An optimized protocol for PrPSc detection in CSF and tear fluid

Introduction:

Prion diseases are fatal neurodegenerative disorders caused by PrPSc accumulation. While CSF RT-QuIC has advanced diagnostics, less invasive biomarkers are needed. Optimized RT-QuIC with FL Hu E200K enables highly sensitive, non-invasive PrPSc detection in TF, improving early diagnosis and monitoring.

Objectives:

To optimize and validate an RT-QuIC protocol using novel recombinant PrP substrates, specifically FL Hu E200K, for sensitive detection of PrPSc in CSF and TF from patients with sporadic and genetic prion diseases, as well as healthy PRNP mutation carriers (HMC).

Methods:

We systematically compared seeding efficiencies of chimeric hamster-sheep, full-length human, and FL Hu E200K recombinant PrP substrates in RT-QuIC assays using CSF and TF samples from prion disease patients, HMC, and controls. Analytical stability and pre-analytical conditions for TF were evaluated, and diagnostic performance was assessed in validation cohorts.

Results/Discussion:

The FL Hu E200K substrate significantly increased RT-QuIC sensitivity for sCJD (92%) and FFI (75%) in CSF, outperforming conventional substrates. In TF, diagnostic sensitivity reached 84% for sCJD and 78% for genetic prion diseases, with abnormal seeding detected in 67% of HMC. Only 1/184 non-prion controls showed a positive TF result. Prion seeding activity in TF remained stable for at least five days at room temperature. Robust signals correlated with later disease stages, supporting the assay’s potential for disease monitoring.

Conclusion:

The optimized RT-QuIC protocol employing FL Hu E200K substrate enables highly sensitive, non-invasive detection of PrPSc in TF. This represents a substantial advance for early diagnosis, surveillance, and therapeutic trial monitoring in prion diseases.