Fig. 1: Establishment of a flow-cytometry based method to assess chimerism exploiting RNA hybridization on KDM5D.

a Expression of KDM5D assessed by ddPCR in peripheral blood mononuclear cells (PBMCs) from male and female healthy donors (n = 5 and 3 respectively) confirms the presence of KDM5D mRNA only in male samples. b Scheme with mRNA KDM5D structure showing the regions where the two probes hybridize. c Representative FACS plot showing validation of KDM5D dual probe system on healthy donors. Mixing of samples was performed on white blood cell (WBC) counts with serial dilution of male sample with female cells. n = 4 independent experiments from a total of eight healthy donors. d Percentage of male chimerism from female, male and male:female mixed samples expressed as percentage on total CD45 + RPL13A+ live cells. e Mean fluorescence intensity (MFI) of KDM5D Alexa647+ cells on total CD45+ cells and in main immune cell subsets from male healthy donor (n = 4). f Graph showing technical reproducibility of three independent 1:1 mixes of the same male and female samples and of the same peripheral blood sample of a transplanted patient with mixed chimerism. Coefficient of variation was calculated for the 1:1 mix (0.051) and for the analyzed patient (0.024).