The Vienna General Hospital and the Medical University of Vienna kindly provides the infrastructure for the Vienna Hepatic Hemodynamic Laboratory. Previous members of the Vienna Hepatic Hemodynamic Laboratory and coworkers should be acknoweledged for their valuable input that helped to continuously improve the methodology of HVPG measurement and transjugular liver biopsy at our institution. In addition, we thank the nurses of the Division of Gastroenterology and Hepatology who are an essential part of the Vienna Hemodynamic Laboratory and continuously provide patients with excellent care.

The protocol described here complies with the guidelines of the human research ethics committee of the Medical University of Vienna.
1. Preparations
<ol>
	<li>Use a specialized room dedicated/approved for procedures using x-ray equipped with a digital x-ray fluoroscopy&#160;system (Figure 1A), a monitor system (Figure 1B), a pressure recorder and transducer (Figure 1C) that are connected to a printer or recording software, and an ultrasound device (Figure 1D). Also, ensure there is sufficient space for a sterile working area (Figure 1E) next to the patient bench.</li>
	<li>Evaluate patients with suspected advanced chronic liver disease (ACLD) for CSPH by measurement of HVPG. In particular, patients with one of the following features might undergo HVPG measurement: thrombocytopenia &#60;150 G/L, portosystemic collaterals on cross-sectional imaging, gastroesophageal varices prior to initiation of non-selective betablocker therapy, ascites, and hepatic tumors that are scheduled for resection.</li>
	<li>Use the following contraindications for the procedure: (i), absence of vascular access due to jugular or caval vein thrombosis, (ii) clinically evident bleeding disorders (e.g. disseminated intravascular coagulation), (iii) contralateral pneumothorax, and (iv) significant cardiac arrhythmias.</li>
	<li>Ensure that the patient is fastened and gave written informed consent after being informed about the purpose and potential risks of the procedure.</li>
	<li>Explain that the procedure must be performed without general anesthesia. However, low doses of midazolam (up to 0.02 mg/kg bodyweight) might be offered to anxious patients.</li>
	<li>Monitor the patient&#8217;s vital signs by non-invasive arterial blood pressure measurement, pulse oximetry, and electrocardiography using a standard monitor system.</li>
	<li>Flush the pressure transducer set using sterile saline solution in a pressure bag.</li>
	<li>Calibrate the pressure transducer (if not precalibrated, calibrate against an external pressure reference before starting measurements, e.g. by using a water column where 13.6 cm H2O equals 10 mm Hg).</li>
	<li>Place the transducer at the level of the right atrium by aiming at the patient&#8217;s midaxillary line.</li>
	<li>Prepare the pressure recorder/recording software to be ready for recording pressure tracings.</li>
	<li>Ensure that all necessary sterile equipment is ready (see Table of Materials).</li>
	<li>Ask the patient to take a supine position on the patient bench.</li>
	<li>Have the operator wash and disinfect her/his hands and forearms.</li>
	<li>Put on surgical cap and face mask, sterile gloves and a sterile coat.</li>
	<li>Use a sterile table cover and prepare a sterile working area for the equipment needed for the central venous access (Figure 2A).</li>
</ol>
2. Central venous access under sterile conditions
<ol>
	<li>Instruct the patient to&#160;turn his/her head slightly to the left side, then disinfect the area of the right anterior and lateral neck with a disinfectant solution.</li>
	<li>Place sterile towels that cover the skin around the carotid triangle.</li>
	<li>Use a ultrasound probe (covered by a sterile US probe cover sheath) to identify the right internal jugular vein and the appropriate puncture site.</li>
	<li>Infiltrate the area of subsequent vascular access with a 21 G needle and apply a local anaesthetic (lidocaine 2%, 5 mL). Then, wait 1&#8211;2 min for its full effect.</li>
	<li>Prepare all materials needed for central venous access (see Table of Materials).</li>
	<li>Using the equipment of the 7.5 F catheter introducer sheath set, place the needle in the right internal jugular vein using ultrasound guidance and advance the guidewire through the needle using the Seldinger technique (Figure 3A). 
	NOTE: If a transjugular biopsy should be performed after HVPG measurement, a larger diameter 10 F liver biopsy introducer sheath set (with extra 18 G needle and guide wire) has to be used.</li>
	<li>Make a 2-3 mm incision along the guidewire using a blade 11 scalpel to ease introduction of the vascular access sheath loaded with the dilatator.</li>
	<li>Insert the vascular access percutaneous sheath&#160;loaded with the dilatator into the internal jugular vein over the guidewire.</li>
	<li>Remove the guidewire and the dilatator of the vascular access sheath.</li>
	<li>Make sure that the vascular access sheath remains in&#160;a stable intravascular position and orient the injection port of the vascular access sheath towards the lateral side (Figure 3B). 
	NOTE: Suturing or taping is not needed.</li>
</ol>
3. Placement of the balloon catheter in a hepatic vein
<ol>
	<li>Prepare all materials needed for placement of the balloon catheter into a&#160;hepatic vein (Figure 2B).</li>
	<li>Flush the balloon catheter with contrast media (dye) and check the integrity of the balloon by repetitive insufflation/deflation with the catheter tip immersed in saline solution. No air bubbles should occur. 
	NOTE: Depending on the visibility of the catheter itself on x-ray, you may also use just sterile saline or diluted dye to flush the vascular lumen of the catheter.</li>
	<li>Moisten the balloon catheter on the outside with sterile saline solution before inserting it into the vascular access sheath.</li>
	<li>Advance the balloon catheter under fluoroscopic guidance into the inferior cava vein (IVC). Aligning the tip of the balloon catheter towards the back of the patient and slight rotations of the catheter usually allows to advance from the right atrium to the IVC. Instructing the patient to inhale deeply might help in some cases (Figure 3B).</li>
	<li>Advance the balloon catheter from the IVC into the hepatic vein. Try to get access to the hepatic veins by repeatedly moving with the tip of the balloon catheter facing to the right towards the suspected area of the junction of the hepatic veins and the IVC (Figure 3C).</li>
	<li>Ensure that the catheter is advanced into a stable position that allows the repeated measurement of the free hepatic venous pressure (FHVP) at a 2-4 cm distance from its opening into the IVC and adequate space for the inflated balloon in the lumen of the hepatic vein in order to record the wedged hepatic venous pressure (WHVP).</li>
	<li>Check for an adequate occlusion of the vein (wedge position) by inflating the balloon (about 2 mL of air into the balloon lumen) and contrast agent&#160;injection (about 5 mL into the vascular lumen) until the hepatic vein distal to the inflated balloon is visualized (Figure 3D).</li>
	<li>Observe the stasis of the contrast media and exclude washout of the contrast media due to insufficient occlusion of the venous lumen by the balloon or due to the presence of vein-vein communications. If significant contrast media washout is observed, try to reposition the balloon catheter.</li>
	<li>Deflate the balloon and flush the lumen of the catheter with saline.</li>
</ol>
4. Hemodynamic readings for assessing the HVPG
<ol>
	<li>Connect the vascular lumen of the balloon catheter to the pressure transducer using an infusion line.</li>
	<li>Start recording the FHVP with the tip of the balloon 2-4 cm from the opening of the hepatic vein to the IVC. The waveform of the curve must be stable without variations over time. 
	NOTE: Stable values are usually obtained after 15 s.</li>
	<li>Inflate the balloon and continue recording the WHVP until the measurement becomes a stable horizontal line with no variations over time. 
	NOTE: Stable tracings of the wedged pressure are usually obtained after &#62;40 s.</li>
	<li>Repeat the measurement of FHVG (&#62;15 s) and WHVP (&#62;40 s) at least three times to obtain triplicate high-quality readings (Figure 3E). 
	NOTE: If significant discrepancies of &#8805;2 mm Hg are observed between the single FHVP/WHVP readings, additional measurements should be obtained. Note the reasons for potential artefacts, such as coughing, moving, or talking at the respective timepoints of hemodynamic recording.</li>
	<li>Record pressure in the IVC at the level of the ostium of the hepatic vein as well as the right atrial pressure (RAP).</li>
	<li>Stop recordings.</li>
	<li>Remove the balloon catheter.</li>
	<li>Calculate HVPG (FHVP subtracted from WHVP) as the mean of 3 measurements. 
	NOTE: If significant differences &#8805;2 mm Hg are noted between the sequentially obtained HVPG readings, additional measurements should be obtained.</li>
	<li>Proceed to transjugular biopsy (step 5.1) or remove the catheter introducer sheath from the internal jugular vein. 
	NOTE: Inserting a stiff guidewire may help to reach the same intrahepatic position for subsequent transjugular liver biopsy.</li>
	<li>Apply pressure on top of the vascular insertion site of the internal jugular vein for at least 5 min using a sterile gauze.</li>
</ol>
5. Preparation for transjugular liver biopsy
NOTE: Two different biopsy methodologies may be used to obtain a transjugular liver biopsy: aspiration (step 6) or core biopsy (step 7). First decide which system to use and then select the appropriate biopsy needle introducer sheath before proceeding with steps 5.1 to 5.3). Alternatively, the needle of the core TJBLX set can also be inserted into the aspiration set (NOTE: use appropriate diameters), which results in a more flexible core biopsy system that may be easier to introduce into a hepatic vein (NOTE: this approach is not described in detail here).
<ol>
	<li>Prepare the respective transjugular liver biopsy (TJLBX) set (aspiration TJLBX: Figure 4A-B; core TJLBX: Figure 4C; see Table of Materials).</li>
	<li>Flush the biopsy needle introducer sheath with sterile saline or, in case of transjugular aspiration liver biopsy, with contrast agent&#160;for better visualization.</li>
	<li>Advance the biopsy needle introducer sheath into a hepatic vein using the same technique as described in 3.4.</li>
	<li>Depending on the biopsy set used, proceed either to step 6.1 for aspiration TJBX&#160;or to step 7.1 for core TJBX.</li>
</ol>
6. Transjugular aspiration liver biopsy
<ol>
	<li>Use a 10 mL Luer-lock syringe to flush the aspiration TJLBX needle with sterile saline, however, leave about 3 mL in the syringe to facilitate aspiration.</li>
	<li>Advance the biopsy needle gently through the biopsy needle introducer sheath until the tip of the needle reaches the end of the introducer sheath. 
	NOTE: Avoid any force or fast movements while advancing the needle. Asking the patient to take a deep breath will decrease the angle between the IVC and the hepatic veins for easier advancement of the biopsy needle.</li>
	<li>Ask the patient to hold his breath.</li>
	<li>Apply suction using the 10 mL Luer-lock syringe and advance the needle into the liver parenchyma.</li>
	<li>Retract the needle while still applying suction (negative pressure).</li>
	<li>Advice the patient to continue breathing normally.</li>
	<li>Remove the needle (but not the needle introducer sheath) and harvest the liver sample. 
	NOTE: The liver sample is usually retained in the syringe, not in the needle (Figure 4D).</li>
	<li>Repeat steps 6.1 to 6.6 for additional needle passages until sufficient liver specimens are obtained.</li>
	<li>Inject 5-10 mL of contrast media over the catheter introducer sheath to rule out perforation of the liver capsule.</li>
	<li>Remove the biopsy needle introducer sheath.</li>
	<li>Remove the 10 F liver biopsy set introducer sheath and apply pressure on top of the vascular insertion site of the internal jugular vein for at least 5 min using sterile gauzes.</li>
</ol>
7. TRANSJUGULAR CORE LIVER BIOPSY
<ol>
	<li>Load the core TJLBX needle by pulling the grip until the shooting mechanism is locked. Advance the core biopsy needle gently through the biopsy needle introducer sheath until the tip of the needle approaches the end of the introducer sheath. 
	NOTE: Avoid any force or fast movements while advancing the needle.</li>
	<li>Ask the patient to hold his/her breath.</li>
	<li>Advance the needle into the liver parenchyma.</li>
	<li>Perform the core biopsy by pulling the trigger of the shooting mechanism.</li>
	<li>Advice the patient to continue breathing normally.</li>
	<li>Remove the needle (but not the needle introducer sheath) and harvest the liver sample. A small needle might help to remove the liver sample (Figure 4D).</li>
	<li>Repeat steps 7.1 to 7.6 for additional needle passages until sufficient liver specimens are obtained.</li>
	<li>Inject 5-10 mL of contrast media over the side port of the catheter introducer sheath to rule out perforation of the liver capsule.</li>
	<li>Remove the biopsy needle introducer sheath.</li>
	<li>Remove the 10 F liver biopsy set introducer sheath and apply pressure on top of the vascular insertion site of the internal jugular vein for about 5 min using sterile gauzes.</li>
</ol>

Thomas Reiberger has received grant and material support by Cook Medical as well as grant support and honoraria for lectures from W.L. Gore &#38; Associates. Philipp Schwabl, Markus Peck-Radosavljevic and Michael Trauner have nothing to disclose in regard to this manuscript. Mattias Mandorfer has received honoraria for lectures from W.L. Gore &#38; Associates.

While HVPG measurements require considerable resources and trained personal with interventional skills and expertise in the reading of pressure tracings, it improves prognostication and might guide treatment decisions, and thus, facilitates personalized medicine. In addition, the opportunity to safely obtain liver biopsy specimens via the transjugular route in the same session is another argument in favor of implementing hepatic hemodynamic laboratories at tertiary care centers. Indeed, guidelines support the use of HVPG...

Patients with cirrhosis are at risk for developing complications mostly related to portal hypertension (PHT), such as ascites or bleeding from gastric or esophageal varices1,2,3. The risk of hepatic decompensation is related to the degree of PHT2. Measurement of the hepatic venous pressure gradient (HVPG) is the gold standard to estimate portal venous pressure in patient with cirrhosis, i.e. assessing the severity of sinusoidal&#160;portal hypertension4. An HVPG of &#8805;6 mm Hg to 9 mm Hg indicates elevated portal pressure (&#39;subclinical portal hypertension&#39;), while an HVPG &#8805;10 mm Hg defines CSPH. This protocol provides a detailed description of the equipment and the procedure and also highlights potential pitfalls and offers advice for troubleshooting.
Clinically, measurement of HVPG is indicated (i) to establish the diagnosis of sinusoidal&#160;portal hypertension, (ii) to identify patients at risk for hepatic decompensation by diagnosing CSPH (HVPG &#8805;10 mm Hg), (iii) to guide pharmacological therapy in primary or secondary prophylaxis of variceal bleeding, and (iv) to assess the risk of hepatic failure after partial hepatectomy2,4. HVPG is used as an established surrogate marker for improvement and/or worsening of liver fibrosis/function, since a decrease in HVPG translates into a clinically meaningful benefit5, whereas higher HVPG values are associated with an increased variceal bleeding risk6. Based on observations on changes in HVPG in patients under non-selective beta-blocker (NSBB) or etiological therapies, a decrease in HVPG of 10% is considered to be clinically relevant7,8.
To date, there are no alternative, non-invasive parameters reflecting the degree of portal pressure with similar accuracy as HVPG. Even if HVPG is actually an &#8216;indirect&#8217; way to measure portal pressure, it strongly correlates and thus accurately reflects &#8216;directly&#8217; measured portal pressure in patients with cirrhosis9. Importantly, HVPG measurements should be performed using a balloon catheter to maximize the assessed amount of liver parenchyma10,11,12. Although HVPG measurements are invasive, resource-intensive, and require interventional skills and expertise in interpreting the reliability and plausibility of pressure readings, this method is the current gold standard for diagnosing and monitoring portal hypertension in patients with cirrhosis13,14,15.
Simple laboratory values, such as platelet count, may help to estimate the likelihood for CSPH. However, platelet count, or non-invasive scores that include platelet count, have limited predictive value16. Imaging modalities showing splenomegaly17 or portosystemic collaterals18 in patients with cirrhosis suggest the presence of CSPH, but are not helpful for quantifying the actual degree of portal hypertension. Novel non-invasive imaging tools, such as elastography of the liver19 and/or of the spleen20 are useful for ruling-in or ruling-out the presence of CSPH. Still, none of the available methods is able to directly measure dynamic changes in portal pressure21.
The prognostic value of HVPG has been underlined by several landmark studies, showing that a HVPG &#8805;10 mm Hg (i.e. CSPH) is predictive for the formation of varices8 (and for the development of complications related to portal hypertension22, while a (pharmacologically-induced) decrease of HVPG modulates the respective risk of variceal growth23 and decompensation7. HVPG-response is the only established surrogate for the effectiveness of NSBBs in preventing (recurrent) variceal bleeding. If HVPG decreases to a value of &#8804;12 mm Hg or is reduced by &#8805;10-20% during NSBB treatment, patients are protected from variceal bleeding and survival is increased24,25. Similarly, HVPG-response also decreases the incidence of ascites and related complications in patients with compensated cirrhosis5,26. Several studies have provided evidence supporting the use of HVPG-guided therapy27,28,29,30,31,32. Thus, in centers with sufficient experience, HVPG-response may&#160;guide treatment decisions, facilitating personalized medicine for patients with portal hypertension.
Moreover, measuring of HVPG might serve as a surrogate endpoint for proof-of-concept studies assessing the effectiveness of novel treatments for cirrhosis and/or portal hypertension being translated from bench to bedside, such as sorafenib33,34, simvastatin35,36, taurine37, or emricasan38. Ultimately, measurements of HVPG can also provide important prognostic information about the risk for development of HCC39 and for liver failure post hepatic resection40.
The infrastructure to measure HVPG should be readily available at secondary and tertiary care centers. Since the technique of HVPG measurement requires specialized training and equipment, it seems rational for academic and transplant centers to establish a hepatic hemodynamic laboratory, facilitating state-of-the-art diagnosis and management of portal hypertension. Large volume centers perform several hundred HVPG measurements per year. Based on our experience, sufficient expertise to perform accurate HVPG measurements is usually obtained after 50-100 supervised HVPG measurements.

<table border="0" cellpadding="0" cellspacing="0" style="width:1628px;" width="1627">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">10 mL Luer-Lock syringe</td>
			<td style="width:335px;">Braun</td>
			<td style="width:317px;">REF 4617100V, LOT 17G03C8</td>
			<td style="width:661px;">Luer-Lock Syringe for connection with the aspiration biopsy set</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL Syringe 2x</td>
			<td>Braun</td>
			<td style="width:317px;">REF 4606108V</td>
			<td>Snonpyrogenic, nontoxic 10 mL syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 F liver biopsy&#160; introducer sheath set</td>
			<td>Cook Medical</td>
			<td style="width:317px;">REF RCFW-10.0-38, REF G07600</td>
			<td>Percutaneous Sheath Introducer Set (TJBX), 10F Port, 13cm, Check-Flo Performer Introducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needle for biopsy introducer sheat</td>
			<td>Arrow International</td>
			<td style="width:317px;">REF AN-04318</td>
			<td>Introducer Needle for TJBX Set, 18 G, 6.35 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 G needle</td>
			<td>Henke Sass Wolf</td>
			<td style="width:317px;">REF 0086, Fine-Ject 21Gx2&#34;</td>
			<td>Sterile injeciton needle, 21 Gx2&#34;, 0.8 x 50 mm, for local anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way channels</td>
			<td>Becton Dickinson</td>
			<td style="width:317px;">BD Connecta Luer-Lok, REF 39402</td>
			<td>Three way channel with Luer-Lok connection system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5 F catheter introducer sheath set</td>
			<td>Arrow International</td>
			<td style="width:317px;">REF SI_09875-E</td>
			<td>Percutaneous Sheath Introducer Set, 7.5 F Port</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aspiration TJLBX set</td>
			<td>Cook Medical</td>
			<td style="width:317px;">REF RMT-16-51.0-TJL, REF G20521</td>
			<td>TJ Liver Access and Biopsy Needle Set (Aspiration Set), 9 F-45 cm, 16 G-50.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Balloon catheter</td>
			<td>Gerhard Pejcl Medizintechnik Austria</td>
			<td style="width:317px;">REF 500765B</td>
			<td>Ferlitsch HVPG Catheter, 7F-65cm, Balloon:2.5mL, Pressure 50-90 kPa, GW: 0.032&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blade 11 scalpel</td>
			<td>Medi-Safe Surgicals</td>
			<td style="width:317px;">MS Safety Scalpel REF /Batch 18012424</td>
			<td>11 blade safety scalpels, retractable, single use, 10 scalpels per package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt tip fill needle</td>
			<td>Becton Dickinson</td>
			<td style="width:317px;">REF 303129</td>
			<td>Sterile blunt tip fill needle, single use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Contrast media (dye)</td>
			<td>Dr. Franz K&#246;hler Chemie GmbH, Bensheim, GER</td>
			<td style="width:317px;">ZNR 1-24112</td>
			<td>Peritrast 300 mg Iod/mL, 50 mL, contrastmedia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Core TJLBX set</td>
			<td>Cook Medical</td>
			<td style="width:317px;">REF RMT-14XT-50.5-LABS-100, REF G08285</td>
			<td>TJ Needle Introducer and Bx-Needle 7 F-53.5 cm, 14 G-53.5 cm/20 mm, 18 G-60 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital x-ray fluoroscope system</td>
			<td>Siemens</td>
			<td style="width:317px;">Model No 07721710</td>
			<td>ARCADIS Varic, mobile x-ray fluoroscope system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disinfectant solution</td>
			<td>Gebro Pharma</td>
			<td style="width:317px;">1-20413</td>
			<td>Isozid-H</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Face mask</td>
			<td>MSP Medizintechnik GmbH</td>
			<td style="width:317px;">REF HSO36984</td>
			<td>Surgical face mask from double fleece, with tie, 50 pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guide wire for biopsy introducer sheat</td>
			<td>Arrow International</td>
			<td style="width:317px;">REF AW-14732</td>
			<td>Marked Spring-Wire Guide, TJBX Set, 0.032&#34;, 0.81 mm, 68 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infusion line</td>
			<td>Rosstec Medical Products b Cardea GmbH &#38; Co</td>
			<td style="width:317px;">REF 220010, 100m</td>
			<td>Infusion line, Luer-Lok for connection of balloon catheter and pressure transducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lidocaine 2%</td>
			<td>Gebro Pharma</td>
			<td style="width:317px;">Xylanaest, ZNR 17.792, 20mg/1mL (2%)</td>
			<td>Sterile vials Xylanest including 2% Lidocain hydrochloride for local anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Midazolame</td>
			<td>Roche</td>
			<td style="width:317px;">Dormicum, ZNr 1-18809, Midazolam 5mg/5mL</td>
			<td>Sterile vials Dormicum including Midazolam for sedation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitor system</td>
			<td>Datex-Ohmeda by GE</td>
			<td style="width:317px;">Type F-CMREC1</td>
			<td>Patient monitoring system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Patient bench</td>
			<td>Silerlen-MAQET</td>
			<td style="width:317px;">Model No 7474.00A</td>
			<td>Mobile patient bench for x-ray fluoroscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure bag</td>
			<td>Ethox Corp</td>
			<td style="width:317px;">REF 4005</td>
			<td>Pressure Infuser Bag 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure recorder</td>
			<td>Edwards Lifesciences</td>
			<td style="width:317px;">Ref T001631A, Lot 61202039</td>
			<td>TruWave 3 cc/150 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure transducer</td>
			<td>Edwards Lifesciences TM</td>
			<td style="width:317px;">REF T001631A</td>
			<td>Pressure Monitoring Set (1x), 3 cc/150 cm, TruWave TM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Recording software&#160;</td>
			<td>Datex Ohmeda by GE</td>
			<td style="width:317px;">Software S/5 is property of Instrumentarium Corp of Datex-Ohmeda TM</td>
			<td>Datex-Ohmeda S/5TM Collect - Software to record pressure tracings of the patient monitor system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile coat</td>
			<td>Lohmann &#38; Rauscher International GmbH &#38; Co</td>
			<td style="width:317px;">REF 19351</td>
			<td>Surgical Gown, Different sizes, e.g. L-130 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile gauze</td>
			<td>Hartmann</td>
			<td style="width:317px;">REF 401798, 10x10cm gauze</td>
			<td>10 x 10 cm sterile gauze, 10 pieces per package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile gloves</td>
			<td>Meditrade</td>
			<td style="width:317px;">REF 9021</td>
			<td>Gentle Skin sterile gloves, different sizes, e.g. 7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile saline solution</td>
			<td>Fresenius Kabi</td>
			<td style="width:317px;">NaCl 0.9%, B009827 REV 03</td>
			<td>Physiologic Saline Solution 0.9% NaCl, 309 mosmol/L, pH-Wert: 4.5-7.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile saline vessel</td>
			<td>KLS Martin</td>
			<td style="width:317px;">REF K8A,&#160; 18/10 Jonas</td>
			<td>Sterilizable Metal Vessel for sterile saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile table cover</td>
			<td>Hartmann</td>
			<td style="width:317px;">REF 2502208</td>
			<td>Table Cover, Foliodrape 150 x 100 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile towel</td>
			<td>BARRIER by M&#246;lnlycke Health Care</td>
			<td style="width:317px;">REF 706900</td>
			<td>Adhesive OP-Towel, 100 x 100 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile US probe cover sheath</td>
			<td>Websinger</td>
			<td style="width:317px;">REF 07014</td>
			<td>Sterile ultrasound probe cover, 20 x 60 cm, inluding two sterie adhesive tapes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stiff guidewire</td>
			<td>Cook Medical</td>
			<td style="width:317px;">REF TSMG-35-180-4-LES, G46729</td>
			<td>Lunderquist Extra Stiff Wire Guide, 0.035&#34;-.89 mm, 180 cm, 4 cm flexible tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical cap</td>
			<td>BARRIER by M&#246;lnlycke Health Care</td>
			<td style="width:317px;">REF 620500, PCS 100, Colour Green</td>
			<td>Surgical Cap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasound device</td>
			<td>FUJIFILM SonoSite</td>
			<td style="width:317px;">Model M-Turbo, REF P17000-17</td>
			<td>Mobile ultrasound system</td>
		</tr>
	</tbody>
</table>

measurement of the hepatic venous pressure gradient and transjugular liver biopsy

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced chronic liver disease followed by an instruction for transjugular biopsy. Under local anesthesia and ultrasound guidance, a catheter introducer sheath is placed in the right internal jugular vein. Using&#160;fluoroscopic guidance, a balloon catheter is advanced into the inferior vena cava (IVC) and inserted into a large hepatic vein. Correct and sufficient wedge position of the catheter is ensured by injecting contrast media while the balloon is blocking the outflow of the cannulated hepatic vein. After calibrating the external pressure transducer, continuous pressure recordings are obtained with triplicate recordings of the wedged hepatic venous pressure (WHVP) and free hepatic venous pressure (FHVP). The difference between FHVP and WHVP is referred to as HVPG, with values &#8805;10 mm Hg indicating clinically significant portal hypertension (CSPH). Before removing the catheter, pressure readings obtained in the IVC at the same level, as well as the right atrial pressure are recorded.
Finally, a transjugular liver biopsy can be obtained via the same vascular route. Different systems are available; however, core biopsy needles are preferred over aspiration needles, especially for cirrhotic livers. Again, under fluoroscopic guidance a biopsy needle introducer sheath is advanced into an hepatic vein. Next, the transjugular biopsy needle is gently advanced through the introducer sheath: (i) in case of aspiration biopsy, the needle is advanced into the liver parenchyma under aspiration and then removed quickly, or (ii) in case of a core biopsy, the cutting-mechanism is triggered inside the parenchyma. Several separate passages can be safely performed to obtain sufficient liver specimens via transjugular biopsy. In experienced hands, the combination of these procedures takes about 30-45 min.

In compensated patients with well-preserved liver function (i.e. without any history of hepatic decompensation, such as ascites or variceal bleeding) the measured HVPG values might be normal or in the range of subclinical portal hypertension (HVPG 6&#8211;9 mm Hg). However, compensated patients might develop CSPH (HVPG &#8805;10 mm Hg) which indicates an increased risk for developing varices or hepatic decompensation. In turn, patients with esophageal or gastric varices, HVPG is usually in the range of CSPH, if not patie...

Watch this Scientific Journal Video about Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy at JoVE.com

Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy

Here, we present a protocol for measurement of hepatic venous pressure gradient (HVPG),the gold standard to diagnose clinically significant portal hypertension. Moreover, we describe how to perform a transjugular liver biopsy within the same session.

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced ...

measurement-hepatic-venous-pressure-gradient-transjugular-liver

Research

JoVE Journal

Medicine

18.9K Views.  Medical University of Vienna. Here, we present a protocol for measurement of hepatic venous pressure gradient (HVPG),the gold standard to diagnose clinically significant portal hypertension. Moreover, we describe how to perform a transjugular liver biopsy within the same session.

Video: Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Quantitative Analysis of Liver Stiffness Distribution

A Three-Dimensional Digital Model for Early Diagnosis of Hepatic Fibrosis Based on Magnetic Resonance Elastography

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the disease. Despite the good progress made with the liver stiffness map (LSM) based on magnetic resonance elastography (MRE), there are still some limitations that need to be overcome, including manual focus determination, manual selection of regions of interest (ROIs), and discontinuous LSM data without structural information, which makes it impossible to evaluate the liver as a whole. In this study, we propose a novel three-dimensional (3D) digital model for the early diagnosis of hepatic fibrosis based on MRE.
MRE is a non-invasive imaging technique that employs magnetic resonance imaging (MRI) to measure the liver stiffness at the scanning site through human-computer interaction. Studies have indicated a significant positive correlation between the LSM obtained through MRE and the degree of hepatic fibrosis. However, for clinical purposes, a comprehensive and precise quantification of the degree of hepatic fibrosis is necessary. To address this, the concept of Liver Stiffness Distribution (LSD) was proposed in this study, which refers to the 3D stiffness volume of each liver voxel obtained by the alignment of 3D liver tissue images and MRE indicators. This provides a more effective clinical tool for the diagnosis and treatment of hepatic fibrosis.

Author Spotlight: Advancing Hepatic Fibrosis Diagnosis Using Magnetic Resonance Elastography and AI 

The objective of this study was to develop a novel three-dimensional digital model for the early diagnosis of hepatic fibrosis, which includes the stiffness of each voxel in the patient's liver and can thus, be used to calculate the distribution ratio of the patient's liver at different fibrosis stages.

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the ...

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...