MATERIALS AND METHODS

Study design
We conducted a prospective observational study investigating protein expression in donor lungs exposed to a standardized EVLP protocol. Approval was granted by our local research ethics committee, and informed consent for research was obtained from donor families and lung transplant recipients (REC 09/H0905/10).

Ex vivo lung perfusion protocol
Adult donor lungs deemed unsuitable for lung transplant by all five UK lung transplant centres but meeting strict EVLP criteria were included in the study (Supplementary Table S1), the results of which were previously published by our group [16]. Lungs were procured in a routine fashion and transported to our institution. The EVLP assessment followed a standardized acellular protocol using the Toronto technique with a closed left atrium and reduced perfusate flow, previously described in detail [3, 16]. Transplant suitability was assessed hourly during perfusion. Lungs meeting transplant criteria (Supplementary Table S2) at two consecutive time points were cooled and transplanted. Lungs deemed to have futile prospects for improvement were taken off the circuit and discarded. Two transplanted and five non-transplanted lungs were perfused ≥5h before a transplant decision was made.

Sample collection and processing
A research BAL was performed for all lungs by wedging an adult bronchoscope in a subsegmental bronchus of the right or left lower lobe [17]. Saline (40 ml) was instilled through the suction channel followed by gentle aspiration and sample collection prior to commencing ventilation at the beginning of EVLP. This process was repeated in the same lobe but in a different subsegmental bronchus before disconnecting the ventilation at the end of perfusion. In addition, hourly perfusate samples of 2.5 ml were collected until the assessment was stopped.

Protein expression analysis
All protein expressions measured in perfusate were adjusted to the predicted total lung capacity (pTLC) of the donor as an estimate of perfused donor lung volume and were reported as corrected perfusate concentrations (pg/ml). The pTLC was calculated in a routine fashion based on donor gender and height [18]. If one lung was deemed unusable due to severe consolidation or extensive contusion on inspection, or if the intended recipient required a single lung transplant on a specific side, only one lung was procured. For single-lung perfusions, the pTLC was adjusted to a factor of 0.55 for right lung and 0.45 for left lung perfusion [19].

Lactate dehydrogenase assay
Lactate dehydrogenase (LDH) levels were measured in perfusate and BALF with a colorimetric LDH cytotoxicity assay kit according to manufacturer’s instructions and reported as arbitrary units (U) (Thermo Fisher Scientific Inc., Rockford, IL, USA).

Multiplex inflammatory cytokine array
Interleukin (IL)-1β, IL-6, IL-8, TNF-α, and IL-10 were analysed with an MSD Multi-Array® (Meso Scale Diagnostics, LLC, Rockville, MD, USA). The assay was performed according to the manufacturer’s instructions. Technical issues prevented reading of perfusate samples from donor lung EVLP03; therefore, these samples were excluded from the analysis.

Enzyme-linked immunosorbent assays
Syndecan-1, IL-33, S100A9 (all R&D Systems, Inc., Minneapolis, MN, USA) and high-mobility group box-1 (HMGB-1) (Shino-Test Corporation, Kanagawa, Japan) were measured with commercially available ELISA kits according to the manufacturers’ instructions.

Statistical analysis
Donor characteristics and physiological parameters are expressed as medians with interquartile ranges and were compared between transplanted and non-transplanted lungs using Mann–Whitney U tests. Paired samples, start-end of perfusion, were compared with Wilcoxon signed-ranks tests. Log protein expressions were compared between transplanted and non-transplanted lungs with multiple t-tests. Correlations between IL-8 and IL-1β expressions and seven post-transplant outcomes (PaO2:FiO2 24-h post-transplant; PGD3 at 72 h post-transplant; ventilation time; intensive care unit stay hospital stay‘; percent of predicted FEV1 at 6 months post-transplant; and percent of predicted FVC at 6 months post-transplant) were analysed by Pearson’s correlation tests. The data were transformed back into non-logged values for reporting as mean (T) for transplanted and mean (NT) for non-transplanted lungs with standard deviations (SD). Multiple testing of donor parameters, protein analyses, and correlations was corrected using the Benjamini-Hochberg false discovery rate (FDR) controlling procedure [20]. Because this was a feasibility study aiming to identify potential markers for further investigation in a validation cohort, an FDR corrected P-value <0.1 was deemed significant. Corrected P-values are reported.
A multiple logistic regression model was fitted using the 11 log transformed protein covariates and their squared counterparts as independent variables and the EVLP outcome as the dependent variable. The optimal model was established by leave-one-out cross-validation using the software package R from R Core Team (2014) (R Foundation for Statistical Computing, Vienna, Austria) [21].