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Prion Proteins Detected With High Sensitivity, Specificity in Blood Samples

NEW YORK (GenomeWeb) – Prions can be detected in blood samples with high sensitivity and specificity, and even before the onset of symptoms, according to new research.

Some 231 people, mostly in the UK and France, have been infected with variant Creutzfeldt-Jakob disease (vCJD). The condition is mostly linked to having eaten beef with bovine spongiform encephalopathy, but four UK cases have been traced to blood transfusions from disease carriers. Recent studies have estimated that as many as 1 in 2,000 people in the UK and 1 in 20,000 in France could be asymptomatic carriers of the disease. But, there is currently no test to diagnose the disease or screen for it in the blood supply.

In a pair of papers appearing today in Science Translational Medicine, research groups from INSERM and the University of Texas Houston Medical School reported using protein misfolding cyclic amplification-based techniques to identify vCJD in blood samples. Both groups reported being able to spot misfolded prion proteins at low concentrations and with high sensitivity and specificity. The INSERM group further said it could detect prions within the blood of asymptomatic individuals.

"In the future, with automation of the test, it could help to ensure the safety of the blood-derived products and further measure the prevalence of asymptomatic carriers in the general population," said INSERM's Daisy Bougard, the first author of one of the papers, in a statement.

Protein misfolding cyclic amplification (PCMA), which was described by UT's Claudio De Soto and his colleagues in 2001, is akin to PCR. In a series of cycles, naturally occurring prion proteins (PrPC) in the blood are converted by any misfolded ones (PrPSc) that are present to that altered form. Fragmentation of any protein aggregates that develop then enables further amplification. The PrPSc signal is detected by western blot. However, there are inhibitors in blood components that halt the conversion process.l

In their new study, Soto, who is also at Universidad de los Andes, and his colleagues added a washing step to remove those inhibitors. Similarly, INSERM's Joliette Coste and her colleagues added a plasminogen bead-capture step to isolate prion proteins from the PMCA inhibitors in their study.

Both approaches appeared to limit the inhibitors to levels that allowed the PMCA reaction to occur.

Soto and his colleagues reported that they could detect PrPSc in blood samples from vCJD patients with 100 percent sensitivity and 100 percent specificity. In particular, they analyzed blood samples obtained from 14 people with vCJD and 153 controls, including healthy people and individuals with sporadic Creutzfeldt-Jakob disease (sCJD) or other neurological disorders.

None of the controls, even those with sCJD, were positive for PrPSc, even after multiple amplification cycles, the researchers reported. Only people with vCJD were positive for PrPSc.

Coste and her colleagues likewise reported 100 percent sensitivity. In a blinded fashion, they analyzed 152 plasma samples from French and British patients, including patients with vCJD, sCJD, or other neurological conditions. They also analyzed samples from 104 healthy blood donors.

Again, their assay correctly identified all 18 vCJD samples. They reported that one sCJD sample tested positive for the variant, giving them a 99.2 percent specificity.

Soto and his colleagues also explored whether they could reduce the amount of blood needed for their assay. A smaller volume of blood, they reasoned, would also limit the amount of PMCA inhibitors present and could allow them to skip the washing step, which they noted was both time consuming and labor intensive. They reported that they could still detect PrPSc within samples a few microliters in size.

Coste and her colleagues also examined whether their approach could detect PrPSc within samples from people not yet exhibiting vCJD symptoms. They assayed samples from two people who developed clinical vCJD who had been regular blood donors — samples from all French blood donors are saved for five years.

Coste and her colleagues found that they could detect PrPSc 31 months before the onset of clinical symptoms in one patient and 16 months before symptom onset in the other, and in all subsequent samples from both. This, they wrote, is "a key step toward the validation of this PMCA technology as a blood-based diagnostic test for vCJD and support its potential for detecting pre-symptomatic patients."

Soto and his colleagues cautioned that both studies were small, with 34 total vCJD blood samples between them, and that future double-blinded studies are needed to validate the assay.

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