Kinetx: a combined flow cytometry assay and analysis software framework to quantitatively measure and categorize platelet activation in real-time

Dunster, J. ORCID: https://orcid.org/0000-0001-8986-4902, Mitchell, J., Mohammed, Y., Taylor, K. ORCID: https://orcid.org/0000-0002-4599-7727, Gibbins, J. ORCID: https://orcid.org/0000-0002-0372-5352 and Jones, C. ORCID: https://orcid.org/0000-0001-7537-1509 (2021) Kinetx: a combined flow cytometry assay and analysis software framework to quantitatively measure and categorize platelet activation in real-time. Journal of Visualized Experiments, 176. e62947. ISSN 1940-087X doi: 10.3791/62947

Abstract/Summary

Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests measure endpoint responses after a defined time period and not the rate of platelet activation. However, the rate at which platelets convert extracellular stimuli into a functional response is an essential factor in determining how efficiently they can respond to injury, bind to a forming thrombus, and signal to recruit other platelets. This paper describes a flow cytometry-based platelet function assay that enables simultaneous data acquisition and sample stimulation and utilizes newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux. Kinetix was developed in R so that users can alter parameters such as degree of smoothing, identification of outlying data points, or time scales. To aid users unfamiliar with the R environment, Kinetix analysis of data can be performed by a single command. Together, this allows real-time platelet activation metrics, such as rate, acceleration, time to peak-rate, time to peak-calcium, and qualitative shape changes, to be accurately and reproducibly measured and categorized. Kinetic measurements of platelet activation give a unique insight into platelets’ behavior during the first stages of activation and may provide a method of predicting the recruitment of platelets into a forming thrombus.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/99880
Identification Number/DOI	10.3791/62947
Refereed	Yes
Divisions	Life Sciences > School of Biological Sciences > Biomedical Sciences
Publisher	MYJoVE Corp
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