The authors acknowledge all the members involved in the Lung Transplant Program at University Hospital Marqu&#233;s de Valdecilla.

These interventions are undertaken at the bedside in the intensive care unit (ICU). This protocol follows the guidelines of the University Hospital Marqu&#233;s de Valdecilla ethics committee and is in accordance with the Spanish legal framework regarding donation procedures. Informed consent was obtained from next of kin for video recording of the procedures for research. cDCD is considered in patients with catastrophic brain damage or a terminal heart or a neurodegenerative disease for whom the decision to withdraw life-sustaining therapy (WSLT) has been made. Exclusion criteria and lung evaluation are the same as with brain death donors (Table 1).
1. Premortem interventions in the donor
<ol>
	<li>Heparinization: administer intravenously a bolus of 300-500 UI/kg of heparin to the donor.</li>
	<li>Cannulation 
	NOTE: Cannulation is performed by the cardiovascular surgeon and is a sterile procedure.
	<ol>
		<li>Prepare a sterile instrumentation table, with all the equipment necessary (ECMO kit and surgical tools), as well as the electrocautery and suction system.</li>
		<li>Prepare a surgical field on the selected groin with disinfectant solution and sterile drapes.</li>
		<li>Make an 8-10 cm longitudinal incision with a no. 23 blade, controlling bleeding with electrocautery and Liga clips. Separate the wound edges with a retractor and proceed with dissection to expose the femoral artery and vein. Embrace both vessels with vessel loops for bleeding control.</li>
		<li>Select the appropriate cannula diameters according to the vessel sizes and large enough to provide a sufficient flow for organ perfusion and to prevent low-flow problems (usually 21 French).</li>
		<li>Cannulate the femoral vein, introducing first a metal wire as a guide, followed by progressive dilators to finally introduce the cannula. If bleeding is observed, perform a peri cannula 4-0 polypropylene purse string suture for control.</li>
		<li>Proceed in the same way with the femoral artery, using in this case a double lumen cannula.</li>
		<li>Cut off a 10 cm piece of the ECMO input line. Insert a straight connector with a Luer lock with a three-way stopcock assembled at one end of the piece and connect the other end of the piece to the arterial cannula (Figure 1).</li>
		<li>Purge the ECMO lines. Employ an irrigation pear with saline to fill the lines while connecting with cannulas. Connect the output ECMO line to the venous cannula and the input ECMO line to the straight connector with the three-way stopcock previously assembled to the arterial cannula. The three-way stopcock can be used for purging the system (Figure 2).</li>
		<li>Keep the ECMO lines clamped. Fix both cannulas to the groin with size 1 silk sutures to avoid displacement during transfer.</li>
		<li>Place a monitored pressure line in the femoral artery cannula and in donor&#39;s left radial artery.</li>
	</ol>
	</li>
	<li>Aortic occlusion balloon placement
	<ol>
		<li>Take, as a reference, the distance between the xiphoid process of the donor and the distal end of the arterial cannula and determine the length of the catheter to be inserted to reach the thoracic descending aorta. Set a reference mark in the balloon with a silk suture or a marker.</li>
		<li>Introduce a metal wire guide through the free lumen of the femoral artery cannula. Continue with the catheter in the same way, guided by the metal wire, and introduce it until the referenced mark.</li>
		<li>Confirm the correct position of the occlusion balloon with portable chest radiography or fluoroscopy (check radiopaque marks of the catheter above the diaphragm).</li>
		<li>Check the correct function of the occlusion balloon by filling it with a 50 cc cone syringe with saline just for 4-5 s, confirming that arterial pressure from the femoral cannula disappears while the pressure from the left radial artery is maintained (Figure 3).</li>
		<li>When the femoral pulse disappears, record the filling volume as the minimum volume to be used to block the thoracic aorta during ARNP. If flow is detected in the femoral cannula, check again for correct positioning or filling.</li>
	</ol>
	</li>
</ol>
2. Withdrawal of life-sustaining therapy (WLST) and declaration of death
<ol>
	<li>Transfer the donor connected to the ECMO system to the operation room. Prepare and drape the donor in a sterile fashion.</li>
	<li>Have the lung and abdominal preservation solutions and lines set and ready. Keep the surgical team scrubbed and sterile and ready in the adjacent operating room.</li>
	<li>Use a chronometer to register warm ischemic times. 
	NOTE: Functional warm ischemic time (FWIT), defined as the time from systolic blood pressure &#60;60 mmHg to ANRP is started for abdominal grafts and the administration of lung preservation solution through the pulmonary artery for lungs (5 min no-touch period is included). Upper limits of 30 min for the liver and pancreas and 60 min for the kidneys and lungs are considered.</li>
	<li>Allow relatives to be with their loved one during WLST until the declaration of death.</li>
	<li>Initiate WLST. Extubating is optional according to the relative&#39;s wishes. After the declaration of death, lead the relatives out from the surgical area.</li>
	<li>After the 5 min non-touch period, fill the aortic occlusion balloon with the previously determined minimum volume that ensures descending thoracic aortic block.</li>
	<li>If pressure from the left radial artery disappears, initiate ANRP. The pressure from the femoral cannula will turn into a continuous non-pulsatile flow provided by the ECMO.</li>
	<li>If flow in the radial line increases parallel to femoral pressure, stop ANRP and check the correct position and filling or clamp the thoracic aorta after another 5 min non-touch period before restoring ANRP. ANRP is not started until aortic occlusion is fully confirmed. 
	&#8203;NOTE: WLST can be performed in the operation room or in the ICU following the preference of relatives and loved ones. If it is performed in the ICU, after the 5 min no-touch period, the balloon is filled and function checked, ARNP is initiated, and the donor is transferred to the operation room where the surgical team is ready to start. If occlusion balloon malfunction is detected, ARNP is stopped until the thoracic aorta is clamped in the operation room.</li>
</ol>
3. Lung recovery and procurement technique
NOTE: Lung recovery and procurement techniques are performed by the thoracic surgeon and the&#160;transplant coordinator (Figure 4).
<ol>
	<li>Perform a medium sternotomy: proceed with a median vertical skin incision from the suprasternal notch to the tip of the xiphoid process. Extend the incision to the pectoral fascia and sternal periosteum using electrocautery.</li>
	<li>Divide the interclavicular ligament and create a plane by finger dissection behind the sternum, both at the level of the suprasternal notch and the xiphoid process. Divide the sternum with an electric saw. Place a sternal retractor and open carefully, releasing the pericardium from the posterior surface of the sternum. Control any bleeding point with electrocautery.</li>
	<li>At the same time, reintubate and ventilate the donor with 100% oxygen and a positive end-expiratory pressure of 5 cm H2O.</li>
	<li>If bronchoscopy was not performed during the donor&#39;s ICU stay as a critical patient management maneuver, it can be performed at this point by the second surgeon of the thoracic team. For the bronchoscopy, introduce a flexible bronchoscope through the endotracheal tube and evaluate the anatomy, mucosal appearance, and clear secretions.</li>
	<li>Open both the pleural cavities by longitudinal incisions in the mediastinal pleura.</li>
	<li>If there are doubts or problems about an adequate block of the supra-aortic vessels with the occlusion balloon, retract the left lung medially to expose and clamp the thoracic aorta as low as possible under direct vision.</li>
	<li>Examine the lungs performing visual and palpatory assessment. Inspect for bullae, contusion, atelectasis, pneumonia, and occult tumors. Deliver 1 L of 4 &#176;C saline in both pleural cavities.</li>
	<li>Reduce the inspired fraction of oxygen to 50%. Open the pericardium with an inverted T incision. Retract laterally the edges of the pericardium with 2-0 silk sutures fixed to the skin with mosquito forceps to expose the heart structures.</li>
	<li>Place a 4-0 polypropylene purse string suture on the main pulmonary artery below the bifurcation. Perform an arteriotomy with a no. 11 blade and dilate with curved mosquito forceps.</li>
	<li>Cannulate the pulmonary artery (PA) with a right-angled straight cannula clamped at the end. Connect the pulmonary artery cannula to the irrigation system line, assembling a straight connector with a Luer lock and a three-way stopcock. Connect the irrigation system to the lung preservation solution. Purge the lines.</li>
	<li>Start flushing 50-60 mL/kg of cold preservation solution in an antegrade fashion. Start flushing 500 &#181;g of prostaglandin diluted in 100 mL of saline at the same time through the three-way stopcock.</li>
	<li>Open the left atrial appendage or left atrium directly to allow free drainage. If areas of atelectasis are found, recruit them with short inspiratory holds at 25-30 cm H2O pressure.</li>
	<li>Once preservation is finished, remove the PA cannula. Announce to the rest of the team the intention to clamp the cava vein and start heart excision.</li>
	<li>Administer 1-1.2 L of saline solution to the donor before clamping the cava veins to avoid a decrease in pump flow due to the loss of blood venous return from the thorax.</li>
	<li>Place a cross-clamp in the inferior cava vein, making sure there is enough stump for the liver. Ligate and divide the inferior cava vein with no. 3 silk strand.</li>
	<li>Tie and divide the superior cava vein caudal to azygous with no. 3 silk strand. Secure the distal stump with a clamp.</li>
	<li>Leave the clamps remaining in the surgical field, being careful not to remove them by accident, as otherwise&#160;ANRP will be compromised. Excise the rest of the heart in a standard fashion.</li>
	<li>After heart excision, remove the lungs following the same procedure as with brain death donors, as described below.
	<ol>
		<li>Divide the inferior pulmonary ligaments, open the posterior pericardium, and expose the esophagus. Free the posterior mediastinal attachments of the lung with blunt dissection, ensuring cautious hemostasis.</li>
		<li>Dissect the pulmonary arteries away from the aorta. Isolate the trachea above the carina and pass a TA stapler around.</li>
		<li>Inflate the lungs to 50%-60% of tidal volume before withdrawing the endotracheal tube and divide the trachea. Remove any remaining attachment and extract the lung block from the donor.</li>
	</ol>
	</li>
	<li>Carefully check the thoracic cavity to detect any bleeding point, especially ligation of the azygos vein and cauterization of the vessels or capillaries from the posterior mediastinum, paratracheal structures, and surrounding tissues. Continuous blood loss may decrease the pump flow.</li>
	<li>Take the lung block to the back table and proceed with bench surgery. Separate the left and right lungs.</li>
	<li>With a Foley catheter with an inflated bulb at the tip, perform sequentially, through each pulmonary vein, a retrograde flush with 0.2-0.25 L of cold preservation solution.</li>
	<li>Pack each lung into a first sterile bag containing cold preservation solution only, surrounded by two other plastic bags, and store in a portable fridge containing ice-cold saline at 4 &#176;C.</li>
	<li>When an ex vivo lung perfusion system is indicated, follow the steps below for device connections to the pulmonary artery and trachea during lung procurement.
	<ol>
		<li>Preserve the main trunk of the pulmonary artery and not only its bifurcation during procurement.</li>
		<li>If it is not possible, take a 3-4 cm piece of the aorta to posteriorly suture it to the pulmonary artery bifurcation to replace the pulmonary artery trunk.</li>
		<li>Divide the trachea four to five rings above the carina to have enough length for intubation.</li>
		<li>Keep and store the lungs in block.</li>
	</ol>
	</li>
</ol>
4. Abdominal normothermic regional perfusion
<ol>
	<li>Initiate ANRP after filling the aortic occlusion balloon and checking for correct function.</li>
	<li>Set the following monitoring target points: pump flow = 2-2.5 L/min, continuous pressure of 60-65 mmHg in the femoral artery cannula, temperature = 37 &#176;C, pH = 7.35-7.45, hematocrit &#62;25%.</li>
	<li>Obtain blood samples from the femoral artery cannula with a 10 mL syringe after starting ANRP and every 30 min for hepatic and renal biochemistry analysis, serum lactate levels, blood arterial gas, and hematocrit values. Maintain ARNP for at least for 90-120 min.</li>
	<li>Discard the liver if alanine transaminase (ALT) or aspartate transaminase (AST) values are more than four times the upper normal limit during ANRP.</li>
</ol>
5. Liver and renal recovery
NOTE: Liver and renal recovery are performed by the hepatic surgeon and kidney surgeon, respectively.
<ol>
	<li>Perform medium laparotomy: proceed with a median vertical skin incision along the linea alba, from the xiphoid process (join to the previous sternotomy) to the pubis, curving the incision around the umbilicus. Use electrocautery to dissect the subcutaneous fat and superficial fascial layers down to the rectus sheath.</li>
	<li>Dissect through the anterior and posterior components of the rectus sheath and open the peritoneum to access the peritoneal cavity. Widen the incision by sticking the fingers into the hole created, taking care not to injure the underlying structures. Place retractors to have adequate exposure of the abdomen.</li>
	<li>Assess the macroscopic quality of the abdominal organs by performing visual and palpatory assessments. A liver biopsy can be taken if any concern is raised, as with brain death donors.</li>
	<li>If the chemistry values are correct and the macroscopic appearance is normal, validate the organs.</li>
	<li>Stop the ECMO device. Flush preservation solution for the abdominal organs via the femoral arterial cannula and use the femoral venous cannula for exsanguination.</li>
	<li>Procure abdominal organs suitable for transplantation in a standard fashion as in DBD27,28.</li>
</ol>

The authors declare there are no conflicts of interest.

Though the use of simultaneous lung cold perfusion with ARNP in cDCD was first published in 2014, very few experiences have been described for this25,26,29. Moreover, the utilization of cDCD lungs, regardless of the technique used, remains low in most countries.
The critical steps within this protocol are the use of premortem interventions; a specific methodology to ensure coronary and cerebral perfusion is not restored with ANRP; the minimization of injury to the grafts due to dual temperature; and the goal of maintaining an adequate flow in the ECMO device to ensure abdominal organ perfusion.
The premortem interventions are contemplated in Spanish protocols. Although they are not essential, premortem heparinization and cannulation can not only reduce the functional warm ischemic time of abdominal organs but also avoid the need for access to the aorta for cannulation after the declaration of death. If there is premortem manipulation, it must be properly explained, and explicit informed consent must be obtained always being respectful of the wishes and values of the patients and their relatives. Initially, both groins were used: one for cannulation and another for aortic occlusion balloon insertion. The use of a double lumen arterial cannula provides both an ECMO entry line and an access port for the occlusion balloon, leaving the contralateral groin free in case of need.
The main ethical concern surrounding the use of ANRP is the possibility of resuscitation. It is mandatory to ensure an adequate lack of flow to aortic arch vessels and avoid any possibility of restoring cerebral and coronary circulation once death has been declared and ANRP is started. To achieve this, aorta clamping (thoracic or abdominal) is performed23,24,30. Aortic occlusion with a balloon was first reported by our group25 and validated31. This methodology ensures an appropriate blockage of the aorta, guaranteeing the absence of perfusion during all the procedure by continuous monitorization of balloon volume and left radial artery pressure. It also avoids the need for prompt access to the aorta, turning a rushed procedure into a calmer one, which could potentially reduce organ damage and losses due to surgical events generated by the hurry32,33.
Initially, the abdomen was simultaneously opened with the thorax. This, added to topical cooling of the lungs with cold saline, favored heat loss. To minimize graft injury due to dual temperature, the thoracic team starts the procedure while the abdomen is left closed during lung procurement. This contributes to maintaining abdominal normothermia and makes the surgical field more comfortable. In addition, only 1 L of cold saline is delivered to each hemithorax for topical lung cooling. With experience, we have noticed this is not mandatory, as ventilated lungs have good tolerance to warm ischemia19,34.
One reason for the low recovery of lungs in cDCD with combined ANRP is the fear of the abdominal teams regarding the poor flow in the pump during lung preservation and procurement. Maintaining pump flow and avoiding volume loss to ensure abdominal organ perfusion are two main goals during the procedure. With the use of the non-touch cava vein technique35, the inferior cava vein is not clamped during lung preservation, improving the blood return to the ECMO circuit. Additionally, a fluid overload is administered to the donor before the cava is clamped to prevent the absence of thoracic venous return. Despite the isolation of the inferior vena cava and descending aorta in the chest, continuous oozing from the intrathoracic blood vessels, especially the azygous vein, during and after lung procurement can lead to volume loss. Ligation of the azygous vein is mandatory, and hemostasis must be careful during lung block removal. The thoracic cavity must be checked for any bleeding points before the thoracic surgeon leaves the surgical field.
Limitations of the method include the following. Premortem interventions are not ethically or legally accepted in many countries. There is wide variability in protocols on cDCD around the world17. Although not essential, these maneuvers have important advantages such as the reduction of FWIT.
This combined procedure increases the complexity of the whole organ procurement process and demands logistical needs. Protocols must be supported by competent authorities and the experience of all the individuals involved is important. Intensive care professionals qualified in cDCD donor management, as well as thoracic and abdominal teams familiar with ECMO systems, are usually concentrated in reference centers, discouraging other small centers to start a program in cDCD. Many hospitals in the national territory of Spain are attached to donation programs but lack the necessary means to put cDCD with ANRP into practice. For this reason, mobile ECMO teams have been set up in several communities to travel and support the preservation and procurement of abdominal organs36,37,38.
Recently, as an evolution of ANRP, in situ thoracoabdominal normothermic regional perfusion has emerged as a novel technique to recover hearts from cDCD donors39,40.
At University Hospital Marqu&#233;s de Valdecilla, located in Cantabria, the region with the highest rate of donation in the Spanish territory, the cDCD program was implemented in 2014. Prior experience with uDCD41 helped us to face this rapid transition to cDCD and embrace this new scenario. As the number of cDCD donors increased, the protocol and technique evolved and were refined. In the last 2 years, 38.4% of lung transplants were performed with cDCD donors (30 of 78), and cDCD lung donation has greatly reduced the time spent on the waiting list (median of 67 days in 2020, 94 days in 2019, 129 days in 2018, and 206 days in 2017), as published in the Spanish National Registry42.
Despite the increased complexity of donor management and organ procurement, this combined retrieval method is feasible, and it is safe for both thoracic and abdominal grafts.

Donation after circulatory death (DCD) started in Spain with uncontrolled donors. In 1996, the first national consensus document on DCD was published as a guide for the practice of uncontrolled donation after circulatory death1 (uDCD), also setting a moratorium on controlled donation after circulatory death (cDCD). In 2012, a new consensus emerged establishing the basis and the legislative framework for the practice of both uDCD and cDCD2. Currently, Spain is one of the most active countries in DCD, reaching the highest rate of donors after circulatory death in the world3. This type of donor represented nearly 35% of the total donors in 2021 in the country, with a marked decrease in uDCD and donors being exclusively cDCD4.
Organ procurement in cDCD is commonly performed using the super-rapid recovery technique5. After the declaration of death and when the non-touch period has elapsed, a rapid sternotomy and laparotomy is performed. The abdominal aorta and pulmonary artery are cannulated and flushed with cold perfusion solutions to preserve the abdominal and thoracic organs, plus topical cooling is conducted before retrieval6. In this situation, cDCD is characterized by the unpredictable consequences of warm ischemia,&#160;after the withdrawal of life-sustaining therapy. The ischemic damage during this period of agonic hypotension and progressive hypoxia, followed by the non-touch period after cardiac arrest, is further exacerbated by the later period of cold ischemia7. This combination of warm and cold ischemia seems to be detrimental, especially to abdominal grafts8,9,10,&#160;generating more reluctance among professionals in the use of these organs from cDCD donors.
To minimize these risks, an in situ preservation model, based on previous experiences from Spanish teams working in uCDC11,&#160;has been developed with growing interest. The use of extracorporeal membrane oxygenation (ECMO) systems to restore blood flow after death and before graft recovery can reverse the metabolic deviations resulting from ischemia and restore cellular physiology12. Abdominal normothermic regional perfusion (ANRP) can improve the quality of ischemic-damaged organs in cDCD13. Organ function can be assessed and improved, allowing a better selection of abdominal grafts for transplantation.
Recent international multicenter experiences provide evidence that ANRP versus the rapid recovery (RR) technique helps overcome traditional limitations in cDCD, reducing rates of post-transplant biliary complications, facilitating successful transplantation of older livers, and improving liver graft survival14,15. In kidneys, it seems to improve short-term outcomes with a lower delayed graft function and higher 1 year graft survival rates16. With this evidence, ANRP in cDCD has gained advantages over the rapid recovery technique for abdominal graft procurement and is now applied in several European countries and other parts of the world17,18.
The use of lungs from cDCD donors, however, was promptly adopted worldwide. A lung functional warm ischemic time of up to 60 min does not seem to affect survival19. In the last decade, several centers and multi-institutional experiences have reported outcomes after lung transplant from cDCD comparable to those from DBD20,21. The RR technique is the routine method for lung procurement: lungs are cooled topically and removed after being flushed with cold preservation solution22.
The first experiences combining ANRP and RR of lungs in cDCD were reported by two United Kingdom groups23,24. Years later, a variation of this technique adding premortem interventions was published25. Results present this dual procurement technique as safe and effective for both abdominal and thoracic grafts26. Obviously, the donation procedure becomes more complex. It requires technological and human resources, sufficient organizational capabilities, and has a higher economic cost. All of this may discourage professionals from starting a program. The aim of this study is to present a protocol especially focused on premortem interventions, cannulation, and aortic occlusion balloon placement, with tips and tricks learned from experience, and comment the different technical details to consider during lung retrieval when ARNP is used. At present, in the Center, cDCD donors have become the main source of grafts for thoracic and abdominal transplantation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vial 5 mL Heparin 1000 UI/mL</td>
			<td>ROVI</td>
			<td></td>
			<td>For donor heparinization</td>
		</tr>
		<tr>
			<td>ECMO KIT (MATERIALS FOR CANNULATION)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Artery pressure lines</td>
			<td>BEXEN MEDICAL</td>
			<td>137.15</td>
			<td>Artery pressure line por radial artery and femoral cannula</td>
		</tr>
		<tr>
			<td>Bandage scissors</td>
			<td>SURGIMEDIC</td>
			<td>BC-881R</td>
			<td>Shear to cut ECMO lines</td>
		</tr>
		<tr>
			<td>Bio-medicus Venous cannula 21 Fr (7.0 mm) x 27.5 in (69.9 cm)</td>
			<td>MEDTRONIC</td>
			<td>96670-121</td>
			<td>Venous cannula</td>
		</tr>
		<tr>
			<td>Clhorhexidine solution 2%</td>
			<td></td>
			<td></td>
			<td>Disinfectant solution</td>
		</tr>
		<tr>
			<td>ECMO device Maquet Rotaflow</td>
			<td>Maquet, Rasttat, Germany</td>
			<td></td>
			<td>ECMO system</td>
		</tr>
		<tr>
			<td>Electrocautery handle</td>
			<td>DEXTRO</td>
			<td>SW12200</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoReturn Arterial Cannula Kit&#160; 21-23F</td>
			<td>Edwards Lifesciences</td>
			<td>ER21B, ER23B</td>
			<td>Arterial cannula with a doble lumen to ECMO connection and to introduce aortic oclussion balloon</td>
		</tr>
		<tr>
			<td>Ethicon LigaClip med/short 20 titanium medium</td>
			<td>ETHICON</td>
			<td>MCS30</td>
			<td>Ligaclips for control bleeding during groin dissection</td>
		</tr>
		<tr>
			<td>Ethicon LigaClip med/short 20 titanium small</td>
			<td>ETHICON</td>
			<td>MCS20</td>
			<td>Ligaclips for control bleeding during groin dissection</td>
		</tr>
		<tr>
			<td>Insertion Kit Bio-medicus 180cm</td>
			<td>MEDTRONIC</td>
			<td>96551</td>
			<td>Insertion Kit for ECMO cannulas, with catheter, metal wire guide and dilators</td>
		</tr>
		<tr>
			<td>Irrigation pear</td>
			<td>MEDLINE</td>
			<td>DYNDE 20125</td>
			<td>Pear to be filled with saline and purge ECMO lines at the site of connection with cannulas</td>
		</tr>
		<tr>
			<td>Luer cone syringe 50cc</td>
			<td>CARDIONATUR</td>
			<td>60ML</td>
			<td>Syringe filled with saline to fill occlusion balloon</td>
		</tr>
		<tr>
			<td>Mersilk no 1, LR-60 CONV , 75 cm</td>
			<td>ETHICON</td>
			<td>W562H</td>
			<td>Silk curved suture for ECMO cannulas fixation</td>
		</tr>
		<tr>
			<td>Prolene 4/0</td>
			<td>ETHICON</td>
			<td>W8355</td>
			<td>polypropylene suture for purse string in femoral vessels or vascular suture</td>
		</tr>
		<tr>
			<td>Prolene 5/0 , 60 cm</td>
			<td>ETHICON</td>
			<td>8325</td>
			<td>polypropylene suture for vascular suture</td>
		</tr>
		<tr>
			<td>Prolene 5/0, 90 cm</td>
			<td>ETHICON</td>
			<td>8720</td>
			<td>polypropylene suture for vascular suture</td>
		</tr>
		<tr>
			<td>Reliant Stent Graft Balloon Catheter&#160;12F</td>
			<td>Medtronic, Ireland</td>
			<td>AB46</td>
			<td>Aortic occlusion balloon introduced through femoral artery. It is used as an endoclamp</td>
		</tr>
		<tr>
			<td>Scalpel blade no 11</td>
			<td>INTRAVEN</td>
			<td>150011</td>
			<td></td>
		</tr>
		<tr>
			<td>Scapel blade no 23</td>
			<td>INTRAVEN</td>
			<td>150023</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tube</td>
			<td>IBERHOSPITEX</td>
			<td>0027224-P</td>
			<td>Silicone tube to connect suction system</td>
		</tr>
		<tr>
			<td>Sofsilk braided silk no 1 strands</td>
			<td>COVIDIEN</td>
			<td>L-12</td>
			<td>Silk strand for ligation or bleeding control</td>
		</tr>
		<tr>
			<td>Sofsilk braided silk no 3 strands</td>
			<td>COVIDIEN</td>
			<td>L-115</td>
			<td>Silk strand for ligation or bleeding control</td>
		</tr>
		<tr>
			<td>straight connector 3/8&#34;x3/8&#34; with Luer lock</td>
			<td>ANDOCOR</td>
			<td>04CS0022</td>
			<td>Piece to connect arterial cannula with ECMO line and the three way stop-cock for pressure line and blood sampling</td>
		</tr>
		<tr>
			<td>Surgical pads pack</td>
			<td>TEXPOL</td>
			<td>146500</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical stapler</td>
			<td>COVIDIEN</td>
			<td>8886803712</td>
			<td>Stapler to close surgical wound</td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>BD CONNECTA</td>
			<td>394501</td>
			<td>Three way stop-cock to connect farterial cannula with pressure line</td>
		</tr>
		<tr>
			<td>Vessel loop large</td>
			<td>MEDLINE</td>
			<td>VLMAXR</td>
			<td>Vascular loop to embrace femoral artery and vein for bleeding control.</td>
		</tr>
		<tr>
			<td>Vessel loop small</td>
			<td>MEDLINE</td>
			<td>VLMINR</td>
			<td>Vascular loop to embrace femoral artery and vein for bleeding control.</td>
		</tr>
		<tr>
			<td>Yankauer suction terminal 50 V</td>
			<td>DEXTROMEDICA</td>
			<td>349701</td>
			<td>Suction terminal for suction while surgical dissection</td>
		</tr>
		<tr>
			<td>SURGICAL TOOLS FOR CANNULATION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adson retractor 20 cm adn 33 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic clamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boyd Scissors 18 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection forceps without jaws 21 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Farabeuf retractor small</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo scissors straight 14 cm and 16 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metzembaum scissors 18 cm, 20 cm and 23 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forceps straigth and curved</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder 18 cm and 23 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Russ dissection forceps 15 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle no 23 and no 21,&#160; 21 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Dissector 23 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATERIALS FOR LUNG PROCUREMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 cc syringe</td>
			<td>BD DISCARDIT</td>
			<td>309110</td>
			<td></td>
		</tr>
		<tr>
			<td>Alprostadil 500 mcgs injectable solution</td>
			<td>PFIZER</td>
			<td></td>
			<td>Prostaglandin injected with lung preservation solution</td>
		</tr>
		<tr>
			<td>Disposable GIA cartridge Steril 6/Ca</td>
			<td>MEDTRONIC</td>
			<td>1141634</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable GIA stapler 60/3.8 3/Ca</td>
			<td>MEDTRONIC</td>
			<td>2802122</td>
			<td>Stapler for trachea and bronquial division</td>
		</tr>
		<tr>
			<td>Foley catheter 18 Ch Folysil</td>
			<td>Folysil, Coloplast</td>
			<td>AA6118</td>
			<td>urinary catheter employed to canulated pulmonary veins for retrograde perfusion</td>
		</tr>
		<tr>
			<td>Lung preservation solution Perfadex 1000 mL</td>
			<td>Medisan, Uppsala, Sweeden</td>
			<td>19811 ( box of 10 units)</td>
			<td>Lung preservation solution</td>
		</tr>
		<tr>
			<td>Mersilk no 1, LR-60 CONV , 75 cm</td>
			<td>ETHICON</td>
			<td>W562H</td>
			<td>Silk curved suture for pericardium sutures</td>
		</tr>
		<tr>
			<td>Paediatric Venous cannula</td>
			<td>SORIN GROUP</td>
			<td>V132-12</td>
			<td>Cannula used for pulmonary artery cannulation</td>
		</tr>
		<tr>
			<td>Prolene 4/0</td>
			<td>ETHICON</td>
			<td>W8355</td>
			<td>polypropylene suture for purse string in pulmonary artery</td>
		</tr>
		<tr>
			<td>Scalpel blade no 11</td>
			<td>INTRAVEN</td>
			<td>150011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sofsilk braided silk no 1 strands</td>
			<td>COVIDIEN</td>
			<td>L-12</td>
			<td>Silk strand to fix arterial cannula with the tourniquet</td>
		</tr>
		<tr>
			<td>Sofsilk braided silk no 3 strands</td>
			<td>COVIDIEN</td>
			<td>L-115</td>
			<td>Silk strand for vessel ligation</td>
		</tr>
		<tr>
			<td>Sterile bags</td>
			<td></td>
			<td></td>
			<td>To keep and store lungs.</td>
		</tr>
		<tr>
			<td>Straigth connector 1,4&#34;/1,4&#34; with luer lock</td>
			<td>ANDOCOR</td>
			<td>04CS0032</td>
			<td>Piece to connect pulmonary artery arterial cannula with preservation line and the three way stop-cock for prostaglandin</td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>BD CONNECTA</td>
			<td>394501</td>
			<td>Three way stop-cock to connect farterial cannula with pressure line</td>
		</tr>
		<tr>
			<td>Uromatic set for irrigation double lead</td>
			<td>MEDISAVE</td>
			<td>TRC4007N</td>
			<td>Irrigation system for lung preservation solution</td>
		</tr>
		<tr>
			<td>Uromatic set for irrigation single lead</td>
			<td>MEDISAVE</td>
			<td>TRC4002</td>
			<td>Irrigation system for lung preservation solution</td>
		</tr>
		<tr>
			<td>SURGICAL TOOLS FOR LUNG PROCUREMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic cross- clamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Battery-powered surgical saw</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooley vascular clamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps 18 cm and 27,9 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finochietto sternal retractor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metzembaum scissors 20 cm and 23 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forceps curved 12,5 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SURGICAL TOOLS FOR ABDOMINAL ORGAN PROCUREMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adson articulated retractors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allis forceps 16 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic cross-clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boyd scissors 17 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo needle holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooley Vascular clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crile forceps curved 18 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Davis retractor 24.5 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DeBakey dissecting forceps 19.7 cm adn 24.1 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DeBakey vascular clamps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps 18 cm and 27.9 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Duval forceps 23 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Farabeuf retractors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kidney Trays 300 cc and 500 cc</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kocher forceps straigth 18 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Langenbeck retractors 21 cm and 23 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo scissors straigth and curved , 17 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito forceps straigth and curved, 12.5 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holders 15 cm, 18 cm, 23 cm and 23 cm.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pean forceps 16 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potts scissors 19cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rochester forceps curved 24 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rochester forceps straigth 24 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Russ dissection forceps 15 cm and 20 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Senn-mueller retractor 16 cm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

lung rapid recovery procurement combined with abdominal normothermic regional perfusion in controlled donation after circulatory death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

We performed a descriptive analysis of 30 lung transplants performed at University Hospital Marqu&#233;s de Valdecilla with lungs obtained from cDCD donors in the last 2 years, 2020 and 2021. Donor and recipient demographic characteristics, technical data, postoperative outcomes, and short-term results are presented here. These results are presented as absolute numbers and percentages for categorical variables and as measures of central tendency and dispersion for continuous variables. The Kolmogorov-Smirnov test was used to test for normal distribution of the data.
Donor characteristics and technical data 
Table 2 shows donors&#39; demographic characteristics and technical data. Half of the donors were males, and the median age was 56.5 years. Only 16.7% had a smoking history. Brain injury was caused in most cases by hemorrhage (53.3%), followed by anoxia (23.3%), and trauma (3.35%). Other causes included the withdrawal of life-sustaining therapy in patients with amyotrophic lateral sclerosis. The median time of donor ICU stay and mechanical ventilation was 6 days. The mean value of final donor partial oxygen pressure in arterial blood over fractional inspired oxygen concentration (PaO2/FiO2) was 427 mmHg. In two cases, ex vivo lung perfusion was necessary for reconditioning the lungs due to the macroscopic appearance of edema.
Lung recipient and transplant-related characteristics 
Table 3 shows the lung recipient and transplant-related characteristics. The percentage of patients with interstitial lung disease (ILD) was the highest (50%), followed by chronic obstructive pulmonary disease (COPD; 40%) and bronchiectasis (10%). Most recipients had a smoking history (83.3%), only 16.7% had systemic hypertension, and 10% had diabetes mellitus (DM). Pulmonary hypertension was present in 14 recipients (46.7%). All procedures were bilateral lung transplants, and none were performed in an urgent situation. One recipient needed intraoperative extracorporeal life support during surgery with ECMO. The median cold ischemic time was 292.5 min for the first graft and 405 min for the second.
Post-transplant complications and short-term outcomes 
There were no intraoperative deaths. The incidence of primary graft dysfunction (PGD) grades is presented in Table 4. Two recipients (6.6%) needed postoperative ECMO support due to PGD3. There was no need for re-thoracotomy due to bleeding or other causes in the postoperative period. The median time for postoperative intubation was 24 h, for ICU stay was 3.1 days, and for hospital stay was 18.9 days. Acute cellular rejection in the first 3 weeks was present in 12 recipients (40%). There was no hospital mortality, and 30 day survival was 100%.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63975/63975fig02.jpg" /> 
Figure 1: Assembly of arterial cannula connections. The figure shows the materials used for arterial cannula connection with ECMO and pressure lines. A 10 cm piece of the ECMO line is cut and used as a bridge between the arterial cannula and a straight connector with Luer lock with a three-way stopcock assembled. The three-way stopcock relates to the pressure line, and the other end of the straight connector is inserted into the ECMO line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/63975fig02large.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/63975/63975fig01.jpg" /> 
Figure 2: Cannulation. (A) The figure shows the double lumen cannula used for femoral artery cannulation. (B) The femoral artery and venous cannulation are performed in one groin by open access. Cannulas are purged, clamped, and connected to the ECMO device. The wound incision is closed, and cannulas are fixed to the skin with silk sutures to avoid displacement. (C) The aortic occlusion balloon is introduced by the free lumen of the femoral artery cannula. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/63975fig01large.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/63975/63975fig03.jpg" /> 
Figure 3: Aortic occlusion balloon. Checking position and function. (A) The aortic occlusion catheter in the correct position checked by chest X-ray (see radiopaque marks above the diaphragm). The femoral pulse in the arterial cannula disappears when the balloon is filled, while the radial pulse is maintained (wave pulse and O2 saturation). The complete disappearance of the femoral pulse indicates the minimum balloon filling volume. (B) If the balloon is not completely full or is too advanced, pressure will be detected in both the femoral and radial arteries. This figure has been modified from Tanaka et al.29. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/63975fig03large.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/63975/63975fig04.jpg" /> 
Figure 4: Scheme of RR lung procurement with ARNP. The figure shows a summary of the procedure. Abbreviations: ECMO = extracorporeal membrane oxygenation; PEEP = positive end-expiratory pressure; ANRP = abdominal normothermic regional perfusion, RR = rapid recovery. This figure has been modified and adapted from Mi&#241;ambres et al.36. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/63975fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Criteria selection for lung donation in cDCD. This table shows the general criteria for lung donor selection in cDCD.&#160;Abbreviations: cDCD = controlled donation after cardiac death; WLST = withdrawal of life-sustaining therapy; FWIT = functional warm ischemic time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/TABLE 1 Selection criteria for lung donation.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Donor characteristics and technical data. This table shows the main donor characteristics and technical data registered. Abbreviations:&#160;cDCD = controlled donation after circulatory death; MV = mechanical ventilation; ICU = intensive care unit; WLST = withdrawal of life-sustaining therapy; CA = cardiac arrest; WIT = warm ischemic time; PaO2/FiO2 = partial pressure of oxygen in arterial blood/fraction of inspired oxygen; EVLP = ex vivo lung perfusion; IQR = interquartile range; SD = standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/TABLE 2 Donor characteristics and technical data.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Lung recipient and transplant-related characteristics. This table shows the main lung recipient characteristics and data registered during transplant surgery. Abbreviations: COPD = chronic obstructive pulmonary disease; ILD = interstitial lung disease; DM = diabetes mellitus; BMI = body mass index; CMV = cytomegalovirus; ECMO = extracorporeal membrane oxygenation; ICU = intensive care unit; MV = mechanical ventilation; PaO2/FiO2 = partial pressure of oxygen in arterial blood/fraction of inspired oxygen; IQR = interquartile range; SD = standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/TABLE 3 Lung recipients and transplant characteristics.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Post-transplant complications and short-term outcomes. This table shows data registered during the hospital stay and short-term results. Abbreviations:&#160;PGD = primary graft dysfunction, ECMO = extracorporeal membrane oxygenation; ICU = intensive care unit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63975/TABLE 4 Post-transplant complications short term outcomes.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death at JoVE.com

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

lung-rapid-recovery-procurement-combined-with-abdominal-normothermic

Nanyang Technological University

Research

JoVE Journal

Medicine

1.7K Views.  University Hospital Marqués de Valdecilla. The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Video: Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.

Breathing-controlled Electrical Stimulation BreEStim for Management of Neuropathic Pain and Spasticity

The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity.

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...