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bioRxiv · 10.1101/2025.04.21.649906

Quantification and Diagnostic Relevance of Blood and Heme-Mediated Inhibition of Prion Detection by RT-QuIC

Piel, R. B.·Schneider, D. A.
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Abstract

Prion diseases are characterized by misfolding of prion protein (PrP) from correctly folded PrPC to a disease-associated form, PrPD. Real-time quaking-induced conversion (RT-QuIC), detects prions by "seeding" reaction mixtures, which contain PrPC, with samples suspected to contain prions, resulting in PrPD amplification. The assay is sensitive to inhibition by tissue constituents, including blood. Heme, a cofactor of hemoglobin (Hb), has been shown to bind PrP in an isoform-specific manner and to affect the stability of other pathogenic amyloids. In the present study, tissue samples from scrapie-positive sheep were used to seed RT-QuIC reactions in the presence of heme--as free hemin, as a cofactor of Hb, and as present in whole blood. At equivalent heme concentrations, the inhibitory action of free heme was the least and that of blood the greatest, suggesting other components of Hb and whole blood have additional inhibitory actions. We also demonstrate that this inhibition of RT-QuIC acts through disruption of the recombinant PrPC assay substrate, rather than destruction of PrPD seeds. Lastly, heme concentrations were measured in several ruminant tissues. Heme levels exceeded inhibitory thresholds in nearly all types of intact tissue but were reduced below inhibitory levels at a 1:1000 dilution of most tissue types, with whole blood being one of a few notable exceptions. Our results suggest that detection of PrPD seeding activity is not precluded by exposure to heme in tissue samples, but that the final heme concentration introduced into the RT-QuIC assay mixture is the critical factor that impacts detection sensitivity.

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Piel, R. B., Schneider, D. A.. 2025-04-22. Quantification and Diagnostic Relevance of Blood and Heme-Mediated Inhibition of Prion Detection by RT-QuIC. https://doi.org/10.1101/2025.04.21.649906

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