Effect of decalcification agents on gene search using FFPE specimens

• Published in: Japanese Journal of Medical Technology Vol. 68; no. 3; pp. 455 - 462
• Main Authors: SERIZAWA, Akihiko, MIYAJIMA, Yoko, SAIKA, Tsubasa, SAKAGUCHI, Shigemi, OYAMADA, Hiroyuki, KATO, Nobuaki, ITOH, Hitoshi
• Format: Journal Article
• Language: Japanese
• Published: Japanese Association of Medical Technologists 25.07.2019
• Subjects: decalcification; DNA fragmentation; FFPE specimen; gene search; molecular target therapeutic drugs
• ISSN: 0915-8669; 2188-5346
• DOI: 10.14932/jamt.17-133

In recent years, gene search from pathological specimens has been indispensable for the development of molecular targeted therapeutic drugs, and the results determine the suitability of therapeutic agents. Recommendations on the type and time of specimen fixation are specified in guidelines and protocols, but detailed descriptions concerning decalcification agents are rarely seen. In this study, we examined the effect of decalcification on gene search. The DNA yield and PCR amplification were compared among FFPE specimens prepared using various decalcification agents and decalcification times. We also determined the effects of various decalcification agents on EGFR mutation. The results showed that DNA yield was low in the strong-acid-type decalcification agents K-CX and Plank-Rychlo, and no amplification by PCR was observed. Also, with the strong-acid-type decalcification and 10% formalin formic acid, the DNA yield decreased as the decalcification time increased. On the other hand, 10% EDTA yielded more DNA, and PCR amplification was good from 100 to 400 bp. Regarding decalcification time, DNA yield did not decrease even after 7 days of treatment, which was a good result. In the study of the EGFR mutation detection kit for pulmonary adenocarcinoma, detection with strong-acid-type decalcification agents and 10% formalin formic acid was impossible regardless of decalcification time, but EGFR mutation can be detected with 10% EDTA treatment for 7 days. When decalcification is required for mutation search, it is essential to use EDTA as a decalcification agent.