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Proposal of an appropriate decalcification method of bone marrow biopsy specimens in the era of expanding genetic molecular study

 Sung-Eun Choi  ;  Soon Won Hong  ;  Sun Och Yoon 
 Journal of Pathology and Translational Medicine, Vol.49(3) : 236-242, 2015 
Journal Title
 Journal of Pathology and Translational Medicine 
Issue Date
Bone marrow ; Decalcification technique ; Ethylenediaminetetraacetic acid disodium salt dehydrate ; Hydrochloric acid ; RDO GOLD
BACKGROUND: The conventional method for decalcification of bone specimens uses hydrochloric acid (HCl) and is notorious for damaging cellular RNA, DNA, and proteins, thus complicating molecular and immunohistochemical analyses. A method that can effectively decalcify while preserving genetic material is necessary. METHODS: Pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl. Pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the EDTA versus HCl comparison group, and 25 cases to the RDO versus HCl comparison group. The decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. For molecular analysis, we randomly selected 5 cases from the EDTA versus HCl and RDO versus HCl groups. RESULTS: The decalcification time for appropriate histomorphologic analysis was the longest in the EDTA method and the shortest in the RDO method. EDTA was superior to RDO or HCl in DNA yield and integrity, assessed via DNA extraction, polymerase chain reaction, and silver in situ hybridization using DNA probes. The EDTA method maintained intact nuclear protein staining on immunohistochemistry, while the HCl method produced poor quality images. Staining after the RDO method had equivocal results. RNA in situ hybridization using kappa and lambda RNA probes measured RNA integrity; the EDTA and RDO method had the best quality, followed by HCl. CONCLUSIONS: The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.
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1. College of Medicine (의과대학) > Dept. of Pathology (병리학교실) > 1. Journal Papers
Yonsei Authors
Yoon, Sun Och(윤선옥) ORCID logo https://orcid.org/0000-0002-5115-1402
Choi, Sung Eun(최성은) ORCID logo https://orcid.org/0000-0002-6955-658X
Hong, Soon Won(홍순원) ORCID logo https://orcid.org/0000-0002-0324-2414
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