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Multicolor flow cytometry in clinical samples for platelet signaling assessment

BACKGROUND: Availability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. OBJECTIVES: To describe a multic...

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Autores principales: Garcia, Cedric, Dejean, Sebastien, Savy, Nicolas, Bordet, Jean-Claude, Series, Jennifer, Cadot, Sarah, Ribes, Agnès, Voisin, Sophie, Rugeri, Lucia, Payrastre, Bernard, Sié, Pierre
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10394564/
https://www.ncbi.nlm.nih.gov/pubmed/37538502
http://dx.doi.org/10.1016/j.rpth.2023.100180
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author Garcia, Cedric
Dejean, Sebastien
Savy, Nicolas
Bordet, Jean-Claude
Series, Jennifer
Cadot, Sarah
Ribes, Agnès
Voisin, Sophie
Rugeri, Lucia
Payrastre, Bernard
Sié, Pierre
author_facet Garcia, Cedric
Dejean, Sebastien
Savy, Nicolas
Bordet, Jean-Claude
Series, Jennifer
Cadot, Sarah
Ribes, Agnès
Voisin, Sophie
Rugeri, Lucia
Payrastre, Bernard
Sié, Pierre
author_sort Garcia, Cedric
collection PubMed
description BACKGROUND: Availability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. OBJECTIVES: To describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen). METHODS: By comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n = 23) or type-1 von Willebrand disease (n = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI–related disease with granule secretion defect). RESULTS: The range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap. CONCLUSION: In complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice.
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spelling pubmed-103945642023-08-03 Multicolor flow cytometry in clinical samples for platelet signaling assessment Garcia, Cedric Dejean, Sebastien Savy, Nicolas Bordet, Jean-Claude Series, Jennifer Cadot, Sarah Ribes, Agnès Voisin, Sophie Rugeri, Lucia Payrastre, Bernard Sié, Pierre Res Pract Thromb Haemost Original Article BACKGROUND: Availability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. OBJECTIVES: To describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen). METHODS: By comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n = 23) or type-1 von Willebrand disease (n = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI–related disease with granule secretion defect). RESULTS: The range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap. CONCLUSION: In complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice. Elsevier 2023-05-16 /pmc/articles/PMC10394564/ /pubmed/37538502 http://dx.doi.org/10.1016/j.rpth.2023.100180 Text en © 2023 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Article
Garcia, Cedric
Dejean, Sebastien
Savy, Nicolas
Bordet, Jean-Claude
Series, Jennifer
Cadot, Sarah
Ribes, Agnès
Voisin, Sophie
Rugeri, Lucia
Payrastre, Bernard
Sié, Pierre
Multicolor flow cytometry in clinical samples for platelet signaling assessment
title Multicolor flow cytometry in clinical samples for platelet signaling assessment
title_full Multicolor flow cytometry in clinical samples for platelet signaling assessment
title_fullStr Multicolor flow cytometry in clinical samples for platelet signaling assessment
title_full_unstemmed Multicolor flow cytometry in clinical samples for platelet signaling assessment
title_short Multicolor flow cytometry in clinical samples for platelet signaling assessment
title_sort multicolor flow cytometry in clinical samples for platelet signaling assessment
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10394564/
https://www.ncbi.nlm.nih.gov/pubmed/37538502
http://dx.doi.org/10.1016/j.rpth.2023.100180
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