1. Figure S1: kinetics of incubation time and temperature MFI Incubation time (min) B Room temperature +4°C D Room temperature +4°C Incubation time (min) S/N ratio A Incubation time (min) S/N ratio GranulocytesLymphocytesMonocytes C S/N ratio +4°C Room Temperature

2. Figure S2: optimal FLAER concentration MFI FLAER concentration (nM) C MFI FLAER concentration (nM) B D % of singlets FLAER concentration (nM) A S/N ratio FLAER concentration (nM)

3. Figure S3: optimal cell amount A % of fluorescence relative to maximum fluorescence 10 4 10 5 10 6 10 7 number of cells non-PNH B % of fluorescence relative to maximum fluorescence 10 4 10 5 10 6 10 7 number of cells PNH C S/N ratio 10 4 10 5 10 6 10 7 number of cells PNH

4. FLAER labeling on Erythrocytes A MFI type I = 18 MFI type III = 14 MFI type I = 20 MFI type III = 15 CD59 FLAER CTRL Figure S4: Impact of the pre-analytical steps on the FLAER staining E GranulocytesMonocytes Lymphocytes +FCS 1%20%1%20%1%20% % of fluorescence intensity relative to fluorescence without FCS F GranulocytesMonocytes Fold decrease of cellular background 9.9 4.3 C FLAER MFI Proportion of red blood cells added to the mix Lyse before stain Whole blood staining FLAER MFI D Proportion of platelets added to the mix Lyse before stain Whole blood staining FLAER labeling on Platelets B CD41a MFI=42MFI=82 CTRLFLAER

5. Figure S5: Impact of the pre-analytical steps on the FLAER staining A S/N ratio volume of whole blood (µl) GranulocytesLymphocytesMonocytes B S/N ratio FLAER concentration (nM) GranulocytesErythrocytes