Investigation of an appropriate decalcifying agent for bone biopsy specimens submitted for histomorphologic analyses and immunohistochemical studies

Decalcification is a critical pre-analytical step in histopathology for mineralized tissues, yet inappropriate agent selection and endpoint control can compromise histomorphologic detail and reduce the interpretability of immunohistochemical (IHC) stains. This study compared the effectiveness of five decalcifying agents for bone biopsy specimens intended for routine histomorphologic evaluation and downstream IHC: 10% hydrochloric acid (HCl), 15% formic acid (FA), Gooding and Stewart (formalin–formic acid), ethylenediaminetetraacetic acid (EDTA), and formic acid–EDTA (FEDTA). Ten bone biopsy specimens were decalcified using each agent, and decalcification endpoints were determined via physical and chemical testing. Morphologic preservation was assessed on hematoxylin and eosin (H&E)–stained sections using structured scoring of cellular and extracellular features, while IHC performance was evaluated using CD34, S100, SATB2, and SMA markers, scored for signal intensity/reactivity. The fastest decalcification endpoint was achieved by 10% HCl (mean 6.30 hours), whereas EDTA required the longest processing time (mean 159.20 hours). Despite slower turnaround, EDTA produced the highest overall H&E preservation score (mean 13.25/15), indicating superior retention of key histomorphologic structures. In contrast, 15% FA demonstrated relatively weaker SATB2 reactivity (mean 1.70), suggesting greater vulnerability of specific antigen targets under stronger acid conditions. Across the five agents, no statistically significant differences were detected among IHC marker outcomes (all p-values > 0.05), although EDTA and FEDTA tended to yield higher mean marker scores in several comparisons. Overall, EDTA is recommended when maximal morphologic preservation and future tissue usability (repeat IHC and ancillary testing) are priorities, while acid-based agents may be reserved for situations where rapid turnaround is essential and modest trade-offs in tissue integrity are acceptable.