Decalcification with Nitrical® Can Preserve DNA/RNA for In Situ Hybridization and Proteins for Immunohistochemmistry in Formalin-Fixed Paraffin Embedded Tissues.
Kristen A Mead, Jamie Tull, Nancy M Drotar, Shengle Zhang. SUNY Upstate Medical University, Syracuse, NY
Background: Decalcification is a common procedure performed on histologic specimens. However, the conventional decalcifier, RDO, does not preserve DNA or RNA well and therefore render failures in various molecular assays, including chromogenic in situ hybridization (CISH) and fluorescent in situ hybridization (FISH). As DNA/RNA based molecular assays are becoming increasingly employed in the practice of pathology, it is important to discover a decalcifier that can preserve DNA/RNA but does not affect proteins for immunohistochemistry (IHC). In this study, we tested Nitrical®, a rapid decalcifier, in its preservation of RNA and DNA for molecular assays as well as proteins for IHC.
Design: Selected tissues of bone marrow, colon or skin were treated in parallel with Nitrical® or RDO for 30 min after formalin fixation and before paraffin-embedding. Commonly-used CISH probes for kappa chain, lambda chain and HPV, and FISH probes for HER2 and EGFR were tested on the treated tissues. In addition, 15 antibodies (pancytokeratin, AE1/AE3, CAM5.2, TTF-1, CDX-2, S100, EMA, P16, SMA, kappa, lambda, LCA CD20, CD34 and CD68) for a variety of proteins were also tested on same tissues. Intensities of in situ signals or IHC stains as well as backgrounds of both decalcifiers were compared.
Results: There were not any CISH or FISH signals identified in the RDO treated tissues. However, both CISH and FISH showed moderate to strong signals in Nitrical® treated tissues with a clean background. Although all the IHC in RDO treated tissues demonstrated staining (1-3+), the intensities in Nitrical® treated tissue are overall stronger (2-3+). It was also noted that background stains of IHC in Nitrical® treated tissue was slightly higher than that of RDO in occasional cases.
Conclusions: Nitrical®, a rapid decalcifier, can preserve RNA, DNA and proteins for CISH, FISH and IHC assays, respectively, in formalin-fixed paraffin embedded tissues. In the age of rapid development of molecular studies, Nitrical® should be considered to replace the conventional decalcifier, RDO, in routine pathology practice.
Monday, February 28, 2011 1:00 PM
Poster Session II # 255, Monday Afternoon