Test di funzione piastrinica

Transcription

1 PIASTRINE 2015 V CORSO NAZIONALE DI AGGIORNAMENTO Milano, novembre Università degli Studi di Milano Test di funzione piastrinica Silvia Riondino IRCCS San Raffaele, Roma Università di Roma Tor Vergata

2 Platelets have a pleiotropic range of biological roles that extend beyond hemostasis Cancer growth Metastasis Dewitte A, Experimental Hematology & Onco

3 Schematic representation of platelet structure and function possible congenital defects Dovlatova, Br J Haematol 2015

4 PLATELET FUNCTION TESTING KEY POINTS Diagnosis and management of patients presenting with bleeding problems rather than thrombosis. Management of perioperative haemostasis Increasingly used for monitoring the efficacy of antiplatelet drugs Transfusion medicine Assess the dynamics of living cells in contrast to test a defined quantity of a clinical biomarker Harrison P, Hematol Oncol Clin N Am 2013, modif.

5 BLEEDING TIME 40 mm Hg Simple, rapid, not expensive.but invasive, poorly reproducible, insensitive to mild platelet defects

6 Tests based on platelet aggregation WHOLE BLOOD PRP TURBIDIMETRIC IMPEDANCE LUMINESCENCE TURBIDIMETRIC/ OPTICAL VerifyNow Chrono-Log Aggregometers Helena PlateletworkS LTA Chrono-Log Lumi-Aggregometers Ledford-Kraemer, MR. The Clotting Times 2004;4(3):2-9

7 LTA 200 g x 10 min at RT PRP /µl

8 LTA according to Born

9 Factors influencing the results of LTA Blood sampling Anticoagulants Platelet count Ph Temperature Hemolyzed samples contain ADP; Lypemic samples interfere with OD; pre-activation during handling Trisodium citrate (109 or 129 mm) (1:10) 200x10 9 /L and 300x10 9 /L DO NOT ADJUST! Storage increases ph of PRP samples Platelet aggregation is suboptimal at T<37 C Aggregometer stirr speed Continuous speed at 1000 rpm Time Skill Within 3-4 hours in order to maintain platelet viability Highly trained personnel

10 70 statements/ 8 sections: (i) clinical usefulness of LTA; (ii) pre-analytical variables; (iii) blood collection; (iv) preparation of PRP and platelet-poor plasma (PPP); (v) assessment of PRP quality; (vi) methodology; include a known control subject (vii) choice of agonists; (viii) evaluation and reporting of results.

11 Whole blood impedance Multiplate Parallel electrodes are immersed in a whole blood suspension and a small current applied. As platelets aggregate in response to the addition of an agonist, they stick to the surface of the electrode, diminish the current intensity and increase the electrical impedance. Sample preparation and manipulation are not required.

12 Whole blood impedance Pros Platelets in their natural milieu Small sample volume Immediate result No manipulation No loss of time No loss of platelet populations No centrifugation/activation Minimal technical training Cons Influenced by: - time - anticoagulant - platelet count - HCT - temperature Requires saline 1:1 dilution Poor LTA correlation

13 Lumiaggregation Detects both platelet aggregation and dense granule secretion (ADP/ATP) at the same time. Firefly luciferin is added to the sample. When ATP is released, it acts upon the luciferin to form luciferase and the amount of luminescence is measured.

14 VerifyNow Fibrinogen-coated polystyrene beads agglutinate in whole blood in proportion to the number of glycoprotein GPIIb-IIIa receptors activated by a specific stimulus, which can be blocked by antiplatelet agents Each cartridge contains: -Fibrinogen coated beads -Agonist (AA; ADP+PGE1; TRAP) -Buffer

15 VerifyNow LT IS CONVERTED INTO ASPIRIN RESPONSE UNITS, P2Y12 RESPONSE UNITS AND PLT AGGREGATION UNITS Whole blood assay- POC test specifically design to monitor antiplatelet therapy Simple, rapid, no blood handling required Influenced by: platelet count, Hct, fibrinogen levels, blood triglycerides

16 Plateletworks POC Whole blood assay Platelet aggregation (assessment of platelet count at baseline and after agonist addition) Simple, rapid, no manipulation Within few minutes of sampling Platelet monitoring during cardiopulmonary bypass

17 Tests based on platelet adhesion under shear stress WHOLE BLOOD PFA 100 IMPACT GLOBAL THROMBOSIS TEST PFA 200 CONE AND PLATE(LET)

18 PFA-100 PFA-200 Built-In Printer LCD screen Soft keys Test Cartridge Cassette Carousel Trigger solution container

19 PFA-100 Whole blood assay POC Shear dependent platelet plug formation (Coll + ADP or Coll + EPI) Simple, rapid, widely used, 800 µl blood no blood handling required by the user. Dependent on: Platelet count Hct vwf levels Blood group O HIGH NEGATIVE PREDICTIVE VALUE

20 IMPACT The cone and plate(let) analyser Whole blood platelet adhesion on a polystyrene plate coated with vwf/ fibrinogen. POC A microscope performs staining and image analysis of the platelets adhering upon the plate under a shear rate applied by a spinning cone Platelet adhesion depends upon plasma VWF, fibrinogen binding to the plastic surface, platelet Gp Ib and Gp IIb/IIIa and platelet activation events. Influenced by: platelet count, Hct

21 GLOBAL THROMBOSIS TEST (GTT)

22 Tests based on viscoelastic methods TROMBOELASTOGRAPHY (TEG/TEM) Haemostatic whole blood test

23 POINT-OF CARE - Near Patient Global Hemostasis Assessment Normal TAO Factor deficit Thrombocytopenia/ Antiplatelet drugs Fibrinolysis Hypercoagulability Quantify the strength and stability of a clot formed by the interaction of platelets, coagulation factors, inhibitors, and fibrinolytic proteins. Both biochemical (clot formation and dissolution) and mechanical properties (strength) of the clot are assessed.

24 Flow cytometry More Physiological Sample manipulation is minimal Multiple platelet receptors can be analysed simultaneously Novel antibodies can be readily incorporated into existing methodologies Small volumes of blood required Can be used in patients with significant thrombocytopenia Normal blood sample Platelets (green) selected on the basis of forward and side light scatter Platelets stained with CD41 (GpIIb) and CD61 (GpIIIa) Platelets stained with CD42b (GpIb) and CD61 (GpIIIa)

25 scd40l sgpvi sgpv sp-selectin CX3CR1 MMPs Platelet-derived microparticles MPV TXA 2 CD40L P-selectin COX-1 GPVI GPV Platelet mrna PS MMPs P-selectin P-selectin βtg PF-4 TSP-1 MRP 8/14 TF TXB 2 Activated GP IIb/IIIa PSGL-1 PMN Urinary excretion Platelet-leukocyte aggregates Urinary 11-dehydro-TXB 2 Platelet-derived biomarkers Ferroni et al. Thromb Haemost 2012

26 Biomarkers of thromboxane biosynthesis Ferroni et al. Antioxid Redox Signal 2012

27 Genotyping The Influence of Gene Profile on Platelet Reactivity and Clinical Implications The enzyme encoded by the CYP2C19 gene is responsible for clopidogrels transformation into an active metabolite. The CYP2C19*2 allele carriers have higher a Platelet Reactivity (PR), as PR inhibition by the drug is insufficient.

28 Platelet mirna MicroRNAs are short ~22-nucleotide noncoding RNA influence the expression of many genes and cellular processes. Platelet mirna expression has been shown to be perturbed during ex vivo storage of platelets and in platelet associated disorders

29 Platelet mirna Gatsiou A, Curr Vasc Pharmacology 2012

30 Platelet mirna - mirnas as potential regulators of signaling pathways in platelets - mirnas as potential markers of platelet-associated disorders - Platelet microparticle-derived mirnas as in vivo biomodulators - mirnas as potential platelet markers of storage and pathogen reduction treatments. Dahiya N, Transfus Med Rev. 2015

31 VASP phosphorylation

32 CONCLUSION Platelet function tests may be of great value depending on the aim they pursue Still recommended for platelet disorders Great interest in POC development Conclusions could be highly dependent on the test used Results from various assays are clearly not interchangeable. Careful evaluation when taking into consideration on-treatment platelet reactivity Tantry US, JACC 2013

33 FUTURE PERSPECTIVES GENOTYPING + PHENOTYPING = PERSONALIZED MEDICINE? Riondino et al. Cancer Genomics and Proteomics