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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
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Metodiche per lo studio della funzione piastrinica
• Diagnosi dei difetti congeniti della funzione piastrinica
• Monitoraggio della terapia antiaggregante (??)
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Diagnosi dei difetti di funzione piastrinica
Test globali dell’emostasi primaria
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Elevata variabilità Operatore-dipendente Impreciso Invasivo Poco sensibile Non utile nell’algoritmo diagnostico
Tempo di emorragia
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Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo
Screening per VWD o Difetti di Funzione Piastrinica
Prolungato Normale
Tempo di emorragia
Paziente con sanguinamenti mucocutanei
Screening per VWD o Difetti di Funzione Piastrinica
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Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo
Screening per VWD o Difetti di Funzione Piastrinica
Prolungato Normale
Tempo di emorragia
Paziente con sanguinamenti mucocutanei
Screening per VWD o Difetti di Funzione Piastrinica
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Algoritmo diagnostico per pazienti con sanguinamento mucocutaneo
Paziente con sanguinamenti mucocutanei
Screening per VWD o Difetti di Funzione Piastrinica
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In Vivo PFA-100
capillary
aperture 150µm
epinephrine or
ADP
FLOW
sub-endothelium
platelet plug
endothelium
- 40 mbar
collagen
membrane
platelet plug
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0
20
40
60
80
100
PCE PCA TE PCE PCA TE PCE PCA TE
PFD VWD
Difetti di coagulazione
75
33 38
86 86
29
14 3 14
14 14 0
Podda et al, JTH 2007
Percentuale di pazienti con valori patologici (tempi prolungati)
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Screening per VWD
Prolungato
PFA-100® C-ADP
Paziente con sanguinamento mucocutaneo
Screening per PFD
VWD No VWD
Screening per PFD
Normale
Screening per VWD
PFD No PFD
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Screening per VWD
Prolungato
PFA-100® C-ADP
Paziente con sanguinamento mucocutaneo
Screening per PFD
VWD No VWD
Screening per PFD
Normale
Screening per VWD
PFD No PFD
?? Da validare!!
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Aggregometria a trasmissione di luce (LTA)
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LTA – PRP citratato
Boneu & Cazenave
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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
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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 109/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 109/L)
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Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate®)
LTA
p=NS p=NS p=NS
Femia et al, JTH 2013
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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
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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
Recommendations: Methodology - 2
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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 0.5-0.7 mg/mL
In case platelet agglutination induced by Ristocetin 1.2 mg/mL is absent, testing should be repeated using Ristocetin 2 mg/mL.
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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
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ADP Collagen AA Ristocetin
Normal
Glanzmann
Bernard -Soulier
SPD
Aspirin
Von Willebrand
temps trans
mis
sion
lum
ineu
se
LTA – PRP citratato
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ADP Collagen AA Ristocetin
Normal
Glanzmann
Bernard -Soulier
SPD
Aspirin
Von Willebrand
temps trans
mis
sion
lum
ineu
se
LTA – PRP citratato
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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; 70:620-623
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Normal Normal + ASA II-4 II-6 PSD
20 2 4
4 20
2
2 4
20
20
4 2
2
4
20
2
4
20
20
Platelet aggregation (upper tracings) and secretion (lower tracings) induced by ADP at the indicated concentrations (μM),
obtained with the lumiaggregometer
δ-SPD
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Algoritmo diagnostico dei difetti di secrezione piastrinica
Secrezione piastrinica
Anormale Normale
STOP Dosaggio del contenuto dei granuli
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Trasporto e localizzazione della serotonina (5HT) nelle piastrine
5HT
SERT
δ-Granule MAO
5HIAA
Normal platelet
VMAT
SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid
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Tests to measure platelet serotonin (5HT) secretion
1. Secretion of 14C-5HT (or 3H-5HT) from pre-loaded 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)
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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 14C-5HT or 3H-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
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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
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Trasporto e localizzazione della serotonina (5HT) nelle piastrine
5HT
SERT
δ-Granule MAO
5HIAA
Piastrina normale
VMAT
SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid
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Trasporto e localizzazione della serotonina (5HT) nelle piastrine
5HT
SERT
VMAT δ-Granule
MAO 5HIAA
MAO 5HIAA
MAO 5HIAA
Piastrina δ–SPD
SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid
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Trasporto e localizzazione della serotonina (5HT) nelle piastrine
5HT
SERT
δ-Granule MAO
5HIAA
Piastrina normale
* * *
*
*
* * *
* VMAT *
SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid
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Trasporto e localizzazione della serotonina (5HT) nelle piastrine
5HT
SERT
δ-Granule
* * *
*
*
* MAO 5HIAA *
*
* VMAT
MAO 5HIAA
MAO 5HIAA *
*
Piastrina δ–SPD
*
SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid
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Trasporto e localizzazione della serotonina (5HT) nelle piastrine
5HT
SERT
δ-Granule MAO
5HIAA
Piastrina normale stimolata
* * *
*
*
* * *
* VMAT
*
* *
*
SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid
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Trasporto e localizzazione della serotonina (5HT) nelle piastrine
5HT
SERT
δ-Granule
* * *
*
*
* MAO 5HIAA *
*
* VMAT
MAO 5HIAA
MAO 5HIAA *
*
Piastrina δ–SPD stimolata
*
*
SERT: serotonin transporter VMAT: vesicular monoamine transporter MAO: monoamine oxidase 5HIAA: 5-hydrocyindolacetic acid
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Aggregazione piastrinica
Metodo impedenziometrico
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firm adhesion and aggregation of platelets on the sensor surface enhances the electrical resistance between the 2 sensor wires
Principle of Multiplate® analysis
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aggr
egat
ion
[AU
]
time [min]
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5
test 1 test 2
MULTIPLATE
Normale
Clopidogrel
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aggr
egat
ion
[AU
]
time [min]
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5
test 1 test 2
Ligh
t tra
nsm
issi
on (%
)
0 0 1 2 3 4 5
time [min]
MULTIPLATE
Normale
Clopidogrel
LTA
Normale
Clopidogrel
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aggr
egat
ion
[AU
]
time [min]
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5
test 1 test 2
Ligh
t tra
nsm
issi
on (%
)
0 0 1 2 3 4 5
time [min]
MULTIPLATE
Normale
Clopidogrel
LTA
Normale
Clopidogrel
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aggr
egat
ion
[AU
]
time [min]
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5
test 1 test 2
Ligh
t tra
nsm
issi
on (%
)
0 0 1 2 3 4 5
time [min]
MULTIPLATE
Normale
Clopidogrel
LTA
Normale
Clopidogrel
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aggr
egat
ion
[AU
]
time [min]
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5
test 1 test 2
Ligh
t tra
nsm
issi
on (%
)
0 0 1 2 3 4 5
time [min]
MULTIPLATE
Normale
Clopidogrel
LTA
Normale
Clopidogrel
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Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate®)
LTA
p=NS p=NS p=NS
Femia et al, JTH 2013
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Effetto della conta piastrinica LTA e Aggregometria a Impedenza (Multiplate®)
LTA
Multiplate®
p=NS p=NS p=NS
p<0.001 p<0.001 p<0.001
Femia et al, JTH 2013
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Correlazione tra aggregazione piastrinica in sangue intero (Multiplate) e ematocrito
Kakouros et al, JTH 2014
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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
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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!
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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 [??]
Validation of laboratory monitoring of clopidogrel treatment
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Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA):
in vivo vs in vitro experiments
Danese et al, JTH in pres
in vivo
in vivo
in vitro
in vitro
-1.5 -1.0 -0.5 0.00
20
40
60
80R2: 0.087
Multiple Electrode Aggregometry (ADP)
CAM (µmol/L)
agg
rega
tion
units
(U)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50
20
40
60
80
100R2:0.580
Multiple Electrode Aggregometry (ADP)
CAM (µmol/L)
aggr
egat
ion
units
(U)
-1.5 -1.0 -0.5 0.00
20
40
60
80R2: 0.175
Multiple Electrode Aggregometry (ADP+PGE 1)
CAM (µmol/L)
aggr
egat
ion
units
(U)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50
20
40
60
80R2: 0.548
Multiple Electrode Aggregometry (ADP+PGE 1)
CAM (µmol/L)
agg
rega
tion
units
(U)
A B
C D
0.05 0.1 0.5 1.0
0.05 0.1 0.5 1.0 5.0 10.0 15.0 0.05 0.1 0.5 1.0
0.05 0.1 0.5 1.0 5.0 10.0 15.0
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Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA):
in vivo vs in vitro experiments
Danese et al, JTH in pres
in vivo
in vivo
in vitro
in vitro
-1.5 -1.0 -0.5 0.00
20
40
60
80R2: 0.087
Multiple Electrode Aggregometry (ADP)
CAM (µmol/L)
agg
rega
tion
units
(U)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50
20
40
60
80
100R2:0.580
Multiple Electrode Aggregometry (ADP)
CAM (µmol/L)
aggr
egat
ion
units
(U)
-1.5 -1.0 -0.5 0.00
20
40
60
80R2: 0.175
Multiple Electrode Aggregometry (ADP+PGE 1)
CAM (µmol/L)
aggr
egat
ion
units
(U)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50
20
40
60
80R2: 0.548
Multiple Electrode Aggregometry (ADP+PGE 1)
CAM (µmol/L)
agg
rega
tion
units
(U)
A B
C D
0.05 0.1 0.5 1.0
0.05 0.1 0.5 1.0 5.0 10.0 15.0 0.05 0.1 0.5 1.0
0.05 0.1 0.5 1.0 5.0 10.0 15.0
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-1.5 -1.0 -0.5 0.00
20
40
60
80R2: 0.087
Multiple Electrode Aggregometry (ADP)
CAM (µmol/L)
agg
rega
tion
units
(U)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50
20
40
60
80
100R2:0.580
Multiple Electrode Aggregometry (ADP)
CAM (µmol/L)
aggr
egat
ion
units
(U)
-1.5 -1.0 -0.5 0.00
20
40
60
80R2: 0.175
Multiple Electrode Aggregometry (ADP+PGE 1)
CAM (µmol/L)
aggr
egat
ion
units
(U)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.50
20
40
60
80R2: 0.548
Multiple Electrode Aggregometry (ADP+PGE 1)
CAM (µmol/L)
agg
rega
tion
units
(U)
A B
C D
0.05 0.1 0.5 1.0
0.05 0.1 0.5 1.0 5.0 10.0 15.0 0.05 0.1 0.5 1.0
0.05 0.1 0.5 1.0 5.0 10.0 15.0
Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with platelet aggregation in whole blood (MEA):
in vivo vs in vitro experiments
Danese et al, JTH in pres
in vivo
in vivo
in vitro
in vitro
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Vasodilator Stimulated Phosphoprotein (VASP) Assay for the Measurement of P2Y12 Antagonism
In the presence of both PGE1 and ADP, VASP-P is directly proportional to the degree of P2Y12 antagonism
Modified from Cattaneo in PLATELETS (Michelson, 2nd ed, 2007)
ADP Antagonist
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-1.5 -1.0 -0.5 0.0 0.5 1.00
25
50
75
100
in vivo study R2: 0.871in vitro study R2: 0.847
CAM (µmol/L)
PRI (
%)
0.05 0.1 0.5 1.0 5.0 10.0
Correlations between plasma concentration of Clopidogrel Active Metabolite (CAM) with PRI (VASP phosphorylation assay):
in vivo vs in vitro experiments
Danese et al, JTH in pres
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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 [??]
Validation of laboratory monitoring of clopidogrel treatment
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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
Validation of laboratory monitoring of clopidogrel treatment