This was a prospective, observational cohort-nested study conducted in four transplant centers in Spain that included adult patients undergoing uni- or bilateral LT between July 2018 and July 2019 and their respective BD or CD donors. The type of death was certified in accordance with Spanish legislation²⁵.

The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Human Research Committees of all participating hospitals (PR [AG]202/2017). All recipients provided written informed consent before being included in the study. Informed consent to participate in the study from donors was included in the donation consent. No deviation from the standard management of lung recipients took place except for serial blood sampling.

LT recipients from DBD and DCD were matched individually for sex, age (±5 years), and indication for LT (Figure 1). We excluded from this analysis patients undergoing lung re-transplantation, cardiopulmonary transplantation or those who had previously undergone or were to receive more than one simultaneous solid organ transplantation, as well as those for whom the graft ischemic time was >420 min. Following previous reports by Koukoulis et al.²⁷ and considering a confidence interval of 95%, statistical power of 80% and patient loss of 15%, a sample size of 36 LT recipients for each group (DCD and DBD) was estimated to detect a difference of at least one SD (15 pg/mL) in IL-6 plasma levels.

The following donor and recipient variables were prospectively collected: demographic (sex, age) and anthropometric (body mass index) measurements; clinical data (cause of death, corticosteroid pretreatment of donors and indication for transplantation of recipients); and surgical data (transfusion of blood products, vasoactive support or application of veno-venous [VV-] or veno-arterial [VA-] extracorporeal membrane oxygenation [ECMO], ischemic times for the first and second lungs and use of cardiopulmonary bypass). In all recipients, we registered the development of PGD (any grade and grade III) within 72 h post-transplantation according to the International Society for Heart and Lung Transplantation (ISHLT) Working Group criteria [8]. Mortality during the first 3 months after the transplant was also recorded.

Blood samples (10 mL) were obtained from donors before skin incision (D0) and before organ perfusion (D1). In recipients, blood samples were obtained in the operating room before implantation surgery (R-1), just after graft reperfusion (R0) and 24 (R24), 48 (R48) and 72 (R72) hours after LT. All blood samples were collected in EDTA tubes and centrifuged at 1,000 g for 10 min at room temperature (22ºC–23°C). Plasma was separated, divided into 2 aliquots of 2 mL each, and immediately stored at −80°C until analysis. At the end of the inclusion period, samples were shipped together in dry ice containers to the central laboratory located at the coordinating site (Hospital Universitari Vall d’Hebron, Barcelona) for cytokine analysis by immunofluorescence assays based on microfluidics using ELLA Simple Plex (Protein Simple, Biotechne, CA, United States) to simultaneously detect IL-6, IL-10, IL-8 and TNF-α. Triplicates of each cytokine result were obtained (the maximum allowed variation among triplicates was 5%).

Data were collected and stored in an ad hoc database on the website of the Organización Nacional de Trasplantes (ONT, Spanish Transplant Organization) and made accessible only to the principal investigator of each participating site. A member of the ONT was commissioned to monitor the study.

The Shapiro-Wilk test was performed as a test of normality for IL level distribution. Categorical variables are expressed as n and percentages. Quantitative data is presented as mean ± SD if normally distributed or as median (Q1, Q3) if not. Chi-square test, or Fisher’s exact test were used to compare categorical variables. Change over time was analyzed with non-parametric 2-way mixed repeated-measures ANOVA. The basic model included a group factor (DBD or DCD), a time factor, and an interaction between group and time. The main effects of group and time were explored for nonsignificant interactions. Bonferroni correction was applied to adjust for multiple comparisons in posthoc tests to determine specific pairwise differences between groups. All statistical analyses were performed using R version 4.3.1 (R Core Team, 2023). A p-value < 0.05 was considered statistically significant.

Figure 1 presents the consort diagram of the study. A total of 156 LT recipients were initially included, but 24 were later excluded because of incomplete data. Of the remaining 132 patients, 106 were DBD recipients and 40 were DCD recipients. Forty of the 106 DBD recipients were matched individually to the 40 DCD ones by sex, age (±5 years) and indication for lung transplantation.

Table 1 contrasts the main characteristics of both donors and recipients. More DBD received corticosteroids and vasoactive support before lung retrieval than DCD (p = 0.02 and p = 0.043, respectively), while more DCD than DBD received transfusions of blood products (p = 0.001). No other significant differences were observed between groups in donor or recipient characteristics.

At D0 and D1 the levels of IL-6, IL-10 and IL-8 were higher in the DBD group than in DCD, and a significant main effect of time with a higher concentrations at D1 compared to D0 with statistical significant increase the IL-10 levels at D1 in DBD group. There was no statistical differences of type or time on TNFα (Table 2; Figure 2).

In recipients, there was no statistical significance of type of donor on IL-6, IL-10, IL-8 and TNF. Before LT (R-1), the plasma level of these cytokines was similar in both groups (DBD and DCD) and immediately after LT (R0), the plasma levels of IL-6, IL-8 and IL-10, increased in both groups of recipients and decreased thereafter during the next 72 h without differeces. There was a statistically significant effect of time on TNFα only for the DBD group (Table 3; Figure 3).

The incidence of PGD of any grade was similar in DCD and DBD recipients (Table 4). Therefore, to further investigate the evolution of systemic inflammation in PGD we merged both groups for analysis. We found that the donor plasma levels of all cytokines were comparable between patients who developed any grade of PGD or not, except for TNF-α, which was higher at D0 and D1 in those recipients with PGD (Table 5; Figure 4). Specific analysis of PGD grade 3 did not show differences between groups (Table 6).

On the other hand, recipients experiencing PGD demonstrated elevated levels of IL-6 at R0 and a trend towards R48 and IL-8 at R0, R48, without differences between groups (Table 7; Figure 5). Furthermore, in the PGD grade 3 group, recipient plasma levels of IL-6 and IL-8 were significantly elevated at R0, R24 and R48 (Table 8).

Post-operative mortality rate was very low and similar in both groups without any additional deaths within 3 months after surgery (Table 4).