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• W2037658163 abstract "The in vitro evaluation of platelet aggregation is useful for diagnosing platelet function disorders 1.Podda G. Femia E.A. Pugliano M. Cattaneo M. Congenital defects of platelet function.Platelets. 2012; 23: 552-63Crossref PubMed Scopus (31) Google Scholar, 2.Cattaneo M. Cerletti C. Harrison P. Hayward C.P.M. Kenny D. Nugent D. Nurden P. Rao A.K. Schmaier A.H. Watson S.P. Lussana F. Pugliano M.T. Michelson A.D. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC/ISTH.J Thromb Haemost. 2013; 11: 1183-9Crossref Scopus (356) Google Scholar; in some laboratories, the test is also used to monitor antiplatelet treatment 3.Cattaneo M. Resistance to antiplatelet drugs: molecular mechanisms and laboratory detection.J Thromb Haemost. 2007; 5: 230-7Crossref PubMed Scopus (175) Google Scholar. Traditionally, platelet aggregation is measured with light transmission aggregometry (LTA), which measures the changes in transmission of a beam of light through a sample of platelet‐rich plasma (PRP) or platelet suspensions in buffer, which occur when platelets change shape and aggregate upon stimulation. As this technique has some disadvantages 2.Cattaneo M. Cerletti C. Harrison P. Hayward C.P.M. Kenny D. Nugent D. Nurden P. Rao A.K. Schmaier A.H. Watson S.P. Lussana F. Pugliano M.T. Michelson A.D. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC/ISTH.J Thromb Haemost. 2013; 11: 1183-9Crossref Scopus (356) Google Scholar, novel methods have been introduced to measure platelet aggregation in vitro. Impedance aggregometry (IA) measures the increase in electrical resistance between two electrodes immersed in a diluted sample of whole blood, PRP, or platelet suspension, which is caused by the adhesion of platelets to the electrodes and the subsequent platelet aggregation 4.Cardinal D.C. Flower R.J. The electronic aggregometer: a novel device for assessing platelet behavior in blood.J Pharmacol Methods. 1980; 3: 135-58Crossref PubMed Scopus (626) Google Scholar. The Multiplate™ analyzer represents a recently refined IA technology 5.Seyfert U.T. Haubelt H. Vogt A. Hellstern P. Variables influencing Multiplate™ whole blood impedance platelet aggregometry and turbidimetric platelet aggregation in healthy individuals.Platelets. 2007; 18: 199-206Crossref PubMed Scopus (153) Google Scholar that is mostly used to monitor the inhibitory effect of antiplatelet drugs 6.Sibbing D. Braun S. Morath T. Mehilli J. Vogt W. Schomig A. Kastrati A. von Beckerath N. Platelet reactivity after clopidogrel treatment assessed with point‐of‐care analysis and early drug‐eluting stent thrombosis.J Am Coll Cardiol. 2009; 53: 849-56Crossref PubMed Scopus (580) Google Scholar. Several preanalytic and analytic variables influence the results obtained with LTA. Although it has traditionally been assumed that the platelet count in PRP influences the results of LTA, it is now clear that this is not true, at least in the range between 150 × 109 L−1 and 600 × 109 L−1 7.Cattaneo M. Lecchi A. Zighetti M.L. Lussana F. Platelet aggregation studies: autologous platelet‐poor plasma inhibits platelet aggregation when added to platelet‐rich plasma to normalize platelet count.Haematologica. 2007; 92: 694-7Crossref PubMed Scopus (118) Google Scholar: for this reason, the platelet count in PRP should not be adjusted to a predefined value with autologous platelet‐poor plasma (PPP) 2.Cattaneo M. Cerletti C. Harrison P. Hayward C.P.M. Kenny D. Nugent D. Nurden P. Rao A.K. Schmaier A.H. Watson S.P. Lussana F. Pugliano M.T. Michelson A.D. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC/ISTH.J Thromb Haemost. 2013; 11: 1183-9Crossref Scopus (356) Google Scholar, because this practice may impair platelet responsiveness to agonists 7.Cattaneo M. Lecchi A. Zighetti M.L. Lussana F. Platelet aggregation studies: autologous platelet‐poor plasma inhibits platelet aggregation when added to platelet‐rich plasma to normalize platelet count.Haematologica. 2007; 92: 694-7Crossref PubMed Scopus (118) Google Scholar. However, considering that the extent of platelet aggregation measured with IA is a function of the volume of platelet mass accumulating on the electrodes, the higher the number of platelets in the sample, the greater may be the size of the platelet aggregate accumulating on the electrodes, increasing electrical impedance. We compared the effect of variations in platelet count between 75 × 109L−1 and 750 × 109L−1 on the results of platelet aggregation measured with the Multiplate™ analyzer and LTA. To this end, we used washed platelet suspensions (WPSs) that were adjusted to predefined platelet counts with suspending buffer, in order to avoid the potential negative interactions of autologous PPP added to PRP or whole blood 7.Cattaneo M. Lecchi A. Zighetti M.L. Lussana F. Platelet aggregation studies: autologous platelet‐poor plasma inhibits platelet aggregation when added to platelet‐rich plasma to normalize platelet count.Haematologica. 2007; 92: 694-7Crossref PubMed Scopus (118) Google Scholar. Healthy subjects (n=19; 11 men; median age of 27 years [22–61]) were recruited among the laboratory personnel and medical students of our institution. Blood samples were collected from an antecubital vein with a 21‐gauge needle, with minimal venostasis. The first 3 mL was collected into K‐EDTA for blood cell counts, and the following 50 mL was anticoagulated with acid citrate dextrose (1 : 7 [v/v]) and used to prepare WPSs 8.Mustard J.F. Perry D.W. Ardlie N.G. Packham M.A. Preparation of suspensions of washed platelets from humans.Br J Haematol. 1972; 22: 193-204Crossref PubMed Scopus (650) Google Scholar and washed red blood cells (RBCs) in Tyrode's solution. The final WPS had a platelet count of 750 × 109 L−1, which was diluted in aliquots at 500 × 109L−1, 250 × 109 L−1, 150 × 109L−1, and 75 × 109 L−1; the final RBC suspension had a hematocrit of 80%, and was added to the WPS to obtain a final hematocrit of 40%. Platelet aggregation was measured with a Multiplate™ analyzer (Roche Diagnostics, Monza, Italy) in WPS with and without RBCs, and with LTA (Chrono‐log series 400; Chrono‐log, Harvertown, PA, USA) in WPS: 20 μm ADP (Sigma‐Aldrich, Milan, Italy), 3.2 μg mL−1 collagen and 32 μm thrombin receptor activator peptide 6 (TRAP‐6) (Verum Diagnostica, München, Germany) were used as agonists. Fibrinogen (0.8 mg mL−1) was added to all suspensions. Results were expressed as area under the curve (AUC) over time (AU*min; Multiplate™ analyzer) and maximal light transmission (LTA). The results of the studies performed with LTA showed that the extent of platelet aggregation induced by collagen or TRAP‐6 was not significantly affected by platelet counts in the range 150–750 × 109 L−1, but was significantly lower at a platelet count of 75 × 109 L−1 (Fig. 1A). Similar results were obtained when platelets were stimulated with ADP, except that the extent of platelet aggregation at 750 × 109 L−1 was lower than that at platelet counts of 150–500 × 109 L−1 (Fig. 1A). The results of platelet aggregation measured with the Multiplate™ analyzer showed that, in both the presence and the absence of RBCs, the extent of platelet aggregation induced by TRAP‐6, collagen or ADP increased linearly with the increase in platelet count from 75 × 109 L−1 to 750 × 109 L−1 (Fig. 1B,C). We calculated a mean increase of 133.7 AU*min with TRAP‐6, 106.0 AU*min with collagen and 50.5 AU*min with ADP for every 50 × 109 L−1 increase in platelet count in WPS. In WPS plus RBCs, the corresponding values were 145.0 AU*min with TRAP‐6, 107.7 AU*min with collagen, and 35.8 AU*min with ADP. Therefore, the results of our study confirm previous observations that a platelet count within the physiologic range does not affect the results of LTA 7.Cattaneo M. Lecchi A. Zighetti M.L. Lussana F. Platelet aggregation studies: autologous platelet‐poor plasma inhibits platelet aggregation when added to platelet‐rich plasma to normalize platelet count.Haematologica. 2007; 92: 694-7Crossref PubMed Scopus (118) Google Scholar; however, it does affect the results obtained with the Multiplate™ analyzer. As compared with our previous experiments with LTA 7.Cattaneo M. Lecchi A. Zighetti M.L. Lussana F. Platelet aggregation studies: autologous platelet‐poor plasma inhibits platelet aggregation when added to platelet‐rich plasma to normalize platelet count.Haematologica. 2007; 92: 694-7Crossref PubMed Scopus (118) Google Scholar, we widened the range of platelet counts studied and used the additional platelet agonist TRAP‐6. Not surprisingly, we observed a decrease in platelet aggregation at the very low platelet count of 75 × 109 L−1. In contrast, the observed decrease in platelet aggregation at 750 × 109 L−1 with ADP was somewhat unexpected, and may tentatively be interpreted as being attributable to the failure of the low concentration of added apyrase 8.Mustard J.F. Perry D.W. Ardlie N.G. Packham M.A. Preparation of suspensions of washed platelets from humans.Br J Haematol. 1972; 22: 193-204Crossref PubMed Scopus (650) Google Scholar to preserve platelet responsiveness to ADP in WPS with very high platelet counts. The experiments with the Multiplate™ analyzer showed that, in both the presence and the absence of 40% autologous RBCs, the increase in platelet aggregation that was associated with platelet count was substantial, and varied with the type of platelet agonist used. Previous studies with the Multiplate™ analyzer demonstrated that there is a correlation between platelet count and extent of platelet aggregation 5.Seyfert U.T. Haubelt H. Vogt A. Hellstern P. Variables influencing Multiplate™ whole blood impedance platelet aggregometry and turbidimetric platelet aggregation in healthy individuals.Platelets. 2007; 18: 199-206Crossref PubMed Scopus (153) Google Scholar, 9.Wurtz M. Hvas A.M. Kristensen S.D. Grove E.L. Platelet aggregation is dependent on platelet count in patients with coronary artery disease.Thromb Res. 2012; 129: 56-61Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, and that the in vitro dilution of whole blood samples with a mixture of PPP and RBCs to reduce the platelet count without affecting the hematocrit caused a reduction in the extent of platelet aggregation 10.Hanke A. Roberg K. Monaca E. Sellmann T. Weber C. Rahe‐Meyer N. Görlinger K. Impact of platelet count on results obtained from multiple electrode platelet aggregometry (Multiplate).Eur J Med Res. 2010; 15: 214-19Crossref PubMed Google Scholar. However, owing to the very nature of their design, these studies failed to establish a causal relationship between platelet count and platelet aggregation, because the effects of all potential confounders were not controlled for. Moreover, in the in vitro experiments, the platelet count was decreased by the addition of PPP, which may inhibit platelet function 7.Cattaneo M. Lecchi A. Zighetti M.L. Lussana F. Platelet aggregation studies: autologous platelet‐poor plasma inhibits platelet aggregation when added to platelet‐rich plasma to normalize platelet count.Haematologica. 2007; 92: 694-7Crossref PubMed Scopus (118) Google Scholar. In our experiments: (i) platelet count was the only parameter that was varied; (ii) we used WPS, which allowed us to dilute the samples with Tyrode's buffer, which does not affect platelet function; and (iii) we explored the whole physiologic range of platelet count, instead of focusing only on the low platelet counts that were obtained by diluting whole blood samples 10.Hanke A. Roberg K. Monaca E. Sellmann T. Weber C. Rahe‐Meyer N. Görlinger K. Impact of platelet count on results obtained from multiple electrode platelet aggregometry (Multiplate).Eur J Med Res. 2010; 15: 214-19Crossref PubMed Google Scholar. The information provided by our study on the effects of platelet counts within the physiologic range on the results obtained with the Multiplate™ analyzer has practical and clinically relevant implications, considering that the test is performed in patients with normal platelet counts in most instances. For instance, the inhibitory effect of antiplatelet drugs on platelet aggregation may be either overestimated or underestimated as a consequence of variations in the platelet count that may transiently occur in different phases of thrombotic disorders. The use of more specific tests, whose results are not influenced by confounders such as platelet count, would be preferable 2.Cattaneo M. Cerletti C. Harrison P. Hayward C.P.M. Kenny D. Nugent D. Nurden P. Rao A.K. Schmaier A.H. Watson S.P. Lussana F. Pugliano M.T. Michelson A.D. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC/ISTH.J Thromb Haemost. 2013; 11: 1183-9Crossref Scopus (356) Google Scholar, 3.Cattaneo M. Resistance to antiplatelet drugs: molecular mechanisms and laboratory detection.J Thromb Haemost. 2007; 5: 230-7Crossref PubMed Scopus (175) Google Scholar. Independently of the usefulness of antiplatelet drug monitoring in general and of the role of the Multiplate™ analyzer in this setting 11.Cattaneo M. Response variability to clopidogrel: is tailored treatment, based on laboratory testing, the right solution?.J Thromb Haemost. 2012; 10: 327-36Crossref PubMed Scopus (47) Google Scholar, we think that the results of our study should be taken into consideration for the correct interpretation of the results and, consequently, for the correct management of patients. E. A. Femia, M. Scavone, and A. Lecchi: platelet aggregation studies, analysis of the data, and final approval of the version to be published. M. Cattaneo: design of the study, analysis of the data, writing of the manuscript, and final approval of the version to be published. M. Cattaneo has received honoraria for lectures/work on advisory boards and research support from Eli‐Lilly, Daiichi‐Sankyo, AstraZeneca, Sanofi‐Aventis, and Evolva. E. A. Femia, M. Scavone and A. Lecchi state that they have no conflict of interest. All reagents, hirudin tubes and test cells were kindly donated by Verum Diagnostica, Munich, Germany." @default.
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• W2037658163 title "Effect of platelet count on platelet aggregation measured with impedance aggregometry (Multiplate™ analyzer) and with light transmission aggregometry" @default.
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