Antigens under cover-The preservation and demasking of selected antigens for successful poststaining immunocytochemistry of effusion, brain smears, and lymph node aspirates

Abstract

Background: In clinical cytology, the applicability of an ancillary test such as immunocytochemistry is too often limited by low sample volume, poor cell representation, and sample preservation. Diagnosticians often read Romanowsky-stained cytology, although specific techniques such as immunocytochemistry are often essential for a definitive diagnosis.

Objectives: The goal of the present study aimed to investigate if immunocytochemistry on previously-stained cytologic specimens was possible. Different pretreatments were examined to determine which treatment preserved antigenicity best.

Methods: One hundred and twenty-two impression smears and 64 fine-needle aspirate preparations of brain and lymph nodes were processed and evaluated microscopically. The impact of staining cytologic preparations with a modified Wright's stain, using a destaining method, performing a coverslipping and decoverslipping process, and subjecting smears to a microwave treatment (MWT) were examined for the immunolabeling of selected nuclear, cytoplasmic, and plasmalemmal antigens, as well as intracellular feline coronavirus (FCoV). Biotinylated secondary antibodies were used, and the bound primary antibody was visualized using an ABC amplification kit.

Results: Cellular antigens were reliably detected with immunocytochemistry after smears were stained with a Romansky stain and were coverslipped early after staining and stayed coverslipped until immediately before immunolabeling. The staining intensity reached the same levels as that of the controls if the films underwent MWT in citrate buffer. In contrast, FCoV antigen detection was abolished after any physicochemical interference.

Conclusions: Poststaining immunocytochemistry represents a practical tool for additional investigations on prestained cytologic specimens when searching for cellular antigens. Paired untreated samples should be kept in case the workup requires testing for more vulnerable viral antigens.

Keywords: coverslip; destaining; feline infectious peritonitis; microwave; neurocytology.