Laboratory of Neurological Infections and Immunity, Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Montana, United States of America.
Bioinformatics and Computational Biosciences Branch, National Institute of Allergy, and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
PLoS Pathog. 2024 Sep 20;20(9):e1012554. doi: 10.1371/journal.ppat.1012554. eCollection 2024 Sep.
Disease associated pathological aggregates of alpha-synuclein (αSynD) exhibit prion-like spreading in synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Seed amplification assays (SAAs) such as real-time quaking-induced conversion (RT-QuIC) have shown high diagnostic sensitivity and specificity for detecting proteopathic αSynD seeds in a variety of biospecimens from PD and DLB patients. However, the extent to which relative proteopathic seed concentrations are useful as indices of a patient's disease stage or prognosis remains unresolved. One feature of current SAAs that complicates attempts to correlate SAA results with patients' clinical and other laboratory findings is their quantitative imprecision, which has typically been limited to discriminating large differences (e.g. 5-10 fold) in seed concentration. We used end-point dilution (ED) RT-QuIC assays to determine αSynD seed concentrations in patient biospecimens and tested the influence of various assay variables such as serial dilution factor, replicate number and data processing methods. The use of 2-fold versus 10-fold dilution factors and 12 versus 4 replicate reactions per dilution reduced ED-RT-QuIC assay error by as much as 70%. This enhanced assay format discriminated as little as 2-fold differences in αSynD seed concentration besides detecting ~2-16-fold seed reductions caused by inactivation treatments. In some scenarios, analysis of the data using Poisson and midSIN algorithms provided more consistent and statistically significant discrimination of different seed concentrations. We applied our improved assay strategies to multiple diagnostically relevant PD and DLB antemortem patient biospecimens, including cerebrospinal fluid, skin, and brushings of the olfactory mucosa. Using ED αSyn RT-QuIC as a model SAA, we show how to markedly improve the inter-assay reproducibility and quantitative accuracy. Enhanced quantitative SAA accuracy should facilitate assessments of pathological seeding activities as biomarkers in proteinopathy diagnostics and prognostics, as well as in patient cohort selection and assessments of pharmacodynamics and target engagement in drug trials.
与疾病相关的α-突触核蛋白（αSyn）病理性聚集物在帕金森病（PD）和路易体痴呆（DLB）等突触核蛋白病中表现出类朊病毒样传播。种子扩增测定（SAA），如实时震颤诱导转化（RT-QuIC），已显示出在来自 PD 和 DLB 患者的各种生物样本中检测蛋白病 αSynD 种子的高诊断灵敏度和特异性。然而，相对蛋白病种子浓度作为患者疾病阶段或预后的指标的有用程度仍未解决。当前 SAA 的一个特征是，其定量不精确性使尝试将 SAA 结果与患者的临床和其他实验室发现相关联变得复杂，这种不精确性通常仅限于区分种子浓度的大差异（例如 5-10 倍）。我们使用终点稀释（ED）RT-QuIC 测定法来确定患者生物样本中的 αSynD 种子浓度，并测试了各种测定变量（如连续稀释因子、重复数和数据处理方法）的影响。使用 2 倍与 10 倍稀释因子以及每个稀释度 12 次与 4 次重复反应，使 ED-RT-QuIC 测定误差降低了多达 70%。这种增强的测定格式除了检测失活处理引起的~2-16 倍种子减少外，还能区分 αSynD 种子浓度低至 2 倍的差异。在某些情况下，使用泊松和 midSIN 算法分析数据提供了对不同种子浓度更一致和更具统计学意义的区分。我们将我们改进的测定策略应用于多个与诊断相关的 PD 和 DLB 生前患者生物样本，包括脑脊液、皮肤和嗅黏膜刷取物。使用 ED αSyn RT-QuIC 作为模型 SAA，我们展示了如何显著提高测定内重现性和定量准确性。增强的定量 SAA 准确性应该有助于将病理性播种活动作为蛋白病诊断和预后的生物标志物进行评估，以及在患者队列选择和药物试验中评估药效学和靶标结合方面。
