Sensitivity and specificity of a seed amplification assay for diagnosis of multiple system atrophy: a multicentre cohort study

Abstract

Background: The pathological hallmarks of multiple system atrophy and Parkinson's disease are, respectively, misfolded-α-synuclein-laden glial cytoplasmic inclusions and Lewy bodies. CSF-soluble misfolded α-synuclein aggregates (seeds) are readily detected in people with Parkinson's disease by α-synuclein seed amplification assay (synSAA), but identification of seeds associated with multiple system atrophy for diagnostic purposes has proven elusive. We aimed to assess whether a novel synSAA could reliably distinguish seeds from Lewy bodies and glial cytoplasmic inclusions.

Methods: In this multicentre cohort study, a novel synSAA that multiplies and detects seeds by fluorescence was used to analyse masked CSF and brain samples from participants with either clinically diagnosed or pathology-confirmed multiple system atrophy, Parkinson's disease, dementia with Lewy bodies, isolated rapid eye movement sleep behaviour disorder (IRBD), disorders that were not synucleinopathies, or healthy controls. Participants were from eight available cohorts from seven medical centres in four countries: New York Brain Bank, New York, USA (NYBB); University of Pennsylvania, Philadelphia, PA, USA (UPENN); Paracelsus-Elena-Klinik, Kassel, Germany (DeNoPa and KAMSA); Hospital Clinic Barcelona, Spain (BARMSA); Universität Tübingen, Tübingen, Germany (EKUT); Göteborgs Universitet, Göteborgs, Sweden (UGOT); and Karolinska Institutet, Stockholm, Sweden (KIMSA). Clinical cohorts were classified for expected diagnostic accuracy as either research (longitudinal follow-up visits) or real-life (single visit). Sensitivity and specificity were estimated according to pathological (gold standard) and clinical (reference standard) diagnoses.

Findings: In 23 brain samples (from the NYBB cohort), those containing Lewy bodies were synSAA-positive and produced high fluorescence amplification patterns (defined as type 1); those containing glial cytoplasmic inclusions were synSAA-positive and produced intermediate fluorescence (defined as type 2); and those without α-synuclein pathology produced below-threshold fluorescence and were synSAA-negative. In 21 pathology-confirmed CSF samples (from the UPENN cohort), those with Lewy bodies were synSAA-positive type 1; those with glial cytoplasmic inclusions were synSAA-positive type 2; and those with four-repeat tauopathy were synSAA-negative. In the DeNoPa research cohort (which had no samples from people with multiple system atrophy), the novel synSAA had sensitivities of 95% (95% CI 88-99) for 80 participants with Parkinson's disease and 95% (76-100) for 21 participants with IRBD, and a specificity of 95% (86-99) for 60 healthy controls. Overall (combining BARMSA, EKUT, KAMSA, UGOT, and KIMSA cohorts that were enriched for cases of multiple system atrophy), the novel synSAA had 87% sensitivity for multiple system atrophy (95% CI 80-93) and specificity for type 2 seeds was 77% (67-85). For participants with multiple system atrophy just in research cohorts (BARMSA and EKUT), the novel synSAA had a sensitivity of 84% (95% CI 71-92) and a specificity for type 2 seeds of 87% (74-95), whereas cases from real-life cohorts (KAMSA, KIMSA, and UGOT) had a sensitivity of 91% (95% CI 80-97) but a decreased specificity for type 2 seeds of 68% (53-81).

Interpretation: The novel synSAA produced amplification patterns that enabled the identification of underlying α-synuclein pathology, showing two levels of fluorescence that corresponded with different pathological hallmarks of synucleinopathy. The synSAA might be useful for early diagnosis of synucleinopathies in clinical trials, and potentially for clinical use, but additional formal validation work is needed.

Funding: Michael J Fox Foundation for Parkinson's Research, Amprion.

Figure 1:. Flowchart showing cohorts, diagnoses and pathologies, and sample analysis

Samples included in this study were kindly provided by the institutions described in this diagram, which included clinically defined CSF samples (green shading), pathology-confirmed CSF samples (red shading), or brain samples (purple shading). Among the clinical samples, three cohorts were classified as research (grey shading) and three as real-life (orange shading) given their different levels of characterisation and expected diagnostic accuracy. Samples were all shipped to Amprion (San Diego, CA, USA) for masked synSAA testing. All the samples were analysed with the novel synSAA (light blue line), and some with sufficient sample volume were also tested with a previously reported synSAA (dark blue line).The novel synSAA is the index test, whereas the previously reported synSAA was used for comparison purposes only; both synSAAs used the clinical diagnosis or pathological findings (if available) as the reference standard. DeNoPa=De Novo Parkinson’s disease at Paracelsus-ElenaKlinik, Germany (de novo Parkinson’s disease cohort). BARMSA=Barcelona MSA at Hospital Clinic Barcelona, Spain. EKUT=Eberhard Karls Universität Tübingen, Germany. KAMSA=Kassel MSA, Paracelsus-Elena-Klinik, Germany. UGOT=Göteborgs Universitet, Sweden. KIMSA=Karolinska Institutet MSA, Sweden. UPENN=University of Pennsylvania, PA, USA. NYBB=New York Brain Bank, Columbia University, NY, USA. PD=Parkinson’s disease. iRDB=isolated rapid eye movement sleep behaviour disorder. HC=healthy controls. NS=non-synucleinopathy. MSA=multiple system atrophy. DLB=dementia with LB. GCI=glial cytoplasmic inclusions. LB=Lewy bodies. AD=Alzheimer’s disease. PSP=progressive supranuclear palsy. CBD=corticobasal degeneration. AGD=argyrophilic grain disease.

Figure 2:. Amplification patterns of brain samples and pathology-confirmed CSF samples

Representative fluorescence traces produced by brain samples with Lewy bodies (A), glial cytoplasmic inclusions (B), non-synucleinopathy (C), and F_max of all replicates (D) from 23 pathology-confirmed brain samples (five Lewy bodies, 12 glial cytoplasmic inclusions, and six non-synucleinopathies). Outliers are represented with an x. Representative fluorescence traces produced by pathology-confirmed CSF samples with Lewy bodies (E), cytoplasmic inclusions (F), non-synucleinopathy (G), and F_max of all replicates (H) from 27 pathology-confirmed CSF samples (seven Lewy bodies, five glial cytoplasmic inclusions, and 15 non-synucleinopathies). Outliers are represented with an x.

Figure 3:. Visualisation of F_max values collected from all evaluated CSF samples

F_max values for all 1254 replicates analysed in this study. (A) F_max from individual replicates (n=81) generated by synSAA analysis of 27 pathology-confirmed CSF samples. (B) F_max from individual replicates (n=837) generated by synSAA analysis of 279 clinically defined CSF samples from research cohorts. (C) F_max from individual replicates (n=336) generated by synSAA analysis of 112 clinically defined CSF samples from real-life cohorts. The y-axis is broken at the 3000 RFU and 45 000 RFU thresholds and each part of the axis is scaled to show dispersion of the measurements within each of the three fluorescence ranges. RFU=relative fluorescence unit.