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Updated: May 26, 2026
Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue
Published on: September 2, 2013
Molecular preservation by extraction and fixation, mPREF: a method for small molecule biomarker analysis and
Jeffrey R Shuster1, Raymond S Lance, Dean A Troyer
1Depts, Of Pathology and Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA, USA. troyerda@evms.edu.
Background:
Histopathology is the standard method for cancer diagnosis and grading to assess aggressiveness in clinical biopsies. Molecular biomarkers have also been described that are associated with cancer aggressiveness, however, the portion of tissue analyzed is often processed in a manner that is destructive to the tissue. We present here a new method for performing analysis of small molecule biomarkers and histology in exactly the same biopsy tissue.
Methods:
Prostate needle biopsies were taken from surgical prostatectomy specimens and first fixed, each in a separate vial, in 2.5 ml of 80% methanol:water. The biopsies were fixed for 24 hrs at room temperature and then removed and post-processed using a non-formalin-based fixative (UMFIX), embedded, and analyzed by hematoxylin and eosin (H&E) and by immunohistochemical (IHC) staining. The retained alcohol pre-fixative was analyzed for small molecule biomarkers by mass spectrometry.
Results:
H&E analysis was successful following the pre-fixation in 80% methanol. The presence or absence of tumor could be readily determined for all 96 biopsies analyzed. A subset of biopsy sections was analyzed by IHC, and cancerous and non-cancerous regions could be readily visualized by PIN4 staining. To demonstrate the suitability for analysis of small molecule biomarkers, 28 of the alcohol extracts were analyzed using a mass spectrometry-based metabolomics platform. All extracts tested yielded successful metabolite profiles. 260 named biochemical compounds were detected in the alcohol extracts. A comparison of the relative levels of compounds in cancer containing vs. non-cancer containing biopsies showed differences for 83 of the compounds. A comparison of the results with prior published reports showed good agreement between the current method and prior reported biomarker discovery methods that involve tissue destructive methods.
Conclusions:
The Molecular Preservation by Extraction and Fixation (mPREF) method allows for the analysis of small molecule biomarkers from exactly the same tissue that is processed for histopathology.
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