Beyond H&E

Abstract

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets. Keywords cancer, ISH, IHC, nucleic acid analysis, molecular pathology, PCR, virology Technical ConsiderationsTissue Handling, Fixation, and Embedding Fixation of tissues in 10% neutral buffered formalin, followed by paraffin embedding (FFPE), has long been the standard approach of tissue preparation for microscopic evaluation. The chemical principle of formalin fixation is based on the crosslinking of proteins. Formalin has been used extensively because it is inexpensive and excellently preserves morphological detail in histological sections. However, the relatively recent development and increasing use of both protein and nucleic acid detection methods have revealed some significant shortcomings associated with the formalin-based fixation process. The effects of formalin fixation on DNA are chemical modification, DNA trapping, and potentially DNA fragmentation. Chemical modification, considered the main effect, results from the direct cross-linking of proteins to DNA. 68 In addition, extensive protein-protein cross-linking and the formation of protein networks result in DNA trapping. The combination of these effects makes it much more difficult to extract DNA from FFPE tissue than from unfixed tissue, especially when methods used for extraction from fresh tissues are used on FFPE tissue without any modifications. 60,83 In addition to interfering with the ability to extract nucleic acid, formalin fixation also causes DNA fragmentation, especially when the formalin used is inadequately buffered, which then leads to formic acid formation during the fixation process. This, in turn, results in depurination of the DNA, which then becomes susceptible to cleavage by hydroxyl ions and results in the formation of single-stranded breaks.60 Similar to DNA, RNA is also degraded and chemically modified as the result of formalin fixation. The most important chemical effect of formalin on RNA is the formation of monomethylol groups with purine bases. 77 This has a significant effect on reverse transcription ...

Ancillary techniques on the evaluation of canine cutaneous mast cell tumors from Brazil

“…The major limitation of this study was the failure of the DNA amplification in seven samples submitted to PCR. There are several pitfalls that prevented or hindered the DNA extraction from formalin fixed and paraffin embedded (FFPE) tissues (MAES et al, 2014). The identification of sample-processing issues and use of good laboratory practices may help overcoming these obstacles.…”

“…The identification of sample-processing issues and use of good laboratory practices may help overcoming these obstacles. This includes adequate sample cleavage (0.5 to 1cm thick), quality and quantity control of the formaldehyde, short fixation time, and quality and temperature control of the paraffin (MAES et al, 2014).…”

A novel RNA-based in situ hybridization to detect Seneca Valley virus in neonatal piglets and sows affected with vesicular disease

“…However, it is well known that PCR detection of a pathogen in a tissue does not mean that the agent caused the lesions or disease. To establish that a pathogen is the causative agent of disease, it is necessary to identify the presence of the pathogen and its association with morphological changes within the tissues [10]. Immunohistochemistry (IHC) and in situ hybridization (ISH) are the main methodologies used for this purpose.…”

“…While IHC identifies a specific protein associated with the pathogen, ISH detects the pathogen’s nucleic acid within the tissue sections. Immunohistochemistry is limited on availability of species-specific antibodies and performance variation among suppliers and batches [10], and a commercial antibody for SVV is lacking.…”

Comparison of FTIR transmission and transfection substrates for canine liver cancer detection

“…While formalin fixation of tissues results in rapid preservation of histological detail by creating cross-links between amino acid residues, the insoluble crosslinks also interfere with efficient biomolecule analysis. Furthermore, formalin fixation fragments including nucleic acids at approximately 400 base pairs significantly limit classical RNA or DNA analysis tools 20 . FTIR spectroscopy can be used to simultaneously detect a variety of macro-molecules in formalin fixed sections and measurements can be acquired within seconds 11 .…”