Multicenter Assessment of Immunohistochemical Methods for Pathological Alpha-Synuclein in Sigmoid Colon of Autopsied Parkinson's Disease and Control Subjects

Abstract

Background: Conflicting results from studies of Lewy-type α-synucleinopathy (LTS) in colonic biopsies of subjects with Parkinson's disease (PD) prompted a two-part multicenter assessment. The first assessment, now published (Acta Neuropathol Commun 4 : 35, 2016), examined archived colonic biopsies and found that none of the tested methods was adequately sensitive or specific.

Objective: As the amount of nervous tissue in typical colonic biopsies may be insufficient, and the clinical diagnosis of PD not completely accurate, the objective of the current study was to use instead full-thickness sections of sigmoid colon from autopsy-proven PD and normal subjects.

Methods: Seven different immunohistochemical (IHC) methods were used, employing five different primary antibodies and four different combinations of epitope exposure and signal development protocols. Specific staining was defined as being restricted to morphological features consistent with neuronal elements. Stained slides from each subject were independently categorized as being positive or negative for LTS, and their density semi-quantitatively graded, by four raters blinded to diagnosis.

Results: Agreement and mean diagnostic performance varied markedly between raters. With the two most accurate raters, 5 methods achieved diagnostic accuracies of 70% or greater; one method had 100% accuracy and 100% inter-rater agreement. The submucosa had the highest prevalence of pathological LTS staining, followed by the muscularis and mucosa.

Conclusions: The major conclusion of this study is that, when sufficient submucosa and LTS is present, and when specific staining is defined as being consistent with neuronal morphology, adequately-trained raters may reliably distinguish PD colon from control using suitable IHC methods.

Keywords: Lewy body; biopsy; diagnosis; enteric nervous system; gastrointestinal tract; pathology.

Figure 1

Photomicrographs of the colon from autopsied PD subjects, stained immunohistochemically for phosphorylated α-synuclein. Panels (a–c) show fibers and dots within the lamina propria, with Method 3 (a) and Method 5 (b and c). Panel (d) shows non-specific staining of epithelial cells with Method 5. Panels (e–g) show fibers and dots within the submucosa, with Method 5 (e) and Method 6 (f and g). Panel (h) shows dots embedded within the vascular wall (graded with the “perivascular dots” template) of a blood vessel in the submucosa, photomicrograph from prior study [27]. Panels (i–l) show fibers and dots within the myenteric plexus, with Method 2 (i and j), Method 5 (k) and Method 6 (l). Specific staining is black, with a red counterstain (panels a, e, i and j), or brown, with a blue counterstain (remainder of panels).

Figure 2

Diagrammatic template (left series of panels) used for density grading of “dots and fibers” type of morphology. On the right series of panels are photomicrographs of sections stained with Method 2, showing examples of each density grade within the myenteric plexus of PD subjects Specific staining is black, with a red counterstain.

Figure 3

Low-magnification photomicrographs (a, e) of the full thickness of the colonic wall from two PD subjects, stained immunohistochemically for phosphorylated α-synuclein with Method 4 (a) and Method 5 (e); boxes show the location of higher magnification features of interest (small arrows in b, c, f, g and h), including a large fiber in the submucosa (b), probable nerve fascicles in the submucosa (c, f and g) and probable nerve fascicles in the intermyenteric plexus (d and h; d located just outside the area depicted in a). Low magnification photographs (i, m) of the colonic wall from a PD subject, double-stained for phosphorylated α-synuclein and neurofilament protein; boxes show the location of higher magnification features of interest in the submucosa (i – l) and muscularis (m – p). Structures staining for phosphorylated α-synuclein are red while those staining for neurofilament are black; the counterstain is light red.