Seed amplification assays: a novel development in synucleinopathy diagnosis

Synucleinopathies are a group of neurodegenerative disorders noted for the abnormal accumulation of misfolded α-synuclein (αSyn) which forms toxic inclusions in cells. This group of diseases, which includes Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple-system atrophy (MSA), is often clinically characterized by movement related issues such as asymmetric resting tremor and bradykinesia.1 Phenotypic characteristics such as these are currently the primary source of clinical diagnoses, with specific disease confirmation still only possible after postmortem examination. However, the recent development of the experimental seed amplification assay (SAA) may provide a valuable alternative approach to synucleinopathy diagnosis.

Aggregates of αSyn found in these diseases tend to be concentrated in the brain but can also be found in the heart, muscle, and other tissues where they propagate to form soluble oligomeric protofibrils. Misfolded αSyn protein aggregates circulating in patient’s biofluids naturally multiply and “seed” further aggregation.2 Samples containing these seed proteins will form larger and larger protein aggregates when provided with additional recombinant αSyn substrate, which can then be detected using fluorescent dyes. This provides a faster, more reliable, biochemical alternative to the current symptom-based approach to diagnosis which necessitates a certain level of disease progression.

The SAA is a method of detection derived from similar techniques targeting prions, where misfolded αSyn “seeds” present in patient biofluids are isolated before undergoing cyclic fragmentation and elongation to achieve in vitro amplification.2 Using the intrinsic self-propagating nature of misfolded αSyn in combination with C-terminally tagged recombinant αSyn substrate and fluorescent dyes such as thioflavin T, researchers can produce a signal reflective of the amount of endogenous αSyn present in a sample. In this way, even small amounts of protein will produce a signal when amplified, to give quantitative evidence for synucleinopathy presence. This provides clinicians with an incredibly valuable biomarker for early diagnosis of these disorders, allowing them to identify the disease before symptoms present themselves and reduce the potential for misdiagnosis.

With numerous reports describing sensitivity and specificity between 85% and 95% in PD, DLB, and MSA patient CSF samples, the improvement from symptom-based diagnostic methods is drastic.2 Importantly, SAA may allow clinicians to differentiate between synucleinopathies, as different aggregates present in PD and MSA will produce a unique signal.2 However, achieving differentiation in practice has been challenging, with some studies demonstrating the capability to differentiate PD from MSA based on the maximum fluorescence of aggregation, while others have been unable to reliably distinguish between them. It is not yet clear if synucleinopathies like DLB or PD dementia can be distinguished on the basis of kinetic parameters.2

Fernandes Gomes et al., used SAA to correctly identify all PD samples in their cohort (100% sensitivity, 70.8% specificity). Similarly robust results were achieved for the identification of MSA vs. healthy controls (92.6% sensitivity, 70.8% specificity), improving on the 62%-79% rates of correct diagnosis for MSA found currently.1,3 However, the specificity for distinguishing MSA from PD was only 7%. Another large study using the Parkinson’s Progression Markers Initiative (PPMI) cohort of 1123 participants found that of 545 PD cases, 87.7% were correctly diagnosed using SAA, with 96.3% of 163 healthy controls correctly judged to be disease-free.4 As the largest analysis of the αSyn SAA for PD diagnosis to date, these high degrees of diagnostic accuracy demonstrate the significant promise of this approach.

Although initial studies focused on the use of SAAs on CSF samples, recent studies have explored the possibility of using more accessible tissue samples such as blood, nasal swabs, and skin.5-8 Development of SAAs to detect αSyn in easily accessible samples could make screening for synucleinopathies viable, as unintrusive as a blood test for adults between 60 and 89 years old when PD is most likely to develop.5 In particular, recent trials using blood serum have found similar high diagnostic performance in SAA, differentiating PD, DLB, and MSA from control samples. αSyn aggregates also retained their disease-specific structural and propagation properties in serum, allowing the differentiation of samples from individuals with PD versus MSA in morphological analyses.5

The application of SAAs to detect αSyn seeds during prodromal stages is one of the most exciting aspects. Some recent reports have shown the ability of SAAs to detect αSyn in isolated rapid eye movement sleep behavior disorder (RBD). Okuzumi, A. et al found that certain assay parameters were correlated with the duration of RBD, as well as with specific binding ratio, suggesting that SAA could provide insights on the phenoconversion of RBD to early PD.5 More long-term data is needed to further investigate this.

The potential of SAA to investigate other neurodegenerative disorders based on protein seeding mechanisms such as that of tau or amyloid in Alzheimer’s disease has also been recognized by researchers, with ongoing research into how SAA can be used to identify these different seed structures and mechanisms.9

Tiago Outeiro, PhD, University Medical Center Göttingen, Göttingen, Germany, discusses the exciting developments in using alpha-synuclein as a biomarker for the early detection of Parkinson’s disease.

There is “tremendous excitement” about the possibilities of SAA as a diagnostic tool from researchers such as Tiago Outeiro, PhD, who we interviewed at MDS 2023. The possibility of “catching the disease early” by analyzing αSyn biomarkers in blood could provide better outcomes for patients and be “important for aiding the selection of patients for clinical trials and with that eventually in aiding the efforts in finding novel therapies”. All of which makes SAA one of the most exciting developments in PD research this year, providing significant optimism for the future of diagnosis and hope for effective treatment of the disease.

Written by Joey Dean

Reviewed by Juliet Lawrence


  1. Fernandes Gomes B, Farris C, Ma Y, et al. α-Synuclein seed amplification assay as a diagnostic tool for parkinsonian disorders. Parkinsonism Relat. Disord. Aug 2023;105807.
  2. Concha-Marambio L, Pritzkow S, Shahnawaz M, et al. Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid. Nat. Protoc. Apr 2023;18(4):1179-1196.
  3. Goh YY, Saunders E, Pavey S, et al. Multiple system atrophy. Pract. Neurol. Mar 2023;23:208-221.
  4. Siderowf A, Concha-Marambio L, Lafontant DE, et al. Assessment of heterogeneity among participants in the Parkinson’s Progression Markers Initiative cohort using α-synuclein seed amplification: a cross-sectional study. Lancet Neurol. May 2023;22(5):407–417.
  5. Okuzumi A, Hatano T, Matsumoto G, et al. Propagative α-synuclein seeds as serum biomarkers for synucleinopathies. Nat. Med. May 2023;29:1448–1455.
  6. Kuzkina A, Rößle J, Seger A, et al. Combining skin and olfactory α-synuclein seed amplification assays (SAA)—towards biomarker-driven phenotyping in synucleinopathies. NPJ Park. Dis. May 2023;9(1):79.
  7. Duan S, Yang J, Cui Z, et al. Seed amplification assay of nasal swab extracts for accurate and non-invasive molecular diagnosis of neurodegenerative diseases. Transl. Neurodegener. Mar 2023;12(1):13.
  8. Liguori R, Donadio V, Wang Z, et al. A comparative blind study between skin biopsy and seed amplification assay to disclose pathological α-synuclein in RBD. NPJ Park. Dis. Mar 2023;9(1):34.
  9. Standke H, Kraus A. Seed amplification and RT-QuIC assays to investigate protein seed structures and strains. Cell Tissue Res. Apr 2023;392(1):323-335.