USES AND LIMITATIONS OF ANCILLARY TECHNIQUES
APPLIED TO CYTOPATHOLOGY

Jeffrey S. Ross, M.D.

TECHNICAL FACTORS INFLUENCING IMMUNOCYTOCHEMISTRY False Negative Staining and Antigen Loss Among the factors that can result in false negative IHC staining due to antigen loss during processing, the most important are: 1) cell drying, 2) improper fixation, 3) poor specimen quality including tumor necrosis, crush artifact and limited intact tumor population, and procedural errors during immunocytochemical preparation1-3  (Table 2). To prevent unwanted antigen loss and false negative staining cytologic smears are best processed for standard immunoperoxidase and avidin-biotin-peroxidase reactions by immediate 95% ethanol fixation. Smears should not be allowed to dry and endogenous peroxidase should be blocked by 0.3% hydrogen peroxide in methanol for 30 minutes. Slides can then be immunostained in the usual way including the use of 1% goat serum, application of monoclonal and polyclonal antibodies, washing and addition of a link system and detection commonly employing the diaminobenzidine chromagen with methyl green or hematoxylin counterstaining. Poorly preserved, sparsely cellular and necrotic tissues, and cells allowed to dry prior to fixation may all result in lack of antibody localization during IHC and a false negative conclusion as to presence of the target antigen. Table 2: Immunocytochemistry in Cytology:               False Negative Staining and Antigen Loss cell drying improper fixation poor specimen quality including tumor necrosis, crush artifact and limited intact tumor population procedural errors during immunocytochemical preparation False Positive Staining and Antibody Specificity Loss Similarly, a variety of factors may result in false positive staining in which the chromagen localization in cytologic preparations is not the true result of antigen production (Table 3). The most common causes of false positive staining are: cell drying, improper fixation, non-specific binding of immunohistochemical reagents and antibodies, an antibody that reacts with multiple antigens due to epitope similarities, insufficient blocking of peroxidase and other enzymes, non-specific avidin binding, endogenous biotin present, and non-specific staining of necrotic tissue, crushed or altered tissue, and acellular debris.1-3  Table 3: Immunocytochemistry in Cytopathology:               False Positive Staining and Antibody Specificity Loss cell drying improper fixation the antibody reacts with multiple antigens due to epitope similarities insufficient blocking of peroxidase and other enzymes non-specific avidin binding endogenous biotin present non-specific staining of necrotic tissue, crushed or altered tissue, and acellular debris Despite the fact that unfixed air-dried smears may be associated with false positive and false negative immunostaining, a method of air-drying followed by weak fixation in 0.1% formal saline has been recently described which resulted in good signal preservation after antigen retrieval and reduced background staining.20  Pitfalls in the Use of Antigen Retrieval Methods As a method to overcome loss of antigen binding sites resulting from specimen fixation and processing, development of antigen retrieval methods has been associated with a new series of pitfalls and interpretation errors.21-24  Microwave oven overheating of cytologic specimens can create non-specific binding of antibody to the target cells creating a false positive interpretation. The use of antigen retrieval methods in quantitative cytology applications such as when the percentage of staining cells in a cell proliferation assay using a cell cycle marker antibody increases with the duration and intensity of the antigen retrieval method (see below). Overheating during microwave retrieval procedures can also destroy an antigen binding site completely and result in a false negative IHC reaction or non-specifically unmask protein binding sites leading to false positive staining.21  Control Specimens In order to reduce the potential impact of false negative and false positive IHC in cytology specimens it is necessary to utilize an antibody specificity control tissue or smear and a method specific control tissue or smear simultaneously stained with the unknown or target specimen. If antigen retrieval techniques are utilized the control specimens must be subjected to the same pH, temperature, time, retrieval solution and heating system as is the target specimen. It has been recommended that in-house immunocytochemistry control samples be maintained using cell culture specimens know to be positive or negative for the particular antigen under study.25  Prestained Smears Although IHC can be performed on decolorized cytologic smears previously stained by the Papanicolaou method26  incomplete decolorization may interfere with slide interpretation after IHC and either xylene coverslip removal and/or excessive decolorization with acid may denature the antigen and prevent antibody binding. There is no consensus as to whether to perform the IHC reaction before or after the destaining procedure which is usually accomplished by use of a dilute (1%) hydrochloric acid-ethanol exposure for 10-15 minutes. Given the potential interpretation errors introduced by destaining cytologic smears, whenever possible, it is recommended that IHC be performed on formalin-fixed paraffin-embedded cell block preparations when they are available and contain sufficient numbers of target cells.27