Vpliv priprave in fiksacije citoloških vzorcev na imunocitokemično dokazovanje diagnostičnih in napovednih dejavnikov malignih tumorjev

Srebotnik Kirbiš, Irena (author), Strojan Fležar, Margareta (mentor)

Ozadje: Za imunocitokemično (ICK) dokazovanje diagnostičnih in napovednih dejavnikov na citoloških vzorcih smo razvili enostaven in učinkovit postopek priprave preparatov, pri katerem citološke vzorce suspendiramo v celičnem mediju in s citocentrifugo pripravimo potrebno število dodatnih preparatov, ki jih fiksiramo in do uporabe shranimo v metanolu. Namen raziskave: Ugotoviti, ali postopek priprave citoloških vzorcev za ICK, ki smo ga razvili, dobro ohrani imunoreaktivnost antigenov in zagotavlja zanesljivo ICK-dokazovanje diagnostičnih in napovednih dejavnikov. Materiali in metode: Vse vzorce za ICK smo suspendirali v celičnem mediju in pripravili citospine. Na humani celični liniji raka dojke (MFC-7) smo najprej potrdili, da fiksacija in shranjevanje citospinov v metanolu, dobro ohrani imunoreaktivnost estrogenskih receptorjev (ER) in določili pogoje ICK-reakcije, nato smo to potrdili še na vzorcih aspiracijskih biopsij (ABTI) raka dojke. Na različnih citoloških vzorcih smo ugotavljali, ali fiksacija tako pripravljenih citospinov v metanolu dobro ohrani tudi imunoreaktivnost označevalcev pripadnosti (CD3, CD10, CD20, CD30, CD31, CD45, CD56, CD68, CD117, CD138), citokeratinov (MNF116, AE1/AE1, CK 7, CK 19, CK 20), drugih intermediarnih filamentov (dezmina, gladkomišičnega aktina, vimentina) in še 20 drugih diagnostično pomembnih označevalcev (α1-fetoproteina, hepatocitnega antigena, HMB45, kalcitonina, kalretinina, kromogranina, Ki67 antigena, MelanA, MOC31, p53 in p16, P504S, PSA, progesteronskih receptorjev, RCC, S100, sinaptofizina, tiroglobulina, TTF1, WT-1). Rezultate ICK-reakcij na citospinih, pripravljenih iz diagnostičnih citoloških vzorcev in citoloških vzorcev, pripravljenih iz svežih histoloških vzorcev, smo primerjali z rezultati imunohistokemičnih (IHK) reakcij na odgovarjajočih histoloških vzorcih, fiksiranih v formalinu in vklopljenih v parafin (FFVP). Za ugotavljanje razlik v deležu ER pozitivnih celic glede na način fiksacije smo uporabili Duncanov test za zvezne spremenljivke, za analizo skladnosti ICK- in IHK-reakcij pa Cohenov kapa koeficient (κ). Rezultati: Na citospinih, fiksiranih v metanolu, smo ugotovili najvišji delež ER pozitivnih celic MCF-7 z najnižjo variabilnostjo (72 ± 5 %) in popolno skladnost rezultatov določanja ER na citoloških in odgovarjajočih FFVP-vzorcih (52/52, 100 %, κ = 1,00). Fiksacija v metanolu je dobro ohranila tudi imunoreaktivnost vseh drugih 38 testiranih antigenov. Skladnost ICK-rezultatov na citospinih pripravljenih iz diagnostičnih citoloških vzorcev in svežih histoloških vzorcev, z IHK-rezultati na FFVP-vzorcih, je bila v obeh skupinah vzorcev zelo visoka (141/146, 97 %, κ = 0,93 in 81/86, 94 %, κ = 0,88). Ugotovitve: Postopek priprave citoloških vzorcev za ICK, ki smo ga uporabili v naši raziskavi, je enostaven, učinkovit in zagotavlja zanesljive ICK-rezultate.

• Language: Slovenian
• Keywords: citologija, imunocitokemija, imunohistokemija, citološki vzorci, priprava preparatov, fiksacija, imunoreaktivnost, estrogenski receptorji
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2018
• PID: 20.500.12556/RUL-102359
• COBISS.SI-ID: 296347136
• Publication date in RUL: 23.08.2018

Secondary language

• Language: English
• Title: The influence of processing and fixation of cytology samples on immunocytochemical detection of diagnostic and prognostic factors in malignant tumours

Abstract

Background: A simple and efficient slide preparation procedure was developed for immunocytochemical (ICC) detection of diagnostic and prognostic factors on cytological samples where required numbers of cytospins fixed and stored in methanol were prepared from samples suspended in a cell medium. Aim: To determine the reliability of ICC detection of different diagnostic and prognostic markers on cytological samples processed according to our slide preparation procedure. Methods: Cytospins for all ICC reactions were prepared from samples suspended in buffered based cell medium. Preservation of immunoreactivity after fixation and storage of cytospins in methanol as well as optimal conditions for reliable ICC detection were first determined for estrogen receptors (ER) on a human breast cancer cell line (MCF-7) and then confirmed on fine needle aspiration biopsies (FNAB) of breast cancer. The same fixation and storage procedure was then evaluated on cytospins prepared from different cytological samples for CD markers (CD3, CD10, CD20, CD30, CD31, CD45, CD56, CD68, CD117, CD138), cytokeratins (MNF116, AE1/AE1, CK 7, CK 19, CK 20), intermediate filaments (desmin, smooth muscle actin, vimentin) and 20 other diagnostic markers (α1-fetoprotein, calcitonin, calretinin, chromogranin, HMB-45, HCC, Ki67, MelanA, MOC31, p53 in p16, progesterone receptors, P504S, PSA, RCC, S100, synaptophysin, thyroglobulin, TTF-1, WT-1). The reliability of ICC reactions was determined evaluating the concordance of ICC reactions between cytospins prepared from diagnostic cytological samples, cytological samples prepared from fresh biopsies, and immunohistochemical (IHC) reactions on corresponding formalin fixed paraffin embedded (FFPE) tissue sections. Differences in percentages of ER positive MCF-7 cells after different fixation and storage procedures were analysed with Duncan test, while Cohen kappa test (κ) was used for analysis of concordance between ICC and IHC reactions. Results: The highest percentage of ER positive MCF-7 cells with the lowest variability (72 ± 5 %) and complete concordance between ICC and IHC assessment of ER on cytological and corresponding FFPE samples (52/52, 100 %, κ = 1,00) was found on cytospins fixed and stored in methanol. Fixation of cytospins in methanol also preserved immunoreactivity of all other 38 markers evaluated. High concordance between ICC results on methanol fixed cytospins prepared from diagnostic cytological samples and cytological samples prepared from fresh biopsies with IHC results on corresponding FFPE tissue sections was found in both tested groups (141/146, 97 %, κ = 0,93 in 81/86, 94 %, κ = 0,88). Conclusion: The cytological sample processing procedure for ICC evaluated in this study is simple, efficient and yield reliable results.

• Keywords: cytology, immunocytochemistry, immunohistochemistry, cytological samples, slide preparation, fixation, immunoreactivity, estrogen receptors