Using a Chemical Biopsy for Graft Quality Assessment
June 17th, 2020
The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.
Chemical biopsy is a new diagnostic solution in organ transplantation for quick and complex graft quality assessment. Currently, there is no other low invasive methods available that enable such analysis. The main advantage of this technique is the possibility of monitoring graft tissue modifications over time of preservation due to the simplicity and low invasiveness of SPME probes.
The small diameter of the probe and a big variety of biocompatible coatings make this method suitable for analysis of transplanted organs and applicable in medical research such as tumor analysis. When attempting this technique for the first time pay attention to details and timing of particular steps because small mistakes may heavily influence the results. The protocol is simple, but it is easier to understand and perform after visual demonstration.
Start by preparing a preconditioning mixture composed of one to one methanol and water. Pipette one milliliter of the solution into each two milliliter glass vial and place one probe in each vial. Agitate the vials on a vortex agitator at 1200 RPM for one hour.
Then rinse the probes with LCMS grade water and sterilize them according to the standard surgical sterilization protocol or in sterile processing department. When ready to extract the sample, open the sterile packaging and insert two probes directly into the kidney cortex for 10 minutes per time point, making sure that the entire length of the coating is covered by the tissue matrix. Make sure to keep track of the time of sampling for each probe.
Retract the probe by pulling it out from the tissue and immediately rinse the coating with LCMS grade water to remove any remaining blood making sure to rinse away from the surgical site. To transport the probes, place them in separate vials and close them. Then place the vials in a box filled with dry ice or liquid nitrogen.
Store the samples at negative 80 degrees Celsius or immediately proceed with desorption. Prepare a desorption solution composed of acetonitrile and water for metabolomic analysis and another composed of isopropanol and methanol for lipidomic analysis. Add 100 microliters of the solution to inserts in the two milliliter labeled vials and place one probe in each vial agitate the vials at 1200 RPM for two hours then remove the probes from the vials and proceed with LCMS analysis.
Liquid chromatography coupled with high resolution mass spectrometry was used to carry out untargeted metabolomic and lipidomic analysis. The data was subjected to principal component analysis to assess quality entertain general insights regarding the results. The quality control samples formed a tight cluster confirming the quality of the analysis.
The studied groups exhibited relatively good separation, allowing for visualization of differences in metabolic and lipidomic profiles before and after transplantation as well as during organ perfusion. A wide spectrum of extracted features was separated on both reversed phase and liquid chromatography columns. Alterations and metabolite levels throughout the experiment were demonstrated with box whisker plots of the selected metabolites.
When performing this procedure, keep in mind the organs heterogeneity. Place the probe in desired location keep the time of sampling identical at all time points, and secure the fiber properly after sampling. The biomarkers discovered with SPME can be quantified using this extraction method coupled to MSMS, LCMS or other analytical instrumentation.
Moreover, since no sample is consumed, routine analysis such as biopsy can be performed. The method can be adapted for other tissue metabolomics and lipidomics analysis without necessity for sample pretreatment. For specific metabolites.
Other coatings can be used to increase selectivity and sensitivity of the essay.
Overview
This protocol outlines a chemical biopsy approach for assessing kidney graft quality prior to transplantation. It emphasizes the use of metabolomic and lipidomic analyses to evaluate grafts effectively.
Key Study Components
Area of Science
- Organ transplantation
- Metabolomics
- Lipidomics
Background
- Chemical biopsy is a novel diagnostic method for graft quality assessment.
- It allows for low-invasive monitoring of graft tissue changes.
- SPME probes are utilized for their small size and biocompatibility.
- Current methods lack the efficiency and simplicity of this technique.
Purpose of Study
- To provide a protocol for assessing kidney graft quality.
- To demonstrate the effectiveness of metabolomic and lipidomic analyses.
- To highlight the advantages of chemical biopsy in organ transplantation.
Methods Used
- Preparation of preconditioning mixture and SPME probes.
- Sampling from kidney cortex using SPME probes.
- Desorption of metabolites for analysis.
- LCMS analysis for metabolomic and lipidomic profiling.
Main Results
- Quality control samples confirmed the reliability of the analysis.
- Distinct metabolic and lipidomic profiles were observed pre- and post-transplantation.
- Box whisker plots illustrated alterations in metabolite levels.
- SPME method allows for routine analysis without consuming samples.
Conclusions
- Chemical biopsy is a promising tool for graft quality assessment.
- The method can be adapted for other tissues in metabolomics studies.
- Future applications may enhance selectivity and sensitivity in analyses.
