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.
Citations
Citations to this article as recorded by
Optimization of Formic Acid-Formalin-Based Decalcification Protocol for Rat Calvarial Bone Histology S. Amitha Banu, Khan Sharun, Merlin Mamachan, Athira Subash, Vadapalli Deekshita, Kirtika Sharma, Karikalan Mathesh, Obli Rajendran Vinodh kumar, Swapan Kumar Maiti, Abhijit M. Pawde, Laith Abualigah, Kuldeep Dhama, Amarpal Journal of Experimental Biology and Agricultural Sciences.2024; 12(2): 218. CrossRef
Effects of fixation and demineralization on histomorphology and DNA amplification of canine bone marrow Gabriella M. L. Diamantino, Janet Beeler-Marfisi, Robert A. Foster, William Sears, Alice Defarges, William Vernau, Dorothee Bienzle Veterinary Pathology.2024; 61(6): 943. CrossRef
In situ metabolomic analysis of osteonecrosis of the femoral head (ONFH) using MALDI MSI Chen Li, Jikun Liu, Yiqi Sheng, Yinghao Wang, Lan Jia, Yinguang Zhang, Jiantao Li, Shuangshuang Di, Honggang Nie, Yehua Han Analytical and Bioanalytical Chemistry.2024; 416(23): 5155. CrossRef
A set of pretreatment reagents including improved formula fixation and decalcification facilitating immunohistochemistry and DNA analyses of formalin-fixed paraffin-embedded bone marrow trephine biopsy Ting Sun, Liming Xu, Hongtian Yao, Jing Zhao, Zhen Chen, Zexin Chen, Bo Wang, Wei Ding Acta Histochemica.2024; 126(8): 152188. CrossRef
Germline and somatic testing for homologous repair deficiency in patients with prostate cancer (part 1 of 2) Andrew J. Armstrong, Amy Taylor, Michael C. Haffner, Wassim Abida, Alan H. Bryce, Lawrence I. Karsh, Scott T. Tagawa, Przemyslaw Twardowski, Anthony V. Serritella, Joshua M. Lang Prostate Cancer and Prostatic Diseases.2024;[Epub] CrossRef
To Freeze or Not to Freeze? Recommendations for Intraoperative Examination and Gross Prosection of Thyroid Glands Fouad R. Zakka, Nicole A. Cipriani Surgical Pathology Clinics.2023; 16(1): 15. CrossRef
Effect of Surface Decalcification With Hydrochloric Acid on the Determination of Estrogen Receptor, Progesterone Receptor, Ki67, and Human Epidermal Growth Factor Receptor 2 Expressions in Invasive Breast Carcinoma Based on Immunohistochemistry and Fluore Wu Ping, Rao Xin, Zhang Li, Chen Yupeng, Song Fangling, Ren Caihong, Hu Shun, Zhang Sheng Applied Immunohistochemistry & Molecular Morphology.2023; 31(4): 232. CrossRef
Diagnostic value of MDM2 RNA in situ hybridization for low-grade osteosarcoma: Consistency comparison of RNA in situ hybridization, fluorescence in situ hybridization, and immunohistochemistry Chen Chen, Xin He, Min Chen, Tianhai Du, Weiji Qin, Wenyi Jing, Hongying Zhang Virchows Archiv.2023; 482(6): 999. CrossRef
Bone marrow fibrosis is associated with non‐response to CD19 CAR T‐cell therapy in B‐acute lymphoblastic leukemia Joshua Anil, Ahab Alnemri, Andrew Lytle, Brian Lockhart, Ashley E. Anil, Michael Baumgartner, Kirubel Gebre, Jared McFerran, Stephan A. Grupp, Susan R. Rheingold, Vinodh Pillai American Journal of Hematology.2023; 98(12): 1888. CrossRef
Epithelioid haemangioendothelioma of the mandible – A case report and review of the literature Ali Rizvi, Tim K. Blackburn, Guy N. J. Betts Oral Surgery.2022; 15(3): 387. CrossRef
Evaluation of EDTA and nitric acid solutions for decalcification of joints in AG/WT, BALB/c, C57, DBA1/J mice, and in Wistar rats Eduarda Correa Freitas, Suelen Pizzolatto Dalmolin, Mateus Müller da Silva, Francine Hehn de Oliveira, Emily Ferreira Salles Pilar Biotechnic & Histochemistry.2022; 97(5): 372. CrossRef
Coupling Lipid Labeling and Click Chemistry Enables Isolation of Extracellular Vesicles for Noninvasive Detection of Oncogenic Gene Alterations Na Sun, Benjamin V. Tran, Zishan Peng, Jing Wang, Ceng Zhang, Peng Yang, Tiffany X. Zhang, Josephine Widjaja, Ryan Y. Zhang, Wenxi Xia, Alexandra Keir, Jia‐Wei She, Hsiao‐hua Yu, Jing‐Jong Shyue, Hongguang Zhu, Vatche G. Agopian, Renjun Pei, James S. Toml Advanced Science.2022;[Epub] CrossRef
The Expressions of CD30 and CD123 of Mastocytosis in Taiwan Ching-Fen Yang, Chih-Yi Hsu Applied Immunohistochemistry & Molecular Morphology.2022; 30(4): 278. CrossRef
Unusual Patterns of HER2 Expression in Breast Cancer: Insights and Perspectives Dora Grassini, Eliano Cascardi, Ivana Sarotto, Laura Annaratone, Anna Sapino, Enrico Berrino, Caterina Marchiò Pathobiology.2022; 89(5): 278. CrossRef
Expert opinion on NSCLC small specimen biomarker testing — Part 1: Tissue collection and management Frédérique Penault-Llorca, Keith M. Kerr, Pilar Garrido, Erik Thunnissen, Elisabeth Dequeker, Nicola Normanno, Simon J. Patton, Jenni Fairley, Joshua Kapp, Daniëlle de Ridder, Aleš Ryška, Holger Moch Virchows Archiv.2022; 481(3): 335. CrossRef
Comparison of bone demineralisation procedures for DNA recovery from burned remains Meghan Mckinnon, Denice Higgins Forensic Science International: Genetics.2021; 51: 102448. CrossRef
A review of the current understanding of burned bone as a source of DNA for human identification Meghan Mckinnon, Maciej Henneberg, Denice Higgins Science & Justice.2021; 61(4): 332. CrossRef
Time is bone — Quantitative comparison of decalcification solvents in human femur samples using dual-X-ray-absorptiometry and computed tomography Joshua Gawlitza, Jakob Steinhäuser, Arno Bücker, Gabriela Krasteva-Christ, Thomas Tschernig Annals of Anatomy - Anatomischer Anzeiger.2021; 235: 151696. CrossRef
Molecular biomarker testing for non–small cell lung cancer: consensus statement of the Korean Cardiopulmonary Pathology Study Group Sunhee Chang, Hyo Sup Shim, Tae Jung Kim, Yoon-La Choi, Wan Seop Kim, Dong Hoon Shin, Lucia Kim, Heae Surng Park, Geon Kook Lee, Chang Hun Lee Journal of Pathology and Translational Medicine.2021; 55(3): 181. CrossRef
Effect of EDTA decalcification on estrogen receptor and progesterone receptor immunohistochemistry and HER2/neu fluorescence in situ hybridization in breast carcinoma Erik Washburn, Xiaoyu Tang, Carla Caruso, Michelle Walls, Bing Han Human Pathology.2021; 117: 108. CrossRef
Performances of single tube nested polymerase chain reaction and GeneXpert ultra on Formalin fixed paraffin embedded tissues in the diagnosis of tuberculous spondylodiscitis Emna Romdhane, Soumaya Rammeh, Chelli Mouna Bouaziz, Hend Riahi, Meriam Rekaya Ben, Meriam Ksentini, Yosra Chebbi, Wafa Achour, Asma Ferjani, Ben Boubaker Ilhem Boutiba, Leila Slim-Saidi, Mohamed Fethi Ladeb Clinical Rheumatology.2021; 40(10): 4317. CrossRef
Molecular Characterization of Prostate Cancers in the Precision Medicine Era Emilio Francesco Giunta, Laura Annaratone, Enrico Bollito, Francesco Porpiglia, Matteo Cereda, Giuseppe Luigi Banna, Alessandra Mosca, Caterina Marchiò, Pasquale Rescigno Cancers.2021; 13(19): 4771. CrossRef
Increased NF-κB Activity in Osteoprogenitor-Lineage Cells Impairs the Balance of Bone Versus Fat in the Marrow of Skeletally Mature Mice Tzuhua Lin, Jukka Pajarinen, Yusuke Kohno, Akira Nabeshima, Laura Lu, Karthik Nathan, Zhenyu Yao, Joy Y. Wu, Stuart Goodman Regenerative Engineering and Translational Medicine.2020; 6(1): 69. CrossRef
Percutaneous CT-guided biopsy of lytic bone lesions in patients clinically suspected of lung cancer: Diagnostic performances for pathological diagnosis and molecular testing Anne-Claire Toffart, Stéphane Asfari, Anne Mc Leer, Emilie Reymond, Adrien Jankowski, Denis Moro-Sibilot, Olivier Stephanov, Julien Ghelfi, Sylvie Lantuejoul, Gilbert R. Ferretti Lung Cancer.2020; 140: 93. CrossRef
Effect of decalcification protocols on immunohistochemistry and molecular analyses of bone samples Elodie Miquelestorena-Standley, Marie-Lise Jourdan, Christine Collin, Corinne Bouvier, Frédérique Larousserie, Sébastien Aubert, Anne Gomez-Brouchet, Jean-Marc Guinebretière, Matthias Tallegas, Bénédicte Brulin, Louis-Romée Le Nail, Anne Tallet, François Modern Pathology.2020; 33(8): 1505. CrossRef
Identifying Opportunities and Challenges for Patients With Sarcoma as a Result of Comprehensive Genomic Profiling of Sarcoma Specimens Margaret A. Hay, Eric A. Severson, Vincent A. Miller, David A. Liebner, Jo-Anne Vergilio, Sherri Z. Millis, James L. Chen JCO Precision Oncology.2020; (4): 176. CrossRef
Comparison of Methods for the Histological Evaluation of Odontocete Spiral Ganglion Cells Tania Ramírez, Simona Sacchini, Yania Paz, Rubén S. Rosales, Nakita Câmara, Marisa Andrada, Manuel Arbelo, Antonio Fernández Animals.2020; 10(4): 683. CrossRef
Molecular Pathology of Primary Non-small Cell Lung Cancer David Ilan Suster, Mari Mino-Kenudson Archives of Medical Research.2020; 51(8): 784. CrossRef
Comparison of ethylenediaminetetraacetic acid and rapid decalcificier solution for studying human temporal bones by immunofluorescence Sumana Ghosh, Mark B. Lewis, Bradley J. Walters Laryngoscope Investigative Otolaryngology.2020; 5(5): 919. CrossRef
A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens Pavla Ticha, Igor Pilawski, Xue Yuan, Jie Pan, Ustun S. Tulu, Benjamin R. Coyac, Waldemar Hoffmann, Jill A. Helms Scientific Reports.2020;[Epub] CrossRef
Accelerating precision medicine in metastatic prostate cancer Joaquin Mateo, Rana McKay, Wassim Abida, Rahul Aggarwal, Joshi Alumkal, Ajjai Alva, Felix Feng, Xin Gao, Julie Graff, Maha Hussain, Fatima Karzai, Bruce Montgomery, William Oh, Vaibhav Patel, Dana Rathkopf, Matthew Rettig, Nikolaus Schultz, Matthew Smith, Nature Cancer.2020; 1(11): 1041. CrossRef
Tissue Morphology and Antigenicity in Mouse and Rat Tibia: Comparing 12 Different Decalcification Conditions Kristofor Bogoevski, Anna Woloszyk, Keith Blackwood, Maria A. Woodruff, Vaida Glatt Journal of Histochemistry & Cytochemistry.2019; 67(8): 545. CrossRef
Cellular and collagen reference values of gingival and periodontal ligament tissues in rats: a pilot study Antoine Alves, Nina Attik, Carine Wirth, Yves Bayon, Alexis Piat, Brigitte Grosgogeat, Kerstin Gritsch Histochemistry and Cell Biology.2019; 152(2): 145. CrossRef
Implementing Precision Medicine Programs and Clinical Trials in the Community-Based Oncology Practice: Barriers and Best Practices Jennifer L. Ersek, Lora J. Black, Michael A. Thompson, Edward S. Kim American Society of Clinical Oncology Educational Book.2018; (38): 188. CrossRef
Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL Zandra C Deans, Jose Luis Costa, Ian Cree, Els Dequeker, Anders Edsjö, Shirley Henderson, Michael Hummel, Marjolijn JL Ligtenberg, Marco Loddo, Jose Carlos Machado, Antonio Marchetti, Katherine Marquis, Joanne Mason, Nicola Normanno, Etienne Rouleau, Ed S Virchows Archiv.2017; 470(1): 5. CrossRef
Protocolo para el estudio de muestras y estandarización del informe patológico de tumores óseos Isidro Machado, José Juan Pozo, David Marcilla, Julia Cruz, Juan C. Tardío, Aurora Astudillo, Sílvia Bagué Revista Española de Patología.2017; 50(1): 34. CrossRef
Extremely Well-Differentiated Papillary Thyroid Carcinoma Resembling Adenomatous Hyperplasia Can Metastasize to the Skull: A Case Report Ju Yeon Pyo, Jisup Kim, Sung-eun Choi, Eunah Shin, Seok-Woo Yang, Cheong Soo Park, Seok-Mo Kim, SoonWon Hong Yonsei Medical Journal.2017; 58(1): 255. CrossRef
Treatment of steroid-induced osteonecrosis of the femoral head using porous Se@SiO2 nanocomposites to suppress reactive oxygen species Guoying Deng, Kerun Niu, Feng Zhou, Buxiao Li, Yingjie Kang, Xijian Liu, Junqing Hu, Bo Li, Qiugen Wang, Chengqing Yi, Qian Wang Scientific Reports.2017;[Epub] CrossRef
Precision Medicine Starts With Preanalytics: Real-Time Assessment of Tissue Fixation Quality by Ultrasound Time-of-Flight Analysis Melissa L. Lerch, Daniel R. Bauer, David Chafin, Abbey Theiss, Michael Otter, Geoffrey S. Baird Applied Immunohistochemistry & Molecular Morphology.2017; 25(3): 160. CrossRef
Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests Jihun Kim, Woong-Yang Park, Nayoung K. D. Kim, Se Jin Jang, Sung-Min Chun, Chang-Ohk Sung, Jene Choi, Young-Hyeh Ko, Yoon-La Choi, Hyo Sup Shim, Jae-Kyung Won Journal of Pathology and Translational Medicine.2017; 51(3): 191. CrossRef
An international survey about nail histology processing techniques Christina Wlodek, Pauline Lecerf, Josette Andre, Beth S. Ruben, David de Berker Journal of Cutaneous Pathology.2017; 44(9): 749. CrossRef
pSTAT5 and ERK exhibit different expression in myeloproliferative neoplasms Ewa Wiśniewska-Chudy, Łukasz Szylberg, Grzegorz Dworacki, Ewa Mizera-Nyczak, Andrzej Marszałek Oncology Reports.2017; 37(4): 2295. CrossRef
How we do: optimizing bone marrow biopsy logistics for sign-out within 2 days I. de Laak–de Vries, A. G. Siebers, L. Burgers, C. Diepenbroek, M. Link, P. Groenen, J. H. J. M. van Krieken, K. M. Hebeda Journal of Hematopathology.2016; 9(2): 67. CrossRef
Do More With Less: Tips and Techniques for Maximizing Small Biopsy and Cytology Specimens for Molecular and Ancillary Testing: The University of Colorado Experience Dara L. Aisner, Mathew D. Rumery, Daniel T. Merrick, Kimi L. Kondo, Hala Nijmeh, Derek J. Linderman, Robert C. Doebele, Natalie Thomas, Patrick C. Chesnut, Marileila Varella-Garcia, Wilbur A. Franklin, D. Ross Camidge Archives of Pathology & Laboratory Medicine.2016; 140(11): 1206. CrossRef
Analysis of the Effects of Bone Marrow Biopsy Decalcification Methods on Histopathological Examination Ji Young Park, Kyung Hee Han The Korean Journal of Clinical Laboratory Science.2016; 48(4): 371. CrossRef
Distinguishing between Microbial Habitats Unravels Ecological Complexity in Coral Microbiomes Amy Apprill, Laura G. Weber, Alyson E. Santoro, Nicole S. Webster mSystems.2016;[Epub] CrossRef
Optimal Fixation and Decalcification Methods for Bone Marrow Biopsy Myung-Sub Choi, Hyunsup Lee, Hyuk-Chul Kwon, Moon-Hwan Bae, Young-Hye Ko, Hee-Jin Kim, Beom-Se Lee, Bon-Kyung Koo Korean Journal of Clinical Laboratory Science.2015; 47(4): 243. CrossRef