Diagnosi dei Difetti dell’Emostasi Primaria

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1 Diagnosi dei Difetti dell’Emostasi Primaria
Marco Cattaneo Unità di Ematologia e Trombosi Ospedale San Paolo DMCO, Università di Milano

2 EMOSTASI Fase vasopiastrinica (Emostasi Primaria)
Fase della coagulazione Fase della fibrinolisi

3 EMOSTASI Fase vasopiastrinica (Emostasi Primaria)
Fase della coagulazione Fase della fibrinolisi

4 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

5 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

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

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

8 Difetti dell’Emostasi Primaria
Piastrinopenie Piastrinopatie Difetti di proteine adesive (afibrinogenemia, malattia di von Willebrand) Anemia (es: uremia)

9 Difetti dell’Emostasi Primaria
Piastrinopenie Piastrinopatie Difetti di proteine adesive (afibrinogenemia, malattia di von Willebrand) Anemia (es: uremia)

10 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)

11 Pseudopiastrinopenia?

12 Pseudopiastrinopenia?
Striscio di sangue periferico in EDTA

13 Normale – Sangue in EDTA

14 Pseudopiastrinopenia – Sangue in EDTA

15 Satellitismo piastrinico

16 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

17 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:

18 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

19 Difetti dell’Emostasi Primaria
Piastrinopenie Piastrinopatie Difetti di proteine adesive (afibrinogenemia, malattia di von Willebrand) Anemia (es: uremia)

20 Patients with bleeding diathesis, no thrombocytopenia:

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

22 Punteggio normali (0→3) basso (4→7) intermedio (8→11) alto (12→19) p
Numero TE (min) (3,0-10,0) ( ) ( ) ( ) PCE (s) (85-244) (88-300) (74-300) (95-300) PCA (s) (67-179) (57-182) (59-300) (73-207) Valori mediani (range) Kruskall-Wallis test

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

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

25 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

26 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

27 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

28 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

29 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

30 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

31 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

32 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

33 Diagnosi di malattia di von Willebrand

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

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

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

37 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

38 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 ( ) R.I.P.A (mg/mL) Decreased (>1.2) Adapted from Castaman et al, 2003

39 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 ( ) R.I.P.A (mg/mL) Decreased (>1.2) Type 2B Adapted from Castaman et al, 2003

40 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 ( ) R.I.P.A (mg/mL) Decreased (>1.2) Plasma High Multimers Type 2B Adapted from Castaman et al, 2003

41 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 ( ) 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

42 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 ( ) 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

43 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

44 Aggregazione piastrinica

45 OUT IN

46 OUT IN OUT IN GPIIb/IIIa

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

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

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

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

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

52 Platelet Aggregation Platelet 1 Platelet 2

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

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

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

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

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

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

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

60 Diagnosi di difetti funzionali piastrinici

61 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

62 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

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

64 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

65 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

66 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 (59%) Primary Secretion Defects (20%) δ-Storage Pool Deficiency (12%) Glanzmann Thrombasthenia (2%) “Aspirin-like” Defects (1.3%) Bernard-Soulier syndrome (1%) P2Y12 defect (0.6%) Incomplete (5%)

67 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 (59%) Primary Secretion Defects (20%) δ-Storage Pool Deficiency (12%) Glanzmann Thrombasthenia (2%) “Aspirin-like” Defects (1.3%) Bernard-Soulier syndrome (1%) P2Y12 defect (0.6%) Incomplete (5%)

68 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:

69 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) U μM Adrenaline 5 μM Ristocetin 1.2 mg/mL (if normal, 0.6 mg/mL) Arachidonic acid 1 mM No agonists (SPA)

70 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

71 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

72 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) U μM 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)

73 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