U.S. flag

An official website of the United States government, Department of Justice.

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

NCJ Number
Journal of Forensic Sciences Dated: February 2022
Jered B. Cornelison PhD; Carolyn V. Isaac PhD; Clara J. Devota BS; Joseph Billian MS; Theodore T. Brown MD; Joyce L. deJong DO; Elizabeth A. Douglas MD; Amanda O. Fisher-Hubbard MD; Wendy L. Lackey-Cornelison PhD; Joseph A. Prahlow MD; Brandy L. Shattuck MD
Date Published
February 2022

Since the extraction of mineral calcium from bone by decalcification is a critical step in the preparation of histological samples for light microscopy, the current study assessed the time required for complete decalcification and the resultant histomorphological preservation of bone histomorphology by three decalcification agents: 7 percent hydrochloric acid (HCl), 5 percent nitric acid, and 10 percent ethylenediaminetetraacetic acid (EDTA).


The goal of this study was to identify which decalcification agent provides the optimal combination of expedient processing and quality histological outcomes of cranial fracture samples. HCl provided the most rapid decalcification ( 𝑋⎯⎯⎯  = 3.57 days), nitric acid followed closely ( 𝑋⎯⎯⎯  = 10.35 days), while EDTA took significantly longer on average ( 𝑋⎯⎯⎯  = 78.97 days) but encompassed a broader range of times. Decalcification agent, sample thickness, sample width, and decedent age were significant predictors of decalcification time. Sample visualization quality, measured for tissues, cells, and nuclei on a five-point Likert scale, was highest for samples decalcified in 10 percent EDTA, second highest using 5 percent nitric acid, and lowest for 7 percent HCl. The quality difference between EDTA and nitric acid was not highly significant for any of the three features. For basic assessments of bone histomorphology, the study results indicate 5 percent nitric acid is suitable for the decalcification of adult specimens and samples thicker than 3 mm. EDTA is a suitable agent for thin samples of the cranial vault (<3 mm) from infants and young children less than three years old, decalcifying samples in a timeframe comparable to nitric acid while providing the best quality and clarity of samples. (publisher abstract modified)

Date Published: February 1, 2022