This disclosure relates to the field of research and clinical usage of extracellular vesicles. More in particular, this disclosure relates to usages of recombinant extracellular vesicles comprising a) a self-assembling protein that directs its own release through vesicles as a luminal membrane-bound protein and b) a heterologous marker. Indeed, the disclosure provides recombinant extracellular vesicles that can be used as a biological reference material in methods to quantify extracellular vesicles in a sample, and/or, that can be used to calibrate a device for sample extracellular vesicles analysis, and/or, that can be used to evaluate the isolation process of extracellular vesicles. This disclosure thus relates to improving the accuracy of an extracellular vesicle-based diagnosis.