Rev. Bras. Hematol. Hemoter. vol.38 número4

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rev bras hematol hemoter. 2016;38(4):310–313

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

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

Determination

of

reference

ranges

for

immature

platelet

and

reticulocyte

fractions

and

reticulocyte

hemoglobin

equivalent

Iuri

Vicente

Camargo

Morkis

∗

_,

_Mariela

_Granero

_Farias,

_Luciana

_Scotti

HospitaldeClínicasdePortoAlegre(HCPA),PortoAlegre,RS,Brazil

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Articlehistory:

Received14April2016 Accepted6July2016

Availableonline6August2016

Keywords:

Referenceinterval Immatureplateletfraction Immaturereticulocytefraction

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Introduction:Theimmatureplateletandimmaturereticulocytefractionsrepresenttheratios ofplateletsandreticulocytesrecentlyreleasedintothecirculationandthuswithhigherRNA content.Theyareconsideredearlyindicatorsofbonemarrowrecovery.

Objective:Theaimofthisstudywastodeterminethereferencerangesfortheimmature plateletandreticulocytefractionsofhematologicallynormalindividualsina university hospital.

Methods:VenousbloodsamplescollectedinethylenediaminetetraaceticacidK3were ana-lyzedusingaSysmexXE-5000TM_analyzer._Individuals_with_platelet_and_reticulocyte_counts

withinthereferenceranges,andabloodcountwithinthelaboratory’sscreeningcriteria wereincluded.Individualswithclinicalconditionsthatcouldaffecthematologicalresults wereexcluded.Theimmatureplateletfraction,high,mediumandlowfluorescence retic-ulocytefractionsandreticulocytehemoglobinequivalentwereevaluated.Thereference rangesweredeterminedaccordingtotherecommendationsoftheInternationalFederation ofClinicalChemistry.

Results:Onehundredandthirty-twooutpatientswereevaluated.The meanagewas44 years(range:13–80years),72(54.5%)werewomentreatedina universityhospital.The meanplateletcountwas250.8×109_/L_and_the_mean_reticulocyte_count_was_0.052_×₁₀9_/L.

Thefollowingreferencerangeswereobtained:immaturereticulocytefraction1.6–12.1%, thehigh,medium andlowfluorescence reticulocytefractionswere0.0–1.7%, 1.6–11.0% and87.9–98.4%,respectively,thereticulocytehemoglobinequivalentwas30.0–37.6%and immatureplateletfractionwas0.8–5.6%.Therewasastatisticallysignificantdifference(p -value=0.006)betweengendersinrespecttotheimmatureplateletfractionwith0.8–4.7%for femalesand0.7–6.1%formales.Theimmaturereticulocytefractionwasdirectlycorrelated withthereticulocytecount.

∗ _{Corresponding}_author_at_:_Hospital_de_Clínicas_de_Porto_Alegre_(HCPA),_Rua_Ramiro_Barcelos,_2350,_90035-903_Porto_Alegre,_RS,_Brazil. E-mailaddress:iurimorkis2@gmail.com(I.V.Morkis).

http://dx.doi.org/10.1016/j.bjhh.2016.07.001

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revbrashematolhemoter.2016;38(4):310–313

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Conclusion: Determining the reference range is critical to the introduction of a new parameter. The reference rangesobtained hereincorroborate those reported in previ-ous publications and will contribute to the clinical and laboratory application of the indices.

©2016Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Recentadvancesinthefield ofhematology analyzersused inclinicallaboratoriesallowthedeterminationofadditional hematologicparameters,providingusefulinformationforthe diagnosisand/ortreatment ofvarious pathologies.1 _Among

theseparametersaretheimmaturefractionsofplateletsand reticulocytes.1–3

Theimmatureplateletfraction(IPF)andimmature retic-ulocytefraction(IRF)areyoungcellsthathaverecentlybeen releasedinto thecirculation, andare consideredindicators of bone marrow recovery.1–3 _They _are _important _in

vari-ousclinical situations, suchas thrombocytopenia, anemia, bonemarrowregenerationfollowingthe transplantation of hematopoieticstemcellsandafterchemotherapy.2,4–13

The IPF and IRF are determined using hematology analyzers with fluorescence and light scatter technology. ImmaturefractionshavelargeramountsofRNAthanmature plateletsandreticulocytes.Thus,theuseoffluorescentprobes thatlabel the RNApermitthe differentiation and quantifi-cationoftheIRFandIPF.1–3,6 _Automated_systems_provide_a

graphicdisplayofthedifferentpopulationsaccordingtothe sizeandamountofRNA.Basedonthis,theindicesare calcu-latedasapercentageoftotalreticulocytesandplatelets.1–3,6

Differentpopulationsofreticulocytesareseparated accord-ingtothesizeandtheamountofRNAandclassifiedasbeing low(LFR),medium(MFR) orhigh(HFR) fluorescence reticu-locytefractions.Thecombinedcountsofthe HFRand MFR correspond to IRF. The reticulocyte hemoglobin equivalent (Ret-He)parametercorrespondstothehemoglobincontentof thereticulocytes,andisstudiedasanindicatorofthe incor-porationofironinreticulocytes.15–18

Advancesinautomationhaveprovidedadditionalblood count parameters that enable new interpretations of the examination. However, clinical application can only occur oncethereferencerangeshavebeendeterminedwith knowl-edgeofpre-analyticalvariables(time,temperature, anticoag-ulants)andstandardizationofthemethodology.1,2,16,17,19–21

Somereferencerangeshavebeencalculatedbythe man-ufacturer,whileothershavenotyetbeendetermined.Thus, itisrecommendedthateachlaboratorycalculatesthe refer-encerangeforitspopulation,takingintoaccounttheroutine andthemethodologyused.22–24_Subjects_should_be_stratified

byageandgenderandmeetstrictcriteriaintheselectionof theindividualsinthepre-analyticalandanalyticalphasesand forstatisticalanalysis.22–24

Thisstudyaimstodeterminethereferencerangesforthe IPF,IRF,LFR,MFR,HFRandRet-HEinhematologicallynormal patientstreatedatauniversityhospital,inordertostudythe rolesoftheparametersaspredictorsofengraftmentinbone

marrow transplantation,oneoftheirmainclinical applica-tions.

Methods

Venousbloodsamplescollectedin ethylenediaminetetraace-ticacidK3(EDTA-K3)wereanalyzedinaSysmexXE-5000TM analyzer(SysmexCorporation,Japan).Tominimizevariations due to sampleage, all assays were performed within four hoursofcollection;thesampleswerekeptatroom temper-atureuntilthetimeofanalysis.Theanalyzerusesthesame reagenttomeasuretheIPFandIRFinthereticulocytechannel. The platelet detection method employs hydrodynamic focusanddirectcurrenttechnology.TheIPFandIRFare deter-mined by fluorescence and light scatter using fluorescent RNA markers.1–3 _The_Ret-He _is_obtained_by_flow_cytometry

technologyusingapolymethinedye,specificforRNA/DNA. Theforwardlightscatterintensitycorrelatestothecellular hemoglobincontent.

Patients withnormalblood countswithinthescreening criteria established by the laboratory such as hemoglobin greaterthan12.0g/dLandplateletandreticulocytecount val-ues withinthe normal ranges (platelet count greater than 150.0×109_/L_and_{reticulocytes}_25–80_×₁₀9_/L)_were_included. Patientswithdiabetes,humanimmunodeficiencyvirus(HIV), cardiovascular diseases,pregnant women,hematologic dis-eases, cancer,and thyroid disease were excluded, asthese conditionsmayaffectthehematologicalresults.19–21

TheIPF,IRF,thereticulocyteratios(HFR,MFRandLFR)and Ret-Hewere evaluated. Assayswereroutinely submittedto internalandexternalqualitycontrols.

Statisticalanalysis

ThereferencerangesweredeterminedusingoftheStatistical PackageforSocialSciencesversion18.0(SPSSInc.,Chicago, IL,USA).Symmetricvariablesareexpressedasthemeanand standarddeviation,whileasymmetricvariablesareexpressed asthemedian,2.5–97.5percentileswithina95%confidence interval,asrecommendedbytheInternationalFederationof ClinicalChemistry(IFCC).22–24_Spearman’s_and_Pearson’s

cor-relationtestswerealsoused.

Results

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Table1–Referenceintervalandmedianforreticulocyte index.

Referencerange Median

LFR(%) 87.89–98.37 94.65

MFR(%) 1.6–11.04 4.9

HFR(%) 0–1.7 0.3

IRF(%) 1.6–12.1 5.35

Ret-He(%) 30.0–37.6 33.8

LFR:lowfluorescenceratio;MFR:mediumfluorescenceratio;HFR: highfluorescenceratio;IRF:immaturereticulocytefraction;Ret-He: reticulocytehemoglobinequivalent.

Table2–Referencerangeandmedianforimmature plateletfraction.

Referencerange Median

Women(%) 0.7–4.7 2.0

Men(%) 0.6–6.1 2.6

Total(%) 0.8–5.5 2.2

concentration was 1.1% (range: 0.6–1.7%) with a count of 52×109_/L_(range:_49–54_×₁₀9_/L).

Themedian valueof the IRF was 5.3% witha range of 1.6–12.1%.ThereferencerangesoftheHFR,MFRandLFRwere 0.0–1.7%, 1.6–11.0%and 87.9–98.4%, respectively. Themean Ret-Hewas33.8%witharangeof30.0–37.6%(Table1).

The median IPF was 2.2% with a range of 0.8–5.6%. A statistically significant difference (p-value=0.006) between genderswas onlyfound forthe IPF, with 2.0%forfemales (range:0.8–4.7%)and2.6%formales(range:0.7–6.1%).TheIRF presentedadirectcorrelation(SpearmanRank-order Coeffi-cient=0.40)withthereticulocytecount(Table2).

Discussion

Althoughnotdirectlyusedinclinicaldecisionmaking,the ref-erencerangeiscriticaltotheintroductionofnewparameters andtheinterpretationoflaboratoryresults.22–24_The_reference

rangeshowsthevariationoflikelyvaluesin‘healthy’ individ-ualswhodonotpresentclinicalconditionsthatcanaffectthe studiedvariables.22–24

Immatureplatelets and immaturereticulocytesare pre-cursor cells recently released into the blood stream that containlarger amounts of RNA. Thus, they can be distin-guished from the mature platelets and reticulocytes and quantifiedusingRNAfluorescentlabeling.1–3 _Several_studies

havedemonstratedthevalueoftheseindicesinthecontext ofhematopoieticstem celltransplantation as indicatorsof hematopoiesis,intransfusionassessmentandthe anticipa-tionofsuccessfulengraftment.1–8_The_Ret-He_is_useful_in_the

evaluationofhematopoiesis,withrespecttotheincorporation ofiron.14–17

ThereferencerangeobtainedfortheIPFwasinaccordance withthestudiesbyTakamietal.(mean2.0%;range:0.5–5.7%),9

Gonzaloetal.(median:2.3%;range:0.6–7.2%),4_Yamaoka_et_al.

(meanof3.0±1%),8_and_Briggs_et_al._(range:_1.1–6.1%).2 _The

studybyKoetal.foundlowerIPFvalues(range:0.5–3.3%).18

ThereferencerangefortheIRFwassimilartothatof Gon-zaloetal.whoobtainedamedianof4.7%(range:1.1–11.4%).4

Althoughtheyusedthesamemethodology,thesestudieswere conducted with the use of a Sysmex XE-2100TM _analyzer. Other authors employedother methodologiestodetermine thereferencerangesfortheIPFandIRFandsotheywerenot comparedwiththeresultsofthecurrentstudy.

Determining the reference range involves several steps, including selectionofsubjects, methodologyand statistical analysis.22–24_It_is_essential_that_it_is_carried_out_for_new

param-etersthatarenotwidelyusedbylaboratoriesandclinics.The referencerangesfortheIRFandIPFreportedinthisstudyare consistentwiththeliteratureandwillcontributetotheclinical applicationoftheseindices.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

We would like tothank the staff atthe Hematology Unit, DepartmentofClinicalPathology,HospitaldeClínicasdePorto Alegre.

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