660 Introduction Transmissible spongiform encephalopathies (TSEs) or prion diseases are invariably fatal neurodegenerative dis- orders affecting humans and other mammals such as sheep, elk, and cattle [36]. These diseases are characterized clin- ically by a rapidly progressive neurological dysfunction and neuropathologically by spongiform degeneration, neuronal loss, gliosis, and sometimes by amyloid plaques, which combine in varying degrees of severity and regional distribution. Biochemically, prion diseases are characterized by the conversion of a constitutively expressed cellular protein, the prion protein (PrPC) into an abnormally folded, insol- uble, partially protease-resistant isoform (PrPSc) [4, 8, 27]. Incubation of a brain homogenate from prion-infected individuals with proteinase K (PK) under conditions lead- ing to a complete degradation of PrPC generates an N- terminal truncated form of PrPSc, commonly referred to as PrP27-30, an established molecular marker for the disease [5, 38]. Sporadic Creutzfeldt–Jakob disease (sCJD), the com- monest form of the human prion diseases, comprises a broad spectrum of clinico-pathological variants [30]. Prion strains are believed to be the main cause of TSE pheno- typic diversity. Prion strains are deﬁned as isolates that show distinct disease phenotypes upon transmission to syngenic animals, persisting on serial transmission [6, 15, 34]. In addition, the host genotype variability in the gene encoding PrPC (PRNP), as determined by polymorphisms and mutations, is another recognized causal factor for phenotypic heterogeneity [2, 12]. The hypothesis that PrPSc itself may encode strain-speciﬁc properties has gained support over the years by the demonstration of distinct physico-chemical subtypes of PrPSc that include differences in the level of glycosylation and in the size of proteinase K-digested PrPSc fragments, possibly reﬂecting distinct protein conformations [1, 3, 9, 10, 14, 22, 28, 29, 32, 39, 41, 42]. Although the ﬁnal proof that conforma- tional variants of PrPSc represent the biological basis of prion strains is lacking, the described physicochemical differences among PrPSc types are increasingly used as surrogate markers of strain properties in a given host. Based on differences in gel mobility and N-terminal sequence of the core fragments (i.e. PrP27-30) generated by PK digestion, Parchi et al. [28, 29, 32] originally identiﬁed two major human PrPSc types: type 1 with a relative molecular mass of 21 kDa and the primary cleavage site at residue 82, and type 2 with relative molecular mass of 19 kDa and the primary cleavage site at residue 97. The two PrPSc types, in conjunction with the codon 129 genotype, signiﬁcantly explained sCJD 123 Acta Neuropathol (2009) 118:659–671 phenotypic variability and provided a molecular basis for disease classiﬁcation [7, 28, 30]. Six subtypes of sCJD with distinctive phenotypic features were identiﬁed, each resulting from a speciﬁc codon 129 genotype/PrPSc type combination with two exceptions. MM1 and MV1 cases were phenotypically indistinguishable and, therefore, merged in one subtype (MM/MV 1), whereas, on the contrary, MM2 subjects were associated with 2 subtypes with distinctive histopathological features in the cerebral cortex and in the thalamus and designated accordingly (MM2-cortical or MM 2C and MM2-thalamic or MM 2T) [30]. By showing that at least some of these sCJD sub- types correspond to speciﬁc agent strain-host genotype combinations, preliminary transmission studies seem to conﬁrm the biological basis of this classiﬁcation [16, 17, 23, 42]. An important aspect of sCJD phenotypic variability, which has not yet been investigated thoroughly, concerns the existence of cases presenting more than one PrPSc type and mixed histopathological features. This issue still represents a potential drawback for the widespread appli- cation of the current disease classiﬁcation for diagnostic and epidemiologic purposes. Since the original report of this phenomenon [30], based on the analyses of a few samples for each brain, the co-occurrence of PrPSc type 1 and 2 in a percentage varying between 12 and 44% of sCJD cases analyzed has been conﬁrmed by a number of subsequent studies [13, 18, 20, 37, 40, 43]. In many of them, the question of how far the proportion of cases with concurrent PrPSc types is a function of the sampling pro- tocol employed was raised. More recently, two studies using novel antibodies recognizing an epitope between residues 82 and 96 (i.e. not detecting type 2) found that all type 2 samples show at least some associated protein migrating in the 21 kDa molecular weight range and concluded that PrPSc type 1 coexists in all type 2 sCJD cases and that the number of cases with concurrent PrPSc types is likely larger than previously thought [35, 45]. It was later shown, however, that the ‘‘type 1 selective’’ antibodies fail to discriminate properly between a bona ﬁde PK-resistant PrPSc core and the partially cleaved fragments generated during digestion due to the several PK cleavage sites included in the PrPSc N-terminus, making difﬁcult the interpretation of the results obtained [25]. To explore the issue of concurrent PrPSc types in sCJD in depth, we carried out a systematic regional study in a large series of cases. To this aim, we applied a reﬁned western blot assay with increased sensitivity for the detection of PrPSc types coexistence [25] and correlated the molecular data with those obtained by clinical and patho- logical examinations.