A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue.
Author(s): Neumeister VM, Parisi F, England AM, Siddiqui S, Anagnostou V, Zarrella E, Vassilakopolou M, Bai Y, Saylor S, Sapino A, Kluger Y, Hicks DG, Bussolati G, Kwei S, Rimm DL
Publication: Lab Invest, 2014, Vol. 94, Page 467-74
PubMed ID: 24535259 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to create a tissue quality index (TQI) based on the change in the immunofluorescence of 34 proteins and phosphoproteins with increasing delays to formalin fixation.
Conclusion of Paper
A positive TQI, calculated based on the automated quantitative analysis (AQUA)scores of cytokeratin and phosphorylated heat shock protein 27 (phospho-HSP27) or phosphorylated 44/42 mitogen activated protein kinase (ERK1/2) and phospho-HSP27, was indicative of a short delay to fixation (<60 min), but as the delay to fixation increased, the TQI decreased, eventually becoming negative. The TQIs were higher for the cohort of breast cancer specimens stored in vacuum sealed bags at 4°C prior to fixation than in the cohort kept at room temperature (assumed not to be in vacuum sealed bags), indicating better preservation of antigenicity at lower temperatures. A positive TQI (indicative of short cold ischemia time) was significantly associated with a higher estrogen receptor (ER) AQUA score in cohorts of 100 breast cancer specimens and 11 healthy breast specimens, but only an insignificant trend was observed with a different cohort of 93 breast cancer specimens.
Studies
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Study Purpose
The purpose of this study was to determine the effects of cold ischemia duration and temperature on the immunofluorescence of 34 proteins in formalin-fixed, paraffin-embedded (FFPE) breast cancer specimens and to identify protein pairs which could serve as tissue quality biomarkers of cold ischemia time. The study consisted of 93 breast cancer specimens with cold ischemia times of 25-415 min on a tissue microarray (2 0.6 mm cores/tumor) and 100 breast cancer specimens that were vacuum sealed and stored at 4°C for 1-72 h before fixation on a tissue microarray (2 1 mm cores/tumor). A tissue microarray containing 0.6 mm cores of 11 normal breast tissue specimens, each of which were exposed to cold ischemia times of 20 min, 2 h, 24 h and 48 h (4 cores/time-point) was also included in the study. TQI was calculated by subtraction of the log2 normalization of the AQUA score of pairs of proteins.
Summary of Findings:
Preliminary analysis using a cohort of 93 breast cancer specimens showed that the TQI based on the combination of phosphotyrosine and phospho-HSP27 was the best predictor of cold ischemia time of more than 60 min followed by TQIs based on cytokeratin and phospho-HSP27 and ERK1/2 and phosphoHSP27. For these markers, a positive TQI was indicative of a short delay to fixation, but as the delay to fixation increased, the TQI decreased. In subsequent analysis, using 11 healthy breast specimens, 100 breast cancer specimens, and a second array of the initial 93 breast cancer specimens, TQI based on phosphotyrosine and phospho-HSP27 was not a good predictor of cold ischemia time; however, the relationship of decrease in TQI based on cytokeratin and phospho-HSP27 and ERK1/2 and phospho-HSP27 with increased cold ischemia time was reproduced. Importantly, the TQIs were higher for the 100 breast cancer specimens which were stored in vacuum sealed bags at 4°C prior to fixation than in the 93 breast cancer specimens which were kept at room temperature (assumed not to be in bags), indicating better preservation of antigenicity at lower temperatures. A positive TQI (indicative of short cold ischemia time) was associated with a higher ER AQUA score in the cohorts of 100 breast cancer specimens (p=0.0311) and 11 healthy breast specimens (p=0.0313), but while a trend was observed with the cohort of 93 breast cancer specimens, it was not significant(p=0.0671).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Protein Tissue microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 20 min
2 h
24 h
48 h
25-415 min
1-72 h
Immunohistochemistry Specific Targeted peptide/protein Cytokeratin
ERK1/2
Phospho-HSP27
Storage Storage temperature 4°C
Room temperature