadsorbedproteinsfor anionicbindingsites.Thus,theadsorbedpositivelycharged Hb molecules are eluted from the column into the liquid phaseataraterelatedtotheiraffinityinthestationaryphase. Thedifferent Hbs elutedfrom the columnare represented graphicallyandautomaticallyquantified.Theresultisa chro-matogramwiththepercentageandretentiontimeofeachHb fraction.Thetimeofelution(retentiontime)ofanynormalor variantHbpresentiscomparedwiththatofknownHbs pro-vidingquantificationofnormalHbs(HbA,HbF,andHbA2) andmanyvariants.ThemostpositivelychargedHbs(e.g.HbS andHbC)havealongerretentiontime.20

Patientsampleswere consideredtocontainHbSand/or

HbCwhenthechromatogramspresentedvariantswith

reten-tiontimesequivalenttotheseHbs.Assuch,thediagnosisof

sicklecellanemiawasmadeonlyifHbS,HbFandHbA2were

present,hemametric indicesof the CBCwere normal, and

theparentspresentednormalCBCswithHbprofileconsistent

withsicklecelltrait.Theserumferritinlevelwasdetermined

bychemoluminescence(ADVIACentaurCPSystem,Siemens).

Ferritinlevels<12ng/mLwereconsideredasirondeficient.21

The deletion of 3.7kb in AT was investigated by

allele-specific polymerase chain reaction (PCR)22 _and _the

participants were identified as heterozygotes (−␣3.7/␣␣),

homozygotes(−␣3.7/−␣3.7)orwildtype(␣␣/␣␣).

Statisticalanalysis

HbA2concentrationsweredescribedaccordingtothetypeof

sicklecelldisease(HbSCorHbSS)together withATstatus

(wild-type,heterozygousorhomozygous)usingcentral

ten-dencyanalysisanddispersion.TheKolmogorov–Smirnovtest

wasusedforanalysisofnormaldistribution.One-wayanalysis

ofvariance(ANOVA)wasusedtoanalyzedifferencesbetween

groups,andtwo-wayANOVAwasusedtoanalyzethe

influ-enceoftheinteractionandconfoundingfactorsbetweentype

ofsicklecellanemiaandpresenceofAT(independent

vari-ables)onHbA2levels(dependentvariable).Posthocanalysis

wasperformedusingTukey’sHonestlySignificantDifference

(HSD)testandtheGames–Howelltesttoidentifysituationsin

whichequalityofvariancewasverified.Duetothereduced

sizeofthegroupwithhomozygousmutations,statistical

sig-nificancewassetatap-value<0.01.

Hb A2 valueswerecategorizedinthreegroupsbasedon

standard reference values: (1) below 2.0%; (2) between 2.0

and3.5%;and(3)above3.5%.Thesecategorieswereanalyzed

accordingtotheATdatausingthechi-squaredtendencytest.

AlldatawereanalyzedusingEPIINFOforWindows(version

3.5.1)andtheStatisticalPackagefortheSocialSciences(SPSS®

–version13.0).

Ethicalconsiderations

The present study was approved by the Ethics

Commit-tee for Research in Human Beings of the Gonc¸alo Moniz

Research Centerfrom the OswaldoCruz Foundation, Bahia

(CEP-CPqGM/FIOCRUZ:protocol#112/2006)and followedthe

researchethicsguidelinesasdefinedinresolution196/96of

theHelsinkideclarationof2008.

Results

Thefinalcohortanalyzedconsistedof242patients(107HbSC

and135HbSS)agedfromtwotosixyearswithanaverageage

of3.05years(±1.01),andmedian(p25–p75)of4(3.0–5.0)years.

Thedistributionof␣3.7-thalassemiaisdescribedinTable1.

There was no significant difference between the

distribu-tion of heterozygous or homozygous AT between the two

typesofhemoglobinopathies (HbSS andHb SC)(2=2.917;

p-value=0.233).

ThemeanHbA2levelinHbSSsamplesindependentlyof

thepresenceofAT(n=135)was3.68%(±0.65);inHbSSsamples

withheterozygousAT(n=28)itwas3.98%(±0.64)andinHbSS

sampleswithhomozygousAT(n=3)itwas4.73%(±0.25).The

meanHbA2inallHbSCsamples(n=107)was4.01%(±0.50);

inHbSCsampleswithheterozygousAT(n=23)itwas4.29%

(±0.41),andinHbSCsampleswithhomozygousAT(n=7)it

was4.91%(±0.22).

WithregardtothemeanHbA2valuesandthesicklecell

diseasetype(HbSSorHbSC),significantlyhigheroverall

val-ues were observedinpatients withHbSC comparedtoHb

SS(p-value<0.001)asshowninTable2.Whenthedatawere

stratifiedaccordingtotheATstatus(wildtype,heterozygous

or homozygous),asignificantdifferencewas observedonly

whensampleswithoutATwerecompared.Valueswerenot

significantforheterozygous(HbSC:4.29±0.41%versusHbSS:

3.98±0.64%;p-value=0.074),orforhomozygousATmutation

(HbSC:4.91±0.22%versusHbSS:4.73±0.25%;p-value=0.267

(4)

Table2–Mean,standarddeviation,maximumandminimumvaluesforHbA2in242childrenaccordingtothepresence of␣3.7-thalassemia,stratifiedbytypeofsicklecelldisease.

␣3.7-Thalassemia n Average SD Minimum–maximum p-Value

<0.001a

␣␣/␣␣ 181 3.68 0.56 1.50–5.20

0.001b

HbSS 104 3.57 0.61 1.50–5.20

HbSC 77 3.84 0.42 2.90–4.80

−␣/␣␣ 51 4.12 0.57 2.30–4.90

0.074b

HbSS 28 3.98 0.64 2.30–4.90

HbSC 23 4.29 0.41 3.30–4.90

−␣/−␣ 10 4.86 0.23 4.50–5.20

0.267b

HbSS 3 4.73 0.25 4.50–5.00

HbSC 7 4.91 0.22 4.60–5.20

Total 242 3.82 0.62 1.50–5.20

<0.001c

HbSS 135 3.68 0.65 1.50–5.20

HbSC 107 4.01 0.51 2.90–5.20

SD:standarddeviation;HbSS:sicklecellanemia;HbSC:SChemoglobinopathy.

a _Comparison_of_average_Hb_A2_values_according_to_presence_of_␣3.7_-thalassemia_(wild-type,_heterozygous_and_homozygous)_{independently}_of typeofhemoglobinopathy–non-parametricKruskal–Wallistest.

b _Comparison_of_average_Hb_A2_values_between_the_types_of_{hemoglobinopathy}_(Hb_SS_or_Hb_SC)_for_each_alpha_thalassemia_{classification}

(wild-type,heterozygousandhomozygous)–non-parametricMann–Whitneytest.

c _Comparison_of_average_Hb_A2_values_between_the_types_of_{hemoglobinopathy}_(Hb_SS_or_Hb_SC)_{independently}_of_alpha_thalassemia

classifi-cation(wild-type,heterozygousandhomozygous)–non-parametricMann–Whitneytest.

0 1 2 3

HbSC HbSS 4 3.57 (0.61) Wild type

Heterozygotes Homozygotes

Hemoglobin A₂ (%) 3.84 (0.42)

3.98 (0.64) 4.29 (0.41)

4.73 (0.25) 4.91 (0.22)

5 6

Figure1–HbA2values(%)accordingto␣3.7-thalassemia

genotypeandsicklecelltype(HbSS/HbSC)in242children.

WhencomparinggroupsaccordingtoATstatusonly

(wild-type,heterozygousorhomozygous),highermeanvalueswere

observed for Hb A2 in patients with the −␣/−␣ genotype

(F=30.985;p-value<0.001–Table2).

Theanalysisoftheinteractionbetweenthetypeofsickle

celldiseaseandATgenotypewascarriedoutafterthe

differ-encebetweentheaverageHbA2valuesforHbSSandHbSC

wasfoundtobesignificant.Therewasnosignificant

inter-action(F=0.583; p-value=0.559) orinfluence ofthe typeof

hemoglobinopathy(F=2.156;p-value=0.43)onthemeanHb

A2values.OnlytheATgenotypeinfluencedthevariationof

themeanHbA2values(F=28.676;p-value<0.001).Theposthoc

analysisindicatedasignificantdifferencebetweenthethree

ATgenotypegroups,showingaprogressiveincreaseinHbA2

levels(Figure1).

UsingthereferenceHbA2valuesdescribedbyOndeietal.in

2007,18_it_was_observed_that_all_homozygous_mutant_samples

had elevatedHb A2 levels (above3.5%),and atendencyfor

increasesinthepercentageofchildrenwithHbA2levelsabove

3.5%fromthewild-type(59.7%)totheheterozygous(88.2%)

andthehomozygous(100%)samples(2_linear_tendency=18.871;

p-valuetendency<0.001),asshowninTable3.

Discussion

Hbdisordersarerecognizedasoneofthemostcommon

inher-ited diseases worldwide. Among the hemoglobinopathies,

sicklecelldiseaseand␤-thalassemiahavethegreatestimpact

onmorbidityandmortality,affectingmillionsofindividuals

worldwide.5

Sicklecelldiseaseshouldbeconsideredasbotha

qualita-tiveandquantitativegeneticdisorderinthatitiscausedby

thepresenceofanabnormalHbvariant–HbS.Homozygosity

forHbSorsicklecellanemiaisthemostcommongenotype;

theother causativegenotypesincludecompound

heterozy-gousstatesofHbSwithHbC(HbSC)or␤-thalassemiavariants

(HbS/␤0-thalassemiaandHbS/␤+-thalassemia).5

Diagnosisofspecificsicklecelldiseasesisaccomplishedby

integratingclinicalandhematologicalparametersalongwith

laboratory Hbanalysis. Combiningtheseelements to

prop-erlydiagnose Hbdisorders isessential forthetreatmentof

anemia,primarypreventionandgeneticcounselingfor

under-lyingdisorders.Inthemajorityofpatients,thepresenceofa

hemoglobinopathycanbediagnosedwithsufficientaccuracy

forclinicalpurposesfromknowledgeofthepatient’sethnical

backgroundandclinicalhistory(includingfamilyhistory)and

theresultsofthephysicalexaminationcombinedwith

rela-tivelysimplebloodtests.Initialinvestigationsshouldinclude

(5)

Table3–ProportionofHbA2above3.5%foundin242accordingtothepresenceof␣3.7-thalassemia.

HbA2

␣3.7-Thalassemia <2.0%

n(%)

2.0–3.5% n(%)

>3.5% n(%)

p-Value

␣␣/␣␣ (n=181) 1(0.6) 72(39.8) 108(59.7)

<0.001a

−␣/␣␣(n=51) – 6(11.8) 45(88.2)

−␣/−␣(n=10) – – 10(100.0)

Total 1(0.4) 78(32.2) 163(67.4)

a _Chi-squared_linear_tendency_test.

detailedexaminationofawell-stainedbloodfilmshouldbe

carriedout.OtherimportantbasictestsareHb

electrophore-sisorchromatography,andthemeasurementofHbA2and

HbF.23_Automated_HPLC,_by_analyzing_large_numbers_of

sam-ples,isincreasinglyreplacingHbelectrophoresisastheinitial

investigativeprocedureinlaboratories.18,23

WhileHPLCisconsideredamethodthatpermitsrapidand

precisedetectionofHbvariants,aswellassensitive

quantifi-cationofHbA2,9 overthelasttwodecadesvariousauthors

have reported the occurrence of falsely elevated Hb A2 in

screeningforHbvariants(HbS,HbC,HbD,HbEandHb

Lep-ore)andforAT.Someoftheseauthorshavecautionedabout

theriskofinterpretationerrorsandthefalsediagnosisofHb

S/␤0-thalassemia.7,10,13,14,16–18,24–26

Accuratediagnosis ofHb disorders isessential insickle

cellsyndromes.ConsideringtheclinicalimportanceofHbA1

andHbA2and/orHbFtogetherwithclinicalandlaboratory

dataforthediagnosisofHbS/␤-thalassemia,thisstudy

ana-lyzedHbA2valuesbyHPLCinconjunctionwithHbSandHb

C,andAT,inapopulationwith alowestimatedfrequency

of␤-thalassemia.27,28_Similar_to_results_observed_in_previous

studies,7,10,13,14,16–18,24–26 _the_Hb_A

2valuesobtainedbyHPLC

wereincreasedinHbSSandHbSCpatientsamples.

␣2-Thalassemia was diagnosed in23.4% of the children

withsickle cell disease,a resultthat agrees withprevious

studies performed inthe population ofthe state ofBahia,

Brazil.29,30_Thus,_as_Suh_et_al.13_and_Ondei_et_al.18_stated,_the

presenceofAThasanimpactonthevalueofHbA2as

quanti-fiedbyHPLCandadditionally,inthepresentstudy,significant

differencesinthemeanlevelsofHbA2wereobserved

accord-ingtothepresenceof␣3.7-thalassemia,withagradualincrease

inthisHbfractionbetweenthethreegroups:wildtypeto

het-erozygoustohomozygous.

Severalstudiesinrecentyears9–12_have_sought_to_compare

differentmethodologiesandsystemsinordertoidentify

pro-cessesinwhichthepresenceofanomalousHbshavetheleast

effectonthemeasurementofHbA2inordertoreachamore

accuratediagnosis.However,studiesseekingtoexplainhow

thepresenceoftheseHbsandATmayresultinfalseHbA2

levelsareextremelyscarce.Eventhemostrecentpublications,

suchasthatofGreeneetal.in20155_seem_to_agree_with_Suh

etal.from199613_in_attributing_the_apparent_increase_in_Hb

A2asquantifiedbyHPLCinsamplescontainingHbstructural

variantstothepresenceofglycosylatedfractionsof

anoma-lousHb(suchasHbS1c)thatco-elutewithHbA2.10,14,17,23

OncomparingtheimpactofthetypeofHbvariantonthe

measurementofHbA2,thegreatestimpactwascausedbythe

presenceofAT,andnotbyHbSorHbC.Headetal.7_defended

thatthe increasedHb A2 levelsobservedintheassociation

ofsicklecelldiseasewithATareexplainableinthatwitha

lowernumberofalphachainsbeingproduced,thesepositively

chargedmoleculescombinewithotherchainsforwhichthey

havegreataffinity,namelythedeltachains.

Theprevalenceof␤-thalassemiaisverylowinthestudied

population,27,28_and_a_careful_{hematological}_study_of_the

par-entsshowednormalerythrogramsandthepresenceofHbS

andHbAconsistentwithheterozygosityforHbS.Giventhese

facts,weconsiderthattheHbA2valuesseeninthepatients

werefalselyelevatedduetointerferenceofthemethodology

usedinthepresenceofstructuralHbvariantsandAT,andas

suchthepatientscanbediagnosedashavingsicklecell

ane-mia,andnotHbS/␤0-thalassemia,eventhoughwerecognizea

limitationbythefactthatwedidnotperformmolecular

anal-ysestoconclusivelyexcludethediagnosisof␤-thalassemia.

WesuggestthatincaseswithapossiblediagnosisofHbS/␤0

thalassemia,theHbprofileshouldnotbeperformedbyHPLC,

orthatifused,thismethodshouldbecomplementedbya

sec-ondconfirmatorytest.Furthermore,whendoubtsstillremain,

amolecularanalysisshouldbecarriedouttoidentifydeletions

orothermutations,inordertoconfirmtheclinicaldiagnosis

andguidegeneticcounseling.5

Conclusion

WeconcludethattheHbA2levelsinsamplescontainingHb

Sand/orHbCcanbeoverestimatedwhenanalyzedbyHPLC,

especiallyinthepresenceofAT,andthatinthesesituations

the referencevaluesofup to3.5%shouldnotbeused.We

reinforcetheimportanceofmulti-centerstudieswhen

estab-lishingpatterns,theneedforindividualevaluationsofcases

inordertoreachadifferentialdiagnosisbetweensicklecell

syndromes,especiallyinregionswithahighprevalenceofthe

differenttypesofhemoglobinopathies,andtheuseof

molec-ularbiologystudiestoclarifyanydoubts.

Funding

CentroNacionaldeDesenvolvimentoCientíficoeTecnológico

(6)

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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