rev bras hematol hemoter. 2015;37(2):73–76
w w w . r b h h . o r g
Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
Scientific
Comment
Comment
on:
Evaluation
of
erythrocyte
and
reticulocyte
parameters
as
indicative
of
iron
deficiency
in
patients
with
anemia
of
chronic
disease
夽
Elisa
Piva
∗AziendaOspedaliera-UniversitàdiPadova,Padova,Italy
a
r
t
i
c
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e
i
n
f
o
Articlehistory:
Availableonline17February2015
Thearticleontheevaluationoftheeffectivenessofmature
redcellandreticulocyteparametersunderthreeconditions:
iron deficiency anemia, anemia of chronic disease (ACD),
andanemiaofchronicdiseaseassociatedwithabsoluteiron
deficiencybyTorinoetal.1isveryvaluable.Automated
retic-ulocytecountsarewidelyusedintheclinicallaboratorydue
totheirgreaterprecision,accuracyandreproducibility
com-pared to those obtained using the microscope. The most
importantbenefitofautomatedmethodsisthegreater
pre-cision of counts. By analyzing a much greater number of
reticulocytes(morethantenthousand),thestatisticalerror
is minimized.2 Visual microscopy is still recommended as
the comparability method for reticulocytes, despite
stud-ies showingthatthe variationcoefficient (VC)ranges from
20% to 40%. Fully automated methods have eliminated
inter-observervariability and subjectivity and substantially
reduced turnaround time. Automated methods employ a
wide variety of reagents for reticulocyte RNA and these
show different sensitivity on binding to RNA.3 Therefore,
although automated flow cytometric analysis has led to a
significant advance in reticulocyte counting, some
limita-tionsstillpersistincomparabilityacrossdifferentlaboratories
夽
SeepaperbyTorinoetal.onpages77–81.
∗ Correspondingauthorat:DipartimentoMedicinadiLaboratorio,AziendaOspedaliera-UniversitàdiPadova,ViaNicolòGiustiniani2,
35128Padova,Italy.
E-mailaddress:elisa.piva@sanita.padova.it
and better methods of standardization and
harmoniza-tionareneeded.4Nevertheless,biologicalandpre-analytical
variations can potentially affect test performance and the
clinical interpretation of laboratory results.5 Pre-analytical
variations represent a major source of inaccurate
labora-toryresults.Reticulocytecountsare significantlydecreased
after 24h of storage at room temperature due to in vitro
maturation of the reticulocytes. At constant temperatures
of 4◦C the counts remain unchanged, with certain
limi-tations for parameters derived or calculated from cellular
volumes.6,7
Automatedreticulocytecountsnotonlyprovideenhanced
precision and accuracy,but alsoperform reliable
measure-ments of mRNA content and of cellular indices such as
volume,hemoglobinconcentrationandcontent.Thesenovel
parameters have prompted interest and studies regarding
theirclinicalusefulness,theutilityofreportingandtheir
inter-pretation.Immaturereticulocytefraction(IRF)assesses
retic-ulocytematurationbytheintensityofthestainingof
reticu-locytes,whichreflectsmRNAcontent.8IRFseemstobeuseful
fortheevaluationofengraftmentinbonemarroworstemcell
transplantation.9
http://dx.doi.org/10.1016/j.bjhh.2015.02.005
1516-8484/©2015Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.PublishedbyElsevierEditoraLtda.Allrights
74
revbrashematolhemoter.2015;37(2):73–76IRFhasbeenproposedasanearlymarkerofengraftment
inbonemarroworhematopoieticstemcelltransplantation
and bone marrow regeneration following chemotherapy.10
SeveralstudieshavedemonstratedthatincreasesintheIRF
are anindicator ofengraftment and precedeother
param-eters,suchasabsoluteneutrophilcounts(ANC),reticulated
plateletorreticulocytecounts.11,12Thus,changesinIRFduring
erythropoiesis-stimulating agent (ESA) therapy are
indica-tiveoftheeffectivenessofstimulation.13Manyauthorshave
reporteddataconcerningtheclinicalutilityofIRFinthe
diag-nosisandmonitoringofanemia.14,15IRFinconjunctionwith
thereticulocyte count,providesessentiallythe same
infor-mationasthereticulocyteproductionindex(RPI),makingits
manual calculation unnecessary. The clinical utility of IRF
hasbeenreportedinavarietyofconditionssuchasinthe
monitoringofanemiatreatment,neonataltransfusionneeds,
prognosisinprematurity, inAIDSanemia, renaltransplant
engraftmentduetoerythropoietinproduction,thedetection
ofoccultorcompensatedhemorrhagesorhemolysis,aplastic
crisisinhemolyticanemiasandtoverifyaplasticanemia.16
TheIRFisapromisingparameterthat needsconsolidation
intotheclinicalpractice.
Meanreticulocytehemoglobin,ameasurementoftheHb
contentofreticulocytesexpressedinpg/cellwasfirst
mea-suredusingBayerH3instrumentsandabbreviatedasCHr.17,18
CHr is the product of the cellular volume and the
cellu-larhemoglobinconcentration.Meanreticulocytehemoglobin
has become available in other fully automated
hemato-logyanalyzersthatprovidereticulocytecountandmaturity.
The methodology developed by Sysmex (Sysmex
Corpora-tion,Kobe,Japan)fortheXE and laterfortheXN seriesof
automated hematology analyzers provides the reticulocyte
hemoglobinorRet-Heparameter,formerlydefinedasRET-Y.
Themean hemoglobincontentofreticulocytes (MCHr)and
themeanhemoglobinconcentrationofreticulocytes(CHCr)
havebecomeavailableintheCELL-DYNSapphireanalyzerof
Abbott(AbbottDiagnostics,SantaClara,CA,USA).The
reticu-locytehemoglobinexpression(RHE)isavailableintheBC6800
Mindrayanalyzerforresearchuseonly(MindrayBioMedical
ElectronicsCo,Shenzhen,P.R.China),whilethereticulocyte
hemoglobincellularcontent (RHCc) isprovidedinthe new
generation ofPentra blood cell analytical systems (Horiba
Medical,MontpellierCedex,France).RHEandRHCcneedto
beevaluatedandcomparisonstudiesshouldbeassessedto
verifyifthenewindicesareclosetothoseobtainedbyother
instruments,thusprovidingreliableresults.
BeckmanCoulter (BeckmanCoulter Inc) providesa new
parameter,theredblood cellsizefactor(RSf),whichseems
tobeinagreementwithCHr.TheRSfparameter,expressedin
fL,joinstogetherthevolumeofmatureredcells(MCV)and
thevolumeofreticulocytes(MRV),accordingtothefollowing
mathematicalformula:RSf=√(MCV×MVR).19
Since the life span of the reticulocytes is four days,
the measurement of reticulocyte hemoglobin content can
directlyreflectthefunctionalavailabilityofironinthattime
frame.20 Reticulocyte hemoglobin content isa reliable and
earlyindicatorofbonemarrowironstatusand maydetect
functional iron deficiency with more sensitivity than
bio-chemicalparameters.21Reticulocytehemoglobincontentmay
optimizeIVirontherapyandindicatetheefficacyofresponses
toanemiatreatmentatanearlystage.Althoughitsreduction
reflectstheimpairmentofhemoglobinproduction,
reticulo-cytehemoglobincontentisnottheappropriatemeasureto
assessironadequacyinthepresenceofgeneticmicrocytosis
suchasthalassemia.22,23Iron-sequestrationsyndromesoccur
in chronicdiseases when iron isnotavailable for
erythro-poiesis,duetoinappropriatelyhigh serumhepcidinvalues,
which determine iron sequestration in reticuloendothelial
systemmacrophages.24Oneofthemajordeterminantsofthe
anemiaofchronicdiseaseisironsequestration.24–27
Several studies have assessed the value of reticulocyte
hemoglobininconjunctionwithotherparameterstodiagnose
iron deficiencystates.Other studies haveassessedthe use
ofbothhepcidinandreticulocytehemoglobininACD.Serum
hepcidin was shownnot tobeclinically usefulor superior
tomorestandardironstatustests,formanagingiron
ther-apyinHDpatientsonESAtreatment;reticulocytehemoglobin
contentandpercentageofhypochromicredbloodcellswere
showntobemoreuseful,eitheraloneorincombinationwith
thetransferrinsaturationratioandferritinlevels.28–30
Theclinicalutilityofreticulocytehemoglobincontenthas
beenwellestablishedasareliablemarkeroffunctionaliron
deficiency in hemodialysis patients, exhibiting high
speci-ficityandsensitivityinthemanagementofIVirontherapy.
In patients with chronic kidney diseases and anemia that
areundergoingESAtreatment,repletionofironstoresshould
be ensured beforeand duringtherapy. Iron levelsmust be
adequate to optimizehemoglobin production in a balance
witherythropoiesisstimulation.31 TheKidney Disease
Out-comesQualityInitiative(NKFKDOQI)TMoftheNationalKidney
Foundation has provided evidence-based clinical practice
guidelines whereCHr is considered an appropriatetest to
assess adequacy ofiron forerythropoiesis.32 Inthe British
Guidelines forLaboratoryDiagnosisofFunctionalIron
Defi-ciency,CHrisoneoftherecommendedtestswithaproposed
cut-offvalueofCHr<29pg.33
Thereticulocytehemoglobincontentpresentssome
diag-nostic limitations. The reticulocyte hemoglobin content is
decreasedinthalassemiasyndromes,wherethereductionin
CHrseemstobecorrelatedwiththedegreeofimpairmentin
betachainsynthesis,andinothermicrocyticanemiasdueto
congenitalhemoglobindiseases.34Itcanalsobeelevatedin
iron-deficientpatientswithconfoundingmegaloblastic
ane-miabecauseofthehighmeanreticulocytevolumeassociated
with megaloblastosis.35 Therefore, it is importantthatCHr
valuesareinterpretedinthecontextofthepatient’soverall
erythrocytephysiology,includingknowledgeofrecentblood
transfusions,irontherapy, vitaminB12or folate deficiency,
chemotherapyand the resultsofhemoglobin analysis.Few
studiesareavailableontheclinicalutilityofreticulocytecell
volume however its usefulness seems tobe similar to the
reticulocyte hemoglobin content inanemia evaluation and
monitoring.35
With the introduction of automated methods, it has
becomemandatorytoreporttheabsolutecountwhichgives
moreaccurateinformationonerythropoiesisthanthesimple
reticulocyte percentage.36 Automated absolute reticulocyte
countshaveresultedinphasingouttheoldfashion
“hema-tocritcorrection”ofreticulocytepercentage.Inaddition,the
revbrashematolhemoter.2015;37(2):73–76
75
reticulocytecountbothforHctandmaturationtime,canbe
replacedwithIRF,whichoffersthesameclinicalsignificance.
Laboratoriesshouldreport thereticulocyte countasthe
absolutenumberofreticulocytes,accompaniedbyproperly
determinedandmethod-specificreferenceranges.The
per-centagevalue may be optional, but it isstill important in
monitoringbonemarrowresponsewhenplasmavolumeis
fluctuating as happens in blood boosting inathletes or in
kidney diseases. The clinical utility of reticulocyte cellular
parameterssuchasIRFandreticulocytehemoglobincontent
hasbeenproven,whileMCVrmaybeoptional,eventhoughit
couldinsomeinstancesprovideusefulinformation.Itmaybe
usefulforlaboratoriestoconsiderprovidinganinterpretation
of the reticulocyte analysis. As an example, if the
abso-lutereticulocytecountandIRFaresimultaneouslyincreased,
an interpretative comment could be added to emphasize
theincreaseoferythropoieticactivity.Thiscomment could
help physicians assess cases of suspected hemolytic
ane-miaorinmonitoringthetreatmentofanemia.Inconclusion,
automatedreticulocytecountsprovideacceptableprecision
and bias while parameters and indices improve the
eval-uation of erythropoiesis. Since a qualitative evaluation is
performedwithreticulocytematurationparametersand
cel-lularindices, externalqualityassessmentprogramsshould
beprovided,andinterpretativereportingshouldbeofferedto
clinicians.37,38Nevertheless,standardizationand
harmoniza-tionshouldbeencouraged.
Conflicts
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interest
Theauthordeclaresnoconflictsofinterest.
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