The effect of decalcification on in situ hybridization

In situ hybridization (ISH) for mRNA polyadenylated sequences was performed on 25 non-neoplastic and neoplastic decalcified, paraffin-embedded tissues using a poly d(T) oligonucleotide probe to assess the efficacy of this molecular diagnostic tool on decalcified tissue samples. Three commercially available decalcifying agents were used, including one EDTA-based solution (Versenate) and two hydrochloric acid-based solutions (S/P Decal, RBD). Before decalcification, the tissues were fixed in formalin for 6, 24, and 72 hours, respectively. The results of ISH performed on decalcified tissues were compared with the results from the nondecalcified control samples for each tissue using a numeric scoring system (0, negative; 4, strong positivity equal to control; 5, stronger than control). There was generally excellent reactivity when using Versenate (mean, 4.15), good reactivity with S/P Decal (mean, 3.17), and fair-to-poor reactivity with RBD (mean, 1.69) (all P values < .0001). The length of time in formalin did not affect the outcome of ISH on these tissues. We conclude that ISH can be performed with success on decalcified, paraffin-embedded tissues when using Versenate, an EDTA-based agent. Although accurate results might be obtained with S/P Decal and RBD, caution should be exercised when using these two hydrochloric acid-based solutions because they might produce false-negative results.