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Counting the platelets: a robust and sensitive quantification method for thrombus formation
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Chemistry.
2016 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 115, no 6, p. 1178-1190Article in journal (Refereed) Published
Resource type
Text
Abstract [en]

Flow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, y- and z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups.

Place, publisher, year, edition, pages
SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN , 2016. Vol. 115, no 6, p. 1178-1190
Keywords [en]
Platelet aggregation; microfluidics; thrombosis; fluorescence microscopy; computer-assisted image processing
National Category
Clinical Laboratory Medicine
Identifiers
URN: urn:nbn:se:liu:diva-130073DOI: 10.1160/TH15-10-0799ISI: 000377237400011PubMedID: 26842994OAI: oai:DiVA.org:liu-130073DiVA, id: diva2:946974
Note

Funding Agencies|Swedish Research Council [K2015-79X-22644-01-3]; Linkoping University

Available from: 2016-07-06 Created: 2016-07-06 Last updated: 2018-09-06
In thesis
1. Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation
Open this publication in new window or tab >>Counting and Tracking: Development and Use of New Methods for Detailed Analysis of Thrombus Formation
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood platelets are a part of the complex system called haemostasis aimed at ensuring our blood’s continuous transport of oxygen and nutrients throughout the body. The transport is ensured by limiting blood loss due to vessel injury and in this process, the platelets form a plug in the damaged area, reinforced by the formation of fibrin. Similar mechanisms may cause thrombus formation, often triggered by atherosclerotic plaque rupture, causing vessel occlusion, embolism or ischemia, which may cause irreversible damage to the heart or the brain.

Platelet research is crucial for improved prevention and treatment of thrombotic disorders. For such research, flow chambers are an interesting tool for studies of platelet adhesion, aggregation and thrombus formation under similar flow conditions as in the blood vessels, which is important, as the flow affects the mechanisms involved in both haemostasis and thrombosis. Flow chambers can be designed for specific purposes, such as for the study of haemostasis at specific flow conditions or to evaluate drugs or biomaterials. In this thesis, our aim has been to improve the usefulness of in-vitro flow chambers and develop a more robust and informative image analysis of such experiments.

Initially, we introduced an internal control within each flow chamber experiment, thereby reducing the experimental variance caused by unknown factors. Furthermore, control and sample were thus exposed to identical experimental settings. By using platelet count as quantification of thrombus formation we introduce a method of analysis with increased or similar sensitivity to today’s standards. The platelet count method facilitated comparison of results obtained in different types of flow chambers by an absolute scale of measurement, independent of user settings. The platelet count method was further developed so that additional parameters could be analysed, providing more information about each individual platelet and the overall thrombus. The parameters analysed included platelet stability, height, movement and contraction. The method was used to evaluate how the pharmacokinetics of a reversible (ticagrelor) and irreversible (prasugrel) platelet ADP-receptor inhibitor affected the overall thrombus formation. Especially, how a non-inhibited platelet fraction, formed between drug administrations of irreversible inhibitors, affected thrombus formation. In addition, we sought to understand the regulation of the thrombin receptor, PAR1, expression in cancer cells. We found the microRNA miR20b to be antioncogenic through its downregulation of PAR1 expression.

This thesis contains numerous flow chamber experiments. However, for further use and full potential of the method increased standardisation is important. Our work regarding the quantification and analysis of flow chamber experiments will contribute to a more robust analysis and maybe even more important, provide new and detailed information on thrombus formation.  

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. p. 54
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1639
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:liu:diva-150960 (URN)10.3384/diss.diva-150960 (DOI)9789176852231 (ISBN)
Public defence
2018-10-05, Berzeliussalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2019-09-30Bibliographically approved

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Claesson, KjerstiLindahl, TomasFaxälv, Lars

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