Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing

Platelet reactivity testing is important for the diagnosis of bleeding disorders, and increasingly to optimise anti-platelet therapy. Traditional light transmission aggregometry is considered the gold standard, whilst 96-well plate aggregometry, founded on similar principles, provides a higher throughput screening method. Despite the widespread use of both, methodologies and outputs vary widely between laboratories. We report a methodological approach towards providing a standardised optical detection of platelet aggregation (optimul method) based upon 96-well plate aggregometry. Individual wells of half-area 96-well plates were coated with gelatine and one of seven concentrations of arachidonic acid (AA), adenosine diphosphate (ADP), collagen, epinephrine (EPI), ristocetin, TRAP-6 amide or U46619, before being lyophilised, vacuum-sealed, foil-packed and stored at room temperature for up to 24 weeks. For platelet testing, 40?µl of platelet-rich plasma was added to each well. Platelet aggregation was determined by changes in light absorbance, release of ATP/ADP by luminescence and release of thromboxane (TX) A₂ by ELISA. Some experiments were conducted in the presence of aspirin (30?µM) or prasugrel active metabolite (PAM; 3?µM). Optimul plates stored for up to 12 weeks permitted reliable detection of concentration-dependent platelet aggregation, ATP/ADP release and TXA₂ production. PAM caused reductions in platelet responses to AA, ADP, collagen, EPI, TRAP-6 and U46619, whilst aspirin inhibited responses to AA, collagen and EPI. We conclude that the optimul method offers a viable, standardised approach, allowing platelet reactivity testing and could provide a broad platelet function analysis without the need for dedicated equipment.     

Clopidogrel anti-platelet effect: an evaluation by optical aggregometry, impedance aggregometry, and the platelet function analyzer (PFA-100)

Platelet aggregation inhibition by clopidogrel may be suboptimal in 4-30% of patients. Traditionally, optical aggregometry is used to assess clopidogrel's anti-platelet effects by inhibition of ADP-induced aggregation in platelet rich plasma. Red blood cells are an important source of ADP and, thus, are known to modulate platelet function. Because the whole blood aggregation by impedance method assesses platelet function in a physiological milieu, we compared clopidogrel response by this method with the optical method in platelet rich plasma (PRP) and the Platelet Function Analyzer (PFA-100). Platelet function studies were performed in 17 healthy subjects at baseline and after 10 days of clopidogrel intake (75 mg/day). Optical and impedance aggregometry were performed after addition of ADP (10 and 20 microM) and collagen (1 and 2 microg/mL). For PFA-100 analysis, whole blood closure time was measured in collagen-coated cartridges with ADP and epinephrine. All subjects except one showed a decrease in ADP-induced aggregation using both aggregation methods. However, ADP-induced platelet aggregation was significantly inhibited when assessed in whole blood as compared to the optical method (71+/- 34% vs. 34.2+/- 23%, p = 0.0002); this suggests that whole blood aggregometry is more sensitive in the detection of clopidogrel effect in the presence of red cells, which are known to modulate platelet function. The PFA-100 ADP closure time was slightly prolonged above the reference interval in only 5/17 (29%) subjects, suggesting that this instrument is not able to detect clopidogrel effect. We conclude that whole blood aggregation appears to be more sensitive in detecting clopidogrel effect compared with the platelet rich plasma method; the PFA-100 was unable to detect clopidogrel effect in the majority of the subjects.     

Variables influencing Multiplate(TM) whole blood impedance platelet aggregometry and turbidimetric platelet aggregation in healthy individuals

We investigated the relationship between impedance platelet aggregometry (IPA) as measured by the Multiplate system and turbidimetric platelet aggregation (TPA) induced by ADP, arachidonic acid (AA), and collagen; blood cell counts; platelet function analyzer (PFA-100) closure times (CT), and von Willebrand factor (VWF) in 120 well-characterized healthy individuals. Pre-analytical and analytical conditions were standardized comprehensively. Analytical reliability of IPA and TPA and the influence of pre-analytical variables on assay results were also examined. IPA and TPA did not change significantly between 0.5 and 5 hours after blood collection when samples were stored at room temperature. TPA and IPA showed significantly greater intra-assay imprecision than respective TPA induced by the same agonists. Intra-individual variation did not differ significantly between IPA and TPA. The lower limits of reference range (2.5th percentiles) of AAIPA, ADPIPA and collagen IPA determined AM were 37, 20 and 40 AU, respectively. ADPIPA showed significantly lower maximum aggregation values than AAIPA and collagen IPA (P < 0.0001). There were no significant differences in any parameter between males and females. No significant differences between blood group 0 and non-0 individuals were noted with respect to IPA and TPA. IPA did not change significantly during the day. In contrast, TPA measured PM was significantly lower than corresponding values determined a.m. (p < 0.0001). CEPI-CT, CADP-CT and leukocyte counts increased significantly from a.m. to p.m. (P = 0.008 and P > 0.0001, respectively). Donors had significantly greater IPA induced by any agonist than non-donors (P-values < 0.0001, 0.0001 and 0.001, respectively), whereas TPA was not significantly different between donors and non-donors. IPA did not correlate significantly with TPA nor with PFA-100 CT. ADPIPA and collagen IPA correlated significantly with platelet count. TPA was not associated with platelet count. An inverse significant correlation was observed between TPA induced by any agonist and leukocyte count, whereas leukocyte count did not influence IPA. CEPI-CT and CADP-CT correlated significantly with VWF:CBA and with each other but not with TPA. We concluded that IPA and TPA measure different aspects of platelet function. IPA results reflect interactions between platelets, red and white cells, while TPA does not. This explains discrepancies in associations of IPA and TPA with cell counts, time of day and blood donation. The clinical significance of IPA determined using the Multiplate device remains to be determined in studies on patients with platelet dysfunction and under treatment with antiplatelet agents.     

Whole blood impedance aggregometry for the assessment of platelet function in patients with congestive heart failure (EPCOT Trial)

OBJECTIVE: Data from small studies have shown the presence of platelet abnormalities in patients with congestive heart failure (CHF). We sought to characterize the diagnostic utility of the whole blood aggregometry (WBA) in a random outpatient CHF population. METHODS: Blood samples were obtained for measurement of whole blood aggregation, shear-induced closure time, platelet contractile force, expression of GP IIb/IIIa, and P-selectin in 100 consecutive patients with CHF. RESULTS: Substantial inter-individual variability of platelet characteristics exists in patients with CHF. There were no statistically significant differences when patients were divided by the incidence of vascular events, emergency revascularization needs, survival, or etiology of heart failure. Surprisingly, aspirin use did not affect instrument readings as well. Whole blood aggregometry correlates well with the closure time (r(2)=0.587), and with GP IIb/IIIa expression (r(2)=0.435). Significant but less strong correlation has been observed for the WBA with platelet P-selectin expression (r(2)=0.295), and no correlation was present for the platelet contractile force measures (r(2)=0.030). CONCLUSIONS: Despite the fact that patients with heart failure enrolled in the EPCOT trial exhibited marginal, sometimes oppositely directed changes, in their platelet characteristics, whole blood impedance aggregometry is indeed capable to serve as a valuable diagnostic tool, and may be successfully used as an established screening device in this population. Ability of the whole blood aggregometry to predict clinical outcomes, or for the monitoring of anti-platelet agents in CHF patients, will be evaluated in the ongoing clinical trials.     

The use of regression analysis in determining reference intervals for low hematocrit and thrombocyte count in multiple electrode aggregometry and platelet function analyzer 100 testing of platelet function

Low platelet counts and hematocrit levels hinder whole blood point-of-care testing of platelet function. Thus far, no reference ranges for MEA (multiple electrode aggregometry) and PFA-100 (platelet function analyzer 100) devices exist for low ranges. Through dilution methods of volunteer whole blood, platelet function at low ranges of platelet count and hematocrit levels was assessed on MEA for four agonists and for PFA-100 in two cartridges. Using (multiple) regression analysis, 95% reference intervals were computed for these low ranges. Low platelet counts affected MEA in a positive correlation (all agonists showed r² ≥ 0.75) and PFA-100 in an inverse correlation (closure times were prolonged with lower platelet counts). Lowered hematocrit did not affect MEA testing, except for arachidonic acid activation (ASPI), which showed a weak positive correlation (r² = 0.14). Closure time on PFA-100 testing was inversely correlated with hematocrit for both cartridges. Regression analysis revealed different 95% reference intervals in comparison with originally established intervals for both MEA and PFA-100 in low platelet or hematocrit conditions. Multiple regression analysis of ASPI and both tests on the PFA-100 for combined low platelet and hematocrit conditions revealed that only PFA-100 testing should be adjusted for both thrombocytopenia and anemia. 95% reference intervals were calculated using multiple regression analysis. However, coefficients of determination of PFA-100 were poor, and some variance remained unexplained. Thus, in this pilot study using (multiple) regression analysis, we could establish reference intervals of platelet function in anemia and thrombocytopenia conditions on PFA-100 and in thrombocytopenia conditions on MEA.     

Immature platelet fraction (IPF) determined with an automated method predicts clopidogrel hyporesponsiveness

The mechanisms for the variability in antiplatelet effects of clopidogrel are not elucidated entirely. Immature (reticulated) platelets may modulate the antiplatelet effects of clopidogrel but must be measured using flow cytometry. Whether new automated detection techniques yield similar results is not known. The objectives of the study to evaluate the role of immature platelets assessed by an automated method in response to the antiplatelet effects of clopidogrel. Twenty-nine healthy volunteers had platelet studies performed before and 1 week after 75 mg daily dosing of clopidogrel. Immature platelet fraction (IPF) was determined using an automated particle counter. Subjects were stratified into tertiles based on the IPF. Platelet studies included light transmission aggregometry (LTA), and vasodilator stimulated phosphoprotein phosphorylation (VASP-P) determined by platelet reactivity index (PRI). Baseline platelet aggregation responses to 2, 5 and 20 μM ADP, were similar in all three tertiles, however they were greater in the upper than in the lower tertile of immature platelets after clopidogrel in response to 5 μM ADP (54% vs. 23%, P = 0.02), with concordant trends for the other two concentrations. PRI was also greater in the upper tertile after clopidogrel (71.2% vs. 57.8%, P = 0.04). The frequency of clopidogrel hyporesponsiveness (aggregation >50% in response to 5 μM of ADP) was also higher in the upper tertile when compared to lower tertile, (60%) versus (10%) respectively (P = 0.0001). Immature platelets measured using an automated method, are associated with impaired response to antiplatelet effects of clopidogrel.     

Relation of body mass index to on-treatment (clopidogrel + aspirin) platelet reactivity

Previous research has suggested that obesity is associated with increased high on-treatment platelet reactivity. We therefore tested platelet reactivity in 251 patients with VerifyNow P2Y12, vasodilator-stimulated phosphoprotein phosphorylation, and light transmission aggregometry with adenosine diphosphate 5 and 20 μM 6 to 24 hours after percutaneous coronary intervention. High on-treatment platelet reactivity was defined as a maximum platelet aggregation ≥46% for light transmission aggregometry with adenosine diphosphate 5 μM or ≥60% for 20 μM, platelet reactivity index ≥50% for vasodilator-stimulated phosphoprotein phosphorylation, and P2Y12 reaction units ≥235 for VerifyNow. The relation between body mass index (BMI) and platelet reactivity values was examined with Spearman coefficients; BMI and high on-treatment platelet reactivity were assessed with Student's t test. Multivariable logistic regression for high on-treatment platelet reactivity was also performed. Average BMI was 30.3 ± 5.9 kg/m2 and 44% of patients had a BMI ≥30 kg/m2. Overall there was very poor correlation between BMI and on-treatment platelet reactivity, with Spearman coefficients ranging from 0.08 to 0.10. BMI was also not associated with the various definitions of high on-treatment platelet reactivity. Multivariable logistic regressions showed no association between BMI and high on-treatment platelet reactivity. In conclusion, and contrary to previous reports, we found no association whatsoever between BMI and on-treatment platelet reactivity as quantified by a variety of platelet function tests.     

Use of specific Lp(a)-lipoprotein as an additional marker in the diagnosis of ischemic heart disease

A total of 1139 representatives of various groups of the USSR population (indigenous inhabitants of Chukotsk and the Altai, migrants to Chukotsk living there for various periods of time and citizens of Novosibirsk) was studied to determine whether the assay of Lp (a)-lipoproteins, a normal genetic marker, can be used as a novel diagnostic test for coronary heart disease. Coronary heart disease was shown to be correlated with the presence of Lp (a)-lipoprotein, with X2 being statistically significant.     

Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia

A new automated method to reliably quantify reticulated platelets, expressed as the immature platelet fraction (IPF), has been developed utilizing the XE-2100 blood cell counter with upgraded software (Sysmex, Kobe, Japan). The IPF is identified by flow cytometry techniques and the use of a nucleic acid specific dye in the reticulocyte/optical platelet channel. The clinical utility of this parameter was established in the laboratory diagnosis of thrombocytopenia due to increased peripheral platelet destruction, particularly autoimmune thrombocytopenic purpura (AITP) and thrombotic thrombocytopenic purpura (TTP). Reproducibility and stability results over 48 h were good. An IPF reference range in healthy individuals was established as 1.1-6.1%, with a mean of 3.4%. Patients in whom platelet destruction might be abnormal, were studied and two of these patients followed serially during the course of treatment. The IPF was raised in several disease states. The most significant increases in IPF values were found in patients with AITP (mean 22.3%, range 9.2-33.1%) and acute TTP (mean 17.2%, range 11.2-30.9%). Following patients during treatment demonstrated that as the platelet count recovered the IPF% fell. These results show that a rapid, inexpensive automated method for measuring the IPF% is feasible and should become a standard parameter in evaluating the thrombocytopenic patient.     

Accelerated platelet inhibition by switching from atorvastatin to a non-CYP3A4-metabolized statin in patients with high platelet reactivity (ACCEL-STATIN) study

Aims CYP3A4-metabolized statins can influence the pharmacodynamic effect of clopidogrel. We sought to assess the impact of switching to a non-CYP3A4-metabolized statin on platelet function among patients receiving clopidogrel and atorvastatin with high on-treatment platelet reactivity (HPR). Methods and results Percutaneous coronary intervention (PCI)-treated patients (n= 50) with HPR [20 μM adenosine diphosphate (ADP)-induced maximal platelet aggregation (MPA) >50%] were enrolled during chronic administration of atorvastatin (10 mg/day) and clopidogrel (75 mg/day) (≥6 months). They were randomly assigned to a 15-day therapy with either rosuvastatin 10 mg/day (n= 25) or pravastatin 20 mg/day (n= 25). Platelet function was assessed before and after switching by conventional aggregometry and the VerifyNow P2Y12 assay. Genotyping was performed for CYP2C19*2/*3, CYP3A5*3, and ABCB1 C3435T alleles. The primary endpoint was the absolute change in 20 μM ADP-induced MPA. After switching, MPAs after stimuli with 20 and 5 μM ADP were decreased by 6.6% (95% confidence interval: 3.2-10.1%; P < 0.001), and 6.3% (95% confidence interval: 2.5-10.2%; P = 0.002), respectively. Fifty-two P2Y12 reaction units fell (95% confidence interval: 35-70; P < 0.001) and the prevalence of HPR decreased (24%; P < 0.001). Pharmacodynamic effects were similar after rosuvastatin and pravastatin therapy. In addition to smoking status, the combination of calcium channel blocker usage and ABCB1 C3435T genotype significantly affected the change of 20 μM ADP-induced MPA. Conclusions Among PCI-treated patients with HPR during co-administration of clopidogrel and atorvastatin, switching to a non-CYP3A4-metabolized statin can significantly decrease platelet reactivity and the prevalence of HPR. This switching effect appears similar irrespective of the type of non-CYP3A4-metabolized statin.