Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test

Abstract Several recombinant thrombomodulin (r-TM) preparations have been developed for different clinical indications. While the in vitro effects of r-TM on blood coagulation parameters are extensively studied, the effect of this agent on platelet function tests such as the adhesion, aggregation, activation and secretion are not fully explored. The purpose of this study was to investigate the effect of a recombinant version of thrombomodulin (ART 123, Asahi, Pharmaceutical, Japan) on various platelet function tests. Platelet aggregation, platelet release and platelet activation by tissue factor (TF) utilizing flow cytometry studies were carried out. In the platelet aggregation studies, citrated whole blood was supplemented with graded amounts of r-TM, in a concentration range of 0–10 ug/ml in the blood of normal healthy volunteers (n=25). Platelet rich plasma (PRP) was prepared by controlled centrifugation (800g) for 15 minutes. Platelet count in the PRP was adjusted to 250,000/ul and aggregation studies were carried out using ADP (5 and 2.5 uM), and alpha thrombin (0.5 U/ml). Platelet activation studies were carried out using flow cytometric method utilizing citrated whole blood and recombinant TF and ADP as activators. In this procedure whole blood was supplemented with TM in a concentration range of 0–10 ug/ml and incubated. TF was then added and further incubated for an additional 2 minutes. Platelets were fixed and incubated with CD 61 and CD 62 antibodies and analyzed using the flow cytometer. Platelet release assays included the 14C serotonin release (SRA) from the washed platelets. In the platelet aggregation assay r-TM did not produce any significant inhibition of agonist induced aggregation with ADP and epinephrine, however, r-TM produced a strong concentration inhibition of thrombin induced aggregation with an IC 50 of 0.42 ug/ml. In the flow studies, r-TM produced an initial augmentation of the generation of microparticles at concentrations up to 0.31 ug/ml (ranges; 5–20%). However at concentrations > 0.31 ug/ml r-TM produced a concentration dependent inhibition of the microparticle formation with an IC 50 of 2.5 ug/ml. At concentrations of >5.0 ug/ml a complete inhibition of TF mediated microparticle formation was noted. Interestingly, r-TM did not produce any inhibition of the p-selectin expression at all concentrations studied. In the SRA, r-TM did not produce any release at concentrations up to 10 ug/ml. However, r-TM produced a strong inhibition of the alpha thrombin induced SRA release. These studies demonstrate that although in the agonist induced platelet aggregation studies r-TM does not produce any modulation of platelet aggregation responses with the exception of thrombin, in the flow cytometric studies it produces a biophasic response. In a concentration range of 0 - .31 ug/ml it produced a slight augmentation of the TF mediated platelet activation. However, at higher concentrations it produced an inhibition of the platelet microparticle formation. Interestingly, there was no effect of r-TM on p-selectin activation. These studies suggest that although r-TM does not produce any inhibition of the agonist induced aggregation of platelets, it can inhibit the TF mediated microparticle formation. Moreover, since r-TM at concentrations of up to 10 ug/ml does not produce any effect on p-selectin expression. It is unlikely to produce any primary hemostatic compromise in a therapeutic range of 2–6 ug/ml.

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Whole Blood Platelet Aggregometry and Platelet Function Testing

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0029-1220325 ◽

2009 ◽

Vol 35 (02) ◽

pp. 168-180 ◽

Cited By ~ 71

Author(s):

David McGlasson ◽

George Fritsma

Keyword(s):

Platelet Function ◽

Whole Blood ◽

Blood Platelet ◽

Platelet Function Testing ◽

Platelet Aggregometry ◽

Function Testing

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In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Blood ◽

10.1182/blood-2011-11-393900 ◽

2012 ◽

Vol 119 (17) ◽

pp. 4066-4072 ◽

Cited By ~ 55

Author(s):

Bethan Psaila ◽

James B. Bussel ◽

Matthew D. Linden ◽

Bracken Babula ◽

Youfu Li ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Whole Blood ◽

Immune Thrombocytopenia ◽

Ex Vivo ◽

Platelet Reactivity ◽

Adenosine Diphosphate ◽

High Dose ◽

Platelet Surface

Abstract The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

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Point-of-care monitoring of platelet function after abciximab therapy is poorly correlated with flow-cytometric measurements of platelet activation

Heart Lung and Circulation ◽

10.1046/j.1443-9506.2003.02290.x ◽

2003 ◽

Vol 12 (2) ◽

pp. A56

Author(s):

Michael J. Ray ◽

J.H.N. Nick Bett ◽

Paul T. McEniery ◽

Darren L. Walters ◽

Con N. Aroney

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Point Of Care ◽

Flow Cytometric

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Platelet-derived microparticles during and after acute coronary syndrome

Thrombosis and Haemostasis ◽

10.1160/th11-11-0779 ◽

2012 ◽

Vol 107 (06) ◽

pp. 1122-1129 ◽

Cited By ~ 49

Author(s):

Mika Skeppholm ◽

Fariborz Mobarrez ◽

Karin Malmqvist ◽

Håkan Wallén

Keyword(s):

Clinical Trials ◽

Acute Coronary Syndrome ◽

Platelet Activation ◽

Platelet Function ◽

Plasma Concentrations ◽

Healthy Controls ◽

Antithrombotic Treatment ◽

Coronary Syndrome ◽

Flow Cytometric ◽

Cell Fragments

SummaryAs microparticles are shedded upon platelet activation, and may be used to assess platelet function, we measured plasma concentrations of platelet-derived microparticles (PMPs) during and after an acute coronary syndrome (ACS). Fifty-one patients with ACS were investigated at admission, within 24 hours (before coronary angiography), and six months later. Sixty-one sex- and age-matched healthy controls were investigated once. PMPs were defined as particles <1.0 μm in size, negative to phalloidin (labels cell-fragments), and positive to CD61. Exposure of phosphatidylserine (PS+), CD62P and CD142 were also measured. Plasma concentrations of PS+PMPs exposing CD61, CD62P and CD142 were elevated 2.5, 6.0-, and 5.0-fold at admission (p<0.001 for all, compared to controls; aspirin only), decreased significantly 24 hours later following initiation of treatment with clopidogrel and subcutaneous anticoagulation (p<0.001 for all), and decreased even further six months later (p<0.01 for all). However, PS+PMPs exposing CD62P or CD142 were still between 1.2-and 2.3-fold higher than in controls (p<0.001 for both). The pattern for PS−PMPs during and after the ACS was very similar to that for PS+PMPs although the numbers were approximately 1/3 lower. In conclusion, PMP concentrations follow the pattern of platelet activation during and after an ACS. Decreased concentrations are observed after initiation of antithrombotic treatment, but PMP exposing CD62P or CD142 are still elevated after six months. Flow cytometric measurements of PMP in frozen-thawed samples enable studies of platelet function in larger clinical trials.

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Flow Cytometric Analysis of Platelet Function in Patients on Antiplatelet Therapy and Suspected Thrombocytopathy

Blood ◽

10.1182/blood-2018-99-110295 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1161-1161

Author(s):

Dana Huskens ◽

Mark Roest ◽

Lisa Florin ◽

Bas De Laat ◽

Katrien Devreese

Keyword(s):

Antiplatelet Therapy ◽

Platelet Function ◽

Assessment Tool ◽

Light Transmission ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Blood Platelet ◽

Flow Cytometric ◽

Healthy Donors ◽

Bleeding Score

Abstract Background: Light transmission aggregometry (LTA) is still the most used test for the identification and diagnosis of platelet function defects (PFD). Disadvantages of LTA include its laborious nature, the large volumes of blood required and the relative insensitivity to small changes in platelet function. We investigated if the flow cytometry-based whole blood platelet activation test (WB-PACT) correlates with LTA or could be used as a complimentary test. Methods: WB-PACT quantifies αIIbβ3 activation and P-selectin expression in response to 3 agonists and VWF binding to platelets in response to ristocetin. In total, 212 patients (51 on dual antiplatelet therapy (acetylsalicylic acid and ADP receptor antagonist), 161 suspected for bleeding diathesis) were tested. For WB-PACT parameters, the 2.5th, 10th and 25th percentiles were determined in 56 healthy donors to score the patient samples. For LTA, maximal aggregation, disaggregation and prolongation of the lagtime in response to 4 agonists and ristocetin was scored. Patients with at least one parameter lower than the 10th percentile measured with WB-PACT in healthy donors or at least one value deviating from the normal range measured with LTA were diagnosed with PFD. A bleeding score (BS) was calculated with the ISTH/SSC bleeding assessment tool (Rodeghiero et al., JTH, 2010). Results: A moderate correlation between LTA versus WB-PACT was found with a R of 0.60 (Figure 1). In total, 95 patients were diagnosed with PFD by both tests (κ=0.40, Table 1) and BS were recorded for 28/95 patients. Of these 28 patients, 25% had an elevated BS (adapted according to gender and age: >5 in women, >3 in men and >2 in children) and 36% had a BS>3. BS were recorded for 30/38 and 22/24 patients who were diagnosed with PFD according to WB-PACT only and LTA only, respectively. Interestingly, 27% of patients with PFD according to WB-PACT had an elevated BS and 40% had a BS>3. For LTA, only 13% of patients with PFD had an elevated BS and 13% had a BS>3. Conclusions: Flow cytometric analysis of platelet function by WB-PACT gives additional value to LTA to determine PFD. Disclosures No relevant conflicts of interest to declare.

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The Changes of Blood Platelet Reactivity in the Presence of Crude Extracts Isolated from Leaves and Twigs of Elaeagnus Rhamnoides (L.) A. Nelson Measuring in Whole Blood

10.21203/rs.3.rs-1171071/v1 ◽

2021 ◽

Author(s):

Bartosz Skalski ◽

Joanna Rywaniak ◽

Jerzy Żuchowski ◽

Anna Stochmal ◽

Beata Olas

Keyword(s):

Phenolic Compounds ◽

Platelet Activation ◽

Whole Blood ◽

Leaf Extract ◽

Platelet Reactivity ◽

Blood Platelets ◽

Blood Platelet ◽

Surface Expression ◽

Sea Buckthorn ◽

Crude Extracts

Abstract Uncontrolled blood platelet activation is an important risk factor of cardiovascular disease (CVDs). Various studies on phenolic compounds indicate that they have a protective effect on the cardiovascular system through different mechanisms, including the reduction of blood platelet activation. One of the plants that is particularly rich in phenolic compounds is sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson). The aim of the present study in vitro was to determine the anti-platelet properties of crude extracts isolated from leaves and twigs of E. rhamnoides (L.) A. Nelson in whole blood using flow cytometric and total thrombus-formation analysis system (T-TAS). The aim of our study was also analyze of blood platelet proteoms in the presence of different sea buckthorn extracts. A significant new finding is a decrease surface expression of P-selectin on blood platelets stimulated by 10 µM ADP and 10 µg/mL collagen, and a decrease surface expression of GPIIb/IIIa active complex on non-activated platelets and platelets stimulated by 10 µM ADP and 10 µg/mL collagen in the presence of sea buckthorn leaf extract (especially at the concentration 50 µg/mL). The twig extract also displayed antiplatelet potential. However, this activity was higher in the leaf extract than in the twig extract in whole blood. In addition, our present findings clearly demonstrate that investigated plant extracts have anticoagulant properties (measured by T-TAS). Therefore, the two tested extracts may be promising candidates for the natural anti-platelet and anticoagulant supplements.

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Effect of Oestrogen Dose on Whole Blood Platelet Activation in Women Taking New Low Dose Oral Contraceptives

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648985 ◽

1994 ◽

Vol 72 (06) ◽

pp. 926-930 ◽

Cited By ~ 12

Author(s):

Lucy A Norris ◽

John Bonnar

Keyword(s):

Arachidonic Acid ◽

Oral Contraceptive ◽

Platelet Activation ◽

Oral Contraceptives ◽

Whole Blood ◽

Contraceptive Use ◽

Blood Platelet ◽

Individual Treatment ◽

Oral Contraceptive Use ◽

Induced Aggregation

SummaryOral contraceptive use is known to cause changes in the haemostatic system. These changes are thought to be related to oestrogen dose and to provide a possible link between the increased risk of thromboembolic disease known to occur in women taking oestrogen containing oral contraceptives. This study measured whole blood platelet activation, serially, in women taking oral contraceptives containing 20 μg and 30 μg ethinyloestradiol combined with desogestrel. Increased levels of ADP and arachidonic acid induced aggregation were observed in women taking the 30 μg ethinyloestradiol combination. Platelet release of β-thromboglobulin (βTG) was also significantly increased. Increased collagen induced aggregation was observed but this failed to reach statistical significance for the individual treatment groups. In women taking the 20 μg ethinyloestradiol combination, a significant increase was only observed when platelets were stimulated with arachidonic acid. Platelet factor 4 (PF4) levels were unchanged in both groups. Significantly higher levels of βTG were observed in women taking the 30 μg ethinyloestradiol combination compared with women taking the 20 μg ethinyloestradiol combination. These results show that oral contraceptive use is associated with platelet activation. Women taking the 20 μg ethinyloestradiol combination show less changes in platelet activation than women taking the 30 μg ethinyloestradiol combination. This lower dose pill may therefore be particularly suitable for high risk women wishing to use oral contraception.

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Cold-stored whole blood platelet function is preserved in injured children with hemorrhagic shock

Journal of Trauma and Acute Care Surgery ◽

10.1097/ta.0000000000002340 ◽

2019 ◽

Vol 87 (1) ◽

pp. 49-53 ◽

Cited By ~ 12

Author(s):

Christine M. Leeper ◽

Mark H. Yazer ◽

Franklyn P. Cladis ◽

Richard Saladino ◽

Darrell J. Triulzi ◽

...

Keyword(s):

Hemorrhagic Shock ◽

Platelet Function ◽

Whole Blood ◽

Blood Platelet

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Comparison of Rapid Platelet Function Assay with Whole Blood Platelet Impedance Aggregometry for the Definition of Aspirin Resistance.

Blood ◽

10.1182/blood.v106.11.4018.4018 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4018-4018

Author(s):

Anna M. Dyszkiewicz-Korpanty ◽

Anne Kim ◽

James D. Burner ◽

Eugene P. Frenkel ◽

Ravindra Sarode

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Blood Platelet ◽

Aspirin Resistance ◽

Impedance Aggregometry ◽

Definition Of ◽

Induced Aggregation

Abstract The reported incidence of aspirin (ASA) resistance ranges from 5 to 30%. Various platelet function assays have been employed to detect aspirin resistance in patients with cardio- and cerebrovascular disease. Such a high proposed incidence of ASA resistance poses a critical need for a rapid point-of -care (POC) platelet function test. Unfortunately, no uniformly accepted definition of ASA resistance exists. Platelet aggregation studies that have been used to define ASA resistance are time consuming and require special technical expertise. The Ultegra Rapid Platelet Function -ASA (RPFA-ASA) has been developed as a POC test that is performed without sample processing. This optical method measures agglutination of fibrinogen-coated beads upon platelet activation with arachidonic acid. In the presence of aspirin effect, however, the agglutination of the beads is inhibited. The described cutoff of ≥ 550 Aspirin Reaction Units (ARU) is termed non-responsiveness to ASA based on a preclinical study and subsequent correlation with epinephrine-induced platelet aggregation in platelet rich plasma. Since RPFA-ASA uses whole blood, we validated its performance characteristics against a classic whole blood platelet aggregation assay (WBA). We studied 50 healthy volunteers, aged 25–75 (24 men, 26 women) with normal CBC, who had not ingested anti-platelet drugs for 14 days prior to the study. Baseline studies included WBA (dual channel aggregometer, Chrono-log Inc., Havertown, PA) using both arachidonic acid (AA -0.5; 0.25 mM) and collagen (1; 2 μg/mL) as well as an RPFA-ASA assay (Accumetrics Inc., San Diego, CA). These studies were repeated after 3 days of ASA (325 mg/d) intake. Based on a review of the literature, we defined an adequate ASA response as a completely inhibited AA-induced platelet aggregation and at least 30% inhibition of collagen-induced aggregation (both concentrations of the agonist). Thus, those with < 30% inhibition of aggregation response to collagen were considered ASA resistant. Eleven subjects were ASA resistant by WBA (20%; 8 females and 3 males (aged 25–63). In contrast, since all 50 subjects achieved ARU values of < 550 ARU, none were recognized as an ASA non-responder by the RPFA-ASA. While the current cutoff of < 550 ARU posed by the Ultegra RPFA-ASA does identify those who have taken ASA, the assay is unable to recognize ASA non-responders. Thus, based on these data, the appropriate cutoff for the recognition of ASA resistance by the RPFA-ASA should be re-adjusted to a significantly lower level to ensure appropriate assay results.

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