BRIEF REPORT |
Correspondence to: R.S.D. Brown, Dept. of Radiotherapy, St Bartholomew's Hospital, West Smithfield, London EC1A 7BE, UK. E-mail: richard.brown@ucl.ac.uk
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Summary |
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Production of paraffin-section material from tissue samples that contain bone requires decalcification. Techniques such as acidic decalcification or EDTA chelation are suitable methods. Acid decalcification is generally quicker than EDTA chelation but studies have suggested that it may result in hydrolysis of DNA. Here we show that limited acid decalcification (less than 24 hr) in 5% formic acid can preserve DNA sufficient for fluorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH) and that prolonged 10% formic acid decalcification results in failure of FISH and only limited retrieval of DNA for CGH studies. (J Histochem Cytochem 50:113115, 2002)
Key Words: bone marrow, comparative genomic, hybridization (CGH), decalcification, DNA, formic acid, fluorescent in situ hybridization, (FISH)
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Introduction |
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Routine acid decalcification (RDO; Apex Engineering Products, Plainfield, IL) for bone marrow trephine and autopsy bone samples has been reported to result in failure to obtain DNA for in situ hybridization (ISH), comparative genomic hybridization (CGH), and flow cytometric studies in trephine and autopsy bone marrow samples from prostate cancer (
We also routinely use acid decalcification for bone marrow trephine specimens (1218 hr) but use a 5% formic acid (Becton Dickinson Laboratory Supplies; Mountain View, CA) solution in distilled water rather than a hydrochloric acid-based product (RDO). This method of decalcification has enabled us to successfully perform FISH on 15 of 15 samples analyzed for androgen receptor (AR) gene copy number in bone metastases from prostate cancer (SpectrumOrange AR FISH probe, SpectrumGreen centromere FISH probe for the X chromosome; Vysis, Downers Grove, IL) (see Fig 1A). AR gene amplification has been implicated in the development of hormone-refractory prostate cancer (HRPC), being found in approximately 30% of locally recurrent hormone-refractory biopsies and rarely in hormone-naïve specimens (
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The FISH studies on decalcified trephine material required an additional pretreatment, i.e., microwave antigen retrieval technique, before standard tissue digestion with pepsin (M. Farquharson, Department of Pathology, Glasgow Royal Infirmary; personal communication).
Formic acid decalcification has also enabled us to successfully hybridize extracted tumor DNA from decalcified bone marrow trephines to normal metaphase chromosomes in five cases studied thus far (Fig 2) for CGH. Degenerate oligionucleotide primer (5'-CGACTCGAGNNNNNNATGTGG-3') polymerase chain reaction (DOP-PCR) products of up to 1500 base pairs in length have been generated from all 15 decalcified trephine samples (Fig 3).
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For larger autopsy bone marrow specimens, we use a 10% formic acid solution for decalcification over a 710-day period (with replenishment of the solution on one to two occasions during this period). Similarly to
Little published information is available on the effects of formic acid decalcification on DNA degradation. We have been unable to find other reports of successful studies using FISH or CGH on formic acid-decalcified bone marrow trephine biopsies (Medline Search 19662001).
We conclude that routine acid decalcification with 5% formic acid can satisfactorily preserve DNA for some types of molecular biological studies (FISH and CGH) on prostate cancer bone metastases taken by trephine biopsy. Longer and stronger acid decalcification resulted in unsuccessful FISH in all autopsy specimens, whereas non-calcified material from the same postmortem samples worked satisfactorily. Decalcification using EDTA appears to offer the best chance of successful DNA retrieval from bone tissue and appears to be the method of choice for decalcification in a prospective study of bone marrow trephine and autopsy samples. Five percent formic acid decalcification avoids major acid hydrolysis of DNA, as judged by our results with FISH and CGH studies on decalcified trephine material. Attempts to use techniques such as FISH and CGH on archival material should not automatically be abandoned if retrospectively collected specimens have been decalcified in formic acid.
Received for publication June 27, 2001; accepted September 5, 2001.
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Literature Cited |
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Alers JC, Krijtenberg P-J, Vissers KJ, van Dekken H (1999) Effect of bone decalcification procedures on DNA in situ hybridization and comparative genomic hybridization: EDTA is highly preferable to a routinely used acid decalcifier. J Histochem Cytochem 47:703-709
Bubendorf L, Kononen J, Koivisto P, Schraml P, Moch H, Gasser TC, Willi N, Mihatsch MJ, Sauter G, Kallioniemi OP (1999) Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays. Cancer Res 59:803-806
Provan AB, Hodges E, Smith AG (1992) Use of paraffin wax embedded bone marrow trephine biopsy specimens as a source of archival DNA. J Clin Pathol 45:763-765[Abstract]
Sarsfield P, Wickham CL, Joyner MV, Ellard S, Jones DB, Wilkins BS (2000) Formic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products. Mol Pathol 53:336
Visakorpi T, Hyytinen E, Koivisto P, Tanner M, Keinanen R, Palmberg C, Palotie A, Tammela T, Isola J, Kallioniemi OP (1995) In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nature Genet 9:401-406[Medline]
Wickham CL, Boyce M, Joyner MV, Sarsfield P, Wilkins BS, Jones DB, Ellard S (2000) Amplification of PCR products in excess of 600 base pairs using DNA extracted from decalcified, paraffin wax embedded bone marrow trephine biopsies. Mol Pathol 53:19-23