Preeclampsia (PE) is a multisystem heterogeneous complication of pregnancy remaining a leading cause of maternal and perinatal morbidity and mortality over the world. PE has a large spectrum of clinical features and symptoms, which make diagnosis challenging. Despite a long period of studying, PE etiology is still unclear and there are no reliable rapid tests for early diagnosis of this disease. During the last decade, it was shown that proteins misfolding and aggregation are associated with PE. Several proteins, including amyloid beta peptide, transthyretin, alpha-1 antitrypsin, albumin, IgG k-free light chains, and ceruloplasmin are dysregulated in PE, resulting in toxic deposition of amyloid-like aggregates in the placenta and body fluids. It is also possible that aggregated proteins induce defective trophoblast invasion, placental ischemia, ER stress, and promote PE manifestation. The fact that protein aggregation is an emerging biomarker of PE provides an opportunity to develop new diagnostic approaches based on amyloids special features, such as Congo red (CR) staining and thioflavin T (ThT) enhanced fluorescence.

BACKGROUND: Preeclampsia is a multisystem complication of pregnancy associated with an increased risk of maternal / perinatal morbidity and mortality. In this regard, the development and following improvement of low-cost and convenient methods for diagnosis of preeclampsia is essential for accurate prediction, quick confirmation of the diagnosis and convenient monitoring of the pathology. AIM: The aim of this study was to optimize a preeclampsia diagnosis test system based on the binding of proteins to the Congo red dye (CRD test). MATERIALS AND METHODS: The study used 70 urine samples obtained from patients diagnosed with preeclampsia (n = 25) and from non-preeclampsia pregnant women (n = 45). The samples were stained with Congo red and the dye retention in the sample on the membrane after washing was calculated. Before staining, protein concentrations in the urine samples were equalized using centrifugal concentrators or the samples were used with the original protein concentrations. To wash the samples from the unbound dye, either methanol or ethanol was used. To compare the effectiveness of four CRD test variants differing in sample preparation, staining, and washing, ROC analysis was performed (IBM SPSS Statistics 20 software). RESULTS: The express CRD test was designed as an optimization of the conventional CRD test. The effectiveness of the express test (the area under the ROC curve being 0.9) was higher than that of the other three test options (the area under the ROC curve ranges from 0.67 to 0.82). The developed express CRD test can provide 95% specificity and 73% sensitivity, which indicates the promise of using this method in clinical diagnostics for the specific detection of preeclampsia patients. CONCLUSIONS: Optimization of the CRD test has provided more effective protocols for diagnosis of preeclampsia from urine samples using Congo red (express CRD test) and has simplified the routine application of this test in clinical practice.

The accumulation of alpha-synuclein (aSyn) is the hallmark of a group of neurodegenerative conditions termed synucleopathies. Physiological functions of aSyn, including those outside of the CNS, remain elusive. However, a reliable and reproducible evaluation of aSyn protein expression in different cell types and especially in low-expressing cells is impeded by the existence of a huge variety of poorly characterized anti-aSyn antibodies and a lack of a routinely used sensitive detection methods. Here, we developed a robust flow cytometry-based workflow for aSyn detection and antibody validation. We test our workflow using three commercially available antibodies (MJFR1, LB509, and 2A7) in a variety of human cell types, including induced pluripotent stem cells, T lymphocytes, and fibroblasts, and provide a cell- and antibody-specific map for aSyn expression. Strikingly, we demonstrate a previously unobserved unspecificity of the LB509 antibody, while the MJFR1 clone revealed specific aSyn binding however with low sensitivity. On the other hand, we identified an aSyn-specific antibody clone 2A7 with an optimal sensitivity for detecting aSyn in a range of cell types, including those with low aSyn expression. We further utilize our workflow to demonstrate the ability of the 2A7 antibody to distinguish between physiological differences in aSyn expression in neuronal and non-neuronal cells from the cortical organoids, and in neural progenitors and midbrain dopaminergic neurons from healthy controls and in patients with Parkinson's disease who have aSyn gene locus duplication. Our results provide a proof of principle for the use of high-throughput flow cytometry-based analysis of aSyn and highlight the necessity of rigorous aSyn antibody validation to facilitate the research of aSyn physiology and pathology.

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