Optimizacija imunocitokemičnih barvanj na citoloških vzorcih z uporabo detekcijskih sistemov OptiView DAB in Envision FLEX

Dremelj, Anja (author), Kos, Janko (mentor), Kloboves Prevodnik, Veronika (comentor)

Imunocitokemija je metoda, pri kateri lahko s pomočjo monoklonskih ali poliklonskih protiteles dokažemo antigene v celičnih vzorcih. Na področju onkološke patologije in citopatologije je postala nepogrešljiva dodatna metoda za opredelitev izvora malignih neoplazem. Vezavo primarnega protitelesa na antigen lahko predstavimo s pomočjo različnih detekcijskih sistemov, kot so na primer iView DAB, OptiView DAB in EnVision FLEX. Danes so za imunocitokemična barvanja v uporabi avtomatski sistemi oziroma barvalci, ki opravijo več zamudnih korakov postopka in izboljšajo ponovljivost reakcij. Standardizacija imunocitokemičnih barvanj je zaradi razlik v pripravi in fiksaciji vzorcev, uporabljenih reagentov in barvalcev težavna, zato je ključno, da vsak citopatološki laboratorij pred začetkom uporabe v rutinski diagnostiki postopke barvanj optimizira in preveri z ustreznimi testiranji. V okviru magistrske naloge smo optimizirali protokole imunocitokemičnih barvanj za 10 tumorskih označevalcev (kalretinin, BER-EP4, MOC-31, CK AE1/AE3, CD68, CK 5/6, LCA, desmin, HBME-1 in WT1), kjer smo uporabili dva nova detekcijska sistema, OptiView DAB in EnVision FLEX. Rezultate ocenjevalnih kriterijev deleža in intenzitete reakcije, ozadja, kontrastiranja in morfologije smo primerjali z rezultati barvanj z detekcijskim sistemom iView DAB, ki ga na Onkološkem inštitutu Ljubljana trenutno uporabljamo v rutini, a se umika iz prodaje. Izbrane protokole smo za vsak tumorski označevalec testirali na 10 pozitivnih in 10 negativnih citoloških vzorcih. Po ocenjevanju pobarvanih preparatov smo prišli do ugotovitve, da so vsi izbrani protokoli s sistemoma OptiView DAB in EnVision FLEX primerni za uporabo v rutinski diagnostiki. Rezultati so bili glede na iView DAB protokole primerljivi ali pri dokazovanju kalretinina, BER-EP4, MOC-31, CK AE1/AE3, CK 5/6 in WT1 celo boljši. V primerjavi z EnVision FLEX se je pri dokazovanju desmina in WT1 protokol z OptiView DAB izkazal za ustreznejšega. Pri protokolih s sistemom OptiView DAB smo se s predobdelavo z raztopino CC1 pri dokazovanju kalretinina, BER-EP4, MOC-31, CK AE1/AE in CK 5/6 izognili ozadju, na ustrezno intenziteto pa je pomembno vplival dodatek stopnje amplifikacije signala. Pri protokolih z EnVision FLEX brez predobdelave moramo biti pazljivi na nespecifično obarvana jedra celic, pri protokolih s predobdelavo z visokim pH pa na obarvanje eritrocitov. Za zanesljivo in kvalitetno postavitev diagnoze je ključno tudi ujemanje med ocenami ocenjevalcev in testiranje dovolj velikega števila citoloških vzorcev.

• Language: Slovenian
• Keywords: imunocitokemija, detekcijski sistem, tumorski označevalci, optimizacija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-142929
• Publication date in RUL: 03.12.2022

Secondary language

• Language: English
• Title: Optimisation of immunocytochemical staining on cytological samples using OptiView DAB and Envision FLEX detection systems

Abstract

Immunocytochemistry is a method that uses monoclonal or polyclonal antibodies to detect antigens in cell samples. In the field of oncological pathology and cytopathology, it has become an indispensable adjunctive method for the identification of the origin of malignant neoplasms. The binding of the primary antibody to the antigen can be visualised using different detection systems, such as iView DAB, OptiView DAB and EnVision FLEX. Automated stainers, used for immunocytochemical staining, have the advantages of performing more time-consuming steps of the procedure and improving the reproducibility of the reactions. Standardisation of immunocytochemical stains is challenging due to differences in sample preparation and fixation as well as in the reagents and stainers used. Therefore, it is essential that every cytopathology laboratory optimises and verifies staining procedures with appropriate testing prior to use in routine diagnostics. In the framework of this master’s thesis, we optimised immunocytochemical staining protocols for 10 tumour markers (calretinin, BER-EP4, MOC-31, CK AE1/AE3, CD68, CK 5/6, LCA, desmin, HBME-1 and WT1) using new detection systems OptiView DAB and EnVision FLEX. The results of the evaluation criteria of reaction percentage and intensity, background, contrast, and morphology were compared with the results of staining with the iView DAB detection system, which is currently routinely used at the Institute of Oncology Ljubljana but is being withdrawn from sale. The selected protocols were tested on 10 positive and 10 negative cytological samples for every tumour marker. After evaluation of the stained slides, we concluded that all selected protocols with OptiView DAB and EnVision FLEX are suitable for routine diagnostic use. The results were comparable or even better for detection of calretinin, BER-EP4, MOC-31, CK AE1/AE3, CK 5/6 and WT1 when compared to the iView DAB protocols. To achieve better results, protocols with OptiView DAB would be prioritised for desmin and WT1 detection over EnVision FLEX. For detection of calretinin, BER-EP4, MOC-31, CK AE1/AE3 and CK 5/6 with OptiView DAB protocols, the background was avoided by pretreatment with CC1 solution, and the appropriate intensity was achieved by the additional step of signal amplification. Attention should be paid to non-specific staining of cell nuclei for EnVision FLEX protocols without pretreatment and to non-specific staining of erythrocytes for protocols with high pH pretreatment. Agreement between assessors' scores and testing of sufficient number of cytological samples are also crucial for a reliable and quality diagnosis.

• Keywords: immunocytochemistry, detection systems, tumour markers, optimisation