ẢNH HƯỞNG CỦA THỜI GIAN THIẾU MÁU LẠNH LÊN SỰ BIỂU HIỆN CỦA CÁC DẤU ẤN ER, PR VÀ KI-67 TRONG UNG THƯ VÚ BẰNG KỸ THUẬT NHUỘM HOÁ MÔ MIỄN DỊCH

Abstract

Background: Immunohistochemical (IHC) assessment of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 plays an essential role for breast cancer diagnosis, prognostication, and treatment decision-making. Because these biomarkers are localized in the nucleus, the accuracy of IHC results depends heavily on antigen preservation. Cold ischemia time is a critical pre-analytical factor, and prolonged delays before fixation may cause protein degradation, attenuated nuclear staining, and misclassification of biomarker status. Objectives: To investigate the impact of cold ischemia time on ER, PR and Ki-67 expression in breast cancer using immunohistochemistry. Subjects and Methods: This exploratory study was conducted on purposively selected breast cancer tissue specimens that met inclusion criteria. Samples were subdivided and subjected to cold ischemia intervals ranging from less than 1 hour to 8 hours before fixation in 10% neutral-buffered formalin (10% NBF; ~4% formaldehyde, phosphate buffer, pH 7.0). After fixation for 8–32 h and tissue processing, sectioned at 3–4 µm, immunohistochemistry for ER, PR, and Ki-67 was performed. Nuclear staining intensity, proportion of positive cells, and rate of nuclear degeneration were analyzed to evaluate the impact of delayed fixation. Results: For ER, the mean staining intensity began to decline at 2 hours, the mean proportion of positive nuclei decreased at 3 hours, and the mean rate of nuclear degeneration increased at 2 hours. For PR, the mean staining intensity decreased at 2 hours, the mean proportion of positive nuclei decreased at 3 hours, and nuclear degeneration increased at 2 hours, followed by a slight reduction at 4 hours and a subsequent increase at 6 hours. For Ki-67, nuclear degeneration began to increase at 2 hours, whereas the proportion of positive nuclei showed only minimal, nonsignificant variation. Conclusions: Prolonged cold ischemia time decreased the staining intensity and proportion of ER and PR expression and increased the rate of nuclear degeneration for ER, PR, and Ki-67. Although the proportion of Ki-67–positive cells was minimally affected, the downward trend in nuclear quality underscores the need for strict preanalytic control. Preanalytic deviations increase the risk of ER/PR under- or over-calling, misclassification of Luminal subtypes, and inappropriate endocrine or chemotherapy decisions. These findings demonstrate a statistically significant association between cold ischemia time and attenuation of ER/PR expression and emphasize adherence ASCO/CAP recommendations: initiate fixation within ≤1 hour of excision to preserve the reliability of IHC.