Non-specific immunohistochemical responses in surgical tissue collected from total joint arthroplasty

Tissues retrieved during revision arthroplasty (n = 25) were processed for routine immunohistochemical techniques to localize the presence of bone resorbing cytokines (IL-1, TNF alpha, and IL-6), and to identify areas of significant necrosis and metallic deposition. Tissues were processed and embedded according to standard laboratory techniques. Several tissue sections were prepared for each sample collected. Several sequential slides were used to demonstrate a thorough histopathological evaluation. Other slides of the same tissues were processed for immunohistochemical evaluation specifically for the localization of cytokines. To identify specific cytokine reactions, antibodies against cytokines were added in working dilutions overnight. Another set of slides were incubated overnight with nonspecific protein and served as negative controls. At the end of the incubation period, all slides were washed in buffer and a second antibody conjugated with peroxidase was added to both sets of slides for one hour. The slides were developed for peroxidase activity. The results revealed that there were several locations on the tissues that appeared highly reactive for cytokines. However, some of these areas turned out to be nonspecific when compared with negative controls and the Hematoxylin and Eosin stained slides. Areas of extreme necrosis or where metal debris was present demonstrated sites of nonspecific reactivity. Areas of necrosis have a high nonspecific affinity for antibodies and these areas of necrosis can be demonstrated on the Hematoxylin and Eosin sections, and were avoided in determining the amount of specific cellular reactivity. The results of this study strongly suggest that the aforementioned factors must be carefully controlled in determining true cellular responses that causes resorption of bone and eventually implant failure.