Platelet Physiology
DRAFT Subcommittee Minutes
24 July 2011
8:00-12:00
Annex Hall 2
Chairman: Marco Cattaneo (IT)
Co-chairmen: Christian Gachet (FR), Paolo Gresele (IT), Paul Harrison (UK), Catherine Hayward (CA), Dermot Kenny (IE), Diego Mezzano (CL), Andrew David Mumford (UK), Diane Nugent (US), Alan T. Nurden (FR)
Dr Paolo Gresele reported on the preliminary results of an online survey on how congenital platelet function disorders are diagnosed worldwide. The survey represents the first step towards the development of methodological guidelines, the main ongoing project of the Platelet Physiology Subcommittee. About 200 centres filled up the online questionnaire, providing information on how they select patients who need to undergo a diagnostic workup for platelet function disorders, what first-step, second-step and third-step diagnostic tests they use. The results were analyzed anonymously to participant identities. As expected, the survey revealed a high variability in diagnostic practices worldwide: as a consequence, methodological standardization is necessary. The information gathered in this survey will be helpful in the development of guidelines.
Dr Dermot Kenny gave a brief review of a 96 well platelet function assay his group developed in 2008. He then outlined a rationale for testing platelet function in disease states other than bleeding disorders and cardiovascular disease. He presented data from assays of platelet function in patients with inflammatory arthritis, patients with HIV, patients with HIV taking the drug abacavir, patients with recurrent miscarriage and finally in patients with pulmonary hypertension. The profiles of platelet function were different in these disease cohorts relative to normal controls and also demonstrated disease specific phenotype relative to disease activity. He concluded that platelet function testing is of interest to the internist.
Dr Diego Mezzano and Dr Cathy Hayward illustrated their most recent personal experience with methods to study platelet secretion. Platelet secretion defects are common platelet function disorders that may, or may not, be associated with aggregation abnormalities. These conditions are emerging to have clinically important bleeding risks and their management sometimes necessitates platelet transfusion.
Dr Mezzano reviewed methods that are used to measure the platelet secretion of serotonin. The method based on loading platelets with radiolabeled (3H or 14C) serotonin has the disadvantage of exposing the operator to radiations and is not feasible in all patients (for instance, in patients who lack platelet delta granules). A method based on the measurement of endogenous serotonin by HPLC by electrochemical detection proved to be reliable, quick and cost-effective, provided that the laboratory is already equipped with the necessary, rather expensive instrumentation.
Dr Hayward presented an update on the diagnosis of platelet function disorders based on dense granule ATP release assays, using light emission triggered by reaction of the released ATP with D-luciferin, catalyzed by firefly luciferase. The released ATP is quantified against an ATP standard. ATP release shows considerably more variability than aggregation assays (Havelow et al, Lab Hematol 2007; 13:59-62; Pai et al, AJCP 2011; in press) and this is reflected by fairly broad reference ranges. Strong agonists (thrombin, collagen) induce more dense granule ATP release than other agonists and show the least variability. Data from a prospective study of ATP release for bleeding disorders (Pai et al, AJCP 2011; in press) indicates that the findings are more predictive of a bleeding disorder, or an inherited platelet function disorder, when abnormalities are present with more than one agonists. Furthermore, the test detects platelet function disorders even when aggregation findings are normal. Receiver operator curve analyses indicate that there is little added value to measuring release with multiple concentrations of an agonist. Among the best agonists for detecting common platelet function disorders due to dense granule release assays are: 6 μM epinephrihne, 5.0 μg/ml collagen and 1 μM thromboxane analogue U46619. When dense granule release is reduced with strong agonists, the differential diagnosis includes dense granule deficiency in addition to secretion defects due to other causes. Overall, ATP release assays can be very helpful to diagnose bleeding disorders. However, caution is warranted in that the reagent commonly used to measure ATP release, Chronolume?, potentiates some aggregation responses that are submaximal. Evidence has now emerged that the reagent can alter platelet aggregation findings for congenital platelet disorders.
Dr Tim Warner described the 96-well platelet aggregometry test that was developed in his laboratory. The test relies upon the detection of changes in light transmission through suspension of platelets, either in platelet rich plasma (PRP) or in washed platelets, caused by platelet agonists; i.e. agonists that cause platelet aggregation lead to a decrease in light scatter through the suspensions and so a decrease in measured absorbance. For 96-well plate aggregation testing, agonists are first placed into the plate, in a similar concentration range to those used for traditional light transmission aggregometry (LTA). Agonists are placed first as there are too many agonist wells to be pipetted after the addition of platelets, followed by the addition of the platelet suspension. The plates can then be placed into a kinetic plate reader which will shake the plates and then read the changes in absorbance as a marker of platelet aggregation over time. The time course of the aggregation is generally slower than that seen in traditional LTA, most probably because the mixing is less vigorous and because the mixing is stopped each time the plate is read, unlike LTA in which mixing and reading take place at the same time. The advantage of 96-well plate aggregometry is that controls, e.g. unstimulated PRP and PPP, and test samples, PRP plus agonists, are run together removing problems of variability in PRP responsiveness over time. In addition, by making use of the many wells available on the plate full agonist curves to multiple platelet agonists can be rapidly developed. If a luminescent plate reader is available the assay can also be used to determine ADP/ATP release by use of luciferase reagents as in LTA, and samples can be retained at the end of aggregation experiments for determination of e.g. the production of TXA2. As samples are already in 96-well format there is very easy transfer to other standard plate based assays. Finally, platelet adhesion in the plates, subject to pre-coating of the plates, can be easily read by the addition of p-nitrophenol phosphate and the detection of the formation of p-nitrophenol by platelet acid phosphatase. In our most recent experiments, we have produced 96-well plates with lyophilized and immobilized platelet agonists. These plates can be stored at room temperature and following the simple addition of PRP can be used for the rapid screening of platelet aggregation responses; in these experiments plates can be vortex mixed for 5 minutes and then the assay read as end point assay of platelet aggregation in any plate reader. This may well be the best use of 96-well plate aggregometry, a rapid across the board screening of platelet responses in a standardized manner, leaving traditional LTA for more in-depth kinetic analyses of platelet function.
