Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrP) associated with variant Creutzfeldt-Jakob disease

Transfusion. 2009 Feb;49(2):376-84. doi: 10.1111/j.1537-2995.2008.01954.x. Epub 2008 Oct 29.

Abstract

Background: Four recent cases of transfusion-related transmission of variant Creutzfeldt-Jakob disease (vCJD) highlight the need to develop a highly sensitive and specific screening test to detect infectivity in the blood of asymptomatic infected individuals. Protein misfolding cyclic amplification (PMCA), a method for the amplification of minute amounts of disease-associated abnormal prion protein (PrP(Sc)) to readily detectable levels, could be incorporated into such a test provided that a suitable substrate source for routine use in human PMCA reactions can be found.

Study design and methods: With the use of seed sources from individuals with variant and sporadic CJD, the use of human platelets (PLTs) as a PMCA substrate source was evaluated. The effects of seed/substrate prion protein gene (PRNP) codon 129 genotype compatibility on amplification efficiency and freeze-thaw on a substrate's ability to support amplification and the degree of amplification achieved by serial PMCA (sPMCA) were investigated.

Results: Seed/substrate PRNP codon 129 compatibility was found to have a major influence on PrP(Sc) amplification efficiency. Individual substrates, of the same PRNP codon 129 genotype, could be pooled and stored frozen for use in subsequent PMCA reactions. A consistent 10-fold increase in PrP(Sc) detection sensitivity was achieved after each round of sPMCA, resulting in a 10,000-fold increase in detection sensitivity after four rounds, with no evidence of de novo PrP(Sc) production detected in the unseeded PLT substrate.

Conclusions: Providing issues of seed/substrate PRNP codon 129 compatibility are taken into consideration human PLTs are a suitable, readily available, renewable substrate source for use in human PMCA applications.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Platelets / metabolism*
  • Brain Chemistry
  • Codon
  • Creutzfeldt-Jakob Syndrome / genetics
  • Creutzfeldt-Jakob Syndrome / metabolism*
  • Humans
  • Immunoassay / methods
  • Nucleic Acid Amplification Techniques
  • Polymorphism, Genetic
  • PrPSc Proteins / analysis
  • PrPSc Proteins / genetics*
  • Protein Conformation
  • Protein Folding
  • Sensitivity and Specificity
  • Substrate Specificity

Substances

  • Codon
  • PrPSc Proteins