The signing of the HIV organ policy equity (HOPE) act into law in 2013 was a revolutionary move in expanding the organ donor pool as it enabled HIV-positive patients well-controlled on highly active antiretroviral therapy (HAART) to be registered as organ donors. People living with HIV (PLWH) are always at a higher risk of end-stage organ disease, more so because HAART has significantly reduced the rates of deadly opportunistic infections. Until recently, having an HIV infection was considered a contraindication for receiving a lung transplant [17]. Researchers have since found out that lung transplant survival rates and postoperative outcomes were similar in HIV-positive recipients as compared to their uninfected counterparts [18, 19]. Solid organ transplantation has therefore become the standard of care for organ failure in PLWH [20]. After clearing the way for HIV-positive transplant recipients, the HOPE act went one step ahead by allowing HIV-positive people to donate their organs to fellow individuals sharing their HIV-positive status.

Once the HOPE act was implemented in 2015, HIV-positive recipients began receiving organs from HIV-positive donors (HIV D+/R+). As of July 2021, there have been 144 true/false HIV-positive donors, which led to 300 kidney and 87 liver transplants [20]. This decision did not pass without its fair share of concerns. Healthcare professionals were worried about several plausible issues upon such transplants, particularly superinfection if the donor's HIV strain did not match the recipients, rate of rejection, and HIV-related organ disease in the allograft [21–23]. As compared to transplanting organs from HIV-negative donor to HIV-positive recipient (HIV D−/R+), the outcomes were similar, with the only real challenge being increased rates of rejection [24].

Heart and lung transplants from HIV-positive donors have been approved and are highly anticipated [20]. Federal HOPE Act guidelines require participating hospitals to carry out five transplants in HIV-positive recipients before they can accept organs from HIV-positive donors. Successful implementation of the HOPE act requires the address of community-level barriers like the lack of awareness, fear of infection in personnel procuring organs from HIV-positive patients, and the fear of revelation of donor/recipient HIV status during transplantation [25]. Challenges faced while coadministering HAART and immunosuppressive therapy also need to be considered. Such studies would lay the foundation for the successful initiation of thoracic organ transplants in HIV-infected individuals from HIV-positive donors. Koval et al. reported outcomes from 29 HIV-infected thoracic transplant recipients (21 heart, 7 lungs, and 1 heart and/or lung) across 14 transplant centers from 2000 through 2016. Compared to an ISHLT registry cohort, similar 1-, 3- and 5-year patient and allograft survival was seen. However, at 1 year, significant rejection rates were high (62%) for heart transplant recipients (HTRs). Pulmonary bacterial infections were high (86%) for lung transplant recipients (LTRs) [19].

#### **6.** *Ex vivo* **lung perfusion (EVLP)**

*Ex vivo* lung perfusion is a technique that allows harvested donor lungs to be explanted into a system that mimics the hemodynamic status of the human body. The general design of the system involves ventilation of the allograft via endotracheal tubes and circulation of a fraction of cardiac output by means of pumps [26]. In cellular EVLP protocols like the lund protocol and organ care system, packed red blood cells mixed with a composite perfusate are allowed to flow through the lungs, while acellular EVLP protocols like toronto protocol do not use blood cells in the perfusate [27]. It enables the assessment of hemodynamic, ventilatory, and gas exchange parameters of the allograft. Biochemical values, imaging studies, bronchoscopic analysis, physical inspection, and palpation can also be performed on these organs [26]. These factors allow EVLP to be used as a tool for analyzing the allograft before transplantation, which is especially useful in the case of marginal lungs facing the threat of being rejected. EVLP thus aims to maximize the utilization of procured donor lungs [26]. In cellular EVLP, Kamal et al. described direCt lung ultrasound evaluation (CLUE) technique as a method for monitoring extravascular lung water in donor lungs during *ex vivo* lung perfusion (EVLP). Significant differences were found between suitable and non-suitable lungs in CLUE scores (1.03 vs. 1.85, p < 0.001), unlike the partial pressure of oxygen/fraction of inspired oxygen ratio. Standard donors had significantly better scores than marginal lungs and proning improved this score, especially in upper lobes [28].

EVLP was mainly developed to tackle the problem of ischemic-reperfusion injury, which increases the risk of development of primary graft dysfunction (PGD) [29]. The current practice involves cold preservation of allografts that facilitates a reduction in the organ's metabolic demand. However, concerns regarding cold ischemic injury have surfaced. EVLP revolves around the principle of retaining the organ in its physiological milieu, at the human body temperature, for as long as possible [26]. Posttransplant outcomes after using EVLP have been consistently similar to traditional techniques in terms of patient and graft survival [30].

While the main role of EVLP remains the analysis of the graft, it is being creatively used to recondition the graft and even make initially rejected organs useful. In edematous lungs, EVLP allows the fluid from extravascular compartments to be drawn out, which can make the graft usable [31]. Antibiotics can be administered in the EVLP system, which ensures targeted therapy, preventing systemic adverse effects [32]. To shift the cytokine profile from pro-inflammatory to anti-inflammatory, Interleukin (IL)-10 gene therapy and stem cell therapies have been tried in porcine models by administering them to the graft using the EVLP systems [26].

Keeping in view the potential of eliminating infections from donor organs, trials began on organs harvested from HCV+ donors. Applying the age-old perspectives from blood product sterilization, researchers irradiated lungs in EVLP systems with ultraviolet C (UVC) light, which has been known for its germicidal properties [33, 34]. Similarly, another light-based sterilization technique called photodynamic therapy (PDT) uses methylene blue irradiated with red light [35]. As compared to standard EVLP, which focused simply on washing the virus away from the organ, PDT was the most effective in reducing HCV load. It cleared 98% of HCV-RNA from the perfusate and 91% from the lung tissue. The UVC group did not show any significant reduction as compared to controls [33, 36]. Despite reduced infectivity of the persistent RNA fragments noticed in *in vitro* analysis, UVC irradiation failed to prevent the development of viremia posttransplant in further trials [37]. Is it

#### *Recent Advances and Outcomes in Heart and Lung Transplantation DOI: http://dx.doi.org/10.5772/intechopen.109068*

hypothesized that this difference arises because of the weaker penetrating power of UVC rays due to wavelength differences as compared to PDT [38]. Additionally, irradiated methylene blue provided better coverage because it could be injected into the vasculature [36].

The potential of EVLP for research and to evaluate and salvage initially rejected lungs is vast and the outcomes after using EVLP are comparable to standard lung transplantation. Multiple trials are demonstrating ingenious ways to utilize the technology clinically [27]. For example, researchers are administering targeted adenosine receptor 2B antagonists via EVLP to abolish its proinflammatory effects [39]. The use of EVLP as a platform for improving the utilization of donated lungs continues to increase.

Recently, Cypel et al. identified 10°C to be an optimal temperature for lung storage, allowing for preservation times of up to 24–36°C. Furthermore, they explored the concept of multiday lung preservation by pairing 10°C lung preservation with short cycles of EVLP in an animal model. For the first time, they were successful 3-days of lung preservation with exceptional immediate posttransplant graft function [40].

Loor et al., in the INSPIRE trial, sought to assess the safety and efficacy of normothermic portable EVLP using the OCS Lung device. It was the first prospective, multicenter, and randomized controlled study in EVLP for standard bilateral lung transplantation*.* The composite primary effectiveness endpoint of the trial was absence of PGD 3 within the first 72 hours after transplant and 30-day survival in the per-protocol population, with a 4% noninferiority margin [41]. The EXPAND trial was used to evaluate the efficacy of normothermic portable organ care system (OCS) lung perfusion and ventilation on donor lung use from extended-criteria donors and donors after circulatory death. OCS resulted in 87% of donor lung use for transplantation with excellent clinical outcomes [42]. A major challenge in lung transplantation is the need for ABO blood group matching. To address this challenge, Wang et al. used two enzymes, FpGalNAc deacetylase and FpGalactosaminidase, to convert blood group A lungs to blood group O lungs during *ex vivo* lung perfusion. The authors demonstrated successful removal of blood group A antigen with no overt changes in lung health. The authors showed reduced antibody and complement deposition, suggesting that this technique may reduce antibody-mediated injury *in vivo* [43].

#### **6.1 Lung transportation**

Conventional cold static preservation of donor's lungs leads to unpredictable lung tissue cooling as also the freezing and thawing cause irreversible cellular damage. The Paragonix LUNGguard donor preservation system provides a stable temperature between 4 and 8ο C for extended preservation times. Hartwig et al. presented their initial report of a study comparing two methodologies of hypothermic storage: patients with donor lungs preserved by the LUNGguard and patients with donor lungs transported by conventional preservation methods (ice protocol) [44].

#### **6.2 Intraoperative use of cardiopulmonary bypass (CPB) vs. ECMO for lung transplantation**

The use of extracorporeal membrane oxygenation (ECMO) for intraoperative cardiopulmonary support has gained traction in recent years. There is growing experience with the preoperative use of ECMO as a bridge to a transplant in patients with refractory respiratory failure. Biscotti et al. compared differences in patient outcomes and operative parameters for extracorporeal membrane oxygenation (ECMO)

versus cardiopulmonary bypass (CPB) in patients undergoing lung transplants. Intraoperatively, CPB group required more cell saver volume, FFP, platelets, and cryoprecipitate. The CPB group had higher rates of primary graft dysfunction at 24 and 72 hours. There were no differences in 30-day and 1-year survivals [45]. Hoetzenecker et al., from the Vienna lung transplant group reported their results of uniform central venoarterial ECMO in patients receiving bilateral lung transplantation. Their median time of mechanical ventilation was 29 hours, 90-day mortality was 3.1%, and 2-year survival was 86%. Thus, routine use of intraoperative ECMO resulted in excellent primary graft function and midterm outcomes in patients undergoing lung transplantation [46]. Magoulitis et al. in their meta-analysis of seven observational studies incorporating 785 patients showed that ECMO support lowered rate of primary graft dysfunction, bleeding, renal failure requiring dialysis, tracheostomy, intraoperative transfusions, intubation time, and hospital stay. However, no difference was reported between operative and ischemic time [47]. Recently, Halpern et al. in their study on patients with no or mild pulmonary hypertension, who underwent bilateral lung transplantation, reported that planned VA ECMO was associated with higher odds of textbook outcomes than planned off-pump support [48].

#### **7. Advances in treatment of post-lung transplant complications**

According to the OPTN annual report in 2020, 14.6% of lung transplant recipients developed acute rejection. 40% of recipients went on to develop bronchiolitis obliterans syndrome (BOS) by the fifth year after transplant, and 24% of recipients developed malignancy in the same period.

Chronic lung allograft dysfunction (CLAD) holds substantial weightage during the discussion of lung transplant because it is a major cause of mortality and morbidity following lung transplantation [49]. According to the ISHLT consensus report 2019, CLAD is a persistent > = 20% decline in FEV1 as compared to the recipient's posttransplant baseline [50]. Decreases in lung functions after three months posttransplant are also attributed to CLAD. Two prominent subtypes of CLAD are BOS

#### **Figure 3.**

*Incidence of acute rejection by 1-year posttransplant among adult lung transplant recipients by induction agent, 2018–2019. Only the first reported rejection event is counted. Cumulative incidence is estimated using the Kaplan– Meier method. IL2-RA, interleukin-2 receptor agonist; TCD, T-cell depleting. (from OPTN/SRTR 2020 annual data report. HHS/HRSA).*

*Recent Advances and Outcomes in Heart and Lung Transplantation DOI: http://dx.doi.org/10.5772/intechopen.109068*

(bronchiolitis obliterans syndrome) and RAS (restrictive allograft syndrome) [49]. Once BOS develops, the mortality rate varies from 25 to 55% [51, 52]. Risk factors of CLAD include autoimmunity, PGD, alloimmune rejections (e.g., acute cellular rejection, antibody-mediated rejection, and lymphocytic bronchiolitis), infections (viral, bacterial, and fungal), persistent bronchoalveolar lavage neutrophilia and pathological gastroesophageal reflux (**Figure 3**) [31].

Newer treatment modalities, such as extracorporeal photopheresis (which uses ultraviolet light to generate anti-inflammatory cytokines), liposomal cyclosporine, and anti-fibrotic agents, such as pirfenidone, are being assessed in the treatment of CLAD in trials.

#### **8. Lung transplantation committee**

Recently, a collaborative effort of transplant centers has led to formation of a committee. The lung transplantation committee of OPTN works regarding lung procurement, allocation, and transplantation, including medical, scientific, and ethical aspects. The goal of the committee's work is to develop evidence-based policies aimed at reducing waitlist mortality in lung transplant candidates, increasing lung utilization, improving access to lung transplantation, and improving the health outcomes of lung transplant recipients.

#### **9. Lung microbiome**

The characteristics of the lung microbiome, its influence on the human body and disease processes, and the techniques to alter the local microbiome, serve as a large reservoir of untapped knowledge. Studies have been constantly trying to understand the differences between the microbiome of a healthy lung and a diseased one. 16S rRNA sequencing studies have disclosed that the lung microbiome is derived majorly from the oropharynx, with prevotella, veillonella, and streptococcus spp. being the most frequent colonizers [53, 54]. Consequently, it has been postulated that disruption of the oropharyngeal microbiome will reflect in the lung and ultimately make it more susceptible to pathogen colonization [55]. It is also being hypothesized that a healthy lung microbiome is based on the property of the organisms to be transient, owing to the dynamicity attributed to breathing and microaspiration. The microbial ecosystem of the lung is maintained delicately by a balance between the entry and active clearance by the cells of the lung. When the mechanisms of clearance fail, the organisms become persistent and disease ensues. It remains to be elucidated as to whether dysbiosis is a cause or effect of the disease [56]. Conventional literature believed that certain disease processes or exposures had a singular effect on the host. Recent studies, while acknowledging its effect on the microbiome independently, go beyond this idea and hypothesize that even without external influences, the microbiome and the host continually modulate each other [57].

Researchers have analyzed the oropharyngeal microbial pattern in patients with end-stage lung diseases, before and after lung transplantation. While waiting for transplantation, it was found that oropharyngeal wash samples of patients with advanced lung diseases had an increased facultative organism dominant microbial burden. Aerobic bacteria were decreased, alongside an overall reduction in richness and diversity. The microbiome stabilized after transplantation briefly, tending toward healthy lung levels. Nevertheless, 6 months after transplantation, it reverted to the severe dysbiosis-like state that existed before transplantation in the diseased lung [55]. If dysbiosis at oral sites indeed precedes/causes dysbiosis in the lungs, then these observations remind us of the increased risk of infections due to pathogen colonization posttransplantation, adding to the problems caused by an immunosuppressed state.

Studies have been working toward establishing and elaborating on the relationship between the altered microbiome and the complications after lung transplant. The most worrisome ones are primary graft dysfunction (PGD), acute rejection (AR), and chronic lung allograft dysfunction (CLAD). Lungs that had enrichment of oral-type taxa, before procurement from the donor and immediately after reperfusion in the recipient, have been seen to develop PGD. Aspiration of these microbes is hypothesized to prime the allografts for PGD [58]. Similarly, it is also observed that higher bacterial load after transplant is correlated with the development of CLAD, although no particular taxa were implicated [59]. These discoveries are promising as the microbiome can prove to be a potentially modifiable risk factor.

It is well known that inflammatory episodes happen in the allograft after transplantation, despite adequate immunosuppression [60]. Long-term immunosuppression and antibiotic therapy posttransplantation may make the local ecosystem more permissive to the overgrowth of certain immunostimulatory bacteria, which otherwise would have been cleared by the host immunity and controlled by the healthy microbiome [61, 62].

Inflammation can also be triggered by imbalances in the levels of normal commensals, which means that it is not necessary that a pathogenic invasion occurs for a disease process to ensue. For example, the Prevotella community is a part of the normal lung microbiome and is generally considered less stimulatory, but its overrepresentation can cause tissue remodeling [63]. In lung transplant recipients, an association between the dominant bacteria and the type of immune reaction was observed. When the dysbiosis was firmicutes or proteobacteria dominant, pulmonary leukocytes expressed inflammatory genes; while pro-remodeling effects were displayed by a Bacteroidetesdominant dysbiosis [63]. Similarly, in the case of pathogens, virulence does not necessarily equate with immunogenicity. S. Pneumoniae, which is the most common cause of community-acquired pneumonia, had a lower stimulatory effect on macrophages in comparison with S.aureus or P.aeruginosa [63]. Hyperammonemia syndrome (HS) is an uncommon but fatal disorder that can affect up to 4% of lung transplant recipients. It is imperative that patients with this syndrome should be screened for ureaplasma species and treated with the appropriate antibiotics for this infection [64].

#### **10. Gut-lung axis**

The effects of the gut microbiome on local and systemic immunity are well studied, and the documentation of its influence on the brain led to the coining of the term "gut-brain axis." Investigators are now trying to find out if such a relationship also exists between the microbiome of the gut and the lung. Several studies indicate that such "gut-lung axis" may indeed exist. For example, gut microbiome dysbiosis caused by antibiotic administration in the early years of life was seen to increase the risk of allergic airway disease [65]. Fecal microbiomes in patients with chronic obstructive pulmonary disease differed from healthy control subjects in terms of taxonomic microbial representation and their metabolites [66]. Murine models that were dietarily supplemented with short-chain fatty acids (SCFA) by adding acetate

#### *Recent Advances and Outcomes in Heart and Lung Transplantation DOI: http://dx.doi.org/10.5772/intechopen.109068*

to their drinking water showed reduced inflammatory infiltrates in their lungs. Au contraire, those receiving a low-fiber diet had lower SCFAs in their circulation and higher airway reactivity [67].

It has been recently found in clinical and animal studies that derangement in the gut microbiome can impact outcomes related to allograft dysfunction after transplantation of the liver and small bowel [68, 69]. To understand whether gut microbiome can impact lung allograft rejection as well mice model studies were conducted. The donors as well as recipients were pretreated with oral antibiotic gavages to reduce microbial load. Those that were pretreated had reduced severity of rejection, while the majority of the controls showed moderate–severe lung allograft rejection. The Bacteroidetes to firmicutes ratio was increased in the gut microbiome of the mice who experienced milder rejection [70]. Previous studies have tried to establish a causal link between CD4+ T lymphocytes and chronic lung allograft rejection in animal models after lung transplantation [71]. This would mean that a depletion of CD4+ T cells would improve the outcomes related to allograft rejection. However, an emerging hypothesis suggests that a reduced dendritic cell activation may instead be the origin of the decreased generation of graft-reactive T cells [70].

Unlike the gut microbiome, where microbial sampling is relatively easier due to their presence in the feces, determining the ecosystem of the lung is complicated by factors like lower biomass, the possibility of contamination during sample collection, interference by environmental factors, and the lack of knowledge about the fungal and viral component of the microbiome. Researchers have been citing the possibility of a metagenomic dark matter due to the presence of a large proportion of unidentified sequences arising during virome studies. This may indicate that we may come across undiscovered species of viruses in the future [72]. For example, recently, metagenomic sequencing from the respiratory tract of healthy as well as sick subjects led to the discovery of a new family of DNA viruses called redondoviridae [73].

More techniques are needed to profile the lung microbiome more accurately, to understand how the interactions affect the host, to establish causality between dysbiosis and specific disease processes, and to observe the resolution of symptoms after the reestablishment of the original microbiome. It would be interesting to see how therapeutic alteration of the microbiome can impact not only the outcomes of lung transplantation but also chronic disease processes in general. Trials are now being proposed to observe the effect of therapies like probiotic administration and fecal microbiota transplantation in the context of respiratory diseases [74]. If endotypes can be targeted specifically, it could open up multiple avenues tailored to deliver a personalized benefit [75]. The prospects of lung microbiome research are certainly promising but long-term studies will be necessary to ascertain the benefits quantitatively.

#### **11. Bioengineering**

Bioengineered lungs could change the landscape of lung transplantation as they counter the challenges posed by the long waitlist [76]. These techniques revolve around harvesting a lung sample and decellularizing it. The scaffold (acellular or artificial or hybrid) is then recellularized with stem or differentiated cells [77]. The matured and functional organ is then implanted into the host. EVLP systems serve as a good conduit to allow the maturation of the engineered graft [76, 78–80]. The fusion of current methods with emerging single-cell technologies and advanced 3D printing modalities could pave way for newer developments [81]. Substantial efforts are necessary to refine the existing engineering techniques and to convert preclinical studies to clinical trials in order to manifest this idea as a utilitarian alternative [76]. The idea of the bioengineered lung which appeared like utopian fiction decades ago could soon become reality [81].

#### **12. Recent advances in heart transplantation**

As a testament to improved survival statistics, heart transplant recipients have had an improved prognosis over the years. Since 2015, adult long-term mortality rates have witnessed a downward trend. Similarly, pretransplant mortality rates have also declined since 2009 [2]. However, this scenario was complicated by the COVID-19 pandemic, where we saw an increase in the 6-month and 1-year mortality rates during the first year of the pandemic. Pretransplant mortality also slightly increased in 2020 (**Figure 4**) [2]. Heart transplant candidates have notable changes in demographic patterns over the years. Candidates over 65 years of age have increased from 11.9% in 2009 to 15.9% in 2020. The prevalence of obesity has increased among heart transplant candidates. As of 2020, more than 40% of candidates have a BMI equal to or more than 30 kg/m2 [2]. Cardiomyopathy remains the most common indication for an adult heart transplant, while congenital heart disease remains the leading diagnosis in the pediatric age group. Notably, candidates receiving transplantation for a diagnosis of coronary artery disease have declined [2]. Continued efforts of the entire healthcare system have helped in improving transplant-related outcomes despite the current COVID-19 pandemic. Fewer candidates have been removed from the waitlist because of death or being too sick to receive transplants as compared to 2018 [2]. The number of multiple organ transplants has also increased, which is appreciated in heart-kidney transplants, while heart-lung transplants have remained stable [2]. In 2020, despite there being lesser new listings for heart transplants, the eventual number of heart transplants performed increased [2].

#### **Figure 4.**

*Patient death among adult heart transplant recipients. All adult recipients of deceased donor hearts, including multi-organ transplants (from OPTN/SRTR 2020 annual data report. HHS/HRSA).*

*Recent Advances and Outcomes in Heart and Lung Transplantation DOI: http://dx.doi.org/10.5772/intechopen.109068*

The number of deceased heart donors continues to increase. The discard rate of recovered organs remains extremely low at 1%, despite a slight rise in 2020. An upward trend is noted in the proportion of IRDs [2]. In the case of heart transplants, the use of mechanical circulatory support (MCS) as a bridge to transplant has dramatically increased over the years. In 2020, 36.4% of candidates had ventricular assist devices (VAD) at listing. This number was just 16.4% in 2010 [2].

To address the issue of waitlist mortality, the year 2018 witnessed new heart allocation criteria, as approved by the united network for organ sharing (UNOS). These criteria prioritized the sickest patients. The older 3-tier system (status 1A, 1B, and 2) was changed to a 6-tier one (status 1–6) [82]. Stable patients supported by LVAD were ranked lower (status 4) as compared to those receiving VA-ECMO and IABP, status 1 and 2, respectively [83].

Traditionally, for prolonged support, ventricular assist devices needed to be placed after securing surgical access. This was especially true when candidates were being bridged to transplant [84]. Recently, more research is being done on assist devices that can be implanted percutaneously. After the UNOS heart allocation policy change, the use of percutaneous devices like Impella jumped from 1% in 2015 to 4% in 2019 among transplant patients [85]. Impella is a catheter-based ventricular assist device for unloading the left ventricle and consequently increasing cardiac output. It was initially used to support patients with cardiogenic shock after acute myocardial infarction [86]. Due to its relative ease of insertion, it is now being considered as an alternative to support candidates waiting for a heart transplant. As compared to recipients who did not receive circulatory support, patients bridged to transplant using Impella 5.0/5.5 showed a similar 30-day and 6-month mortality [84, 87, 88].

#### **13. Donation after circulatory death, organ care system, and normothermic regional perfusion**

The standard cold ice transport of donor's hearts results in unpredictable myocardial cooling leading to irreversible suppression of diastolic function and protein denaturation. The recently introduced Paragonix SherpaPak Cardiac Transport System incorporates a novel nested canister system in concert with proprietary thermal cooling to provide physical and thermal protection for donor's hearts. Results of 1-year transplant survival analysis of GAURDIAN registry showed that despite longer ischemic times and higher proportions of temporary MCS devices, numerically higher 1-year survival was seen in the cohort with sherapak usage [89]. Dhittal et al. pioneered the technique of DCD heart procurement. Their technique allows for rapid decompression of the systemic venous system and subsequent cardioplegia administration. Following infusion of supplemented cardioplegia, the heart is explanted first and instrumented onto the portable extracorporeal TransMedics organ care system (OCS), where its viability and suitability for transplantation can be assessed [90].

#### **13.1 Organ care system**

An OCS heart is a portable extracorporeal heart perfusion and monitoring system. The aorta is connected to the aortic line, the pulmonary artery is connected to the pulmonary artery line (PA line), the left atrium is opened, and the left ventricle is vented via a left ventricle vent. The superior and inferior vena cava are ligated. Blood from the PA line and LV vent goes to the reservoir and is pumped to the gas exchanger

where it is oxygenated, passed through a warmer, and returned to the heart through the aortic line. It allows continuous monitoring of aortic pressure, lactate level, and coronary blood flow of the graft. The advantages of OCS are:

In the EXPAND trial, out of 93 such marginal hearts, 75 hearts were transplanted (81% utilization rate). The mean cross-clamp time and OCS perfusion time were 381 minutes and 279 minutes, respectively. In this study, the 30-day survival was 95%, the incidence of severe primary graft dysfunction within 24 hours of transplant was 11%, and the overall and graft survival at 24 months was 82% and 95%, respectively.

4.It provides a platform to assess DCD hearts prior to implantation.

However, the present limitation of the OCS is the additional cost of resources and personnel required for its application as well as its transportation is more complicated [91].

#### **13.2 Normothermic regional perfusion**

The alternate method of DCD heart procurement is normothermic regional perfusion (NRP) wherein VA-ECMO is utilized (by cannulating aorta and right atrium) to maintain the thoracic organ perfusion to allow them to recover warm ischemia. The advantages of NRP are:

1.It restores heart function by reducing myocardial injury.

2.Reduces time spent by the organ in warm ischemia.

3.Allows visual assessment of heart and other organs prior to procurement.

The utilization of NRP for DCD requires significant resources and coordination between the donor hospital, procurement teams, and organ procurement organizations [92].

#### **14. Post-heart transplant rejection surveillance**

Between 2018 and 2019, 23.6% of recipients of heart transplants suffered from acute rejection in one year [2]. Even though the gold standard for posttransplant rejection surveillance is an endomyocardial biopsy, there have been exciting developments in the past decade in molecular noninvasive diagnostic surveillance. Such novel molecular techniques have the capacity to facilitate early diagnosis, improve

the accuracy of diagnosis and decrease the frequency of invasive interventions for the diagnosis of allograft rejection [93].

#### **14.1 Microarray technology**

This technology examines endomyocardial biopsy samples at a molecular level to diagnose acute cellular rejection (ACR) as well as antibody-mediated rejection (AMR) [94]. Molecules called rejection associated transcripts (RAT) are identified to assess mRNA expression. Probabilities of developing ACR and AMR are then deduced. This technique was first implemented in patients who underwent kidney transplants. Halloran et al. later found out that similar RAT expression was observed in heart biopsy specimens, thus leading to the extrapolation of the idea in heart transplantation. This technique aims to tackle the main drawback of traditional histopathologic biopsies, which is interobserver variability in reporting findings [93, 95].

Currently, microarray technology is employed by the molecular microscope diagnostic system, which does an unsupervised archetypal analysis and predicts a normal, ACR, AMR, or injury pattern based on the assignment of scores [95]. This technology has been found to be effective in predicting molecular rejection [96]. The utility of intragraft mRNA transcripts as an addendum to the histopathology reports of endomyocardial biopsies can pave way for a newer modus operandi in diagnosing cardiac transplant rejection [93].

#### **14.2 Gene expression profiling**

Gene expression profiling (GEP) is done using peripheral blood leukocytes. It is done after 55 days posttransplant and has a high negative predictive value for moderate to severe rejection [93, 97]. Thus, it is used as an effective screening tool in stable posttransplant patients for diagnosis of rejection [93]. Allomap (CareDx) is the presently utilized gene expression profiling test in the clinical domain, which assigns a score of 0–40. A watershed score of > = 34 signals a higher probability of ACR [93, 98]. According to both IMAGE and E-IMAGE (early-IMAGE) clinical trials, GEP was found non-inferior to routine endomyocardial biopsy surveillance techniques, thus demonstrating similar clinical composite outcomes and rates of rejection [99, 100]. However, its use is limited only to the detection of ACR. Additionally, it also harbors a poor positive predictive value [93]. The CARGO-II trial proposed that a score less than 34 can imply a low risk of rejection [101]. Allomap can be utilized to time an early steroid weaning in patients with a lower risk of rejection [93].

#### **14.3 Donor-derived cell-free DNA**

This noninvasive modality of investigation is based on the cell death of the graft tissue, which releases donor-derived cell-free DNA (dd cfDNA) in the recipient's blood. The percentage of donor-derived cell-free DNA is titrated against the total cell-free DNA in the recipient's blood, detecting both ACR and AMR. It has also been found that dd cfDNA leads to earlier detection of acute rejection in asymptomatic patients compared to endomyocardial biopsy. The figure given below depicts the correlation between the percentage of dd cfDNA with ACR, AMR, and echocardiographic findings [93].

A study was conducted in Canada during the global COVID-19 pandemic utilizing noninvasive surveillance techniques for heart transplant rejection, thus ensuring minimal hospital exposure to patients. The study used cell-free DNA and gene expression

profiling as a substitute for the invasive endomyocardial biopsy. The results were then harnessed for personalized titration of immunosuppressive therapy in patients. Noninvasive rejection surveillance was associated with the ability to lower immunosuppression, increase satisfaction, and reduce anxiety in HT recipients, minimizing exposure for patients and providers during a global pandemic [102].

#### **15. Xenotransplantation**

Xenotransplantation could act as a bridge to allotransplantation to tide over the crisis posed by the ever-growing waitlist. Recent developments have been in the domain of genetically engineered pigs functioning as donors for human recipients. Naturally occurring human antibodies commonly target 3 carbohydrate antigens in pigs among which galactose-a1,3-galactose is postulated to be particularly important.  With the advent of novel gene editing techniques, double and triple-knockout pigs, engineered after the elimination of multiple carbohydrate coding genes were created.

Relatively species-specific complement pathway regulatory proteins (CPRP) like CD55, CD59, and CD56 attenuated complement-mediated injury and improved graft survival in various nonhuman primate models. These were introduced into the genome of pigs. Despite the addition of hCPRP (human complement pathway regulatory proteins) modification in GTKO pigs; the grafts were affected by microvascular thrombosis. This was probably due to a disharmony of thromboregulatory molecules like pig thrombomodulin and pig endothelial protein C receptor, which could be of a lower antithrombotic efficacy than their human counterparts. CD47 functions as a self-recognition marker in humans, which serves to inhibit phagocytosis. The Introduction of CD47 in the genome of pigs has shown better outcomes. Recently on January 7, 2022, the University of Maryland performed the first clinical heart xenotransplantation in a 57-year-old man, from a genetically engineered pig having 10 genetic modifications. He lived with a reasonable heart function for 2 months following the transplant but went on to develop graft dysfunction and ultimately, death. This event has however infused euphoria in the field of xenotransplantation and thoracic organ transplant academia. Similarly, in June/July 2022, Moazami et al. from New York University Langone successfully performed two further xenotransplants (pig hearts into humans) utilizing 10 genetic modifications, including four porcine gene "knockouts" to prevent rejection and abnormal organ growth as well as six human transgenes ("knock-ins") to promote expression of proteins that regulate important biologic pathways that can be disrupted by incompatibilities between pigs and humans.

### **Conflict of interest**

The authors have nothing to disclose.

#### **Abbreviations**

*Recent Advances and Outcomes in Heart and Lung Transplantation DOI: http://dx.doi.org/10.5772/intechopen.109068*

### **Author details**

Akshay Kumar1 \*, Sania Thite2 , Varad Wazarkar2 , Kamal Ayyat1,3, Jesus Gomez Abraham4 and Suresh Keshavamurthy5

1 Department of Thoracic and Cardiothoracic Surgery, Heart, Vascular and Thoracic Institute, Cleveland Clinic, Cleveland, Ohio, United States

2 Lokmanya Tilak Municipal Medical College, Mumbai, India

3 Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States

4 Department of Cardiothoracic Surgery, Newark Beth Israel Medical Center, Newark, New Jersey

5 Division of Cardiothoracic Surgery, Surgical Director Lung Transplantation, University of Kentucky College of Medicine, Kentucky, United States

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Recent Advances and Outcomes in Heart and Lung Transplantation DOI: http://dx.doi.org/10.5772/intechopen.109068*

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#### **Chapter 8**

## Machine Perfusion Strategies in Liver and Renal Transplantation

*Stella Vasileiadou, Nikolaos Antoniadis, Athanasios Kofinas, Konstantina-Eleni Karakasi, Georgios Katsanos and Georgios Tsoulfas*

#### **Abstract**

Transplantation is the only treatment for patients with end-stage renal and hepatic disease but unfortunately, it is limited worldwide due to the organ shortage. The need to expand the donor pool with the use of donors after cardiac death (DCD) and extended criteria donors (ECD) has led to major improvements in organ preservation. From cold static storage and preservation solutions to different types of machine perfusion, the possibility of successfully transplanting a marginal organ becomes reality. In this chapter, we examine the machine perfusion methods and the advantages of new technology in minimizing ischemic injury and improving the transplant outcome. Τhe establishment of protocols with the use of biomarkers in order to assess the transplant suitability of the graft will eventually provide the ideal opportunity to intervene and improve the quality of the organ.

**Keywords:** machine perfusion, liver transplantation, kidney transplantation, organ preservation, hypothermic machine perfusion, normothermic machine perfusion

#### **1. Introduction**

In this chapter, after a brief introductory reference to the definition and history of machine perfusion in transplantation, their contemporary necessity is discussed. For this purpose, two important topics such as ischemia–reperfusion injury and marginal grafts are analyzed. Subsequently, the machine perfusion strategies in liver and kidney transplantation are mentioned separately.

The review of the literature was conducted using the combination of various keywords (liver transplantation, kidney/renal transplantation, machine perfusion, hypothermic/normothermic/subnormothermic machine perfusion, ischemia–reperfusion injury, marginal grafts, extended criteria donors, donors after cardiac death, cold static storage, and organ preservation) in PubMed research database. The inclusion criteria of the articles were the English language and their relevance to the topic, which was judged by the authors after studying the abstracts.

Transplantation is the only treatment for patients with end-stage renal and hepatic disease. Despite the recent U.S. milestone of 1 million transplants until 2022, the

number of patients on waiting lists remains high [1]. The great need for increment of the donor pool leads to the utilization of allografts from extended criteria donors (ECD) and donors after circulatory death (DCD). The vulnerability of these marginal grafts in ischemia–reperfusion injury (IRI) [2] revealed all the weak points of standard preservation methods and resulted in poor transplant outcomes. Moreover, the ECD and DCD kidney grafts have increased immunogenicity and as a result higher rates of rejection [3].

The combination of nonideal donors with limitations in graft evaluation results in discarded organs that further exacerbate the transplantation gap. In an attempt to find solutions, the interest of scientific community turned to preservation strategies, particularly the application of machine perfusion methods to these organs, in order to assess and make them suitable for transplantation [4, 5].

The concept of machine perfusion (MP) in the field of transplantation is not new. In 1885, the initial idea of extracorporeal circulation with the construction of a closed circulatory system [6] and 52 years later the transition from blood to chemical perfusion solutions, in order to keep organs "alive" [7], laid the foundations for the development of organ preservation. Machine perfusion in a human kidney was performed for the first time in 1967 by Belzer. The first liver transplantation in humans was performed by Starzl, with the application of a hypothermic low-flow perfusion method that used diluted blood [8, 9]. Thus, although the original concept involved room temperature and dynamic perfusion, in 1969 Collins GM changed completely the perspective, with the 12-hour preservation of canine kidneys in an iced solution, which later evolved into Collins solution with a longer preservation time [10]. Nowadays, preserving the organ in an ice box at 0–4°C, after flushing it with a cold preservation solution (usually the University of Wisconsin or Histidine-Tryptophan-Ketoglutarate solution), is the most common method worldwide. Static cold storage (SCS) has been established as the gold standard due to its simplicity, efficacy, and low cost [11]. The idea of machine perfusion was abandoned but, in the beginning of this century, gained interest again in clinical practice.

Machine perfusion is an *ex-vivo* platform where organs are connected to a pump and perfused with the solution in a controlled flow, constantly, until implantation. The main purpose is to provide essential substances and remove waste products in order to maintain cellular metabolism in a predetermined way. There is variation in machine perfusion methods in terms of temperature, oxygen and nutrient supply, preservation solutions, delivery method, duration, and time-point of perfusion. Depending on the temperature, there are three types of machine perfusion: normothermic (35.5–37.5°C), hypothermic (1–8°C), and subnormothermic (20–35.5°C) temperature with the latter sought in fewer studies [12]. Also, the variation of temperatures differs slightly in the literature.

Hypothermia reduces cellular metabolism while at the same time, the preservation solution enhances ATP production [13]. Also, the addition of oxygen restores mitochondrial oxidative activity by electron transport [14]. Hypothermic machine perfusion (HMP) has the significant advantage of safety, as in case of machine failure, the graft can easily be preserved in SCS.

The aim of reducing cold ischemia time (CIT), which is an independent risk factor for delayed graft function (DGF), has led to the development of normothermic machine perfusion (NMP). During this method, the reperfusion phase after ischemia time takes place in an artificial environment as close as possible to physiological conditions (temperature and oxygen), in order to avoid the inflammatory process and the destructive injury, it causes to the graft. Normothermia has the important

#### *Machine Perfusion Strategies in Liver and Renal Transplantation DOI: http://dx.doi.org/10.5772/intechopen.111436*

advantage of assessing the graft in real-time, as bile and urine are produced by liver and kidney grafts, respectively [15]. On the other hand, it is a more complicated and costly procedure as it requires a sufficient amount of oxygen, oxygen carriers, and necessary substances for the graft function [16]. In an effort to limit the use of human blood products with red blood cells or plasma, the development of extracellular oxygen carriers began [17].

Subnormothermic machine perfusion (SNMP) is an intermediate state between hypothermic and normothermic conditions, maintaining low cellular energy demands and mitochondrial activity, while the organ is still "alive." This method is simpler than NMP as red blood cells are not necessary [18]. Although SNMP started as a promising technique, disappointing results in a porcine kidney model prevented its further clinical application. The SNMP group had increased rates of renal and tubular injury than the NMP group [19]. Currently, it is still at an experimental stage in both liver and kidney transplantation with important clinical prospects.

Controlled oxygenated rewarming (COR) is an alternative perfusion method that focuses on transitioning the graft from hypothermia to normothermia by gradually increasing the temperature (from 8–20°C). Mitochondrial function can be affected by rapid temperature change when the graft is reperfused after cold ischemic preservation. In particular, the opening of the mitochondrial permeability transition pore contributes in a destructive way to the occurrence of IRI. COR allows smooth recovery of mitochondrial function and studies have shown its feasibility in clinical application in liver and kidney transplantation [20, 21].

In the last decade, many studies have been published on different models of machine perfusion and large clinical trials in humans have been performed, changing the concept of preservation in transplantation. From procurement to transplantation, the reasons for this development, in addition to the increasing use of ECD or DCD grafts, are the comprehension of the complicated biological pathways and phenomena such as IRI.

#### **1.1 Ischemia: Reperfusion injury**

During organ procurement, the blood supply to the organs is interrupted, and after implantation or *ex-situ* normothermic machine perfusion of the grafts, it is restored. This process, defined as ischemia–reperfusion injury, leads to cell death in an oxidative environment and production of reactive oxygen species (ROS) [2].

IRI consists of three phases and each of them causes a different effect on the organ. First, it is the ischemic phase, when the blood and oxygen supply of the organ is inhibited. Dramatic depletion of ATP unregulates sodium-potassium ATPases in cell membranes, resulting in increased calcium concentration and acidic pH [22, 23]. It is of notice that the type of donor (donation after brain death or after circulatory death) plays an important role in ischemia injury to the graft [24]. Second, it is the reperfusion phase, when ischemic tissues are reoxygenated and as a result release ROS, that is mainly formed in complex 1 - mitochondrial respiratory chain [25]. Furthermore, in liver grafts, disorders in the mitochondrial permeability transition pore (MPTP) allow the release of danger-associated molecular patterns (DAMPs) from the nucleus [26], which interact with Toll-like receptors (TLRs) and activate Kupffer cells [27]. Finally, a prolonged period of inflammation is established where important liver microstructures are deranged. For example, damage to the endothelial glycocalyx or the combination of increased vasoconstrictive agents and decreased nitric oxide levels deteriorates the microcirculation and raises the risk

of early allograft dysfunction (EAD) [28, 29]. Third is the late injury phase, which follows the release of ROS and continues the cascade of cytotoxic events with the activation of resident and recruited immune cells [30].

IRI is globally independent risk factor in transplantation and is related to acute rejection, postreperfusion syndrome, DGF, and primary non-function (PNF) [31]. The standard static cold storage was an inferior preservation method for kidneys [32] and failed to protect marginal livers from the effects of ischemia–reperfusion injury [33], so the need for a different strategy was imperative. Continuous machine perfusion circulation of metabolic substances and removal of inflammatory products, such as toxins and cytokines [34], reduce the degree of graft injury, for example, to the liver's deep peribiliary glands and stroma [35].

#### **1.2 Marginal grafts**

As already mentioned, the use of suboptimal grafts is constantly increasing due to the transplant gap between the donor pool and the recipient waiting list. Marginal liver grafts are organs with an increased chance of failure or PNF after transplantation. Also, the definition includes cases where diseases can be transmitted from the donor to the recipient [36]. Usually, high risk liver grafts come from donors with factors such as: liver macrosteatosis, cardiac death, advanced age, long stay in the intensive care unit, infections and malignancies.

Macrosteatosis is not a prohibitive factor for transplantation if the percentage is <30% but the fatty liver is considered to have worse postoperative outcomes than grafts without steatosis [37]. DCD livers due to prolonged warm ischemia time are susceptible to ischemic-type biliary lesions. Microcirculation disorders, during IRI, cause damage to the arterial plexus of the biliary tree, so there is high risk of biliary strictures after the transplantation [38]. Advanced age has negative effects on liver reserves, so injury after reperfusion is more serious and increases the risk of posttransplant complications [39].

Machine perfusion techniques appear to benefit these borderline livers most, with characteristics such as macrosteatosis >30%, DCD donors, donors >60 years old, cold ischemia time > 8-10 h, warm ischemia time > 30 min, insufficient *in situ* perfusion, prolonged organ procurement, and generally discarded grafts due to eliminated transaminases or any nonvascular problem [40, 41].

Different scoring systems have been developed to predict graft survival: Survival outcomes following liver transplantation (SOFT), donor risk index (DRI), Eurotransplant-DRI, etc. Clinical parameters (cardiac arrest >15 min, age > 55 years, BMI >30), laboratory results (AST, ALT, HBV, and HCV), ICU conditions (days, drugs, and infections) and of course histology after biopsy are included and considered in the transplant decision. Finally, nonheart beating and DCD donors are ECDs by definition [42].

The definition of borderline kidneys, which actually includes donor risk factors for kidney transplants, is derived from living donors, as in the early stages were mainly associated with risks to the recipient [43]. The Kidney donor risk index (KDRI) is a practical tool for predicting the posttransplant outcome. Factors such as weight, height, race, history of diabetes, hepatitis C status, and DCD are included [44]. The OPTN-approved criteria for ECD kidneys consider the following donor parameters: age, cerebrovascular accident as the cause of death, history of hypertension, and creatinine level [45].

Also, acute kidney injury or extracorporeal membrane oxygenation (ECMO) prior to organ retrieval significantly affects the graft quality [46, 47].

#### **2. Machine perfusion of the liver**

#### **2.1 Hypothermic machine perfusion**

During hypothermic machine perfusion, the liver graft is perfused with a preservation solution, mainly University of Wisconsin (UW) or Vasosol (modified UW), usually at 4–11°C. In this way, the graft takes essential substances and eliminates waste products [40, 48]. The pO2 level is 20 kPa, as no additional oxygen is provided. HMP clinical series was first published in 2010, as a safe and effective perfusion system by James Guarrera [49].

In hypothermic conditions, deceleration of cellular metabolism and cryoprotection, prepare the liver graft for the time of reperfusion. HMP provides protection to the graft in a complicated and not completely clarified way, as far as ischemia– reperfusion injury is concerned. Studies have shown that it has positive effects on mitochondrial complexes and increases cellular energy, leading to metabolism of molecules such as succinate [50, 51].

Also, livers perfused under hypothermic conditions had less of the ultrastructural effects seen in SCS. The number of CD68+ macrophages and, in general, inflammatory injury agents were significantly decreased, as shown by measurements at the end of preservation and after graft reperfusion [51, 52].

During hypothermic oxygenated machine perfusion (HOPE), the pO2 level is 60–100 kPa, and in this technique additional perfusate oxygen is provided to the graft. Oxygen is diluted in the perfusion fluid so there is no need for oxygen carriers [53]. Oxygenation in hypothermic conditions restores mitochondrial function and promotes metabolic activity. As a result, the reduction of ROS and succinate does not lead to further damage in mitochondrial function [54, 55].

HOPE was initially used in a clinical setting in 2014, where Maastricht Type III-DCD liver grafts were treated and subsequently assessed [56]. A matched case analysis of DCD livers compared HOPE treatment with standard SCS. 25 HOPE livers had fewer biliary complications and better 1-year graft survival than 50 SCS livers, but with longer cold ischemic time in the last group [57]. HOPE-DCD livers showed a reduced risk of acute rejection and improved 5-year graft survival in a recent study with the same comparators (94% in HOPE DCD vs. 78% in SCS DCD). They also have better laboratory results (INR and lactates on the first day) and fewer postoperative complications [58]. Moreover, HOPE treatment proved beneficial in macrosteatotic livers and has a positive impact on graft function and survival compared to the unperfused group [59]. Similarly, grafts from donors with advanced age seem to have good results with HOPE treatment. In two transplantations with octogenarian grafts, this perfusion technique promoted the recovery process after IRI and reduced hospitalization [60].

The hypothermic machine perfusion uses either the portal vein alone or the portal vein and hepatic artery together. The dual perfusion with oxygen is called dual flow hypothermic oxygenated perfusion (D-HOPE) and attempted to optimize the oxygenation of the biliary system. D-HOPE DCD livers had low peak ALT and bilirubin levels compared with SCS, but no difference in patient and 1-year graft survival. Perfusion pressure is also a technical detail of great importance during machine perfusion. High pressure leads to additional damage at endothelial cells, so the desired levels are 20–30 mmHg in the hepatic artery and 3–5 mmHg in the portal vein during HMP [61].

A meta-analysis of 12 studies comparing HMP and SCS in liver grafts showed the superiority of HMP in reducing posttransplant AST and ALT serum levels, early allograft dysfunction, total biliary complications, and ischemic cholangiopathy. No statistical significance was observed in outcomes for primary nonfunction, hepatic artery thrombosis, postreperfusion syndrome, patient survival, and liver graft survival (1 month, 6 months, and 1 year) [62]. An additional advantage of HMP is that in case of pump failure, the organ can be stored in cold static conditions, so it is a safe perfusion method.

The viability assessment during HMP is challenging because of metabolic deceleration and absence of bile production. However, there is a growing interest in the literature for potential markers during HMP and HOPE. Peak AST after transplantation was found to be associated with AST and LDH levels after 2 h of perfusion [49]. Also, cellular ATP concentration during D-HOPE appears to be related to liver posttransplant function at a biochemical level [61]. A recent study by Muller et al. discovered a new mitochondrial biomarker that can be counted by spectroscope after 30–45 min of HOPE. Flavin mitochondrial mononucleotide (FMN) is easily detected in HOPE perfusate and can predict graft function and possible complications before implantation [63].

#### **2.2 Normothermic machine perfusion**

Normothermic machine perfusion uses temperatures between 35 and 38°C and components from blood products such as erythrocytes. Oxygenators are necessary and dual perfusion (portal and arterial) is performed. The aim is to mimic the physiologic environment so that the metabolism and function of the liver graft being maintained. The first clinical introduction was in 2016 when a phase-1 clinical trial was published [64]. Minimizing clod ischemia time enables long preservation of the liver graft without injury. In fact, a recent protocol described a metabolic, active, and functional human liver after 7 days of NMP [65].

The combination of HOPE and NMP versus NMP in nontransplanted livers was studied by the Birmingham group. The HOPE/NMP livers showed lower indicators of inflammation and better recovery of their metabolic rate [66]. According to the first prospective multicenter randomized controlled trial, the use of NMP (121 livers) instead of SCS (101 livers) increases the utilization of grafts by 50%. NMP despite the longer preservation time (11.9 vs. 7.7 h) reduced the grade of graft injury as seen by the low levels of AST and EAD, in both DCD and DBD livers. However, there was no superiority of NMP in PNF, biliary complications, ischemic cholangiopathy, graft, and patient survival [67].

The great advantage of NMP is the ability to evaluate the graft during preservation time. Observation of liver parameters such as bile production with laboratory test results, such as transaminases, lactate, glucose, and pH, have created scoring systems to aid in the transplant decision. Perfusate lactate, bile production, vascular flows, and liver appearance of five rejected livers were evaluated during NMP (after SCS), and finally transplanted with good outcomes [68]. In a study in 23 human livers, biochemical parameters of bile were examined after 6 hours of NMP and compared with the grade of bile duct injury. Biliary bicarbonate, pH, glucose, and bile/perfusate glucose ratio were identified as potential biomarkers of posttransplant cholangiopathy [69].

#### **2.3 Subnormothermic machine perfusion**

Subnormothermic machine perfusion is a method that uses temperatures between HMP and NMP, usually 21–25°C, and an oxygenated preservation solution such as HOPE. This subphysiologic temperature, on one hand, reduces the metabolic rate but, on the other hand, the graft is warm enough to allow viability assessment [70].

Three hours of SNMP in seven discarded human livers showed satisfactory bile production and liver function [71] but the study did not include posttransplant outcomes in order to determine the favorable effect of SNMP.

#### **2.4 Controlled oxygenated rewarming**

In an experimental animal study, a gradual raise in temperature (up to 20°C) with simultaneous oxygenated perfusion was performed for 90 min after a period of end-ischemia HMP. Posttransplant peak transaminase level was lower, and 6 months of patient survival was higher in the COR group compared with the control group [72]. Combined protocols, such as DHOPE-COR-NMP used in discarded livers, have significantly increased the number of transplantable grafts [73].

#### **2.5 Therapeutics in machine perfusion methods**

During machine perfusion, in addition to the standard enrichment of the preservation solution (glucose, heparine, insulin, antibiotics, maybe oxygen, and nutrients), agents targeting graft recondition can be added. Machine Perfusion therapeutic agents aim to improve the marginal graft to follow a successful transplantation. The unique advantage of this *ex vivo* procedure is that the agents are washed out of the graft and cannot interact with the recipient's immune system. Defatting cocktails that increase lipolysis, vasodilators with vascular protective effects, and gene therapies with specific targets and without side effects are just several possible treatments for a marginal liver graft.

#### *2.5.1 Defatting agents*

In a porcine model, 48 h of NMP in steatotic livers resulted in higher metabolism (triglyceride, glucose, and urea in the perfusate) and less lipid detection in the tissue (histologically demonstrated) [74]. In another rodent model with steatotic grafts, 3 hours of NMP with the addition of a defatting cocktail showed a reduction of lipids in the cells with a raise in lipid mobility at the gene level [75]. The defatting cocktail was tested on discarded human grafts with good outcomes compared to the control group. Triglycerides accumulation and the grade of macrovesicular steatosis were significantly reduced [76].

#### *2.5.2 Vasodilators*

Prostaglandin E1 is a vasodilator with fibrin clot dissolution activity, which has been shown to meliorate microcirculation injury. Rodent studies where PGE1 is used in NMP verified the beneficial effect on treated livers. The levels of aspartate and alanine aminotransferase were decreased and the bile production was higher [77, 78]. Nitroglycerin and prostaglandin E1 were contained in the Vasosol solution used in the first clinical trial of HMP in human livers [79]. Apart from PGE1, prostacyclin had the same advantages in treated porcine livers [80]. Another porcine transplant study researched the effect of BQ123 and verapamil as vasodilators and noticed that treated livers had better flow in the hepatic artery [81].

#### *2.5.3 Gene modulation agents*

Antisense oligonucleotides (ASOs) and siRNA are auspicious gene agents that can have specific targets without the need for viral convection. Silencing of miRNA-122

by ASO resulted in reduced HCV activity in a porcine study [82]. Despite the fact that antiviral medication is widespread and efficient, this finding is important in the evolution of MP therapeutics. siRNAs can have multiple targets such as Fas pathway, p53, RelB, TNF-a, and proapoptotic caspases. All these targets are associated with different aspects of IRI and can potentially protect livers, as shown in animal models under normothermic or hypothermic conditions [83–85].

#### *2.5.4 Others*

Anti-inflammatory agents (alprostadil, n- acetylcysteine, carbon monoxide, and sevoflurane) used under subnormothermic or hypothermic conditions, especially the NLRP3 inflammasome inhibitor mcc950 [86], enkephalin, which is a Δ-opioid agonist treated rat livers under NMP [87], and human liver stem cells in an extracellular form [88], significantly reduced IRI markers.

#### **3. Machine perfusion of the kidney**

#### **3.1 Hypothermic machine perfusion**

HMP has been established as a safe and effective preservation method with benefits in kidney transplantation. In one of the first RCTs with 336 deceased donors, nonoxygenated HMP was superior to CSC in reducing DGF and increasing 1-year graft survival, regardless of donor type [32]. This publication integrated HMP into daily clinical practice. Afterward, the good short-term results (DGF and 1-month kidney function) of HMP were also confirmed in DCD kidneys. Regarding long-term outcomes, the 164 kidney transplants (82 HMP vs. 82 SCS) had no significant difference in 1-year graft survival [89].

The addition of oxygen and the development of HOPE showed promising results. Application of HOPE in a rodent study significantly decreased markers of reperfusion injury (IL-6, ENO, and TNF-alpha) after kidney transplantation [90]. In a recent meta-analysis, PNF and DGF were significantly reduced in HMP kidneys compared with SCS, but no difference in acute rejection was observed. Also, one-year graft survival was longer in the HMP group [91].

Although the benefit of HMP over SCS has been proven, a large randomized clinical trial COPE-POMP, which evaluated ECD kidneys stored on ice and then perfused in oxygenated hypothermic conditions for 2 hours before implantation, showed no advantages in graft survival (3 months and 1 year) and function (PNF and DGF). These results may be associated with the use of HMP in different centers [92]. Another recent RCT, COPE-COMPARE, compared oxygenated HMP with nonoxygenated HMP in Maastricht III DCD kidneys (50 years and older). The first group improved renal function while decreasing posttransplant complications and acute rejections [93].

The ideal duration and timepoint of HMP are under research, with most studies using end-ischemia time. This phase is practical as the graft is transported on ice and assessed prior to transplantation. In a clinical study of 66 ECD kidneys subjected to end-ischemic perfusion for 369 minutes, the percentage of DGF was 0 versus 9.3% in the contralateral kidneys preserved in SCS [94].

Several potential biomarkers for graft assessment have been studied. The levels of glutathione S-transferase (GST), N-acetyl-beta-D- glucosaminidase (NAG),

heart-type fatty acid-binding protein (H-FABP), or LDH measured in the perfusate during MP and are related to DGF [95–97]. Parameters in HMP, such as resistance and flow, are used in scoring systems to assess kidney quality and risk of DGF [98]. It is noteworthy that CIT, as an independent risk factor in graft function after transplantation, increases the percentage of DGF as hours of cold ischemia pass. As the study of the data from the "Machine Perfusion Trial" showed, if CIT was above 10 hours, DGF was not reduced by HMP [99].

#### **3.2 Normothermic machine perfusion**

The first clinical application of NMP (35 min after 11 hours of SCS) in human kidney transplantation was in 2011 with good postoperative results [100]. A larger clinical study compared 18 ECD kidneys that underwent NMP (for approximately 63 min) with matched ECD kidneys that were preserved on ice. DGF was lower in the NMP group (5.6 vs. 36.2%) but 1-year graft survival was comparable in both groups [101].

The advantages of preserving a kidney in normothermic conditions are many. Cold ischemic injury is minimized as aerobic metabolism and renal functions are restarted [102]. The preservation time is investigated in the literature. In a recent study, discarded human kidneys underwent 24 h of NMP using urine recirculation with stable or slightly better tubular function after histopathological assessment [103].

The observation of the graft is feasible and provides the opportunity for assessment.

Parameters, such as total urine output, renal blood flow, and macroscopic perfusion during NMP, are used in score systems to evaluate the graft quality and "transplantability" [104]. Of course, therapies and agents that improve the condition of the graft prior to transplantation are feasible in normothermic preservation. A functional organ in NMP is more easily modified than in hypothermic conditions where cellular mechanisms are under-functioning.

#### **3.3 Therapeutics in machine perfusion**

#### *3.3.1 Hypothermic conditions*

Mesenchymal stem cells (MSC) and their extracellular vesicles, which have been used in a rat model where the kidneys underwent HMP, have a potential protective role in the perfusion process. The results showed increased activation of enzymes related to cell metabolism and membrane permeability [105]. Carbon monoxidereleasing molecule 401 (CORM-401) decreased IRI markers and improved graft function [106]. Anticoagulative agents, such as thrombalexin and heparin conjugate, have been tested to improve microcirculation, with positive outcomes in the kidneys [107, 108].

#### *3.3.2 Normothermic conditions*

Application of MSCs to NMP had a suppressive role in the inflammatory process, in human kidneys [109]. Erythropoietin and its derivative, cyclic helix B peptide (CHBP), can reduce inflammatory agents, such as caspase-3 and IL-1β, providing renoprotection against IRI and improving urine production of the kidney. Recently, a mouse kidney model defined the role of endoplasmic reticulum stress (ERS) in IRI and pointed out the therapeutic potential of CHBP with CHOP (an indicator of ERS) as a biomarker

[110–112]. Also, nanoparticles can be used as drug-delivered agents in blocking or preventing events of inflammation at a molecular level. For example, a possible target is the vascular cells of the graft's endothelium that are mainly damaged by IRI and performed antidonor antibodies. Nanoparticles, with the presence of anti-CD 31 antibodies, can target endothelial cells and deliver therapeutic agents during NMP [113].

#### **4. Conclusion**

Banking organs on a worldwide scale after quality tests would be the ideal method of organ preservation in order to select the perfect-match recipient and then proceed to scheduled surgery [114]. This goal has been achieved in stem cell transplantation and important achievements are taking place in solid organ transplantation, through the development of organ preservation with machine perfusion. Starting with animal experiments and, subsequently, with human clinical studies and randomized controlled trials, machine perfusion is an alternative that still has a lot to offer in solid organ transplantation.

The advantages of MP in short-term outcomes of transplantation are dependent on the preservation method but also the beneficial effects in long-term organ function are continuously demonstrated. It is a platform where, beyond quality, the immunogenicity of the graft could be studied and modified according to the profile of the recipient [103]. The specific targets and the lower dosages, which can be achieved using MP therapeutics agents compared to conventional systemic pharmacological agents, open a new chapter in marginal graft utilization. This may in the future help change the transplant process to a more personalized medicine approach.

New MP technology reveals new perspectives in transplant logistics. Graft evaluation leads to more transplantable organs perhaps in more complex recipients. The prolonged time of preservation provides the opportunity for preoperative planning and elective surgery in day time.

"*Which perfusion technique enables the highest utilization rate of otherwise discarded livers with the best available outcomes*, *regarding complications*, *graft*- *and patient survival*" [115]. This question, which could concern the kidney as well, remains to be answered. So far, there is no evidence of the superiority of a preservation strategy. Randomized clinical trials with combined perfusion protocols may provide the answer to the ideal approach in solid organ transplantation.

*Machine Perfusion Strategies in Liver and Renal Transplantation DOI: http://dx.doi.org/10.5772/intechopen.111436*

#### **Author details**

Stella Vasileiadou\*, Nikolaos Antoniadis, Athanasios Kofinas, Konstantina-Eleni Karakasi, Georgios Katsanos and Georgios Tsoulfas Organ Transplant Unit, Hippokration General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Georgios Tsoulfas*

Organ donation and transplantation is a highly specialized and challenging field of medicine that has witnessed tremendous advancements during the last few decades. This book provides a comprehensive overview of the challenges involved in organ donation and transplantation, including ethical, legal, and medical issues, as well as the technical and immunological problems facing experts involved in the care of transplant patients. Over three sections, chapters address such topics as the different types of donations and transplants, technical and surgical aspects of transplantation, and ways to achieve transplant tolerance.

Published in London, UK © 2023 IntechOpen © Rosendo Serrano Valera / iStock

Current Challenges and Advances in Organ Donation and Transplantation

Current Challenges and

Advances in Organ Donation

and Transplantation

*Edited by Georgios Tsoulfas*