{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T02:49:17Z","timestamp":1790822957313,"version":"4.1.0"},"reference-count":25,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T00:00:00Z","timestamp":1780617600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>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 \u2014 fixation duration, cold ischemia time, and fixative preparation (4% neutral buffered versus 4% non-buffered formaldehyde) \u2014 impact the preservation and evaluation of tissue biomarkers in invasive breast cancer.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>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\u2009=\u200940%) who had not received neoadjuvant chemotherapy. Fifty microsamples (5\u201315 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 receptor-ER, progesterone receptor-PR) and Ki67 expression were evaluated by IHC using the Allred scoring system and current international recommendations.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Fixative preparation had a statistically significant, yet small, impact on biomarker evaluation. The mean percentage of ER-positive cells was 96.89\u2009\u00b1\u20090.74% with neutral buffered formaldehyde compared to 94.32\u2009\u00b1\u20091.51% with non-buffered formaldehyde (p\u2009=\u20090.011). Similar trends were seen for PR (94.89\u2009\u00b1\u20090.95% vs. 92.63\u2009\u00b1\u20091.67%, p\u2009=\u20090.027) and staining intensity. However, Allred scores remained unchanged. Fixation duration was significantly correlated with biomarker expression (Spearman \u03c1 between \u22120.60 and \u22120.83, p\u2009\u2264\u20090.007), with stable values from 0.5 to 48 h and a significant decline beyond 72 h (one-way ANOVA across fixation windows: all p\u2009&lt;\u20090.01). 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\u2009&lt;\u20090.05).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec004\">\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>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 multi-patient 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\u201310%) or heterogeneous expression.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pone.0343185","type":"journal-article","created":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T17:22:56Z","timestamp":1780680176000},"page":"e0343185","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["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 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