Lumipulse

CSF was obtained by lumbar puncture and collected in polypropylene tubes following international recommendations [17] (link). Samples were processed (centrifuged 2000 g at 4°C, during 10 min) and aliquoted into polypropylene tubes within the first two hours after lumbar puncture. Aliquots were then stored at -80°C until analysis. CSF levels of core AD biomarkers (Aβ1-42, Aβ1-40, tTau, and pTau181) were measured in the Lumipulse fully-automated platform using commercially available kits (Fujirebio Europe, Ghent, Belgium), as previously described and according to the provider's instructions [18, (link)19] (link).
Following the AT(N) classification system [20] (link), we used previously validated cutoffs [19] (link) for CSF Aβ1-42/Aβ1-40 as a marker of β amyloid deposition (A+ < 0.062), and CSF pTau181 (T+ > 63pg/ml) as a marker of Tau pathology, and we classified all participants as A+T+, A+T-, A-T+ or A-T-.
We used NfL in CSF as a marker of the (N) category. Levels were measured using a commercially available ELISA kit (NFlight, UmanDiagnostics, Umeå, Sweden,) as previously described [16, (link)21, (link)22] (link).

Platelet tau was characterized using our novel WAN-GEL (Western Blot-native Agarose gel elution) model developed by us recently (Humpel, 2022 submitted) . Native Western Blots were performed as described above but directly after the gel run, different molecular weight bands were dissected with a scalpel with a size between 80 and 0 kDa related to the color molecular weight marker. For the elution, 2.5 mL sample vials (PE, 14 mm, Roth 5863.1) were used and 1 mL 4% agarose was pipetted at the bottom of the tubes and cooled. Then, the cut Western Blot bands were carefully placed on top of the hardened agarose. Next 1 mL 1% handwarm agarose was pipetted onto the extracted bands and cooled to harden. In the next step, the bottom of the vial was cut, and the tube closed with a Millicell culture insert (3 µm, 12 mm, PITP01250, Merck). Next 1 mL of a MES/histidine puffer was carefully pipetted into the tube using a syringe and 10 µL bromphenobuffer was pipetted to follow up the elution. These tubes were placed into the agarose gel chamber and proteins eluted for 20-60 min to the cathode. After the run, the buffer with the eluted proteins was collected and analyzed by lumipulse assay or mass spectometry. Total tau levels were measured using automated lumipulse technology (Fujirebio G600II); see https://www.fujirebio.com/en/productssolutions/lumipulse-g600ii.