Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

Lordkipanidzé, Marie; Lowe, Gillian; Kirkby, Nicholas S.; Chan, Melissa; Lundberg, Martina H.; Morgan, Neil V.; Bem, Danai; Nisar, Shaista P.; Leo, Vincenzo C.; Jones, Matthew L.; Mundell, Stuart J; Daly, Mary E.; Mumford, Andrew D; Warner, Timothy D.; Watson, Steve P.

doi:10.1182/blood-2013-08-520387

Cited by 60 publications

(54 citation statements)

References 36 publications

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“…A total of 2.5 ml PRP is needed to compare all agonist concentrations on the plate, which is the equivalent of approximately 6 ml blood compared to the approximate 50 ml that would be required to produce the same analysis through LTA (32). The low blood volume required makes this method of particular benefit in cases where blood sample is limited, such as in patients with advanced disease and in neonates.…”

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%

“…Because the Optimul method requires only standard equipment and the ability to prepare and pipette PRP, it may be easily run by routinely trained staff. In addition, a wider range of agonists and concentrations can be studied which provides a ‘global portrait’ of platelet responsiveness that may prove beneficial in diagnosing the 1% of the population with a mild bleeding disorder (32). Indeed, in collaboration we have compared LTA and Optimul aggregometry in samples from participants with suspected bleeding disorders within the Genotyping and Phenotyping of Platelets study and demonstrated that LTA was able to detect platelet defects in 54% of these people while the Optimul method was able to detect 66% (32).…”

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%

Section: Lyophilized Agonist Platelet Aggregationmentioning

confidence: 99%

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96-well plate-based aggregometry

2018

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“…As all other parameters were identical these differences can only be explained by differences in the mechanical/physical environments of the two assays (3). LTA is fairly standardized with respect to the mixing of platelets with a stirrer bar turning at 1000–1200rpm.…”

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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“…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.…”

Section: Introductionmentioning

confidence: 99%

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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Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

Abstract: Key Points The Optimul 96-well platelet aggregation assay has high levels of sensitivity and specificity for detecting platelet defects. The requirement for a small volume of blood, straightforward nature, and speed make Optimul a promising screening test in bleeding patients.

Cited by 60 publications

References 36 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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