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. 2022 Dec 20;12:21973. doi: 10.1038/s41598-022-26580-6

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Discrimination of EVs based on HLA phenotype. (a) Representative scatter plots of an HLA-A3 + PPP sample (left) and an HLA-A3- PPP sample (right) stained with anti-HLA-A3 (X-axis) and anti-CD9-APC (Y-axis) before and after detergent treatment (top and bottom row, respectively). (b) Visual examination of events representative for each fluorescent population demonstrated the selection and subsequent analysis of particles showing single spot fluorescence (no coincidence events), indicating the selection and analysis of single EVs. (c,d) Concentrations of HLA-A3 + and CD9 + HLA-A3 + fluorescent events in all pre-transplantation donor, recipient, and buffer only (control) samples before and after detergent treatment, and after isotype staining (grey, orange, and blue boxes, respectively). HLA-A3 + events were found to not be indicative of EV signals and were excluded from analysis. Double-positive events were found to be indicative of EV signals, as well as to be discriminative between HLA-A3 + and HLA-A3- PPP samples.