OMIP-097: High-parameter phenotyping of human platelets by spectral flow cytometry.

Using spectral flow cytometry, we developed a 16-color panel for analysis of platelet phenotype and function in human whole blood. The panel contains markers of clinical relevance and follows an optimized protocol for the high-parameter phenotyping of (phosphatidylserine positive) procoagulant platelets. Inclusion of established markers, such as CD62P and PAC-1, allows the subsetting of classic (proinflammatory and proaggregatory) phenotypes, while addition of novel markers, such as TLR9, allows the resolution of platelets with nonclassic functions.

Clinical Cytometry for Platelets and Platelet Disorders.

Clinical flow cytometry tests for inherited and acquired platelet disorders are useful diagnostic tools but are not widely available. Flow cytometric methods are available to detect inherited glycoprotein deficiencies, granule release (secretion defects), drug-induced thrombocytopenias, presence of antiplatelet antibodies, and pharmacodynamic inhibition by antiplatelet agents. New tests take advantage of advanced multicolor cytometers and allow identification of novel platelet subsets by high-dimensional immunophenotyping.

Platelet Phenotyping by Full Spectrum Flow Cytometry.

Platelets play key roles in hemostasis, immunity, and inflammation, and tests of platelet phenotype and function are useful in studies of disease biology and pathology. Full spectrum flow cytometry offers distinct advantages over standard tests and enables the sensitive and simultaneous detection of many biomarkers. A typical assay provides a wealth of information on platelet biology and allows the assessment of in vivo activation and in vitro reactivity, as well as the discovery of novel phenotypes.

Comprehensive phenotyping of human platelets by single-cell cytometry.

Platelets are small anucleate blood cells that contribute to hemostasis, immunity, and inflammation. Circulating platelets are heterogeneous in size, age, receptor expression, and reactivity. They inherit many features from megakaryocytes and are further modified on exposure to bioactive substances in the bloodstream.

Immunophenotypic Analysis of Platelets by Flow Cytometry.

Platelets are small but very abundant blood cells that play a key role in hemostasis, contributing to thrombus formation at sites of injury. The ability of platelets to perform this function, as well as functions in immunity and inflammation, is dependent on the presence of cell surface glycoproteins and changes in their quantity and conformation after platelet stimulation. In this article, we describe the characterization of platelet surface markers and platelet function using platelet-specific fluorescent probes and flow cytometry.

Platelet Immunophenotyping by High-Dimensional Mass Cytometry.

Platelets are small blood cells that contribute to hemostasis, immunity, and inflammation. Characterization of platelet surface markers allows for differentiation of activated platelets from resting platelets, diagnosis of platelet disorders, and investigation of platelet biology and pathology. In this article, we describe the use of mass cytometry or "CyTOF" (mass spectroscopy detection of metal-tagged antibodies on individual cells) to measure a large number of markers on each platelet and to identify platelet subsets based on the shared expression of multiple markers.

Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin.

Background: Robust platelet activation leads to the generation of subpopulations characterized by differential expression of phosphatidylserine (PS). Prostacyclin (PGI ) modulates many aspects of platelet function, but its influence on platelet subpopulations is unknown.

Objectives And Methods: We used fluorescent flow cytometry coupled to multidimensional fast Fourier transform-accelerated interpolation-based t-stochastic neighborhood embedding analysis to examine the influence of PGI on platelet subpopulations.

Effectiveness at 24 Months of Single-Source Generic Carbamazepine, Lamotrigine, or Levetiracetam in Newly Diagnosed Focal Epilepsy.

Background: Kaiser Permanente advocates using single-source generics for brand-name drugs. We compared the effectiveness of 3 different-generation generic antiepileptic drugs (AEDs) in patients with focal epilepsies.

Objective: To compare the effectiveness of the 3 most commonly used AEDs (carbamazepine [CBZ], lamotrigine [LTG], and levetiracetam [LEV]) after 24-month monotherapy.

Thrombospondin-1 promotes hemostasis through modulation of cAMP signaling in blood platelets.

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost.

Platelet mass cytometry: Optimization of sample, reagent, and analysis parameters.

Platelets mediate key biological processes, including hemostasis, immunity, and inflammation. Although platelets are often treated as a homogeneous cell population, they are known to be heterogeneous in size, age, surface receptor expression, and response to agonist stimulation, raising the possibility that distinct platelet subsets perform specialized functions and that such subsets may be altered in disease settings. Attempts to identify platelet subsets by flow cytometry have had limited success due in part to limits on the number of probes that can be used at the same time and due to the challenges of compensating for probes that have large spectral overlap.

Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations.

Platelet function is regulated by finely tuned phosphoprotein signals. Subtle aberrations in signaling can cause platelet hyperactivity and severe cardiovascular events. Mapping phosphorylation profiles in health and disease could accelerate antiplatelet discovery and enhance cardiovascular management, but traditional assays are ill-suited to clinical application as they are laborious and low throughput.

Atherogenic lipid stress induces platelet hyperactivity through CD36-mediated hyposensitivity to prostacyclin: the role of phosphodiesterase 3A.

Prostacyclin (PGI) controls platelet activation and thrombosis through a cyclic adenosine monophosphate (cAMP) signaling cascade. However, in patients with cardiovascular diseases this protective mechanism fails for reasons that are unclear. Using both pharmacological and genetic approaches we describe a mechanism by which oxidized low density lipoproteins (oxLDL) associated with dyslipidemia promote platelet activation through impaired PGI sensitivity and diminished cAMP signaling.

Platelet Flow Cytometry: Instrument Setup, Controls, and Panel Performance.

Platelet flow cytometry is widely used in cardiovascular medicine as the platelet surface is rich in clinical biomarkers. Surface profiling is critical in disease management, but current assays can abet clinical errors as they are suboptimal and prone to bias. Accordingly, the technical and analytical advances that can be used to create high quality assays with minimal error and maximal sensitivity were reviewed.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding.

High-Throughput Signaling Profiling in Blood Platelets by Multiplexed Phosphoflow Cytometry.

Multiplexed phosphoflow cytometry is a novel method that provides rapid and quantitative readouts on intracellular phosphoprotein signaling. In this approach, flow cytometry is combined with fluorescent cell barcoding (FCB) to facilitate high-throughput analyses of signaling events. After stimulation, fixed and permeabilized platelets are labeled with distinct dye intensities to create unique fluorescent signatures for individual samples.

Corrigendum: The Effect of a Simulated Commercial Flight Environment With Hypoxia and Low Humidity on Clotting, Platelet, and Endothelial Function in Participants With Type 2 Diabetes - A Cross-Over Study.

[This corrects the article on p. 26 in vol. 9, PMID: 29487564.

Platelet function following induced hypoglycaemia in type 2 diabetes.

Aim: Strict glycaemic control has been associated with an increased mortality rate in subjects with type 2 diabetes (T2DM). Here we examined platelet function immediately and 24hours following induced hypoglycaemia in people with type 2 diabetes compared to healthy age-matched controls.

Methods: Hyperinsulinaemic clamps reduced blood glucose to 2.

The Effect of a Simulated Commercial Flight Environment with Hypoxia and Low Humidity on Clotting, Platelet, and Endothelial Function in Participants with Type 2 Diabetes - A Cross-over Study.

Aims: To determine if clotting, platelet, and endothelial function were affected by simulated short-haul commercial air flight conditions (SF) in participants with type 2 diabetes (T2DM) compared to controls.

Methods: 10 participants with T2DM (7 females, 3 males) and 10 controls (3 females, 7 males) completed the study. Participants were randomized to either spend 2 h in an environmental chamber at sea level conditions (temperature: 23°C, oxygen concentration 21%, humidity 45%), or subject to a simulated 2-h simulated flight (SF: temperature: 23°C, oxygen concentration 15%, humidity 15%), and crossed over 7 days later.

Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets.

Background: Dissecting the signaling events that contribute to platelet activation will increase our understanding of platelet function and aid in the development of new antiplatelet agents. However, high-throughput methodology for the quantitative analysis of platelet signaling events is still lacking.

Objective: To develop a high-throughput assay for the analysis of platelet signaling events in whole blood.

Acute hypertriglyceridemia induces platelet hyperactivity that is not attenuated by insulin in polycystic ovary syndrome.

Background: Atherothrombosis is associated with platelet hyperactivity. Hypertriglyceridemia and insulin resistance (IR) are features of polycystic ovary syndrome (PCOS). The effect of induced hypertriglyceridemia on IR and platelet function was examined in young women with PCOS.

Evaluation of maturation and function of visual pathways in neonates: role of flash visual-evoked potentials revisited.

The purpose of our study was to establish the guidelines for interpreting neonatal flash visual-evoked potentials (FVEPs) by examining the correlation between maturation of the waveforms and conceptual age (CA). We retrospectively analyzed 220 consecutive neonatal FVEPs performed on premature and full-term infants. The CA of the participants ranged from 28 to 52 weeks.

Midline spikes.

Midline spikes are characterized by spike foci recorded at Cz, Fz, or Pz with amplitude ranging from 20 to 350 microvolts. Out of 7,929 EEGs performed at the Neurodiagnostics Laboratory, Kaiser Permanente Medical Center, Anaheim, California, between 1996 and 2006, 17 EEGs (0.21%) were identified as having interictal midline spikes with or without other epileptiform discharges.