ordinul nr. 1217

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ORDINULNR.1217/2010privindaprobareaGhidurilordepracticmedicalpentruspecialitateaanatomiepatologic.Anexa1:TEHNICIORIENTATIVEDELUCRUPENTRUPRELUCRARESICOLORAREAPREPARATELORCITOPATOLOGICESIHISTOPATOLOGICE

07.11.2013/MinisterulSntii

PRINCIPIIGENERALEDETEHNICAHISTOPATOLOGICA

Principiultehniciihistopatologiceimpunetransformareaunuiprelevatdintrunorgansauesutdintromasatisularopacntrunpreparatfin,translucidcarepermitevizualizareamicroscopicaadetaliilorstructuraletisulare.Pentruarealizaaceastaprelevatulsufertransformrisuccesive:

1.Fixareaprevinealterareasiautolizaesutului

2.prelucrareahistopatologic(deshidratare,clarificareiimpregnarecuparafin)

3.Includereainblocdeparafina

4.Secionarealamicrotomaprelevatuluiinclusinparafina

5.Etalareaseciunilorpelame(obinereapreparatuluihistopatologicnecolorat)

6.Colorarealamelor

7.Montarealamelorpermiteproteciasiconservareapreparatuluihistopatologiccolorat

Produsfinalpreparatulhistopatologicpermanentcarepermiteinterpretareacorectainvedereastabiliriidiagnosticuluianatomopatologic.

Protocoaleledelucruhistopatologiesuntorientativepotfifolosiireactividiferii,timpuldeaciuneiconcentraiilemenionatenreetesuntorientativeiseadapteaznfunciedecondiiilelocale(temperaturambiental,duritateaipHulapeiderobinetetc).Tehnicafolositesteconsideratcorectdacseobinrezultatelepreconizateprincoloraiarespectiv.Lareeteledecoloraiirecomandatesuntadmisevariaii,nfunciedestructuracaresedoreteevideniatdeasemenea,potfifolositealtecoloraiispecialepentruevideniereaaceleiaistructuri(deexemplucoloraiepentrucolagenRouSirius,coloraiemixtuzualipentrucolagenHematoxilineozinsafran,mucicarminpentrumucopolizaharideacide,auraminpentrubaciliacidalcoolorezisteniialteleasemenea)saucoloraiispecialepentrusubstanecarenupotfiidentificateprinprocedeelerecomandate(deexemplucoloraievonKossapentrucalciu,coloraieScharlach,SudanIII,negruSudansaurouuleiOpentrulipide,coloraiecurodaninpentrudepozitedecupruialteleasemenea).

0miplace



Pentrutehniciledeimunohistochimietimpuldeexpunerelaanticorpiprimariisecundaripoatefimodificatnfunciedediluiaanticorpuluideasemeneametodeledepretratamentpotfimodificatefaderecomandareaproductorului(deexemplufierberentamponcitratnlocdetratamentenzimaticpentrupancitokeratinaAE1AE3)cucondiiaobineriiderezultateconcordantecustructurileevideniatepelamemartor.

PROTOCOLDELUCRU

prelucrareapieseloranatomopatologice(piesechirurgicale,biopsii,fragmentetisularerecoltatelanecropsii)

1.Verificareadocumentelordeinsotireamaterialuluibiopticsevaverificadacapefisadeinsotiresunttrecutecorectnumelesiprenumelepacientului,varsta,numarulfoiideobservatie,sectiacaresolicitaexamenulhistopatologic,diagnosticulclinicprezumptiv,denumireamaterialuluibioptictrimis,datarecoltarii,fixatorulutilizat,semnaturasiparafamediculuicaresolicitadiagnosticulhistopatologic,numelesisemnaturaceluicareaadusmaterialulbioptic/citologic

2.Verificareamaterialuluibioptic.Sevaverificadacamaterialulbioptic/citologicesteinrecipient.Sevaverificadacamaterialulbiopticesteadusfixat(informolsaualtasubstantafixatoare)sauesteproaspat.Sevaverificadacadinpunctdevederecalitativmaterialulbioptic/citologicnuestecorespunzator(dimensiunifoartemicisi/saucuariiintinsedenecroza).sevaverificaconcordanaeidocumentelordeinsotireamaterialuluibiopticrecipientmaterialbioptic

3.Inregistrareacordareadenumar/cazunuicazsepoateacordaunulsaumaimultenumeredacunuicaziseacordunsingurnumrincursulorientriimacroscopiceserecolteazmaimultefragmente,acesteavorfietichetateindividualcunumeresauliteresuplimentare(deexemplunumrcaz1000dincaresepreleveaz3fragmentecunumrul1000/1,1000/2i1000/3sau1000/A,1000/Bi1000/C)nregistrareacazuluisepoatefacenaintesauncursulorientriimacroscopice

4.Orientaremacroscopic/fasonaredescriereapieselor,secionarealorirecoltareadefragmentepentruprelucrarehistopatologicnconcordancuparticularitilefiecruicazorientareamacroscopicsepoatefacefiepepieseproaspete(nefixate),fiedupofixareprealabilde2472ore

5.Fixarenformol10%tamponat,minimum24htimpuldefixarepoatefiredusnfunciedetimpuldefixareprealabil(anterioarorientriimacroscopice)afragmentelortisulare

6.Decalcifieredacserecolteazfragmenteosoasesaucalcificate

7.Prelucrarehistopatologicpebateriemanualsauprocesareautomat(histochinet,histoprocesor,autotehnicon)etapeleprelucrriihistopatologicedifernfunciedetipuldeesutprelucrat,reactiviipreferaiideaparaturaexistentcelmaifrecventsefolosescagenidedeshidratare(alcooletilic,eventualaceton,altesubstaneasemenea),agenideclarificare(alcoolizopropilic,alcoolamilic,benzen,toluen,xilensaualtesubstaneasemenea),impregnarecuparafinperioadadeprocesarevariazntre17ore(procesareautomat)i5zilelucrtoare(procesaremanual)

8.Includereinparafindacseformeazblocurideparafin,acesteavorfifasonateietichetatedacseincludepecasete,casetelevorfifasonateisevaverificaetichetareaacestora



9.Sectionareablocurilordeparafinalamicrotom.

10.Etalareaseciunilorpelame

11.Deparafinareairehidratarealamelorsevaefectuaprintreceriprinbisuccesivedeagentdedeparafinare(benzen,toluen,xilensaualtesubstaneasemenea),alcooletiliciapderobinetnumrulbiloritimpuldeexpunerevariaznfunciededesfurareamanualsauautomataprocesului(minimum25deminuteautomat,1ormanual)

16.Coloraredupreetacoloraieirespective

17.Deshidratare(eventualuscare),clarificare(benzen,toluen,xilensaualtesubstaneasemenea),

18.Montare(cumediudemontare)

19.Etichetare.

PROTOCOLDELUCRU/CITOLOGII

Materialprimit:broaj,punciemamara,puncietiroidiana,lichidascitic,lichidpleural,lichidsinovial,urina,frotiuricervicovaginale.

Frotiugataintins:1.Uscare

2.Fixarealcooletilic96saualcoolmetilic

3.Uscare

4.ColorareGiemsa,HE,Papanicolaou

Recipientcuproduspatologic:

1.Centrifugare

2.Intinderefrotiuri

3.Uscare

4.Fixarealcooletilic96saualcoolmetilic

5.Uscare

6.ColorareGiemsa,HE

COLORAII:sepotfolosireactivipreparaidinpulberisaukiturigatapreparate.Timpiiiconcentraiilemenionatenreetesuntorientativeiseadapteaznfunciedecondiiilelocale(temperaturambiental,duritateaipHulapeiderobinetetc).Coloraiaesteconsideratcorectdacseobinrezultatelepreconizate

1.COLORATIIUZUALE

1.1.Coloratiahematoxilinaeozina

I.Deparafinare



II.HematoxilinMayer25minute

III.Splare

IV.Difereniererapidnalcoolacidclorhidric

V.Splare

VI.Eozin1015secunde

VII.Deshidrataretreibaidealcooletilic(70,96,absolut)

VIII.Clarificare35baitoluen

IX.Montare.

Reactivi:

HematoxilinaMayer:hematoxilina1.5g+alaundepotasiu75g+iodatdesodiu0.3g+apadistilata1litru.

Eozina:eozinagalbena6g+eozinaalbastra6g+orangeG0.4g+alcooletilic70700ml+solutiacarbonatdelitiu80ml+acidaceticglacial3ml.

Alcoolacidclorhdric:99mlalcooletilic70+1mlacidclorhidric1N

Rezultate:nucleiialbastru,citoplasmarosu.

2.COLORATIISPECIALE

2.1ColoratiaVanGieson

I.Deparafinare

II.Hidratare

III.Spalare

IV.ColorarecuhematoxilinWeigert(2prihematoxilin+1partemordant)5minute

V.Spalare

VI.Diferentiererapidainalcoolacidclorhidric.

VII.Spalare

VIII.ContrastarensolutiesaturatadeLi2CO3

IX.Spalare

X.Colorarecupicrofucsin25minute

XI.Spalare

XII.Deshidratare

XIII.Clarificare



XIV.Montare

Reactivi:

HematoxilinWeigert:99mlalcool96oC+1ghematoxilinlatermostattimpde24ore

Mordant:4mlsolutiehexaclorohidratdefiertrivalent29%+1mlacidclorhidric1N+95mlapadistilata

Picrofucsin:100mlacidpicricsolutiesuprasaturata+10mlfucsinacid1%

rezultat:nucleiinegri,citoplasmagalben,colagenrosu

2.2.ColoratiaPeriodicAcidSchiff(PAS)

I.Deparafinare

II.Hidratare

III.Apdistilat

IV.Oxidarecuacidperiodic0,51%510minute

V.Apdistilat

VI.ReactivSchiff515minute

VII.Aprobinet10minute

XI.HematoxilinMayer25minute

XII.Aprobinet

XIII.ContrastarensolutiesaturatadeLi2CO3

XIV.Aprobinet

XV.Deshidratare

XVI.Clarificare

XVII.Montare

Reactivi:

ReactivSchiffseaduc100mlapdistilatlafierbere.Sedizolv0,5gfuxinbazicmojarat(diamantfuxin).Sercetela50oCiseadaug10mlacidclorhidric1N.Sercetela25oCiseadaug0,5gmetabisulfitdenatriusaupotasiu.Seetaneazvasul,senveletenhrtieneagrisepstreaz24hlantuneric(latemperaturacamereisaulafrigiderla4oC).Adouaziseadaug0,51gcrbuneactiv,seagitiselas2minutenrepaus.Sefiltreaz.Sepstreaznsticlebrunelafrigider.Culoareareactivuluitrebuiesfiedelagalbenpailaincolor.

*Vaseletrebuiesafiecltitecuapdistilatiperfectuscatenuselucreaznatmosferdeformol.



Rezultate:nucleialbastru,polizaharidesaucomplexdepolizaharidemagenta,membranebazalerou

2.3ColoratiaGiemsa

I.Deparafinare

II.Hidratare

III.Apdistilat

IV.Giemsa(1parteGiemsaKIT/4priap)2030minute

V.Splarenapdistilatncaresepun78picturideacidaceticglacialla100mlapdistilat

VI.Diferenierecualcool96%(pnpreparatuldevineroz)

VII.Deshidrataresiclarificareinalcoolizopropilic

IX.Clarificarentoluen

X.Montare

Rezultate:nucleiviolet,citoplasmabazofilalbastru,citoplasmaacidofilarou,mastociteviolet,Helicobacterpilorialbastrunchis

2.4ColoratiaPerls

I.Deparafinare

II.Hidratare

III.Apdistilat

IV.Imersiensoluie:sol.ferocianurdepotasiu2%

sol.acidclorhidric2%

(raportsol.=1/1incubaretimpde30minute,5660gradeC)

V.Apdistilat10minute

VI.SupracolorarecuKernechtrot3minutesau

fuxinacid1%1minutsau

rouneutru1%5minute

VII.Splare

VIII.Deshidratare

IX.Clarificare

X.Montare



Reactivi

Sepreparextemporaneu1,5gferocianurdepotasiun75mlapdistilati48mlacidclorhidric1Ncu27mlapdistilat.

Rezultate:depozitedefieralbastru,nucleirosu

2.5.ColoratiaZiehlNielsen

I.Deparafinare/Imersiafrotiuluinapdistilat

I.Carbolfucsin1020minute(incalzirelaintervaledetimpegalepanalaemisiadevapori)

II.Splare

III.Tratarecudecolorantacidalcool3minute

IV.Splare

V.Contracolorarecusolutiealbastrudemetilen1%sauverdelumin3060secunde

VI.Spalare

VII.Deshidratare

VIII.Clarificare

IX.Montare.

Rezultate:bacilialcoolorezistentirosii,fondalbastrusauverde

2.6ColoratiaMasson

I.Deparafinare

II.Apdistilat

III.HematoxilinWeigert710minute

IV.Splarenap,Li2CO35secunde

V.Ponceaufucsin4minute

(2priPonceau1%,1partefuxinacid1%,1parteacidaceticglacial)

VI.Splare(napdistilat)

VI.Acoperirecuacidfosfomolibdenic1%10minute

VII.Anilinorangesauverdelumin5minute(lamanusespalnainte)

VIII.Splare(napdistilat)

IX.Deshidratarerapid(nalcoolabsolut1%)

X.Clarificare



XI.Montare

Reactivi:

Ponceau1%:RouPonceau1gn99mlapdistilat

Fucsinacid1%:1gfucsinacidn99mlapdistilat

Acidfosfomolibdenic1%:1gacidfosfomolibdenicn99mlapdistilat

Anilinorange:0,5ganilinblue,2gorangeG,2,5mlacidaceticglacial,95mlapdistilat

verdelumina:0,1g.verdeluminain100ml.acidacetic1%.

Rezultate:nucleiialbatri(negri),colagen,mucusalbastru/verde,citoplasmaroucrmiziu,eritrociterouportocaliu

2.7.ColoratiaGmri

I.Deparafinare

II.Hidratare

III.Ap

IV.Oxidarecupermanganatdepotasiu1%(sefiltreaz)

V.Splare

VI.Decolorarerapidacumetabisulfitdenatriusaupotasiu2%

VII.Splare

VIII.Mordansarecualaunferiamoniacal2%2minute

IX.Splare

X.Impregnarecusoluieargentoamoniacal(Tollens)

XI.Apdistilat2birapide

XII.Reducereformolneutru10%

XIII.Ap

XIV.Reducerecumetabisulfitdenatriu/potasiu2%1minutfacultativ

XV.Ap

XVI.Fixaretiosulfatdenatriu1%1minut

XVII.Splare

XVIII.Deshidratare

XIX.Clarificare



XX.Montare

Reactivi:

Soluiaextemporaneuargentoamoniacal(Tollens):secntresc500mgAgNO3+5mlapdistilat+1mlhidroxiddenatriu10%+sol.amoniac(picturcupictur)pnladizolvareacompletaprecipitatului.Sedubleazvolumulsoluieicuapdistilat.

Rezultate:fibreledereticulinanegrubrun

2.8.ColoratiaRosudeCongo

I.Deparafinare

II.Hidratare

III.Splare

IV.ColorarecusoluieRoudeCongo0,5%1030minute

V.Splarenapdistilat

VI.DiferenierensoluieKOH/NaOH0,2%

VII.Splareapdistilat

VIII.Hematoxilin25minute

IX.Splare

X.ContrastarensoluiesuprasaturatdeLi2CO3

XI.Splare

XII.Deshidratare

XIII.Clarificare

XIV.Montare

Reactivi:

SoluieRoudeCongo0,5%:0,5gRoudeCongon100mlalcooletilic50%

Rezultate:nucleialbatri,amiloidroucrmiziu(birefringenverdenluminpolarizat)

2.9.ColoratiaVanGiesonelastic

I.Deparafinare

II.Hidratare

III.Imersierapidainalcooletilic96o

IV.Imersiesolrezorcinfucsin(30minute1h)



V.Splareapdistilat

VI.Difereniere(alcool96o)

VII.ColoraiaVanGieson

Rezultate:fibreelasticenegru,nucleinegru,colagenrou,muschigalben

2.10ColoratiaAlbastruAlcian

I.Deparafinare

II.Hidratare

III.SolutieAlbastrualcian20minute

IV.Spalare

V.ContracolorarecuHematoxilinaMayer25minute

VI.Spalare

VII.Diferentiererapidacualcoolacidclorhidric

VIII.Spalare

IX.Deshidratare

X.Clarificare

XI.Montare

Reactivi:

SolutieAlbastrualcianpH1:Albastrualcian8GX1g+99mlapadistilata+acidclorhidric1NpanalapH1.

SolutieAlbastrualcianpH2,5:Albastrualcian8GX1g+50mlalcooletilicabsolut+50mlapadistilata+acidaceticglacialpanalapH2,5

Rezultate:nucleialbastru,polizaharideacidealbastru

2.11.ColoratiaPapanicolaou

I.Hidratare

II.Spalare

III.HematoxilinaHarris15minute

IV.Spalare

V.Diferentiereacidclorhidric0,25%(1020secunde)

VI.Spalare

VII.OrangeG30secunde4minute



VIII.Alcooletilic96o2minute

IX.EA50sauEA51115minute

X.Alcool96odouabaide3,respective2minute

XI.Alcooletilicabsolut2minute

XII.Clarificare

XIII.Montare

2.12.ColoratiaMassonFontana

I.Deparafinare

II.Hidratare.

III.Spalare

IV.Imersietimpde824hlaintunericinsolutieargentoamoniacala

V.Spalare

VI.Fixarecutiosulfatdesodiu0,51%5060secunde

VII.Spalare.

VIII.Contracolorarecusolutiefucsinaacida0,1%3060secunde

`IX.Uscare

X.Clarificare

XI.Montare

Reactivi:solutieargentoamoniacala:5mlsolutieazotatdeargint10%+hidroxiddeamoniu25%panaladisparitiaprecipitatuluideoxiddeargint+azotatdeargint10%panalaopalescenta.Seaducela50mlcuapadistilata.

Rezultate:pigmentmelanic,hemosiderinnegru

2.13.ColoratiaGram

I.Deparafinare

II.Hidratare.

III.Violetdegentiana1%1minut.

IV.Spalare

V.SolutieLugol2minute

VI.Decolorarecualcoolacetona.

VII.Contracolorarecusolutiefucsinabazica0,1%1minut



VIII.Uscare

IX.Clarificare

X.Montare

Reactivi:Kitdecolorare.

Preparare:Violetdegentiana1g.+100ml.apadistilata

SolutieLugol:ioduradepotasiu1g+iod1g.+300ml.apadistilata

solutiefucsinabazica:carbofuxina1ml.+alcoolabsolut10ml.+apadistilata50ml.

Rezultate:bacteriigramnegativerosu,bacteriigrampozitivealbastru

PROTOCOLDELUCRUEXAMENULEXTEMPORANEU

1.Severificdocumenteledeinsotireafragmentuluitisularifragmentultisularsimilarprotocoluluidelucrupentruprelucrarehistopatologic.nplusseverificadacafragmentultisularestededimensiunisuficienteexamenuluiextemporaneu

2.senregistreazcazul

3.seorienteazmacroscopicpiesasedimensioneazasisedescriefragmentultisularsepreleveazafragmentetisularepentruexamenulextemporaneu

4.secongeleazfragmentuluitisularsemonteazafragmentultisularpesuportulmetalicisengheancriostatlatemperatursubminus40Cpndevineperfectopac

5.sesecioneazlacriomicrotomseniveleazablocultisularngheat,efectuandusesectiunila210

6.Sectiunileobtinuteseetaleazpelamedesticlacuajutorulunuiportacsaudirectcuajutorulplceiantirol

7.Lamelecumaterialhistopatologicsectionatlagheatasecoloreazacuhematoxilineozinsausolutiedealbastrudetoluidina(coloratiiuzuale)sauScharlach(coloratiespecialapentrulipide).

8.Semonteazanap,glicerinsaumediidemontaresolubilensolveniorganici

PROTOCOLDELUCRUIMUNOHISTOCHIMIE

(METODATRISTADIALINDIRECT,EVIDENIERECUDAB)

1.deparafinare

2.splarecutamponTBSsauPBS,pH7.4,1020min(23bi)

3.pretratamentenfunciedespecificaiilefiecruianticorpprimar.


5.inhibareaperoxidazeiendogene(HydrogenPeroxide3%),timpde15min

ordinul nr. 1217

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