Effects of on-slide fixation on the cell quality of cytocentrifuged equine bronchioalveolar lavage fluid

Abstract

Background: During bronchoalveolar lavage fluid (BALF) sample preparation in horses, several technical aspects can affect sample variability. To date, the effects of different fixatives on prepared equine BALF films have been insufficiently investigated.

Objectives: This study aimed to determine the effect of various on-slide fixation methods on cell quality, including spray fixation of wet films, and acetone and methanol fixation of air-dried samples in comparison with unfixed, air-dried films.

Methods: Cytocentrifuged BALF samples from 5 horses were fixed in a wet state using a commercially available fixation spray. They were also fixed with acetone or methanol after air-drying using standard protocols or were air-dried with no fixation. After different postfixation storage durations and temperatures, the samples were stained with May-Grünwald Giemsa or immunocytochemistry stains. Subsequently, differential cell counts (DCCs) were performed, cell areas were measured, and cell morphologies and immunocytochemical staining intensities were assessed semiquantitatively.

Results: Optimal cell morphology results were achieved with the wet-spray fixation method. Acetone and methanol fixation, especially when performed at -20°C, caused reduced cell morphology quality, thereby significantly altering DCCs. For storage of unstained samples for 1 week at room temperature, no significant changes in cell morphology were observed for either fixation method. Wet-spray fixation resulted in enhanced preservation of macrophage, granulocyte, and mast cell sizes compared with air-drying techniques. Immunocytochemical staining of unfixed and acetone-fixed samples was the most intense.

Conclusions: Wet-spray fixation resulted in the best preservation of cellular morphology and less cell shrinkage compared with unfixed specimens and is, therefore, recommended for BALF cytology.

Keywords: acetone fixation; cell morphology; differential cell count; immunocytochemistry; methanol fixation; spray fixation.