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Background: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). Multiple factors may concur to explain this, including increased amount of highly reactive immature platelets., Objectives: To investigate the association between immature platelets and reactivity determined with multicolour flow cytometry using the SYTO-13 dye in STEMI patients., Methods: We conducted an observational study of 59 patients with acute STEMI. Blood samples were obtained within 24 h after admission and after loading doses of dual antiplatelet therapy. For comparison, samples were obtained from 50 healthy individuals. Immature platelets and platelet reactivity were investigated using multicolour flow cytometry including the SYTO-13 dye that binds to platelet RNA and thus provides a method for subdividing platelets into immature and mature platelets. Additionally, we assessed platelet aggregation, serum-thromboxane B 2 levels and standard immature platelet markers., Results: Immature platelets were more reactive than mature platelets in both STEMI patients and healthy individuals (p-values < 0.05). STEMI patients had lower platelet aggregation and thromboxane B 2 levels than healthy individuals. We found a positive association between automatically determined immature platelet markers and CD63 expression on activated platelets (Spearman's rho: 0.27 to 0.58, p-values < 0.05)., Conclusions: Our study shows that immature platelets identified with a multicolour flow cytometric method using the SYTO-13 dye are more reactive than mature platelets in patients with acute STEMI and in healthy individuals. The presence of immature platelets may be important for the overall platelet reactivity, which may have implications for the effect of antiplatelet therapy., Competing Interests: Declaration of competing interest None related to the present study. The authors report the following general conflict. ELG has received speaker honoraria or consultancy fees from AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Pfizer, Novo Nordisk, MSD, Lundbeck Pharma and Organon. He is investigator in clinical studies sponsored by AstraZeneca, Idorsia or Bayer and has received unrestricted research grants from Boehringer Ingelheim. AMH has received unrestricted research support from CSL Behring. SDK is coordinating national investigator in a clinical trial sponsored by Idorsia. OBP, PHN and LP have no conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Flow cytometry is an increasingly used method for platelet function analysis because it has some important advantages compared with other platelet function tests. Flow cytometric platelet function analyses only require a small sample volume (3.5 mL); however, to expand the field of applications, e.g., for platelet function analysis in children, even smaller volumes are needed. Platelets are easily activated, and the size of the needle for blood sampling might be of importance for the pre-activation of the platelets. Moreover, to use flow cytometry for investigation of platelet function in clinical practice, a reference interval is warranted. The aims of this work were 1) to determine if small volumes of whole blood can be used without influencing the results, 2) to examine the pre-activation of platelets with respect to needle size, and 3) to establish reference intervals for flow cytometric platelet function assays. To examine the influence of sample volume, blood was collected from 20 healthy individuals in 1.0 mL, 1.8 mL, and 3.5 mL tubes. To examine the influence of the needle size on pre-activation, blood was drawn from another 13 healthy individuals with both a 19- and 21-gauge needle. For the reference interval study, 78 healthy adults were included. The flow cytometric analyses were performed on a NAVIOS flow cytometer (Beckman Coulter, Miami, Florida) investigating the following activation-dependent markers on the platelet surface; bound-fibrinogen, CD63, and P-selectin (CD62p) after activation with arachidonic acid, ristocetin, adenosine diphosphate, thrombin-receptor-activating-peptide, and collagen. The study showed that a blood volume as low as 1.0 mL can be used for platelet function analysis by flow cytometry and that both a 19- and 21-gauge needle can be used for blood sampling. In addition, reference intervals for platelet function analyses by flow cytometry were established.

This letter to the editor discusses the challenges of diagnosing platelet disorders and presents a case study of a patient with a δ-granule storage pool disease. The patient had a history of bleeding symptoms and was diagnosed using flow cytometry analysis of CD63 expression on activated platelets and liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement of plasma serotonin release after platelet activation. The study suggests that these methods may be reliable for diagnosing δ-granule defects in laboratories with the necessary equipment. The authors emphasize the importance of using multiple methods for accurate diagnosis and highlight the limitations of light transmission aggregometry (LTA) in detecting δ-granule defects. [Extracted from the article]

Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, "SYTO-high") corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, "SYTO-low") corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity. [ABSTRACT FROM AUTHOR]

Increased platelet activity is an important predictor for recurrent cardiovascular events in patients with acute coronary syndromes (ACS). Flow cytometry is an advanced method for evaluation of platelet activity. We aimed to summarize the current literature on dynamic changes in platelet activity analyzed by flow cytometry in patients with ACS. Employing the guidelines of Preferred Report Items for Systematic Reviews and Meta-Analyses (PRISMA), we searched PubMed and Embase on October 26, 2021, and identified studies measuring platelet activity with flow cytometry in ACS patients in the acute phase (baseline) and at follow-up in a more stable phase. In the 12 included studies, fibrinogen receptor, α-granule secretion, platelet reactivity index, monocyte-platelet aggregates, neutrophil-platelet aggregates, and reticulated platelets were measured. The fibrinogen receptor and α-granule secretion were either unchanged or lower during follow-up measurements than in the acute phase. Platelet reactivity index showed inconsistent results. Values of monocyte-platelet aggregates and neutrophil-platelet aggregates were lower at follow-up than at baseline ( p -values <0.05). Reticulated platelets were either unchanged ( p -value >0.64) or lower at 1 to 2 months follow-up ( p -value 0.04), and also lower at 5 months to 1-year follow-up ( p -value >0.005) compared with baseline. Overall, flow cytometric analyses of platelet function in ACS patients showed that platelet activity was lower at follow-up than at baseline. However, in some patients, platelet activity remained unchanged from baseline to follow-up, possibly indicating a sustained high platelet activity that may increase the risk of recurrent cardiovascular events. [ABSTRACT FROM AUTHOR]

Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard–Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard–Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count. [ABSTRACT FROM AUTHOR]