Inter-laboratory assessment of PrPSc typing in creutzfeldt-jakob disease: a Western blot study within the NeuroPrion Consortium - PubMed (original) (raw)

Multicenter Study

doi: 10.1111/j.1750-3639.2008.00187.x. Epub 2008 Jul 2.

Silvio Notari, Petra Weber, Heinz Schimmel, Herbert Budka, Isidre Ferrer, Stéphane Haik, Jean-Jacques Hauw, Mark W Head, James W Ironside, Lucia Limido, Agustin Rodriguez, Thomas Ströbel, Fabrizio Tagliavini, Hans A Kretzschmar

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Multicenter Study

Inter-laboratory assessment of PrPSc typing in creutzfeldt-jakob disease: a Western blot study within the NeuroPrion Consortium

Piero Parchi et al. Brain Pathol. 2009 Jul.

Abstract

Molecular typing is of considerable importance for the surveillance and epidemiology of human transmissible spongiform encephalopathies (TSEs). It relies on the detection of distinct protease-resistant prion protein (PrP(Sc)) core fragments that differ in molecular mass and/or glycoform ratio. In this collaborative study, we tested the inter-laboratory agreement in TSE molecular typing. Sixteen characterized brain specimens from sporadic TSEs and variant Creutzfeldt-Jakob disease (vCJD) cases were distributed blindly to seven laboratories for molecular characterization by a defined protocol and classification. Agreement between laboratories in the classification of samples was excellent. In particular, there were no differences in the distinction between PrP(Sc) type 1, type 2A, and type 2B with one exception, which eventually was identified as a case with types 1 and 2 co-occurrence. This shows that the general technique and particular classification system used here are robust and represent a reliable basis for diagnostic and epidemiologic purposes. The subtle further distinction of subtypes among type 1 and type 2 groups requires high-sensitivity gel electrophoresis protocols that are unsuitable for routine diagnostic needs and must be reserved for research investigations. Further research is necessary on the identification and significance of co-occurrence of PrP(Sc) types 1 and 2 within one brain.

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Figures

Figure 1

Figure 1

Immunoblot analysis of PrPSc extracted from frontal cortex homogenates of blindly coded human TSE brain samples. All samples were treated according to given instructions (see Materials and Methods) and were separated in a conventional gel electrophoresis system [12% Tris–glycine sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with 6.5‐cm separation distance]. Panel A shows an example of the first analyses, while panel B illustrate an example of the second analyses, during which all cases identified as having the same PrPSc type were run together.

Figure 2

Figure 2

Immunoblot analysis of PrPSc extracted from frontal cortex homogenates of blindly coded human TSE brain samples. All samples were treated according to given instructions (see Materials and Methods) and were separated in a high‐resolution gel electrophoresis system [15% Tris–gylcine sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with 15‐cm separation distance]. The samples, previously analyzed in a conventional gel electrophoresis system (see Figure 1), were diluted or concentrated to even the signal intensities of the samples and were loaded either onto gel 1 (A) or gel 2 (B) according to their migration pattern (ie, type 1 or type 2) determined in the conventional gel electrophoresis system.

Figure 3

Figure 3

Western blot analyses (laboratory 4) on case RV18 showing a co‐occurrence of PrPSc types 1 and 2. One PrPSc type 1 (RV17) and two PrPSc type 2 samples (RV22 and RV23) are included for comparison.

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