Quantification and diagnostic relevance of blood and heme-mediated inhibition of prion detection by RT-QuIC

Authors: PIEL, ROBERT - Washington State University; Schneider, David

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/15/2025
Publication Date: 12/18/2025
Citation: Piel, R.B., Schneider, D.A. 2025. Quantification and diagnostic relevance of blood and heme-mediated inhibition of prion detection by RT-QuIC. Journal of Clinical Microbiology. 64(1). Article e00615-25. https://doi.org/10.1128/jcm.00615-25.
DOI: https://doi.org/10.1128/jcm.00615-25

Interpretive Summary: Prion diseases in sheep, goats, elk, and deer are fatal, infectious neurodegenerative diseases. Sensitive detection of infection is important for disease management efforts because these diseases have long pre-clinical incubation periods. Prions can be found in multiple tissues of infected animals. Blood is an inherent part of any tissue sample and is a known inhibitor of certain assays that otherwise produce ultrasensitive detection of prions in tissues. These inhibitory effects on the Real-Time Quaking-Induced Conversion assay were studied and quantified. Whole blood and its major components, hemoglobin and heme, significantly inhibit the sensitivity of this assay at concentrations found in some tissues. Detection was inhibited at the level of the assay mechanism, rather than through destruction of the prions in the blood-containing tissue. Thus, blood-contaminated samples may still be useful for sensitive detection by developing a process that separates prions from blood contaminants.

Technical Abstract: Prion diseases are characterized by misfolding of the prion protein (PrP) from the correctly folded, cellular form (PrP-C) to a disease-associated form (PrP-D). The disease form propagates, ultimately forming pathogenic aggregates, by acting as a template for additional misfolding of PrP-C to PrP-D. Ultrasensitive amplification assays, including the real-time quaking-induced conversion (RT-QuIC) assay, exploit this mechanism for prion detection by “seeding” reaction mixtures, which contain PrP-C substrate, with samples suspected to contain prions. Seeded conversion of the PrP-C assay substrate results in PrP-D concentrations being amplified to readily detectable amounts. RT-QuIC is known to be sensitive to inhibition from high concentrations of tissue homogenate constituents, including blood. Heme, an iron-containing porphyrin cofactor of hemoglobin (Hb), has been shown to bind to PrP in an isoform-specific manner and has also been demonstrated to affect the stability of other pathogenic biological amyloids such as those associated with Alzheimer’s disease, Parkinson’s disease, and lysozyme amyloidosis. In this study, tissue samples from scrapie-positive sheep were tested by RT-QuIC in the presence of various concentrations of whole blood, Hb, apoHb, and heme. The inhibitory contributions of hemin, apoHb, and other whole blood components were each found to be significant and to be additive. We also demonstrate that blood-mediated inhibition of RT-QuIC acts through disruption of rPrP-C in the assay substrate rather than destruction of the PrP-D seeds present in test samples. Lastly, heme concentrations were measured in panels of potential diagnostically relevant tissue types. Heme levels were found to exceed inhibitory thresholds in nearly all types of intact tissue, but to mostly fall below inhibitory levels by the commonly applied 1:1000 sample dilution, with whole blood being one of a few notable exceptions. Our results suggest that recovery of PrP-D 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 which may impact detection sensitivity.