Platelet Function Tests: Precision and Reliability in Healthy Volunteers and Donors on Daily Antiplatelet Agent Therapy [Hot Topic]

karon
Brad Karon, M.D., Ph.D.

Mechanical circulatory support (MCS) is increasingly used for patients with end-stage heart failure, though complications such as bleeding and thrombosis are common. Protocols for MCS often call for monitoring and titration of antiplatelet agents in the perioperative period. Dr. Brad Karon describes a study to identify the optimal platelet function tests for this purpose.

Presenter and Credentials:
Brad Karon, M.D., Ph.D., Consultant in the Division of Clinical Core Laboratory Services at Mayo Clinic in Rochester, Minn., and Associate Professor of Laboratory Medicine and Pathology in the College of Medicine.

Transcript

Our speaker for this program is Dr. Brad Karon, a Consultant in the Division of Clinical Core Laboratory Services at Mayo Clinic in Rochester, Minnesota. Dr. Karon is also Associate Professor of Laboratory Medicine and Pathology in the College of Medicine.Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Today our topic is platelet function tests: precision and reliability in healthy volunteers and donors on daily antiplatelet agent therapy.

Thank you Cara, it’s a pleasure to spend a few minutes today to discuss the results of our recent study comparing different platelet function tests.

I have no disclosures related to this talk or subject.

Our study of platelet function tests was originally motivated by an internal practice need to support the mechanical circulatory support group at Mayo.  Mechanical circulatory support, which includes use of devices such as left ventricular assist devices and total artificial hearts, is increasingly being used as a bridge to cardiac transplant, a bridge to recovery in patients with severe myocardial damage, and a destination therapy for patients with end-stage heart failure who are not candidates for cardiac transplant.

Although MCS therapy has improved the outcome in many patients with very serious heart disease, use of these devices is not without complications and risks.  Up to 60% of patients will experience excess bleeding in the perioperative period; while at the same time 5 to 20% of patients may experience device thrombosis.  Balancing the risk of bleeding vs. clotting (that is to say either device, thrombosis or systemic thrombosis, like a stroke) during the perioperative period is one of the greatest challenges in mechanical circulatory support or MCS.  To accomplish this, many published protocols for MCS placement, such as the Berlin or Arizona anticoagulation protocols, call for monitoring and titration of antiplatelet agents in the perioperative period.  Laboratory tests of arachidonic acid-induced platelet function are suggested to titrate aspirin therapy; while tests of ADP-induced platelet function are often used to titrate agents that inhibit ADP-dependent platelet function.

With that background, we designed a study to answer the question, “Which platelet function tests are appropriate for monitoring and titrating antiplatelet agent therapy in the critical care setting?”, or in other words, which platelet function tests are appropriate for monitoring and titrating antiplatelet agent therapy over relatively short periods of time, a few days.

We performed a method comparison of five tests of arichodonic-acid and ADP-induced platelet function.  Those tests are platelet mapping by thromboelastography (or TEG PM), VerifyNow whole blood cassette-based aggregation system, conventional light transmission aggregometry (or LTA), the Multiplate whole blood impedance aggregometry system, all of which measure both arachidonic acid-induced and ADP-induced platelet function, and vasodilator stimulated phosphoprotein (or VASP) flow cytometry, which provides only a measure of ADP-induced platelet activity.  The assays were compared for their ability to discriminate platelet function in healthy volunteers with normal platelet function from platelet function in donors who are taking daily aspirin or clopidogrel therapy and were expected to have reduced or inhibited platelet function

For our study we recruited 40 healthy adult volunteers who were screened for any history of bleeding disorders and were not taking antiplatelet medications.  Blood was drawn from these volunteers for testing by all methods in duplicate.  24 of these volunteers came back within 24 hours for a second blood draw to repeat testing by all methods again in duplicate.  In the same manner 10 to 13 donors on daily aspirin and/or clopidogrel therapy were also recruited for 2 blood draws within 24 hours, with testing in duplicate by all methods after each blood draw.

We compared the tests using a number of conventional and some not so conventional calculations and ways to display quantitative data.  We constructed scatter plots to compare platelet function results in healthy volunteers to donors on antiplatelet therapy; and also compared the mean and standard deviation of platelet function results for each of these groups.  We also performed receiver operating characteristic, or ROC, sensitivity analysis to compare the ability of different tests to discriminate healthy volunteers from donors on antiplatelet agent therapy. In the interest of time, I will not be showing the ROC analysis during this presentation.  Because there is no generally accepted reference method for measuring platelet function, and because the various tests all measure slightly different aspects of platelet function or activity, there was no obvious way to measure or compare the accuracy of the various tests. Thus the tests are compared primarily on their ability to discriminate platelet function in healthy volunteers from that observed in donors on daily antiplatelet agent therapy; and on various measures of precision and reliability important to interpreting changes in these tests over short periods of time.

To compare the tests in their utility for assessing changes in platelet function over short periods of time, we measured intra-assay and interassay precision measured coefficient of variation, and reliability coefficient.  Intra-assay precision represents the analytic precision of the various methods and was calculated from all sets of duplicate testing performed in both healthy volunteers and donors on antiplatelet agent therapy.  Interassay precision was calculated from the quadruplicate set of results from each volunteer and donor when blood was collected twice within a 24-hour period.  Interassay precision represents average within person variability, and is impacted by both analytic variability of the methods themselves, preanalytic variability caused by platelet activation that occurs during blood drawing and processing for these tests, and biologic variability in platelet function.  By collecting blood twice within 24 hours we purposefully minimized biologic variability, in order to compare the tests for the sum of analytic and preanalytic variability.  Finally we calculated the reliability coefficient for each test for repeat measurement in both healthy volunteers and donors on antiplatelet agent therapy.  Reliability coefficient is another way to measure within person variability, in this case comparing it to between person variability.  The reliability coefficient varies from 0 to 1.  Tests with reliability coefficients of 0.8 to 1 are considered to have very good or nearly perfect reliability, while 0.6 to 0.8 represents substantial reliability, and 0.4 to 0.6 moderate reliability, tests with reliability of 0.2 to 0.4, or less than 0.2 represent fair or poor reliability, respectively.

Based on an extensive review of existing literature and knowledge of platelet function testing, we came up with a set of acceptance criteria for measurement of platelet function in healthy volunteers and donors on antiplatelet agent therapy.  Among healthy volunteers intraassay precision of less than or equal to 10% and interassay precision of less than or equal to 15% were considered acceptable.  Among donors on antiplatelet agent therapy, who were expected to have lower values for platelet function, intraassay precision of less than or equal to 20% and interassay precision of less than or equal to 30% was considered acceptable.  Based upon a previous study, reliability coefficients of 0.40 or above, representing moderate or greater reliability were also considered acceptable.

Looking at the distribution of arachidonic acid-induced platelet function among healthy volunteers and donors on antiplatelet agent therapy, one can observe that all of the tests were able to distinguish healthy volunteers with normal platelet function from donors on daily aspirin therapy to some extent.  For TEG platelet mapping, light transmission aggregometry or LTA, and Multiplate whole blood impedance aggregometry, mean values among donors on daily aspirin therapy were approximately 5-fold lower than arachidonic acid-induced platelet function among healthy volunteers.  In contrast, mean values by VerifyNow among donors on daily aspirin therapy were less than 2-fold lower than the values among healthy volunteers.  In addition, a handful of healthy volunteers had very low arachidonic acid-induced platelet function when measured by LTA and this was not observed by the other methods.

Looking at intra- and interassay precision among methods used to measure arachidonic acid-induced platelet function, the VerifyNow device demonstrated the best precision.  Light transmission aggregometry did not meet acceptance criteria for interassay precision among donors on daily aspirin therapy, demonstrating a coefficient of variation of approximately 38%.  TEG PM was the least precise device, with coefficient of variation of approximately 100% among aspirin-treated donors.

Looking at the reliability coefficient for tests of arachidonic acid-induced platelet function, only Mulitplate whole blood impedance aggregometry met acceptance criteria of moderate or better reliability for both healthy volunteers and donors on daily aspirin therapy.

To summarize our findings of tests of arachidonic acid-induced platelet function: TEG PM or TEG Platelet Mapping, LTA, and Multiplate all differentiated platelet function between healthy volunteers and donors on daily aspirin therapy, with mean values among aspirin-treated donors approximately 5-fold lower than those observed in healthy volunteers. TEG PM and LTA demonstrated poorer precision among aspirin-treated donors; while Multiplate was the only platelet function method that demonstrated acceptable precision and reliability among both healthy volunteers and aspirin-treated donors.

This slide shows the distribution of all results for ADP-induced platelet function in healthy volunteers and donors on daily clopidogrel therapy. LTA (Light Transmission Aggregometry), VerifyNow, and Multiplate all produced mean values for ADP-induced platelet function among the clopidogrel-treated donors that were approximately half that observed among healthy volunteers. This is the expected result based upon previous studies of clopidogrel effect on platelet function. VASP flow cytometry best differentiated platelet function between the healthy volunteers and clopidogrel-treated donors; while TEG Platelet Mapping showed the least sensitivity to the effects of clopidogrel on platelet function.

All methods for ADP-induced platelet function met predefined acceptability criteria for intra- and interassay precision, with again the VerifyNow showing the best overall precision of the various methods.

The reliability coefficients for tests of ADP-induced platelet function all met acceptability criteria with the exception of TEG PM (TEG Platelet Mapping) among the healthy donors.

To summarize our results for tests of ADP-induced platelet function, TEG PM was the least optimal test for distinguishing the effect of clopidogrel on platelet function; while VASP flow cytometry was the best able to distinguish platelet function in healthy volunteers from that observed in donors on daily clopidogrel therapy.  Because VASP requires sample fixation and expertise in flow cytometry, its application in the critical care setting may be limited.  Beyond VASP, all the tests with the exception of TEG Platelet Mapping may be appropriate for short-term monitoring of ADP-induced platelet function.

In summary, Multiplate whole blood impedance aggregometry was the only method studied that met acceptability criteria for precision and reliability for both arachidonic acid-induced and ADP-induced platelet function among both healthy volunteers and donors on daily antiplatelet agent therapy. Multiplate is a whole blood method offering rapid turnaround time and requiring little user expertise.  Because VerifyNow demonstrated the best precision of all the methods, its use may also be appropriate, especially in situations which require precise measurement of platelet function from day to day.  TEG Platelet Mapping is not an appropriate method for monitoring platelet function among patients placed on antiplatelet agent therapy in the critical care setting.

Thank you for viewing this presentation on platelet function testing. I hope you have found it valuable and informative.

mmledu

MML Education

This post was developed by our Education and Technical Publications Team.