diagnosi dei difetti dellemostasi primaria marco cattaneo unità di ematologia e trombosi ospedale...

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

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