Comparison of Three Different Platelet Function Assays to Determine Aspirin Sensitivity.

Abstract In large studies of patients with cardiac disease, it was observed that up to 9% are ASA resistant (ASAR) and 23% are ASA semi responders using the platelet function analyzer (PFA-100), which measures whole blood platelet adhesion and aggregation under high shear. The prevalence of ASAR is of great clinical importance. With an estimated 20 million patients in the USA taking ASA for prevention of atherosclerotic events, even a 10% incidence equates to more than two million patients receiving inadequate anti-platelet therapy. Given the widespread use of ASA, more reliable and rapid methods to measure aspirin sensitivity are needed. Importantly, future large scale studies to determine the effect of monitoring ASA sensitivity to optimize therapy are compromised due to current lack of uniformity in assessing ASAR from center to center. The classical method of platelet aggregometry is labor-intensive and not readily adaptable to the clinical setting. Moreover, platelet aggregometry measures platelet responses under low shear, which does not simulate the high shear conditions expected to be involved in arterial thrombosis. In contrast, the PFA-100 does measure thrombus formation under high shear. The new TEG platelet coagulation assay uses whole blood, includes high shear, and is a point of care method. We compared these three techniques to assess aspirin effects on platelet function and clot formation: PFA-100 with collagen-epinephrine cartridges, TEG platelet coagulation assay, and standard optical aggregation in platelet rich plasma using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists. RESULTS: We found significant differences between the PFA-100, TEG platelet function, and standard optical aggregation. Twelve normal individuals previously defined as ASA sensitive or ASAR, using the PFA-100, were studied at baseline and following three days of oral aspirin at 81mg/day. We found that all four patients determined to be ASAR by PFA-100 were found to be sensitive in TEG and aggregometer assays when using AA as the agonist. Furthermore, some participants showed a gain of platelet function following ASA when studied on the TEG and aggregometer using ADP as the agonist. Currently, it is unclear which response to ASA treatment is most important to predict cardiovascular complications in normal individuals or patients placed on aspirin. Gum et al, showed that increased urinary secrection of thromboxane metabolites, suggesting ASA insensitivity, is associated with a higher incidence of cardiovascular events. Unfortunately, this test may simply indicate poor patient compliance rather than true ASAR, so a clear demonstration of platelet sensitivity will also be necessary. Our data confirms the need for a collaborative trial comparing different platelet assays to assess true ASA sensitivity together with concurrent measurements of urinary thromboxane metabolite levels. Knowing which assay best links aspirin sensitivity to disease outcome will allow physicians to better manage normal individuals and patients at risk for significant cardiovascular disease.

Download Full-text

Sensitivity and Efficacy in Measurement of Anti-Platelet Medications.

Blood ◽

10.1182/blood.v106.11.1253.1253 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1253-1253

Author(s):

Rachelle Blain ◽

Peggy Nakagawa ◽

Melissa Belvedere ◽

Monika Scherer ◽

Diane Nugent

Keyword(s):

Vascular Disease ◽

Platelet Function ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Large Population ◽

Adenosine Diphosphate ◽

Full Effect ◽

Normal Individuals ◽

Coagulation Assay ◽

Selection Of

Abstract INTRODUCTION: Large prospective studies must be performed to measure the true effect of clopidogrel (Plavix) or aspirin (ASA) in prevention of vascular disease. An easy and accurate point of service instrument will facilitate these studies and confirm effectiveness and compliance with medications. With such a variety of equipment available for analysis of platelet dysfunction, it is important to understand the sensitivity and efficacy of each assay when evaluating patients. We have found significant differences in our comparative studies of the PFA-100, optical aggregation, and Thrombelastograph (TEG) platelet coagulation in normal individuals using Plavix or ASA. METHODS: We have compared these three techniques to evaluate their effectiveness in assessing ASA and Plavix inhibition of platelet function. Eighteen normal individuals, who had been examined previously for ASA effect on these instruments, were studied at baseline and after three days of Plavix 75mg daily. All samples were drawn and run simultaneously. The TEG platelet coagulation assay used whole blood in a sodium heparin tube instead of a 3.2 % citrate tube which was used for aggregation and the PFA-100. The PFA-100 whole blood assays included both collagen-epinephrine (EPI) and collagen-adenosine diphosphate (ADP) cartridges. The TEG platelet coagulation assays were run with arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists. The optical aggregation used platelet rich plasma with AA and ADP agonists. RESULTS: We found significant differences between the PFA-100, TEG platelet function, and standard optical aggregation in measurement of Plavix effect. Optical aggregation and the TEG platelet assay did show the effects of Plavix, with the aggregometer being more sensitive than the TEG platelet assay although much more time consuming. In additional studies, the Plavix inhibition became more striking in the TEG by extending treatment to 6 days, suggesting that future studies should be done after a week (rather than 3 days) to see full effect. In contrast, Plavix effect was absent on the PFA-100. All three assays did show the effects of ASA in previously identified aspirin- responsive individuals. Ideally, one instrument could be used to assess response to both agents. However, the sensitivity of the instrument and the pharmacology of these and future medications must play an important role in the selection of which assay will be most informative for these large population studies in the prevention of vascular disease. Figure Figure

Download Full-text

Assessment of ADP-induced platelet aggregation with light transmission aggregometry and multiple electrode platelet aggregometry before and after clopidogrel treatment

Thrombosis and Haemostasis ◽

10.1160/th07-07-0478 ◽

2008 ◽

Vol 99 (01) ◽

pp. 121-126 ◽

Cited By ~ 181

Author(s):

Siegmund Braun ◽

Stefan Jawansky ◽

Wolfgang Vogt ◽

Julinda Mehilli ◽

Albert Schömig ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Whole Blood ◽

Light Transmission ◽

Platelet Rich Plasma ◽

Platelet Aggregometry ◽

Light Transmission Aggregometry ◽

Before And After ◽

Standardized Method ◽

Multiple Electrode

SummaryThe level of platelet aggregation, measured with light transmission aggregometry (LTA) in platelet rich plasma (PRP), has been shown to predict outcomes after percutaneous coronary intervention (PCI). However, measuring parameters of platelet function with LTA is time consuming and weakly standardized. Thus, a fast and standardized method to assess platelet function after clopidogrel treatment would be of great value for clinical practice. A new method, multiple electrode platelet aggregometry (MEA), to rapidly measure platelet aggregation in whole blood has recently been developed. The aim of this study was to assess parameters of platelet function with MEA and LTA before and after administration of 600 mg clopidogrel. Blood samples from 149 patients scheduled for coronary angiography were taken after clopidogrel treatment; in addition, in 60 of the patients samples were available before clopidogrel treatment. ADP-induced platelet aggregation was measured with LTA and simultaneously in whole blood with MEA on the Multiplate analyzer. Platelet aggregation measured with MEA decreased significantly after clopidogrel treatment (P<0.0001). ADP-induced platelet aggregation assessed with MEA and LTA correlated significantly (Spearman rank correlation coefficient=0.71; P<0.0001).The results of MEA, a fast and standardized method to assess the platelet response to ADP prior to and after clopidogrel treatment, correlate well with LTA.

Download Full-text

Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood

Thrombosis and Haemostasis ◽

10.1160/th09-07-0505 ◽

2010 ◽

Vol 104 (08) ◽

pp. 392-401 ◽

Cited By ~ 7

Author(s):

Katleen Broos ◽

Alexandre Fontayne ◽

Tímea Szántó ◽

Changgeng Ruan ◽

Alan Nurden ◽

...

Keyword(s):

Platelet Function ◽

High Throughput ◽

Whole Blood ◽

Large Scale ◽

Platelet Rich Plasma ◽

Sandwich Elisa ◽

Elisa Assay ◽

Platelet Response ◽

Healthy Donors ◽

Diagnostic Values

SummaryPlatelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or αIIbβ3 and GPIbα were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.

Download Full-text

Enhanced platelet aggregation and activation under conditions of hypothermia

Thrombosis and Haemostasis ◽

10.1160/th07-03-0189 ◽

2007 ◽

Vol 98 (12) ◽

pp. 1266-1275 ◽

Cited By ~ 41

Author(s):

Ruben Xavier ◽

Ann White ◽

Susan Fox ◽

Robert Wilcox ◽

Stan Heptinstall

Keyword(s):

Cardiac Surgery ◽

Platelet Aggregation ◽

Platelet Function ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Baseline Levels ◽

Spontaneous Aggregation ◽

High Concentrations ◽

Induced Aggregation ◽

P2y12 Antagonist

SummaryThe effects on platelet function of temperatures attained during hypothermia used in cardiac surgery are controversial. Here we have performed studies on platelet aggregation in whole blood and platelet-rich plasma after stimulation with a range of concentrations of ADP, TRAP, U46619 and PAF at both 28°C and 37°C. Spontaneous aggregation was also measured after addition of saline alone. In citrated blood, spontaneous aggregation was markedly enhanced at 28°C compared with 37°C. Aggregation induced by ADP was also enhanced. Similar results were obtained in hirudinised blood. There was no spontaneous aggregation in PRP but ADP-induced aggregation was enhanced at 28°C. The P2Y12 antagonist AR-C69931 inhibited all spontaneous aggregation at 28°C and reduced all ADP-induced aggregation responses to small, reversible responses. Aspirin had no effect. Aggregation was also enhanced at 28°C compared with 37°C with low but not high concentrations of TRAP and U46619. PAF-induced aggregation was maximal at all concentrations when measured at 28°C, but reversal of aggregation was seen at 37°C. Baseline levels of platelet CD62P and CD63 were significantly enhanced at 28°C compared with 37°C. Expression was significantly increased at 28°C after stimulation with ADP, PAF and TRAP but not after stimulation with U46619. Overall, our results demonstrate an enhancement of platelet function at 28°C compared with 37°C, particularly in the presence of ADP.

Download Full-text

Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIbα

Thrombosis and Haemostasis ◽

10.1160/th06-07-0367 ◽

2006 ◽

Vol 96 (12) ◽

pp. 774-780 ◽

Cited By ~ 18

Author(s):

Luca Pontiggia ◽

Beat Steiner ◽

Hans Ulrichts ◽

Hans Deckmyn ◽

Marc Forestier ◽

...

Keyword(s):

Monoclonal Antibody ◽

Thrombin Generation ◽

Platelet Rich Plasma ◽

Annexin V ◽

High Shear ◽

Double Labeling ◽

Platelet Aggregometry ◽

Microparticle Formation ◽

Arterial Stenoses ◽

Von Willebrand

SummaryWe studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIbα-von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a coneand-plate viscometer (shear: 5,000 s−1 for5 min) in the presence of antagonists to GPIbα (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared fromACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIbα with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ∼30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIbα, explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIbα with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.

Download Full-text

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

Download Full-text

Development Of a Novel Method To Assess Neonatal Platelet Function

Blood ◽

10.1182/blood.v122.21.4740.4740 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4740-4740

Author(s):

Kristina M. Haley ◽

Michael Recht ◽

Owen J.T. McCarty

Keyword(s):

Cord Blood ◽

Platelet Function ◽

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Platelet Response ◽

Primary Hemostasis ◽

Platelet Aggregometry ◽

Age Dependent ◽

Static Conditions

Background The hemostatic system is developmentally regulated, resulting in qualitative and quantitative differences in the mediators of primary and secondary hemostasis as well as fibrinolysis. Age-dependent values of pro- and anti-coagulant proteins have been determined. However, the task of defining age-dependent normal values of neonatal platelet function has been met with challenges owing to difficulties in obtaining adequate blood volumes for functional assays and inconsistent results amongst varying testing methods. In order to overcome many of these challenges, cord blood is often used as a source of neonatal platelets. Platelet aggregometry comparing adult and cord blood derived platelets has demonstrated a near lack of platelet response to epinephrine, collagen, and thromboxane in cord blood samples. In contrast, other studies of platelet function, such as flow cytometry, have failed to demonstrate this phenotypic difference. Assays of primary hemostasis reveal that neonatal blood mediates primary hemostasis as effectively as adult blood. In order to overcome the challenges associated with studying neonatal platelets, we have developed a novel platelet function assay employing small volumes of blood obtained directly from the neonate in order to assess platelet adhesion, activation, and aggregation simultaneously. Methods Eight-well slide chambers were coated with either fibrillar collagen or fibrinogen and allowed to adsorb at room temperature for one hour. Blood was obtained from healthy adult controls via venipuncture and neonatal samples via heelstick into sodium citrate. The blood was separated into two 200 µl aliquots, and TRAP (Thrombin Receptor Activating Peptide: 30 mM) was added to one aliquot. 100 µl of plain whole blood was added to both a collagen and a fibrinogen coated well and 100 µ of whole blood plus TRAP was added to a fibrinogen coated well. The samples were then incubated at 37°C for 30 minutes. Non-adherent cells were washed three times with modified HEPES-Tyrode buffer. FITC-P-selectin was then added (10 µg/ml), and the samples were incubated at 37 oC for 10 minutes and subsequently washed. Samples were imaged with differential interference contrast (DIC) and fluorescence microscopy on a Zeiss Axiovert 200 M microscope. Results Platelet adhesion, activation, and aggregation were assessed for 3 neonatal samples and 3 adult control samples. Both adult and neonatal platelets adhered to fibrinogen and collagen equally. Exposure to collagen and fibrinogen (+/- TRAP) resulted in alpha granule release and P-selectin expression in both neonatal and adult platelets. In addition, both adult and neonatal platelets were observed to undergo the characteristic cytoskeletal changes that result in platelet spreading on fibrinogen (+/- TRAP) and collagen surfaces. Both neonatal and adult platelets were observed to form platelet aggregates on both surfaces under static conditions. (Figure 1) Conclusions We have successfully developed a novel platelet function assay using small volumes of whole blood to assess three key platelet functions: adhesion, activation, and aggregation. This is the first study to demonstrate that neonatal platelets spread on adhesive and extracellular matrix proteins and suggests that neonatal platelets contain the cytoskeletal machinery necessary to undergo this change in platelet formation. This assay fills a critical need in clinical pediatric hematology where efforts to diagnose and treat neonatal platelet dysfunction are often met with technical challenges related to conventional platelet function assays. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text