Changes in exosome/microvesicle concentrations associated with the onset of disease can also be assessed. In addition, the technology offers the ability to discriminate target exosomes/microvesicles associated with a specific disease and cellular origin from the host of naturally occurring exosomes/microvesicles that are present due to the naturally occurring physiological processes within the body.
The ability to work in fluorescence mode potentially bypasses the requirement to purify clinical samples (by ultracentrifugation/size exclusion chromatography/chromatography, etc.) and avoids the potential difficulties and variables presented by the method of purification itself.
The suitability of FNTA to study syncytiotrophoblast microvesicles (STBMs) associated with preeclampsia was recently evaluated. STBMs are present in normal pregnancy but found in significantly elevated levels in preeclampsia. Researchers were able to prove the accuracy of FNTA first through particles of a known and controlled size, i.e., reference particles.
Accurate measurement of a simple mono-dispersed 400 nm bead (sized at 401 nm) was demonstrated, moving on to prove that in a bimodal population of 100 and 300 nm particles, accurate sizing could still be demonstrated in a sample that more closely mimics the natural spread of sizes that might be found in a microvesicle preparation.
Accuracy of particle counting was demonstrated (again using reference beads) in the range of 2x108 to 2x1010 particles per mL, with only a slight deviation in the measured concentration from the calculated concentration. The study also measured preparations of STBMs generated by a placental perfusion showing a bimodal distribution (peaks at 100 nm and 160 nm with a tail out to 500 nm) postulated to be a mixed population of exosomes and microvesicles.
Researchers were able to fluorescently label this population using quantum dots antibody labelled to placental alkaline phosphatise (NDOG2), showing an almost identical match between the fluorescently labeled sample and the sample measured under light scatter. The distributions were subsequently confirmed by electron microscopy.
This research confirmed the ability of FNTA to both measure the size and size distribution of exosomes and microvesicles, measure their concentration, and when fluorescently labeled confirm the phenotype of the microvesicles measured, with the goal of linking the observations to a clinically relevant observation, in this case preeclampsia.