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Journal of Histochemistry and Cytochemistry, Vol. 47, 703-710, May 1999, Copyright © 1999, The Histochemical Society, Inc.

TECHNICAL NOTE

Effect of Bone Decalcification Procedures on DNA In Situ Hybridization and Comparative Genomic Hybridization: EDTA Is Highly Preferable to a Routinely Used Acid Decalcifier

Janneke C. Alersa, Pieter-Jaap Krijtenburga, Kees J. Vissersa, and Herman van Dekkena
a Department of Pathology, Erasmus University Rotterdam, Rotterdam, The Netherlands

Correspondence to: Janneke C. Alers, Dept. of Pathology, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: Alers@PATH.FGG.EUR.NL

Decalcification is routinely performed for histological studies of bone-containing tissue. Although DNA in situ hybridization (ISH) and comparative genomic hybridization (CGH) have been successfully employed on archival material, little has been reported on the use of these techniques on archival decalcified bony material. In this study we compared the effects of two commonly used decalcifiers, i.e., one proprietary, acid-based agent (RDO) and one chelating agent (EDTA), in relation to subsequent DNA ISH and CGH to bony tissues (two normal vertebrae, six prostate tumor bone metastases with one sample decalcified by both EDTA and RDO). We found that RDO-decalcified tissue was not suited for DNA ISH in tissue sections with centromere-specific probes, whereas we were able to adequately determine the chromosomal status of EDTA-decalcified material of both control and tumor material. Gel electrophoresis revealed that no DNA could be successfully retrieved from RDO-treated material. Moreover, in contrast to RDO-decalcified tumor material, we detected several chromosomal imbalances in the EDTA-decalcified tumor tissue by CGH analysis. Furthermore, it was possible to determine the DNA ploidy status of EDTA- but not of RDO-decalcified material by DNA flow cytometry. Decalcification of bony samples by EDTA is highly recommended for application in DNA ISH and CGH techniques. (J Histochem Cytochem 47:703–709, 1999)

Key Words: bone, comparative genomic hybridization, decalcification, DNA flow cytometry, EDTA, in situ hybridization, prostate adenocarcinoma


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