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- Banerjee, Meenakshi, et al.
(author)
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Prospective, International, Multisite Comparison of Platelet Isolation Techniques for Genome-Wide Transcriptomics : Communication from the SSC of the ISTH
- 2024
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836.
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Journal article (peer-reviewed)abstract
- Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the ISTH SSCs, we assessed how three of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALLTM filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting the techniques are transferrable and reproducible. Moreover, all three isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ3 activation is reduced by anti-CD45 bead isolation compared to washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.
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| 2. |
- Boknäs, Niklas, et al.
(author)
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Contact activation : important to consider when measuring the contribution of tissue factor-bearing microparticles to thrombin generation using phospholipid-containing reagents
- 2014
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836. ; 12:4, s. 515-518
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Journal article (peer-reviewed)abstract
- Background: A commercial MP reagent containing phospholipids is used for thrombin generation (TG) measurements to estimate the procoagulant activity of microparticles (MPs). Previous reports have shown that contact activation affects TG when TF levels are low, and that addition of phospholipids might augment this effect.Objectives: To quantify the impact of contact activation on TG in the presence of phospholipids and low/no TF, as is the case using a commercially available MP-reagent. Methods Thrombin generation was analyzed using MP- or platelet-rich plasma (PRP)-reagent in the presence and absence of corn trypsin inhibitor and anti-TF antibodies, respectively. To quantify the impact of different experimental parameters on contact activation, microparticle-depleted plasma was analyzed in the presence of different concentrations of phospholipids, TF and/or contact activating agents (kaolin).Results: Even with low contact activating blood collection tubes, substantial thrombin generation was observed with the MP-reagent, but this was completely inhibited by addition of corn trypsin inhibitor. Control experiments illustrate that the phospholipids in the reagent play a major role in enhancing TG initiated by FXIIa. Even with the PRP-reagent, which is recommended for determining the content of phospholipids from MPs, TG was partly dependent on contact activation.Conclusions: Contact activation plays a major role in TG when using reagents/samples containing phospholipids but little or no tissue factor. This needs to be considered and accounted for in future clinical studies using TG to assess the procoagulant activity of MPs.
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| 4. |
- Frelinger, Andrew L., 3rd, et al.
(author)
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Consensus recommendations on flow cytometry for the assessment of inherited and acquired disorders of platelet number and function : Communication from the ISTH SSC Subcommittee on Platelet Physiology
- 2021
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836. ; 19:12, s. 3193-3202
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Journal article (peer-reviewed)abstract
- Flow cytometry is increasingly used in the study of platelets in inherited and acquired disorders of platelet number and function. However, wide variation exists in specific reagents, methods, and equipment used, making interpretation and comparison of results difficult. The goal of the present study was to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet disorders. A modified RAND/UCLA survey method was used to obtain a consensus among 11 experts from 10 countries across four continents, on the appropriateness of statements relating to clinical utility, pre-analytical variables, instrument and reagent standardization, methods, reporting, and quality control for platelet flow cytometry. Feedback from the initial survey revealed that uncertainty was sometimes due to lack of expertise with a particular test condition rather than unavailable or ambiguous data. To address this, the RAND method was modified to allow experts to self-identify statements for which they could not provide expert input. There was uniform agreement among experts in the areas of instrument and reagent standardization, methods, reporting, and quality control and this agreement is used to suggest best practices in these areas. However, 25.9% and 50% of statements related to pre-analytical variables and clinical utility, respectively, were rated as uncertain. Thus, while citrate is the preferred anticoagulant for many flow cytometric platelet tests, expert opinions differed on the acceptability of other anticoagulants, particularly heparin. Lack of expert consensus on the clinical utility of many flow cytometric platelet tests indicates the need for rigorous multicenter clinical outcome studies.
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| 5. |
- Josefsson, Emma C., et al.
(author)
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Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets : communication from the Scientific and Standardization Committee of the ISTH
- 2023
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836. ; 21:8, s. 2291-2299
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Journal article (peer-reviewed)abstract
- BACKGROUND: Procoagulant platelets are a subpopulation of highly activated platelets that promote coagulation through surface-exposed, negatively charged phospholipids, especially phosphatidylserine (PS). Procoagulant platelets are important for clot stabilization during haemostasis and an increased number of these platelets is associated with thrombotic risk. There is a need for harmonisation in this area since many of the markers and methods used to assess procoagulant platelets are not specific when used in isolation but are also associated with platelet apoptosis.OBJECTIVE: We initiated this project to identify a minimum set of markers and/or methods that can detect and distinguish procoagulant platelets from apoptotic platelets.METHODS AND RESULTS: The study design involved a primary panel with twenty-seven international experts participating in an online survey and moderated virtual focus group meetings. Primary and secondary panel members were then invited to provide input on themes and statements generated from the focus groups. This led to a recommendation to use flow cytometry and a combination of the following three surface markers to differentiate procoagulant from apoptotic platelets: P-selectin (CD62P), PS (recognized by annexin V), and a platelet-specific receptor GPIX (CD42a) or αIIb integrin (CD41, GPIIb).CONCLUSION: Procoagulant platelets are expected to be positive for all three markers, while apoptotic platelets will be positive for annexin V and the platelet specific surface receptor(s) but negative for P-selectin.
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| 6. |
- Jourdi, Georges, et al.
(author)
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Consensus report on flow cytometry for platelet function testing in thrombocytopenic patients : communication from the SSC of the ISTH
- 2023
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In: Journal of Thrombosis and Haemostasis. - : John Wiley & Sons. - 1538-7933 .- 1538-7836. ; 21:10, s. 2941-2952
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Journal article (peer-reviewed)abstract
- Background: Platelet count alone does not reliably predict bleeding risk, suggesting platelet function is important to monitor in patients with thrombocytopenia. There is still an unmet need for improved platelet function diagnostics in patients with low platelet count in many clinical situations. Flow cytometry is a promising tool allowing reliable platelet function study in this setting.Objectives: The goal of this joint project between the International Society on Thrombosis and Haemostasis (ISTH) Scientific Standardization Committee (SSC) Subcommittees on Platelet Physiology and Platelet Immunology is to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet function, particularly activation, in patients with low platelet counts.Methods: A literature review was performed to identify relevant questions and areas of interest. An electronic expression of interest form was thereafter announced on the ISTH webpage, followed by a survey encompassing 37 issues regarding preanalytical, analytical, postanalytical, and performance aspects. Areas of disagreement or uncertainty were identified and formed the basis for 2 focus group discussions.Results: Consensus recommendations relative to patient sample collection, preanalytical variables, sample type, platelet-count cutoff, any potential specific modification of the standard flow cytometry protocol, and results expression and reporting are proposed based on the current practices of experts in the field as well as on literature review.Conclusion: The proposed consensus recommendations would allow standardization of protocols in upcoming clinical studies. The clinical utility of platelet function testing using flow cytometry to predict bleeding risk still needs rigorous multicenter outcome studies in patients with thrombocytopenia.
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