Pitfalls in diagnostic immunohistology

The integration of immunostaining as an often-essential component of morphologic assessment makes it necessary that pathologists are familiar with the many technical and interpretive pitfalls that may arise because of the wide variety of factors that can significantly influence the ability to demonstrate relevant antigens in paraffin-embedded tissue sections. Pre-analytical variables that affect immunostaining include fixation, type of fixative, duration, temperature, and pH of fixation, variables in tissue processing, antigen loss resulting from delays in fixation, tissue necrosis, and levels of antigen expression. Analytical factors relate to the complex and sometimes capricious immunolabeling procedure that has as variables the specificity and sensitivity of the antibody clone, reagent dilution, detection system, and chromogen, and importantly, the method of antigen retrieval, which has its own set of variables such as time, temperature, method of heat generation, retrieval solution pH and molarity, and the synergistic action of proteolytic digestion. To obtain the highest diagnostic yield from immunostaining the correct questions must be asked and the pathologist must have familiarity with the characteristics of the antibody, its cross reactivity, if any, and localization of antigen in the cell to understand thresholds and cut-off levels and to recognize false-positive staining. Proper utilization of immunostaining requires that it is employed as a morphology-based technique and not interpreted in isolation.