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

## TL;DR

This study examines how blood and heme interfere with prion detection using the RT-QuIC assay and identifies ways to mitigate this interference.

## Contribution

The study quantifies and explains the inhibitory effects of heme and blood on prion detection and identifies dilution thresholds for reliable results.

## Key findings

- Heme in blood disrupts the RT-QuIC assay by affecting the recombinant PrP substrate, not the prion seeds.
- Whole blood causes the most inhibition, while free heme causes the least at equivalent concentrations.
- Heme levels in most tissues drop below inhibitory thresholds at a 1:1,000 dilution, except for whole blood.

## 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
recombinant PrP, with samples suspected to contain prions, resulting in
amplification of misfolded PrP. 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. Herein, 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
PrP 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 1:1,000 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.

Real-time quaking-induced conversion (RT-QuIC) is an ultrasensitive
amplification assay for the detection of prions. The assay has shown
exceptional performance in optimal laboratory conditions, on par with
bioassay, and far surpassing current immunoassay diagnostics. However,
efforts to apply RT-QuIC as a real-world diagnostic have been hampered by
inconsistencies and unexpectedly low sensitivity in some field samples. This
study aims to quantify and characterize the mechanism of inhibition from
blood and its constituent parts, hemoglobin and heme—omnipresent
components of most sample types. Such systematic evaluations of RT-QuIC
inhibitory factors represent necessary steps toward the consistent and
sensitive performance necessary for a field-applicable diagnostic assay.