Effect of prolonged formalin fixation on the immunohistochemical reactivity of breast markers

Prolonged formalin fixation is known to reduce the immunohistochemical reactivity of many paraffin section antibodies. Before the common use of heat induced epitope retrieval methods, vimentin reactivity was proposed as a marker of antigen preservation. To evaluate the effect of formalin fixation on breast tumor markers, multitumor blocks of tissue fixed in formalin for varying time intervals from 33 different infiltrating breast carcinomas were analyzed for the expression of estrogen receptor (ER), progesterone receptor (PR), c-erb-B2, Ki-67, p27, and vimentin. The mean/median length of the longest fixation time per specimen was 53/42 days (range 7 days-154 days). Formalin fixation did not significantly reduce immunoreactivity for Ki-67, p27, or vimentin, even in tissue fixed for 154 days. Of 23 ER-positive cases, a significant reduction in immunoreactivity (2 grades or more) was identified in three samples, occurring at 57 to 64 days. For 21 PR-positive cases, only one showed a significant reduction (from 3+ to 1+) at 120 days. Of nine c-erb-B2 positive (2 + or 3+) cases, four became negative (1+ or 0) at 20, 42, 49, and 99 days. The immunoreactivity of some breast prognostic markers is reduced by formalin over-fixation, but only after extensive fixation that may not be clinically relevant. The loss of antigen preservation is not accompanied by a loss of vimentin immunoreactivity, making vimentin a suboptimal marker for ER, PR, or c-erb-B2 preservation.