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Abc‐immunoperoxidase staining of cytologic preparations: Improvement of specificity

Immunoperoxidase (IP) methods perfected on formalin‐fixed, paraffin‐embedded tissues (FFPE) and then applied to aspirate smears may result in high background staining and, a significant number of false‐positive results. This is especially true if polyclonal primary antibodies are used or if aspirates and fluids contain a high interstitial or serum protein content. Because of a recurring problem with antiserum to alpha‐fetoprotein (AFP), AFP was selected as the primary test antibody with which to evaluate our avidin‐biotin complex (ABC)‐IP method. The same method, with diaminobenzidine (DAB) or aminoethylcarbazole (AEC) chromogens, was performed on six types of cytologic preparations of a fresh liver specimen. The liver did not stain for AFP in FFPE and frozen tissue; therefore, it could be used to evaluate potential false‐positive staining of direct touch imprints, washed aspirate smears, and cytospins that were both air‐dried and alcohol‐carbowax‐fixed. Initial chromogen incubation times were standardized to give identical results on AFP‐positive fixed hepatoma and fetal liver controls. Cytologic preparations immunostained with ABC‐IP with AEC chromogen resulted in varying background and hepatocyte staining. In comparison, the ABC‐IP method using DAB chromogen resulted in no false‐positive results and a clean background. The ABC‐IP method with AEC standardized for sensitivity on a fixed tissue control required a markedly shortened chromogen incubation time to preclude significant false‐positive staining of cytology specimens. It appears that use of AEC chromogen for this antibody with incubation time standardized on a FFFE tissue control and then applied to cytologic preparations also amplifies nonspecific and background staining, contributing difficulty in assessing a true‐positive result. In contrast, the ABC‐IP method with DAB chromogen standardized on fixed tissues can be directly translated to various types of cytologic preparations. Knowledge of this pitfall should be especially useful in trouble‐shooting other immunoreactions since it is open difficult to obtain and store cytologic quality‐control materials for immunohistochemistry.