Using six-colour flow cytometry to analyse the activation and interaction of platelets and leukocytes--A new assay suitable for bench and bedside conditions.
BACKGROUND
Platelets are main effector cells in haemostasis and also promote inflammation. Platelet-leukocyte complexes are key mediators in a variety of thromboinflammatory disorders and consecutive organ failure. Cell-specific epitopes and activation markers on platelets and leukocytes can be measured using flow cytometry. However, until recently a major restriction has been a paucity in antibody combinations and lack of detection strategies. We aimed to develop a six-colour flow cytometry method which depicts multiple aspects of platelet and leukocyte interactions in human whole blood.


METHODS
Platelets, including microparticles and aggregates, were detected in flow cytometry using a platelet-specific anti-CD41-FITC antibody and size-defined regions. The morphology of platelet-leukocyte complexes (including granulocyte and monocyte content) were depicted using anti-CD45-PerCP, anti-CD66b-PE-Cy7, and anti-CD14-APC antibodies in a single sample. Expression of platelet and leukocyte activation markers P-selectin and CD11b were detected using anti-CD62P-PE and anti-CD11b-BV421 antibodies, respectively.


RESULTS
The sensitivity of this assay to detect the effects of various agonists (TRAP-6, ADP, collagen, epinephrine, TNF-α and LPS) is demonstrated. Furthermore, the assay is shown to detect platelet and leukocyte activation induced by extracorporeal circulation in vitro. The suitability of this assay for bedside analysis is demonstrated exemplarily in a patient treated with mechanical circulatory life support.


CONCLUSIONS
Using the concurrent assessment of multiple parameters, this method gives detailed insights into the complexity and dynamics of platelet-leukocyte interactions. This assay carries the potential to increase our understanding of the mechanisms and pathophysiology of platelet-leukocyte interaction in the research laboratory and clinical setting.
Department of Anaesthesiology and Intensive Care Medicine, Tübingen University Hospital, Eberhard-Karls University Tübingen, Germany. Electronic address: tiago.granja@uni-tuebingen.de.
Tiago
T
Granja
Department of Anaesthesiology and Intensive Care Medicine, Tübingen University Hospital, Eberhard-Karls University Tübingen, Germany. Electronic address: jessica.schad@student.uni-tuebingen.de.
Jessica
J
Schad
Department of Anaesthesiology and Intensive Care Medicine, Tübingen University Hospital, Eberhard-Karls University Tübingen, Germany. Electronic address: patricia.schuessel@student.uni-tuebingen.de.
Patricia
P
Schüssel
Department of Anaesthesiology and Intensive Care Medicine, Tübingen University Hospital, Eberhard-Karls University Tübingen, Germany. Electronic address: Claudius.Fischer@med.uni-tuebingen.de.
Claudius
C
Fischer
Department of Anaesthesiology and Intensive Care Medicine, Tübingen University Hospital, Eberhard-Karls University Tübingen, Germany. Electronic address: Helene.Haeberle@med.uni-tuebingen.de.
Helene
H
Häberle
Department of Anaesthesiology and Intensive Care Medicine, Tübingen University Hospital, Eberhard-Karls University Tübingen, Germany. Electronic address: peter.rosenberger@med.uni-tuebingen.de.
Peter
P
Rosenberger
Department of Anaesthesiology and Intensive Care Medicine, Tübingen University Hospital, Eberhard-Karls University Tübingen, Germany. Electronic address: andreas.straub@uni-tuebingen.de.
Andreas
A
Straub
D003116:Color; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D015539:Platelet Activation / Q000502:physiology*; D010974:Platelet Aggregation / Q000502:physiology*
20.130: Inflammation, leukocytes, and thrombosis; 20.190: Platelets; 20.210: Platelet agonists and adhesion receptors; 20: Platelets and Cell Biology