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) TROMBOSI 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

FASE VASO-PIASTRINICA sottoendotelio Lesione di continuo w w w w w sottoendotelio

FASE VASO-PIASTRINICA w w w w w w w w

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)

PIASTRINOPENIA Classificazione PIASTRINOPENIE EREDITARIE PIASTRINOPENIE ACQUISITE: - Da ridotta o difettosa produzione midollare - Da aumentata distruzione/consumo periferico su base immune su base non immune - Da sequestro od anomalo pooling - Da emodiluizione PSEUDOPIASTRINOPENIA (riduzione in vitro della conta piastrinica, per agglutinazione EDTA-dipendente)

Pseudopiastrinopenia?

Pseudopiastrinopenia? Striscio di sangue periferico in EDTA

Normale – Sangue in EDTA

Pseudopiastrinopenia – Sangue in EDTA

Satellitismo piastrinico

Pseudopiastrinopenia? Ereditaria o Acquisita? Striscio di sangue periferico in EDTA Sì No STOP Ereditaria o Acquisita? Conta piastrinica normale in passato? Familiarità? Anomalie morfologiche e/o funzionali? Ereditaria Acquisita

Inherited Thrombocytopenias: a Proposed Diagnostic Algorithm 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/difettosa Produzione di MK/plts Aumetata distruzione/consumo Sequestro o pooling 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 12 TE (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) 0.870 PCE (s) 130 (85-244) 142 (88-300) 152 (74-300) 153 (95-300) 0.004 PCA (s) 85 (67-179) 91 (57-182) 89 (59-300) 95 (73-207) 0.457 Valori mediani (range) Kruskall-Wallis test

Patients with bleeding diathesis, no thrombocytopenia: Screening tests: PT, aPTT, BT (or PFA-100) 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

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

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

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 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

Work-up for hemophilia Work-up for vWD 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

Work-up for hemophilia Work-up for vWD 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

Work-up for hemophilia Work-up for vWD 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 for hemophilia Work-up for vWD 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 for Platelet Function Disorders

Diagnosi di malattia di von Willebrand

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Present Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Present Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Present plasma VWF:RCo Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Present Proportionate (0.7 – 1.2) Type 1 plasma VWF:RCo Rco:Ag Discrepant (<0.7) Type 2 Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Present Proportionate (0.7 – 1.2) Type 1 plasma VWF:RCo Rco:Ag Discrepant (<0.7) Type 2 Increased (0.2-0.8) R.I.P.A (mg/mL) Decreased (>1.2) Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Present Proportionate (0.7 – 1.2) Type 1 plasma VWF:RCo Rco:Ag Discrepant (<0.7) Type 2 Increased (0.2-0.8) R.I.P.A (mg/mL) Decreased (>1.2) Type 2B Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Present Proportionate (0.7 – 1.2) Type 1 plasma VWF:RCo Rco:Ag Discrepant (<0.7) Type 2 Increased (0.2-0.8) R.I.P.A (mg/mL) Decreased (>1.2) Plasma High Multimers Type 2B Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Present Proportionate (0.7 – 1.2) Type 1 plasma VWF:RCo Rco:Ag Discrepant (<0.7) Type 2 Increased (0.2-0.8) R.I.P.A (mg/mL) Absent Type 2A Decreased (>1.2) Plasma High Multimers Type 2B Present Type 2M Adapted from Castaman et al, 2003

Diagnostic Flow Chart of VWD Types plasma VWF:Ag Absent Type 3 Platelet VWF Present Proportionate (0.7 – 1.2) Type 1 Proportionate Plasma FVIII:C/vWF:Ag plasma VWF:RCo Rco:Ag Discrepant Discrepant (<0.7) Type 2 Type 2N FVIII binding assay Increased (0.2-0.8) R.I.P.A (mg/mL) Absent Type 2A Decreased (>1.2) Plasma High Multimers Type 2B Present Type 2M Adapted from Castaman et al, 2003

Work-up for hemophilia Work-up for vWD 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 for Platelet Function Disorders

Aggregazione piastrinica

OUT IN

OUT IN OUT IN GPIIb/IIIa

Epinephrine Thrombin TxA2 ADP 5HT Shear OUT IN OUT IN GPIIb/IIIa

Epinephrine Thrombin TxA2 ADP 5HT Shear OUT IN OUT IN GPIIb/IIIa

OUT IN OUT OUT IN IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN

OUT IN OUT IN OUT IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN Adesive protein

OUT IN OUT OUT IN IN OUT IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN Adesive protein OUT IN

Platelet Aggregation Platelet 1 Platelet 2

Strong agonists Weak agonists Aggregation TxA2 Synthesis Secretion ADP Low [Ca2+]o + + TxA2 Synthesis + Secretion + ADP

OUT IN OUT OUT IN IN OUT IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN Adesive protein OUT IN

OUT IN OUT OUT IN IN OUT IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN Adesive protein OUT IN

OUT IN OUT OUT IN IN OUT IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN Adesive protein OUT IN

OUT IN OUT OUT IN IN OUT IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN Adesive protein OUT IN

OUT IN OUT OUT IN IN OUT IN Epinephrine Thrombin TxA2 ADP 5HT Shear GPIIb/IIIa OUT IN Adesive protein OUT IN

Strong agonists Weak agonists Aggregation TxA2 Synthesis Secretion ADP Low [Ca2+]o + + TxA2 Synthesis + Secretion + ADP

Diagnosi di difetti funzionali piastrinici

Inherited Platelet Function Disorders Laboratory Diagnosis - 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 Disorders Laboratory Diagnosis - 2 Where? Specialized institutions (importance of pre-analytical variables!) First-step screening tests: Simple Rapid Cost-effective Sensitive to the most common disorders

Inherited Platelet Function Disorders First-step screening tests Light microscopy of whole-blood smear: platelet size and morphology

Inherited Platelet Function Disorders that can be suspected by inspection of the blood smear 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 Laboratory 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 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 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%)

HK Nieuwenhuis, JW Akkerman, JJ Sixma Blood 1987; 70:620-623 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

Inherited Platelet Function Disorders First-step screening tests Light microscopy of whole-blood smear: platelet size and morphology 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)

Platelet aggregation (upper tracings) and secretion (lower Normal II-7 Normal + ASA II-4 II-6 III-1 20 2 4 Platelet aggregation (upper tracings) and secretion (lower tracings) induced by ADP at the indicated concentrations (μM), obtained with the lumiaggregometer

Inherited Platelet Function Disorders that can be identified by the first screening step 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 First-step screening tests Light microscopy of whole-blood smear: platelet size and morphology 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) Clot retraction (save serum for TxB2 assay, to confirm abnormalities of AA pathway or rule out NSAID)

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