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 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.
Clinical relevance
While most aspects of cardiac transplantation have evolved significantly since the first heart transplant in 1967, cold storage (CS) remains the standard for donor heart preservation1. CS exposes the organ to a period of cold ischemia that limits the safe preservation interval (4–6 hours) and increases the risk of primary graft dysfunction2,3,4. Due to the static nature of CS, assessments of function or therapeutic interventions are not possible in the time between the organ procurement and transplantation. This is a particular limitation in extended criteria donors including hearts donated after circulatory death (DCD), creating an obstacle for overcoming the considerable gap between demand and the current donor pool5,6. To address this limitation, ex situ heart perfusion has been proposed as a novel, semi-physiologic method of preserving donated hearts, minimizing exposure to cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart during preservation time1,7,8.
Ex situ heart perfusion
One of the most frequently used methods for ex situ examination of the isolated heart is Langendorff perfusion. In this method, introduced by Oskar Langendorff in 1895, the blood flows into the coronary arteries and out the coronary sinus of the isolated heart, with the heart in an empty and beating state9,10. Clinical ESHP in a Langendorff mode with the Transmedics Organ Care System apparatus (OCS) has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts1, and has facilitated the clinical transplantation of DCD hearts11. However, there are concerns about the ability of the device to evaluate organ viability, as a number of donor hearts initially thought to be transplantable were discarded after perfusion on the OCS3. The OCS supports the heart in the Langendorff (non-working) mode, and thus possesses a limited capacity for evaluation of the pumping function of the heart3,12. A growing body of evidence suggests that functional parameters offer a better way to assess organ viability, suggesting that assessments of cardiac function may become a reliable tool for the evaluation and selection of hearts for transplantation during ESHP3,12,13,14, Furthermore, our studies on ex situ perfused porcine hearts suggest that ESHP in working mode provides enhanced functional preservation of the heart during the perfusion interval15,16.
An ESHP apparatus capable of preserving the heart in a working mode must possess a level of automation to safely and precisely maintain preload, afterload and flow rates. Also, such a system should possess the flexibility to facilitate comprehensive assessments of cardiac function to be undertaken. The ESHP apparatus used here is equipped with custom software that 1) provides and maintains desired aortic (Ao) and left atrial (LA) pressure/flow and 2) provides real-time analysis of functional parameters and visual evaluation of pressure waveforms with minimal need for supervision. Pressure data is acquired with standard fluid-filled pressure transducers, and flow data is acquired with transit-time doppler flow probes. These signals are digitized with a bridge and analog input, respectively. The heart is positioned horizontally with a slight elevation to the great vessels on a soft silicone membrane. The cannulation attachments pass through the membrane, incorporating a compliance chamber for dampening ventricular ejection. The goal of this work is to provide researchers in the field of cardiac transplantation with a protocol for ex situ perfusion and evaluation of the heart, under normothermic, semi-physiologic conditions in working mode, in a large mammal (Yorkshire pig) model.
All the procedures in this manuscript were performed in compliance with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The protocols were approved by the institutional animal care committee of the University of Alberta. This protocol has been applied in female juvenile Yorkshire pigs between 35–50 kg. All individuals involved in ESHP procedures had received proper biosafety training.
1. Pre-surgical Preparations
2. ESHP Software Initialization and Adjustments
NOTE: The ESHP apparatus used here is equipped with a custom software program to allow control of pump speed in order to achieve and maintain desired LA and Ao pressures. The software also analyzes functional parameters and provides a visual evaluation of pressure waveforms (Figure 4).
3. Preparations and Anesthesia
4. Blood Collection and Heart Procurement
5. Placement of the Heart onto the ESHP Apparatus and Initiation of Perfusion
6. Metabolic Support During ESHP
NOTE: Organ perfusion solutions, including Krebs-Henseleit buffer solution, typically contain glucose as the primary energy substrate.
7. Anti-microbial and Anti-inflammatory Agents
8. Assessment of Function
NOTE: The ESHP controlling software automatically calculates and records steady-state hemodynamic and functional indices every ten seconds.
9. Metabolic Assessment of the Ex Situ Perfused Heart
10. Removing the Heart from ESHP Apparatus at the End of Perfusion
At the start of the perfusion (in non-working mode), the heart will normally resume a sinus rhythm when the temperature of the system and perfusate approaches normothermia. When entering working mode, as the LA pressures are approaching the desired values, ejection on the Ao pressure tracing should be observed and the LA flow (a reflection of cardiac output) should increase gradually. In a Yorkshire pig model (35–50 kg) and a starting heart weight of 180–220 grams, the initial LA flow will be ~2,000 mL/min, and this will typically approach ~2,750 mL/min during the first hour of perfusion in working mode. Figure 7 displays trends in Ao pressure (A) as well as LA and pulmonary arterial flow (B) over 12 h of perfusion.
During ESHP in the physiologic working mode, various metabolic assessments of the heart are also possible. Blood gas analysis/metabolic assessments performed on the perfusate samples obtained during ESHP provide extensive information on the metabolic status of the heart over time (Tables 1 and 2) and (Figure 8A, B)20. In addition to blood gas analysis, perfusate samples can be collected and assessed for different biomarkers such as brain natriuretic peptide and troponin-I; however, it should be noted that ESHP occurs in a closed system, with no exchange of perfusate solution. In the absence of the organs that naturally metabolize/clear these factors (e.g. kidneys), the accumulation of biomarkers over time in the perfusate solution is typically observed (Figure 9).
Functional assessment of the heart using this platform may include both load-dependent parameters [including myocardial performance (cardiac index, CI), LVSW, maximum and minimum rates of pressure change (dp/dt max and min)], and load-independent parameters (PRSW) (Table 3). Figure 10 demonstrates the evaluation of LV PRSW during a computer-controlled linear reduction in the LA pressure13. In our experience with ESHP of >200 porcine hearts and >10 human hearts, the use of an automated ESHP software program has been in association with the development of standard operating procedures resulting in minimal inter- and intra-operator variability in the functional parameters. The ESHP apparatus and software system used here have been designed to maintain the desired pressures and collect the functional parameters with minimal need for manual adjustments, and we have observed an interclass correlation coefficient (ICC) ≥0.9 for all of the assessed parameters (e.g. LVSW, and dP/dt max and min) that accounts for excellent inter-rater, intra-rater and test-retest reliability. In this system, the electrocardiographic monitoring of the heart during perfusion can also take place using two electrodes as described in the protocol, providing information on the heart rate and rhythm during perfusion (Figure 4).
The assessment of the heart during ESHP may be extended to different imaging modalities. Echocardiography during ESHP can provide additional information on myocardial function (e.g. ventricular ejection fraction) and anatomical parameters (Figure 11 and Figure 12). Moreover, an assessment of the coronary vasculature is possible with angiographic imaging21.
Performing a linear regression analysis identifies which parameters best correlated with myocardial performance (cardiac index: mL·min–1·g–1) during ESHP. We previously showed that despite the significant variation in the ability of the measured functional parameters to predict myocardial performance, overall, functional parameters exhibit a high correlation with cardiac output. The best functional predictors included systolic stroke work [coefficient of determination (R2) = 0.759], for systolic function, and minimum dP/dt, (R2 = 0.738) for diastolic function. Interestingly, metabolic parameters alone show a very limited ability to predict myocardial performance (oxygen consumption: R2 = 0.28; coronary vascular resistance: R2 = 0.20; lactate concentration: R2 = 0.02).13 Perfusion of the heart in a normothermic working mode offers the opportunity to obtain comprehensive metabolic and functional assessments of the heart during organ preservation. A clinical ESHP device with the ability to support the donor heart in working mode will provide the healthcare team with the opportunity to made decisions about organ viability based on objective data before transplantation.
Figure 1: The silicone support membrane for the heart. Support membrane pictured with integrated aortic cannula (A), left atrial cannula (B), and pulmonary artery cannula (C). Please click here to view a larger version of this figure.
Figure 2: The ESHP circuit. (A) Schematic figure of the ESHP circuit. (B) ESHP apparatus used in our setting. A = organ chamber and silicone support membrane, B = reservoir, C = arterial line filter, D = left atrial pump, E = aortic pump, F = membrane oxygenator and heat exchanger, G = gas mixer, H = tube flow sensor, I = pressure sensor, J = stopcock/luer lock. Please click here to view a larger version of this figure.
Figure 3: De-airing the pumps by positioning the pump outlet to a higher level. Please click here to view a larger version of this figure.
Figure 4: Screen shot from the running ESHP software program showing cardiac functional parameters. Please click here to view a larger version of this figure.
Figure 5: Screen shot from the initialized ESHP software program. Please click here to view a larger version of this figure.
Figure 6: The magnetic left atrial cannula secured to the posterior aspect of the left atrium. Please click here to view a larger version of this figure.
Figure 7: Monitoring pressures and flows during the perfusion. (A) Trends in the aortic pressure during 12 h of ESHP. (B) Trends in the left atrial and pulmonary artery flows during 12 h of ESHP Please click here to view a larger version of this figure.
Figure 8: Trends over time. (A) Myocardial oxygen consumption and (B) venous lactate concentration during 12 h of ESHP Please click here to view a larger version of this figure.
Figure 9: Trends over time in perfusate concentration of cardiac troponin-I during 12 h of ESHP. Please click here to view a larger version of this figure.
Figure 10: Assessment of preload recruitable stroke work a poorly-functioning heart (grey) versus a well-functioning heart (black). Please click here to view a larger version of this figure.
Figure 11: Representative two-dimensional echocardiographic images. Please click here to view a larger version of this figure.
Figure 12: Representative M-mode echocardiographic images. Please click here to view a larger version of this figure.
Aortic (arterial) parameters | PA (venous) parameters | ||||||
T1 | T5 | T11 | T1 | T5 | T11 | ||
Blood Gas values | |||||||
pH | 7.28 | 7.44 | 7.33 | 7.25 | 4.42 | 7.30 | |
pO2 (mmHg) | 123.00 | 149.00 | 141.00 | 44.00 | 55.40 | 57.80 | |
pCO2 (mmHg) | 38.00 | 33.90 | 42.50 | 43.00 | 37.10 | 46.10 | |
Oximetry Values | |||||||
Hb (g/dL) | 4.20 | 4.10 | 3.90 | 4.20 | 4.10 | 3.90 | |
sO2 (%) | 100.00 | 100.00 | 100.00 | 64.00 | 95.50 | 92.00 | |
Electrolyte Values | |||||||
K+ (mmol/L) | 4.20 | 4.60 | 5.20 | 4.20 | 4.60 | 5.20 | |
Na+ (mmol/L) | 142.00 | 144.00 | 149.00 | 142.00 | 144.00 | 149.00 | |
Ca2+ (mmol/L) | 1.02 | 1.20 | 1.40 | 1.02 | 1.20 | 1.40 | |
Cl- (mmol/L) | 107.00 | 109.00 | 114.00 | 107.00 | 109.00 | 114.00 | |
Osm (mmol/kg) | 291.30 | 292.50 | 302.40 | 291.90 | 292.90 | 302.40 | |
Metabolite values | |||||||
Glucose (mmol/L) | 7.00 | 5.30 | 5.10 | 7.00 | 5.20 | 5.00 | |
Lactate (mmol/L) | 3.00 | 2.30 | 2.00 | 3.10 | 2.40 | 1.90 | |
Acid Base status | |||||||
Hco3– (mmol/L) | 17.60 | 23.10 | 21.90 | 18.50 | 23.70 | 22.40 |
Table 1: A case of the blood gas analysis performed during the ex situ heart perfusion. Ca2+, calcium ion; Cl–, chloride ion; Hb, hemoglobin; HCO3–, bicarbonate ion; K+, potassium ion; Na+, sodium ion; Osm, osmolarity; paCO2, arterial partial pressure of carbon dioxide; paO2, arterial partial pressure of oxygen; sO2, oxygen saturation; T1, 1 h of ex situ perfusion (early perfusion); T5, 5 h of ex situ perfusion (mid-perfusion); T11, 11 h of ex situ perfusion (late perfusion)
Time | |||
Metabolic Parameters | T1 | T5 | T11 |
MVO2 mL/min/100 g | 6.68 | 2.44 | 1.77 |
Venous Lactate mmol/L | 3.1 | 2.4 | 1.9 |
Venous – Arterial lactate difference mmol/L | 0.1 | 0.1 | -0.1 |
Glucose Utilization g/h | 1.23 | 0.6 | 1.14 |
Table 2: Metabolic parameters calculated using the blood gas analysis data. MVO2, myocardial oxygen consumption; T1, 1 h of ex situ perfusion (early perfusion); T5, 5 h of ex situ perfusion (mid-perfusion); T11, 11 h of ex situ perfusion (late perfusion)
Time | |||
Functional Parameters | T1 | T5 | T11 |
CI (mL/min/g) | 10.26 | 9.66 | 7.50 |
SW (mmHg*mL) | 2253 | 1965 | 1323 |
dP/dT max (mmHg/s) | 1781 | 1783 | 1482 |
Sys p (mmHg) | 128 | 121 | 91 |
ME (%) | 6.69 | 16.85 | 21.68 |
PRSW | 399 | 348.38 | 248.63 |
dP/dT min (mmHg/s) | -1444 | -2350 | -844 |
Table 3: A case of Left ventricular functional parameters assessed during ex situ heart perfusion. CI, cardiac index; dP/dT max, maximum rate of pressure change; dP/dT min, minimum rate of pressure change; ME, mechanical efficiency; PRSW, preload recruitable stroke work; SW, stroke work; Sys p, systolic pressure; T1, 1 h of ex situ perfusion (early perfusion); T5, 5 h of ex situ perfusion (mid-perfusion); T11, 11 h of ex situ perfusion (late perfusion).
Successful perfusion is defined according to the aims of the study; however, this should include uninterrupted ESHP for the desired amount of time and complete collection of the data on cardiac function during the perfusion. For this purpose, a few critical steps in the protocol must be followed.
The heart is an organ with high oxygen and energy demands, and minimizing the ischemic time before cannulation and perfusion is an important principle that must be followed. The process of procurement, mounting the heart on the ESHP apparatus, and initiating perfusion should not exceed 20–30 min.
For efficient perfusion and reliable functional assessment, the process of mounting the heart on the apparatus bears critical importance. Proper anatomical alignment of the great vessels plays an important role in this regard. The heart should be procured with an adequate length of PA and Ao arch branches so that these vessels are not stretched when attached to the representative cannulae. From the start of the perfusion, efficient coronary perfusion plays a pivotal role in the protection of the heart during ex situ perfusion. After starting of the perfusion in non-working mode, the Ao pressure should be monitored and adjusted on at least 30 mmHg to support the coronary perfusion efficiently. The appearance of a dark deoxygenated perfusate in the PA line is a reflector of the reestablishment of coronary flow. After switching to the working mode, the Ao pressure should be adjusted to 40 mmHg to provide adequate coronary perfusion pressure for the working heart.
Deairing the heart chambers and Ao is essential for successful ESHP. At the time of attaching the LA cannula, squeezing the chambers will help in deairing the heart. Any air remaining inthe LV that is ejected should recirculate through the purge line in the innominate artery, which minimizes the risk of coronary air embolism. However, if substantial air remains in the left heart at the time of switching to the working mode, coronary air embolism is possible leading to a significant decline in myocardial function.
The goal of the presented approach is to provide a reproducible and reliable platform for experimental ESHP studies in large mammal models. Such a system provides the opportunity for perfusion in a physiologic working mode, and for extensive evaluation of the perfused heart. This provides an opportunity to evaluate cardioprotective protocols aimed at resuscitating dysfunctional donor organs. This system facilitates simple and reproducible assessments of cardiac functional parameters alongside metabolic parameters during ESHP, providing objective data that can be used to identify viable organs for transplantation. Such a comprehensive assessment is of particular importance when evaluating extended criteria donated hearts and hearts donated after circulatory death. Moreover, according to our observations in the setting of experimental ESHP, hearts perfused in a working mode display superior preservation of systolic and diastolic function over time compared to hearts preserved in a Langendorff mode and may help extend the safe preservation time.
ESHP in a working mode is an efficient method to preserve the donated heart and assess its viability, yet it is an artificial setting, lacking many of physiologic aspect of the body (e.g. real-time hormonal and nutritional balance/support, and free radical scavenging systems). The heart is an organ with sophisticated energy/metabolic demands. Thus, providing consistent, efficient metabolic support to the heart perfused is critically important. We have observed a decline in the function of the ex situ perfused heart, particularly during extended perfusion times22. Such a decline may be reflective of metabolic inefficiencies affecting the function of working mode-perfused heart. More studies are warranted to characterize the optimal metabolic support for the heart during ESHP. An additional challenge is the complexity of working mode heart perfusion. Despite the enhanced simplicity of ESHP in this system, working mode perfusion should be performed by well-trained personnel.
The ESHP apparatus with the capacity to perform a comprehensive functional and metabolic assessment of the hearts in a large mammal model, offers great potential to develop translational therapeutic protocols to improve dysfunctional/suboptimal donated hearts. ESHP may serve as a platform to administer therapeutic interventions targeting a wide range of conditions (e.g. ischemia reperfusion injury), and evaluate their effects on the metabolic and functional parameters of the perfused heart12. Moreover, working mode ESHP may facilitate extension of the safe preservation interval, which may help to overcome geographic limitations of organ donation and facilitate better allocation of donated hearts.
The authors have nothing to disclose.
This work was supported by grants from the Canadian National Transplant Research Program. SH is the recipient of a Faculty of Medicine and Dentistry Motyl Graduate Studentship in Cardiac Sciences. DHF is a recipient of a Collaborative Research Projects (CHRP) grant in aid from the National Sciences and Engineering Research Council and Canadian Institutes of Health Research.
Debakey-Metzenbaum dissecting scissors | Pilling | 342202 | |
MAYO dissecting scissors | Pilling | 460420 | |
THUMB forceps | Pilling | 465165 | |
Debakey straight vascular tissue forceps | Pilling | 351808 | |
CUSHING Gutschdressing forceps | Pilling | 466200 | |
JOHNSON needle holder | Pilling | 510312 | |
DERF needle holder | Pilling | 443120 | |
Sternal saw | Stryker | 6207 | |
Sternal retractor | Pilling | 341162 | |
Vorse tubing clamp | Pilling | 351377 | |
MORRIS ascending aorta clamp | Pilling | 353617 | |
Surgical snare (tourniquet) set | Medtronic | CVR79013 | |
2-0 SILK black 12 X 18" strands | ETHICON | A185H | |
3-0 PROLENE blue 18" PS-2 cutting | ETHICON | 8687H | |
Biomedicus pump drive (modified) | Medtronic | 540 | Modified to allow remote electronic control of pump speed |
Biomedicus pump | Maquet | BPX-80 | |
Membrane oxigenator D 905 | SORIN GROUP | 50513 | |
Tubing flow module | Transonic | Ts410 | |
PXL clamp-on flow sensor | Transonic | ME9PXL-BL37SF | |
TruWave pressure transducer | Edwards | VSYPX272 | |
Intercept tubing 3/8" X 3/32" X 6' | Medtronic | 3506 | |
Intercept tubing 1/4" X 1/16" X 8' | Medtronic | 3108 | |
Heated/Refrigerated Bath Circulator | Grant | TX-150 | |
ABL 800 FLEX Blood Gas Analyzer | Radiometer | 989-963 | |
5F Ventriculr straight pigtail cathter | CORDIS | 534550S | |
5F AVANTI+ Sheath Introducer | CORDIS | 504605A | |
Emerald Amplatz Guidewire | CORDIS | 502571A | |
Dual chamber pace maker | Medtronic | 5388 | |
Defibrilltor | CodeMaster | M1722B | |
Infusion pump | Baxter | AS50 | |
Surgical electrocautery device | Kls Martin | ME411 | |
Gas mixer | SECHRIST | 3500 CP-G | |
Medical oxygen tank | praxair | 2014408 | |
Cabon dioxide tank | praxair | 5823115 | |
Bovine serum albumin | MP biomedicals | 218057791 |