title: Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization.
abstract: Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral (Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents (Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.
authors.affiliation: Institute of Pathology and Forensic Veterinary Medicine, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria.
authors.forename: Meike M
authors.initials: MM
authors.lastname: Mostegl
authors.forename: Barbara
authors.initials: B
authors.lastname: Richter
authors.forename: Nora
authors.initials: N
authors.lastname: Dinhopl
authors.forename: Herbert
authors.initials: H
authors.lastname: Weissenböck
mesh_terms: D000818:Animals; D002863:Chromogenic Compounds; D017925:Circovirus / Q000302:isolation & purification*; D003458:Cryptosporidium / Q000302:isolation & purification; D005557:Formaldehyde / Q000737:chemistry*; D017403:In Situ Hybridization / Q000379:methods*; D019316:Porcine respiratory and reproductive syndrome virus / Q000302:isolation & purification*; D011529:Protozoan Infections, Animal / Q000469:parasitology; D012911:Snakes; D013552:Swine; D013553:Swine Diseases / Q000821:virology; D016707:Tissue Fixation; D014318:Tritrichomonas / Q000302:isolation & purification
chemical_list: D002863:Chromogenic Compounds; D005557:Formaldehyde