fig2

Figure 2. Efficiency and precision of NDEV isolation. (A) NDEV isolation was performed on unprocessed plasma samples (Round 1, N = 4, aliquoted from the same plasma pool). After the first round of ExoSORT, supernatants were retained and subjected to an additional round of capture with fresh ExoSORT beads (Rounds 2 and 3, see Supplementary Figure 3). NRGN mRNA was measured by TaqMan QPCR. Ct values are shown. (B) Tau-rich EVs generated by iPSC-derived neurons were spiked into plasma aliquots. The recovery of spiked NDEVs in three consecutive rounds of ExoSORT was measured as above, and residual NDEV amounts after each round were estimated. Fold change differences between initial plasma pools (Round 1) and the two consecutive rounds are shown (Rounds 2 and 3). The signals in Rounds 2 and 3 were 8.7 ± 5.5% and 0.02 ± 0.01% in Round 1, respectively. (C) EVs isolated from a culture of human iPSC-differentiated cortical neurons were spiked into 300 ml plasma aliquots in 4 different concentrations based on known amounts of Tau. EV recovery was calculated by the following formula: $$ \text { Recovery }=\frac{\text { Tau concentration in spiked sample-Tau concentration in non-spiked sample }}{\text { Tau input }} $$ Tau recovery after ExoSORT remained uniform for a range of spiked-in EV-associated Tau amounts and was much higher than with IgG control (P = 0.026, two-way ANOVA). (D) Identical EV amounts from a culture of human iPSC-differentiated cortical neurons were spiked into plasma samples from 11 healthy individuals and six dementia patients. The variability of recovery was < 13% for all samples, with no difference between groups (P = 0.6). (E-G) Five different ExoSORT procedures were performed on different days, using aliquots derived from the same two plasma samples (QC1 and QC2, respectively). The levels of p181-Tau, total-tau, and Aβ42 were measured in the same assay (Luminex-based Milliplex), yielding variability estimates of 20.3%, 22.7%, and 14.9% for p181-Tau, Tau, and Aβ42, respectively. (H) ExoSORT was performed in triplicate technical replicates by two operators on plasma from 4 distinct donors, and the results were compared using Tau levels as an output. We note similar variations between donors and similar levels determined by different operators for each donor (P = 0.83).