Metodiche per lo studio della funzione piastrinica

Transcription

1 Metodiche per lo studio della funzione piastrinica Marco Cattaneo Medicina 2, ASST Santi Paolo e Carlo Dipartimento di Scienze della Salute Università degli Studi di Milano Milano, Italy

2 Metodiche per lo studio della funzione piastrinica Diagnosi dei difetti congeniti della funzione piastrinica Monitoraggio della terapia antiaggregante (??)

3 Diagnosi dei difetti di funzione piastrinica Test globali dell emostasi primaria

4 Tempo di emorragia Elevata variabilità Operatore-dipendente Impreciso Invasivo Poco sensibile Non utile nell algoritmo diagnostico

5 Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo Paziente con sanguinamenti mucocutanei Tempo di emorragia Prolungato Normale Screening per VWD o Difetti di Funzione Piastrinica Screening per VWD o Difetti di Funzione Piastrinica

6 Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo Paziente con sanguinamenti mucocutanei Tempo di emorragia Prolungato Normale Screening per VWD o Difetti di Funzione Piastrinica Screening per VWD o Difetti di Funzione Piastrinica

7 Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo Paziente con sanguinamenti mucocutanei Screening per VWD o Difetti di Funzione Piastrinica

8 endothelium sub-endothelium platelet plug In Vivo PFA-100 epinephrine or ADP - 40 mbar aperture 150µm membrane collagen platelet plug FLOW capillary

9 Percentuale di pazienti con valori patologici (tempi prolungati) PFD VWD Difetti di coagulazione PCE PCA TE PCE PCA TE PCE PCA TE Podda et al, JTH 2007

10 Paziente con sanguinamento mucocutaneo PFA-100 C-ADP Prolungato Normale Screening per VWD Screening per PFD VWD No VWD PFD No PFD Screening per PFD Screening per VWD

11 Paziente con sanguinamento mucocutaneo PFA-100 C-ADP?? Prolungato Da validare!! Normale Screening per VWD Screening per PFD VWD No VWD PFD No PFD Screening per PFD Screening per VWD

12 Aggregometria a trasmissione di luce (LTA)

13 LTA PRP citratato Boneu & Cazenave

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15 Recommendations LTA is clinically useful for the study of subjects with bleeding disorders LTA should NOT be used for the identification of subjects at risk for thrombosis LTA should NOT be used to monitor subjects on anti-platelet therapy

16 Recommendations: assessing the quality of PRP Grossly hemolyzed samples should be discarded If the sample tested is lipemic, the final report should indicate this It is necessary to check the platelet count of the PRP sample tested The results of LTA studies could be inaccurate when the platelet count in the PRP samples is lower than 150 x 10 9 /L, therefore, caution should be taken when interpreting abnormal results in samples with low platelet counts PRP with low platelet counts may be tested to exclude severe platelet function disorders (BSS, type 2B and platelet type von Willebrand disease) Platelet count of PRP samples should NOT be adjusted to a standardized value with autologous PPP (uncertain for PRP samples with platelet counts > 600 x 10 9 /L)

17 Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate ) LTA p=ns p=ns p=ns Femia et al, JTH 2013

18 Recommendations: Methodology - 1 LTA studies must include a known normal subject, run in parallel with the subject(s) under study After centrifugation, PRP samples should be allowed to sit at room temperature for 15 min before testing PRP should be used to set 0% light transmission in the aggregometer Autologous PPP should be used to set 100% light transmission in the aggregometer LTA studies should be performed at 37 C During LTA testing, PRP samples should be constantly stirred at 1,000 rpm using a disposable stirrer, unless otherwise specified by the manufacturer of the aggregometer

19 Recommendations: Methodology - 2 Before adding an agonist, baseline tracings for LTA should be observed for oscillations and stability for at least 1 minute The volume of agonist added for LTA should be consistent, and never more than 10% of the total sample volume Platelet aggregation should be monitored for: - a minimum of 3 minutes after adding an agonist - a minimum of 5 minutes after adding an agonist that does not cause maximal aggregation by 3 minutes with most control samples - a minimum of 10 minutes after adding an agonist that does not cause maximal aggregation by 5 minutes with most control samples LTA studies should be completed within a maximum of 4 hours after blood sampling

20 Recommendations: agonists The following platelet agonist should be used for diagnostic LTA studies: ADP: 2 µm (higher concentrations if abnormal results with 2 µμ) Epinephrine: 5 µm (higher concentrations if abnormal results with 5 µμ) Collagen: 2 µg/ml (Horm collagen) (higher concentrations if abnormal results with 2 µg/ml) Thrombin Receptor Activating Peptide (TRAP): 10 µm (higher concentrations if abnormal results with 10 µμ) The thromboxane A2 mimetic U46619: 1 µμ (higher concentrations if abnormal results with 1 µμ) Arachidonic acid: 1 mμ (higher concentrations if abnormal results with 1 mμ) Ristocetin: 1.2 mg/ml In case platelet agglutination induced by Ristocetin 1.2 mg/ml is normal, testing should be repeated using Ristocetin mg/ml In case platelet agglutination induced by Ristocetin 1.2 mg/ml is absent, testing should be repeated using Ristocetin 2 mg/ml.

21 Recommendations: evaluation and reporting of results The platelet aggregation tracing should be evaluated based on: - presence of shape change - length of the lag phase - slope of aggregation - maximal amplitude or % aggregation - amplitude or % aggregation at the end of the observation - disaggregation - visual examination of the aggregation tracings The presence of a "secondary wave induced by epinephrine should be evaluated Studies completed more than 4 hours after blood collection should be reported with a comment of this Clinical laboratories must establish an appropriate reference interval and validate test performance with each lot of reagents

22 LTA PRP citratato Normal transmission lumineuse ADP Collagen AA Ristocetin temps Glanzmann Bernard -Soulier SPD Aspirin Von Willebrand

23 LTA PRP citratato Normal transmission lumineuse ADP Collagen AA Ristocetin temps Glanzmann Bernard -Soulier SPD Aspirin Von Willebrand

24 Patients with a prolonged bleeding time and normal aggregation tests may have storage pool deficiency: studies in one hundred six patients HK Nieuwenhuis, JW Akkerman, JJ Sixma Blood 1987; 70:

25 Normal δ-spd Normal + ASA II-4 II-6 PSD Platelet aggregation (upper tracings) and secretion (lower tracings) induced by ADP at the indicated concentrations (μm), obtained with the lumiaggregometer

26 Algoritmo diagnostico dei difetti di secrezione piastrinica Secrezione piastrinica Anormale Normale Dosaggio del contenuto dei granuli STOP

27 Trasporto e localizzazione della serotonina (5HT) nelle piastrine SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid 5HT SERT δ-granule VMAT MAO 5HIAA Normal platelet

28 Tests to measure platelet serotonin (5HT) secretion 1. Secretion of 14 C-5HT (or 3 H-5HT) from preloaded platelets (considered the gold standard) 2. Tests to measure secreted endogenous 5HT: Fluorimetric assay using ortho-phtalaldehyde ELISA HPLC, coupled to electrochemical or fluorescence detection Liquid chromatography tandem-mass spectrometry (LC-MS)

29 Tests to measure platelet serotonin (5HT) secretion General principles 1. Both total platelet content of 5HT and secreted 5HT should be measured (total and secreted radioactivity in case of 14 C-5HT or 3 H-5HT) 2. Values expressed as percent secretion of total content 3. Platelet stimulation should be performed in the presence of a SERT inhibitor (usually, imipramine), in order to prevent the reuptake of secreted 5HT

30 Tests to measure platelet serotonin (5HT) secretion advantages and disadvantages Method Advantages Disadvantages Radiolabeled 5HT - gold standard - simple - use of radioisotopes - not suitable for patients with delta granules deficiency o-phthaldehyde - low cost - time consuming ELISA - expensive HPLC - accurate, precise - expensive instrumentation - experienced personnel LC-MS - accurate, precise - expensive instrumentation - experienced personnel

31 Trasporto e localizzazione della serotonina (5HT) nelle piastrine SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid 5HT SERT δ-granule VMAT MAO 5HIAA Piastrina normale

32 Trasporto e localizzazione della serotonina (5HT) nelle piastrine SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid 5HT SERT δ-granule VMAT MAO 5HIAA MAO 5HIAA MAO 5HIAA Piastrina δ SPD

33 Trasporto e localizzazione della serotonina (5HT) nelle piastrine SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid δ-granule VMAT 5HT SERT 5HIAA MAO Piastrina normale

34 Trasporto e localizzazione della serotonina (5HT) nelle piastrine SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid 5HT δ-granule VMAT SERT 5HIAA MAO MAO 5HIAA MAO 5HIAA Piastrina δ SPD

35 Trasporto e localizzazione della serotonina (5HT) nelle piastrine SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid δ-granule VMAT 5HT SERT 5HIAA MAO Piastrina normale stimolata

36 Trasporto e localizzazione della serotonina (5HT) nelle piastrine SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid 5HT δ-granule VMAT SERT 5HIAA MAO MAO 5HIAA MAO 5HIAA Piastrina δ SPD stimolata

37 Aggregazione piastrinica Metodo impedenziometrico

38 Principle of Multiplate analysis firm adhesion and aggregation of platelets on the sensor surface enhances the electrical resistance between the 2 sensor wires

39 aggregation [AU] test 1 test time [min] 4 5 Normale MULTIPLATE Clopidogrel

40 aggregation [AU] test 1 test time [min] 4 5 Normale MULTIPLATE Clopidogrel Light transmission (%) LTA Normale Clopidogrel time [min]

41 aggregation [AU] test 1 test time [min] 4 5 Normale MULTIPLATE Clopidogrel Light transmission (%) LTA Normale Clopidogrel time [min]

42 aggregation [AU] test 1 test time [min] 4 5 Normale MULTIPLATE Clopidogrel Light transmission (%) LTA Normale Clopidogrel time [min]

43 aggregation [AU] test 1 test time [min] 4 5 Normale MULTIPLATE Clopidogrel Light transmission (%) LTA Normale Clopidogrel time [min]

44 Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate ) LTA p=ns p=ns p=ns Femia et al, JTH 2013

45 Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate ) LTA p=ns p=ns p=ns Multiplate p<0.001 p<0.001 p<0.001 Femia et al, JTH 2013

46 Correlazione tra aggregazione piastrinica in sangue intero (Multiplate) e ematocrito Kakouros et al, JTH 2014

47 Diagnostic performance for PFD in 109 children with bleeding history: LTA vs Multiplate Test N. of patients with abnormal results LTA 15 Multiplate 3 Haas et al, Platelets 2018

48 Response variability to Clopidogrel The solution? Laboratory monitoring of antiplatelet treatment : increase the dose of Clopidogrel (use another drug) in patients with HTPR (based on the results of platelet function tests) A MEANS to be used if of proven efficacy and safety!

49 Validation of laboratory monitoring of clopidogrel treatment 1. Identification of the most accurate laboratory test 2. Standardization of pre-analytical and analytical variables [??] 3. Identification of universal cut-off values [??] 4. Clinical validation [??]

50 Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA): in vivo vs in vitro experiments A aggregation units (U) Multiple Electrode Aggregometry (ADP) in vivo R 2 : B aggregation units (U) Multiple Electrode Aggregometry (ADP) in vitro R 2 : CAM (µmol/l) CAM (µmol/l) C aggregation units (U) Multiple Electrode Aggregometry (ADP+PGE 1 ) in vivo R 2 : D aggregation units (U) Multiple Electrode Aggregometry (ADP+PGE 1 ) in vitro R 2 : CAM (µmol/l) CAM (µmol/l) Danese et al, JTH in pres

51 Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA): in vivo vs in vitro experiments A aggregation units (U) Multiple Electrode Aggregometry (ADP) in vivo R 2 : B aggregation units (U) Multiple Electrode Aggregometry (ADP) in vitro R 2 : CAM (µmol/l) CAM (µmol/l) C aggregation units (U) Multiple Electrode Aggregometry (ADP+PGE 1 ) in vivo R 2 : D aggregation units (U) Multiple Electrode Aggregometry (ADP+PGE 1 ) in vitro R 2 : CAM (µmol/l) CAM (µmol/l) Danese et al, JTH in pres

52 Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA): in vivo vs in vitro experiments A aggregation units (U) Multiple Electrode Aggregometry (ADP) in vivo R 2 : B aggregation units (U) Multiple Electrode Aggregometry (ADP) in vitro R 2 : CAM (µmol/l) CAM (µmol/l) C aggregation units (U) Multiple Electrode Aggregometry (ADP+PGE 1 ) in vivo R 2 : D aggregation units (U) Multiple Electrode Aggregometry (ADP+PGE 1 ) in vitro R 2 : CAM (µmol/l) CAM (µmol/l) Danese et al, JTH in pres

53 Vasodilator Stimulated Phosphoprotein (VASP) Assay for the Measurement of P2Y 12 Antagonism Antagonist ADP In the presence of both PGE 1 and ADP, VASP-P is directly proportional to the degree of P2Y 12 antagonism Modified from Cattaneo in PLATELETS (Michelson, 2nd ed, 2007)

54 Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with PRI (VASP phosphorylation assay): in vivo vs in vitro experiments PRI (%) in vivo study R 2 : in vitro study R 2 : CAM (µmol/l) Danese et al, JTH in pres

55 Validation of laboratory monitoring of clopidogrel treatment 1. Identification of the most accurate laboratory test [??] 2. Standardization of pre-analytical and analytical variables [??] 3. Identification of universal cut-off values [??] 4. Clinical validation [??]

56 Validation of laboratory monitoring of clopidogrel treatment 1. Identification of the most accurate laboratory test [??] 2. Standardization of pre-analytical and analytical variables [??] 3. Identification of universal cut-off values [??] 4. Clinical validation