FLEX Monoclonal Mouse Anti-Human Progesterone Receptor Clone PgR 636 Ready-to-Use (Link) Koda IR068. Predvidena uporaba
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1 FLEX Monoclonal Mouse Anti-Human Progesterone Receptor Ready-to-Use (Link) Koda IR068 Predvidena uporaba Druga poimenovanja antigena Povzetek in razlaga Za diagnostično uporabo in vitro. FLEX Monoclonal Mouse Anti-Human Progesterone Receptor,, Ready-to-Use (Link), je namenjen za uporabo v imunohistokemiji skupaj z vizualizacijskim kompletom EnVision FLEX+, High ph in instrumenti Autostainer Link pri polkvantitativnem zaznavanju človeških progesteronskih receptorjev v humanem karcinomu dojk, fiksiranem v formalinu in vklopljenem v parafin. To protitelo označi pozitivne progesteronske receptorske celice in se uporablja pri oceni stanja progesteronskih receptorjev v humanih karcinomih dojk. Specialistična interpretacija vsakršnega obarvanja oziroma njegove odsotnosti mora biti podprta z morfološkimi analizami ob izvajanju primernih kontrol in mora biti ovrednotena v okviru pacientove anamneze ter drugih diagnostičnih testov, ki jih opravi usposobljen patolog. PR Vloga receptorjev steroidnih hormonov pri raku dojke je dobro znana (1, 2). Odsotnost estrogenskih receptorjev (ER) in progesteronskih receptorjev (PR) pri pacientkah z rakom dojke napoveduje zgodnjo ponovitev in manjšo verjetnost preživetja (3 6). Prisotnost ER in PR v tumorjih pa napoveduje pozitivne učinke zdravljenja s tamoksifenom in drugih terapij, ki delujejo na endokrini sistem. Vrednosti ER in PR je mogoče določati polkvantitativno z imunohistokemičnimi metodami ali kvantitativno s testom DCC (z dekstranom, vezanim na aktivno oglje) ali encimsko imunskimi testi. Korelacija med polkvantitativnimi in kvantitativnimi ocenami vrednosti PR je med 73 in 91 %, odvisno od laboratorija in uporabljenih protiteles (7 9). Glejte General Instructions for Immunohistochemical Staining (Splošna navodila za barvanje v imunohistokemiji) družbe Dako ali navodila sistema za zaznavanje v postopkih IHC, kjer so navedene informacije glede: 1) načela postopka, 2) potrebnih materialov, ki niso priloženi, 3) shranjevanja, 4) priprave vzorca, 5) postopka barvanja, 6) nadzora kakovosti, 7) odprave napak, 8) interpretacije obarvanja in 9) splošnih omejitev. Priloženi reagent Za uporabo pripravljena mišja protitelesa v tekoči obliki v pufru s stabilizacijskim proteinom in NaN 3 s koncentracijo 0,015 mol/l. Klon: PgR 636 (10) Izotip: IgG1, kapa Imunogen V formalinu fiksiran rekombinantni receptor polnega človeškega progesterona A (10). Specifičnost Prikazano je bilo, da Anti-PR, PgR 636 pri prenosu Western celotnih celičnih ekstraktov reagira z oblikama PR-A in PR-B ter da reagira tako s prostim in hormonsko vezanim PR (10). Epitop je bil preslikan na aminoterminalno domeno, ki je skupna PR-A in PR-B. Previdnostni ukrepi 1. Za diagnostično uporabo in vitro. 2. Za profesionalno uporabo. 3. Izdelek vsebuje natrijev azid (NaN 3), kemikalijo, ki je v čisti obliki zelo toksična. Čeprav niso klasificirane kot nevarne, lahko natrijev azid v koncentracijah v izdelku reagira s svinčenimi in bakrenimi cevmi, pri čemer se tvorijo zelo eksplozivni kovinski azidi. Pred odstranjevanjem sperite z obilico vode, da preprečite nalaganje kovinskih azidov v ceveh. 4. Kot pri vseh izdelkih, pridobljenih iz biološkega porekla, je treba slediti pravilnim postopkom za ravnanje. 5. Nosite primerno osebno zaščitno opremo, da preprečite stik z očmi in kožo. 6. Neuporabljeno raztopino je treba zavreči skladno z lokalnimi, nacionalnimi in zveznimi predpisi. Shranjevanje Shranjujte pri temperaturi 2 8 C. Ne uporabljajte po izteku roka uporabnosti, ki je odtisnjen na viali. Če se reagenti shranjujejo pri pogojih, ki so drugačni od navedenih, mora pogoje preveriti uporabnik sam. Ni očitnih znakov za nestabilnost tega izdelka. Zato je treba skupaj z vzorci pacientov opravljati tudi pozitivne in negativne kontrole. Če opazite nepričakovano obarvanje, ki ga ni mogoče razložiti s spremembami laboratorijskih postopkov, in če sumite na težave s protitelesom, se obrnite na tehnično podporo družbe Dako. ( ) SL_004 str. 1/5
2 Priprava vzorca, vključno s potrebnim materialom, ki ni priložen Postopek barvanja vključno s potrebnim materialom, ki ni priložen Interpretacija barvanja Omejitve, specifične za izdelek Značilnosti delovanja Parafinske rezine: protitelesa se lahko uporabljajo za označevanje tkivnih rezin, fiksiranih v formalinu in vklopljenih v parafin. Vzorci tkiva morajo biti razrezani na debelino približno 4 µm. Predobdelava: tkivne rezine, ki so fiksirane v formalinu in vklopljene v parafin, je treba predobdelati. Rezultati bodo optimalni, če za predobdelavo tkiv s postopkom HIER uporabite razredčeno raztopino EnVision FLEX Target Retrieval Solution, High ph (50x) (kodi K8000/K8004). Za deparafinizacijo, rehidracijo in razkrivanje epitopov lahko uporabite instrument Dako PT Link (koda PT100/PT101/PT200). Podrobnosti najdete v uporabniškem priročniku instrumenta PT Link. Za PT Link uporabite naslednje parametre: temperatura predogrevanja: 65 C; temperatura in trajanje za razkrivanje epitopov: 97 C za 20 minut (±1); ohlajanje na 65 C. Okvir s p reparati vzemite iz rezervoarja PT in stekla nemudoma potopite v posodo/rezervoar (npr. PT Link Rinse Station (koda PT109)) z razredčeno raztopino EnVision FLEX Wash Buffer (20x) (koda K8007) na sobni temperaturi. Stekelca v raztopini Wash Buffer pustite 1 5 minut. Tkivne rezine se med predobdelavo ali poznejšim imunohistokemičnim barvanjem ne smejo posušiti. Za močnejši oprijem tkivnih rezin na objektna stekla priporočamo stekla FLEX IHC Microscope Slides (koda K8020). Po barvanju morajo biti rezine dehidrirane, prečiščene in pripravljene z medijem za trajno pripravo preparata. Vizualizacija: priporočeni sistem za vizualizacijo je EnVision FLEX+, High ph (Link) (koda K8002). Program: koraki barvanja in inkubacijski časi so že programirani v programski opremi Autostainer Link. Priporočeni volumen reagenta je 1 x 200 µl ali 2 x 150 µl na steklo. Podrobna navodila o vlaganju preparatov in reagentov najdete v ustreznem uporabniškem priročniku sistema Autostainer Link. Če protokolov ni mogoče najti na uporabljeni platformi Autostainer Link 48, se obrnite na Dako Technical Services. Vse inkubacijske korake je treba opravljati na sobni temperaturi. Nasprotno barvanje: priporočeno nasprotno barvilo je EnVision FLEX Hematoxylin (Link) (koda K8008). Priporoča se brezvodni medij za trajno pripravo preparata. Kontrole: pozitivna in negativna kontrola morata potekati hkrati in po enakem protokolu kot vzorci pacientov. V idealnem primeru bi morala pozitivna kontrola vključevati tkivo karcinoma dojk, ki izraža nizko vrednost PR. Kot nadomestna možnost se lahko uporabi tudi benigni maternični vrat. Pri celicah/strukturah morajo biti v vseh pozitivnih vzorcih razvidni enaki reakcijski vzorci, kot so za to tkivo opisani v razdelku "Značilnosti delovanja". Priporočeni negativni kontrolni reagent je FLEX Negative Control, Mouse (Link) (koda IR750). Vzorec celičnega obarvanja je. Če se opazi citoplazemsko obarvanje, je treba to šteti kot nespecifično. Rezultat je pozitiven, če ima najmanj 1 % tumorskih celic obarvana jedra ne glede na intenzivnost barvanja. To je skladno s priporočenimi mejnimi rezultati ASCO/CAP v višini najmanj 1 % pozitivnih tumorskih celic za pozitivno oceno (11). 1. Zaradi postopne degradacije antigena v tkivih lahko pride do lažnih negativnih rezultatov. Vzorce je treba obarvati v dveh mesecih od prepariranja tkiv, če so bili shranjeni pri sobni temperaturi (12). 2. Zaradi reagenta FLEX Monoclonal Mouse Anti-Human PR lahko pride do obarvanja pri limfocitih in stromalnih celicah, pri čemer se to ne sme razlagati ali šteti kot obarvanje PR pri tumorskih celicah. 3. Za optimalne in obnovljive rezultate je za protein PR potrebna vrnitev cilja, če so tkiva rutinsko fiksirana (nevtralno puferiran formalin) in vklopljena v parafin. 4. Uporaba reagenta Dako Anti-PR, na tkivih z drugimi fiksativi namesto formalina ni odobrena. Natančnost: za testiranje so bile odvzete zaporedne rezine iz 12 različnih blokov s karcinomom dojk, fiksiranih v formalinu in vklopljenih v parafinu, kar je predstavljalo dinamičen razpon izražanja PR. Testiranje je potekalo, kot je opisano v nadaljevanju: Natančnost znotraj serije: v skladu s standardnim protokolom EnVision FLEX+, High ph so bile tri rezine iz vsakega tkivnega bloka obarvane z reagentom Anti-PR,. Hkrati je bila ena rezina iz vsakega bloka obarvana z negativnim kontrolnim reagentom. Natančnost med serijami: zgornji postopek je bil ponovljen v petih nezaporednih dneh, pri čemer je bila obarvana ena rezina iz vsakega bloka. Hkrati je bila ena rezina iz vsakega tkivnega bloka obarvana z negativnim kontrolnim reagentom. Natančnost med instrumenti: zgornji postopek so izvedli trije različni upravljavci s tremi različnimi instrumenti Autostainer, pri čemer so bile skupno obarvane tri rezine iz vsakega tkivnega bloka. Hkrati je bila ena rezina iz vsakega tkivnega bloka obarvana z negativnim kontrolnim reagentom. Testi natančnosti z reagentom Anti-PR, so pokazali skladnost rezultatov znotraj serije, med serijami in med instrumenti. Testni pogoji so se ohranjali v celotnem poteku raziskave, reagenti pa so bili med opravljanjem testov shranjeni pri temperaturah od 2 do 8 C. Normalna tkiva: tabela 1 vsebuje povzetek imunoreaktivnosti priporočene skupine normalnih tkiv z reagentom Anti-PR,. Vsa tkiva so bila fiksirana v formalinu in vklopljena v parafin ter obarvana z reagentom Anti- PR, v skladu s priloženimi navodili. Citoplazemsko obarvanje z reagentom Anti-PR, Clone PgR 636 je bilo opaženo pri več različnih elementih tkiva, vključno z epitelijem, stromo, intersticijskimi celicami in vnetnimi celicami. Čeprav je bilo opaženo citoplazemsko obarvanje, se to ne šteje kot diagnostično glede na predvideno uporabo tega protitelesa. ( ) SL_004 str. 2/5
3 Tabela 1: Povzetek reaktivnosti normalnega tkiva z reagentom Anti-PR, Vrsta tkiva Pozitivni elementi tkiva Vrsta tkiva Pozitivni elementi tkiva (št. testov) (št. testov) Nadledvična žleza (3) 1/3 celice v glomerulozni coni Živci, periferni (3) 0/3 (50 %), 1/3 celice v glomerulozni coni (50 %), Jajčnik (3) 3/3 stromalne celice (50 70 %), Kostni mozeg (3) 0/3 Trebušna slinavka (3) 2/3 Langerhansovi otočki (50 90 %), Dojka (3) 2/3 žlezne epitelijske celice (50 90 %), Obščitnice (3) 3/3 žlezne epitelijske celice (1 10 %), Mali možgani (3) 0/3 Hipofiza (3) 3/3 hipofizne žlezne celice (1 40 %), Veliki možgani (3) 1/3 celice možganske ovojnice (100 %), Prostata (3) 3/3 stromalne celice (30 80 %), Maternični vrat (3) 3/3 epitelijske celice (50 90 %), Žleza slinavka (3) 3/3 žlezne epitelijske celice (< 1 60 %), 3/3 stroma, vključno z vnetnimi Koža (3) 0/3 celicami (50 %), Kolon (3) 1/3 limfoidne/vnetne celice (10 %), Tanko črevo (3) 3/3 stromalne in vnetne celice (30 50 %), 1/3 limfoidne/vnetne celice (10 %), Vranica (3) 0/3 Požiralnik (3) 1/3 stromalne celice (50 %), Želodec (3) 1/3 intersticijske celice (20 %), Ledvice (3) 3/3 intersticijske celice (1 5 %), Moda (3) 3/3 intersticijske celice (5 80 %), Jetra (3) 0/3 Timus (3) 0/3 Pljuča (3) 2/3 intersticijske celice (1 10 %), Ščitnica (3) 0/3 2/3 vnetne celice (1 10 %), Tonzila (3) 0/3 Mezotelijske celice (2) 0/2 Maternica (2) 2/2 žlezne epitelijske celice (100 %), Mišica, srčna (3) 3/3 miociti (30 %), perinuklearni 2/2 miometrijske stromalne celice (100 %), Mišica, skeletna (3) 0/3 Primerjava metod: testiranje z reagentom Monoclonal Mouse Anti-Human PR, je bilo opravljeno s kompletom EnVision FLEX+, preštevanje pa je bilo opravljeno v skladu s smernicami ASCO/CAP (mejni rezultati v višini najmanj 1 %) (11). Testiranje z reagentom Anti-PR (Clone 1294) je bilo opravljeno s kompletom Dako ER/PR pharmdx, preštevanje pa je bilo opravljeno v skladu s smernicami preštevanja, opisanih v priloženih navodilih. Podatki o primerjavi metod so prikazani v tabeli 2. Na podlagi teh smernic preštevanja se je reagent Monoclonal Mouse Anti-Human PR, ujemal s komponento Anti-PR kompleta Dako ER/PR pharmdx, pri čemer je izražal vrednosti za skupno, pozitivno in negativno ujemanje v višini 94,5 %, 95,8 % oziroma 93,1 %. Tabela 3 prikazuje primerjavo obeh testov pri preštevanju v skladu s smernicami ASCO/CAP. Tabela 2: Ujemanje med reagentom Anti-PR, (ASCO/CAP) in komponento Anti-PR kompleta ER/PR pharmdx (Allred) Komponenta Anti-PR kompleta ER/PR pharmdx Kit Monoclonal Mouse Anti- Human PR, pozitivno negativno skupaj Odstotek pozitivnega ujemanja = 95,8 % (95 % CI: 91,1 96,8) Odstotek negativnega ujemanja = 93,1 % (95 % CI: 90,5 96,7) Odstotek skupnega ujemanja = 94,5 % (95 % CI: 90,8 96,8) ( ) SL_004 str. 3/5
4 Tabela 3: Ujemanje med reagentom Anti-PR, (ASCO/CAP) in komponento Anti-PR kompleta ER/PR pharmdx (ASCO/CAP) Komponenta Anti-PR kompleta ER/PR pharmdx Kit Monoclonal Mouse Anti- Human PR, pozitivno negativno skupaj Odstotek pozitivnega ujemanja = 99,1 % (95 % CI: 93,0 98,0) Odstotek negativnega ujemanja = 93,3 % (95 % CI: 92,8 98,0) Odstotek skupnega ujemanja = 96,2 % (95 % CI: 93,0 98,0) Obnovljivost med laboratoriji: testiranje obnovljivosti reagenta Anti-PR, je bilo opravljeno v treh testnih laboratorijih v petih nezaporednih dneh na 21 edinstvenih vzorcih raka dojke, preštevanje pa je bilo opravljeno za skupno 315 ocen v skladu s smernicami ASCO/CAP (mejni rezultati v višini najmanj 1 %). V tabelah 4, 5 in 6 je podrobno prikazana obnovljivost testa med laboratoriji. Izračuni povprečnega odstotka pozitivnega, negativnega in skupnega ujemanja podpirajo visoko stopnjo obnovljivosti rezultatov testa PR (PgR 636) za določanje stanja PR v kliničnem okolju. Tabela 4: Primerjava obnovljivosti reagenta Anti-PR, med laboratorijem 1 in laboratorijem 2 Laboratorij 2 Laboratorij 1 pozitivno negativno skupaj Povprečni odstotek pozitivnega ujemanja = 96,5 % Povprečni odstotek negativnega ujemanja = 95,8 % Tabela 5: Primerjava obnovljivosti reagenta Anti-PR, med laboratorijem 1 in laboratorijem 3 Laboratorij 3 Laboratorij 1 pozitivno negativno skupaj Povprečni odstotek pozitivnega ujemanja = 99,2 % Povprečni odstotek negativnega ujemanja = 98,9 % Tabela 6: Primerjava obnovljivosti reagenta Anti-PR, med laboratorijem 2 in laboratorijem 3 Laboratorij 3 Laboratorij 2 pozitivno negativno skupaj Povprečni odstotek pozitivnega ujemanja = 95,7 % Povprečni odstotek negativnega ujemanja = 94,7 % ( ) SL_004 str. 4/5
5 Reference 1. Henderson C. Breast cancer. V: Harrison s principles of internal medicine, 12. izdaja, Wilson JD, Braunwald E, Isselbacher KJ, Petersdorf RG, Martin JB, Fauci AS, Root RK (ur.). McGraw-Hill, Inc. New York, Fuqua SAW. Estrogen and progesterone receptors and breast cancer. Diseases of the Breast, Harris et al, ur. Lippincott-Raven, Str McGuire WL, Clark GM. The prognostic role of progesterone receptors in human breast cancer. Sem Oncol 1983;10:2. 4. Clark GM, McGuire WL, Hubay CA, Pearson OH, Marshall JS. Progesterone receptors as prognostic factor in stage II breast cancer. N Engl J Med 1983;39: Ravdin PM, Green S, Dorr TM, McGuire WL, Fabian C, Puch RP, et al. Prognostic significance of progesterone receptor levels in estrogen receptor-positive patients with metastatic breast cancer treated with tamoxifen: Results of a prospective southwest oncology group study. JCO 1992;10: Chevallier B, Heintzmann F, Mosseri V, Dauce JP, Bastit P, Graic Y, Brunelle P, Basuyay JP, Comoz M, Asselain B. Prognostic value of estrogen and progesterone receptors in operable breast cancer: Results of a univariate and multivariate analysis. Cancer 1988; 62: Page DL, Jensen RA, Simpson JF. Routinely available indicators of prognosis in breast cancer. Breast Can Res Treat 1998;51: Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998;11: Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, et al. Prognostic factors in breast cancer. College of American Pathologists consensus statement. Arch Pathol Lab Med 2000;124: Press M, Spaulding B, Groshen S, Kaminsky D, Hagerty M, Sherman L, et al. Comparison of different antibodies for detection of progesterone receptor in breast cancer. Steroids 2002; 67: Hammond MEH, Hayes DF, Dowsett M, Allred C, Hagerty KL, Badve S, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer. Arch Pathol Lab Med 2010;134: Clinical and Laboratory Standards Institute (nekdaj NCCLS). Quality assurance for immunocytochemistry; Approved guideline. CLSI document MM4-A ( )- CLSI, 940 West Valley Road, Suite Wayne, PA USA Katalo ka tevilka Temperaturne omejitve Proizvajalec Koda serije Dako North America, Inc Via Real Carpinteria, California USA Dako Denmark A/S Produktionsvej 42 DK-2600 Glostrup Denmark Rok uporabe PT0020/ Rev C Glejte navodila za uporabo Tel Fax Technical Support Customer Service Tel Fax Izdaja 11/15 ( ) SL_004 str. 5/5
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