Optimizacija protokolov imunocitokemičnih reakcij za vzorce, shranjene v različnih transportnih medijih in pripravljene s kompletom BD Totalys SlidePrep

Cerar, Špela (author), Kloboves Prevodnik, Veronika (mentor)

Z imunocitokemično (ICK) reakcijo s pomočjo primarnih protiteles določimo antigene (Ag) v vzorcih, odvzetih z aspiracijsko biopsijo s tanko iglo (ABTI), v izlivih, tkivnih vzorcih dezintegriranih bezgavk in drugih citoloških vzorcih. Za dodatna ICK barvanja vzorce lahko pripravimo s citocentrifugo (citospini) ali z enim od aparatov za pripravo tekočinskih citoloških vzorcev. Vzorce, ki jih pripravljamo z aparatom za pripravo tekočinskih citoloških vzorcev, pred pripravo fiksiramo v posebnem transportnem mediju. Z nalogo smo želeli primerjati učinkovitost ICK barvanja citospinov in preparatov, pripravljenih iz citoloških vzorcev, shranjenih v različnih transportnih medijih. Za pripravo slednjih smo uporabili aparat BD Totalys SlidePrep, testirali pa smo 3 transportne medije (CytoRich Red, CytoRich Blue in SurePath). V prvem delu naloge smo na celični liniji MCF-7 testirali rezultate ICK barvanj dveh označevalcev; estrogenski receptor (ER) in CK AE1/AE. Na podlagi rezultatov smo se za nadaljevanje naloge odločili za transportni medij CytoRich Red. V drugem delu naloge smo na 10 citoloških vzorcih z ICK metodo določili izraženost 8 označevalcev (ER, CK AE1/AE3, kalretinin, MOC-31, LCA (CD45), TTF-1, MM koktejl, p40). Rezultate ICK barvanj smo ocenili štirje ocenjevalci. Ocenjevali smo reakcijo, intenziteto barvanja, morfologijo celic, kontrastiranje in ozadje. Primerjali smo ohranjenost morfologije in rezultate ICK reakcij na citospinih (C), in na z BD Totalys SlidePrep pripravljenih preparatih (BD). Statistično značilne razlike v ICK reakcijah med C in BD v večini primerov nismo dokazali, zato sklepamo, da so vsi postopki primerni za rutinsko ICK dokazovanje Ag.

• Language: Slovenian
• Keywords: citologija, imunocitokemija, tekočinska citologija, optimizacija protokolov, celični označevalci
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2021
• PID: 20.500.12556/RUL-130177
• COBISS.SI-ID: 76999939
• Publication date in RUL: 11.09.2021

Secondary language

• Language: English
• Title: Optimization of immunocytochemistry reaction protocols for samples stored in different fixation media and processed by BD Totalys SlidePrep

Abstract

Immunocytochemical (ICK) reactions are used for detection of cellular antigens with specific primary antybodies on fine needle aspiration biopsy (FNA) samples, effusions, disintegrated lymph nodes tissue samples and other cytological samples. Samples for additional ICK staining can be prepared by cytocentrifuge (cytospins) or by liquid-based cytology (LBC) specimen slide processors. The aim of the study was to compare the efficiency of ICK staining on cytospins and slides prepared from LBC samples stored in three different transport media: CytoRich Red, CytoRich Blue and SurePath, and prepared by BD Totalys SlidePrep slide processor. In the first part of the study, we tested the ICK determination of estrogen receptor (ER) and cytokeratin AE1/AE on the MCF-7 cell line cytospins and LBC slides. Based on the results, we choose CytoRich Red transport medium for further experiments. In the second part of the study, the expression of 8 biomarkers (ER, CK AE1/AE3, calretinin, MOC-31, LCA (CD45), TTF-1, MM cocktail, p40) was determined immunocytochemically on 10 cytological samples . The results of ICK staining were evaluated by four evaluators. Reaction (percentage of stained cells), staining intensity, cell morphology, contrast, and nonspecific background were evaluated. Comparison of results of ICK reactions on cytospins (C) and on BD Totalys SlidePrep-prepared preparations (BD) was performed. We did not prove statistically significant differences in ICK reactions between C and BD in most cases, so we conclude that LBC sampes stored in CytoRich Red and prepared by BD Totalys Slide Prep slide processor are suitable for routine ICK staining.

• Keywords: citology, immunocytochemistry, liquid-based citology, cellular antigens, optimization of protocols