Abstract 4281: Overcoming preanalytical variabilities for peripheral blood mononuclear cells and exosome profiling in the IMMUcan multicentre study
Authors: Mustapha Rami , Pomella Valentina , Vicencio Jose , Lopez-Lastra Silvia , Bahlawane Christelle , Ammerlaan Wim , Hong Henoch S. , Morfouace Marie , Vallely-Gilroy Caoimhe , Soumelis Vassili , Romano Emanuela , Tejpar Sabine , Ng Tony , Betsou Fay ,   IMMUcan consortium
Published: September 2020

Abstract
IMMUcan is a joint public-private partnership to advance our understanding of how tumors and the immune system interact and the effect of therapeutic interventions. The project will collect tumor biopsies and peripheral blood from up to 3,000 patients across 13 European countries for multi-omic analyses.
Here, we assessed the impact of sample collection and processing conditions on the quality of peripheral blood mononuclear cells (PBMCs). We also compared circulating exosomes obtained from plasma and serum to determine the ideal source for the study.
We collected peripheral blood from 9 healthy donors across 3 centers in different countries. PBMC isolation and freezing were either performed locally at 3, 8, 24, 48 and 72 hours delays or alternatively shipped to a designated biobank for central processing at 8, 24, 48 and 72 hours delays. PBMCs were assessed for yield, viability and frequencies of immune cell populations by flow cytometry. Serum vs plasma derived exosomes were compared using nanoparticle tracking analysis for size distribution and immuno-dotblot for protein levels of HER1, HER2, HER3, cMET, P4HA1, S1000A9, PD-L1, and ALIX.
Centralized processing yielded more consistent results with an overall improvement in PBMC numbers and viability. The delay between blood withdrawal and PBMC isolation had a noticeable impact on PBMC quality when it exceeded 24 hours. Between 24 and 48 hours, we observed a significant and donor dependent decrease in viability and a major increase in granulocytic contamination. Relative frequencies of NK cells were the most sensitive to processing delays during the first 24 hours. Despite a slight global decrease in viability at 24 hours, the relative proportions of other major immune subsets remained stable. Regarding exosomes, plasma-exosomes had a significantly higher modal size compared to serum-derived. Particle abundance was minimally higher in the serum-exosomes. There were no significant differences in the levels of most proteins except for HER3 and c-MET, which were significantly upregulated in plasma exosomes.
We opted to centralize PBMC processing to one biobank to minimize variability. As it was logistically impossible to ensure shipments and processing within 24 hours for all recruitment sites, we limited sample inclusion up to 48 hours. This will be accounted for by tracking the number of granulocytes in future CyTOF measurements and including the delay durations in subsequent multi-variate analyses. Exosomes from serum and plasma will be included in the study as the data showed that both sources produced exosomes of adequate quality yet were different enough in protein levels to merit further investigation.
Funding: IMI2 JU grant agreement 821558, supported by EU's Horizon 2020 and EFPIA.
Citation Format: Rami Mustapha, Valentina Pomella, Jose Vicencio, Silvia Lopez-Lastra, Christelle Bahlawane, Wim Ammerlaan, Henoch S. Hong, Marie Morfouace, Caoimhe Vallely-Gilroy, Vassili Soumelis, Emanuela Romano, Sabine Tejpar, Tony Ng, Fay Betsou, IMMUcan consortium. Overcoming preanalytical variabilities for peripheral blood mononuclear cells and exosome profiling in the IMMUcan multicentre study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4281.