Decalcification of bone specimens using the incubation method: A feasible option for reducing the turnaround time

Objectives: Bone decalcification is an essential step in the processing of surgical bone samples. This process is slow and takes days to weeks, depending on various factors. Heat acceleration enhances decalcification. Our objective was to compare the turnaround time and histomorphology of conventional and incubation methods of decalcification using 10% formic acid and 10% nitric acid. Materials and methods: Formalin-fixed tissues of 27 surgical samples that need decalcification were included. The turnaround time (TAT) for both methods of decalcification – conventional and incubation (45⁰C) by two decalcifying fluids- 10% nitric acid and 10% formic acid was calculated. The quality of the incubation method (at 45⁰C) and conventional method of decalcification using both decalcifying fluids was evaluated based on histomorphology. A pathologist scored the histomorphological details, including staining intensity, nuclear morphology, microtomy artifacts, and tissue integrity, as poor, good, and excellent. Statistical analysis: Comparison of time duration for decalcification between conventional and incubation methods was done using an independent students t test/Mann Whitney U test and Kruskal-Wallis test and expressed as median with interquartile range. The comparison of histomorphological details between the groups was done using the chi-square test/Fisher’s exact test. Results: The decalcification time taken by the incubation method is a median of 5 h with an interquartile range (IQR) of 2.7-10 h, compared to a decalcification time of a median of 9 h with an IQR of 6-19.6 h by the conventional method. Decalcification by the incubation method using 10% nitric acid took the minimum time. A 10% formic acid solution in the incubation method yields excellent staining intensity. The nuclear morphology by the incubation method (both formic acid and nitric acid) is superior to the conventional method. Conclusions: Using an incubator for decalcification significantly reduces TAT of decalcification with superior nuclear morphology compared to the conventional method.