Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry

Fox, Sue; Sasae, R; Janson, Sven; May, Jane; Heptinstall, Stan

doi:10.1080/09537100310001645979

Cited by 38 publications

(33 citation statements)

References 17 publications

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“…Such losses of single platelets have been previously reported as measures of platelet aggregation. [10][11][12] By using enumeration beads to quantify the single platelet count per volume, it was possible to accurately quantify depletion and make comparisons between samples. Using this approach, we recorded concentration-dependent losses of the single platelet population (Figure 2D-F).…”

Section: Resultsmentioning

confidence: 99%

“…Prasugrel, like clopidogrel, is a blocker of the ADP-receptor P2Y 12 . [18][19][20] It is well-established that P2Y 12 blockade has significant effects upon platelet responsiveness to collagen, U46619, and PAR4 agonists consistent with its antithrombotic effect in man.…”

Section: Detection Of the Antiplatelet Effect Of P2y 12 Receptor Blocmentioning

confidence: 99%

“…They have crucial roles in certain bleeding disorders and are the targets of antithrombotic therapies, notably aspirin and P2Y 12 receptor blockers such as clopidogrel. [1][2][3] Numerous assays have been developed to investigate platelet responses with an understandable focus on analyzing human clinical samples.…”

Section: Introductionmentioning

confidence: 99%

“…Several studies have demonstrated platelet counting to be a viable alternative method for monitoring platelet aggregation because single platelets are "consumed" into larger aggregates upon activation. [10][11][12] Similarly, it has also previously been demonstrated that flow cytometry can be used to determine platelet counts in ;5 mL of blood by reference to known amounts of enumeration beads. 13 With flow cytometry now widely available, such an approach provides an attractive and viable solution for determining platelet aggregation in low volumes of blood.…”

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confidence: 97%

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Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

Armstrong

Kirkby

Chan

et al. 2015

Blood

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Key Points• Low-volume, high-throughput whole blood aggregometry will facilitate future mouse platelet function research.• Application of this approach identifies ICAM-1 as a novel mediator of platelet-monocyte interaction through fibrinogen binding.Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. (Blood. 2015;126(10):e11-e18) IntroductionPlatelets are key mediators of hemostasis. They have crucial roles in certain bleeding disorders and are the targets of antithrombotic therapies, notably aspirin and P2Y 12 receptor blockers such as clopidogrel. [1][2][3] Numerous assays have been developed to investigate platelet responses with an understandable focus on analyzing human clinical samples. 4,5 These assays require relatively large volumes of blood, with the majority requiring a sample volume in excess of 200 mL per test. 6 Mouse models are widely used in cardiovascular research, particularly for phenotyping of genetic modifications and testing novel therapeutic agents. However, platelet function testing in mice remains difficult because a complete and terminal blood collection provides at most 1 mL of blood. Further, commonly used strategies for extending the volume available such as dilution or platelet washing can introduce artifactual changes in platelet responses and thus does not permit study of platelet interactions with other blood cells and constituents. As a result, both the number and type of platelet tests that can be performed in samples from mice are severely limited, diminishing the experimental value of each mouse and making detailed study of mouse platelets impractical. There is therefore a clear and pressing experimental demand for murine-focused and optimized ...

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Section: Resultsmentioning

confidence: 99%

Section: Detection Of the Antiplatelet Effect Of P2y 12 Receptor Blocmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 97%

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Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

Armstrong

Kirkby

Chan

et al. 2015

Blood

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“…Secondary aggregation, also known as macroaggregation, is an irreversible reaction with thromboxane A 2 (TXA 2 ) formation and granule secretion, and is induced by the feedback amplification of primary aggregation when the agonist is at a supraphysiological concentration (e.g., 10 lM ADP) (16)(17)(18).…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric assessment of platelet aspirin resistance using light scattering

Won

Yang

Kim

et al. 2007

Cytometry Part B Clinical

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Background: A simple and reliable assay is needed for the prediction of the clinical efficacy of antiplatelet aspirin therapy. We have devised another method for the evaluation of platelet function and aspirin resistance (AR), via conventional flow cytometry (FCM).Methods: To devise an optimized protocol for the assessment of platelet AR, various analytic variables of FCM were investigated. Using this devised protocol, AR was assessed in healthy subjects as an example.Results: The protocol utilized herein is as follows: citrate-anticoagulated platelet-rich plasma was mixed 1:1 with HEPES-buffered saline in two tubes, one of which is treated with aspirin. During the acquisition of FCM, arachidonate is added to the tube. The response of the aspirin-treated platelets is then compared with those of nontreated ones on a time/forward scatter plot. In the 61 total subjects, none was identified as aspirin resistant by this assay.Conclusions: Using light scattering, platelet aggregation and aspirin's antiplatelet effects can be readily and cost-effectively detected. According to this assay, biochemical AR appears to be rare. This new protocol may prove useful in a clinical setting or as a research tool. q 2007 Clinical Cytometry Society

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Platelet preparation for function testing in the laboratory and clinic: Historical and practical aspects

Hechler

Dupuis

Mangin

et al. 2019

Research and Practice in Thrombosis and Haemostasis

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Laboratory tests of platelet function are instrumental in studying platelet physiology and inherited or acquired platelet abnormalities. Light transmission aggregometry, developed in the early 1960s, is still considered the gold standard for the identification and diagnosis of platelet function disorders. Since then, novel techniques have been developed, including flow‐based assays and flow cytometry. In this tutorial, we describe the basic methodologies for the preparation of citrated platelet‐rich plasma and washed platelet suspensions and discuss their respective advantages and limitations as well as important factors to consider to perform high‐quality tests of platelet function. In addition, the methodologies of the main platelet function tests (light transmission aggregation, flow‐based assays, and flow cytometric assays) are described, and their respective strengths and limitations are discussed to assess various aspects of platelet biology.

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Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry

Cited by 38 publications

References 17 publications

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

Flow cytometric assessment of platelet aspirin resistance using light scattering

Platelet preparation for function testing in the laboratory and clinic: Historical and practical aspects

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