Cytological analysis of equine bronchoalveolar lavage fluid. Part 3: The effect of time, temperature and fixatives.

This research investigates how time, temperature, and the use of different fixatives affect the results of cytology analyses in equine bronchoalveolar lavage fluid. The researchers’ objective is to provide a guided protocol for the optimal storage conditions for these fluid samples to facilitate more accurate clinical diagnoses.

Key objectives of the research

• The main aim of this study was to evaluate the influences of time, temperature, and two different kinds of fixatives — formalin-based and alcohol-based — on the cytological results of bronchoalveolar lavage fluid (BALF) from horses.
• The knowledge obtained from this research can be utilized to establish guidelines for the most suitable storage conditions for equine BALF samples, ultimately leading to more precise clinical diagnoses.

Methodology

• The study was conducted by observing changes in several parameters over a time period of 72 hours. These include total nucleated cell count (TCC), differential cell counts (DCC), absolute cell counts (ACC), cell viability, cell morphology, and bacterial growth.
• Samples were kept under varying conditions: at temperatures of 4, 18 (with and without formalin or alcohol-based fixatives) and 38 degrees Celsius.

Key Findings

• The research found that the time taken for significant reductions in TCC and cell viability in non-fixed BALF samples was reduced as the storage temperature increased. In other words, high temperatures make the cells in the samples die faster.
• At any of the experimented temperatures, there was no significant difference in the DCC or ACC over the 72-hour time period.
• Without fixatives, BALF samples showed a significant growth of bacteria by 24 hours at 4 degrees Celsius and 8 hours at both 18 and 38 degrees Celsius, suggesting that storage temperature affects the rate of bacterial growth.
• Poor cell morphology was seen in non-fixed BALF samples by 48 hours at 4 degrees Celsius, 24 hours at 18 degrees Celsius, and 8 hours at 38 degrees Celsius. Evidence that storage temperature affects the shape and structure of cells in the samples.
• BALF samples that had been fixed showed poor morphology when analysed with Leishman’s stain compared to non-fixed samples, indicating that fixing has an impact on how cells appear under staining.