Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry
Lisa H Cazares1,2,3, Sean A Van Tongeren4, Julie Costantino5
1Henry M. Jackson Foundation, Maryland, USA. lisa.h.cazares.ctr@mail.mil.
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.
Insights
Heat fixation effectively inactivates viral and bacterial pathogens in tissue samples, ensuring sample stability for proteomic analysis. This method is crucial for safe handling of infectious samples in research settings.
Area of Science:
- Proteomics
- Pathogen Inactivation
- Mass Spectrometry Imaging
Background:
- Timely ex-vivo tissue fixation is essential to prevent proteomic variations.
- Infectious samples require pathogen inactivation before proteomic analysis.
- Heat fixation offers rapid enzymatic degradation elimination but pathogen inactivation is unstudied.
Purpose of the Study:
- To evaluate heat fixation's efficacy in inactivating viral and bacterial pathogens.
- To assess the suitability of heat-fixed tissue for Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-MSI).
Main Methods:
- Infected mouse tissues and a tissue mimetic model were used.
- Samples were divided into heat-fixed and untreated groups.
- Pathogen viability was assessed via plaque assays and colony-forming unit (CFU) counts.
- MALDI-MSI was performed on heat-fixed tissues.
Main Results:
- Heat fixation completely inactivated all tested viral (Venezuelan Equine Encephalitis virus) and bacterial (Burkholderia, Escherichia, Klebsiella, Acinetobacter) pathogens.
- Untreated samples showed viable pathogen presence.
- MALDI-MSI of heat-fixed tissues accurately reflected molecular distributions.
Conclusions:
- Heat fixation is a validated method for inactivating diverse viral and bacterial pathogens in tissue.
- This workflow is compatible with proteomic analysis, including MALDI-MSI.
- Enables safe analysis of bio-safety level 3 infectious samples for proteomic and imaging applications.
