ABSTRACT
Advances in spatial proteomics and protein colocalization are a driving force in the understanding of cellular mechanisms and their influence on biological processes. New methods in the field of spatial proteomics call for the development of algorithms and open up new avenues of research. The newly introduced Molecular Pixelation (MPX) provides spatial information on surface proteins and their relationship with each other in single cells. This allows for in silico representation of neighborhoods of membrane proteins as graphs. In order to analyze this new data modality, we adapted local assortativity in networks of MPX single-cell graphs and created a method that is able to capture detailed information on the spatial relationships of proteins. The introduced method can evaluate the pairwise colocalization of proteins and access higher-order similarity to investigate the colocalization of multiple proteins at the same time. We evaluated the method using publicly available MPX datasets where T cells were treated with a chemokine to study uropod formation. We demonstrate that adjusted local assortativity detects the effects of the stimuli at both single- and multiple-marker levels, which enhances our understanding of the uropod formation. We also applied our method to treating cancerous B-cell lines using a therapeutic antibody. With the adjusted local assortativity, we recapitulated the effect of rituximab on the polarity of CD20. Our computational method together with MPX improves our understanding of not only the formation of cell polarity and protein colocalization under stimuli but also advancing the overall insight into immune reaction and reorganization of cell surface proteins, which in turn allows the design of novel therapies. We foresee its applicability to other types of biological spatial data when represented as undirected graphs.
Subject(s)
Membrane Proteins , Humans , Membrane Proteins/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Proteomics/methods , Algorithms , Rituximab/pharmacology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Single-Cell Analysis/methodsABSTRACT
The spatial distribution of cell surface proteins governs vital processes of the immune system such as intercellular communication and mobility. However, fluorescence microscopy has limited scalability in the multiplexing and throughput needed to drive spatial proteomics discoveries at subcellular level. We present Molecular Pixelation (MPX), an optics-free, DNA sequence-based method for spatial proteomics of single cells using antibody-oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. The relative locations of AOCs are inferred by sequentially associating them into local neighborhoods using the sequence-unique DNA pixels, forming >1,000 spatially connected zones per cell in 3D. For each single cell, DNA-sequencing reads are computationally arranged into spatial proteomics networks for 76 proteins. By studying immune cell dynamics using spatial statistics on graph representations of the data, we identify known and new patterns of spatial organization of proteins on chemokine-stimulated T cells, highlighting the potential of MPX in defining cell states by the spatial arrangement of proteins.
Subject(s)
Proteomics , Single-Cell Analysis , Proteomics/methods , Single-Cell Analysis/methods , Humans , T-Lymphocytes/metabolism , Sequence Analysis, DNA/methodsABSTRACT
INTRODUCTION: To evaluate the effect of repeating test failures using an automated, non-sequencing based non-invasive prenatal testing test on a general-risk population in Finland. MATERIAL AND METHODS: A total of 545 samples from women who represent the average-risk population in Oulu, Finland were analyzed with Vanadis® non-invasive prenatal testing. Repeat testing of test failures was performed using a second sample. Results before and after repeat testing were compared with the reference outcome, as determined by clinical examination of neonates. RESULTS: There were eight test failures after first-pass analysis, representing 1.5% of samples (95% CI 0.6%-2.9%). Seven out of eight failures could be resolved by analysis of a second sample, thereby reducing the test failure rate from 1.5% to 0.2% (95% CI 0.0%-1.0%). CONCLUSIONS: Repeating test failures with a second plasma sample could significantly reduce the effective failure rate, thereby providing a way to effectively minimize test failures and further improving clinical utility and test performance.
Subject(s)
Diagnostic Errors/statistics & numerical data , Genetic Testing , Prenatal Diagnosis , Trisomy 13 Syndrome/diagnosis , Trisomy 18 Syndrome/diagnosis , Adult , Female , Finland , High-Throughput Nucleotide Sequencing , Humans , Pregnancy , Young AdultABSTRACT
OBJECTIVE: To evaluate clinical performance of a new automated cell-free (cf)DNA assay in maternal plasma screening for trisomies 21, 18, and 13, and to determine fetal sex. METHOD: Maternal plasma samples from 1200 singleton pregnancies were analyzed with a new non-sequencing cfDNA method, which is based on imaging and counting specific chromosome targets. Reference outcomes were determined by either cytogenetic testing, of amniotic fluid or chorionic villi, or clinical examination of neonates. RESULTS: The samples examined included 158 fetal aneuploidies. Sensitivity was 100% (112/112) for trisomy 21, 89% (32/36) for trisomy 18, and 100% (10/10) for trisomy 13. The respective specificities were 100%, 99.5%, and 99.9%. There were five first pass failures (0.4%), all in unaffected pregnancies. Sex classification was performed on 979 of the samples and 99.6% (975/979) provided a concordant result. CONCLUSION: The new automated cfDNA assay has high sensitivity and specificity for trisomies 21, 18, and 13 and accurate classification of fetal sex, while maintaining a low failure rate. The study demonstrated that cfDNA testing can be simplified and automated to reduce cost and thereby enabling wider population-based screening.
Subject(s)
Noninvasive Prenatal Testing/methods , Trisomy/diagnosis , Chromosomes, Human, Pair 13 , Chromosomes, Human, Pair 18 , Chromosomes, Human, Pair 21 , Female , Humans , PregnancyABSTRACT
Cell-free DNA analysis is becoming adopted for first line aneuploidy screening, however for most healthcare programs, cost and workflow complexity is limiting adoption of the test. We report a novel cost effective method, the Vanadis NIPT assay, designed for high precision digitally-enabled measurement of chromosomal aneuploidies in maternal plasma. Reducing NIPT assay complexity is achieved by using novel molecular probe technology that specifically label target chromosomes combined with a new readout format using a nanofilter to enrich single molecules for imaging and counting without DNA amplification, microarrays or sequencing. The primary objective of this study was to assess the Vanadis NIPT assay with respect to analytical precision and clinical feasibility. Analysis of reference DNA samples indicate that samples which are challenging to analyze with low fetal-fraction can be readily detected with a limit of detection determined at <2% fetal-fraction. In total of 286 clinical samples were analysed and 30 out of 30 pregnancies affected by trisomy 21 were classified correctly. This method has the potential to make cost effective NIPT more widely available with more women benefiting from superior detection and false positive rates.
Subject(s)
Cell-Free Nucleic Acids/blood , Down Syndrome/diagnosis , Prenatal Diagnosis/methods , Single Molecule Imaging/methods , Aneuploidy , Case-Control Studies , Cost-Benefit Analysis , Female , Humans , Pregnancy , Prenatal Diagnosis/economics , Prospective Studies , Single Molecule Imaging/economicsABSTRACT
INTRODUCTION: Functional imaging could be utilized for visualizing pancreatic islets of Langerhans. Therefore, we present a stepwise algorithm for screening of clinically available positron emission tomography (PET) tracers for their use in imaging of the neuroendocrine pancreas in the context of diabetes. METHODS: A stepwise procedure was developed for screening potential islet imaging agents. Suitable PET-tracer candidates were identified by their molecular mechanism of targeting. Clinical abdominal examinations were retrospectively analyzed for pancreatic uptake and retention. The target protein localization in the pancreas was assessed in silico by -omics approaches and the in vitro by binding assays to human pancreatic tissue. RESULTS: Six putative candidates were identified and screened by using the stepwise procedure. Among the tested PET tracers, only [(11)C]5-Hydroxy-tryptophan passed all steps. The remaining identified candidates were falsified as candidates and discarded following in silico and in vitro screening. CONCLUSIONS: Of the six clinically available PET tracers identified, [(11)C]5-HTP was found to be a promising candidate for beta cell imaging, based on intensity of in vivo pancreatic uptake in humans, and islet specificity as assessed on human pancreatic cell preparations. The flow scheme described herein constitutes a methodology for evaluating putative islet imaging biomarkers among clinically available PET tracers.