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High-Dimensional Single-Cell Mass Cytometry Demonstrates Differential Platelet Functional Phenotypes in Infants With
Sean X Gu1, Brian S Marcus2, Vivian W Gu3
1Department of Laboratory Medicine (S.X.G.), Yale University School of Medicine, New Haven, CT.
Insights
Infants with congenital heart disease (CHD) show altered platelet subpopulations, suggesting exhaustion from persistent stimulation. This may explain bleeding and clotting complications in CHD patients.
Area of Science:
- Hematology
- Pediatric Cardiology
- Immunology
Background:
- Congenital heart disease (CHD) is linked to hematologic issues, including bleeding and clotting risks.
- Previous studies show conflicting results on platelet activation in CHD patients.
- Understanding platelet behavior in infants with CHD is crucial for managing complications.
Purpose of the Study:
- To develop a mass spectrometry method for characterizing single platelets in infants with CHD.
- To identify the reasons behind conflicting findings on platelet activation in CHD.
- To analyze platelet subpopulations and their functional characteristics in infants with CHD.
Main Methods:
- Enrolled 19 infants with CHD and 21 controls.
- Utilized single-cell mass cytometry to analyze platelet surface markers in whole blood.
- Performed plasma cytokine analysis for 52 vascular and inflammatory markers.
Main Results:
- Infants with CHD displayed significant differences in platelet activation and functional markers compared to controls.
- Identified 8 platelet subpopulations, with P1, P4, and P5 being more abundant in CHD infants and showing reduced aggregation but altered secretion.
- Observed increased hypogranular platelets and elevated plasma cytokines (e.g., IL-6, IL-8, VWF) in CHD infants.
Conclusions:
- Developed a mass cytometry approach to reveal platelet phenotypic heterogeneity in infants with CHD.
- Found distinct platelet subpopulations with reduced aggregation and secretory dysfunction in infants with CHD.
- Hypothesize that platelets in infants with CHD may be exhausted due to chronic stimulation, explaining vascular complications.
Background:
Congenital heart disease (CHD) is a group of complex heart defects associated with hematologic abnormalities, including increased risk of thrombotic and bleeding events. Past studies have observed evidence of platelet hyperreactivity, while other studies showed decreased platelet activation in patients with CHD. The goal of this study was to develop a mass spectrometry approach to characterize single platelets in infants with CHD and identify potential etiology for such discrepant results.
Methods:
We enrolled 19 infants with CHD along with 21 non-CHD controls at Yale New Haven Children's Heart Center. A single-cell high-dimensional mass cytometry method was developed to quantitatively interrogate platelet surface markers in whole blood. Additionally, plasma cytokine analysis was performed through a multiplexed panel of 52 vascular and inflammatory markers to assess for platelet releasates.
Results:
We found that infants with CHD had significant differences in platelet activation and functional markers by mass cytometry compared with non-CHD controls. Based on cell surface markers, we classified the platelets into 8 subpopulations (P0 to P7). Distinct subpopulations of platelets (P1, P4, and P5) exhibiting decreased aggregatory phenotype but altered secretory phenotypes were also identified and found to be more abundant in the blood of infants with CHD. Electron microscopy identified increased proportion of hypogranular platelets in CHD. Moreover, cytokine analysis demonstrated an overall increase in plasma cytokines and biomarkers in CHD, including IL (interleukin)-6, IL-8, IL-27, RANTES (regulated upon activation, normal T cell expressed and secreted), and VWF (von Willebrand factor), which are expressed in platelet granules and can be released upon activation.
Conclusions:
We developed a robust mass cytometry approach to identify platelet phenotypic heterogeneity. Infants with CHD had alterations in distinct subpopulations of platelets with overall reduced aggregatory phenotype and secretory dysfunction. These findings suggest that platelets in infants with CHD may be exhausted due to persistent stimulation and may explain both bleeding and thrombotic vascular complications associated with CHD.
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