Alers J C, Krijtenburg P J, Vissers K J, van Dekken H
Department of Pathology, Erasmus University Rotterdam, Rotterdam, The Netherlands.
J Histochem Cytochem. 1999 May;47(5):703-10. doi: 10.1177/002215549904700512.
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.
脱钙是含骨组织组织学研究的常规操作。尽管DNA原位杂交（ISH）和比较基因组杂交（CGH）已成功应用于存档材料，但关于这些技术在存档脱钙骨材料上的应用报道较少。在本研究中，我们比较了两种常用脱钙剂，即一种专利的酸性试剂（RDO）和一种螯合剂（EDTA），对后续骨组织DNA ISH和CGH的影响（两个正常椎体，六个前列腺肿瘤骨转移灶，其中一个样本用EDTA和RDO进行脱钙）。我们发现，在使用着丝粒特异性探针的组织切片中，RDO脱钙组织不适合进行DNA ISH，而我们能够充分确定对照和肿瘤材料中EDTA脱钙材料的染色体状态。凝胶电泳显示，无法从RDO处理的材料中成功提取DNA。此外，与RDO脱钙的肿瘤材料相比，我们通过CGH分析在EDTA脱钙的肿瘤组织中检测到了几种染色体失衡。此外，通过DNA流式细胞术可以确定EDTA脱钙材料而非RDO脱钙材料的DNA倍体状态。强烈建议使用EDTA对骨样本进行脱钙，以应用于DNA ISH和CGH技术。
