Standardised optical multichannel (optimul) platelet aggregometry using high-speed shaking and fixed time point readings

Chan, Melissa; Warner, Timothy D.

doi:10.3109/09537104.2011.603066

Cited by 33 publications

(41 citation statements)

References 7 publications

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“…The lyophilization of the plates and subsequent vacuum packing produces plates that may be stored at room temperature for up to 12 weeks before use. We have also removed the variation in shaking type, speed and time by recommending a standard plate shaker as well as reducing the volume of PRP needed for each test to 40 μl (18,31). …”

Section: Lyophilized Agonist Platelet Aggregationmentioning

confidence: 99%

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96-well plate-based aggregometry

2018

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While there are many bench and bedside tests to assess platelet reactivity, ex vivo light transmission aggregometry (LTA) remains the gold standard. LTA, however, is expensive, time-consuming and requires dedicated equipment and staff, making it impractical in many situations. In addition, there is significant variability between data generated at different testing sites meaning that tests often need to be repeated if a patient is transferred to the care of a different hospital. As such, there is clearly an unmet need for standardization of platelet testing.Using the principles of LTA, aggregometry can be conducted in 96-well plates with readings being made in a standard plate reader. This approach allows for the assessment of multiple concentrations of agonists, since the volume of platelets required for each test is significantly lower than for LTA. Furthermore, the lyophilization of a set panel of agonists to a 96-well plate to produce a stable assay substrate allows the production of portable, standardized plates that can be used to generate reproducible tests at multiple sites.In this review, we will discuss the methods and uses of 96-well plate aggregometry for both research and the clinic.

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Section: Lyophilized Agonist Platelet Aggregationmentioning

confidence: 99%

“…We and others, however, have demonstrated that the effect of anti-platelet drugs can be elucidated using this method (18,31,32). …”

Section: Lyophilized Agonist Platelet Aggregationmentioning

confidence: 99%

96-well plate-based aggregometry

2018

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“…We have coupled this with half-area 96-well plates pre-coated with a standardized panel of agonists where the operator needs only to pipette PRP and PPP into the appropriate wells. Aggregation can then be calculated easily from a set formula (6,7). Furthermore, the large number of replicates and broad range of agonist concentrations allow 96-well plate aggregometry to be used to construct concentration-response curves, which permits deeper analyses of platelet function, useful to the characterization of the effects of antiplatelet drugs or genetic variations.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the large number of replicates and broad range of agonist concentrations allow 96-well plate aggregometry to be used to construct concentration-response curves, which permits deeper analyses of platelet function, useful to the characterization of the effects of antiplatelet drugs or genetic variations. While this is a standardized assay, we have previously demonstrated how mixing the 96-well plates within a kinetic plate reader produces different results than mixing the same plates on the external vortex mixer (7), another illustration of the strong influences of the mechanical environment upon platelet aggregation.…”

Section: Discussionmentioning

confidence: 99%

Not all light transmission aggregation assays are created equal: qualitative differences between light transmission and 96-well plate aggregometry

et al. 2018

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In this short article, submitted as part of the review on platelet function testing, we illustrate the quantitative and qualitative differences between classical light transmission aggregometry (LTA) and 96-well plate aggregometry.We show that responses to platelet agonists and antagonists differ depending upon the method of aggregation testing. For example, in 96-well aggregometry, responses to collagen are strongly inhibited by P2Y12 receptor antagonists while in LTA they are much less affected. Furthermore, we explore the importance of differences in the mechanical environment upon platelet aggregation.We emphasize that LTA and 96-well aggregometry are not interchangeable assays. These two assays are best used as complementary tests to explore platelet function in depth.

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“…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 functional assays.We previously developed the Optimul assay for measuring platelet aggregation in low volumes (40 mL) of platelet-rich plasma (PRP) 7,8 and have very recently reported its utility in the testing of platelet function patients with bleeding disorders.9 However, the Optimul system relies on optical measurement of sample turbidity to assess platelet aggregation, making it unsuitable for analysis of platelet function in whole blood. 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.…”

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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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Standardised optical multichannel (optimul) platelet aggregometry using high-speed shaking and fixed time point readings

Cited by 33 publications

References 7 publications

96-well plate-based aggregometry

96-well plate-based aggregometry

Not all light transmission aggregation assays are created equal: qualitative differences between light transmission and 96-well plate aggregometry

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

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