Effects of Fixation and Tissue Processing on Immunohistochemical Demonstration of Specific Antigens

Abstract

Identification of biomarkers in archival tissues using immunochemistry is becoming increasingly important for determining the diagnosis and prognosis of tumors, for characterizing preinvasive neoplastic changes in glandular tissues such as prostate, for evaluating the response of tumors and preinvasive neoplastic changes to certain therapies (i.e., as a surrogate intermediate end point), for selecting patients who are candidates for specific therapies (e.g., immunotherapy) and for retrospective studies. For detecting specific biomarkers it is important to understand the limitations imposed by the fixation methods and processing of the tissues. This study was designed to determine the effects of fixation on the detection in archival paraffin blocks of selected antigens postulated to be important in tumor biology. We evaluated the antigens TGF alpha, p185erbB-2, broad spectrum keratins, p53, and TAG-72 (B72.3). Fixatives evaluated included standard preparations of neutral buffered formalin, acid formalin, zinc formalin, alcoholic formalin, ethanol, methanol, and Bouin's fixative. We found that in general neutral buffered formalin is the poorest fixative for maintaining antigen recognition by immunohistochemistry and that no single fixative was best for all antigens. The dehydrating (coagulant) fixatives (e.g., ethanol and methanol) preserved immunorecognition of p53 and broad spectrum keratins best while the slow cross-linking fixatives (e.g., unbuffered zinc formalin) were best for demonstrating TGF alpha and p185erbB-2. Fixatives other than neutral buffered formalin produced equivalent recognition of the epitope of TAG-72 by B72.3. In formalin fixed archival tissues, only a portion of the antigen signal can be detected by routine immunohistologic methods.

Zinc-Based Fixative Improves Preservation of Genomic DNA and Proteins in Histoprocessing of Human Tissues

“…However, most fixatives have in common that they are originally designed for an optimal histologic examination. For immunorecognition there is no best fixative of all antigens; rather an antigen and antibody clone and a fixative-processing method must be matched to optimize immunorecognition (Arnold et al, 1996). This fact probably explains why some proteins in this study are better preserved in ZBF, whereas others are better preserved in NBF.…”

Suggested Guidelines for Immunohistochemical Techniques in Veterinary Diagnostic Laboratories

“…Formalin fixation produces methylene bridges between free amino groups and other functional groups of proteins, resulting in conformational changes in the tertiary and quaternary structures within proteins and cross-linking between tissue proteins, thus modifying the epitopes recognized by antibodies. 3,15,21,22,43,71 However, it needs to be emphasized that adequate fixation is essential in IHC. Underfixation of tissues for IHC testing is one of the major problems in human pathology.…”

When Tissue Antigens and Antibodies Get Along

“…130 The immunoreactivity of antigens in fixed and paraffinembedded tissue varies markedly depending on the antigen and more specifically on the epitope recognized by the antibody. 8,59 In a comparison of the effect of different fixatives (strong cross-linkers, weak cross-linkers, coagulant, and combination coagulant/cross-linkers) on antigen immunoreactivity in mouse tissue, formaldehyde, followed by the combination fixatives, performed better than the other types. 59 Substitution of a different fixative in an IHC test validated for formalin-fixed tissue demands a new validation study.…”