Analysis of the Effects of Bone Marrow Biopsy Decalcification Methods on Histopathological Examination

Decalcification is routinely performed to obtain a pathological diagnosis using bone marrow biopsy. During the decalcification process using a conventional acidic solution, such as HCl, the antigenicity of tissue is damaged. Especially DNA and RNA in the bone marrow are impaired. Hence, there is the need for a standardized decalcification protocol that preserves the antigenicity of tissue. To this end, we compared the effects of two commonly used decalcifiers: Commercial decalcifier (Calcl-Clear Rapid, HCl) and the EDTA (12.5%, pH 7.0). Bone marrow biopsies sampled from 71 patients were decalcified in accordance with the protocols of respective groups—HCI versus EDTA. The differences of decalcification protocols were analyzed with respect to Hematoxylin & Eosin staining, Gomori’sreticulum staining, and immunohistochemical staining and molecular analysis. Immunohistochemical staining used Ki-67, CD20 and CD138 as primary antibodies and molecular analysis was conducted through the DNA concentration analysis, in situ hybridization (ISH) and immunoglobulin heavy chain (IGH) gene rearrangement. On the routine histopathology analysis, there was no difference between HCl and EDTA. Moreover, in case of immunohistochemical staining, the cytoplasmic membrane or cytoplasmic CD markers was well preserved. However, nuclear proteins, such as Ki-67, were stained with low quality. Conversely, according to the molecular analysis, the EDTA protocol preserved the DNA and RNA compared with the HCI. The differences of DNA quantity and quality were statistically significant between protocols of HCl and EDTA. We used 38 cases in HCl and 12 cases in EDTA. Consequently, the EDTA protocol maintains the antigenicity of the protein on tissue and is acceptable for examination with molecular base analysis. Decalcification of bone marrow biopsy by EDTA is highly recommended for the examination of immunohistochemical staining and molecular analysis.