Pre-analytical variables affecting breast cancer biomarker expression: A controlled single-specimen study of fixation duration, cold ischemia time, and fixative preparation in a low-resource setting

RESEARCH ARTICLE Pre-analytical variables affecting breast cancer biomarker expression: A controlled singlespecimen study of fixation duration, cold ischemia time, and fixative preparation in a low-resource setting Clément Parfait Ndengue 1*, Paul Jean Adrien Atangana1,2, Gilbert Roger Ateba1, Samuel Honoré Mandengue3, Emile Telesphore Mboudou1,4, Carole Else Eboumbou Moukoko2 1 Douala Gynaeco-Obstetric and Pediatric Hospital (DGOPEH), Douala, Cameroon, 2 Faculty of Medicine and Pharmaceutical Sciences, University of Douala, Douala, Cameroon, 3 Faculty of Sciences, University of Douala, Douala, Cameroon, 4 Faculty of Medicine and Biomedical Sciences, University of Yaounde I, Yaounde, Cameroon * Abstract Background OPEN ACCESS Citation: Ndengue CP, Atangana PJA, Ateba GR, Mandengue SH, Mboudou ET, Eboumbou Moukoko CE (2026) Pre-analytical variables affecting breast cancer biomarker expression: A controlled single-specimen study of fixation duration, cold ischemia time, and fixative preparation in a low-resource setting. PLoS One 21(6): e0343185. https://doi. org/10.1371/journal.pone.0343185 Editor: Tomasz W. Kaminski, Versiti Blood Research Institute, UNITED STATES OF AMERICA Received: March 5, 2026 Accepted: May 20, 2026 Published: June 5, 2026 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal. pone.0343185 Optimal pre-analytical management of breast tissue specimens, particularly formalin fixation, is essential for accurate immunohistochemical (IHC) biomarker assessment in invasive breast cancer. Although international guidelines suggest using 4% neutral buffered formalin with controlled fixation time, many laboratories in low-resource settings deviate from these standards. This study aimed to determine whether three pre-analytical variables — fixation duration, cold ischemia time, and fixative preparation (4% neutral buffered versus 4% non-buffered formaldehyde) — impact the preservation and evaluation of tissue biomarkers in invasive breast cancer. Methods We conducted an exploratory, proof-of-concept, experimental study using fresh mastectomy tissue from a 34-year-old patient with invasive ductal carcinoma (pT4, hormone receptor-positive, HER2-negative, Ki67 = 40%) who had not received neoadjuvant chemotherapy. Fifty microsamples (5–15 mm in length, approximately 1 mm in diameter) were obtained using a 14-gauge core needle biopsy device and divided into four cohorts: (1) 19 samples fixed in 4% neutral buffered formaldehyde for 0.5 to 144 hours; (2) 19 samples fixed in 4% non-buffered formaldehyde for 0.5 to 144 hours; (3) 6 samples with delayed fixation (0.5 to 8 hours) then fixed in neutral buffered formaldehyde for 10 hours; (4) 6 samples with delayed fixation (0.5 to 8 hours) then fixed in non-buffered formaldehyde for 10 hours. Hormone receptors (estrogen PLOS One | https://doi.org/10.1371/journal.pone.0343185 June 5, 2026 1 / 14 Copyright: © 2026 Ndengue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data availability statement: All relevant data are within the manuscript and its Supporting Information files. Raw immunohistochemistry scoring data, complete fixation and cold ischemia time parameters, descriptive statistics, and statistical analysis outputs (including the added Spearman correlations and one-way ANOVA across fixation-duration windows performed for this revision) are provided in the S1 Data file. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Abbreviations: ASCO/CAP: American Society of Clinical Oncology/College of American Pathologists, CI: Confidence interval, DGOPEH: Douala Gynaeco-Obstetric and Pediatric Hospital, ER: Estrogen receptor, FFPE: Formalin-fixed paraffin-embedded, HER2: Human epidermal growth factor receptor 2, ICC: Intraclass correlation coefficient, IHC: Immunohistochemistry, IS: Intensity score, LMIC: Low- and middle-income countries, NBF: Neutral buffered formaldehyde, NB-F: Non-buffered formaldehyde, PR: Progesterone receptor, PS: Proportion score, SEM: Standard error of the mean. receptor-ER, progesterone receptor-PR) and Ki67 expression were evaluated by IHC using the Allred scoring system and current international recommendations. Results Fixative preparation had a statistically significant, yet small, impact on biomarker evaluation. The mean percentage of ER-positive cells was 96.89 ± 0.74% with neutral buffered formaldehyde compared to 94.32 ± 1.51% with non-buffered formaldehyde (p = 0.011). Similar trends were seen for PR (94.89 ± 0.95% vs. 92.63 ± 1.67%, p = 0.027) and staining intensity. However, Allred scores remained unchanged. Fixation duration was significantly correlated with biomarker expression (Spearman ρ between −0.60 and −0.83, p ≤ 0.007), with stable values from 0.5 to 48 h and a significant decline beyond 72 h (one-way ANOVA across fixation windows: all p < 0.01). Cold ischemia time was strongly correlated with decreased biomarker expression regardless of fixative preparation. Hormone receptor expression and Ki67 remained stable with minimal Allred score changes for up to 2 hours of cold ischemia, but significantly decreased after 2 hours, with scores decreasing in proportion to the duration of ischemia (p < 0.05). Conclusions In this single-specimen controlled experiment, non-buffered formaldehyde preserved tissue biomarkers with small but measurable differences relative to neutral buffered formaldehyde for IHC analysis, although these findings require validation in multipatient studies. Consistent with current guidelines, a cold ischemia time of up to 1 hour maintained adequate biomarker preservation. These preliminary results may be relevant for pathology laboratories in resource-limited settings where neutral buffered formalin may not be easily accessible, and warrant further investigation across diverse tumor types and baseline expression levels, particularly tumors with ER-low-positive (1–10%) or heterogeneous expression. Introduction Breast cancer remains the most commonly diagnosed cancer and the leading cause of cancer death among women worldwide, with an estimated 2.3 million new cases and 685,000 deaths in 2020 [1]. In sub-Saharan Africa, breast cancer incidence is rising rapidly, with patients often presenting at advanced stages and experiencing poorer outcomes compared to high-income countries [2,3]. Optimal management requires accurate assessment of predictive and prognostic biomarkers, including estrogen receptor (ER), proges (...truncated)