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Indian J Pathol Microbiol. 2019 Jul-Sep;62(3):423-429. doi: 10.4103/IJPM.IJPM_853_18.

A novel approach to decalcification in histopathology laboratory: An adaptation from the Hammersmith protocol.

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Currently working in Department of Pathology, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha, India.
Department of Pathology, Jawaharlal Institute of Post graduate Medical Education and Research (JIPMER), Puducherry, India.



Utility of modified Hammersmith protocol in the deacalcification and/or softening of tissues and samples in a histopathology laboratory were studied. The object of the study was to prepare a novel method for softening/decalcifying tissue for histopathology.

Materials and Methods:

All the hard tissues received in the histopathology section were received in 10% neutral buffered formalin and then placed in freshly prepared combination of 10 mL of concentrated formaldehyde and 5 mL of 10% formic acid in 85 mL distilled water was used for decalcification. The tissue was checked for evidence of adequate decalcification/softening every 6 hours. Those which were decalcified/softened were sent for routine tissue processing and staining, while those which were not, were again placed in formalin. The process was repeated until the tissue was ready for further processing. The routine sections of these slides were reviewed for morphology and stain quality along with special stains and immunohistochemistry performed. The time taken for decalcification, the variables most likely to affect decalcification, the morphology and staining characteristics were documented. Statistical analysis was done to determine the effect of softening/decalcification process on each variable.


A total of 201 blocks in 119 specimens from humans including 61 males and 58 females were studied. Time taken was found to have a significant correlation only with the nature of the tissue (bone vs nonbone) and not with any other parameter viz. age, gender, specimen size, type of bone, and nature of pathology.


This novel and modified method has circumvented the common problems of overdecalcification, preserved morphology, and produced consistent results without interfering with special stains and immunohistochemistry.


Bone; bone pathology; bone tumors; fixation; immunohistochemistry

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