662 MM cases with either a dominant type 1 or a dominant type 2. Overall, we found that, compared to the latter, the ﬁrst approach is associated to a relatively minor overestimation of type 2 protein. In particular, the difference was almost negligible for the group of samples with dominant type 1 (69/31% on non-glycosylated isoforms vs. 72/28% after deglycosylation, n = 20), and slightly more evident in the group of samples with dominant type 2 (29/71% on non- glycosylated isoforms vs. 37/63% after deglycosylation, n = 30). To express quantitatively with a single number the rel- ative amount of type 1 and type 2 in a given case (total type 1 or type 2 ‘‘load’’) we calculated the mean of the relative amounts of either type 1 or type 2, expressed in % of total PrPSc signal, in the different brain regions examined. Molecular genetic analysis Genomic DNA from all subjects was used to amplify the coding region of PRNP in the polymerase chain reaction (PCR). PRNP open reading frame was ampliﬁed as previ- ously described [11]. The PCR product was visualized on a 1% agarose gel to detect potential insertion mutations or deletions. Potential point mutations were revealed by dHPLC analyses. Mutations were also ruled out by direct sequencing of PRNP open reading frame in about 50% of cases. Finally, the codon 129 genotype was examined by digestion with the restriction endonuclease Nsp 1. Neuropathology Semiquantitative evaluation of spongiosis and gliosis was carried out in all cases by comparing hematoxylin and eosin stained sections obtained from the same 21 brain regions evaluated biochemically. Spongiosis was rated on a 0–4 scale (not detectable, mild, moderate, severe, and status spongiosis), whereas gliosis was scored on a 0–3 scale (not detectable, mild, moderate, and severe). A lesion proﬁle for each patient was obtained by averaging the two scores. For PrP immunohistochemistry, parafﬁn sections from formalin-ﬁxed and formic acid treated blocks of the areas selected for neuropathologic examination were processed using the monoclonal antibodies 3F4, according to pub- lished protocols [30]. PrP deposits were described as diffuse or synaptic, perivacuolar, coarse or patchy and plaque-like. Clinical analysis Clinical data were available in most cases, and medical records always included at least one neurological exami- nation. Duration of symptoms was calculated from the time 123 Acta Neuropathol (2009) 118:659–671 of presentation of neurological signs suggesting an organic cause. Prodromal symptoms were not taken into account. Clinical signs were classiﬁed ‘‘at onset’’ when observed within the ﬁrst quarter of the mean duration of symptoms of the group to which the patient belonged (i.e. 1 month if the mean duration was 4). Results PrPSc detection and typing In the present study, we tried to maximize the detection sensitivity of PrPSc type co-occurrence in sCJD-affected brains. At variance with previous studies, we typed PrPSc after a detailed topographic analysis of 21 brain regions and using the method recently described by Notari et al. [25], which has shown increased sensitivity for the detec- tion of bona-ﬁde concurrence of PrPSc types 1 and 2. Furthermore, we probed selected immunoblots with 1E4, a recently characterized monoclonal antibody with a higher afﬁnity for type 2 than for type 1 [44]. The latter approach allowed us an increased protein loading in the gel and a better identiﬁcation of PrPSc type 2 when present in rela- tively low amounts (Fig. 1a) in the protein types mixture. The results obtained in the largely unselected series of 200 cases after using both 3F4 and 1E4 antibodies are shown in Table 1. The overall prevalence of PrPSc types 1 and 2 concurrence was 35% (32% when only the results with 3F4 were considered). The same incidence of mixed cases was also found in the two largest groups of subjects from Italy and USA when considered individually. Although the protein mixture was detected in each codon 129 genotype (Fig. 1b), PrPSc types 1 and 2 co-occurred more frequently in MM than in MV or VV subjects. A PrPSc proﬁle comprising two distinct bands also charac- terized the MV2 subtype with kuru plaques (MV 2K) in many brain regions, but the relative size of the associated band (i.e. 20 kDa) distinguished these proﬁles from bona- ﬁde types 1 and 2 concurrence (Fig. 1b), as previously shown [24, 25]. Regional variation and relative ‘‘load’’ of PrPSc Types 1 and 2 concurrence MM subjects The regional distribution of co-existing PrPSc types 1 and 2 in MM subjects reproduced features previously observed in the pure MM1 and MM2-cortical (MM 2C) sCJD subtypes [30], although quantitatively the relative proportion of type 1 and type 2 accumulation in these cases varied exten- sively. Most cases had PrPSc type 1 in all areas and PrPSc