Name: Video: Using a Chemical Biopsy for Graft Quality Assessment
Uploaded: 2020-06-17T13:30:00.000+00:00
Duration: 5 min
Description: 5.1K Views. Nicolaus Copernicus University in Torun, Collegium Medicum in Bydgoszcz. 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.

The study was supported by grant Opus UMO-2017/27/B/NZ5/01013 from National Science Centre. The authors would like to acknowledge MilliporeSigma, a business of Merck KGaA, Darmstadt, Germany for providing SPME devices. The life science business of Merck operates as MilliporeSigma in the U.S. and Canada. Also, the authors want to thank Thermo Fisher Scientific for the access to Q-Exactive Focus orbitrap mass spectrometer. The authors would like to thank Dr. Aleksandra Woderska-Jasin&#769;ska and the personnel of the Department of Transplantology and General Surgery in Bydgoszcz for their kind assistance in the project. BB want to thank Prof. Janusz Pawliszyn for the opportunity of sample collection at Toronto General Hospital during her stay at University of Waterloo.

All animals received humane care in compliance with the &#8216;&#8216;Principles of Laboratory Animal Care&#8217;&#8217; formulated by the National Society for Medical Research and the &#8216;&#8216;Guide for the Care of Laboratory Animals&#8217;&#8217; published by the National Institute of Health, Ontario, Canada. The Animal Care Committee of the Toronto General Research Institute approved all studies. The research involved human subjects was approved by the Bioethical Committee at Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Torun.
NOTE: Obtain approval from appropriate ethical boards. Remember to always wear safety gloves. Do not touch the extraction phase of SPME probes. The use of deactivated glass vials is recommended for lipidomic analyses.
1. Preparation of probes
<ol>
	<li>Prepare titanium-nickel alloy probes (40 mm length; 0.2 mm diameter) coated with 7 mm mix-mode sorbent. The number of probes depends on the time points targeted during the entire procedure and the number of replicates (3 is recommended per time point). 
	NOTE: The length and type of extraction phase may be adjusted based on the mode of study, the polarity of metabolites, and the sample matrix.</li>
	<li>Prepare a cleaning mixture composed of 2:1 chloroform:methanol (v/v). Pipet 1.0 mL of the solution to each 2.0 mL glass vial and place one probe, previously pierced through the cap, in each vial. 
	NOTE: Before use, clean all probes to remove contaminating particles.</li>
	<li>Put the vials on the agitator and set the agitation speed at 1,200 rpm. After 45 min, stop the device and rinse the coatings with LC-MS grade water.</li>
	<li>As coatings must undergo a preconditioning step to activate them, prepare a preconditioning mixture composed of 1:1 methanol:water (v/v). Pipet 1.0 mL of the solution to each 2.0 mL glass vial and place one probe, previously pierced through the cap, in each vial.</li>
	<li>Put the vials on the vortex agitator and set the agitation speed at 1,200 rpm.</li>
	<li>After 60 min, stop the agitator and rinse the coatings with LC-MS grade water.</li>
	<li>Sterilize probes according to the standard surgical equipment sterilization protocol.</li>
</ol>
2. Extraction
<ol>
	<li>Open the sterile package right before sampling to ensure a sterile environment.</li>
	<li>Insert two probes directly into the kidney cortex for 10 min at each time point. The entire length of the coating must be covered by the tissue matrix; no specific angle is required, but ca. 90 deg is usually used. 
	NOTE: The entire medical procedure follows standard protocols in given institution. No modification concerning SPME sampling is considered. The procedure involves the six following sampling time points: 
	a) before kidney resection, in vivo from donor; 
	b)-e) after 1 h, 3 h, 5 h, 7 h of kidney perfusion, ex vivo in organ chamber; 
	f) after reperfusion, in vivo from recipient.</li>
	<li>Retract the probes by pulling it out from the tissue and then rinse coatings with LC-MS grade water to clear any remaining blood from the coating surface. Rinse away from the surgical site, and immediately after removal of the probes.</li>
</ol>
3. Transport and storage
<ol>
	<li>Place probes in separate vials and close them.</li>
	<li>Place vials in a Styrofoam box filled with dry ice or in liquid nitrogen for transport.</li>
	<li>Store samples in a freezer (-80 &#176;C), or immediately commence the desorption step.</li>
</ol>
4. Desorption
<ol>
	<li>Prepare desorption solutions composed of 80:20 acetonitrile:water (v/v) for metabolomic analysis, and 1:1 isopropanol:methanol (v/v) for lipidomic analysis.</li>
	<li>Pipet 100 &#956;L of the solution to inserts placed into 2.0 mL labeled vials and place one probe, previously pierced through the cap, in each vial.</li>
	<li>Put the vials on the vortex agitator and set the agitation speed at 1,200 rpm for 120 min.</li>
	<li>Remove probes from the vials. Obtained extracts are now ready for instrumental analysis.</li>
</ol>
5. LC-MS analysis
<ol>
	<li>Place vials containing extracts in the autosampler of the LC-MS system. 
	NOTE: Liquid chromatography (RP, HILIC) coupled with high resolution mass spectrometry and an orbitrap mass analyzer were used for this study. For metabolomic analysis parameters, go to step 5.2. For lipidomic analysis parameters, go to step 5.6.</li>
	<li>Use a pentafluorophenyl (PFP) column (2.1 mm x 100 mm, 3 &#956;m) for reversed phase separation.
	<ol>
		<li>Set the flow rate to 300 &#181;L/min, and autosampler and column temperatures to 4 &#176;C and 25 &#176;C, respectively.</li>
		<li>Prepare mobile phases according to the following proportions: mobile phase A: water:formic acid (99.9:0.1, v/v), and mobile phase B: acetonitrile:formic acid (99.9:0.1, v/v). Set the mobile phase flow according to the following parameters: starting mobile phase flow (0-3 min): 100% A followed by a linear gradient to 10% A (3-25 min), ending with isocratic flow of 10% A until 34 min, followed by 6 min of column re-equilibrium time.</li>
	</ol>
	</li>
	<li>For HILIC separation, use a HILIC column (2.0 mm x 100 mm, 3 &#956;m, 200A). Set the flow rate to 400 &#956;L/min.
	<ol>
		<li>Prepare mobile phases according to the following proportions: mobile phase A: acetonitrile:ammonium acetate buffer (9:1, v/v, effective salt concentration 20 mM), mobile phase B: acetonitrile:ammonium acetate buffer (1:1, v/v, effective salt concentration 20 mM).</li>
		<li>Set the mobile phase flow according to the following parameters: starting mobile phase flow (0-3 min) at 100% A, hold for 3.0 min and then ramp to 100% B within 5 min. Hold with 100% B until 12 min, followed by 8 min of column re-equilibrium time.</li>
	</ol>
	</li>
	<li>Set the scan range to 85-1000 m/z. Set HESI ion source parameters in positive ionization mode to: spray voltage 1,500 V, capillary temperature 300 &#176;C, sheath gas 40 a.u., aux gas flow rate 15 a.u., probe heater temperature 300 &#176;C, S-Lens RF level 55%.</li>
	<li>Run QC samples composed of 10 &#181;L of each analyzed sample (regularly, every 10-12 samples) to monitor instrument performance.</li>
	<li>For reversed phase separation, use a C18 column (2.1 mm x 75 mm, 3.5 &#956;m).
	<ol>
		<li>Set the flow rate to 200 &#181;L/min, and autosampler and column temperatures to 4 &#176;C and 55 &#176;C, respectively. Prepare mobile phases according to the following proportions: mobile phase A: H2O:MeOH (60:40, v/v), 10 mM ammonium acetate and 1 mM acetic acid; mobile phase B: IPA:MeOH (90:10, v/v), 10 mM ammonium acetate and 1 mM acetic acid.</li>
		<li>Set the mobile phase flow according to the following parameters: 0-1 min (20% B), 1-1.5 min (20-50% B), 1.5-7.5 min (50-70% B), 7.5-13 min (70-95% B), 13-17 min (95% B), 17-17.1 min (95-20% B), 17.1-23 min (20%).</li>
	</ol>
	</li>
	<li>For HILIC separation, use a HILIC column (100 x 2.1 mm, 3 &#956;m).
	<ol>
		<li>Set the flow rate to 400 &#181;L/min, and autosampler and column temperatures to 4 &#176;C and 40 &#176;C, respectively.</li>
		<li>Prepare mobile phases according to the following proportions: mobile phase A: ACN; mobile phase B: 5 mM ammonium acetate in water. Set the mobile phase flow according to the following parameters: 0-2 min (96% B), 2-15 min (96-80% B), 15-15.1 min (80-96% B), 15.1-21 min (96% B).</li>
	</ol>
	</li>
	<li>Set HESI ion source parameters in positive ionization mode to spray voltage 3,500 V, capillary temperature 275 &#176;C, sheath gas 20 a.u., aux gas flow rate 10 a.u., probe heater temperature 300 &#176;C, S-Lens RF level 55%.</li>
	<li>Run QC samples composed of 10 &#181;L of each analyzed sample (every 10-12 samples) to monitor instrument performance.</li>
	<li>Perform data acquisition in software compatible with the instrument.</li>
</ol>
6. Data analysis
<ol>
	<li>Perform data processing, putative identification, and statistical analysis with the use of software dedicated to untargeted metabolomics and lipidomics analysis. 
	NOTE: Principal components analysis (PCA) and box-whisker plots can be obtained to visualize data structure.</li>
</ol>

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The authors would like to acknowledge MilliporeSigma (Merck KGaA, Darmstadt, Germany) for providing SPME devices and Thermo Fisher Scientific for access to the Q-Exactive Focus Orbitrap mass spectrometer.

Evaluation of organ quality remains a large challenge for physicians, who must make quick informed decisions regarding whether a given organ is viable for transplantation or whether it must be discarded. Multiple factors, such as donor age, duration of ischemia, and infections and inflammatory processes, can affect the long-term graft outcome. While diverse methods have been developed to date to diagnose kidney allograft function, histopathological inspection remains the gold standard in that matter3,4,12. Although the biopsy procedure can yield significant information regarding pre-existing donor disease and vascular changes, it is not free of flaws. Sampling errors associated to interobserver variability and sampling of insufficient glomeruli for comprehensive information regarding organ function remain typical concerns in this regard. Moreover, specimen preparation brings some issues such as an incomplete appraisal of the graft in case of frozen sections, and extension of procedure time for paraffin sectioning. However, the increased risk of hemorrhage, which may appear acutely as microscopic or gross hematuria, is the major life-threatening complication associated with the biopsy procedure. For this reason, the number of allowable biopsies is strictly limited in transplantation procedures, a factor that hampers the capture of dynamic changes and time series analyses via this method12,13,14. The benefits of a histological analysis must be weighed against the risks associated with the methodology. The value of histological findings is indisputable, but they do not explain the molecular mechanisms of the aberrations.
Metabolomics and lipidomics are the youngest domains of the &#8220;-omics&#8221; scientific family. The complete set of low molecular (&#60;1,200 Da) human metabolites and lipids connected within a metabolic network is defined as a human metabolome. The genome remains relatively constant throughout its lifetime, with slight modifications caused by mutations occurring infrequently. The metabolome is the product of gene expression, which is highly sensitive to changes in all biological processes as well as environmental factors. The dynamic nature of metabolites and lipids makes them perfect indicators of current organ condition7,8,15,16. The SPME method proposed in the abovementioned protocol enables detection of changes occurring in the organ during its preservation, starting from organ removal from the donor&#8217;s body till revascularization at the recipient&#8217;s. The small diameter of the probe (~200 &#181;m) provides minimal invasiveness and allows for several samplings from the same organ without causing any damage to the tissue. Conducting studies using kidney, as the most frequently transplanted organ, allows for a better understanding and further characterization of the metabolic pathways responsible for declining the quality and function of grafts. The possibility of monitoring modifications over time certainly is an important advantage of the technique as compared to conventional invasive methods such as biopsy. The currently presented analysis identified altered concentrations of various groups of lipids and metabolites, especially of essential amino acids, purines, purine nucleosides, and glycerophospholipids. These results are consistent with previous tissue analysis reports5,6,17,18,19,20. To date, the majority of scientific reports utilizing metabolomics or lipidomics to explain processes inducing complications after transplantation or ischemia/reperfusion injury (IRI) phenomena have been limited to analysis of biofluids21,22,23.
Each clinical application requires optimization of the sampling protocol to ensure that the performance of the analytical method meets expected criteria. In this regard, the benefit of utilizing SPME is the possibility of adjusting conditions for various experimental designs. The variety of accessible extraction phases provides a broad spectrum of extracted metabolites with diversified polarities. At the same time, this might be considered as a limitation of the method due to the fact that each sorbent provides selectivity towards specific features and does not extract all compounds present in the sample matrix. It should be noted that SPME coatings extract only via free molecules, and simply do not interact with a bound fraction of the analyte. The biocompatibility of the coatings does not introduce toxicity to the tissue while restraining the extraction of large molecules such as proteins; as a consequence, the enzymatic processes are inhibited already at the stage of sample collection and the presence of artefacts is minimized, which is a great advantage over alternative sampling methods. The length of the coating influences the efficiency of extraction (i.e., the length of the coating designates the surface area and the extraction phase volume); thus, longer coatings yield higher recoveries. On the other hand, shorter coatings enable higher spatial resolution. For reliable results, it is crucial to submerge the probe to the exact same depth of kidney cortex. Insertion too deep causes the risk of entering the kidney medulla. The time of the extraction is also proportional to the extraction efficiency. Therefore, selection of optimum extraction time is one of the most critical steps in SPME method development. The accuracy of time measurement provides the highest repeatability. In biological applications such as the one discussed, there is always a compromise between the sensitivity and repeatability of the analytical protocol and the restrictions of the medical procedure. While equilibrium extraction provides the highest sensitivity, for safety reasons, pre-equilibrium conditions are often used in such applications, as extraction time should not affect the total duration of the surgery. The efficiency of desorption is determined by the time of the process and the composition of the desorption solvent, which should be compatible with the mobile phase used for chromatographic separation9,10,11.
One of the major requirements for diagnostic instrumentation used for intra-surgical assessments is time of analysis. Current attempts are being made to develop a rapid tool for in vivo SPME extraction coupled directly to a mass spectrometer via microfluidic open interface (MOI)24 or coated blade spray (CBS)25. Such approaches would allow for the disclosure of analytical results in real or close to real time. The use of such methods for pre-intervention analyses of metabolic and lipidomic profiles could enhance the decision-making process during transplantation procedures, enabling the best possible personalized approach and fast response in case of organ failure.
As a summary, it is hypothesized that the proposed protocol will enable attainment of full metabolic and lipidomic profiles of kidney grafts, which in turn would provide a comprehensive assessment of organ quality and characterization of the processes responsible for ischemia-reperfusion injury. The novelty of the project includes the utilization of solid-phase microextraction (SPME), offering low invasive sampling of living systems, in combination with one of the most innovative technologies available for metabolomics and lipidomics analysis (e.g., the Orbitrap high resolution mass spectrometer). SPME combines sample collection, extraction, and quenching of metabolites in one step, therefore making it a perfect tool for rapid analysis. It is expected that this protocol will help to answer questions related to what pre-transplant conditions of the kidney are responsible for delayed organ function or its dysfunctions after transplantation, as well as to how the graft preservation protocol influences the biochemistry of the organ. Such knowledge would not only have significant impact on the prevention of possible complications related to transplantation, but may help to improve current graft preservation protocols, minimizing loss of viable transplant tissue as well as loss of life. The proposed solution will open the door to further investigations in this field, including the validation of specific potential biomarkers and improvement of therapeutic outcomes in transplantology.

An erratum was issued for: <a href="https://jove.unicartagenaproxy.elogim.com/t/60946/using-a-chemical-biopsy-for-graft-quality-assessment">Using a Chemical Biopsy for Graft Quality Assessment</a>. One of the affiliations was&#160;updated.
The third affiliation was&#160;updated from:
Department of Transplantation and General Surgery, University Hospital, University of Nicolaus Copernicus Torun&#34;
to:
Department of Transplantology and General Surgery, Collegium Medicum in Bydgoszcz, Antoni Jurasz University Hospital No. 1 in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland

An erratum was issued for: <a href="https://jove.unicartagenaproxy.elogim.com/t/60946/using-a-chemical-biopsy-for-graft-quality-assessment">Using a Chemical Biopsy for Graft Quality Assessment</a>. One of the affiliations was&#160;updated.

Erratum: Using a Chemical Biopsy for Graft Quality Assessment

According to the United States&#8217; Organ Procurement and Transplantation Network, there were 94,756 patients waiting for kidney transplants in the US in 2019; while in Europe in 2018, that number was 10,791. Every ten minutes, someone is added to the national transplant waiting list in the U.S., and it is estimated that 20 people die each day waiting for a transplant1,2. Kidney transplantation is a life-saving treatment for a large number of people suffering with end-stage renal dysfunction worldwide. The procedure is associated with increased survival rate and greater quality of life when compared to conventional dialysis.
However, transplantation faces many serious problems, such as organ shortage or lack of effective tools for organ quality assessment. The standard protocols are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information regarding the quality of the graft. While a visual appraisal allows for identification of tumors visible to the eye, anatomical abnormalities, or extensive damage to the grafts, this approach is very subjective, varying in its effectiveness according to the experience of the observers. Biopsy, on the other hand, can provide valuable information regarding pre-existing renal disorders, and is thus considered to be a method of objective and evidenced value in determining graft outcomes. However, the biopsy procedure is not free of flaws; there is the risk of potential complications such as bleeding and an additional 4-5 hours of sample preparation is required, which significantly prolongs the cold ischemic time. Therefore, especially in Europe, the use of direct tissue analysis is limited to expanded criteria donors (ECD) and donors after circulatory death (DCD)3,4.
Metabolomics and lipidomics have been recently recognized as promising approaches to attaining a better understanding of the changes in biochemical pathways occurring during organ preservation. Metabolomic and lipidomic profiling enables monitoring of immediate responses of the system to sudden environmental changes related to organ removal with subsequent consequences: ischemia, oxidative stress, or inflammatory responses5,6,7,8. The kidney is an organ that is largely associated with metabolic processes, thus measurements of metabolites and lipids concentrations may permit identification of potential organ quality biomarkers and enable better predictions of graft outcome.
Given the above complications and limitations associated with current organ quality assessment methods, a less invasive diagnostic solution is needed for quick and complex organ quality assessment. Solid phase microextraction (SPME) complies with these requirements as a minimally invasive analytical method that enables coverage of a broad spectrum of metabolites and lipids. The technique is based on the insertion of a thin (~200 &#181;m), biocompatible, titanium-nickel alloy probe covered with a selective extraction phase into the examined organ for a short time. It should be emphasized that SPME prevents protein extraction, and hence enables metabolism inhibition already at the stage of sample collection, which is a significant advantage over alternative methods. Moreover, the miniaturization of the device allows for the execution of repetitive and simultaneous analyses of few structures of the organ9,10,11.

<table>
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>5330010050</td>
			<td>Mobile phase additive</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Alchem</td>
			<td>696-34967-4X2.5L</td>
			<td>HPLC solvent</td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Merck</td>
			<td>5330040050</td>
			<td>Mobile phase additive</td>
		</tr>
		<tr>
			<td>BENCHMIXER XL MULTI-TUBE VORTEXER</td>
			<td>Benchmark Scientific</td>
			<td>BV1010</td>
			<td>Vortex mixer</td>
		</tr>
		<tr>
			<td>Caps</td>
			<td>Perlan Technologies</td>
			<td>5183-2076</td>
			<td>Blue scrw tp, pre-slit PTFE/Si spta, 100PK</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>1024441000</td>
			<td></td>
		</tr>
		<tr>
			<td>Discovery HS F5 Supelguard Cartridge, 3 &#956;m, L &#215; I.D. 2 cm &#215; 2.1 mm</td>
			<td>Merck</td>
			<td>567570-U</td>
			<td>HPLC guard column</td>
		</tr>
		<tr>
			<td>Discovery HS F5, 2.1 mm x 100 mm, 3 &#956;m</td>
			<td>Merck</td>
			<td>567502-U</td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Alchem</td>
			<td>497-94318-50ML</td>
			<td>Mobile phase additive</td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Perlan Technologies</td>
			<td>5182-0714</td>
			<td></td>
		</tr>
		<tr>
			<td>HILIC Luna 3 &#956;m, 200A, 100 x 2.0 mm</td>
			<td>Shim-Pol</td>
			<td>PHX-00D-4449-B0</td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>HILIC SecurityGuard Cartridge, 3 &#956;m, 4 x 2.0 mm</td>
			<td>Shim-Pol</td>
			<td>PHX-AJ0-8328</td>
			<td>HPLC guard column</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Alchem</td>
			<td>231-AL03262500</td>
			<td>HPLC solvent</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Alchem</td>
			<td>696-34966-4X2.5L</td>
			<td>HPLC solvent</td>
		</tr>
		<tr>
			<td>Nano-pure water</td>
			<td>Merck</td>
			<td>1037281002</td>
			<td>HPLC solvent</td>
		</tr>
		<tr>
			<td>Q Exactive Focus hybrid quadrupole-Orbitrap MS</td>
			<td>Thermo Scientific</td>
			<td>Q Exactive Focus</td>
			<td>Mass Spectrometer</td>
		</tr>
		<tr>
			<td>SeQuant ZIC-cHILIC 3&#181;m,100&#197; 100 x 2.1 mm</td>
			<td>Merck</td>
			<td>1506570001</td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>SeQuant ZIC-HILIC Guard Kit 20 x 2.1 mm</td>
			<td>Merck</td>
			<td>1504360001</td>
			<td>HPLC guard column</td>
		</tr>
		<tr>
			<td>SPME LC fiber probes, mixed mode</td>
			<td>Supelco</td>
			<td>prototype fibers</td>
			<td></td>
		</tr>
		<tr>
			<td>UltiMate 3000 HPLC systems</td>
			<td>Thermo Scientific</td>
			<td>UltiMate 3000</td>
			<td>HPLC system</td>
		</tr>
		<tr>
			<td>Vial inserts (deactivated)</td>
			<td>Perlan Technologies</td>
			<td>5181-8872</td>
			<td></td>
		</tr>
		<tr>
			<td>XSelect CSH C18 3.5&#956;m 2.1x75mm</td>
			<td>Waters</td>
			<td>186005644</td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>XSelect CSH C18 VanGuard Cartridge 3.5&#956;m, 2.1x5mm</td>
			<td>Waters</td>
			<td>186007811</td>
			<td>HPLC guard column</td>
		</tr>
	</tbody>
</table>

using a chemical biopsy for graft quality assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

Sample collection was performed according to the SPME method described above, using probes coated with a 7 mm mixed-mode extraction phase. Sampling was conducted directly from the graft tissue (in vivo before transplantation), ex vivo in kidney chamber after 1 h, 3 h, 5 h, and 7 h of perfusion, and in vivo after revascularization in the recipient. Untargeted metabolomic and lipidomic analyses were carried out with the use of liquid chromatography (RP, HILIC) coupled with high resolution mass spectrometry, with the mass analyzer set in positive ionization mode. To assess data quality and attain general insights regarding the results, data was subjected to principal component analysis (PCA). As shown in Figure 1, QC samples formed a tight cluster, confirming the quality of the analyses. The studied groups exhibit relatively good separation, allowing for visualization of differences in metabolic and lipidomic profiles before and after transplantation, as well as during organ perfusion (Figure 2). SPME sampling permits metabolic profiling of the organ over time. Box-whisker plots of selected metabolites serve to exemplify alterations in metabolite levels throughout the experiment (Figure 3). The wide spectrum of extracted features separated on both RP and HILIC columns is shown in Figure 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60946/60946fig01.jpg" /> 
Figure 1: Principal component analysis showing clustering of quality control samples for metabolomic reversed-phase (A), HILIC (B) and lipidomic reversed-phase (C), HILIC (D) analyses.&#160;The quality control is necessary in untargeted analysis to monitor any possible changes induced by instrumental instability. Tight cluster of QC samples ensures that all changes observed are of biological origin. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60946/60946fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60946/60946fig02.jpg" /> 
Figure 2: Principal component analysis showing metabolomic (A) and lipidomic (B) differences between particular sampling points.&#160;Untargeted profiling of kidney with SPME-LC-HRMS enables to observe biochemical differences in organs at subsequent steps of their preservation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60946/60946fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60946/60946fig03.jpg" /> 
Figure 3: Box-whisker plots of selected compounds showing alterations in metabolites (A, B) and lipids (C, D) during the peritransplant period.&#160;After selection and identification of discriminating compounds box-whisker plots enables to monitor changes in levels of these compounds at subsequent steps of the protocol and compare the metabolites levels in organs subjected to different preservation protocols or harvested from different donors e.g. heart beating donors or donors after cardiac death. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60946/60946fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60946/60946fig04.jpg" /> 
Figure 4: Ion map (m/z versus retention time) of features obtained by LC-MS analysis with (A) reversed-phase and (B) HILIC separation.&#160;The plot enables to estimate overall number of features obtained with the proposed protocol (from extraction to detection) and assess analyte coverage based on the polarity. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60946/60946fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using a Chemical Biopsy for Graft Quality Assessment at JoVE.com

Using a Chemical Biopsy for Graft Quality Assessment

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.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

using-a-chemical-biopsy-for-graft-quality-assessment

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

5.1K Views.  Nicolaus Copernicus University in Torun, Collegium Medicum in Bydgoszcz. 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.

Video: Using a Chemical Biopsy for Graft Quality Assessment

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.

Substernal thyroid lesions are common, and need to be differentiated from malignancy. Obtaining percutaneous fine needle biopsy is not possible due to its retrosternal location. This article proposes a protocol for biopsy of substernal thyroid lesions using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA).

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Synergizing Antegrade Endoscopic with Bridging Vein Harvesting for Improvement of Great Saphenous Vein Graft Quality from the Lower Leg

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative wound complications. This standardized protocol for antegrade endoscopic vein harvesting (EVH) from the lower leg has the potential to be introduced to routine coronary artery bypass grafting (CABG). Patients undergoing CABG surgery are positioned on a surgical table with two additional foam rollers below the extended legs, enabling antegrade EVH from the lower leg. Following minimally invasive surgical access through a bridging vein harvest technique, an endoscopic optical dissector is inserted antegrade into the wound. The main vessel and side branches are dissected under continuous optical control of vein quality status and the working channel. After, an endoscopic optical retractor is inserted with an internal bipolar electrocoagulation device for precise, safe, and tissue-protective interruption of side branches. After release of the vein, the vessel is cut off at the proximal and distal ends under optical control, retrieved from the wound, then cannulated and flushed with heparinized saline. Finally, all side branches of the vein graft are double-clipped. Vascular histology is analyzed in a randomized selection of vein samples. After applying this standardized EVH protocol, the learning curve was shown to be steep, and graft quality was sufficient for coronary artery bypass grafting in every case. There was no conversion to surgical harvesting and low risks for tissue damage and bleeding. Leg positioning and synergizing EVH with bridging vein harvesting improved procedural success and vein graft quality. In our hands, antegrade EVH from the lower leg was feasible, demonstrating straightforward graft dissection as well as adequate macroscopic and microscopic graft quality with preserved endothelial integrity. In conclusion, the introduced technique is safe, shows excellent vein autograft quality, and illustrates feasibility for elective and urgent isolated CABG and combined CABG scenarios.

Presented here is a protocol for antegrade endoscopic vein harvesting from the lower leg, which can safely be introduced in routine coronary artery bypass grafting. Vein grafts present excellent graft quality following this standardized protocol with positioning of the legs, minimally invasive access to the vein, and antegrade endoscopic vein harvesting.

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative ...