A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue

Abstract

While efforts are made to improve tissue quality and control pre-analytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a Tissue Quality Index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative Immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, pERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on 2 independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts, as well as an association with loss of ER expression levels on all 3 breast cohorts. Using expression levels of 3 epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.

Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

“…Preanalytic variables (specimen handling, shipping, and storage procedures) can have a significant impact on the reliability of biomarker measurements in the laboratory, and phosphoproteins involved in dynamic responses of signaling pathways are notoriously labile during specimen collection due to ischemia and other factors (23)(24)(25). The stability of full-length MET and its phosphospecies was characterized in biopsies of SNU5 xenografts subjected to increasing ischemia time, which was defined as the total time needed for core needle biopsy sampling, specimen handling, and flash freezing.…”

Predictive Biomarkers and Companion Diagnostics. The Future of Immunohistochemistry

“…The use of internal or intrinsic proteins has been proposed as a method for qualifying a specimen as suitable for IHC staining 7,8,22 . This approach also may be extended to form a more extensive system of ‘quantifiable internal reference standards’ (QIRS), as a means of rendering a quantitative measurement by IHC 7,8 .…”

“…This proposal parallels ‘routine practice’ in molecular testing, whereby measurement of a ‘housekeeping gene’ or product (e.g. actin) serves as a quality control 21,22 and under specified conditions may serve as a calibration or reference standard 7,8 .…”

Recommendations for Clinical Biomarker Specimen Preservation and Stability Assessments

“…Measures to check the quality of specimens received for analysis may also be developed. One such approach involves development of tissue stability indicator protein tests that rely on evaluation of a combination of labile (e.g., phosphoproteins) and stably expressed (e.g., metabolic enzymes) housekeeping proteins [16].…”