Dataset Information

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples.

ABSTRACT: A recently released kit (PerFix EXPOSE) was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12). The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24?h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP) of PGE1-induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets.

SUBMITTER: Mallouk N

PROVIDER: S-EPMC6128249 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples.

Mallouk N N Varvat J J Berger A A Epinat M M Accassat S S Garcin A A Montmartin A A Li G G Garnier P P Mismetti P P Lambert C C

Practical laboratory medicine 20180216

A recently released kit (PerFix EXPOSE) was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12). The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24  ...[more]

PMID: 30202779

Similar Datasets

Project description:BackgroundUpon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane.ObjectiveTo evaluate procoagulant PS-exposing platelets by imaging flow cytometry.MethodsPlatelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbβ3, CD62P, and PS exposure.ResultsA subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and αIIbβ3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs.ConclusionsInnovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

Project description:Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less). Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing enrichment, and the ability to align cell subsets across multiple data samples for comparative analysis. In this manuscript, we develop hierarchical modeling extensions to the Dirichlet Process Gaussian Mixture Model (DPGMM) approach we have previously described for cell subset identification, and show that the hierarchical DPGMM (HDPGMM) naturally generates an aligned data model that captures both commonalities and variations across multiple samples. HDPGMM also increases the sensitivity to extremely low frequency events by sharing information across multiple samples analyzed simultaneously. We validate the accuracy and reproducibility of HDPGMM estimates of antigen-specific T cells on clinically relevant reference peripheral blood mononuclear cell (PBMC) samples with known frequencies of antigen-specific T cells. These cell samples take advantage of retrovirally TCR-transduced T cells spiked into autologous PBMC samples to give a defined number of antigen-specific T cells detectable by HLA-peptide multimer binding. We provide open source software that can take advantage of both multiple processors and GPU-acceleration to perform the numerically-demanding computations. We show that hierarchical modeling is a useful probabilistic approach that can provide a consistent labeling of cell subsets and increase the sensitivity of rare event detection in the context of quantifying antigen-specific immune responses.

Dataset Information

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples.

Publications

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets