| 1. |
- 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. |
- Allentoft, Morten E., et al.
(author)
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Population genomics of post-glacial western Eurasia
- 2024
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In: Nature. - 0028-0836 .- 1476-4687. ; 625:7994, s. 301-311
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Journal article (peer-reviewed)abstract
- Western Eurasia witnessed several large-scale human migrations during the Holocene1–5. Here, to investigate the cross-continental effects of these migrations, we shotgun-sequenced 317 genomes—mainly from the Mesolithic and Neolithic periods—from across northern and western Eurasia. These were imputed alongside published data to obtain diploid genotypes from more than 1,600 ancient humans. Our analyses revealed a ‘great divide’ genomic boundary extending from the Black Sea to the Baltic. Mesolithic hunter-gatherers were highly genetically differentiated east and west of this zone, and the effect of the neolithization was equally disparate. Large-scale ancestry shifts occurred in the west as farming was introduced, including near-total replacement of hunter-gatherers in many areas, whereas no substantial ancestry shifts happened east of the zone during the same period. Similarly, relatedness decreased in the west from the Neolithic transition onwards, whereas, east of the Urals, relatedness remained high until around 4,000 bp, consistent with the persistence of localized groups of hunter-gatherers. The boundary dissolved when Yamnaya-related ancestry spread across western Eurasia around 5,000 bp, resulting in a second major turnover that reached most parts of Europe within a 1,000-year span. The genetic origin and fate of the Yamnaya have remained elusive, but we show that hunter-gatherers from the Middle Don region contributed ancestry to them. Yamnaya groups later admixed with individuals associated with the Globular Amphora culture before expanding into Europe. Similar turnovers occurred in western Siberia, where we report new genomic data from a ‘Neolithic steppe’ cline spanning the Siberian forest steppe to Lake Baikal. These prehistoric migrations had profound and lasting effects on the genetic diversity of Eurasian populations.
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| 3. |
- 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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| 4. |
- Ivert, Torbjorn, et al.
(author)
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Platelet function one and three months after coronary bypass surgery in relation to once or twice daily dosing of acetylsalicylic acid
- 2017
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In: Thrombosis Research. - : Elsevier. - 0049-3848 .- 1879-2472. ; 149, s. 64-69
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Journal article (peer-reviewed)abstract
- Introduction: Current guidelines recommend acetylsalicylic acid (ASA) treatment after coronary artery bypass grafting (CABG) to reduce thrombotic vein graft occlusion. The optimal dosage of ASA is not known. Materials and methods: Forty-two patients undergoing elective CABG were randomized to receive either ASA 75 mg or 160 mg once daily (OD) or 75 mg twice daily (BID) after the operation. Platelet function testing was performed before, and one and three months after the operation. Results: White blood cell counts increased during the initial postoperative days whereas platelet countswere initially slightly reduced after the operation but increased after one month without any major changes of mean platelet volumes. Serum thromboxane B-2 was more effectively suppressed at one and three months after the operation with ASA 75 mg BID or 160 mg OD than with 75 mg OD (p < 0.001). ASA 75 mg BID and 160 mg OD were equally effective. Adenosine diphosphate stimulated platelet aggregation in whole blood (Multiplate (R)) was increased one and three months after the operation, and this was counteracted by ASA 75 mg BID but not by 75 or 160 mg OD. Arachidonic acid-induced aggregation was more effectively inhibited by 75 mg BID or 160 mg OD compared to 75 mg OD at three months. Conclusions: Less effective inhibition of platelet activation was obtained with ASA 75 mg OD than with ASA 160mg OD or 75mg BID up to three months after CABG. Especially the latter dose is of interest for further studies of efficacy and clinical outcomes after CABG.
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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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