Comparison between Conventional Decalcification and a Microwave-Assisted Method in Bone Tissue Affected with Mycetoma

Abstract

Mycetoma is a lifelong granulomatous disease of subcutaneous tissues and bones. Histopathology is a substantiated indicative method based on the assumption of a definitive diagnosis of mycetoma. It requires efficient processing of tissues including bone decalcification. The decalcification process must ensure complete removal of calcium and also a proper preservation of tissue and microorganisms’ staining ability. Objectives. To compare the conventional method used in decalcification with the microwave method using different decalcification solutions. Different characteristics were tested, including the speed of decalcification and morphological and fungal preservation in bone tissue affected with mycetoma. Materials and Methods. Three decalcification solutions were employed to remove calcium from 50 bone tissue samples affected with mycetoma, including 10% neutral buffered EDTA (pH 7.4), 5% nitric acid, and 5% hydrochloric acid. Conventional and microwave methods were used. Haematoxylin-eosin (HE) stain, Gridley’s stain, and Grocott hexamine-silver stain were employed to evaluate the bone and fungi morphologies. Results. The decalcification time of the conventional method compared with the microwave method with 10% EDTA (pH 7.4) took 120 hours and 29 hours, while 5% hydrochloric acid and 5% nitric acid took 8 hours and 3 hours, separately. Also, 10% EDTA is the best decalcifying agent for HE staining and fungal stains. 5% hydrochloric acid and 5% nitric acid can be used for fungal staining. Conclusion. The current study investigated the effects of different decalcifying agents as well as two decalcification procedures on the preservation of the bone structure and fungal staining, which will help to develop suitable protocols for the analyses of the bone tissue affected with mycetoma infection.

A comparison of three decalcification agents for assessments of cranial fracture histomorphology

“…According to our assessment, EDTA provides the best outcome for the preservation and clarity of the tissues, cells, and nuclei in the bones of the human cranial vault; however, nitric acid is a close second (Figure 3). The results of the Likert scoring mirror the consensus of the scientific community that HCl is a poor decalcification agent for bone histology [4,7,12]. The fast‐acting HCl leaves more room for potential over‐decalcification and subsequent damage to the bone’s microanatomy.…”

“…Acids rapidly extract calcium by reacting with hydroxyapatite to form soluble calcium salts, while chelating agents gradually diminish hydroxyapatite crystals by bonding to calcium ions to form stable agent‐Ca 2+ complexes [3]. EDTA, being the slowest decalcification solution compared to its acid counterparts, is well represented throughout the literature [4,5,7,8,10,12,14,16,17,20]. The general linear model for nitric acid and EDTA developed in this study, from which HCl was excluded due to an insufficient sample size, indicates the factors of decedent age, sample thickness, and width significantly impact decalcification time.…”

“…also influence the length of decalcification [1,[4][5][6]. Overall, strong acids are the most expedient, requiring only a few days [4,[7][8][9][10][11][12], while bone submerged in a chelating agent such as EDTA may take several months to fully decalcify [1,4,8,9,[13][14][15]. Under normal circumstances, forensic case reports are required to be completed in 30 to 60 days; thus, processing time is a decisive factor in the substance chosen for demineralization.…”

“…Numerous studies have performed comparative analyses of histological preservation by decalcifying agents, with a general consensus that slow decalcifications in EDTA provides superior outcomes for staining and cell and tissue morphology at the cost of lengthy immersion times [1,4,7,[11][12][13]15,16]. However, these inquiries are predominately based on animal models [4,5,7,8,11,[16][17][18] and human dentition [9,13,15], rather than human cranial bone.…”