Bone tissue is critically lagging behind soft tissues and biofluids in our effort to advance precision medicine. The main challenges have been accessibility and the requirement for deleterious decalcification processes that impact the fidelity of diagnostic histomorphology and hinder downstream analyses such as fluorescence in-situ hybridization (FISH). We have developed an alternative fixation chemistry that simultaneously fixes and decalcifies bone tissue. We compared tissue morphology, immunohistochemistry (IHC), cell signal phosphoprotein analysis, and FISH in 50 patient matched primary bone cancer cases that were either formalin fixed and decalcified, or theralin fixed with and without decalcification. Use of theralin improved tissue histomorphology, whereas overall IHC was comparable to formalin fixed, decalcified samples. Theralin-fixed samples showed a significant increase in protein and DNA extractability, supporting technologies such as laser-capture microdissection and reverse phase protein microarrays. Formalin-fixed bone samples suffered from a fixation artifact where protein quantification of β-actin directly correlated with fixation time. Theralin-fixed samples were not affected by this artifact. Moreover, theralin fixation enabled standard FISH staining in bone cancer samples, whereas no FISH staining was observed in formalin-fixed samples. We conclude that the use of theralin fixation unlocks the molecular archive within bone tissue allowing bone to enter the standard tissue analysis pipeline. This will have significant implications for bone cancer patients, in whom personalized medicine has yet to be implemented.
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We thank Jennifer Freeland, Sharmini Muralitharan, and Haiping Liu for their helpful guidance and discussions regarding FISH protocols. This work was supported in part by George Mason University, Inova Fairfax Hospital, Rizzoli Orthopedic Institute, and two National Institutes of Health grants to LAL (R21CA125698-01A1 and R33CA157403-01) from the National Cancer Institute program “Innovations in cancer cample preparation”.
Conflict of interest
LAL and VE are inventors of the fixation technology and, as university employees, may receive patent royalties per university policies related to US patent 8,460,859 B2: Tissue preservation and fixation method.
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Tissue Morphology and Antigenicity in Mouse and Rat Tibia: Comparing 12 Different Decalcification Conditions
Journal of Histochemistry & Cytochemistry (2019)