A Tale of Three Fixatives: Detection of Middle East Respiratory Syndrome Coronavirus in Paraffin‐Embedded Tissues Preserved in Three Fixatives

Abstract

Middle East respiratory syndrome coronavirus (MERS‐CoV) is a zoonotic pathogen endemic to dromedary camels known to cause a respiratory illness in humans that is fatal in 35% of cases. As such, inactivated tissues provide a safer format when testing for this virus. Neutral buffered formalin (NBF), pathology’s gold standard for tissue fixation, preserves histomorphology and inactivates many pathogens. However, NBF causes the crosslinking and fragmentation of proteins and nucleic acids in tissues. This results in a rapid decline in biomolecular quality after just 24 hours and damage increases thereafter. NBF is also a volatile organic compound and carcinogen. Two novel fixatives, phosphate‐buffered 70% ethanol (BE70) and BE70 with added guanidium salts (BE70G), are alternative preservatives that cause less damage to biomolecules, even in tissues immersed for longer than 3 months. These ethanol (EtOH)‐based fixatives have equivalent histomorphology and inactivation profiles as well as better human safety profiles. We hypothesized that existing anti‐MERS‐CoV immunohistochemistry (IHC) and in situ hybridization (ISH) protocols would work on tissues preserved with these EtOH‐based fixatives.Roughly equivalent tracheal and nasal turbinate samples from eight alpacas experimentally infected with MERS‐CoV were fixed in 10% NBF, BE70, and BE70G, processed into paraffin and sectioned for hematoxylin and eosin staining or immunolabeling. Anti‐MERS‐CoV nucleocapsid IHC using polymer‐based detection and MERS‐CoV specific ISH using RNAscope® 2.5 HD brown detection were conducted on all samples. Because many EtOH‐based fixatives do not require antigen retrieval (AR), ISH and IHC were run with and without AR on the EtOH‐based fixed tissues.Although MERS‐CoV IHC and ISH appropriately labeled target molecules in all samples, signal strength varied across the fixatives. The strongest labeling was seen in NBF, followed by BE70G, and finally BE70. This result was not unexpected as the protocols were optimized for use with NBF tissues. Interestingly, both BE70 and BE70G required AR to successfully visualize the targets by both IHC and ISH, although BE70G did have weak ISH signal without AR. Histomorphology for all three fixatives without AR was very similar. However, AR blurred cell borders and reduced overall cellular definition in the EtOH‐based fixed samples, particularly BE70G.Taken together, these results support the use of these novel EtOH‐based fixatives as alternatives to NBF, particularly when biomolecule preservation for downstream molecular biology assays is critical. Finally, BE70 and BE70G increase human safety by inactivating pathogens with less harmful chemicals as well as provide a more flexible fixation time frame. This is particularly useful for high containment work as it would allow safe transfer of inactivated tissues to lower biosafety level labs while better preserving the samples for downstream analysis.Support or Funding InformationThis work was funded by the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University and the USDA APHIS NBAF Scientist Training Program.Histomorphology, Immunohistochemistry (IHC), and In Situ Hybridization (ISH) of NBF‐, BE70‐, and BE70G‐Fixed Tissues Column 1 is hematoxylin and eosin staining, Column 2 is anti‐MERS‐CoV nucleocapsid IHC, and Column 3 is MERS‐CoV‐specific ISH in fixed, paraffin‐embedded nasal turbinates. All immunolabeled tissues were counterstained with hematoxylin. Row 1 is NBF‐fixed tissue. Row 2 is BE70‐fixed tissue. Row 3 is BE70G‐fixed tissue. The bar for all main images is 100 microns.Figure 1