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P11. Decalcification of tissue with formic acid remarkably reduces the performance of FISH: EDTA could be the new preferable agent

Mads Anæus Brogaard, Anne Oehlenschlæger Christensen, Marie Lorans and Pia Boelskifte Jensen

Institute of Pathology, Aalborg University Hospital, Denmark.

Background: Preservation of intact DNA throughout pre-analytical procedures is essential for successful detection of chromosomal abnormalities by Fluorescence In Situ Hybridization (FISH). Formic acid is routinely used for decalcification of tissue, but is known to cause degradation of DNA, leaving the tissue less suitable for FISH. EDTA is a more gentle decalcifying agent, but the rate of calcium extraction by EDTA chelation is more time consuming.

Objective: The aim of this study is to investigate the impact of formic acid and EDTA as decalcification methods in combination with fixation time on the performance of FISH.

Methods: Thirteen soft tissue samples were formalin-fixed for 6, 24 or 72h and subjected to decalcification with either formic acid for 6h, 12h (in microwave oven at 37 ̊C) or 24h or EDTA for 24h or 48h and FISH was performed according to routine HER2 protocol. The performance of FISH was assessed by evaluation of signal intensity, nuclei integrity and rejection criteria according to ASCO/CAP HER2 guidelines. Success rate was defined as the fraction of FISH results for each specific pre-analytical procedure that passed the rejection criteria, showed acceptable signal intensity and preserved nuclei integrity. 

Results: Decalcification by EDTA resulted in stronger signal intensities, better preservation of nuclei integrity and higher success rate. Decalcification with EDTA on 72h fixed tissue allowed evaluation of all samples whereas only 17% could be evaluated when decalcified with formic acid in combination with microwaves, regardless of fixation time.

Conclusion: EDTA decalcification resulted in unequally better FISH performance. Regarding FISH performance prolonged fixation time led to a decrease using formic acid and an increase using EDTA.

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KONXION A/S