Tissue is alive: New technologies are needed to address the problems of protein biomarker pre-analytical variability

Abstract

Instability of tissue protein biomarkers is a critical issue for molecular profiling. Pre-analytical variables during tissue procurement, such as time delays during which the tissue remains stored at room temperature, can cause significant variability and bias in downstream molecular analysis. Living tissue, ex vivo, goes through a defined stage of reactive changes that begin with oxidative, hypoxic and metabolic stress, and culminate in apoptosis. Depending on the delay time ex vivo, and reactive stage, protein biomarkers, such as signal pathway phosphoproteins will be elevated or suppressed in a manner which does not represent the biomarker levels at the time of excision. Proteomic data documenting reactive tissue protein changes post collection indicate the need to recognize and address tissue stability, preservation of post-translational modifications, and preservation of morphologic features for molecular analysis. Based on the analysis of phosphoproteins, one of the most labile tissue protein biomarkers, we set forth tissue procurement guidelines for clinical research. We propose technical solutions for (i) assessing the state of protein analyte preservation and specimen quality via identification of a panel of natural proteins (surrogate stability markers), and (ii) using multi-purpose fixative solution designed to stabilize, preserve and maintain proteins, nucleic acids, and tissue architecture.

Figure 1

Ideal tissue stabiliziation/preservation scheme for biomarker analysis. A core needle biospy is immediately placed in a multi-purpose stabilization/preservation solution at room temperature. The fixed biopsy specimen could subsequently undergo cryosectioning, processing in standard formalin or ethanol tissue processors, or flow cytometric analysis. The fixed samples could be archived as frozen sections/blocks or paraffin-embedded blocks. The samples would be compatible with standard protein/nucleic acid methodologies.

Figure 2

A multi-purpose chemical solution for stabilization and preservation of proteins and RNA, and maintenance of histomorphology. (A) Frozen uterine leiomyoma tissue reveals on-going phosphoproteomic changes post excision. A reverse phase protein microarray analysis of tissue analyzed from a deep area of the tissue block (1.0–2.0 mm from surface) showed reactive protein changes as compared with the time zero sample (100% value, 10 min post excision, outermost tissue surface). Reactive proteins constituted a variety of molecular cascades: apoptotic pathway, stress/inflammation pathway, hypoxia/ischemia pathway, transcription factors, proliferation/survival pathway, and adhesion/cytoskeleton proteins. (B) RNA stability time course of T47D-cultured cells. T47D human mammary adenocarcinoma cell lines were cultured at 37°C, 5% CO₂. Medium was removed and cells washed three times with cold phosphate-buffered saline. The cells were scraped from the flasks, combined and aliquots were incubated with RNAlater (Qiagen) (black square) or our multi-purpose fixative (black triangle) at 4°C for 1, 2, 4, 8, 24, 48, and 72 h. An untreated cell culture aliquot was used as the time zero control (open circle). RNA was extracted using the RNA Mini Kit (Qiagen) and RIN were determined using a Bioanalyzer 2100 (Agilent) for duplicate samples at each time point. (C and D) Tissue stabilized at room temperature in a multi-purpose chemical solution yields histomorphology similar to formalin fixed tissue. (C) Human breast tumor epithelium fixed in a multi-purpose stabilization solution containing phosphatase and kinase inhibitors, alcohol, and a permeation enhancer, was processed via a standard histology technique. H&E staining showed well-delineated nuclear membranes and chromatin clumping comparable to an adjacent tissue sample fixed in 5% formalin (D). (UltraLight Histology™ processing courtesy of Dr. Thomas Donndelinger, Bi-Biomics).