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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