title: Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging.
abstract: BACKGROUND
  Tissue samples should be fixed and permanently stabilized as soon as possible ex-vivo to avoid variations in proteomic content. Tissues collected from studies involving infectious microorganisms, must face the additional challenge of pathogen inactivation before downstream proteomic analysis can be safely performed. Heat fixation using the Denator Stabilizor System (Gothenburg, Sweden) utilizes conductive heating, under a mild vacuum, to rapidly eliminate enzymatic degradation in tissue samples. Although many studies have reported on the ability of this method to stop proteolytic degradation and other sample changes immediately and permanently, pathogen inactivation has not been studied.
  
  
  RESULTS
  We examined the ability of the heat fixation workflow to inactivate bacterial and viral pathogens and the suitability of this tissue for Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-MSI). Mice were infected with viral or bacterial pathogens representing two strains of Venezuelan Equine Encephalitis virus (VEEV) and two strains of Burkholderia. Additionally, a tissue mimetic model was employed using Escherichia, Klebsiella and Acinetobacter isolates. Infected tissue samples harvested from each animal or mimetic model were sectioned in half. One half was heat fixed and the other remained untreated. Lysates from each sample were checked for organism viability by performing plaque (infectivity) assays or plating on nutrient agar for colony forming unit (CFU) calculation. Untreated infected control tissue demonstrated the presence of each viable pathogen by positive plaque or colony formation, whereas heat fixation resulted in complete inactivation of both the viral and bacterial pathogens. MALDI-MSI images produced from heat fixed tissue were reflective of molecular distributions within brain, spleen and lung tissue structures.
  
  
  CONCLUSIONS
  We conclude that heat fixation inactivates viral and bacterial pathogens and is compatible with proteomic analysis by MALDI-MSI. This treatment will enable the use of infected tissue from studies performed in bio-safety level 3 laboratories with VEEV and Burkholderia to be safely used for proteomic, small molecule drug detection, and imaging mass spectrometry analysis.
authors.affiliation: Henry M. Jackson Foundation, Maryland, USA. lisa.h.cazares.ctr@mail.mil.|DoD Biotechnology High Performance Computing Software Applications Institute (BHSAI/TATRC), Frederick, Maryland, USA. lisa.h.cazares.ctr@mail.mil.|Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. lisa.h.cazares.ctr@mail.mil.
authors.forename: Lisa H
authors.initials: LH
authors.lastname: Cazares
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. sean.a.vantongeren.ctr@mail.mil.
authors.forename: Sean A
authors.initials: SA
authors.lastname: Van Tongeren
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. julie.costantino@lonza.com.
authors.forename: Julie
authors.initials: J
authors.lastname: Costantino
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. tara.a.kenny.ctr@mail.mil.
authors.forename: Tara
authors.initials: T
authors.lastname: Kenny
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. nicole.l.lackemeyer.ctr@mail.mil.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Garza
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. ginger.c.donnelly.ctr@mail.mil.
authors.forename: Ginger
authors.initials: G
authors.lastname: Donnelly
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. doug.j.lane.ctr@mail.mil.
authors.forename: Douglas
authors.initials: D
authors.lastname: Lane
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. rekha.g.panchal.civ@mail.mil.
authors.forename: Rekha G
authors.initials: RG
authors.lastname: Panchal
authors.affiliation: Division of Molecular and Translational Sciences, US Army Medical Research Institute for Infectious Diseases (USAMRIID) Fort Detrick, Maryland, USA. sina.bavari.civ@mail.mil.
authors.forename: Sina
authors.initials: S
authors.lastname: Bavari
mesh_terms: D000818:Animals; D015169:Colony Count, Microbial; D003264:Containment of Biohazards; D004203:Disinfection / Q000379:methods*; D006358:Hot Temperature; D051379:Mice; D050296:Microbial Viability / Q000528:radiation effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*; D010948:Viral Plaque Assay