Impact of Preanalytical Factors on the Measurement of Tumor Tissue Biomarkers Using Immunohistochemistry

Abstract

Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute’s Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12–24 hr to avoid confounding effects of these preanalytical factors on IHC.

Assessment of the Impact of Alternative Fixatives on HER2 Detection in Breast Cancer and Gastric Cancer Tumor Specimens

“…Discordant results of HER2 status within indications such as BC and GC can be further explained, to a certain degree, by preanalytical and analytical factors such as delay to, and prolonged, formalin fixation, 19 tumor heterogeneity, 9 , 37 the diversity of kits and antibodies, 38 and data interpretation. 39 Although IHC testing methods have steadily improved with the introduction of automated staining platforms and improved antigen retrieval and detection systems, the need for assay validation and ongoing monitoring has not diminished given that results are still affected by various preanalytic and analytic factors.…”

“…Other laboratories continued to assess HER2 by IHC using the scoring guidelines included in the test kit rather than the ASCO/ CAP scoring criteria. 10,12 Although several studies report on the effects of some preanalytical variables such as cold ischemic time (delay to formalin fixation) [14][15][16][17][18][19] and prolonged formalin fixation for biomarker assay development, 14,[18][19][20][21] few have addressed the technical aspects of fixative choice to define best practices for clinical studies of HER2 status. 22,23 The buffer's qualities (ie, concentration and pH levels) in the formalin solution used for fixing tissue specimens have been shown to affect the quality of immunostaining.…”

“…Although several studies report on the effects of some preanalytical variables such as cold ischemic time (delay to formalin fixation)14–19 and prolonged formalin fixation for biomarker assay development,14,18–21 few have addressed the technical aspects of fixative choice to define best practices for clinical studies of HER2 status 22,23…”

Considerations for Whole-Slide Analysis of Murine Xenografts Experiments

“…Furthermore, when preparing the samples for immunohistochemical analysis of drug action, caution must be taken with pre-analytic factors. 17 Bagchi et al (2021) reported when preparing organs for immunohistochemistry, the delay to fixation and the time in the fixative reagent can have significant implications on many cancer biomarkers. With a longer delay to fixation (>2 hours), some biomarkers were reduced (c-MET, p53 and pAKT), whilst others were increased (EGFR and CD10), when compared to a shorter time (1 hour).…”

“…The authors suggest that the delay to fixation should be below 1 hour and the time in the fixative reagent should be 12-24 hours to prevent bias from these pre-analytical factors. 17 Therefore, when designing and conducting a tumor experiment, factors such as the tumor age, location of the tumor and pre-analytic factors should be considered.…”