Multicolor flow cytometry in clinical samples for platelet signaling assessment

Cedric Garcia; Sebastien Dejean; Nicolas Savy; Jean-Claude Bordet; Jennifer Series; Sarah Cadot; Agnès Ribes; Sophie Voisin; Lucia Rugeri; Bernard Payrastre; Pierre Sié

doi:10.1016/j.rpth.2023.100180

Multicolor flow cytometry in clinical samples for platelet signaling assessment

Res Pract Thromb Haemost. 2023 May 16;7(4):100180. doi: 10.1016/j.rpth.2023.100180. eCollection 2023 May.

Authors

Cedric Garcia^{1

2}, Sebastien Dejean³, Nicolas Savy³, Jean-Claude Bordet^{4

5}, Jennifer Series², Sarah Cadot², Agnès Ribes^{1

2

6}, Sophie Voisin¹, Lucia Rugeri^{4

7}, Bernard Payrastre^{1

2

6}, Pierre Sié^{1

2

8}

Affiliations

¹ CHU de Toulouse, Laboratoire d'Hématologie, Toulouse, France.
² Institut des Maladies Métaboliques et Cardiovasculaires INSERM U1048, Université de Toulouse, Toulouse, France.
³ Université Paul Sabatier Toulouse III, Institut de Mathématiques, CNRS UMR 5219, Toulouse, France.
⁴ Laboratoire d'Hématologie, Hospices Civiles de Lyon, Lyon, France.
⁵ EA 4609-Hémostase et Cancer, Université Claude Bernard Lyon 1, Lyon, France.
⁶ Faculté de Médecine, Université Paul Sabatier Toulouse III, Toulouse, France.
⁷ Hospices Civils de Lyon, Unité d'Hémostase clinique, Bron, France.
⁸ Université Paul Sabatier Toulouse III, Faculté de Pharmacie, Toulouse, France.

Abstract

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.

Keywords: blood platelet disorders; fluorescent barcoding; multicolor flow cytometry; phosphoproteins; platelet signaling.