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

2008 ◽  
Vol 99 (01) ◽  
pp. 121-126 ◽  
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.

Multiple electrode aggregometry: A new device to measure platelet aggregation in whole blood

2006 ◽  
Vol 96 (12) ◽  
pp. 781-788 ◽  
Author(s):  
Andreas Calatzis ◽  
Sandra Penz ◽  
Hajna Losonczy ◽  
Wolfgang Siess ◽  
Orsolya Tóth
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Blood Platelet ◽  
Platelet Inhibition ◽  
New Device ◽  
Platelet Aggregometry ◽  
Spontaneous Platelet Aggregation ◽  
Induced Aggregation ◽  
Blood Platelet Aggregation ◽  
Multiple Electrode

SummarySeveral methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p<0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications.

Enhanced platelet aggregation and activation under conditions of hypothermia

2007 ◽  
Vol 98 (12) ◽  
pp. 1266-1275 ◽  
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.

Standardization of Light Transmission Aggregometry for Diagnosis of Platelet Disorders: An Inter-Laboratory External Quality Assessment

2019 ◽  
Vol 119 (07) ◽  
pp. 1154-1161 ◽  
Author(s):  
Karina Althaus ◽  
Barbara Zieger ◽  
Tamam Bakchoul ◽  
Kerstin Jurk ◽  
Keyword(s):  
Platelet Function ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Reference Ranges ◽  
Technical Problems ◽  
Platelet Function Disorders ◽  
Light Transmission Aggregometry ◽  
Laboratory Survey ◽  
Definition Of

AbstractSeveral in vitro platelet function tests are available for the diagnosis of inherited platelet function disorders. Currently, the light transmission aggregometry (LTA) is recommended as one of the first-step tests. LTA is available in most specialized hemostasis laboratories. Although the LTA is accepted as a ‘gold standard’ assay for the evaluation of platelet function, its standardization in the clinical practice is still challenging. The GTH-based THROMKID-Plus Study Group has performed an inter-laboratory trial in Germany and Austria. Five different agonists were selected according to the Scientific and Standardization Committee/International Society on Thrombosis and Haemostasis recommendations and shipped in 3 different sets (one should represent a healthy control and two should simulate platelet function disorders) to 15 specialized laboratories in Germany and Austria. Agonists were analyzed by APACT or PAP4/8 aggregometer using platelet-rich plasma from healthy donors. In addition, laboratory-internal platelet agonists were tested in platelet-rich plasma from a healthy donor. All laboratories (9 used APACT, 6 used PAP4/PAP8) showed very consistent data regarding the maximum percentage of aggregation induced by the tested agonists and identified the differential diagnosis of the simulated platelet function disorders with one exception, which was due to technical problems. In contrast, there was a high variability of the laboratory-internal inductors regarding reagent type, concentrations and pathological cut-off values. Our study showed that the shipment of agonists is suitable for an inter-laboratory survey of LTA. However, there is still a remarkable need for standardization of agonist reagents and their concentration as well as for definition of reference ranges.

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3901-3901
Author(s):  
Rachelle Blain ◽  
Peggy Nakagawa ◽  
Thomas Kunicki ◽  
Melissa Belvedere ◽  
Diane Nugent
Keyword(s):  
Platelet Function ◽  
Whole Blood ◽  
Large Scale ◽  
Classical Method ◽  
Platelet Rich Plasma ◽  
High Shear ◽  
Aspirin Sensitivity ◽  
Normal Individuals ◽  
Platelet Aggregometry ◽  
Coagulation Assay

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.

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

Blood ◽  
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 &lt; 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 &lt; 550 ARU, none were recognized as an ASA non-responder by the RPFA-ASA. While the current cutoff of &lt; 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.

Platelet Aggregation Induced By Formaldehyde

1981 ◽  
Author(s):  
J A Zeller ◽  
K Eurenius ◽  
R E Dayhoff ◽  
R S Ledley ◽  
L S Rotolo
Keyword(s):  
Platelet Aggregation ◽  
Small Volume ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Microscopic Analysis ◽  
Formaldehyde Concentration ◽  
Microscopic Method ◽  
Light Transmission Aggregometry ◽  
Quantitative Image ◽  
Aggregation Analysis

Formaldehyde causes platelet aggregation (or agglutination) which varies with dosage. Aggregation was studied in ten blood samples drawn from normal volunteers. A small volume of formaldehyde was added to platelet rich plasma in a light transmission aggregcmeter to produce final formaldehyde concentrations between 0.1% and 8%. Measurements were made by three methods: (1) light transmission aggregometry, (2) visual semi-quantitative microscopic analysis, and (3) quantitative image analysis (Computerized Platelet Aggregation Analysis) . Using the visual semi-quantitative microscopic method, the dose response curve (expressed as the percentage of platelets involved in aggregates) increased from 11% at a formaldehyde concentration of 0.1% to 41% at a 0.5% formaldehyde concentration; it then decreased to 11% at a formaldehyde concentration of 8%. This curve may reflect the influence of two different formaldehyde effects: an aggregating effect which increases until about 0.5% concentration, whereafter a fixative effect may predominate. Light transmission aggregometry recordings did not provide a reliable indicator of the presence and degree of aggregation. A comparison of the visual semi-quantitative method and CPAA show similar detection of aggregating effects. In sunmary, formaldehyde has an aggregating effect on normal platelets. The physiologic significance of this effect is unknown; however, because formaldehyde is used to process platelets in studies of platelet aggregation, this effect may be of importance.

Platelet Aggregation in Whole Blood - Studies with a Platelet Counting Technique - Methodological Aspects and Some Applications

1989 ◽  
Vol 61 (03) ◽  
pp. 423-428 ◽  
Author(s):  
C Falcon ◽  
J Arnout ◽  
J Vermylen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
High Sensitivity ◽  
Optimal Concentration ◽  
Counting Technique ◽  
Platelet Function Disorders ◽  
Methodological Aspects ◽  
Storage Times

SummaryWe describe a method for measuring platelet aggregation in whole blood by single platelet counting. The importance of a low stirring speed (100 rpm) to obtain agonist-specific aggregation is stressed. Despite this low stirring speed, the sensitivity to agonists equals that of the turbidometric technique in platelet-rich plasma. The optimal concentration of formaldehyde for fixing the aggregates, the effects of storage times and anticoagulant are studied. Applicability to the study of platelet function inhibitors or of inherited platelet function disorders is illustrated. It is concluded that this technique, used under the appropriate conditions, combines the advantage of measuring platelet aggregation in a more physiologtc environment with the advantages of the turbidometric technique such as high sensitivity.

Evaluation of Platelet Dysfunction In Children: Improved Detection and Efficiency

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1446-1446
Author(s):  
Diane J. Nugent ◽  
Ryan Roberts ◽  
Peggy Nakagawa
Keyword(s):  
Platelet Function ◽  
Mutational Analysis ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Clot Formation ◽  
Activity Levels ◽  
Food History ◽  
Platelet Dysfunction ◽  
Light Transmission Aggregometry ◽  
Arachadonic Acid

Abstract Abstract 1446 Currently, there is no single assay that will detect platelet function abnormalities in all individuals. We prospectively studied 369 patients with a strong history of bruising and mucosal membrane bleeding for possible platelet dysfunction following documentation of normal Von Willebrand antigen and activity levels. In an effort to evaluate platelet function under more diverse conditions we chose to simultaneously evaluate 1) aggregation using platelet rich plasma and light transmission aggregometry (LTA), 2) adhesion under high shear using the Platelet Function Analyzer (PFA-100, ADP-collagen, and Epinephrine-collagen cartridges) and 3) platelet initiated clot formation using heparinized whole blood and the two standard mapping agonists, arachadonic acid (AA) and ADP (Hemascope Thromboelastograph Analyzer). Of the 369 platelet evaluations performed, 87 patients (24%) were found to be normal in all three assays with all agonists. On repeated assays with increased attention to medication and food history, an additional 152 patients were felt to have a transient or acquired dysfunction which improved or normalized on further testing. Leaving 130 patients with persistent bleeding and documented abnormalities on one or more of the assays used. There were no patients with only Collagen (CN) or arachadonic acid (AA) aggregation alone on LTA. Using LTA, there was one patient with combined CN and ADP aggregation defect only, another with AA and EPI absent aggregation only, and one with only AA and Thrombin receptor agonist peptide (TRAP) aggregation abnormalities. A summary of the remaining 127 patients are displayed in the table below:Abnormal AssayLTA EPILTA ADPLTA ADP + EPILTA ADP, EPI CNLTA ADP, EPI, AALTA ADP, EPI CN, AAPFA and PLT Mapping ONLYNumber201240841825M/F3M/17F6M/6F16M/24F1M/7F1M/3F8M/10F12M/13FPFA1M/2F4M7M/4F01M01M/3FPlt Mapping2M/2F1M/2F3F1F009M/7FBoth PFA and Mapping1M0001F2M/9F2M/3F Summary: By using a combination of three assays, we were able to identify 25 additional patients with significant platelet dysfunction detected with abnormal platelet mapping or PFA-100 despite normal light transmission aggregometry. Patients with the most abnormalities on aggregation, also demonstrated abnormal adhesion and platelet initiated clot formation. However, the use of PFA-100 and/or platelet mapping alone would miss the majority of patients with aggregation defects. In the future, the unique combination of platelet function defects as measured by these assays, and future technologies, will not only improve detection, but also facilitate phenotype to genotype associations and expedite mutational analysis. Disclosures: Off Label Use: Rituximab to treat ITP.

Effect of Heparin:PMX60056 Complexes on Platelet Aggregation Induced by ADP or HIT Antibody

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4386-4386
Author(s):  
Walter Jeske ◽  
R. Eric McAllister ◽  
Jeanine M. Walenga ◽  
Michelle Paulus ◽  
Jawed Fareed
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Anticoagulant Activity ◽  
Platelet Factor ◽  
Induce Platelet Aggregation ◽  
Heparin Induced Thrombocytopenia ◽  
Platelet Aggregometry ◽  
Positively Charged

Abstract Abstract 4386 Introduction: A neutralization of anticoagulant activity occurs when heparin binds to a variety of positively charged substances such as protamine and platelet factor 4 (PF4). PMX60056 (PolyMedix, Radnor, PA) is a novel compound that is being developed as a heparin antagonist. Since heparin-PF4 complexes are antigenic, with antibodies against this complex activating platelets to trigger thrombin generation, thrombus formation and associated morbidities, it is of interest to determine whether heparin:PMX60056 complexes affect platelet function. This study compares the effect of PMX60056 and protamine, alone and complexed to heparin, on platelet function as assessed by platelet aggregometry. Materials and Method: Whole blood, collected from 10 healthy individuals, was anticoagulated with 3.2% sodium citrate and centrifuged to make platelet rich plasma (PRP). PRP was supplemented with 10 μ g/ml heparin (~1.5 IU/ml), 10 μ g/ml heparin antagonist (protamine or PMX 60056) or a complex of 10 μ g/ml heparin and 10 μ g/ml heparin antagonist. Platelet aggregation was stimulated by the addition of ADP (5 or 10 μ M final concentration) or serum from a patient with heparin-induced thrombocytopenia. Result: ADP-induced platelet aggregation was not affected by the addition of heparin, protamine, PMX 60056, or complexes of heparin with heparin antagonist. In the HIT system, heparin + HIT serum led to a significant increase in platelet aggregation vs. saline (46.7 ± 3.2 % vs. 8.2 ± 2.9%). HIT serum + heparin antagonist did not induce platelet aggregation (PMX60056: 10.2 ± 4.4%; protamine: 11.6 ± 3.5%). The aggregation responses to HIT serum + heparin (46.7 ± 3.2%), HIT serum + heparin:PMX60056 (43.6 ± 5.8%) and HIT serum + heparin:protamine (47.8 ± 3.8%) were not significantly different. Conclusion: When mixed at equigravimetric amounts, protamine and PMX60056 do not prevent formation of immune complexes consisting of HIT antibody and heparin which lead to platelet activation. Previous data from human trials has suggested that smaller amounts of PMX60056 (less than equigravimetric) may effectively neutralize heparin. Thus, it is speculated that smaller heparin:PMX60056 complexes may induce less antibody formation than larger heparin:protamine complexes. Validation of this hypothesis in animal models or clinical studies is warranted. Disclosures: McAllister: PolyMedix, Inc.: Employment.

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