Breast cancer is the most common malignancy among women in the developed world, and it is curable if detected early. Women with a positive hormonal status, whose tumors express hormone receptors such as estrogen (ER) and progesterone receptors (PR), are additionally treated with targeted hormonal therapy. The hormonal status can be rapidly determined on cytospins using immunocytochemistry (ICC); however, with the introduction of new ICC staining platforms such as the Dako Omnis, one of the newest on the market, it is essential to optimize and validate ICC protocols for ER and PR determination. The aim of this master’s thesis was to optimize and validate ICC protocols for ER and PR determination on cytological samples using the Dako Omnis platform. In the first part of the study, we tested various ICC staining conditions (primary antibody incubation time, pretreatment at low or high pH at 65 °C or 97 °C, and the addition of an ICC reaction enhancer). As a control and reference protocol for comparison, we used the ICC protocol for ER and PR on the BenchMark ULTRA platform, routinely applied in cytopathological diagnostics at the Institute of Oncology Ljubljana. Among all tested conditions, the most optimal ICC staining results for ER and PR, compared with the reference protocol, were achieved with a protocol including 20-minute incubation of the primary antibody, pretreatment at low pH and 97 °C, and the addition of a commercial enhancer (mouse linker, Agilent Technologies). In the second part of the study, the selected optimized protocol was validated on 20 breast cancer samples, confirming very high concordance with the reference protocol as well as with ER and PR expression levels in the corresponding histological samples, demonstrating that the optimized Dako Omnis protocol is suitable for routine use in the cytopathology laboratory.
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