Fast and Molecular Friendly Heat and Mechanical Agitation Based EDTA Decalcification of Bone Marrow Trephine Biopsies Provides High Quality DNA with Preserved Histology and Immunohistochemistry
Menon M, Whiteley L, Beher J, Karmo N, Fletcher F, Adamczyk K, Gomez-Gelvez J, Inamdar K, Cankovic M, Chitale DA, and Zarbo R. Fast and Molecular Friendly Heat and Mechanical Agitation Based EDTA Decalcification of Bone Marrow Trephine Biopsies Provides High Quality DNA with Preserved Histology and Immunohistochemistry. Lab Invest 2018; 98:814.
Background: Modern hematopathology relies heavily on molecular studies as diagnostic, prognostic and predictive ancillary studies, especially with the advent of next generation sequencing (NGS). While bone marrow (BM) aspirate smears continue to be used as the primary DNA source, hemodilution and BM fibrosis might make them non-representative of native BM including lack of potential sub clones. Traditional Hydrochloric acid (HCL) based decalcification (HD) makes bone marrow trephine biopsy non-amenable to DNA studies. We demonstrate a temperature and magnetic stirring based EDTA decal (ED) method that preserves DNA and provides a reasonable turnaround time. Design: BM biopsies were received in formalin and treated with HCL (RDO rapid decal, Apex, Aurora, IL) or 10% EDTA (Mol- Delcalcifier and BoneStation, Milestone Medical, Kalamazoo, MI) (at 37 and 50 °C) followed by regular processor vs. rapid processor (Tissue Tek VIP 300E vs. Tissue-Tex Xpress x50, Sakura, Torrance, CA). Immunohistochemistry (IHC) was performed for CD20, CD3, CD34, TdT, CD138 and Tryptase. DNA was evaluated by spectrophotometry and amplification of control size ladder mix generating amplicons of 100, 200, 300, and 400 base pairs (bp) (Invivoscribe, San Diego, CA). Student T test was used for statistical analyses. Results: BM histology and IHC was unaltered by ED (37 °C or 50 °C) or use of regular vs. rapid processor. When comparing ED method across all conditions, best results were obtained with ED at 50 °C + regular tissue processor with reliable amplification up to 300 bps. For 400 bp, this method reliably generated amplicons albeit at lower peak heights (as compared to 100-300 bp) while other conditions mostly failed to generate any 400 bp amplicons. In contrast, HD amplified DNA only up to 100 bp although mean peak amplicon height for 100 bp was substantially less than ED method (13732 vs. 24275). Surprisingly, DNA quality of ED was superior to clot section. ED (50 °C) with regular processor demonstrated statistically significant superiority over use of rapid processor at either 37 or 50 °C for 100- 300 bp (p<0.05). Conclusions: We demonstrate that increasing temperature to 50 °C (with magnetic stirring) during ED does not alter histology or immunohistochemistry and yields good quality DNA with reasonable turnaround time (approximately 5 hours decalcification) as compared to the historically long decalcification periods (up to 24 hours) of EDTA based methods. NGS studies are in progress to further validate this process.