After decoding we had identified 60 to 70% of the positive samples with no false positive samples. Once the infection status of all samples was known we found that a rolling ball correction with a radius of 2 pixel and cut-off of 500 intensity units could discriminate 100% of the positive samples from the negative samples. By using a 2 pixel background selection, only particles with a radius smaller than 2 pixel, i.e. 660 nm were counted as PrP aggregates. By specifying a high intensity cut-off of 500 fluorescence units only bright particles were counted. We cannot exclude the existence of larger PrP aggregates in blood, but they could not be differentiated from the background. The best correlation between disease and PrP aggregates in Publications Using Abomle PDTC plasma was clearly achieved by filtering small and bright aggregates. At least two factors contribute to the size Publications Using Abomle R428 distribution of the PrP aggregates: the genuine in vivo size distribution and the break-down of larger aggregates by the ultrasonication step during the preparation from plasma. We estimate a particle size for the aggregates selected for discrimination in Figure 5B of about a micrometer, after subtracting 50 to 100 nm for the size of the fluorescent antibody label. Particle sizes in the literature were derived from nanofiltration studies investigating the removal of infectivity from blood or plasma. Summarizing the studies applying different filters, preparation procedures and solution conditions lower limits of the particle sizes between 15 and 200 nm were estimated.Thus the lower limit found in filtration experiments and the upper limit reported here do not contradict each other. Sonication can affect PrP aggregate size, but does not break down the aggregates below the size of infectious particles. A rough estimation of the number of PrP molecules in PrP aggregates as observed in plasma leads to 105 to 106 molecules, which corresponds to an earlier estimation of PrP molecules per infectious unit in brain. While our estimates of particle size are derived from natural PrP aggregates in plasma, it must be pointed out that all of the precedent filtration studies on plasma have, by necessity, employed brain or spleen derived tissue homogenates as a source of PrPSc and infectivity. This is because there has not been an assay sensitive enough to detect diseasespecific PrP in blood. This is the first direct measurement of the size of endogenous disease-specific PrP aggregates in plasma. It should be noted that particular, detergent-including preparation of infectious particles from brain led to determination of smaller infectious units.