140308 Immune thrombocytopenias ISTH EDU course Cascais 2014.pptc.ymcdn.com/sites/ · PDF...

S. Felix

1456 1856

Medizinische Klinik der königlichen Universität Greifswald

Andreas Greinacher

Institut für Immunologie und TransfusionsmedizinUniversität Greifswald, Germany

Immune Thrombocytopenias

Disclosures forAndreas Greinacher

In compliance with COI policy, ISTH requires the following disclosures to the session audience:

Research Support/P.I. Boehringer-Ingelheim; Bayer Healthcare

Employee No relevant conflicts of interest to declare

Consultant Schering-Plough; Mitsubishi Pharma; Instrumentation Laboratories

Major Stockholder No relevant conflicts of interest to declare

Speakers Bureau No relevant conflicts of interest to declare

Honoraria Merck, Schering-Plough, Mitsubishi Pharma, GSK, Bayer

Scientific Advisory Board Boehringer-Ingelheim

• Autoimmune-Thrombocytopenia

• Alloimmune-Thrombocytopenia

• Drug-Induced-Immune- Thrombocytopenia– drug induced autoimmune TP– drug dependent TP– GP IIb/IIIa inhibitor induced TP– heparin-induced TP

Immune Thrombocytopenias

Definition of ITP

Primary immune thrombocytopenia

Platelet count <100,000/µL

No other cause of thrombocytopenia

No clinically evident secondary form

Secondary ITP: SLE, CLL, HIV, Hepatitis C

Rodeghiero et al. Blood (2009); 113:2386

Provan et al. Blood (2010); 115: 168

• Newly-diagnosed ITP: within three months from

diagnosis

• Persistent ITP: between 3 to 12 months from

diagnosis

• Chronic ITP: lasting for more than 12 months

Phases of the Disease

Rodeghiero F et al. Blood, 2009

1

Petechien

Haematoma

Clinical Symptoms in ITP

2

Doan, et al (1960) Ann Intern Med. 53:861-876

Demographics of ITP

Abrahamson, et al. European Journal of Haematology (2009) 83: 83

circa 1960 circa 2010

Cines DB, Blanchette VS. N Engl J Med. 2002;346:995-1008

Pathophysiology of ITP in 2002

New concept:

in part ITP is also caused by

impaired platelet production

McMillan, R. et al. Blood 2004;103:1364

Suppression of megakaryocyte production by ITP plasma

Kosugi, et al. (1996) British J Haem. 93:704

Plasma TPO levels in ITP

Megakaryocyte Ultrastructure

This image cannot currently be displayed.

Houwerzijl EJ, et al. Blood. 2004;103:500.

normal (para)apoptosis

Olsson, et al (2003) Nat Med 9:1123-1124

- Platelet antibodies not detected in ~1/3 patients- Impaired thrombopoesis

T-cell mediated platelet cytotoxicity

antibodies I T cellsChang M et al, Blood 2003McMillan R et al, Blood 2004 Olsson B et al, Nat Med 2003

Olsson B et al, Blood 2008Bao W et al, Blood 2010

direct lysisNardi M et al, Cell 2001

Fc-receptor mediatedphagocytosis

complement activation(lysis or phagocytosis)

Hauch TW & Rosse WF, Blood 1977McMillan R & Martin M, Br J Haematol 1981

Myers TJ et al, Blood 1982Winiarski J & Holm G, Clin Exp Immunol 1983

Kurata Y et al, Br J Haematol 1985Tsubakio T et al, Br J Haematol 1986

Harrington WJ et al, Ann Int Med 1953Shulman NR et al, Ann NY Acad Sci 1965

antibodies II

Firkin BG et al, Blood 1969Handin RI & Stossel TP, New Engl J Med 1974

McMillan R et al, New Engl J Med 1974Tsubakio T et al, Act Haematol 1983

Clarkson SB et al, New Engl J Med 1986Fujimoto TT et al, Br J Haematol 2007

Cornelis van 't Veer & Tom van der PollNature Medicine 14, 606 - 608 (2008

Refrigeration up-regulates glycosidases to the surface of platelets.

Jansen A J G et al. Blood 2012;119:1263-1273

©2012 by American Society of Hematology

The mechanism by which AAbs in ITPpatients lead to platelet clearance differbetween individuals.

Deglycosylation of human autoantibodies (AAb)

25 kDa

50 kDa

Ponceau S(Protein)

AA

bs

47 kDa

AA

bs

de-A

Abs

LCA(N-Glycan)

cell

num

ber

antibody binding to platelets (FITC)0 0

AAbs de-AAbsctl ctl

de-A

Abs

Asn297

EndoF

N-Glycans

AAb (IgG) de-AAb (IgG)

AAb-mediated phagocytosisplatelets (CMFDA) monocyte membrane (CD14) nucleus (Hoechst 33342)

control IgG AAb de-AAb

1

2Intraperitoneal injection of

antibodies of interest

3

Flow analysis(number, function)

Periodical collection ofblood samples

4

Introduction of human PRP into the retroorbital plexus

Platelet destruction in NOD/SCID mice

Boylan B et al, Blood 2006; Bakchoul T et al, Blood 2013

Survival of human platelets

control IgG(n = 4)

non complement-activating AAb

(n = 8)

complement-activating AAb

(n = 7)

0 60 120 180 240 3000

20

40

60

80

100

0 60 120 180 240 3000

20

40

60

80

100

0 60 120 180 240 3000

20

40

60

80

100

0 60 120 180 240 3000

20

40

60

80

100

0 60 120 180 240 3000

20

40

60

80

100

0 60 120 180 240 3000

20

40

60

80

100

time [min]

plat

elet

sur

viva

l [%

]AAb

de-AAb

p = 0.27 p = 0.003

in the absence or presence of complement

- Hereditabilty

- Infection

- Molecular mimicry

(- Epitope spread)

Origination of ITP

Heritability of ITP

-Incidence-PARC-ITP: 10/445 (2.3%) of pediatric patients + family hx1

-MHC-HLA-DRw22

-Decreased HLA-DRB1* and DQB103* (non-H. pylori)5

-Linkages at 1q22-23 and 11-13 (severe SLE)6

-FcgRIIc (OR 2.4)7

1. Ped Blood Ca 2006; 46:678; 2. J Clin Invest 1979;63:1085; 3. Blood 1998;91:3616; 4. Hematol. 2002;7:119

5. Blood 2002;100:1925; 6. Blood 2003;101:997; 7. Blood 2008;111:1029; 8. Br J Haem 2001;115:125

-Response genes-FcgRIIIA: 158V/V to prednisone 158F/F to splenectomy8

-DRB1*0410 - to prednisone (Japanese)3

-HLA-A2 - to splenectomy (females)4

If more than 1 family member has chronic thrombocytopenia

hereditary platelet disorders are most likely!!MYH-9, GATA-1, von Willebrand type IIB

Bernard Soulier syndrome

Greifswald experience:90 patients with MYH-9 disorder

5 patients with BSS4 patients with vWD Type IIB

1 family with ITP

Estimated prevalence of 2o ITP in US

Cines et al. Blood 2009;113:6511

SECONDARY IMMUNE THROMBOCYTOPENIA

• Autoimmune:– Lupus (SLE)– Antiphospholipid

antibody syndrome (APLS)

– Immune Thyroid Disease

– Evan’s Syndrome

• Lymphoproliferative:– CLL & WDLL– Hodgkin’s– LGL

• Infectious

Immune thrombocytopenia post-infection

HIV HCV H.pylori

HOW CAN AN INFECTIOUS AGENT INDUCE THROMBOCYTOPENIA?

• Infection of megakaryocytes: Decreased production or platelets by infected megakaryocytes.

• Immune complex disease: Immune complexes binding to platelet FcR and then secondary clearance by macrophages.

• Immune dysregulation: Loss of recognition of self with the development of anti-platelet antibodies.

• Antigenic mimicry: Antibodies against antigens of the infectious agent cross-react with platelet epitopes.

Immune thrombocytopenia post-infection:

Molecular mimicry GP IIIa (aa 49-66)

HIV Platelet

Nardi. M. et al.PNAS (1997);94:7589

HCV

Zhang et al.Blood (2009);113:4086

Takahasi, T. et al. Br H Haem (2004);124:91

Cag A

H. pylori

THROMBOCYTOPENIA WITH CHRONIC HEPATITIS C

• Thrombocytopenia (< 150 x 109/L): 151 of 368 (41%) patients with chronic HCV– 10 of 53 (19%) with chronic HBV.

• Thrombocytopenia (< 50 x 109/L): 9% of HCV infected patients

Nagamine et al. J Hepatol 1996; 24: 135

Characteristics of HCV(+) vs. HCV(-) ITP

• Older and equally distributed between the sexes

• Bleeding at higher platelet counts

• Corticosteroids are less effective than in non-HCV ITP and may increase viral reproduction

• ITP may respond to IFN

Rajan S et al Br J Haematol 2005; 129:819

RESPONSE TO TREATMENTHCV+ vs. HCV- ITP

TREATMENT MODALITY HCV+ N (%) HCV- N (%)Corticosteroids Ref:1,3,4

9/33 (20.7%) 145/266 (50.4%)

Immunoglobulin Anti-Rh DCombined Ref:4

8/9 10/1118/20 (90%) 90/98 (92%)

Splenectomy Ref: 1, 3 7/12 (58%) 42/65 (65%)

Interferon alpha Ref: 2,4,5

16/22 (73%) 0

1. Sakuraya et al. Eur J Haematol 2002; 68: 49 ; 2. Garcia-Suarez et al. Br J Haematol 2000;p 110: 98 ; 3. Zang et al. Eur J Haematol 2003; 70: 196 ; 4. Rajan et al. Br J Haematol 2005; 129: 819; 5. Dufour et al. Eur J Gastroenterol Hepatol 2009; 21: 245

IFN treatment causes thrombocytopenia.Low platelet count is a frequent cause to stop IFN

treatment of HCV infection prematurely

McHutchison JG et al. N Engl J Med. 2007;357:2227-36.

Phase II Study of Eltrombopag in Hepatitis C Thrombocytopenia

Stasi, et al. Blood (2009) 113:1231-1240

Heterogeneity in ITP response to eradication of H. pylori

Response (%)

SECONDARY IMMUNE THROMBOCYTOPENIA

• Autoimmune:– Lupus (SLE)– Antiphospholipid

antibody syndrome (APLS)

– Immune Thyroid Disease

– Evan’s Syndrome

• Lymphoproliferative:– CLL & WDLL– Hodgkin’s– LGL

• Infectious:– HIV– HCV– H. pylori

Antinuclear antibodies in ITP

Author Year n (+)ANA SLEPerez 1985 18 6 4

Anderson 1985 117 24 5Panzer 1989 45 7 0Kurata 1994 66 29 0Leung 2001 220 76 3

Li 2005 545 39 3/2*

Altinas 2007 108 36 1*

Abbasi 2007 41 10 01160 227 (20%) 16 (1.4%)

Incidence of APLA in ITP

Incidence APLANomura Ann Hematol ‘94 13/56 (23%) CL

Stasi Blood, ‘94 69/149 (46%) CL, LAArfors Eur J Hematol ‘96 12/30 (30%) CLLipp Eur J Hematol ‘96 52/71 (70%) CL, PSSung Plts ‘99 6/57 (11%) b2GPIDiz Blood. ‘01 31/82 (38%) CL, LA

Bidot Br J Haem ‘04 26/40 (66%) multiple

Total 196/485 (43.1%)

Prevalence of APLA, ANA, ATA in ITP

ANAAPLA 30-40%

25-50%ATA

15-25%

ITP Anotherautoantibody

DAT 1-5%

Thrombosis in ITP with APLAStudy APLA (+) APLA (-)

Fanauchi ‘97 5/7 (71%) 3/20 (15%)

Diz-Kuckkaya ‘01

14/31 (45%) 3/51 (2.3%)

Bidot ‘04 8/19 (42%) -----

Pierrot-Deseilligny ‘08

4/55 (7%) 10/160 (6%)

Treatment of ITP: When? Why? How?

BT

Platelet transfusionTPO-receptor agonists

steroidsi.v. IgG(2g/kg bw over 2 or 4 days)anti-Dsplenectomie

anti-CD20(375 mg/m2/week,over 4 weeks)

platelets spleen/RES Immune system

Platelets<20,000

Platelets20,000-30,000

Platelets30,000-50,000

No symptoms (treatment) wait and watch wait and watch

Petechiae or ecchymoses

treatment treatment wait and watch

Bleeding treatment treatment treatment

Prednisone 1-2 mg/kg/d (4-6 W) or high dose-dexamethasone (oral 40 mg/day for 4 days)

ivIgG 0.4 g/kg/d for 1-5 days

Anti-D i.v./s.c.

Treatment

Rodeghiero et al. Blood, 2009

40

Definite Indication for Treatment

wet purpura

Life threatening bleeding- Platelet concentrates- rFVIIa (25 µg/kg bw)

Severe bleeding- 100 mg prednisolone per day or- high dose-dexamethasone (40 mg/d for 4d)

- combined with 2 g/kg b.w. ivIG (over 2 or 4d)

Patient

• 46 year old man.• 10 year history of ITP: platelet count 20-

40,000/µL; no major bleeding.• wait and watch strategy, short courses of

prednisone in case of increased bleeding symptoms. Good response documented.

• Admitted to ICU after bicycle accident with intracranial hemorrhage. Platelet count 11,000/µL.

Male 46 ysChronic ITP ~20-40,000/µL, bicycle accident

Initial Management

Platelet count 11.000/µL• Platelet transfusions until bleeding stops?• i.v. IgG 1g/kg bw• Prednisone i.v.• No drugs which inhibit platelet funtion• No heparin

48h after admission: platelet count 40,000 µL. CT scan no

increase in bleeding. Which is the appropriate management?

a. Platelet transfusions until platelet counts are >50,000/µL?

b. Third course of i.v. IgG 1g/kg bwc. Prednisone i.v.d. No drugs which inhibit platelet function e. No heparin

Patient

• Day 1 five platelet concentrates transfused until plt count increased to 35,000/µL

• i.v. IgG 1g/kg bw, day 1 and 2• Prednisone 100 mg/day• No heparin• Antiepileptic drug to prevent

seizures:levetiracetam (Keppra)• Day 5: pulmonary embolism

Patients with thrombocytopenia require thrombosis prophylaxis in risk

situations for DVT

Prevent thrombosis – PE!

Splenectomie

- second line treatment (after steroids) in USA, GB- Less frequently used in Germany

Kojouri et al, Blood 2004

CR 3506/5087 = 69%CR (5 Jahre) 779/1159 = 67%relapse nach CR 15%

Surgical complications:open surgery 318/2465 = 12.9%laparoscopic 88/921 = 9.6%open surgery, fatal 48/4955 = 1.0%Laproscopic, fatal 3/1301 = 0.2%

49

Splenectomie

- adverse effects:- increased risk for sepsis (life long)- increased risk for atherosclerosis- increased risk for pulmonary hypertension

SepsisSchilling, Ann Int Med 1995

226 families with hereditaryspherocytosis

fatal infections = 0.73 / 1000 years(CI, 0.015-1.5)

AtherosclerosisSchilling, Lancet 1997

144 splenectomized patients22 events

Hazard ratio 5.6 (CI, 1.7-19)

Rituximab (Anti-CD20)

- 375 mg x m-2 x wk-1 for 4 weeks- remission ~ 50%

Symptomatic treatment

N-plate, Romiplostim; AMG 531 (Amgen)- Weekly 2-10 µg x kg-1s.c

Eltrombopag (SB-497115-GR) (GlaxoSmithKline)- TPO-receptoragonist (small molecule)- oral 50-75 mg/day

Laboratory Diagnosis of ITP

ITP

Macro TP EDTA- pseudoTP satellitism

Free and cell-bound anti-platelet antibodies

autoantibodies

platelet

HLA

1. auto-antibodies in AITP are adsorbed by the patient´s platelets

2. platelets store IgG in the alpha granules and bind IgG via FcRIIa

0

0.2

0.4

0.6

0.8

1.0 S

ENS

ITIV

ITY

0 0.2 0.4 0.6 0.8 1.0

1-SPECIFICITY

20%50%

Total PAIgG

Surface PAIgG (two-stage)

Surface PAIgG I-Staphylococcal protein A

Surface PAIgG I-Mo-anti-IgG

0.45 OD

125

125

HIT Assays

MAIPA/ACE

PAIgG Assays

SRAELISA 1/50

Operating Characterstics of Platelet-Antibody Assays

Warkentin & Greinacher, 2001

MAIPA V.Kiefel et al. Blood 1988

(Monoclonal antibody immobilization of platelet antigens)

anti-human Abauto Ab

murine Ab against GPIIb

anti-mouse Ab

cell-lysis

platelet

I. II.

HLA Ab

HLAHLA

PAIgG: eluate (pH 2.8) testing [PIFT,GP-immunoassay]

*Kiefel et al., Ann. Hematol. 1996;72:280-285

patients with AITP 20/41 (sensitivity 48.8%)patients with NI-TP 0/24 (specificity > 95%)*

This technique is equivalent to GP-PAIgG testing (GPs IIb/IIIa, Ib/IX), but requiresmore platelets (100 x 106)

Platelet aab testing required?

Aab testing usually not required patients with acute, probable “post-infectious” AITP, esp. in

childhood

patients with uncomplicated AITP responsive to corticosteroids, IVIG, …

Aab testing useful patients with comorbidities which can cause TP, e.g.

hematologic malignancies

patients with refractory TP especially before institution of invasive (splenectomy), expensive/experimental therapy

Platelet Membrane Glycoprotein Polymorphisms that can cause Alloimmune Thrombocytopenia

According to PJ Newman 2001Adapted from Humphries and Mould

Science 294:316, 2001

Br(Glu505Lys)

AM

GPIa-IIa (21)

SitBrb variant(Thr799Met)

A vWf-like A(dhesion) domain (blue ball) is insertedbetween propellers 2 and 3 of the subunit.

The Br polymorphism is located betweenpropellers 5 and 6 and is homologous to vSer323

GPIIb-IIIa (IIb3)

PlA(Leu33Pro)

Pen(Arg143Gln)

Bak(Ile843Ser)

MaxBakb variant

(Val837Met)

PlA2 variant(Leu40Arg)

Mo(Pro407Ala)

Ca/Tu(Arg489Gln)

Sr(Arg636Cys)

Oea

PlA2 variant(Lys611)

Duv(Thr140Ile)

La(Arg62Gln)

Gro(Arg633His)

-TD

Ko(Thr145Met)

Iy(Gly15Glu)

Alloimmune-TPs• Neonatal AITP• Post-Transfusion Purpura (PTP)• acute thrombocytopenia after

transfusion of platelet antibodies• Platelet transfusion refractoriness

• important glycoproteins:

GP IIb/IIIa, GP Ia/IIa, GP Ib/IX, Gov

• Major problem: HLA-antibodies• detection of: sensitivity specificity

free abs (plts.): ++/(+) +-free abs (GP-spec): ++ ++PaIgG: +- --Plt-GP spec. abs +(-) ++genotyping!!!

Alloimmune TPs

Fetal and neonatal alloimmune thrombocytopeniaFNAIT

HPA-1 a a b b

Kiefel et al. Blood 2006

Effect of random PC on platelet counts in acute NAIT

Epitope-specific monoclonal antibodies as treatment

Fetal

platelets SZ21

maternal anti-HPA-1a IgG

Bakchoul T et al. Transfusion 2009

SZ21NGM-SZ21

N-glycan blot

17±1 days of

gestation

1-8 hours after

birth 0 60 120 180 240 3000

20

40

60

80

100 maternal anti-HPA-1a + NGM-SZ21maternal anti-HPA-1a + NGM-AP2maternal anti-HPA-1a alone

Time (min)

Plat

elet

sur

viva

l %

Bakchoul T, et al. BLOOD 2013

IgG F(ab)`2 deglykosylated-SZ21

Phagocytosisbinding to FcγR

Transport to the childbinding to FcRn

Potential applicationpostnatal

prenatal

Transfusion induced alloimmune thrombocytopenia

• Abrupt decrease of platelet counts following platelet or plasma transfusion

• Fever, nausea, no obvious other reason• Blood donor (usually woman with > 2

pregancies) had platelet allo-antibodies which are transferred with the plasma = passive immunization

0

100

200

300

400

500

0 1 2 3 4 5 6 7 8 9 10

Postoperative day (day 0 = day of surgery)

Pla

tele

t co

unt

(x10

9 /L)

bacteria contaminated transfusion

transfusion of blood product

passive alloantibody TP

Post-Transfusion Purpura

• 99.8% women preimmunized against a platelet alloantigen (usually HPA-1a)

• 7-14 days after transfusion of HPA-1a positive blood (RBCs, platelets):

• Platelet counts <10,000/µl, bleeding• Pathogenesis: epitope spreading?• Boosted allo-antibodies crossreact with HPA-

1a negative autologous platelets• Treatment: ivIgG 2g/kg b.w.

Lubenow et al. Thromb Research, 2000;100:115-25

Transfusion Refractoriness

• Most frequent immune thrombocytopenia• Patient develops antibodies (HLA>>HPA)

which bind to transfused platelets and cause platelet destruction in the RES

Drug-induced Immune-Thrombocytopenias

• Drug-induced AITP• Drug-dependent TP• GP IIb/IIIa-antagonist-induced TP• Heparin-induced TP

Drug-induced ITP

• Patients treated with gold develop ITP• Clinical presentation, diagnosis, treatment as in

ITP• Antibody titers decrease slowly after cessation of

gold. Gold treatment may cause relapse. A.von dem Borne et al. Brit J Haematol,1986;63:509-10

• GPV is the major platelet glykoprotein involvedGarner SF et al. Blood. 2002 Jul 1;100(1):344-6

Richard H. Aster and Daniel W. BougieN Engl J Med 2007; 357:580-587

0

100

200

300

400

500

600

700

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46

days

plat

elet

s (x

10

9 /l)

LMWH dalteparin

Danaparoid

Piperacillin/tazobactam Piperacillin

Hip surgery

Pla

tele

ts x

109 /

L

Pneumonia HIT antigen test positive

Lubenow N, Hron G, Greinacher A, unpublished

0

0,5

1

1,5

2

2,5

extin

ctio

n

donor platelets 1-4without piperacillin

0

0,5

1

1,5

2

2,5

extin

ctio

n

normal serum

patient serum

donor platelets 1-4without piperacillin

donor platelets 1-4with piperacillin

donor platelets 1-4with piperacillin

Drug-Metabolite

Ab-class TMP-SMX

Ex-vivo none

Patient 1 - IgG - ++++ -

Patient 1 - IgM - - -

Patient 2 - IgG ++++ - -

Patient 2 - IgM ++++ ++ -

Kiefel et al. Transfusion 1987;27:262-265

Abciximab (ReoPro®):(chimeric Fab-fragments)

Tirofiban (Aggrastat®):(nonpeptide tyrosinderivate)

Eptifibatide (Integrilin®):(cyclic RGD-mimetic)

Papain

Thrombocytopenia after GPIIb/IIIa-inhibitor treatment

0

50

100

150

200

250

300

0 25 50 75 100

h (after abciximab-bolus)

plat

elet

s (

x 1

000

/µl)

patient R15patient R25

platelet-transfusion

~50% are pseudothrombocytopenia

Immune ThrombocytopeniasAITP PTP Drug

dependent TP GP IIb/IIIainhibitor

TP

HIT TTP

Platelet count

variable<20.000

<10.000 <10.000 <10.000 40-80.000

10-30.000

Bleeding symptoms

(+) - +++ ++++ ++++ (+) - - - --

Onset chronic day 7-14 after

transfusion

day 7-14 after start of drug

(day 1 in case of reexposure)

day 1 of GPIIb/IIIa treatment(delayed onset)

day 5-14 acutedeteriorating

Thrombosis -- (+) -- -- -+depends

on treatment

++++ ++

Drug dependent TP: clinical management`The Friday evening Consultant Call´

• female 67 years old, diabetic coma, renal failure, rhabdomyolysis, platelet count < 5000/µl

• Multiple blood transfusions during the last 2 weeks, 2 pregnancies

• DD: HIT? drug-dependent TP? post transfusion purpura?• Intrafusin, structolipid, glucose, voluven, paspertin,

sufenta, actrapid, liquemin, lasix, tracutil, cernevit, antra, acetylcystein, konakion, ebrantil, nitro, ciprobay, vancomycin, aterenol, vicogant, glucerna, decortin

• Stop all drugs but electrolytes, vitamins, hormons • Start alternative antibiotics• Start i.v. IgG and exclude PTP as soon as possible• Reintroduce drugs sequentially after platelet counts

raised