Standardization of tissue handling in veterinary pathology: How fixation affects morphological and molecular...

Variables in the fixation and processing of histological specimens and storage of formalin-fixed paraffin-embedded (FFPE) tissues affect the outcomes of morphological and molecular examinations. The present study investigated the influence of unbuffered formalin fixation and prolonged fixation on histomorphology, immunohistochemistry, polymerase chain reaction (PCR) analysis, and DNA quality in FFPE tissues, which are essential issues in veterinary diagnostic pathology. Fixation of tissues with 10% unbuffered formalin resulted in increased intercellular spaces and decreased color contrast in hematoxylin and eosin-stained sections, attenuated Ki-67 immunolabeling, advanced DNA fragmentation, and lower PCR amplification efficiency compared with 10% neutral-buffered formalin. Prolonged fixation resulted in attenuated immunolabeling for multiple antigens, advanced DNA fragmentation, and lower PCR amplification efficiency. Preserving formalin-fixed tissue in 70% ethanol prevented loss of immunolabeling by prolonged fixation. Regarding storing FFPE tissues, coating FFPE blocks with paraffin prevented chronological DNA fragmentation. Based on the results obtained in this study and previous publications, the authors propose a standard preanalytic tissue handling guideline for histology and molecular examinations using FFPE tissues in veterinary medicine. In particular, fixation with 10% neutral-buffered formalin within 72 hours is recommended to obtain high-quality FFPE samples widely applicable for precision molecular diagnostics in routine veterinary histological examinations.