Background The lack of standardized guidelines for protein analysis of archival tissues (AT) seems to have hampered the use of such samples in molecular analyses. IMPACTS is a new European consortium aiming to establish guidelines for AT analyses. Here we focus on the evaluation of protein analysis in AT, fixed with formalin or new fixatives, for research and diagnostic use. Methods Tissue samples were fixed with formalin (FFPE) or a new fixative (FineFix, Milestone Srl) and inspected by histology. Five sections from three matched samples of each tissue were distributed within the consortium in order to compare the efficiency of protein recovery by immunohistochemistry, Western blot, and protein lysate microarray technology. Results By applying an optimized protocol, proteins can be successfully extracted from FFPE and FineFix tissues. Moreover, we could show a correlation between immunohistochemical, Western blot, and protein lysate microarray results for each of the tissue fixation procedures. Differences between methods, however, were observed in the total protein yield and staining intensities for antibodies detecting membrane proteins, including E-cadherin, EGFR, and HER2. Conclusion(s) Analysing proteins from fixed tissue samples can provide critical information for clinical decision making. Although long-term studies for formalinsubstitutes such as FineFix are still missing, formalin-free tissue fixation represents a reliable alternative for protein preservation, regarding quality and quantity, as compared to FFPE tissue. The IMPACTS consortium is working towards evaluating AT sample processing for protein analysis and tap into the vast treasure chest of archived samples.
Impacts: planning a European initiative for standardized protein analysis of archival clinical tissues
ZAMO', Alberto;
2009-01-01
Abstract
Background The lack of standardized guidelines for protein analysis of archival tissues (AT) seems to have hampered the use of such samples in molecular analyses. IMPACTS is a new European consortium aiming to establish guidelines for AT analyses. Here we focus on the evaluation of protein analysis in AT, fixed with formalin or new fixatives, for research and diagnostic use. Methods Tissue samples were fixed with formalin (FFPE) or a new fixative (FineFix, Milestone Srl) and inspected by histology. Five sections from three matched samples of each tissue were distributed within the consortium in order to compare the efficiency of protein recovery by immunohistochemistry, Western blot, and protein lysate microarray technology. Results By applying an optimized protocol, proteins can be successfully extracted from FFPE and FineFix tissues. Moreover, we could show a correlation between immunohistochemical, Western blot, and protein lysate microarray results for each of the tissue fixation procedures. Differences between methods, however, were observed in the total protein yield and staining intensities for antibodies detecting membrane proteins, including E-cadherin, EGFR, and HER2. Conclusion(s) Analysing proteins from fixed tissue samples can provide critical information for clinical decision making. Although long-term studies for formalinsubstitutes such as FineFix are still missing, formalin-free tissue fixation represents a reliable alternative for protein preservation, regarding quality and quantity, as compared to FFPE tissue. The IMPACTS consortium is working towards evaluating AT sample processing for protein analysis and tap into the vast treasure chest of archived samples.
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