Platelet Function Tests and Flow Cytometry to Monitor Antiplatelet Therapy

ABSTRACT

There are numerous methods to measure platelet function in specialized laboratories. They provide valuable results for research or small patient studies, but none of these tests is currently used to guide antiplatelet therapy in routine clinical practice. Aggregometry is the most commonly used test, but this technique is time-consuming and preanalytical preparation, choice of anticoagulant, and agonist have not been standardized. The Rapid-Platelet-Function-Analyzer uses whole blood to measure glycoprotein (GP) IIb/III inhibitor activity. This test is quick and simple and results for abciximab correlate closely with aggregometry. The platelet function analyzer PFA-100 is a good screening method to detect platelet function defects. The closure time of the PFA is also sensitive for aspirin-resistance that affects many patients. Flow cytometry can monitor a whole panel of platelet activation markers and platelet interactions with other cells. It can quantitate receptor expression and inhibition. Although cytometry is time-consuming and expensive, it is a valuable tool for research.

Platelets have many different functions. This requires specific tests optimized for each of the different functions. Each test has advantages and disadvantages and should be interpreted only in concert with a thorough history and physical examination. The ideal test should be simple, quick, inexpensive, and available on a 24-hour basis.

KEYWORDS

Platelet function - aggregometry - Rapid-Platelet-Function-Analyzer - PFA-100 - flow cytometry

REFERENCES

• 1 Roth G J, Stanford N, Majerus P W. Acetylation of prostaglandin synthase by aspirin.  Proc Natl Acad Sci USA. 1975;  72 3073-3076
• 2 Marcus A J. Aspirin as an antithrombotic medication.  N Engl J Med. 1983;  309 1515-1517
• 3 Duke W W. A simple instrument for determining the coagulation-time of the blood.  Arch Intern Med. 1912;  9 258-261
• 4 Gold H K, Gimple L W, Yasuda T et al.. Pharmacodynamic study of F(ab')2 fragments of murine monoclonal antibody 7E3 directed against human platelet glycoprotein IIb/IIIa in patients with unstable angina pectoris.  J Clin Invest. 1990;  86 651-659
• 5 Cox D. Methods for monitoring platelet function.  Am Heart J. 1998;  135 S160-S169
• 6a Jürgens R, Braunsteiner H. Zur Pathogenese der Thrombose.  Schweiz Med Wochenschr. 1950;  80 1388-1394
• 6b Born G VR. Aggregation of blood platelets by adenosine diphosphate and its reversal.  Nature. 1962;  194 927-929
• 7 O'Brien J R. Platelet aggregation. Part I. Some effects of the adenosine phosphates, thrombin, and cocaine upon platelet adhesiveness. Part II. Some results from a new method of study.  J Clin Pathol. 1962;  15 446-455
• 8 Trip M D, Cats V M, van Capelle F L J, Vreeken J. Platelet hyperreactivity and prognosis in survivors of myocardial infarction.  N Engl J Med. 1990;  323 1549-1554
• 9 Yasuda T, Gold H K, Fallon J T et al.. Monoclonal antibody against the platelet glycoprotein/GP) IIb/IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs.  J Clin Invest. 1988;  81 1284-1291
• 10 Cannon C P, McCabe C H, Borzak S et al.. Randomized trial of an oral platelet glycoprotein IIb/IIIa antagonist, sibrafiban, in patients after acute coronary syndrome.  Circulation. 1998;  97 340-349
• 11 Chambless L E, McMahon R, Finch A et al.. ARIC hemostasis study-III. Quality control. Atherosclerosis Risk in Communities.  Thromb Haemost. 1993;  70 588-594
• 12 Richards E M, Baglin T P. Quantitation of reticulated platelets: methodology and clinical application.  Br J Haematol. 1995;  91 445-451
• 13 Breddin H K, Heptinstall S, Mascelli M, Harder S, Jeske W, Walenga J M. Concerted action to improve the validation of platelet function tests [abstract].  Thromb Haemost. 2001;  86(suppl) 387
• 14 Breddin K, Ziemen M, Bauer O et al.. Time and temperature dependent changes of ADP- and collagen-induced “spontaneous” aggregation.  Thromb Res. 1980;  19 621-638
• 15 Mascelli M A, Worley S, Veriabo N J et al.. Rapid assessment of platelet function with a modified whole-blood aggregometer in percutaneous transluminal coronary angioplasty patients receiving anti-GPIIb/IIIa therapy.  Circulation. 1997;  96 3860-3866
• 16 Thompson N T, Scrutton M C, Wallis R B. Particle volume changes associated with light transmittance changes in the platelet aggregometer-dependence upon aggregating agent and effectiveness of agent.  Thromb Res. 1986;  41 615-626
• 17 Storey R F, Wilcox R G, Heptinstall S. Differential effects of glycoprotein IIb/IIIa antagonists on platelet microaggregate and macroaggregate formation and effect of anticoagulation on antagonist potency.  Circulation. 1998;  98 1616-1621
• 18 Gabbasov Z A, Popov E G, Gavrilov I Y, Pozin E Y. Platelet aggregation: the use of optical density fluctuations to study microaggregation formation in platelet suspension.  Thromb Res. 1989;  54 215-223
• 19 Ozaki Y, Satoh K, Yatomi Y, Yamamoto T, Shirasawa Y, Kume S. Detection of platelet aggregates with a particle counting method using light scattering.  Anal Biochem. 1994;  218 284-294
• 20 Fusegawa Y, Handa S. Platelet aggregation induced by ADP or epinephrine is enhanced in habitual smokers.  Thromb Res. 2000;  97 287-295
• 21 Miyamoto S, Kawano H, Sakamoto T et al.. Formation of platelet microaggregates correlates with adverse clinical outcome in patients with coronary artery disease.  Thromb Haemost. 2003;  89 681-686
• 22 Coller B S, Lang D, Scudder L E. Rapid and simple platelet function assay to assess glycoprotein IIb/IIIa receptor blockade.  Circulation. 1997;  95 860-867
• 23 Smith J W, Steinhubl S R, Lincoff A M et al.. Rapid platelet-function assay. An automated and quantitative cartridge-based method.  Circulation. 1999;  99 620-625
• 24 Steinhubl S R, Kottke-Marchant K, Moliterno D J et al.. Attainment and maintenance of platelet inhibition through standard dosing of abciximab in diabetic and nondiabetic patients undergoing percutaneous coronary intervention.  Circulation. 1999;  100 1977-1982
• 25 Steinhubl S R, Talley D, Braden G A et al.. Point-of-care measured platelet inhibition correlates with a reduced risk of an adverse cardiac event after percutaneous coronary intervention. Results of the GOLD (AU-Assessing Ultegra) Multicenter Study.  Circulation. 2001;  103 2572-2578
• 26 Matzdorff A C, Kühnel G, Kemkes-Matthes B, Voss R. Comparison of GPIIb/IIIa inhibitors and their activity as measured by aggregometry, flow cytometry, single platelet counting, and the rapid platelet function analyzer.  J Thromb Thrombolysis. 2001;  12 129-139
• 27 Frelinger III A L, Mattson J C, Furman M I et al.. Monitoring tirofiban therapy in patients: effect of ex vivo anticoagulant [abstract].  Thromb Haemost. 2001;  86(suppl) 377
• 28 O'Neill W W, Serruys P, Knudtson M for the EXCITE Trial Investigators et al.. Long-term treatment with a platelet glycoprotein-receptor antagonist after percutaneous coronary revascularization.  N Engl J Med. 2000;  342 1316-1324
• 29 The SYMPHONY Investigators . Comparison of sibrafiban with aspirin for prevention of cardiovascular events after acute coronary syndromes: a randomised trial.  Lancet. 2000;  355 337-345
• 30 Kratzer M A, Born G V. Simulation of primary haemostasis in vitro.  Haemostasis. 1985;  15 357-362
• 31 Fressinaud E, Veyradier A, Sigaud M, Boyer-Neumann C, Le Boterff C, Meyer D. Therapeutic monitoring of von Willebrand disease: interest and limits of platelet function analyser at high shear rates.  Br J Haematol. 1999;  106 777-783
• 32 Angstwurm M, Sander A, Schwarz S, Spannagl M, Schramm W. Analysis of platelet activation within the devices of PFA-100^® using cytometry [abstract].  Ann Hematol. 2001;  80(suppl 1) A4
• 33 Hézard N, Metz D, Nazeyrollas P et al.. Use of the PFA-100^™ apparatus to assess platelet function in patients undergoing PTCA during and after infusion of cE3 Fab in the presence of other antiplatelet agents.  Thromb Haemost. 2000;  83 540-544
• 34 Schultz C, Hoppensteadt D A, Ma Q, Jeske W P, Khan E, Fareed J. Comparative studies on the antiplatelet effects of various glycoprotein IIb/IIIa inhibitors as measured by aggregometry and a rapid point-of-care platelet function analyzer (Ultegra RPFA) [abstract].  Blood. 2000;  96(suppl 1, part 2) 69b
• 35 Mattson J C, Farrah D, Westley S, O'Neill W W. Effect of PPACK anticoagulant on platelet function analyzer (PFA-100) closure times at baseline and following GPIIB-IIIA blockade [abstract].  Thromb Haemost. 2001;  86(suppl) 1227
• 36 Jilma B. Platelet function analyzer (PFA-100): a tool to quantify congenital or acquired platelet dysfunction.  J Lab Clin Med. 2001;  138 152-163
• 37 McKee S A, Sane D C, Deliargyris E N. Aspirin resistance in cardiovascular disease: a review of prevalence, mechanisms, and clinical significance.  Thromb Haemost. 2002;  88 711-715
• 38 Andersen K, Hulren M, Arnesen H, Seljeflot I. Aspirin non-responsiveness as measured by PFA-100 in patients with coronary artery disease.  Thromb Res. 2003;  108 37-42
• 39 Hézard N, Metz D, Nazeyrollas P et al.. PFA-100k and flow cytometry: can they challenge aggregometry to assess antiplatelet agents, other than GPIIbIIIa blockers, in coronary angioplasty?.  Thromb Res. 2003;  108 43-47
• 40 Gear A L R. Rapid reactions of platelets studied by a quenched-flow approach: aggregation kinetics.  J Lab Clin Med. 1982;  100 866-886
• 41 Matzdorff A C, Kühnel G. A simple and rapid method to determine GPIIb/IIIa-receptor inhibitor activity in whole blood.  Hämostaseologie. 1999;  19 134-138
• 42 Storey R F, May J A, Wilcox R G, Heptinstall S. A whole blood assay of inhibition of platelet aggregation by glycoprotein IIb/IIIa antagonists: comparison with other aggregation methodologies.  Thromb Haemost. 1999;  82 1307-1311
• 43 Storey R F, Wilcox R G, Heptinstall S. Differential effects of glycoprotein IIb/IIIa antagonists on platelet microaggregate and macroaggregate formation and effect of anticoagulant on antagonist potency.  Circulation. 1998;  98 1616-1621
• 44 Xiao Z, Theroux P, Frojmovic M M. Modulation of platelet-neutrophil interaction with pharmacological inhibition of fibrinogen binding to platelet GPIIb/IIIa receptor.  Thromb Haemost. 1999;  81 281-285
• 45 McEver R P, Martin M N. A monoclonal antibody to a membrane glycoprotein binds only to activated platelets.  J Biol Chem. 1984;  259 9799-9804
• 46 Becker R C, Tracy R P, Bovill E G, Mann K G, Ault K. The clinical use of flow cytometry for assessing platelet activation in acute coronary syndromes. TIMI-III thrombosis and anticoagulation group.  Coron Artery Dis. 1994;  5 339-345
• 47 Ault K A, Cannon C P, Mitchell J et al.. Platelet activation in patients after acute coronary syndrome: results from the TIMI-12 trial. Thrombolysis in Myocardial Infarction.  J Am Coll Cardiol. 1999;  33 634-639
• 48 Nurden A T, Macchi L, Bihour C, Durrieu C, Besse P, Nurden P. Markers of platelet activation in coronary heart disease patients.  Eur J Clin Invest. 1994;  24(suppl I) 42-45
• 49 Cahill M R, Macey M G, Dawson J R, Newland A C. Platelet surface activation antigen expression at baseline and during elective angioplasty in patients with mild to moderate coronary artery disease.  Blood Coagul Fibrinol. 1996;  7 165-168
• 50 Wahba A, Black G, Koksch M et al.. Cardiopulmonary bypass leads to a preferential loss of activated platelets.  Eur J Cardiothorac Surg. 1996;  10 768-773
• 51 Michelson A D, Barnard M R, Hechtmann H B et al.. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.  Proc Natl Acad Sci USA. 1996;  93 11877-11882
• 52 Ferroni P, Pulcinelli F M, Lenti L, Gazzaniga P P. Is soluble P-selectin determination a more reliable marker of in vivo platelet activation than CD62p flow cytometric analysis?.  Thromb Haemost. 1999;  81 472-473
• 53 Born G R V, Richardson P D. Activation time of blood platelets.  J Membr Biol. 1980;  57 87-90
• 54 O'Brien J R. Exhausted platelets continue to circulate.  Lancet. 1978;  2 1316-1317
• 55 Quinn M, Deering A, Stewart M, Cox D, Foley B, Fitzgerald D. Quantifying GPIIb/IIIa receptor binding using 2 monoclonal antibodies: discriminating abciximab and small molecular weight antagonists.  Circulation. 1999;  99 2231-2238
• 56 Konstantopoulos K, Wu K K, Udden M M, Bañez E I, Shattil S J, Hellums J D. Flow cytometric studies of platelet responses to shear stress in whole blood.  Biorheology. 1995b;  32 73-93
• 57 Schenk J F, Morgenstern E, König J, Seyfert U T, Heinrich W, Wenzel E. Reliability of platelet retention measurement using the platelet retention test homburg (“RT-H”) [abstract].  Thromb Haemost. 2001;  86(suppl) 384
• 58 Varon D, Dardik R, Shenkman B et al.. A new method for quantitative analysis of whole blood platelet interaction with extracellular matrix under flow conditions.  Thromb Res. 1997;  85 283-294
• 59 Grotemeyer K H. The platelet-reactivity-test: a useful by-product of the blood-sampling procedure?.  Thromb Res. 1991;  61 423-431
• 60 Grotemeyer K H. Two-year follow-up of aspirin responder and aspirin non responder. A pilot-study including 180 post-stroke patients.  Thromb Res. 1993;  71 397-403
• 61 Hoffman M, Monroe D M. A cell-based model of hemostasis.  Thromb Haemost. 2001;  85 958-965
• 62 Abrams C, Shattil S J. Immunological detection of activated platelets in clinical disorders.  Thromb Haemost. 1991;  65 467-473