Laboratory safety

Do nucleic acid extracts carry prion seeding activity?

Until recently this was assumed rather than tested. Gresch and colleagues tested it in two laboratories independently, the University of Minnesota Center for Prion Research and Outreach and the Canadian Food Inspection Agency, using filter-based and magnetic bead-based commercial DNA extraction kits on tissues from CWD-positive and negative white-tailed deer and on prion-infected and control Syrian hamster brains. Seeding activity in the DNA eluates and in the source tissue was measured by RT-QuIC.

The Minnesota laboratory found substantial to almost perfect agreement between seeding activity in DNA eluates from both extraction methods and seeding activity in the source tissue homogenate. The Canadian laboratory optimised RT-QuIC to a 30-hour run and reached 74 percent sensitivity and 94 percent specificity across 88 archived deer DNA samples. Both concluded that commercial kits do not eliminate PrPSc, which carries over into the eluate. Their recommendation is that, until infectivity is resolved by animal bioassay, DNA from PrPSc-positive tissue should be handled under biosafety protocols appropriate to the originating prion disease, with matching decontamination and containment.

The limitation is stated in that recommendation. Seeding activity in an eluate is not demonstrated infectivity, and no bioassay result is reported. The practical consequence is that archived DNA collections derived from prion-positive animals or patients, including genotyping archives held for other purposes, sit outside the containment assumptions under which they were created.

What containment do human prion assays need?

The Montpellier group’s cerebrospinal fluid assay for variant CJD is the clearest published statement of requirement. Their 2018 report describes discriminating a heterozygous methionine/valine variant CJD case from sporadic CJD cases using an ultrasensitive protein misfolding cyclic amplification assay on cerebrospinal fluid. The method as published runs untreated CSF against brain substrate from humanised transgenic mice over many cycles of incubation and sonication, and the authors state that it requires a biosafety level 3 laboratory and highly experienced personnel, and make no claim to a validated clinical test.

Amplification is the aggravating factor. An assay that multiplies seeding activity by design produces, by the end of a run, material with more seeding activity than the clinical sample that went in. Containment planning for these assays has to account for the product as well as the input, and Ferrer’s 2025 review makes the same point for tissue handling: because positive PMCA products and PrPSc are present in several tissues in CJD, special care and biosafety conditions must be applied when managing and processing human samples from suspected prion disease. That review also states that the seeding behaviour of RT-QuIC products needs further experimental study.

Can fixation reduce the handling burden?

Formalin fixation does not inactivate prions, but it changes what can be done with a sample and what waste it generates. Nicholson and colleagues asked whether aqueous extraction methods previously used to recover PrPSc from formalin-fixed paraffin-embedded tissue for western blot and ELISA also work for RT-QuIC. They report complete agreement between known TSE status and RT-QuIC results seeded with aqueous extracts of fixed embedded tissue, and note that the method avoids organic solvents, which would otherwise create mixed chemical and biological waste for disposal.

For laboratories that hold fixed archives, this widens the diagnostic options without opening frozen material. It does not reduce the containment class of the work.

What should a laboratory take from this?

The sequence worth checking in a local risk assessment is where prion-positive material leaves the recognised prion workflow: into a genotyping run, into a histology archive, into a shared automated extractor, or into a waste stream validated against conventional pathogens. The published evidence above shows seeding activity following material into the first of those, and no bioassay yet tells anyone how much infectivity travels with it.

Related: biosafety, tissue and organs, food chain and the regulations atlas.