diagnosi dei difetti dellemostasi primaria marco cattaneo unità di ematologia e trombosi ospedale...
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Diagnosi dei Difetti dell’Emostasi Primaria
Marco Cattaneo
Unità di Ematologia e Trombosi
Ospedale San Paolo
DMCO, Università di Milano
EMOSTASI
• Fase vasopiastrinica (Emostasi Primaria)
• Fase della coagulazione• Fase della fibrinolisi
EMOSTASI
• Fase vasopiastrinica (Emostasi Primaria)
• Fase della coagulazione• Fase della fibrinolisi
EMOSTASI
TROMBOSI
• Meccanismo di difesa che arresta il sanguinamento da soluzioni di continuo dell’albero vascolare• Dipende da una complessa interazione tra flusso ematico, parete vascolare e sangue (cellule e proteine del plasma)
Forma malregolata o inappropriata di emostasi
Tutti i test di laboratorio che esplorano l’emostasi primaria sono utili solamente per la diagnosi dei difetti dell’emostasi
Nessuno di essi e’ utile per la definizione del rischio trombotico o per il monitoraggio della terapia antitrombotica
vaso
Lesione di continuosottoendotelio
wwwww
FASE VASO-PIASTRINICA
sottoendotelio
wwwww
w w w
FASE VASO-PIASTRINICA
Difetti dell’Emostasi Primaria
• Piastrinopenie• Piastrinopatie• Difetti di proteine adesive
(afibrinogenemia, malattia di von Willebrand)
• Anemia (es: uremia)
Difetti dell’Emostasi Primaria
• Piastrinopenie• Piastrinopatie• Difetti di proteine adesive
(afibrinogenemia, malattia di von Willebrand)
• Anemia (es: uremia)
PIASTRINOPENIAClassificazione
• PIASTRINOPENIE EREDITARIE• PIASTRINOPENIE ACQUISITE:
- Da ridotta o difettosa produzione midollare- Da aumentata distruzione/consumo periferico
su base immunesu base non immune
- Da sequestro od anomalo pooling- Da emodiluizione
• PSEUDOPIASTRINOPENIA (riduzione in vitro della conta piastrinica, per agglutinazione EDTA-dipendente)
PIASTRINOPENIA
Pseudopiastrinopenia?
PIASTRINOPENIA
Pseudopiastrinopenia?•Striscio di sangue periferico
in EDTA
Normale – Sangue in EDTA
Pseudopiastrinopenia – Sangue in EDTA
Satellitismo piastrinico
STOP Ereditaria o Acquisita?•Conta piastrinica normale in passato?
•Familiarità?•Anomalie morfologiche e/o funzionali?
Ereditaria Acquisita
Sì No
PIASTRINOPENIA
Pseudopiastrinopenia?•Striscio di sangue periferico
in EDTA
Inherited Thrombocytopenias: a Proposed DiagnosticAlgorithm from the Italian Gruppo di Studio delle Piastrine
CL Balduini, M Cattaneo, F Fabris, P Gresele, A Iolascon,FM Pulcinelli, A Savoia, on behalf of the Italian
Gruppo di Studio delle Piastrine
Haematologica 2003, 88: 582-592
PIASTRINOPENIA ACQUISITA
•[Emocromo, MPV]•[Striscio di sangue periferico]
•Anamnesi farmacologica e trasfusionale•Valutazione splenomegalia
•Markers virus epatite, Herpes, HIV•Elettroforesi sieroproteica•Analisi aspirato midollare
(obbligatorio se >60 anni e se anomalie sangue periferico)•Ricerca ANA
Ridotta/difettosaProduzione di MK/plts
Aumetatadistruzione/consumo
Sequestro opooling anomalo
Difetti dell’Emostasi Primaria
• Piastrinopenie• Piastrinopatie• Difetti di proteine adesive
(afibrinogenemia, malattia di von Willebrand)
• Anemia (es: uremia)
Patients with bleeding diathesis, no thrombocytopenia:
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
Punteggio normali (0→3) basso (4→7) intermedio (8→11) alto (12→19) p
Numero 41 53 22 12TE (min) 5.3 (3,0-10,0) 5.0 (2.3-14.3) 5.0 (2.0-20.0) 4.8 (3.3-20.0) 0.870PCE (s) 130 (85-244) 142 (88-300) 152 (74-300) 153 (95-300) 0.004PCA (s) 85 (67-179) 91 (57-182) 89 (59-300) 95 (73-207) 0.457
Valori mediani (range) Kruskall-Wallis test
P
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P
N
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT
N aPTT, BT PT, BT BT - aPTT
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT
N aPTT, BT PT, BT BT - aPTT
Work-up for hemophilia
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT
N aPTT, BT PT, BT BT - aPTT
Work-up for hemophilia
FVII FXI, FIX FV, FX, FII FV FVII FVIII Fibrinogen Fibrinogen
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT PTT, BT BT -
N aPTT, BT PT, BT BT - aPTT PT PT, aPTT PT, aPTT, BT
Work-up for hemophilia
FVII FXI, FIX FV, FX, FII FV FVII FVIII Fibrinogen Fibrinogen
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT PTT, BT BT -
N aPTT, BT PT, BT BT - aPTT PT PT, aPTT PT, aPTT, BT
Work-up for hemophilia Work-up for vWD
FVII FXI, FIX FV, FX, FII FV FVII FVIII Fibrinogen Fibrinogen
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT PTT, BT BT -
N aPTT, BT PT, BT BT - aPTT PT PT, aPTT PT, aPTT, BT
Work-up for hemophilia Work-up for vWD
FVII FXI, FIX FV, FX, FII FV FVII vWD FVIII Fibrinogen Fibrinogen
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT PTT, BT BT -
N aPTT, BT PT, BT BT - aPTT PT PT, aPTT PT, aPTT, BT
Work-up for hemophilia Work-up for vWD
FVII FXI, FIX FV, FX, FII FV FVII vWD negative FVIII Fibrinogen Fibrinogen
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT PTT, BT BT -
N aPTT, BT PT, BT BT - aPTT PT PT, aPTT PT, aPTT, BT
Work-up for hemophilia Work-up for vWD
FVII FXI, FIX FV, FX, FII FV FVII vWD negative FVIII Fibrinogen Fibrinogen
Work-up forPlatelet Function Disorders
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
Diagnosi di malattia divon Willebrand
Adapted from Castaman et al, 2003
plasma VWF:Ag Absent
Present
Diagnostic Flow Chart of VWD Types
Adapted from Castaman et al, 2003
plasma VWF:Ag Absent
Present
Type 3
Diagnostic Flow Chart of VWD Types
Adapted from Castaman et al, 2003
plasma VWF:Ag Absent
Present
Type 3
Diagnostic Flow Chart of VWD Types
plasma VWF:RCo
Adapted from Castaman et al, 2003
plasma VWF:Ag Absent
Present Proportionate(0.7 – 1.2)
Discrepant(<0.7)
Type 2
Type 1
Type 3
Diagnostic Flow Chart of VWD Types
plasma VWF:RCo Rco:Ag
Adapted from Castaman et al, 2003
plasma VWF:Ag Absent
Present Proportionate(0.7 – 1.2)
Discrepant(<0.7)
R.I.P.A (mg/mL)Increased(0.2-0.8)
Decreased(>1.2)
Type 2
Type 1
Type 3
Diagnostic Flow Chart of VWD Types
plasma VWF:RCo Rco:Ag
Adapted from Castaman et al, 2003
Type 2B
plasma VWF:Ag Absent
Present Proportionate(0.7 – 1.2)
Discrepant(<0.7)
R.I.P.A (mg/mL)Increased(0.2-0.8)
Decreased(>1.2)
Type 2
Type 1
Type 3
Diagnostic Flow Chart of VWD Types
plasma VWF:RCo Rco:Ag
Adapted from Castaman et al, 2003
Type 2B
plasma VWF:Ag Absent
Present Proportionate(0.7 – 1.2)
Discrepant(<0.7)
R.I.P.A (mg/mL)Increased(0.2-0.8)
Decreased(>1.2)
Plasma High Multimers
Type 2
Type 1
Type 3
Diagnostic Flow Chart of VWD Types
plasma VWF:RCo Rco:Ag
Adapted from Castaman et al, 2003
Type 2B
Type 2M
Type 2A
plasma VWF:Ag Absent
Present Proportionate(0.7 – 1.2)
Discrepant(<0.7)
R.I.P.A (mg/mL)Increased(0.2-0.8)
Decreased(>1.2)
Plasma High Multimers
Absent
Present
Type 2
Type 1
Type 3
Diagnostic Flow Chart of VWD Types
plasma VWF:RCo Rco:Ag
Adapted from Castaman et al, 2003
Type 2B
Type 2M
Type 2A
plasma VWF:Ag Absent
Present Proportionate(0.7 – 1.2)
Discrepant(<0.7)
R.I.P.A (mg/mL)Increased(0.2-0.8)
Decreased(>1.2)
Plasma High Multimers
Absent
Present
Type 2
Type 1
Type 3
Proportionate
Discrepant
Type 2N
Platelet VWF
Plasma FVIII:C/vWF:Ag
FVIII binding assay
Diagnostic Flow Chart of VWD Types
plasma VWF:RCo Rco:Ag
P PT aPTT PT, aPTT PT, aPTT, BT PT, BT PTT, BT BT -
N aPTT, BT PT, BT BT - aPTT PT PT, aPTT PT, aPTT, BT
Work-up for hemophilia Work-up for vWD
FVII FXI, FIX FV, FX, FII FV FVII vWD negative FVIII Fibrinogen Fibrinogen
Work-up forPlatelet Function Disorders
Patients with bleeding diathesis, no thrombocytopenia:Screening tests: PT, aPTT, BT (or PFA-100)
Aggregazione piastrinica
OUT
IN
GPIIb/IIIaOUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
GPIIb/IIIaOUT
IN
OUT
IN
GPIIb/IIIaOUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
GPIIb/IIIaOUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Adesive protein
GPIIb/IIIaOUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Adesive protein
GPIIb/IIIaOUT
IN
OUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Platelet 1
Platelet 2
Platelet Aggregation
Strong agonists
Aggregation
TxA2 Synthesis
Secretion
ADP
Low [Ca2]o
Weak agonists
Adesive protein
GPIIb/IIIaOUT
IN
OUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Adesive protein
GPIIb/IIIaOUT
IN
OUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Adesive protein
GPIIb/IIIaOUT
IN
OUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Adesive protein
GPIIb/IIIaOUT
IN
OUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Adesive protein
GPIIb/IIIaOUT
IN
OUT
IN
OUT
IN
Epinephrine Thrombin TxA2 ADP 5HT Shear
OUT
IN
Strong agonists
Aggregation
TxA2 Synthesis
Secretion
ADP
Low [Ca2]o
Weak agonists
Diagnosi di difetti funzionali piastrinici
1. Whom?• Patients with positive history for abnormal bleedings
(particularly if mucocutaneous and/or associated with prolonged BT or PFA-100 CT), in whom the following conditions were excluded:
• Thrombocytopenia• vWD• Afibrinogenemia• Drugs known to affect platelet function
• Patients with positive history for abnormal bleedings (any type), in whom other hemostatic abnormalities were ruled out
Inherited Platelet Function DisordersLaboratory Diagnosis - 1
Inherited Platelet Function DisordersLaboratory Diagnosis - 2
• Where?– Specialized institutions (importance of pre-
analytical variables!)
• First-step screening tests:– Simple– Rapid– Cost-effective– Sensitive to the most common disorders
1. Light microscopy of whole-blood smear: platelet size and morphology
Inherited Platelet Function DisordersFirst-step screening tests
• Bernard-Soulier syndrome• Pseudo-vWD• Bolin-Jamieson syndrome• Glanzmann thrombasthenia• GPIa/IIa deficiency• GPVI deficiency• P2Y12 defects• TxA2 receptor defects• α2-receptor defect• δ-Storage Pool Deficiency• α,δ -Storage Pool
Deficiency
• Grey-Platelet Syndrome• Quebec Platelet disorder• Paris-Trousseau-Jacobsen’s
Syndrome• Defects of signal
transduction• Scott syndrome• Stormorken syndrome• Primary Secretion Defects• Macrothrombocytopenia
with glycophorin expression• Wiskott-Aldrich syndrome• Montreal Platelet syndrome
Inherited Platelet Function Disorders that can be suspected by inspection of the blood smear
Inherited Platelet Function DisordersLaboratory Diagnosis - 2
• Where:– Specialized laboratories (importance of
pre-analytical variables!)
• First-step screening tests:– Simple– Rapid– Cost-effective– Sensitive to the most common disorders
Patients screened for Platelet Function Disorders at the A. Bianchi Bonomi Hemophilia and Thrombosis
Ctr, University of Milano - From 1990 to 2002
• Total number of screened patients: 318• Diagnosis:
• No abnormalities 187 (59%)
• Primary Secretion Defects 63 (20%)
• δ-Storage Pool Deficiency 38 (12%)
• Glanzmann Thrombasthenia 5 (2%)
• “Aspirin-like” Defects 4 (1.3%)
• Bernard-Soulier syndrome 3 (1%)
• P2Y12 defect 2 (0.6%)
• Incomplete 15 (5%)
Patients screened for Platelet Function Disorders at the A. Bianchi Bonomi Hemophilia and Thrombosis
Ctr, University of Milano - From 1990 to 2002
• Total number of screened patients: 318• Diagnosis:
• No abnormalities 187 (59%)
• Primary Secretion Defects 63 (20%)
• δ-Storage Pool Deficiency 38 (12%)
• Glanzmann Thrombasthenia 5 (2%)
• “Aspirin-like” Defects 4 (1.3%)
• Bernard-Soulier syndrome 3 (1%)
• P2Y12 defect 2 (0.6%)
• Incomplete 15 (5%)
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
1. Light microscopy of whole-blood smear: platelet size and morphology
2. Lumiaggregometry on citrated PRP: explores platelet aggregation and secretion simultaneously:
• ADP 2 μM (if abnormal, ADP 10 μM)• Collagen 2 μg/mL (if abnormal, 10 μg/mL)• U46619 1μM• Adrenaline 5 μM• Ristocetin 1.2 mg/mL (if normal, 0.6 mg/mL)• Arachidonic acid 1 mM• No agonists (SPA)
Inherited Platelet Function Disorders First-step screening tests
NormalNormal II-7II-7 Normal + ASANormal + ASA II-4II-4 II-6II-6 III-1III-1
20202244
442020
22
22
44
2020
2020
4422
22
44
2020
22
44
2020
2020
Platelet aggregation (upper tracings) and secretion (lowertracings) induced by ADP at the indicated concentrations (μM),
obtained with the lumiaggregometer
• Bernard-Soulier syndrome• Pseudo-vWD• Bolin-Jamieson syndrome• Glanzmann thrombasthenia• GPIa/IIa deficiency• GPVI deficiency
• P2Y12 defects
• TxA2 receptor defects
• α2-receptor defect
• δ-Storage Pool Deficiency• α,δ -Storage Pool Deficiency
• Grey-Platelet Syndrome• Quebec Platelet disorder• Paris-Trousseau-
Jacobsen’s Syndrome• Defects of signal
transduction• Scott syndrome• Stormorken syndrome• Primary Secretion Defects• Macrothrombocytopenia
with glycophorin expression• Wiskott-Aldrich syndrome• Montreal Platelet syndrome
Inherited Platelet Function Disorders that can be identified by the first screening step
1. Light microscopy of whole-blood smear: platelet size and morphology
2. Lumiaggregometry on citrated PRP: explores platelet aggregation and secretion simultaneously
• ADP 2 μM (if abnormal, ADP 10 μM)• Collagen 2 μg/mL (if abnormal, 10 μg/mL)• U46619 1μM• Adrenaline 5 μM• Ristocetin 1.2 mg/mL (if normal, 0.6 mg/mL)• Artachidonic acid 1 mM• No agonists (SPA)
3. Clot retraction (save serum for TxB2 assay, to confirm abnormalities of AA pathway or rule out NSAID)
Inherited Platelet Function Disorders First-step screening tests
Raccomandazioni
• Test dell’emostasi primaria: no per trombosi• Sospettare pseudopiastrinopenia (striscio!)• Anticorpi antipiastrine: inutili• Escludere VWD e farmaci prima di indagare
difetti funzionali piastrinci• Diagnosi di difetti funzionali piastrinici:
lumiaggregometria• Diagnosi di difetti funzionali piastrinici: solo in
Centri specializzati