P1‐250: rapid and ultra‐sensitive detection of α‐synuclein seeds in brain and cerebrospinal fluid by α‐syn rt‐quic: an aid to diagnosis for pd and dlb

Disease-associated forms of α-Synuclein (αSynD) may contribute to Parkinson's Disease, DLB and related disorders. Two recent αSynD seed amplification tests demonstrated ultrasensitive and specific detection of aSynD in CSF. We have developed a-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay with high sensitivity and specificity, but much faster than prior assays (1-2 days vs 5-13 days). Lumbar CSF was obtained under IRB-approved protocols from well-characterized subjects with PD (n=12, including 8 de novo PD), DLB (n=17), AD (n=16), Tauopathy (n=3), elderly controls (n=12). Over 50% of the AD and DLB cases and all 3 tauopathies had autopsy confirmation. Brain tissue from controls, PD, DLB, AD and tauopathy was used as + and - controls. For RT-QuIC reactions, brain homogenates or CSF were added to microplate wells in quadruplicate. Wells contained 98 μL of reaction mix: phosphate buffer (pH 8.0), NaCl, 0.1 mg/ml recombinant αSyn with the K29Q mutation (rαSYN), 10 μM thioflavin T (ThT). Plates were sealed and incubated at 42°C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking and 1 min rest. ThT fluorescence was measured every 45 min. Average ThT fluorescence was calculated over the first 10 hours for each sample and a threshold determined as mean + 3 S.D.s; a positive reaction occurred when sample fluorescence exceeded this threshold. PD and DLB brain tissue yielded positive reactions, while AD and tauopathy were negative. End-point dilution analyses allowed quantitation of relative amounts of αSynD seeding activity in CSF. Positive ThT fluorescence was detectable in 24-48 hours. Blinded analysis of the clinical samples yielded 93% diagnostic sensitivity and 100% specificity for synucleinopathy diagnoses. We estimated that femtogram/μL levels of seeding capable αSynD may exist in the CSF of PD or DLB patients.