Immunostaining of Cytologic Preparations: A Review of Technical Problems

Immunostaining of cytologic preparations has not achieved the level of success attained by the same procedure performed in paraffin sections. This deficiency has been largely attributed to the difficulties in interpretation arising from false positivity, high background staining, and inadequacy of material for immunocytochemical analysis. Many of these setbacks have now been overcome by recent developments that include the preparation of cell blocks, rehydration of smears, and the use of a weak fixative. Contrary to the current practice of using ethanol, cold acetone, B5, and other fixatives for immunocytochemistry, we recommend the preparation of thin, evenly spread air-dried smears (up to 24 h) followed by fixation in 0.1% formal saline (2–14 h). Air-drying minimizes the loss of cells and the weak formalin preserves cellular antigens and also removes proteinaceous fluid as well as causing lysis of red blood cells, both of which contribute to background staining in cytologic preparations. Brief post-fixation in 100% ethanol (10 M) improves cytomorphology. Immunostaining is enhanced by the routine application of heat-induced antigen retrieval. The adoption of all these technical procedures will eliminate the impediments to the realization of the full potential of immunocytochemistry in diagnostic cytology.