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TABLE A(hCEKQW]cioAutoList61.1.1.1.1.1.1.1."1.(hCEKQW]cioAutoList71.1.1.1.1.1.1.1.$1.(9 Z(Times New Roman Level 1Level 2Level 3Level 4Level 5 > 'USUS.,  _EXERCISE4:_Reticulocyte_ԀCount_MLAB_Ԁ1415Hematology  AY) xd3ExA$35;AGMU]c1I.A.1.a.(1)(a)i)a)(hCEKQW]cioAutoList81.1.1.1.1.1.1.1.@*1.TABLE D'dxd Level 1 Level 2 Level 3 Level 4 Level 5(-2$ !USUS.,  ($ (    ) 2341M << deUUXX%XX ReticulocytesXXOXXXV#XXYVofXXWV reticulocytesXX VcountedXXuVinXXiV1XXV,XXV000XXVredXXVcellsXX G10 UFUNC{%~Reticulocytes~=~{\#~of~reticulocytes~counted~\in~1,000~red~cells}over{10}}TABLE C X($     3=1A<< c d(.3$ !USUS.,  _]3`aE<<CLevel 1Level 2Level 3Level 4Level 5($$   1  YYYY)!dxdxr3]Inline Te&xt" <<CLevel 1Level 2Level 3Level 4Level 5aY 234O  , deUU !USUS.,  _(8XXdd8  XX  Oa_RETICULOCYTE_ԀCOUNT  s XX  LABOBJECTIVE  s Thestudentwillperform,within20%accuracyofinstructorsvalues,two_reticulocyte_Ԁcounts _  onwholebloodspecimens.OneofthecountswillbeusingtheMillerocularandtheother K  withouttheocular.Thestudentwillcalculatethe_reticulocyte_Ԁvalue,corrected_reticulocyte_Ԁand 7  the_reticulocyte_Ԁproductionindex. #   PRINCIPLE    _Reticulocytes_Ԁareimmature_RBCs_Ԁthatcontainremnantcytoplasmicribonucleicacid(RNA)and t  organellessuchasmitochondriaandribosomes._Reticulocytes_Ԁarevisualizedbystainingwith `  vitaldyes(suchasnewmethyleneblue)thatprecipitatetheRBCandorganelles.Thisstain L  causestheribosomalandresidualRNAto_coprecipitate_Ԁwiththefewremainingmitochondria 8  and_ferritin_Ԁmassesinlivingyoungerythrocytestoformmicroscopicallyvisibledarkblue $  clustersandfilaments(reticulum).AnerythrocytestillpossessingRNAisreferredtoasa   _reticulocyte_.The_reticulocyte_Ԁcountisameansofassessingthe_erythropoietic_Ԁactivityofthe o  bonemarrow.Thenumberof_reticulocytes_Ԁisexpressedasapercentageofthetotalnumber_od_ [ erythrocytescounted. G  SPECIMEN   Wholebloodthatis_anticoagulated_Ԁwitheither_EDTA_Ԁorheparinissuitable.Capillaryblood   drawninto_heparinized_Ԁtubesorimmediatelymixedwithstainmayalsobeused.Redblood  cellsmuststillbelivingwhenthetestisperformedthereforeitisbesttoperformitpromptly p afterbloodcollection.Bloodmaybeusedupto8hoursaftercollection.Stainedsmearsretain \ theircolorforaprolongedperiodoftime. H  QUALITYCONTROL    Threeslidesshouldbemadeforeach_retic_Ԁperformed.Finalcalculated_retic_%sfromtwoslides   countsshouldagreewithin15%.Ifthisagreementisnotreached,countthethirdslide. k   REAGENTS,SUPPLIESANDEQUIPMENT  C"  (5h83 "3"  3w 25h  1  .3  0    Commerciallypreparedliquidnewmethylenebluesolution.Itshouldbestoredina /# brownbottle.Ifprecipitateisaproblemonthesmear,thestainshouldbefilteredpriorto $  use.3w ݌%!(#(# Ќ  "3"  325h  2  .3  0    Microscopeslides3L݌%"(#(# Ќ  "3"  325h  3  .3  0    Microscope3&݌&l #(#(# Ќ  "3"  325h  4  .3  0    10x75mmtesttubes3݌'X!$(#(# Ќ  "3"  325h  5  .3  0    Pasteur_pipets_Ԁ(withbulbif_pipets_Ԁareglass)3݌(D"%(#(# Ќ  "3"  325h  6  .3  0    Capillarytubes3݌)0#&(#(# Ќ  "3"  325h  7  .3  0    Millerocular(ifavailable)3݌*$'(#(# Oa    ь    PROCEDURE K Preparationofsmears  7  ! ! (Eh88 "3"  32Eh  1  .3  0    Labela10x75mmtesttubewithpatientnameandID.3݌# (#(# Ќ  "3"  32Eh  2  .3  0    Add5dropsofnewmethylenebluesolutionto5dropsofthoroughlymixed_EDTA_   _anticoagulated_Ԁbloodtoaglass10x75mmtesttube.3݌ (#(# Ќ  "3"  32Eh  3  .3  0    Mixthecontentsbygentlyshakingandallowtoincubateatroomtemperaturefora  t minimumof10minutes.3݌ `(#(# Ќ  "3"  32Eh  4  .3  0    Attheendof10minutes,gentlymixtheblood/stainsolution.3݌L (#(# Ќ  "3"  32Eh  5  .3  0    43݌8 (#(# Ќ  "3"  32Eh  6  .3  0    Usingthewedgesmeartechnique,makeacceptablesmearsnottoothickorthin.3݌$ (#(# Ќ  "3"  32Eh  7  .3  0    Labeltheslideswithpatientname,ID#anddate.3݌ (#(# Ќ  "3"  32Eh  8  .3  0    Allowtoairdry.(Donotblowtohastentodrying.)3݌o (#(# Ќ  ManualmethodofcountingwithouttheMillerdisc : G  ! ! (Uh88"3"  32Uh  1  .3  0    Placethefirstslideonthemicroscopestageand,usingthelowpowerobjective(10x), 7 findanareainthethinportionofthesmearinwhichtheredcellsareevenlydistributed # andarenottouchingeachother.Carefullychangetotheoilimmersionobjective(100x)  andfurtherlocatedanareainwhichthereareapproximately100redcellsperoil  immersionfield.3݌t(#(# Ќ  m 0  0` kkDothisbyfindingafieldwherethecellsareevenly ` distributedandmentallydividethefieldinto4quadrants. L Countthecellsin1quadrant.Ifthereareabout25,youare 8 inagoodarea.Therewillbealotofopenspacebetween $ theredcells.` k` k "3"  m 3#2Uh  2  .3  0    Whenyouhaveselectedaproperareaforcounting,the_retics_Ԁmaybecounted.Besureto o countallcellsthatcontainabluestainingfilamentoratleast2ormorediscreteblue [ aggregatesofreticulumintheerythrocyte.(Seeprocedurenotesforinclusionsthatmay G  beconfusedwithreticulum.)3#$݌3!(#(# Ќ  "3"  3"&2Uh  3  .3  0    Count1000redcellsinconsecutiveoilimmersionfields.Recordthenumber " _reticulocytes_Ԁseen.Youmaycount500cellsontwoslideseach.Theyshouldagree  # within15%ofeachother.Iftheydonot,repeatthe_reticulocyte_Ԁcountonthethird #  smear.3"&O&݌$p!(#(# Ќ     k*#' Ї CalculationUsingtheManualMethod _ ! ! Calculatethepercentof_reticulocytes_Ԁasfollows: K 576N>:z X p 0 @ H@E7 tt7 t 7  EXAMPLE: D  _Retics_Ԁcounted=47in_1000_Ԁerythrocytes 0  ̀%_Retics_Ԁ=47󀀀=4.7%  Ѐ10   MethodusingtheMillerdisc  Y   (eh88"3"  3,2eh  1  .3  0    Usea100xobjectiveanda10xocularsecuredwithaMillerdisc.Seefigure3115on E  page799oftextbook.TheMillerdiscimposestwosquares(one9timestheareaofthe 1  other)ontothefieldofview.Findasuitableareaofthesmear.Agoodareawillshow3   10_RBCs_ԀinthesmallersquareoftheMillerdisc.3,E,݌| (#(# Ќ  "3"  3y.2eh  2  .3  0    Countthe_reticulocytes_Ԁwithintheentirelargesquareincludingthosethataretouchingthe h linesontheleftandbottomoftheruledarea.Count_RBCs_Ԁinthesmallersquarewhether T theycontainstainedRNAornot.A_retic_Ԁinthesmallersquareshouldbecountedasan @ RBCanda_retic_.RecordRBC#countedand_retic_Ԁ#countedseparately.3y..݌,(#(# Ќ  "3"  3s12eh  3  .3  0    Continuecountinguntilaminimumof111_RBCs_Ԁhavebeenobserved(usually1520  fields).Thiswouldcorrespondto999_RBCs_Ԁcountedwiththestandardprocedure.3s11݌(#(# Ќ  CalculationUsingtheMillerDiscMethod  i ̀%_Reticulocytes_Ԁ=Total_retics_ԀinlargersquareX100 A ЀTotalRBCinsmallersquareX9 - EXAMPLE: x  _Retics_Ԁcountedinlargesquare=20 d! _RBCs_Ԁcountedinsmallsquare=111 P" ̀%_retics_Ԁ=򀀀20X100=2.02% ($ Ѐ111x9 %   Calculationofcorrected_reticulocyte_  &y " The_reticulocyte_Ԁcountismostoftenexpressedasapercentageoftotalredcells.Instatesof 'e!# anemia,the_reticulocyte_Ԁpercentageisnotatruereflectionof_reticulocyte_Ԁproduction.A (Q"$ correctionfactormustbeusedsoasnottooverestimatemarrowproduction,becauseeach )=#% _reticulocyte_Ԁisreleasedintowholebloodcontainingfew_RBCs_Ԁalow_hematocrit_Ԁ(_Hct_)thus *)$& _relativelyincreasingthepercentage.Thecorrectedreticulocytecountmybecalculatedbythe s followingformula: _ Correctedreticulocytecount=Retic%x򀀀patientsHct% 7  ЀAveragenormalhct* #  ̀*Averagenormalhctis45formenand42forwomen.   EXAMPLE:  ` Uncorrectedretic%=5.0% L  Patienthct=25.0%(male) 8  Averagenormalhctformale=45% $  ̀Correctedretic%=5.0%x25%=2.8% o  Ѐ45% [   Calculationofthereticulocyteproductionindex(RPI)  3 EstimatingRBCproductionbyusingthecorrectedreticulocytecountmayyielderroneouslyhigh  valuesinpatientswhenthereisaprematurereleaseofyoungerreticulocytesfromthemarrow   (owingtoincreasederythropoietinstimulation).Theprematurereticulocytesarecalled stressor  shiftreticulocytes.Theseresultwhenthereticulocytesofthebonemarrowpoolareshiftedto p thecirculationpooltocompensateforanemia.Theyoungerstressreticulocytespresentwith \ morefilamentousreticulum.Thematurereticulocytemaypresentwithgranulardots H representingreticulum.Normally,reticulocyteslosetheirreticulumwithin24to27hoursafter 4 enteringtheperipheralcirculation.Theprematurestressreticshaveincreasedreticulumand   require2to2.5daystolosetheirreticulum,resultinginalongerperipheralbloodmaturation   time. k * aY!YYYYu ddd Xdd Xdd X(#(#,'dd ,dd +  $C C  $&g %XXMaturationTime ' 4 'Hematocrit  4 % !  1day 5+ "x   F@45F@545 4*("x!  F@45 F@ 41.5days 5+ W$"  A@35A@535 4*(W$#  A@35 A@ 42days 5+ %P$  9@259@525 4*(%P%  9@25 9@ 43days 5+ /' &  .@15.@5158.,/' '  .@15  .@ 8XX %&gTheperipheralbloodsmearshouldbereviewedcarefullyforthepresenceofmany )"( polychromatophilicmacrocytes,thusindicatingstressreticulocytesandtheneedforcorrection )#) forboththeRBCcountandthepresenceofstressreticulocytes.Thevalueobtainediscalled_the *t$* _reticulocyte_Ԁproductionindex(_RPI_).An_RPI_Ԁequaltoorgreaterthan3representsanadequate s responsetoanemiabythebonemarrow,whereasan_RPI_Ԁoflessthan2isconsideredan _ inadequateresponseof_erythopoiesis_Ԁbythebonemarrowtoastateofanemia. K Calculationofthe_RPI_: #  _RPI_Ԁ=Corrected_retic_Ԁcountin%   ЀMaturationtimeindays  t EXAMPLE: L  Patientscorrected_retic_Ԁ=12% 8  Patients_hct_Ԁ=25% $  _RPI_Ԁ=12%=6 o  Ѐ2 [   PROCEDURENOTES  G   Interpretationofresults  3 ! ! "(h88"3"  3'K2h  1  .3  0    The_reticulocyte_Ԁcountiselevated:1)inpatientswithhemolyticanemia,2)inthosewith  hemorrhage(acuteandchronic),3)followingtreatmentofirondeficiencyanemiaandthe   _megaloblastic_Ԁ_anemias_,and4)inpatientswithuremia.3'KTK݌(#(# Ќ  "3"  3bM2h  2  .3  0    The_reticulocyte_Ԁcountisdecreasedincasesof:1)aplasticanemia,2)aplasticcrisesof p hemolytic_anemias_,3)ineffective_erythropoiesis_Ԁasseeninthalassemia,pernicious \ anemiaand_sideroblastic_Ԁanemia.3bMM݌H(#(# Ќ  "3"  3O2h  3  .3  0    _Reticulocytopenia_Ԁinthepresenceofasuggestedhemolyticanemiamayoftenmake 4 diagnosisdifficult.Thediagnosisofahemolyticanemiacanbemadebecausethe   combinationofbothhemolysisand_reticulocytopenia_Ԁresultsinarapidlyfalling   hemoglobinand_hematocrit_.3OO݌k(#(# Ќ  Sourcesoferror  C  ! ! $(h88""3"  3R2h  1  .3  0    A_refractile_Ԁappearanceoferythrocytesshouldnotbeconfusedwith_reticulocytes_. /! _Refractile_Ԁbodiesareduetopoordryingowingtomoistureintheair.Toruleout " _refractile_Ԁmaterial,finefocusupanddown.The_refractile_Ԁmaterialwillgetshinyand # reticulumwillremainblue.3RS݌# (#(# Ќ  "3"  3U2h  2  .3  0    Filtrationofthestainisnecessarywhenprecipitatedmaterialispresentwhichcan $l! resemblea_reticulocyte_.3UU݌%X"(#(# Ќ  "3"  3W2h  3  .3  0    Erythrocyteinclusionsshouldnotbemistakenfor_reticulocytes_.HowellJollybodies &D # appearasoneorsometimetwo,deeppurpledensestructures.Heinzbodiesstainalight '0!$ bluegreenandareusuallypresentattheedgeoftheerythrocyte._Pappenheimer_Ԁbodies ("% aremoreoftenconfusedwith_reticulocytes_Ԁandarethemostdifficulttodistinguish. {)#& Thesepurplestainingirondepositsgenerallyappearasseveralgranulesinasmallcluster. g*#' If_Pappenheimer_Ԁbodiesaresuspected,stainwithWright_Giemsa_Ԁtoverifytheirpresence. s HemoglobinHinclusionswillappearasmultiplesmalldotsineverycell.3W;W݌_(#(# Ќ  "3"  3f[2h  4  .3  0    Falselydecreased_reticulocyte_Ԁcountscanresultfrom_understaining_Ԁthebloodwithnew K methyleneblue.Besurethestain/bloodmixtureincubatesthefull10minutes.3f[[݌7 (#(# Ќ  "3"  3)]2h  5  .3  0    Highglucoselevelscancause_reticulocytes_Ԁtostainpoorly.3)]V]݌# (#(# Ќ  "3"  3N^2h  6  .3  0    Thereishighdegreeofinaccuracyinthemanual_reticulocyte_Ԁcountowingtoerror(2%)   inlowcountsand7%inhighcounts)andalackofreproducibilitybecauseofthe   inaccuracyofthebloodfilm.Thisinaccuracyhasbeenovercomebytheuseofautomated  t instrumentsusingflow_cytometry_.3N^{^݌ `(#(# Ќ  "3"  3`2h  7  .3  0    Ifno_reticulocytes_Ԁareobservedafterscanningatleasttwoslides,report noneseen.3``݌L (#(# Ќ  REPORTINGRESULTS  $   Normalvalues    Newborn(02weeks):0  2.56.0%o (#(# Adult:0  0` (#(#0 ` (#` (#0 (# (#0.5_Ԅ2.5%_[ (#(#  REFERENCES  3 &XXXX_Harmening_.,Denise,ClinicalHematologyandFundamentalsof_Hemostasis_,3rdedition,pp.599  601.   _Turgeon_,MaryLouise,ClinicalHematologyTheoriesandProcedures,#XXX&Xkc#  3rdedition,pp324_Ԅ326._   XX&g %XX<IrStudentsname:____________________________________________Date:________________  _Retic_Ԁstainlot#:_____________________________ExpirationDate:______________  Gradingcriteria:Slidequality,slidelabeling,accuracyofresult. S8 Adultnormalrange:_____________________Newbornnormalrange:_________________ +   Showyourcalculations    * :aY;YYYYud d'dd 'dd  aY!"",Sdd ,Fdd , dd , dd ,dd ,dd +     Patientname/ID#/ ? $  gender/age     Cellscounted  ? $  _Hct_  ? $  Calculation  ? $  Result  ? $ Normal?  ? $     Slide1_____  h  Slide2_____ 3  (Slide3_____)   Average:_____  x    h     h     h     h    _D$ Slide1_____ % Slide2_____ ' (Slide3_____) _D) Average:_____  +   ,   -   .  /  XX %&g Showcalculations #&g %XXk# 0 XX %&g* +aY-YYYYuddSdd SFdd F dd  dd dd dd :aY;"",Sdd ,dd , dd , dd +  1 #&g %XXGl#PatientID  ?$2 _Retic_Ԁ  ?$3 Corrected_Retic_  ?$4 _Reticulocyte_ԀProductionIndex  ?$5 1stpatient 6   "<   = Calculation: > Result:______________  "D Calculation: E Result:____________  "K XX %&g2ndpatient '$ L   )%R   '$ S Calculation: '$ T Result:______________  )%Z Calculation: '$ [ Result:____________ )%a  #&g %XXo# &&g % %&g& %&&g   ,(c <Ir <XXX <  STUDYQUESTIONS#&&g %&7q#&XX %&&g   Vq  Name________________________________  Date_________________________________ k Pointspossible=_29#XXX&Xq#_ W  #&g %XXq#  (X,""(XX %&g1pt#&g %XXs#   ! ! *(h88$"3"  XX %&g3t2h  1  .3  0   Whatisthe_reticulocyte_Ԁusedtoassess?3tt݌  " " Ќ    t 2pt  L  "3"  3u2h  2  .3  0   Whatcriterionisusedtoidentify_reticulocytes_?3uv݌ L  " " Ќ  _   <      D      L _ $  1pt   "3"  3w2h  3  .3  0   Whatisreticulumcomposedof?3ww݌  " " Ќ   g  1pt ?  "3"  3x2h  4  .3  0   Whatistheratioofbloodtostainintheusual_retic_Ԁprocedure?3xy݌?  " " Ќ    1pt \ "3"  3-z2h  5  .3  0   Howlongmustthebloodincubatewiththenewmethylenebluebefore \ makingthe_retic_Ԁsmear?3-zZz݌H " " Ќ    4 1pt   "3"  3{2h  6  .3  0   Whatisa stress_reticulocyte_?3{{݌  " " Ќ    3pt c "3"  3|2h  7  .3  0   List3conditionsinwhichthe_reticulocyte_Ԁcountisincreased.3|+}݌c " " Ќ   A. O     <      D      L    !  B. '"  C. #l  $X  3pt &0" "3"  3D2h  8  .3  0   List3conditionsinwhichthe_reticulocyte_Ԁcountisdecreased.3Dq݌&0" " " Ќ   A. '#   B. )$   C. _+&   7-( Ї_6pt  "3"  3=2h  9  .3  0   List3RBCinclusionsthatcouldbeconfusedwithreticulocytesonaretic  smearandstatehowyoucoulddifferentiatethemfromretics.3=j݌ " " Ќ   A. W  B. /   C.  t 1pt  L "3 "  3(2h  10  .3  0   Howcanyoudistinguishbetweenreticulumandartifact?3(U݌ L " " Ќ   $  2pt   "3 "  3Y2h  11  .3  0   Statethenormalrangeforthefollowingpatientpopulations:3Y݌  " " Ќ   Adult:_______________ g   Newborn:____________ ?  + 2pt p "3 "  32h  12  .3  0   Calculatethefollowingreticcountwhichwasdoneusingthemanualmethod. p Showyourcalculations.3)݌\ " " Ќ   1stslide=15reticscountedin500RBCs H  2ndslide=17reticscountedin500RBCs 4   1pt c "3 "  32h  13  .3  0   Correctthereticcountobtainedinquestion#12.Thepatientisanadult c femalewithahematocritof25%.Showyourcalculation.3B݌O " " Ќ   "l 2pts $D  "3 "  32h  14  .3  0   CalculatethefollowingreticcountwhichwasdoneusingtheMillerdisc $D  method.Thepatientisanadultmale.Showyourcalculation.3׉݌%0! " " Ќ   #reticscountedinlargersquare:26 &"  #RBCsinsmallersquare:111 '#   _*%  2pts 7,'  "3"  3ы2h  15  .3  0   WhywouldadoctorrequestanRPI?