Background: Variant of Creutzfeldt-Jakob disease (vCJD), the human counterpart of BSE, is a neurodegenerative infectious disorder, characterised by the prominent accumulation of the prion protein pathological isoform (PrPTSE) in the brain and lymphoid tissues. This distribution raised concern about potential inter-individual transmission of PrPTSE by blood transfusion. Since the publication in the UK of 4 apparent cases following red blood cell transfusions and 1 apparent case following factor VIII injection, the presence of vCJD infectivity in the blood cannot be excluded. The estimated femtomolar sensitivity level required to detect PrPTSE in the plasma of a donor in the pre-clinical phase of infection has been a caveat towards the development of blood screening assays. Objectives: To develop a sensitive and specific test that enables the confirmation of a PrPTSE positive blood sample. Methods: The assay comprises 3 steps: 1) selective capture of PrP molecules by ligand-coated beads; 2) in vitro amplification of PrPtse by Protein Misfolding Cyclic Amplification (PMCA), in which minute amounts of the captured PrPtse are amplified in a cyclic process using brain of transgenic mice as substrate; 3) detection by Western Blot of the amplified PrPtse after PK digestion. Optimisation of the assay was performed with plasma samples spiked with serial ten-fold dilutions of 10% vCJD infected brain homogenate. Results: After capture of PrPtse spiked in plasma, and 3 rounds of PMCA, a 5log increase in sensitivity was obtained in comparison with western blot detection only. Discussion: We have developed a test that combines a concentration step and in vitro amplification of PrPTSE, which reaches the sensitivity required for the detection in blood. The future prospects are: 1) to realise epidemiological studies on at risk population samples; 2) to analyse any repeatedly positive result that would obtained after screening of blood donations for confirmation.