A Novel α – helix /β -sheet ratio as a Potential Metric For Studying Thermal Effects of Increased Formaldehyde Temperatures on Native Tissues

Formaldehyde is a commonly used fixative in histological studies, with fixation outcomes significantly influenced by physical factors such as temperature. This study investigates the impact of varying fixation temperatures on tissue morphology and protein structural integrity using formaldehyde as the fixative. Ten (10) Wistar rats were sacrificed via cervical dislocation, and their liver, lungs, and kidneys were extracted. These organs were fixed in 10% Neutral Buffered Formalin at 25°C, 37°C, and 60°C for 24 hours. Standard tissue processing was followed, with 4 µm sections prepared for histological analysis using hematoxylin and eosin staining to assess tissue morphology. Additionally, 20 µm sections were analyzed using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy to examine protein spectral characteristics, employing the Agilent Cary 630 spectrometer in the 4000–600 cm⁻¹ IR range. Results revealed that tissues fixed at 25°C and 37°C showed superior preservation, while those at 60°C exhibited significant distortion for most of the tissues. Findings also showed that there was an elevation in beta-sheets as formalin temperature increases to 60°C compared with alpha-helixes on liver, lungs, and kidneys. However, performance test using receiver’s operating characteristics (ROC) curve showed potential of α – helix /β -sheet ratio to monitor effect of formalin-temperature increase on tissues and lung tissues appeared to be a perfect model for this study showing 100% accuracy, 100% sensitivity and 100% specificity to tell apart changes occuring to protein secondary structures between 25°C controlled formalin fixation and those at higher fixed at 37°C. These findings underscore the importance of optimizing fixation temperature to balance fixation speed and tissue integrity, drawing implications for antigen recovery in immunohistochemical studies.