Multiparameter platelet function analysis of bleeding patients with a prolonged platelet function analyser closure time

The antiestrogenic drug tamoxifen, used in patients with breast cancer, is associated with an increase in arterial and venous thrombotic events, the mechanism of which is not clearly understood. We report a case of a lady who presented with new bruising and prolonged bleeding following a tooth extraction 4–6 weeks after starting tamoxifen. Investigations were consistent with an acquired platelet storage pool disorder. Repeat platelet function analysis was normal, performed 3 months after discontinuation of tamoxifen. We present a previously clinically unreported effect of tamoxifen on platelet function.

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The influence of the haematocrit on primary haemostasis in vitro

Thrombosis and Haemostasis ◽

10.1160/th05-06-0424 ◽

2005 ◽

Vol 94 (12) ◽

pp. 1213-1218 ◽

Cited By ~ 70

Author(s):

Marco Eugster ◽

Walter H. Reinhart

Keyword(s):

Platelet Function ◽

Type I Collagen ◽

Inverse Correlation ◽

Type I ◽

Membrane Pore ◽

Closure Time ◽

Function Analyzer ◽

Platelet Function Analyzer

SummaryPrimary haemostasis consists of platelet adhesion to subendothelial collagen, their activation and aggregation and finally the formation of a platelet plug. Erythrocytes are involved in this process because they flow in the center of the vessel and push platelets towards the site of action on the vessel wall and enhance shear forces, which activate platelets. In the platelet function analyzer PFA-100® (Dade Behring, Düdingen, Switzerland), the in vivo situation is simulated in vitro with blood being aspirated at high shear rates (5000s-1) through a capillary into a membrane pore with a diameter of 150 μm coated with type I collagen and either epinephrine or adenosine diphosphate. Aggregating platelets plug the pore and stop the flow, which is measured as the closure time. We analysed the influence of erythrocytes on platelet function analyzer measurements by systematic variation of the haematocrit (20,30,40,and 50%) at constant platelet counts of 289±61 ×103/μl plasma, or 152±30 ×103/μl blood, 96±9 ×103/μl blood and 54±5 ×103/μl blood, respectively. An inverse correlation was found between haematocrit and closure time under all circumstances. A decrease of the platelet count by 50 ×103 /μl could be compensated for by a 10% increase in haematocrit. The haematocrit must, therefore, be taken into consideration for the correct interpretation of PFA-100® measurements. Our data also provide a pathophysiological rationale to reduce the risk of bleeding in patients with thrombocytopenia and anaemia by normalizing the haematocrit with erythrocyte transfusions.

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Safe removal of an epidural catheter 72 hours after clopidogrel and aspirin administrations guided by platelet function analysis and thromboelastography

Journal of Anaesthesiology Clinical Pharmacology ◽

10.4103/0970-9185.105813 ◽

2013 ◽

Vol 29 (1) ◽

pp. 99 ◽

Cited By ~ 3

Author(s):

Henry Liu ◽

Eric Glenn ◽

Judson Mehl ◽

FrancisA Rosinia

Keyword(s):

Epidural Catheter ◽

Platelet Function ◽

Function Analysis

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Monitoring of Clopidogrel Action: Comparison of Methods

Clinical Chemistry ◽

10.1373/clinchem.2004.047050 ◽

2005 ◽

Vol 51 (6) ◽

pp. 957-965 ◽

Cited By ~ 133

Author(s):

Jörg Geiger ◽

Lino Teichmann ◽

Ralf Grossmann ◽

Barsom Aktas ◽

Udo Steigerwald ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Function ◽

Significant Proportion ◽

Coronary Intervention ◽

Platelet Response ◽

Closure Time ◽

Vasp Phosphorylation ◽

Function Analyzer ◽

Potent Drug ◽

Platelet Function Analyzer

Abstract Background: Clopidogrel is a potent drug for prevention of adverse effects during and after coronary intervention. Increasing experience indicates that a significant proportion of patients do not respond adequately to clopidogrel. Because failure of antiplatelet therapy can have severe consequences, there is need for a reliable assay to quantify the effectiveness of clopidogrel treatment. Methods: Of 24 healthy volunteers admitted to the study, 18 were treated for 1 week with clopidogrel (300-mg loading dose and 75-mg maintenance dose), and 6 with placebo. Platelet function was monitored by 2 assays, based on flow cytometry and enzyme immunoassay, that measure the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) and by aggregometry, flow cytometry of P-selectin, and the platelet function analyzer at baseline, on days 1–5, and on day 9 of treatment. Results: Aggregometry and VASP phosphorylation revealed a loss of platelet response to ADP within 12 h after clopidogrel intake. The phosphorylation status of VASP correlated with the inhibition of platelet aggregation. In contrast, neither P-selectin expression nor PFA-100 closure time was a clear indicator of clopidogrel effects on platelets. Conclusions: VASP phosphorylation assays are reliable for quantifying clopidogrel effects. Because the VASP assay directly measures the function of the clopidogrel target, the P2Y12 receptor, the assay is selective for clopidogrel effects rather than effects of other platelet inhibitors commonly in use.

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Ionising Irradiation Influences Platelet Adhesion and Aggregation under High Flow Conditions.

Blood ◽

10.1182/blood.v106.11.1894.1894 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1894-1894

Author(s):

Robert Loncar ◽

Fatima Araba ◽

Christof Schmuling ◽

Folker Wenzel ◽

Rudiger E. Scharf

Keyword(s):

Gamma Irradiation ◽

Platelet Function ◽

Standard Dose ◽

Poor Response ◽

Immunocompromised Host ◽

Rapid Onset ◽

Response To Treatment ◽

Blood Components ◽

Test Analysis ◽

Closure Time

Abstract Transfusion-associated graft-versus-host disease (GVHD) is a dramatic immunologic reaction mediated by transfused immunocompetent lymphocytes directed against an immunocompromised host. GVHD shows a rapid onset and is characterized by severe symptoms and a poor response to treatment (mortality rate > 90%). Prophylactic gamma irradiation of cellular blood components is the most efficient and reliable way to prevent GVHD. The currently recommended standard dose of gamma irradiation of blood products is 25 Gy. While irradiated blood components including platelet concentrates have been extensively transfused over the past 25 years, only few studies (with controversy results) evaluated the effect of ionising irradiation on platelet morphology and function. In this study, we examined the effect of a standard irradiation dose of 25Gy on platelet function using a platelet function analyzer (PFA-100). Citrate-anticoagulated blood was obtained in duplicate from twelve healthy blood donors. One sample was immediately irradiated according to a standard protocol (25 Gy), the other specimen was used as control. Platelet function of irradiated and control samples was evaluated using PFA-100. This device applies a high shear flow system to simulate the conditions under which platelets are subjected at the site of a damaged blood vessel wall. Anticoagulated whole blood was passed through the membranes coated with either collagen and epinephrine (Col/Epi) or collagen and ADP (Col/ADP) under hemodynamic conditions similar to those of small capillaries. Platelets adhere and aggregate across the aperture until the flow ceases, evaluated by the closure time (CT, sec). Statistical analysis was performed using a standard software (Sigma Plot v. 8; Student’s paired t test). Analysis of platelet function with Col/Epi cartridges showed that irradiated samples had a prolonged closure time compared to corresponding non irradiated controls (144 ± 21 sec vs. 120 ± 18 sec, p<0.05). The averaged CT prolongation was 19±5%. Analysis of CT using Col/ADP cartridges showed also prolongated CT (averaged prolongation 7%) in seven of nine irradiated samples compared to non irradiated controls (97 ± 14 sec vs. 103 ± 13 sec, p<0.05). Our results suggest that a standard dose of ionising irradiation for prevention of GVHD has a significant influence on platelet function. The biochemical nature by which this platelet dysfunction following irradiation is caused, remains to be assessed in further detail.

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Rapid Assessment of Platelet Function Using Thromboelastography and Small Volumes of Citrated Whole Blood.

Blood ◽

10.1182/blood.v106.11.3995.3995 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3995-3995 ◽

Cited By ~ 1

Author(s):

Fred G. Pluthero ◽

Margaret L. Rand ◽

Victor S. Blanchette ◽

Walter H. Kahr

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Whole Blood ◽

Function Analysis ◽

Maximum Amplitude ◽

Ease Of Use ◽

Platelet Response ◽

Platelet Function Disorders ◽

Wide Range

Abstract Platelet function disorders are a key cause of abnormal bleeding, and diagnosis is challenging because: platelet abnormalities are diverse, affecting many aspects of function; variability in platelet function testing in clinical laboratories makes it difficult to compare results; large blood volumes required for platelet function analysis make it difficult to perform in neonatal patients; manipulation of platelet rich plasma used for platelet aggregation can lead to test variability; platelet aggregation curves are difficult to interpret in thrombocytopenic patients. We describe a method of testing platelet function using citrated whole blood and thromboelastography (TEG) that overcomes some of these limitations. Commercially-available platelet mapping kits allow the effects of the platelet agonists adenosine diphosphate (ADP) and arachidonic acid (AA) to be assessed via a TEG assay where reptilase and activated factor XIII produce fibrin clots independent of thrombin in heparinized whole blood. The activation and aggregation of platelets is quantified by measuring the difference in maximum amplitude (MA) between unstimulated samples, which form weak fibrin-only clots, and samples with agonists added, which form stronger clots containing fibrin and activated/aggregated platelets. Platelet mapping was used as the basis for a TEG assay which can be used to assess platelet responses to a wide range of stimuli - including ADP, AA, epinephrine, collagen, U46619 (thromboxane-A2 receptor agonist), SFLLRN (PAR-1 thrombin receptor activating peptide) and AYPGKF (PAR-4 activating peptide) - in small samples (330μL) of citrated native (CN) blood or plasma to which heparin is added to a concentration of 20U/mL. Samples were recalcified by adding calcium chloride to 10mM (necessary for the function of reptilase and FXIIIa), and other reagent volumes were the same as in platelet mapping assays, with fibrin activator prepared at 1/2 regular strength. The concentrations of platelet agonists were: collagen 51μg/ml, epinephrine 0.27μM, ADP 5.4μM, arachidonic acid 135μg/mL, U46619 2.6μM, SFLLRN 6.76μM and AYPGKF 34μM. These concentrations produced TEG MA values in heparinated fibrin-activated CN blood from a panel of normal individuals comparable to those obtained from recalcified CN blood in the absence of heparin (the fibrin/platelet response control). The platelet response was rapid with maximum amplitudes reached within 10 minutes for all agonists except collagen, which required >30 minutes to produce maximum amplitude. We have found this TEG platelet-response assay to be useful in detecting platelet function abnormalities, producing results which correlate with and extend those of other platelet function tests. For example in one patient a weak response to epinephrine corresponded to similar platelet aggregation results, and in another the TEG assay detected a weak PAR-1 response not specifically detected in other tests. The assay has also proven useful in assessing platelet function in blood and plasma having low platelet concentrations (<50 x 10E9/L) from experimental or pathological causes (e.g. thrombocytopenia), in titrating platelet responses to agonists and in assessing the effects of antiplatelet agents in vivo and in vitro. Thus this TEG platelet function assay has the advantages of speed, ease of use, flexibility, adaptability to low platelet concentrations and sample economy, requiring small volumes of citrated blood which can be used for other coagulation assays and platelet response tests.

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The Aptamer ARC1779 Potently and Specifically Inhibits the Excessive von Willebrand Factor (vWF) Activity and vWF-Mediated Ex Vivo Platelet Function of Patients with Thrombotic Thrombocytopenic Purpura (TTP).

Blood ◽

10.1182/blood.v110.11.279.279 ◽

2007 ◽

Vol 110 (11) ◽

pp. 279-279 ◽

Cited By ~ 1

Author(s):

Bernd Jilma ◽

Florian B. Mayr ◽

Paul Knoebl ◽

Alexander O. Spiel ◽

Patricia G. Merlino ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Ex Vivo ◽

Healthy Controls ◽

Citrate Anticoagulation ◽

Closure Time ◽

A1 Domain ◽

Plug Formation ◽

Novel Therapeutic

Abstract Background: ARC1779 is an aptamer which blocks the binding of the vWF A1 domain to platelet GPIb receptors. In TTP there is an excess of ultra-large multimers of vWF which are especially avid for binding GPIb and give rise to disseminated platelet thrombi which are fibrin-poor and vWF-rich in composition. ARC1779 is being evaluated for use as front-line therapy of acute TTP in conjunction with plasma exchange. ARC1779 has already been demonstrated in healthy volunteers to inhibit vWF activity and vWF-dependent platelet function. ARC1779 has no anticoagulant effect and does not inhibit other pathways of platelet activation. ARC1779 is expected to normalize platelet dysfunction and prevent the thrombotic end-organ complications of TTP based upon the mechanism of action defined for ARC1779 and the mechanism of thrombosis defined for TTP. Methods: We first assessed vWF activity (vWF:RiCO) and platelet function in blood samples taken from TTP patients and age-matched, healthy controls. We then studied the ex vivo dose response curves for ARC1779 on vWF activity (free A1 domain sites) and on platelet function assessed by the Platelet Function Analyzer (PFA-100®), cone and plate analyzer (IMPACT®), and agonist-induced impedence platelet aggregometry (Multiplate®) of TTP patients (N=10, 2 in acute phase and 8 in remission) and healthy age-matched controls (N=23). Results: vWF:RiCO activity (p=0.002) and vWF-dependent platelet plug formation (p=0.001) were increased in TTP patients relative to healthy controls, but agonist-induced platelet aggregation (ADP, arachidonic acid, collagen, TRAP) was not. ARC1779 fully blocked platelet plug formation as measured by PFA-100® with an IC100 of ∼ 1 mcg/mL with citrate anticoagulation, and ∼ 3–4 mcg/mL with hirudin anticoagulation in both TTP patients and in healthy controls. ARC1779 fully blocked shear-dependent platelet adhesion measured by the IMPACT® analyzer with an IC100 of ∼ 1 mcg/mL with citrate anticoagulation in both TTP patients and in healthy controls. ARC1779 fully blocked vWF activity (free A1 domain sites) with an IC90 of ∼ 6 mcg/mL in TTP patients and ∼ 2 mcg/mL in young controls (p<0.001 between groups). ARC1779 did not inhibit platelet aggregation by ADP, collagen or arachidonic acid at concentrations (10mcg/mL) that fully inhibited vWF dependent platelet function. Conclusions: ARC1779 potently and specifically inhibits vWF activity and vWF dependent platelet function in the setting of TTP where vWF activity is increased. ARC1779 represents a novel therapeutic principle (vWF antagonism) and a novel therapeutic class (aptamers) with potential for the treatment of TTP. vWF Activity and Platelet Function in TTP Patients and Age-Matched Controls Population Healthy Controls TTP Patients values shown as mean +/− standard deviation Sample Size N=23 N=10 vWF:RiCO (%) 94.9 +/− 60.4 153.3 +/− 55.9 PFA-100® Closure Time in Citrate (sec) 90.9 +/− 16.0 66.8 +/− 12.7 PFA-100® Closure Time in Hirudin (sec) 84.0 +/− 12.9 64.6 +/− 11.9 ARC1779 IC100 PFA in Citrate (ARC1779 mcg/mL) 0.9 +/− 0.4 1.4 +/− 0.6 ARC1779 IC100 PFA in Hirudin (ARC1779 mcg/mL) 3.2 +/− 1.5 4.4 +/− 2.7 IC100 IMPACT in Citrate (ARC1779 mcg/mL) 0.8 +/− 1.2 0.8 +/− 0.8 IC90 vWF free A1 domain sites (ARC1779 mcg/mL) 1.8 +/− 0.8 6.2 +/− 2.7

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The Relationship between ABO Groups, Factor VIII, von Willebrand Factor Levels and Platelet Function Analysis Using Cone and Plate(let) Analyzer.

Blood ◽

10.1182/blood.v110.11.4024.4024 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4024-4024

Author(s):

Maria Lourdes Barjas Castro ◽

Aline Crucello ◽

Heloise P. Fernandes ◽

Norma C. Sousa ◽

Joyce M. Annichino-Bizzacchi ◽

...

Keyword(s):

Factor Viii ◽

Blood Group ◽

Platelet Function ◽

Von Willebrand Factor ◽

Function Analysis ◽

World Health ◽

Enzyme Linked Immunosorbent Assay ◽

Abo Blood Group ◽

Von Willebrand ◽

Willebrand Factor

Abstract ABO blood group has been described to influence levels of von Willebrand factor (VWF), as well as factor VIII. Individuals carrying O allele have significant lower plasma levels of these factors. Indeed, recently non-O individuals have been described to have increased risk for both, arterial and venous thrombotic disease. VWF mediate platelet interaction with areas of damage blood vessel wall. Thus, it could be interesting to evaluate the possible influence of the ABO group in this interaction, particularly in situations in which low levels of VWF are close to those found in VW disease (such in O group). Cone and plate(let) analyzer (CPA) represent a simple and fast method, that allow the evaluation of platelet function (adhesion as well aggregation) in whole blood under shear conditions, closer to physiological conditions. In this method, no platelet agonists are needed and interaction with fibrinogen and VWF is particularly evaluated. The aim of the present study was to evaluate the influence of ABO group in platelet function using CPA. Samples from 15 male blood donors with no history of drug intake, were submitted to ABO serology and molecular analysis, VWF:Ag, FVIII dosages, and CPA analysis using Impact-R (Diamed - Switzerland), according to manufacturer’s instructions. ABO phenotypes were determined by agglutination test using monoclonal and polyclonal anti-A, B and AB antibodies (Asem-NPBI, São Paulo Brazil; DiaMed SA, Suisse; DiaMed Latino América, Brazil). H antigen was determined using anti-H lectin from Ulex europaeus (DiaMed Latino América, Brazil). ABO genotyping was performed by polymerase chain reaction (PCR) amplification of exons 6 and 7 of the ABO gene, followed by diagnostic restriction enzyme digestion. Factor VIII coagulant was measured by a one stage clothing method using a factor-VIII deficient substrate. VWF:Ag was measured by an enzyme linked immunosorbent assay (ELISA) using polyclonal antiserum (Dako, Denmark). Lyophilised commercial reference preparations of VWF:Ag, and FVIII, standardized against the World Health Organization standard, were used as the standards in this study. The age of the donors ranged from 27–65 years (median = 42 years). The donors were distributed according to ABO groups: 5 = OO; 5 = AB; 5 = AO. Median levels of factor VIII, according to blood group were: OO= 79% (70–142%); AO= 87% (80–140%); AB= 112% (98–200%). Median levels of VWF, according to blood group were: OO= 79% (50–99%); AO= 82% (73–120%); AB= 169% (92–250%). CPA analysis presented the following results: median AS in μm2 (average size) - OO= 24 (23–42); AO= 33 (24–42); AB= 23 (21–24) - median SC in % (surface coverage) - OO= 7.1 (4–13); AO= 8 (5–8); AB= 6.9 (4.8–8). No significant differences using Wilcoxon’s rank sum test were found among groups, when platelet function was analyzed. In conclusion, our results suggest that, although O allele carriers present lower levels of both factor VIII and VWF, the use of platelet function analysis does not seem to predict the risk for bleeding or thrombosis, according to individual ABO blood group.

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Comprehensive Quality Management of Multiple Electrode Platelet Aggregometry.

Blood ◽

10.1182/blood.v114.22.4463.4463 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4463-4463

Author(s):

Michael Spannagl ◽

Andrea Dick ◽

Andreas Calatzis

Keyword(s):

Quality Control ◽

Quality Management ◽

Correlation Coefficient ◽

Platelet Function ◽

Function Analysis ◽

Management Approach ◽

Platelet Function Tests ◽

Control Material ◽

Test Cells ◽

Multiple Electrode

Abstract Abstract 4463 Platelet function analysis provides quantitative results which may reveal platelet disorders, platelet inhibition during anti-platelet therapy or anti-platelet drug resistance. The results may have important consequences on patients therapy. As in all laboratory methods, a comprehensive quality management approach is crucial and increasingly demanded by regulatory authorities. In platelet function methods quality control is hampered by the fact that platelets are not stable over longer time periods and loose their functional activities after freezing and freeze-drying. Therefore for most platelet function tests no control materials are available. When no biological quality control material is available, it is even more important to install and maintain a quality management approach, which covers as many influence factors and sources of error as possible. Here we present the quality management procedures of Multiple Electrode Aggregometry (MEA) a relative new platelet function test based on the analysis of whole blood (Multiplate analyzer, Dynabyte medical, Munich, Germany). In the MEA device temperature of the measurement system is controlled by the analyser and can be verified by an external QC kit. The signal reaction of this method is based on the rise of electrical resistance induced by the adhesion and aggregation of activated blood platelets on metal sensor electrodes in a disposable test cell. In order to control possible sensor inconsistencies and improve precision, the test cell incorporates two independent sensor units, each consisting of 2 silver-coated highly conductive wires. The duplicate sensors thus serve as an internal control. During each measurement Pearson's correlation coefficient of single measurements of the curves assessed by the two electrode pairs and the difference of the two AUCs are calculated automatically by the analyzer's software. The result is flagged if the values are outside of the acceptance range (correlation coefficient <0.98, difference to the mean curve >20%). The instrument has an integrated procedure for an electronic control which checks the function of the electronic amplifier in the analyzer. In addition liquid controls are available, based on solutions with different ional strength. Using these solutions the instrument, pipettor and test cells are controlled. Using blood from a healthy individual, users can control qualitatively all aspects of the analysis (instrument, test cells, reagents, pipettor). Abnormal control reagents are available, containing either aspirin, a GpIIbIIIa antagonist or prostaglandin E1, which can produce an abnormal result when added to a normal blood sample before the analysis. The instrument provides an electronic pipettor with interactive software-guided operation procedures, which help standardise the analysis and minimize user-related errors. Using artificial liquid control materials a pilot external QC was performed in 6 individual centers and the results were centrally analyzed. Level I control was determined as 125+-6 aggregation units (AU, mean+-sd), level II control was 64+-5 AU. Coefficients of variation of all determinations were 4.6% and 7.3% respectively. In conclusion it is shown that while a stable biological control material for platelet function analysis is not available, it is possible to perform quality controls covering many parts of the analytical procedure. Manufacturers and users of platelet function tests should try to implement control procedures that cover as many aspects of the technology they apply as possible to ensure correct performance of the tests over the lifetime of the instrument, test cells and reagents. As long as stable biological quality control materials for platelet function analysis are not available, qualitative biological controls using normal blood or plasma should be combined with artificial control materials or electronic test procedures according to the analytical reaction which is performed. If the biological reaction cannot be quantitatively controlled, then at least the physical process leading to the signal of the respective test procedure should be verified (measurement of pressure, optical density or electrical impedance). Using a step by step approach comprehensive quality management of platelet function analysis is feasible and should be implemented in routine. Disclosures: Calatzis: Dynabyte Medical : Equity Ownership, Patents & Royalties.

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