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

Choi SE, Hong SW, Yoon SO - J Pathol Transl Med (2015)

Bottom Line: A method that can effectively decalcify while preserving genetic material is necessary.Staining after the RDO method had equivocal results.The EDTA protocol would be the best in preserving genetic material.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

The quality, quantity, and feasibility of real time PCR study is compared between EDTA, RDO, and HCl protocols. The first row demonstrates EDTA versus HCl, and the second row RDO versus HCl (A, D, DNA yield; B, PCR result of EDTA; E, PCR result of RDO; C, F, PCR results of HCl). D, PCR result of RDO. PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid disodium salt dehydrate; RDO, RDO GOLD; HCl, hydrochloric acid; PNA, peptide nucleic acid.
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f1-jptm-49-3-236: The quality, quantity, and feasibility of real time PCR study is compared between EDTA, RDO, and HCl protocols. The first row demonstrates EDTA versus HCl, and the second row RDO versus HCl (A, D, DNA yield; B, PCR result of EDTA; E, PCR result of RDO; C, F, PCR results of HCl). D, PCR result of RDO. PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid disodium salt dehydrate; RDO, RDO GOLD; HCl, hydrochloric acid; PNA, peptide nucleic acid.

Mentions: DNA quantity, purity, and Ct values of internal controls after real-time PCR in the EDTA versus HCl group and RDO versus HCl group are depicted in Table 3 and Fig. 1. Although differences were not statistically significant, the DNA yield of the EDTA protocol was about 2 times higher than the HCl protocol. In addition, the Ct value of the former protocol was significantly lower than that of the latter (p<.001) with the estimated difference being about 7. Furthermore, the Ct values of EDTA processed samples were lower than 30, demonstrating that the amount of intact DNA feasible for PCR with the EDTA protocol is better preserved by a factor of 2 [7] than the HCl protocol.


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

Choi SE, Hong SW, Yoon SO - J Pathol Transl Med (2015)

The quality, quantity, and feasibility of real time PCR study is compared between EDTA, RDO, and HCl protocols. The first row demonstrates EDTA versus HCl, and the second row RDO versus HCl (A, D, DNA yield; B, PCR result of EDTA; E, PCR result of RDO; C, F, PCR results of HCl). D, PCR result of RDO. PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid disodium salt dehydrate; RDO, RDO GOLD; HCl, hydrochloric acid; PNA, peptide nucleic acid.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440935&req=5

f1-jptm-49-3-236: The quality, quantity, and feasibility of real time PCR study is compared between EDTA, RDO, and HCl protocols. The first row demonstrates EDTA versus HCl, and the second row RDO versus HCl (A, D, DNA yield; B, PCR result of EDTA; E, PCR result of RDO; C, F, PCR results of HCl). D, PCR result of RDO. PCR, polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid disodium salt dehydrate; RDO, RDO GOLD; HCl, hydrochloric acid; PNA, peptide nucleic acid.
Mentions: DNA quantity, purity, and Ct values of internal controls after real-time PCR in the EDTA versus HCl group and RDO versus HCl group are depicted in Table 3 and Fig. 1. Although differences were not statistically significant, the DNA yield of the EDTA protocol was about 2 times higher than the HCl protocol. In addition, the Ct value of the former protocol was significantly lower than that of the latter (p<.001) with the estimated difference being about 7. Furthermore, the Ct values of EDTA processed samples were lower than 30, demonstrating that the amount of intact DNA feasible for PCR with the EDTA protocol is better preserved by a factor of 2 [7] than the HCl protocol.

Bottom Line: A method that can effectively decalcify while preserving genetic material is necessary.Staining after the RDO method had equivocal results.The EDTA protocol would be the best in preserving genetic material.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus