The goal of this observational study is to analyze the expression of liver-derived miRNAs in different types of liver donors to gain in depth knowledge about distinctive physiologic features within donors and find potential biomarkers for graft quality assessment. The main questions it aims to answer are:

Is there a distinctive miRNA profile between donors after brain-stem death and donors after circulatory death?

Is heparinase I treatment necessary to overcome miRNA quantification interference in heparinized liver donor samples ?

Researchers will compare miRNA expression in donors after brain-stem death and donors after circulatory death with I) a subgroup of donors with unusable grafts due to significant steatosis, 2) and with a subgroup of healthy controls undergoing elective cholecystectomy to set the reference miRNA profiles in extreme conditions.

Between October 2019 and May 2021, perfusates, liver biopsies and serum samples were collected prospectively from adult deceased organ donors in Vall d'Hebron University Hospital, Barcelona, Spain.

Samples from adult human brain death donors (n=10) and donors after circulatory death (n=10) were analyzed for the presence of miR-122, miR-148, miR-155, miR-22, miR-222 and ratio miR122/miR222, target biomarkers of liver injury selected from the literature.

To set the reference miRNA profiles, a subgroup of donors with unusable grafts due to significant steatosis (n=3) were analyzed.

Donors for re-transplantation, pediatric, and split were excluded to avoid biopsy-related injury.

As there were no living donor inclusions in the study period, serum specimens from a subgroup of healthy controls undergoing elective cholecystectomy (n=7) were analyzed.

This study is part of a PhD thesis and data collection was done by the main investigator based in organ donor availability in one tertiary hospital centre in Barcelona, Spain. Based on resource limitation constraints, the sample size was limited to the consecutive samples obtained in a 2-year period.

Written informed consent was obtained from patients and relatives of donors included in the study. Samples and data from patients were provided by the Vall d'Hebron University Hospital Biobank (PT20/00107), integrated in the Spanish National Biobanks Network, and they were processed following standard operating procedures. The study protocol was approved by the medical ethics committee of the Vall d'Hebron University Hospital [PR(AG)46/2019] and conforms to the principles outlined in the Declaration of Helsinki.

Statistical analysis:

To optimize the quality of data, readings with extreme Ct values (Ct less than 11 or Ct greater than 39) were discarded. The mean of the Cts and the standard deviation for each of the miRNAs in the different samples analyzed (technical triplicates) were calculated. To improve the quality of the data analyzed, in triplicates with high standard deviation (Ct SD greater than 0.3), the Ct value furthest from the mean was eliminated, leaving a total of at least two values per miRNA per sample. Likewise, for each miRNA, samples with less than two valid replicates were excluded. To correct for potential RNA input or RT efficiency biases, Ct values were normalized using the average Ct of endogenous references (miR-103a, miR-191, miR-30c, miR-16 and let7a). miRNA relative quantification (RQ) levels were analyzed using the Livak et al. method 2^(-ΔΔCt), calculated as follows ΔCt (miRNA Ct target - averaged endogenous control Ct) and the difference (ΔΔCt) between comparison groups (ΔCt comparison - ΔCt reference sample. Group comparisons were performed using T-test or U-Mann Whitney (non-parametric) for continuous data and Fisher test for qualitative. P-values less than 0.05 were considered significant.

Methodology substudy:

A subgroup of adult human brain death donors (n=4) and donors after circulatory death (n=4) were analyzed for the presence of miR-122 and miR-148. Heparin is known to have an inhibiting effect in RNA qPCR analysis and liver donor samples are prone to have heparin traces, as it's used to avoid thrombus formation during organ procurement. Heparinase-I has been used in the literature to overcome the heparin inhibiting effect.

In this substudy, the samples were treated with 0 IU, 6 IU or 12 IU of heparinase-I to evaluate the counteracting effect of heparinase I on miRNA detection levels by RT-qPCR.

I) Main donor groups (DBD and DCD):

Inclusion Criteria:

• Adult deceased liver donors

Exclusion Criteria:

• Pediatric donors (less than18 year-old)
• Donors for re-transplantation cases
• Donors who underwent split or partition liver technique to obtain two small grafts for two recipients (adult and pediatric).

II) Discarded DBD grafts:

Inclusion Criteria:

- Unusable grafts for transplantation due to moderate (30% to 60%) or severe (more than 60%) steatosis in liver biopsies.

Exclusion Criteria:

• Unusable grafts discarded for other reasons.
• Pediatric donors (less than18 year-old)
• Donors for re-transplantation cases
• Donors who underwent split or partition liver technique to obtain two small grafts for two recipients (adult and pediatric).

III) Control group:

Inclusion criteria:

- Same inclusion criteria as the living liver donor program in our center: 18 to 60 years old, normal liver function, ultrasound without signs of liver disease and steatosis less than 30%.

Exclusion criteria:

- Patients with preoperative deranged liver function tests.

Fourteen combined perfusates, liver biopsies and serum samples were collected prospectively from brain death donors (DBD, n=14) and donors after circulatory death (DCD, n=14).

Three combined perfusates, liver biopsies and serum samples were collected prospectively from brain death donors discarded for transplantation due to significant steatosis (DBDd, n=3).

Seven serum samples were collected prospectively from healthy controls undergoing elective cholecystectomy (Control, n=7).

Single centre study with samples collected at Vall d'Hebron University Hospital, Spain, between October 2019 and May 2021.