Key words : Antegrade perfusion, Heart transplant, Ischemic reperfusion injury, Myocardial protection, Primary graft dysfunction

Introduction

Around 2000 heart transplants are performed in Europe every year. Primary graft dysfunction (PGD) rates in Europe are among the highest in the world. The increasing use of marginal donor organs has been suggested as a contributing cause. This has resulted in a renewed interest in perfecting myocardial protection techniques to increase the yield of organs retrieved. With increasing demand for organs and a limited supply of donors, novel techniques such as the use of ex vivo normothermic perfusion have also been introduced, although the cost of the equipment has limited its widespread use. Despite these factors, the incidence of PGD remains unchanged.¹ Improving myocardial protection may play a role in reducing the incidence of PGD and improving early mortality rates.

Wheeldon and colleagues noted that up to 55% of the surveyed transplant centers used some form of reperfusion during heart transplant.² This includes intermittent cold blood or oxygenated crystalloid cardioplegia during implantation of the heart followed by administration of “hot shots” as described by Buckberg.³

We present a cohort of patients who underwent heart transplant at our unit in which we used a novel implantation technique aimed to reduce PGD. We used a national cohort of patients (UK wide) who received heart transplants within this period as controls. Our coprimary outcome measures were incidence of PGD and all-cause mortality at 1 year.

Materials and Methods

Description of the Glasgow operative technique

All recipients at our center receive inhaled nitric oxide at 20 ppm during induction of anesthesia. On arrival to the operating theatre, the donor heart is removed from cold static storage and placed in an ice slush basin. An aortic cross clamp is applied onto the donor ascending aorta. An aortic root “DLP” cannula (model 23009, Medtronic; 11F, 9 gauge, 13.3 cm) is inserted and secured in the ascending aorta, close to the aortic root anteriorly (Figure 1).

An antegrade infusion of adenosine (12 mg), followed by 700 mL of blood cardioplegia and St. Thomas solution (4:1 dilution) at 4 ºC and then by cold oxygenated blood (4-6 °C), is infused to achieve a mean aortic root pressure of 60 to 70 mm Hg with flows of 200 to 300 mL/min (Figure 2). Flow is achieved by the use of a constant pressure-variable CPB flow pump through an in situ leukocyte-depleting filter (Pall LeukoGuard LG arterial filter; Pall Biomedical).

This continuous antegrade perfusion is maintained throughout the left atrium and aortic anastomosis with a left ventricular vent in situ (Figure 3). After completion of the aortic anastomosis, the Y-limb of the DLP cannula is then connected to a de-airing channel for de-airing of the ascending aorta and aortic root followed by gradual rewarming of infused blood before removal of the recipient aortic cross clamp (Figure 4).

Systemic perfusion is initiated with continued aortic root and left ventricle venting. The remaining anastomoses are carried out in the usual fashion sequentially with pulmonary artery, inferior vena cava, and superior vena cava with normothermic systemic perfusion and ventricular pacing.

Statistical analyses

We compared our experience with the Glasgow method versus a national UK cohort (2015-2016) of patients (control group). We excluded transplanted hearts that had been procured using normothermic ex vivo perfusion (TransMedics organ care system). Continuous variables are presented as mean ± SD. Categorical variables are expressed as a percentage. We compared baseline characteristics between the Glasgow experience group and the control group using t test and Mann-Whitney U test as appropriate and chi-square test or the Fisher exact test for categorical variables. Variables with significance of P < .1 in the unadjusted analysis were initially introduced as candidate variables in a multivariable logistic regression model for the probability of PGD and removed by stepwise backward elimination. Other variables of interest that contributed to PGD were also included in the multivariable analysis. We used Minitab 17 statistical software (2010) for analysis.

Results

Our study included 194 patients; 28 patients were in the Glasgow group and 166 in the control group. The mean age was 47.5 ± 13.7 years for recipients and 38.2 ± 12.1 years for donors.

The incidence of PGD was 39.2% (n = 76 of 194) with 14.9% (n = 29 of 194) of patients having severe PGD requiring institution of advanced mechanical circulatory support within 24 hours of transplantation. Demographic details showed more recipients with diabetes in the control group and a higher incidence of preoperative urgent-listed patients in the Glasgow group (Table 1). All other donor and recipient demographic parameters were comparable across the groups (Table 1 and Table 2).

Postoperative outcomes showed similar total ischemic times for both groups and similar institution rates for use of mechanical circulatory support and intraaortic balloon pump (IABP) (Table 3). The cold ischemic time was longer in the Glasgow group than in the control group as part of the operative technique, although this did not reach statistical significance (P = .093). Bypass times were also longer in the Glasgow cohort than in the control group (P = .013).

Patients in the Glasgow experience group had lower pulmonary capillary wedge pressure and mean pulmonary arterial pressure values than the control group with an overall reduction in postoperative PGD rates. The mortality at 1 year was not significantly different between the groups.

We did not include the pulmonary capillary wedge pressure, mean pulmonary arterial pressure, and central venous pressure results in the multivariable analysis as they were part of the diagnostic criteria of PGD. Parameters included in the multivariable analysis to assess independent predictors of PGD included bypass time, donor age, recipient diabetes status, and urgent listing status in groups (Glasgow vs control). The results of the multivariable analysis are presented in Table 4.

Discussion

Functional warm ischemia time

This novel technique utilizes a minimization in functional warm ischemic time by antegrade per-fusion of the donor graft using cold blood cardioplegia alongside topical cooling. Cardioplegia negates the electromechanical energy consumption associated with myocyte contraction, thereby reducing myocar-dial oxygen demand. In addition, the topical cooling reduces enzymatic reactions, which in turn reduce cellular metabolism.⁵ Several studies have shown similar findings with prolonged warm ischemia linked with an increased incidence of PGD. Avtaar Singh and colleagues¹ highlighted the role of prolonged warm ischemia time as an independent predictor of PGD. Marasco and colleagues noted that warm ischemic time in excess of 80 minutes was associated with reduced survival compared with warm ischemic time of less than 60 minutes.⁶ Both studies also noted donor age to be a significant independent predictor of PGD and survival as noted in our cohort.

In contrast, prolonged cold ischemic time was not associated with PGD in our cohort. These findings were also noted by the UCLA group in which cold ischemic times of >300 minutes were not associated with adverse outcomes.⁷ This could be due to attenuation of ischemic-reperfusion injury (IRI), which is thought to be the pathophysiological basis of PGD. Registry data from the International Society for Heart and Lung Transplantation are difficult to interpret given the overlapping results of warm and cold ischemic time, commonly presented as total ischemic time.⁸ Other solid-organ transplant studies have shown a direct correlation between increased warm ischemic time and IRI.^9,10 Ischemia promotes expression of proinflammatory factors and bioactive agents, such as leukocyte adhesion molecules, cytokines, endothelin, and thromboxane A2 while repressing “protective” factors like constitutive nitric oxide synthase, thrombomodulin, and prostacyclin.¹¹

The use of continuous antegrade oxygenated cold blood perfusion ensures universal cooling of the heart. The oxygen delivery is impaired at hypothermia as the dissociation curve of hemoglobin is displaced toward the left, and oxygen delivered to the tissues is mainly transported in the dissolved form. However, as opposed to cold crystalloid, oxygenated blood has a buffer capacity of the imidazole nucleus of the hemoglobin molecule.¹² Use of cold blood cardiop-legia has also resulted in lower postoperative cardiac enzyme levels compared with cold crystalloid cardioplegia in general cardiac procedures.¹³

Ischemic-reperfusion injury

The length of ischemia has a direct effect on cell dysfunction leading to cell injury or death, with reperfusion being the ideal treatment option. Reperfusion restores oxygen delivery to facilitate aerobic ATP generation while flushing out hydrogen accumulation from anaerobic states. Jennings and colleagues initially described an accelerated state of necrosis on reperfusion in 1960, coining the term reperfusion injury.¹⁴ It arises due to rapid generation of reactive oxygen species, increased intracellular calcium due to attenuated ATP-dependent exchangers, opening of mitochondrial permeable transition pores that dissipate mitochondrial membrane potential, and endothelial dysfunction accompanied by activation of inflammatory pathways, leading to a vicious cycle of increasing tissue injury.¹⁵

Inhaled nitric oxide

We hence utilized targeted therapeutic strategies to attenuate IRI. Inhaled nitric oxide may play a role as a preconditioning factor alongside a pulmonary vascular dilator. Nitric oxide has antioxidant properties not related to alterations in neutrophil migration or adhesion but by reduction by nitric oxide of superoxide anion-mediated tissue toxicity, which may account for much of the protective effect of nitric oxide during IRI.¹⁶ It also acts as an oxygen radical scavenger.¹⁷ It attenuates the effects of reperfusion by competitively binding to cytochrome-c oxidase. This prevents abrupt resumption of the electron transport chain during reperfusion with oxygen-rich blood, which would result in generation of oxygen radicals.¹⁸ Its role in reducing right ventricular afterload is well established in the literature^19,20 as is its role as a coronary vasodilator.²¹ The thin-walled right ventricle is more compliant, thus accommodating right-sided venous return well but struggling against acute increases in afterload, resulting in dilation and impaired contractility.^22,23 Reducing the afterload may therefore assist the rewarming of right ventricle contraction and prevent dilation. The coronary vasodilatory effect may also allow greater reperfusion and control rewarming of the donor graft on release of the aortic cross clamp.

Leukocyte-depleting filter

We use a leukocyte-depleting filter in all patients to prevent leukocyte-mediated IRI. Leukocyte activation results in chemotaxis, leukocyte-endothelial cell adhesion, and transmigration. Activated leukocytes release toxic oxygen radicals, proteases, and elastases, resulting in increased microvascular permeability, edema, thrombosis, and parenchymal cell death while activating a positive feedback loop resulting in increased transmigration.²⁴ The role of the leukocyte-depleting filter in reducing myocardial reperfusion injury in general cardiac surgery is well understood with reductions in biochemical markers and postoperative inotropic requirements without any difference in clinical outcomes.²⁵ In heart transplant procedures, it has been shown to reduce creatinine phosphokinase-MB and thromboxane B2 after reperfusion.²⁶ Another trial showed similar results with reduced markers of reperfusion injury, easier weaning from cardiopulmonary bypass, and lower need of inotropic support postoperatively.²⁷

Ischemic preconditioning has been shown to improve ventricular function and reduce myocardial neutrophil accumulation and apoptosis in experi-mental models.²⁸ It is associated with an increase in extracellular adenosine production with experi-mental studies highlighting the role of A1, A2a, and A3 adenosine receptor involvement in endogenous cardioprotective responses.^29,30 The administration of adenosine to the donor graft during perfusion permits the preconditioning effect and enhances coronary vasodilation while slowing the sinoatrial nodal pacemaker rate, delaying atrioventricular nodal impulse conduction, and reducing atrial contractility, all of which may contribute to ensuring the heart is arrested while cardioplegia is adminis-tered after removal from cold storage. In heart valve replacement operations, it has been shown to reduce cardiac troponin I, interleukin 6, and interleukin 8 release, resulting in less myocardium injury in ultrastructure after surgery.³¹

Intraaortic balloon pump

Despite having a similar number of IABP insertions, the moderate PGD rate was lower in the Glasgow cohort than in the control group. This resulted from use of the IABP in conjunction with extracorporeal membranous oxygenation support to allow offloading of the left ventricle. The counter pulsation of IABP results in a mildly reduced end-diastolic volume decrease and a decreased afterload due to the vacuum effect of balloon deflation.³² The net effect is an increase in stroke volume. This allows adequate emptying of the left ventricle with regular opening of the aortic valve.

Limitations

The study has some limitations. Because of the multicenter nature of the control group, regional variations between patient management strategies, such as inotropic preference and choice of immuno-suppression, could not be addressed. Some standar-dized criteria are established by the Cardiothoracic Advisory Group and the National Health Service Blood and Transplant to keep the variations to a minimum. The current experience at our center with the nascent method is relatively new, hence the smaller numbers in the Glasgow cohort. We were unable to collect biomarkers to assess the bioche-mical changes postulated by this method.

Conclusions

This novel approach is associated with significant reductions in PGD rates and a trend toward improved 1-year survival. Larger studies are needed to show differences after further adjustment for known confounders of PGD. We believe this novel technique is safe, cost-effective, and reproducible.

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