STANDARDIZATION OF IMMUNOHISTOCHEMISTRY BASED ON ANTIGEN RETRIEVAL TECHNIQUE FOR ROUTINE FORMALIN-FIXED TISSUE-SECTIONS

Worldwide use of the antigen retrieval (AR) technique has served to en hance the value of immunohistochemistry (IHC) for routine surgical pat hology and has provided promise of improved reproducibility of MC stai ning. However, after the initial introduction of the AR method, many v ariations and ''improvements'' have been proposed, including different heating methods, heating times, and buffer solutions, with strong adv ocates of different variations of the method. Our approach has been to seek to standardize the end result of AR and IHC staining, rather tha n the details of the technical procedure per se. This approach focuses on determining the AR method that leads to optimal results for each i ndividual laboratory and each antibody; we refer to this optimal resul t as ''maximal retrieval.'' To explore the possibility of standardizat ion of IHC through achieving ''maximal retrieval,'' we used a ''test b attery'' that examines the principal technical variables for archival paraffin tissue sections. Tests were conducted using monoclonal antibo dies to AE1, MIB1, p53 (Pab-1801, DO7, and BP53-12-1) for AR-IHC on fo rmalin-fixed, paraffin-embedded tissues of renal carcinoma and breast carcinoma, that had been fixed in formalin for different periods: 4, 1 2, and 24 hours and 3, 7, 14, and 30 days. Using the ''test battery'' approach, varying the buffer solution, pH, and heating time, an optima l protocol of AR could be established. For MTB1, Tris-HCl buffer of pH 1 yielded maximal retrieval for various tissues tested; for all other antibodies tested, both Tris-HCl buffer of pH 1 and pH 10 may yield m aximal retrieval results for all tested tissue sections. Establishing optimal or maximal retrieval is a significant step toward the standard ization of IHC on archival formalin paraffin tissues.