Collagen-epinephrine Cartridges Research Articles

PFA-100 System: A New Method for Assessment of Platelet Dysfunction.

This is a celebratory reprint of a historical paper published in STH in 1998. The original Abstract follows.The PFA-100 system is a platelet function analyzer designed to measure platelet-related primary hemostasis. The instrument uses two disposable cartridges: a collagen/epinephrine (CEPI) and a collagen/ADP (CADP) cartridge. Previous experience has shown that CEPI cartridges detect qualitative platelet defects, including acetylsalicylic acid (ASA)-induced abnormalities, while CADP cartridges detect only thrombocytopathies and not ASA use. In this seven-center trial, 206 healthy subjects and 176 persons with various platelet-related defects, including 127 ASA users, were studied. The platelet function status was determined by a platelet function test panel. Comparisons were made as to how well the defects were identified by the PFA-100 system and by platelet aggregometry. The reference intervals for both cartridges, testing the 206 healthy subjects, were similar to values described in smaller studies in the literature (mean closure time [CT] of 132 seconds for CEPI and 93 seconds for CADP). The use of different lot numbers of cartridges or duplicate versus singleton testing revealed no differences. Compared with the platelet function status, the PFA-100 system had a clinical sensitivity of 94.9% and a specificity of 88.8%. For aggregometry, a sensitivity of 94.3% and a specificity of 88.3% were obtained. These values are based on all 382 specimens. A separate analysis of sensitivity by type of platelet defect, ASA use versus congenital thrombocytopathies, revealed for the PFA-100 system a 94.5% sensitivity in identifying ASA users and a 95.9% sensitivity in identifying the other defects. For aggregometry, the values were 100% for ASA users and 79.6% for congenital defects. Analysis of concordance between the PFA-100 system and aggregometry revealed no difference in clinical sensitivity and specificity between the systems (p > 0.9999). The overall agreement was 87.5%, with a Kappa index of 0.751. The two tests are thus equivalent in their ability to identify normal and abnormal platelet defects. Testing 126 subjects who took 325 mg ASA revealed that the PFA-100 system (CEPI) was able to detect 71.7% of ASA-induced defects with a positive predictive value of 97.8%. The overall clinical accuracy of the system, calculated from the area under the receiver operating characteristic curve, was 0.977. The data suggest that the PFA-100 system is highly accurate in discriminating normal from abnormal platelet function. The ease of operation of the instrument makes it a useful tool to use in screening patients for platelet-related hemostasis defects.

Platelet Dysfunction in Blood Donors Detected by Platelet Function Analyzer PFA-100™.

BackgroundPlatelet transfusions may be indicated to prevent and treat bleeding in patients with quantitative or qualitative platelet defects. Millions of platelet components are transfused worldwide. It is well known that platelet dysfunction predicts blood loss after surgery. Hence, the quality of the platelet donation and the resulting platelet concentrate are critical for the transfusion. The aim of this study is to assess platelet function in well-qualified blood donors.MethodologyBlood samples from 275 blood donors were collected in 0.129 M (3.8%) sodium citrate tubes prior to routine blood donation. Platelet function was assessed by measuring the closure time (CT) on the platelet function analyzer (PFA-100™; Siemens Health Diagnostics, Marburg, Germany) using collagen/epinephrine (CEPI) and collagen/ADP (CDP) cartridges.ResultsUsing the PFA-100™, 20.4% of donors had an abnormal platelet function test, of whom 9.4% had prolonged CT with two cartridges, 7% had only prolonged CEPI CTs consistent with aspirin-like defect, and 4% had prolonged CADP CTs only. We found no closure (>300 seconds) in 6.54% of donors, including 1.45% with the CEPI cartridge, 2.9% with the CADP cartridge, and 2.18% with CEPI and CADP cartridges. Level of von Willebrand factor ristocetin cofactor (vWF: RCo) activity was 112% (56-168%). Of the factors examined (age, sex, cigarette smoking, blood donation type, ABO, and Rhesus blood group), only blood group O was significantly linked with impaired platelet function test in qualified blood donors (p = 0.023; odds ratio = 1.981; 95% confidence interval (1.091-3.595)).ConclusionsSome qualified blood donors present abnormal platelet function results. More research is required to provide greater insight into the impact of platelet dysfunction in blood donors on the clinical efficacy of their platelet components. This study has confirmed that the influence of ABO blood group on the CT PFA-100™is not wholly dependent on vWF.

Variables that Influence Platelet Function Analyzer-100™ Closure Times in Healthy Algerian Adults.

Context:The Platelet Function Analyzer (PFA-100™) assesses primary hemostasis in vitro under high shear stress to simulate the conditions to which platelets are exposed at the site of an injured blood vessel wall.Aims:We investigated preanalytical variables in healthy Algerian adults, and we also assess the performance of the test.Subjects and Methods:Closure time (CT) was measured in 302 well-characterized healthy Algerian adults with the collagen/epinephrine (CEPI) and the collagen/adenosine diphosphate (CADP) cartridges.Results:Age and sex did not affect CT values. Blood group O was associated significantly with longer CEPI CT and CADP CT than non-O groups (P ≤ 0.0001 for both). CTs were shorter in samples collected in the morning vs the afternoon (P < 0.0001 for both). We found a strong positive correlation between CT CEPI and CT CADP with r = 0.72 and P < 0.001 and an inverse mean correlation between von Willebrand factor level and CT with r = −0.56, r = −0.45 for CT CEPI and CT CADP, respectively P < 0.001. Duplicate analysis of PFA-100™ CT revealed a mean difference of 3.6% ±19.1% for the CEPI cartridge and 1.5% ±10% for the CADP cartridge. The mean coefficient of variation was 7.4% for the CADP CT and 7.6% for the CEPI CT. No marked difference between test positions.Conclusions:The PFA-100™ showed good reproducibility. The variables influencing the test in healthy Algerian adults are similar to Western and Asian populations. Standardization of preanalytical and analytical conditions is essential for obtaining reliable PFA-100™ results.

P4571PFA-100, a test of platelet adhesion/aggregation, predicts cardiovascular events after an acute coronary syndrome and can help in the decision-making for dual antiplatelet extension

Abstract Background The dual antiplatelet therapy (DAPT) duration is a matter of great interest among cardiologists. Because of the conflicting evidences and the necessity to balance the reduction in major cardiac events (MACE) occurrence and the risk of major bleedings after an acute coronary syndrome (ACS), there is a general consensus on prolonging DAPT on an individual basis. There is less consensus on which parameters are to be evaluated. Nowadays tests of platelet reactivity are not included in the decision-making. Few data are available on the prognostic value of aspirin response tests that are sensitive to other mediators of platelet adhesion and aggregation in vivo under flow conditions. Purpose To demonstrate the role of the Platelet Function Analyzer (PFA-100) Collagen/Epinephrine (CEPI) cartridge, which is very sensitive to von Willebrand factor (VWF) levels, an emerging vascular risk factor, in risk stratification in ACS patients undergoing percutaneous coronary intervention (PCI). Methods We measured platelet reactivity by PFA-100 CEPI cartridges in a prospective cohort of 928 patients admitted for ACS between January 2006 to December 2009 and urgently treated by PCI at day 6±1 after admission. All the patients were treated with aspirin and clopidogrel according to current standard of that time. Results High platelet reactivity (HPR) defined as PFA-100 values &lt;190”was found in 307 (33%) patients. No significant differences were found in demographic, angiographic and biochemical characteristics of both populations (PFA-100 &lt;190” and PFA-100&gt;190”). At a mean follow up of 5±1 years patients with HPR had a significant increase in cardiac death: 12.3% vs 2.6% (hazard ratio 6.05; 95% confidence interval: 3.34–10.95; p&lt;0.0001) and recurrence of myocardial infarction (MI): 49% vs 17% (hazard ratio 4,29; 95% confidence interval: 3,27–5,64; p&lt;0.0001). The trend in events incidence in our cohort of patients, in line with recent evidences, was maintained beyond the first year of follow-up Conclusions Using a multivariable Cox-proportional hazard model, HPR was found to be an independent predictor of MACE. These results indicate that PFA-100 CEPI cartridge, which correlates well with VWF levels, may be a useful point-of-care test to stratify the cardiovascular risk after an ACS. This also underlines the additional value of this test in the decision-making about the correct extension of DAPT.

Assessment of ‘on-treatment platelet reactivity’ and relationship with cerebral micro-embolic signals in asymptomatic and symptomatic carotid stenosis

IntroductionThe relationship between on-treatment platelet reactivity and cerebral micro-embolic signals (MES) is unknown, and has not been previously simultaneously assessed in asymptomatic and symptomatic carotid stenosis patients. MethodsConsecutive eligible patients with ≥50% asymptomatic or recently symptomatic carotid stenosis (≤4weeks following TIA/ischaemic stroke) were recruited to this pilot study. Symptomatic patients were followed up to the ‘late’ phase (≥3months) following symptom onset or carotid intervention; longitudinal data were analysed from symptomatic patients with data available at both time-points. Platelet function/reactivity was assessed with the PFA-100® to measure collagen-ADP (C-ADP) and collagen-epinephrine (C-EPI) closure times in citrate-anticoagulated whole blood. Bilateral simultaneous 1-hour transcranial Doppler ultrasound (TCD) monitoring of the middle cerebral arteries was performed to classify patients as MES +ve or MES −ve. Results31 patients with ≥50% asymptomatic and 46 with early symptomatic carotid stenosis or occlusion were included. 35 symptomatic patients were followed up to the late phase (23 following carotid intervention). Prevalence of ‘high on-treatment platelet reactivity’ (HTPR) on the C-EPI cartridge did not differ between asymptomatic and symptomatic patients overall, but was lower in ‘symptomatic post-intervention’ than asymptomatic patients on aspirin monotherapy (10% vs. 50%; p=0.03). The prevalence of HTPR on the C-EPI cartridge decreased between the early and late phases in symptomatic patients (63% vs. 34%; p=0.017), including those on aspirin monotherapy (p=0.016). There were no significant differences in HTPR status between asymptomatic vs. early or late symptomatic MES +ve or MES −ve patients. DiscussionCarotid interventional treatment, presumably in combination with resolution of the acute phase response, may decrease the prevalence of HTPR in patients with recently symptomatic carotid stenosis over time. Preliminary subgroup analysis suggests that successful intervention may reduce the prevalence of aspirin-HTPR in symptomatic patients to lower levels than asymptomatic medically-treated patients on aspirin monotherapy. Larger, longitudinal studies are warranted to reassess the impact of more intensive secondary preventive treatment on ex vivo platelet function at different levels of shear stress in carotid stenosis patients.

Effect of chronic kidney disease on platelet reactivity to dual-antiplatelet therapy in patients treated with drug-eluting stents

We investigated the effect of chronic kidney disease (CKD) on platelet function in patients receiving dual-antiplatelet therapy after drug-eluting stent (DES) implantation. We examined 19 patients with CKD and 18 patients without CKD who underwent percutaneous coronary intervention (PCI) with DES. All of the patients had been on chronic aspirin treatment. Blood samples were obtained 20-24 h after a loading dose (300 mg) of clopidogrel. Platelet function was evaluated by measuring the closure time (CT) of a collagen/epinephrine (CEPI) or collagen/adenosine diphosphate (CADP) cartridge in the PFA-100 system. Maximum aggregation of agonist (epinephrine, ADP, collagen, or ristocetin)-induced platelet aggregation was also examined by light transmittance aggregometry. The frequency of the poor responders among CKD (n = 9, 47.4%) patients was significantly higher than that among non-CKD patients (n = 2, 11.1%, P = 0.016) as assessed using a CEPI-CT cartridge. Multiple logistic regression analysis revealed that CKD (odds ratio 7.39, 95% confidence interval 1.38-62.09, P = 0.0183] was the only independent determinant of the poor responders. There were no significant differences between the two groups in the value of CADP-CT or in maximum aggregation of epinephrine-, ADP-, collagen-, and ristocetin-induced platelet aggregation. Compared with non-CKD patients, CKD patients had lower response to aspirin therapy as assessed by the PFA-100 system with a CEPI cartridge. On the other hand, the platelet response to dual-antiplatelet therapy was not different between CKD and non-CKD patients.

Effects of Tipranavir, Darunavir, and Ritonavir on Platelet Function, Coagulation, and Fibrinolysis in Healthy Volunteers

The use of HIV protease inhibitors (PIs) as part of antiretroviral therapy in the treatment of HIV-1 infection may be associated with an increased risk of bleeding. This prospective, randomized, open-label trial in healthy volunteers compared the effects of tipranavir/ritonavir (TPV/r), darunavir/ritonavir (DRV/r), and ritonavir (RTV) alone on platelet aggregation after a single dose and at steady-state concentrations. Subjects were selected on the basis of normal platelet aggregation and arachidonic acid (AA)-induced platelet aggregation inhibition after administration of a single 325-mg dose of aspirin. All 3 PI therapies were administered twice daily for 10 days.In some but not all subjects, TPV/r inhibited AA-induced platelet aggregation and prolonged PFA-100® closure time with collagen-epinephrine cartridge, which was of lesser magnitude and consistency compared with aspirin, but greater when compared to DRV/r and RTV. At least 2 subjects in each treatment arm showed complete inhibition of AA-induced platelet aggregation on treatment, and the magnitude of change in all platelet-function tests did not correlate with PI plasma concentrations. Effects of TPV/r on platelet aggregation were reversed 24 hours after the last TPV/r dose. None of the PI treatments tested were associated with increases in bleeding time, decreases in plasma coagulation factors, or increase in fibrinolysis. There was large inter-patient variability in antiplatelet effect for all PI treatments, ranging from no effect to complete inhibition of AA-induced platelet aggregation.

Enhanced ex vivo inhibition of platelet function following addition of dipyridamole to aspirin after transient ischaemic attack or ischaemic stroke: First results from the TRinity AntiPlatelet responsiveness (TrAP) study

Ex vivo dipyridamole 'non-responsiveness' has not been extensively studied in ischaemic cerebrovascular disease. Platelet surface marker expression, leucocyte-platelet complex formation and inhibition of platelet function at high shear stress as detected by the PFA-100® Collagen-Adenosine-diphosphate (C-ADP) and Collagen-Epinephrine cartridges was assessed in 52 patients within 4 weeks of transient ischaemic attack (TIA) or ischaemic stroke on aspirin, and then 14 d (14 d) and >90 d (90 d) after adding dipyridamole. A novel definition of 'Dipyridamole non-responsiveness' was used. The median C-ADP closure time increased following addition of dipyridamole, remained elevated at 90 d (P ≤ 0·03), and was unaffected by aspirin dose. 59% at 14 d and 56% at 90 d were 'dipyridamole non-responders' on the PFA-100. The proportion of non-responders at 14 and 90 d was similar (P= 0·9). Compared with baseline (4·6%), median monocyte-platelet complexes increased at 14 d (5·0%, P= 0·03) and 90 d (4·9%, P= 0·04). Low C-ADP closure times were associated with increased monocyte-platelet complexes at 14 d (r= -0·32, P= 0·02) and 90 d (r= -0·33, P = 0·02). Monocyte-platelet complexes increased in the subgroup of dipyridamole non-responders on the PFA-100 (P≤ 0·045), but not in responders (P ≥ 0·5), at 14 and 90 d versus baseline. Additional inhibition of platelet function has been detected with the PFA-100 when dipyridamole is added to aspirin. Elevated monocyte-platelet complexes may contribute to ex vivo dipyridamole non-responsiveness.

Use of the PFA-100 closure time to predict cardiovascular events in aspirin-treated cardiovascular patients: a systematic review and meta-analysis.

PFA-100 is a point-of-care assay that evaluates platelet reactivity in high-shear-stress conditions by measuring the closure time (CT) of a membrane aperture. When determined with a collagen/epinephrine cartridge (CEPI), the CT is usually prolonged by aspirin. Studies of the predictive value of a short PFA-100CT(CEPI) for ischemic events in aspirin-treated patients have given variable results. To conduct a systematic review and meta-analysis of studies on the clinical predictive value of a short PFA-100CT(CEPI) in aspirin-treated cardiovascular patients. Relevant studies were identified by scanning electronic databases. Studies were selected if they included aspirin-treated patients with symptomatic atherosclerosis, measured the PFA-100CT(CEPI), used a CT cut-off value to define aspirin 'responders' and 'non-responders', and reported ischemic events. We selected seven non-prospective studies (1466 patients) and eight prospective studies (1227 patients). In non-prospective studies, the PFA-100CT(CEPI) was performed after the ischemic clinical endpoint, and a publication bias was identified. In prospective studies, the global odds ratio (OR) for the recurrence of an ischemic event in 'aspirin non-responders' relative to 'aspirin responders' was 2.1 [95% confidence interval (CI) 1.4-3.4, P < 0.001]. Pooled analysis with a random effect model revealed no heterogeneity (Q Cochran P = 0.36 and I(2) = 9.4%). A short PFA-100CT(CEPI) is associated with increased recurrence of ischemic events in aspirin-treated cardiovascular patients. This finding needs to be confirmed in stable ischemic patients, and the PFA-100CT(CEPI) cut-off needs to be refined in these patients.

Resistance to platelet antiaggregants: an important cause of very late thrombosis of drug eluting stents? Observations from five cases

Very late thrombosis of drug eluting stents is a rare complication that might be triggered by resistance to platelet antiaggregants (PAAs). Following an initial case where clinical data strongly suggested resistance to PAAs, we carried out a prospective systematic analysis of platelet aggregation in four subsequent cases of late thrombosis. Resistance to aspirin was investigated with the PFA-100 test employing a collagen-epinephrine cartridge (Platelet Function Analyzer; Dade Behring). Resistance to clopidogrel was determined by flow cytometry of intraplatelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. All four cases showed resistance to either aspirin or clopidogrel, and two cases showed dual resistance to both of these PAAs. Analysis of platelet function in a patient with late stent thrombosis is useful and may allow adaptation of subsequent patient management. The value of monitoring platelet function after implantation of a drug eluting stent should be evaluated in prospective studies.

Laboratory Response to Intranasal Desmopressin in Women with Menorrhagia and Underlying Platelet Dysfunction.

Intranasal desmopressin (IN-DDAVP) is used for home treatment of menorrhagia in women with inherited bleeding disorders. The effect of IN-DDAVP on laboratory hemostatic parameters in women with menorrhagia and associated platelet dysfunction is unknown. We evaluated the effects of IN-DDAVP on hemostatic parameters in women with menorrhagia and platelet dysfunction and correlated them with menstrual blood flow. Eleven women (aged 18–45) with menorrhagia and abnormal platelet aggregation and/or platelet adenosine tri-phosphate (ATP) release had determination of factor VIII coagulant activity (FVIII: C), von Willebrand factor antigen (VWF: Ag), von Willebrand factor ristocetin cofactor (VWF: RCo) activity, platelet aggregation, and platelet ATP release pre and 60 minutes post IN-DDAVP. Eight of eleven women underwent Platelet Function Analyzer (PFA-100) closure time determination with collagen/epinephrine (CEPI) and collagen/adenosine diphosphate (CADP) cartridge pre and post treatment. IN-DDAVP was administered during two consecutive menstrual cycles. Menstrual blood flow was assessed during each cycle using a pictorial blood assessment chart (PBAC). Following administration of IN-DDAVP there was a 2–3 fold increase in FVIII: C, VWF: Ag, and VWF: RCo observed in most patients over baseline levels. Mean FVIII:C increased from 130% ± 34.8% to 218% ± 96.5% (P < 0.007), VWF: Ag increased from 86.9% ± 35.1% to 128.7% ± 64.3% (P < 0.011), and VWF: RCo increased from 89.8% ± 42.8% to 139.6% ± 86.5% (P < 0.02). The median baseline PBAC score of 235 decreased to 136 following IN-DDAVP. In addition, there were significant inverse correlations between changes in PBAC score and changes in VWF: Ag (rs= − 0.85, p = 0.02), VWF: RCo (rs= −0.81, p = 0.029), and FVIII:C (rs= 0.92, p = 0.003) with IN-DDAVP. Mean PFA-100 closure time with the CEPI cartridge shortened from 166.8sec ± 85sec to 90.9sec ± 49.3sec (p< 0.02), and the closure time with the CADP cartridge shortened from 104.5sec ± 34.3sec to 64.8sec ± 40.4sec (p <0.007). There were significant inverse correlations between post IN-DDAVP PFA-100 CADP closure time and post IN-DDAVP FVIII:C (rs= −0.86, p = 0.005), VWF: Ag (rs= −0.72, p = 0.04), and VWF: RCo (rs= −0.80, p = 0.01). In addition there were also significant inverse correlations between post IN-DDAVP PFA-100 CEPI closure time and post IN-DDAVP FVIII:C (rs= −0.73, p = 0.03), and VWF: RCo (rs= −0.74, p = 0.03), but not VWF: Ag (rs= −0.66, p = 0.07). In-vitro platelet aggregation and platelet ATP release response did not correct and did not correlate with changes in menstrual blood flow. Our results demonstrate a correlation between VWF parameters and menstrual blood flow following IN-DDAVP in women with menorrhagia and underlying platelet dysfunction.

Comparison of a rapid platelet function assay – Verify Now™ Aspirin – with whole blood impedance aggregometry for the detection of aspirin resistance

The reported incidence of aspirin resistance or non-responsiveness ranges between 5 and 30% [1]. There is neither a uniformly accepted definition of aspirin resistance nor an established standard method to assess aspirin non-responsiveness in the laboratory. The PFA-100™ has been most often used to assess aspirin effect. Aspirin non-responsiveness by the PFA-100™ is defined as an absence of a prolongation of the Collagen–Epinephrine cartridge closure time [2]. The second most widely used method is Born's optical platelet aggregometry, a labor intensive, time-consuming method that requires technical expertise.

Identification of ADRA2A Polymorphisms Related to Shear-Mediated Platelet Function by the PFA-100® System.

[Background]α 2A adrenergic receptor (ADRA2A) on platelets interacts with epinephrine, which has a key role in regulating platelet functions. There is familial clustering of inter-individual variations in the epinephrine-induced platelet aggregation, the molecular basis of which, however, has not been fully understood. In this study, we screened the sequence variations in the transcriptional region of ADRA2A gene and analyzed the relationship between the two common polymorphisms and platelet function using collagen/epinephrine (CEPI) cartridge in the platelet function analyzer (PFA)-100® system.[Methods and Results]Written informed consent was obtained from all study subjects who were genetically unrelated healthy Japanese males (n=255). Of the 255 subjects, an initial screening for the sequence variation(s) in the ADRA2A transcriptional region was performed on 44 subjects enrolled in 2003. Subsequently, relationship between ADRA2A polymorphisms and platelet function was investigated in 211 subjects enrolled between 2004 and 2005. In the screening, we observed 16 single nucleotide polymorphisms (SNPs) including 5 novel variations. We next examined the association between the ADRA2A polymorphisms and epinephrine-mediated platelet function using the PFA-100® system under high shear conditions (5000–6000/sec). According to the pilot study on the 16 SNPs, we focused on 1780A/G and 2732A/G (the first nucleotide of the open reading frame is nt#1, which corresponds to nt#31586326 of the GenBank NT_030059). We measured platelet function, as assessed by closure time (CT) using CEPI cartridge. A longer CEPI-CT indicates lower platelet function in the interaction with collagen and epinephrine. Because the manufacturer's instructions for the PFA-100® report a mean CEPI-CT value of 132 s, study subjects were divided in two groups: a higher function group with a CEPI-CT < 132 s (n=90) and a lower function group with a CEPI-CT ≥ 132 s (n=121). The frequency of the 1780GG genotype in the lower function group was significantly higher than that in the higher function group (p=0.0478) whereas no association between the CEPI-CT and the 2372A/G polymorphism was observed (p=0.1164). We also observed the effect of the combination of 1780GG+2372AA genotypes for shorter CT (p=0.0319). A multiple logistic regression model to adjust the reported confounding factors (VWF: RCo, platelet count, and hematocrit) revealed an adjusted odds ratio of 2.10 (p=0.0274) for the genotypic combination or 1.82 (p=0.0757) for the 1780A/G alone. Plasma VWF:RCo level was also an independent predictor for CEPI-CT. These observations suggested the enhanced effect of the genotypic combination as compared with the 1780A/G alone.[Conclusion]Of the 16 sequence variations of the transcriptional region of the ADRA2A gene, the combination of the 1780A/G and 2372A/G polymorphism was associated with collagen/epinephrine-mediated platelet function by PFA-100® system.

Platelet aggregation and platelet function analyzer 100 (PFA-100) closure time in camels—a comparative study with humans

Despite the very active coagulation system in camels, there are no previous studies on camel platelet functions. It is our aim to study camel platelet function using aggregometry, Platelet Function Analyzer (PFA100), and flow cytometry. A total of 103 camels, 19 males and 84 females, were studied. Their ages ranged from 5 to 20 years (mean±SD: 6.4±4.4 years). The results obtained were compared with healthy humans. Platelet aggregometry was undertaken in platelet-rich plasma in response to adenosine diphosphate (ADP), adrenaline, collagen, arachidonic acid, and ristocetin. Camel platelet function in whole blood was also tested using the PFA-100 and by flow cytometry using three human monoclonal antibodies (CD42, CD61, and CD62). Camel platelets failed to respond to arachidonic acid, adrenaline, and ristocetin. However, responses to ADP and collagen were obtained but were less than the human values. The addition of human plasma caused some enhancement of the aggregation responses to adrenaline and collagen but not ristocetin or arachidonic acid. However, the presence of human serum or heparin resulted in a very marked enhancement of the camel platelet aggregation responses to all agonists, except arachidonic acid. PFA-100 closure times of the collagen–ADP and the collagen–epinephrine cartridges were markedly longer than in humans. In the flow cytometry studies, camel platelets failed to respond to any of the human monoclonal antibodies with or without activation by ADP, thrombin, human plasma, or serum. This first study on camel platelet functions uncovered the distinction between camel and human platelet functions. The lack of platelet responses to certain aggregating agonists, their enhancement with human plasma and serum, as well as the prolongation of the PFA-100 closure times, add other unique characteristics to the biology of this interesting creature.

Influence of Systemic Inflammation on the Interpretation of Response to Antiplatelet Therapy, Monitored by PFA-100

Recently it was shown that inflammation adversely influences results obtained with the Platelet Function Analyzer System PFA-100, hypothesizing that inflammation could confound interpretation of platelet function results. We investigated the clinical relevance of these results in patients with peripheral arterial occlusive disease (PAOD), with and without signs of systemic inflammation. In 98 PAOD patients, all treated with acetylsalicylic acid (ASA), we measured PFA-100 closure times with the collagen-epinephrine test cartridges. C-reactive protein (CRP) values were measured as an indicator for systemic inflammation. Mean CRP levels were elevated in 23 patients (23%). There was no difference in mean PFA-100 results between patients with elevated CRP-levels and those without. During clinical use of the PFA system, systemic inflammation had no major influence on the performance of the collagen-epinephrine cartridge. With respect to the response to antiplatelet therapy with ASA, the results suggest that the platelet inhibitory effect of ASA is not reduced under inflammatory conditions.

Platelet Dysfunction in Primary Thrombocythemia Using the Platelet Function Analyzer, PFA-100

We measured platelet function by standard aggregometric tests and by the PFA-100 (Dade Behring, Newark, DE) in samples from 55 patients with primary thrombocythemia (PT) and 26 healthy volunteers. Platelet function was evaluated in platelet-rich plasma by aggregation tests. PFA-100 studies (closure time) were performed in citrated whole blood using collagen–adenosine diphosphate (ADP) and collagen-epinephrine cartridges. Plasma levels of von Willebrand factor (vWF) also were measured. The mean ± SD closure time for patients vs volunteers for the collagen-epinephrine cartridge was prolonged (210.8 ± 62.2 vs 118.1 ± 19.6 seconds; P < .001); results were abnormal for 38 patients (69%). Results with the collagen-ADP cartridge also were abnormal in patients (134.3 ± 58.4 seconds) vs volunteers (87.3 ± 15.6 seconds; P < .001); closure time was prolonged in 23 patients (42%). A decreased response to epinephrine (38.4% ± 34.2% vs 82.5% ± 10.3%; P < .001), the main defect detected by platelet aggregation tests, affected 32 patients (58%). Platelet response to collagen also was abnormal (52.0% ± 34.6% vs 86.0% ± 10.1%; P < .01) but affected only 21 patients (38%). vWF levels for patients were normal. The results seem to confirm that platelet function in patients with PT is abnormal and show that platelet function can be assessed by an easy, reproducible, and sensitive method, the PFA-100. Closure time usually was prolonged; this feature could be applied in the diagnosis of PT.

Platelet Dysfunction in Primary Thrombocythemia Using the Platelet Function Analyzer, PFA-100

We measured platelet function by standard aggregometric tests and by the PFA-100 (Dade Behring, Newark, DE) in samples from 55 patients with primary thrombocythemia (PT) and 26 healthy volunteers. Platelet function was evaluated in platelet-rich plasma by aggregation tests. PFA-100 studies (closure time) were performed in citrated whole blood using collagen-adenosine diphosphate (ADP) and collagen-epinephrine cartridges. Plasma levels of von Willebrand factor (vWF) also were measured. The mean +/- SD closure time for patients vs volunteers for the collagen-epinephrine cartridge was prolonged (210.8 +/- 62.2 vs 118.1 +/- 19.6 seconds; P < .001); results were abnormal for 38 patients (69%). Results with the collagen-ADP cartridge also were abnormal in patients (134.3 +/- 58.4 seconds) vs volunteers (87.3 +/- 15.6 seconds; P < .001); closure time was prolonged in 23 patients (42%). A decreased response to epinephrine (38.4% +/- 34.2% vs 82.5% +/- 10.3%; P < .001), the main defect detected by platelet aggregation tests, affected 32 patients (58%). Platelet response to collagen also was abnormal (52.0% +/- 34.6% vs 86.0% +/- 10.1%; P < .01) but affected only 21 patients (38%). vWF levels for patients were normal. The results seem to confirm that platelet function in patients with PT is abnormal and show that platelet function can be assessed by an easy, reproducible, and sensitive method, the PFA-100. Closure time usually was prolonged; this feature could be applied in the diagnosis of PT.

Are the Pfa-100® Closure Time and the Skin Bleeding Time Useful in the Diagnostic Work-Up of Patients with Bleeding Diathesis? Correlation with the Severity of Bleeding Symptoms and Sensitivity for Known Defects of Primary Hemostasis.

We compared the performance of PFA-100® closure time (CT) and the skin bleeding time (BT) in the diagnostic work-up of 128 consecutive patients referred to our Centre from June 2002 through June 2003 to be screened for bleeding disorders, due to the presence of bleeding symptoms or the casual finding of abnormal screening tests of hemostasis. The correlation with the severity of the bleeding symptoms and the sensitivity of BT and PFA-100® CT for known defects of primary hemostasis were evaluated. All patients underwent a careful medical interview and were assigned a “bleeding score”, based on the number, type, frequency and severity (development of anemia, need for blood transfusion and/or surgical or medical intervention) of bleeding symptoms. In addition, all patients underwent a first-line screening, which included prothrombin time, activated partial thromboplastin time (APTT), BT and PFA-100® CT (with both the collagen-ADP and the collagen-epinephrine cartridges). The search for Von Willebrand disease (VWD), platelet function disorders (PFD), clotting factor defects and abnormalities of fibrinolysis was performed according to the results of the first line screening tests and the severity and type of bleeding history. Seven (6%) patients had type-1 VWD, 12 (9%) PFD, 29 (23%) defects of clotting factors, 18 (14%) had prolongations of the APTT due to abnormalities that are not associated with bleeding (factor XII deficiency and lupus anticoagulant), while in 63 (49%) all tests gave normal results. The sensitivity of PFA-100® for VWD was 86% (both cartridges), for PFD 75% (collagen-epinephrine) and 8% (collagen-ADP). The sensitivity of BT for VWD was 29%, for PFD 33%. The CT of the PFA-100® CT collagen-epinephrine cartridge was mildly prolonged in 38% of patients with clotting factor deficiency. After dividing the patient population in four quartiles of distribution, according to the severity of the bleeding score (null, low, intermediate and severe), only the CT of the PFA-100® CT collagen-epinephrine cartridge showed a progressive and significant prolongation from the first to the fourth quartile (p=0.04). No association of BT and collagen-ADP CT with the severity of bleeding history was found. In conclusion: 1) CT of the PFA-100® collagen-epinephrine cartridge was significantly associated with the severity of the bleeding history; 2) PFA-100® CT showed a better sensitivity than BT for VWD and PFD; 3) at variance with the BT, PFA-100® could help to discriminate between VWD (prolongation of CT of both cartridges) and PFD (prolongation of CT of the collagen-epinephrine cartridge only).

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

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 the O’Brien Filter Test and the PFA-100 Platelet Analyzer in the Laboratory Diagnosis of von Willebrand’s Disease

Von Willebrand's disease (vWD) is the most common congenital haemorrhagic diathesis, characterized by the quantitative or qualitative disorder of von Willebrand factor (vWF). A number of methods have been used for the diagnosis of the disease, and the bleeding time determination is widely accepted as a screening test in spite of its low sensitivity. Our aim was to evaluate and compare the performance of two high shear systems (the O'Brien filter test and the PFA-100 device) in the screening and diagnosis of vWD. Thirty patients (n=13 type 1 with mild symptoms, n = 9 type 1 with severe symptoms, n = 2 type 2A, n = 3 type 2B and n = 3 type 3 vWD) and twenty controls were investigated. In mild vWD the platelet retention in the second phase of the filter test with citrated blood showed the highest sensitivity (91.6%). The sensitivity of the PFA-100 method with collagen-epinephrine cartridges in this group was 76.9%, while the bleeding time was prolonged only in 15.4% of the cases. In severe type 1, in type 2A and type 3 all functional tests reflected the bleeding tendency of the patients. In type 2B disease the bleeding time was prolonged only when the patient was thrombocytopenic, but both high shear systems revealed the disease independently of the presence of thrombocytopenia. The overall sensitivity of the bleeding time determination was 50% compared to the 80-90% sensitivity of the O'Brien filter test and the PFA-100 system. The sensitivity values of the filter test and the PFA-100 device with collagen-epinephrine cartridges were in the same range, but the collagen-ADP cartridges showed a lower (65.5%) sensitivity, though the results were specific and had high positive predictive value. We conclude that both high shear systems are suitable for the screening of vWD, and that they are superior to the traditional bleeding time determination in case of mild disease or type 2B vWD.