Coronary Angiography During Ex-Situ Heart Perfusion in a Porcine Model

Podsumowanie

Screening for coronary artery disease could increase the procurement of donor hearts with extended criteria. A protocol for performing reproducible coronary angiography during ex-situ heart perfusion is described herein in a porcine model.

Streszczenie

Heart transplantation is the gold standard treatment for advanced heart failure. The procurement of extended criteria donors (ECD) increases due to the current organ shortage. Coronary angiography is recommended in ECD at risk for coronary artery disease but is not systematically performed. These hearts are, therefore, either declined for transplant or procured without screening for coronary artery disease. Coronary angiography during normothermic ex-situ heart perfusion (NESP) could be an interesting approach to enhance the rate of ECD procurement and to reduce the risk of primary graft failure in the absence of coronary angiography in ECD. The present protocol aims to provide material details along with optimal imaging views for coronary angiography during NESP. Reproducible angiographic views were observed, including one dedicated to the right coronary artery, two for the left anterior descending artery, two for the circumflex artery, and a spider view. Continuous lactate extraction was observed in all procedures with a final median concentration of 1.10 mmol/L (0.61-1.75 mmol/L) two hours after coronary angiography, consistent with myocardial viability. The median contrast agent volume used for ex-situ imaging of the isolated perfused heart was 48 mL (38-108 mL). This protocol was reproducible for coronary artery imaging and did not impair myocardial viability during NESP.

Wprowadzenie

Heart transplantation is the gold standard therapy for advanced heart failure^1,2. However, this treatment is limited by the current organ shortage, leading to increased allocation of extended criteria donors (ECD), who are more frequently exposed to coronary artery disease (CAD). Coronary angiography is recommended in France to prevent the risk of primary graft failure related to CAD if the donor's age is greater than 55 years or 45 years with two cardiovascular risk factors³. Even though coronary angiography increases the chances of graft acceptance by 9%, this procedure is performed in only 33% of cases in France⁴. Hearts from ECD at risk for CAD are therefore either declined for transplant or procured without screening for coronary artery disease.

The Organ Care System (OCS) is the only clinically available machine perfusion (MP) for Normothermic Ex-situ heart perfusion (NESP). This technology allows for longer preservation before transplantation by perfusing the donor heart with warm, oxygenated blood. Continuous metabolic assessment is performed during NESP by monitoring the lactate profile⁵. The efficacy and safety of NESP are now demonstrated by several studies, including a recent meta-analysis^6,7,8. Performing coronary angiography during NESP would be of major interest to increase the probability of transplanting hearts from ECDs at risk for CAD.

Previously published experiences of ex-situ coronary angiography^9,10,11 suggest that conventional views are not suitable due to the inverted position of the heart in the perfusion module. In addition, OCS components visible with X-rays may impair imaging findings. Lastly, the toxicity of contrast agents on the myocardium during isolated heart perfusion remains questionable. The present report aims to provide a safe and reproducible approach to performing coronary angiography during NESP.

Protokół

All pigs received humane care in compliance with the 'Principles of Laboratory Animal Care' formulated by the National Society for Medical Research and the 'Guide for the Care and Use of Laboratory Animals' prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (Eighth Edition, 12910. National Academies Press, Washington, D.C., 2011). This study design was approved by the French Ministry of Research for the use of animals for scientific purposes (protocol APAFiS#23716-2020012112432923v1). Eight large-white male pigs (60 kg, 6 months) were used for the present study. The animals were housed for one week before surgery with congeners at room temperature with a day-night light shift. Food and water were freely available. The reagents and equipment used for this study are listed in the Table of Materials.

1. Animal preparation

1. Premedicate animals with an intramuscular injection of Tiletamine and Zolazepam (2 mg/kg).
2. Complete anesthesia with an intravenous injection of propofol (3 mg/kg).
3. After mask ventilation using 100% FiO₂, perform oro-tracheal intubation with a 7.5 mm probe using a laryngoscope dedicated to large animals.
4. Maintain general anesthesia by inhalation of 2% isoflurane, continuous infusion of sufentanil (10 µg/kg/h), and intermittent injections of cisatracurium (0.3 mg/kg every 30 min).
5. Perform a transthoracic echocardiogram to look for any cardiac disease that may contraindicate ex-situ perfusion of the heart. Significant aortic regurgitation does not allow for efficient NESP in Langendorff mode since coronary flow will be impaired. Retrograde aortic flow will mainly go to the left ventricle in this condition.
   NOTE: Animals presenting with cardiac abnormalities, including significant aortic regurgitation, should be excluded from the protocol (the anesthesia is stopped for excluded animals to allow transfer to the animal facility).

2. Heart retrieval

1. Perform a midline sternotomy. Use a scalpel (No. 23) to open the skin. Open the subcutaneous tissues using a monopolar cautery. Open the sternum using a semilunar sternal saw and complete the sternotomy using Mayo scissors.
   1. Dissect the thymus, open it, and expose the pericardium with Metzenbaum scissors to avoid ventricular fibrillation induced by the electric field of the monopolar cautery.
2. Inject Heparin (300 IU/kg) directly into the right atrium.
3. Perform purse-string stitches using 4/0 polypropylene sutures on the right atrium and aortic root to prepare for cannulation.
4. Carefully detach the aorta from the main pulmonary artery to prepare for clamping of the aorta.
5. Control the superior and inferior vena cava with a surgical loop.
6. Introduce a 9-French antegrade long cannula into the aortic root 3 min after heparin administration.
7. Purge the cardioplegia line and connect it to the aortic cannula, then clamp the cardioplegia line before blood collection.
   NOTE: Blood collection results in a significant volume drop that may impair appropriate cannulation of the ascending aorta. Therefore, it is easier to do it before blood collection.
8. Canulate the right atrium with a 32-French two-stage venous cannula.
9. Connect a sterilized bag previously heparinized (10,000 IU) to the right atrium cannula and collect 1500 mL of blood.
   NOTE: The collected blood is leukodepleted by another operator using a leukocyte filter and then placed directly in the machine perfusion circuit.
10. Clamp the aorta and infuse 1000 mL of cold (4 °C) cardioplegia solution (Table 1).
11. Secure the superior and inferior vena cava with a surgical loop.
12. Stop mechanical ventilation. Open the inferior vena cava and the left superior pulmonary vein to unload the heart. Place a cold saline solution in the pericardium.
13. Remove the aortic and venous cannulas. Explant the heart by sectioning the left azygos vein, the two vena cavae, the distal ascending aorta, the pulmonary artery trunk, and the four pulmonary veins.
    NOTE: The left azygos vein is specific to porcine anatomy. This vein goes directly to the coronary sinus. The left azygos vein must be transected between two ligatures before the heart is harvested.

3. Heart preparation for NESP

1. Place the heart in 4 °C saline solution.
2. Investigate the aortic valve to confirm normal coaptation and close a patent foramen ovale if present using a 4-0 polypropylene suture.
3. Complete the separation between the aorta and the pulmonary artery.
4. Place four pledgeted sutures (4/0 polypropylene) around the distal section of the ascending aorta.
5. Insert the appropriate aortic cannula and fix it with a hose clamp. Four sizes of aortic cannula are available: 3/4 inch, 7/8 inch, 1 inch, and 5/4 inch.
6. Insert the 30 Fr pulmonary artery cannula and secure it with a 4/0 purse-string suture.
7. Place a 20-French intracardiac sump through the mitral orifice to vent the left ventricle.

4. Normothermic ex-situ heart perfusion

NOTE: A detailed composition of the cardioplegia solution, priming solution, epinephrine, and adenosine infusions is provided in Table 1.

1. Prime the machine perfusion (MP) with the collected blood (1500 mL) and add the priming solution (500 mL).
2. Oxygenate with a membrane oxygenator and warm the blood to 34 °C using a heater-cooler.
3. Place a pressure probe on the aortic line above the aorta.
4. Place a flowmeter around the pulmonary artery cannula.
   NOTE: Continuous infusion of epinephrine (0.5 mL/h) is initiated after connection to the aortic line. Adenosine infusion will be adjusted to maintain mean aortic pressure between 65 mmHg and 80 mmHg.
5. Increase the pump speed to reach a flow rate of 2500 mL/min.
6. Collect an initial blood sample for blood gas analysis, ionogram, and lactate level. Adjust potassium, calcium, glycemia, and pH if necessary.
   NOTE: All these steps are performed simultaneously with heart retrieval.
7. Before positioning the heart in the MP, reduce the pump flow to 1000 mL/min and remove the shunt between the aortic and venous lines.
8. De-air the aorta and connect the aortic cannula to the aortic line.
9. Place the heart for optimal NESP (following the manufacturer's instructions). The postero-inferior ventricular wall will be in front of the operator, and the pulmonary artery cannula will be placed behind the aortic cannula.
10. Perform gentle cardiac massage until the heart is warmed.
11. Deliver a 20 J shock in case of ventricular fibrillation.
12. Pace the heart at 80 beats per minute with an external pacemaker.
13. Slowly increase pump flow to reach an aortic pressure of 65 mmHg.
14. Close the inferior vena cava with a 4/0 polypropylene running suture.
15. Purge and connect the pulmonary artery cannula to the venous line to continuously assess coronary flow.
    NOTE: The coronary flow should be between 650 mL/min and 850 mL/min. The mean aortic pressure should be maintained between 70 mL/min and 85 mmHg. Assess blood gas and ionogram every hour and lactates every 30 min. If venous lactate is higher than arterial lactate, the first step is to ensure the normality of the ionogram and correct any biochemical abnormality. If pressure and coronary flow are low, increase the pump speed. If the pressure is high and the coronary flow is low, increase adenosine to vasodilate the coronary arteries. Stop adenosine infusion if aortic pressure is below 70 mmHg.

5. Coronary angiography during NESP

1. Experimental setup
   NOTE: The setup is depicted in Figure 1.
   1. Ensure sufficient space is available for the machine perfusion (MP) system.
   2. Elevate the MP by approximately 25 cm using a platform. Rotate the MP 90° to the left from the initial position.
2. Perform coronary angiography (Figure 2).
   1. Flush the 5 French introducer and the 5 French Judkins right 3.5 coronary angiography catheter with serum.
   2. Insert a 5 French introducer through the valved orifice of the MP.
      NOTE: The distal part of the introducer has to be placed just above the aorta.
   3. Advance the guide wire into the coronary angiography catheter up to the distal orifice. Insert the catheter and guide wire into the introducer up to the aortic root.
   4. Perform catheterization of the right coronary artery (RCA) using the following views: RAO 35/ CAU 44 or RAO 135/ CAU 62. Capture images of the RCA network using the views described in Table 2.
   5. Perform catheterization of the left coronary artery with the following view: RAO 135/ CAU 62. Capture images of the left coronary artery network using the views described in Table 2.
   6. Remove the coronary angiography catheter and the introducer.
      NOTE: Use approximately 50 mL of Iomeprol without exceeding 100 mL.

Wyniki

The outlined protocol was designed for easy reproducibility in clinical practice. Minimal modifications were made to the available module for Normothermic Ex-situ Heart Perfusion (NESP). The only adjustments were made to connect the module to an extracorporeal life support pump, an oxygenator, and a heater-cooler unit to replace the machine perfusion (MP).

Two test procedures were initially conducted to assess the coronary angiography views used in this protocol. Six coronary angiogra...

Dyskusje

This study describes, for the first time, a protocol for coronary angiography on an isolated beating heart without compromising myocardial metabolic viability during Normothermic Ex-situ Heart Perfusion (NESP). The views were reproducible, allowing for a comprehensive anatomical analysis of the coronary network. The required contrast agent volume was approximately 50 mL and rarely exceeded 100 mL. In comparison, the average volume used for in vivo coronary angiography is twice as much, ranging between 1...

Materiały

Name	Company	Catalog Number	Comments
3T Heater Cooler System	Liva Nova, Châtillon, France	IM-00727 A	Extracorporeal Heater Cooler device
4-0 polypropylene suture	Peters, bobigny, France	20S15B	sutures
5-0 polypropylene suture	Peters, bobigny, France	20S10B	sutures
Adenosine	Efisciens BV, Rotterdam, Netherlands	9088309	Drugs for the ex-vivo perfusion
Adrenaline	Aguettant, Lyon, France	600040	Drugs for the ex-vivo perfusion
ADW software	General Electric Healthcare, Chicago, Ill, USA	ADW_IGS730/ M5173653	Software used for the coronary angiography visualization
Atracurium	Pfizer Holding France, Paris, France	582547	Drugs for the induction of the anesthesia
CG4 +	Abbott, Chicago, Ill, USA	03P85-25	Lactates and blood gas cartridge for the blood analyzer machine
CG8 +	Abbott, Chicago, Ill, USA	03P88-25	Ionogram cartridge for the blood analyzer machine
DeltaStream	Fresenius Medical Care, L’Arbresle, France	MEH2C4024	Extracorporeal blood pump
Discovery IGS 7	General Electric Healthcare, Chicago, Ill, USA	Discovery IGS7.3 / M5173652	Angiography system for hybrid operating room
DLP Aortic Root Cannula	Medtronic Inc. Minneapolis, Minneapolis, USA	ref. 11012, 12.5 inch	Aortic Root Cannula
External pacemaker	Medtronic Inc. Minneapolis, Minneapolis, USA	5392	Pacemaker device
Fresinus Agilia injectomat	Fresenius vial , Brezins, France	018010/20640320	Syringe driver
Glucose 5%	B.Braun Melsungen AG, Melsungen, Germany	3400891780017	Drugs for the priming solution
Haemonetics leukocyte filter	Haemonetics, Boston, Massachusetts, USA	RS1VAE	Leukocyte Filter
Heart Perfusion Set, Organ Care System	Transmedics, Andover, MA, USA	Ref#1200	Normothermic ex-vivo heart perfusion device, including, the perfusion module, pulmonary artery and aortic cannula
Intellivue MX550	Philips Healthcare, Suresnes, France	NA	Permanent monitoring system
Iomeron 350	Bracco imaging, Massy, France	3400933794606.00	Iomeprol contrast agent product
istat 1	Abbott, Chicago, Ill, USA	714336-03O	Blood Analyzer machine
Judkins Right 3.5	Boston Scientific,  Marlborough, Massachusetts, USA	H74934358202	Coronary angiography catheterization probe
Magnesium	Aguettant, Lyon, France	564 780-6	Drugs for the cardioplegia
Magnesium Sulfate	Aguettant, Lyon, France	600111	Drugs for the cardioplegia
Mannitol 20%	Macopharma, Mouvoux, France	3400891694567.00	Drugs for the cardioplegia
Methylprednisolone	Mylan S.A.S, Saint Priest, France	400005623	Drugs for the priming solution
MWI software	General Electric Healthcare, Chicago, Ill, USA	NA	software used for the Ultrasound echocardiographic machine
Orotracheal probe	Smiths medical ASD, Inc., Minneapolis, Minneapolis, USA	100/199/070	probe for the intubation during anesthesia
Potassium chloride 10%	B.Braun Melsungen AG, Melsungen, Germany	3400892691527.00	Drugs for the cardioplegia
Propofol	Zoetis France, Malakoff, France	8083511	Drugs for the induction of the anesthesia
Quadrox-I small Adult Oxygenator	Getinge, Göteborg, Sweden	BE-HMO 50000	Extracorporeal blood oxygenator
Ringer solution	B.Braun Melsungen AG, Melsungen, Germany	DKE2323	Drugs for the cardioplegia
Sodium Bicarbonate	Laboratoire Renaudin, itxassou, France	3701447	Drugs for the cardioplegia
Sodium chloride	Aguettant, Lyon, France	606726	Drugs for the priming solution
Swan Ganz Catheter	Merit Medical, south jordan, utah, USA	5041856	Right pressure and cardiac output probe
Tiletamine	Virbac France, Carros, France	3597132126021.00	Drugs for the induction of the anesthesia
Two staged Venous Cannula	Medtronic Inc. Minneapolis, Minneapolis, USA	91240, 32 french	Venous Cannula
Vivid E95 ultraSound Machine	General Electric Healthcare, Chicago, Ill, USA	NA	Ultrasound echocardiographic machine
Xylocaïne 2%	Aspen, Reuil-malmaison, France	600550	Drugs for the cardioplegia
Zolazepam	Cardinal Health LLC, Waukegan, Illinois, USA	31050522	EKG detection electrodes
Zolazepam	Virbac France, Carros, France	3597132126021.00	Drugs for the induction of the anesthesia