A severe case of immune thrombocytopenia associated with anti-HLA antibodies induced by pregnancy

1. The Christ HospitalThe Christ Hospital
April 2012April 2012
Diana Girnita MD, PhDDiana Girnita MD, PhD
Internal MedicineInternal Medicine
Grand RoundsGrand Rounds

2. Case PresentationCase Presentation

3. Chief complainsChief complains
72 yo Caucasian F72 yo Caucasian F
ConfusionConfusion
Back pain in the lumbar areaBack pain in the lumbar area
Easy bruisingEasy bruising
Multiple ecchymosesMultiple ecchymoses

4. What would you like to know?What would you like to know?

5. HPIHPI
May 2011: fell on the floor and hit her right hip;May 2011: fell on the floor and hit her right hip;
has seen a chiropractorhas seen a chiropractor
For 1 month tried a herbal product, calledFor 1 month tried a herbal product, called
“Willow Bark” (aspirin substitute), did not help“Willow Bark” (aspirin substitute), did not help
Occasionally Ibuprofen 1-2 tablets/ day PRN forOccasionally Ibuprofen 1-2 tablets/ day PRN for
many yearsmany years
Two months after fallTwo months after fall
– easy bruising with minimal aggressionseasy bruising with minimal aggressions
– fatiguedfatigued
– weight loss (6-8 Lbs)weight loss (6-8 Lbs)
July 2011: in the ED due to confusion, severeJuly 2011: in the ED due to confusion, severe
back pain rated as 8/10, irradiated in the groinback pain rated as 8/10, irradiated in the groin
area, hips and LEsarea, hips and LEs

6. ROSROS
General/ constitutional:General/ constitutional: weight loss 8lbs, + fatigueweight loss 8lbs, + fatigue, no f/c/s, no f/c/s
Skin:Skin: + petechiae, purpura on UEs &LEs and and+ petechiae, purpura on UEs &LEs and and
ecchymoses lumbar and sacral areasecchymoses lumbar and sacral areas
HEENT:HEENT: no recent changes in vision/ ear pain, occasionalno recent changes in vision/ ear pain, occasional
epistaxisepistaxis with ibuprofen intakewith ibuprofen intake
Cardiovascular: no palpitations, chest pain, orthopnea,Cardiovascular: no palpitations, chest pain, orthopnea,
Respiratory: no dyspnea, cough, hemoptysisRespiratory: no dyspnea, cough, hemoptysis
Gastrointestinal: no N/V/C/D, melena, hemetemesisGastrointestinal: no N/V/C/D, melena, hemetemesis
GenitourinaryGenitourinary:: no dysuria, changes in urine color, odor orno dysuria, changes in urine color, odor or
flowflow
Musculoskeletal: no muscle weaknessMusculoskeletal: no muscle weakness
Neurologic: no focal neurologic changesNeurologic: no focal neurologic changes

7. PMHPMH: none, have not seen a doctor in 20 years: none, have not seen a doctor in 20 years
HOME MEDSHOME MEDS: MULTIVITAMIN po: MULTIVITAMIN po
ALLERGIESALLERGIES: Codeine (Nausea,Vomiting ): Codeine (Nausea,Vomiting )
SURGICAL HX:SURGICAL HX: nonenone
FHXFHX::
Cancer, diabetes -MotherCancer, diabetes -Mother
Cancer, HTN -FatherCancer, HTN -Father
SOCIAL HXSOCIAL HX: Married, G3P3, non-smoker,: Married, G3P3, non-smoker,
occasionally alcoholoccasionally alcohol

8. Physical examinationPhysical examination

9. VS: temp 97.9/ HR 57/ RR18/VS: temp 97.9/ HR 57/ RR18/BP 168/78BP 168/78/ weight/ weight
231lbs231lbs
General:General: obese, well developed, NADobese, well developed, NAD
HEENT:HEENT: PERRLA, EOMI, throat non-edematous/PERRLA, EOMI, throat non-edematous/
erythematous, normal mucoses ,erythematous, normal mucoses , no epistaxisno epistaxis
Neck:Neck: normal ROM, nontender, trachea midline,normal ROM, nontender, trachea midline, LNLN
nonpalpablenonpalpable/ nontender, no JVD, no carotid bruit/ nontender, no JVD, no carotid bruit
CV:CV: RRR, normal S1, S2, no m/r/gRRR, normal S1, S2, no m/r/g
Resp:Resp: CTA bilaterally without r/r/wCTA bilaterally without r/r/w
Abd:Abd: soft, ND/NT, + BS,soft, ND/NT, + BS, no HSMno HSM
Ext:Ext: 2+ pulses, without clubbing, cyanosis, or edema2+ pulses, without clubbing, cyanosis, or edema

10. Physical examinationPhysical examination
Skin:Skin: warm, dry,warm, dry,
– multiple petechiae on her UE &LEmultiple petechiae on her UE &LE
– purpurapurpura
– ecchymoses more on the UEs, few on LEs, aecchymoses more on the UEs, few on LEs, a
large ecchymose on the R lumbar arealarge ecchymose on the R lumbar area
Neuro:Neuro: AOx 2 (person, time), CN II-XIIAOx 2 (person, time), CN II-XII
grossly intact, motor and sensory functiongrossly intact, motor and sensory function
intact with no focal deficitsintact with no focal deficits

11. What would you like for work up?What would you like for work up?
LabsLabs
CBCCBC
BMPBMP
PT, INR, aPTT, bleeding timePT, INR, aPTT, bleeding time
TSHTSH
Liver profileLiver profile

12. Results- admission at OSHResults- admission at OSH
CBC:CBC:
– Hb=10.4; Ht=31.5, MCV 89Hb=10.4; Ht=31.5, MCV 89
– WBC =6000/mm3WBC =6000/mm3
– Platelets =2000/mm3Platelets =2000/mm3
INR =1.03INR =1.03
TSH -1.54 (WNL)TSH -1.54 (WNL)
ESR =30/1hESR =30/1h
BMPBMP
138
3.8
103
23
0.8
11

13. DifferentialDifferential
diagnosisdiagnosis

14. CLL / lymphomaCLL / lymphoma
MDSMDS
ITPITP
Collagen disease (APLS, SLE)Collagen disease (APLS, SLE)
TTP, DIC/sepsisTTP, DIC/sepsis
Drugs (NSAIDs, Willow bark/ASA)Drugs (NSAIDs, Willow bark/ASA)
Splenic sequestration/ hypersplenismSplenic sequestration/ hypersplenism
Pseudothrombocytopenia-unikelyPseudothrombocytopenia-unikely
Dilutional s/p massive transfusionDilutional s/p massive transfusion

15. ImagingImaging
CT head
MRI head
CXR
CT chest/ abdomen/pelvis
Xray of lumbar spine
PET scan
US abdomen – spleen

16. CT head w/out contrastCT head w/out contrast
Hyperdense
intracranial masses
compatible with
metastatic disease/
possible that these
are hyperdense due
to blood

17. MRI headMRI head

18. MRI impressionMRI impression
Intracranial lesions do not demonstrateIntracranial lesions do not demonstrate
significant contrast enhancement assignificant contrast enhancement as
expected for metastatic diseaseexpected for metastatic disease
Have characteristics compatible withHave characteristics compatible with
acute/early subacute blood lossacute/early subacute blood loss
Multifocal intraparenchymal brainMultifocal intraparenchymal brain
hemorrhagehemorrhage

19. CXRCXR

20. CT chest, abdomen, pelvisCT chest, abdomen, pelvis

24. CT chest, abdomen, pelvisCT chest, abdomen, pelvis
impressionimpression
No mass or adenopathy in the chest,No mass or adenopathy in the chest,
abdomen, pelvisabdomen, pelvis
Bilateral pulmonary ground glass infiltratesBilateral pulmonary ground glass infiltrates
Likely alveolar hemorrhageLikely alveolar hemorrhage

25. US spleen -normalUS spleen -normal

26. X-ray lumbar spineX-ray lumbar spine
No fracture, Mild scoliosisNo fracture, Mild scoliosis
Degenerative disc disease:L2-L3, L4-L5Degenerative disc disease:L2-L3, L4-L5

27. PET scanPET scan
nono
evidenceevidence
of skeletalof skeletal
metastaticmetastatic
diseasedisease

28. What do we know so far?What do we know so far?
Critical platelets countsCritical platelets counts
Multiorgan hemorrhagesMultiorgan hemorrhages

29. OSH treatmentOSH treatment
8U of platelets8U of platelets
Decadron iv 4mg Q6HDecadron iv 4mg Q6H
One dose of IVIG =1mg/kg (1000mg)One dose of IVIG =1mg/kg (1000mg)
Platelets remained in the 2-3000/mm3Platelets remained in the 2-3000/mm3
Patient was transferred to TCHPatient was transferred to TCH

30. TCH work-upTCH work-up
CBC:CBC:
–WBC=6000/mm3,WBC=6000/mm3,
–H/H = 8.2/24.8,MCV =91H/H = 8.2/24.8,MCV =91
–Platelets =3000/mm3Platelets =3000/mm3
BMP -normalBMP -normal

31. Anemia work-upAnemia work-up
Retic count -110976 (H)Retic count -110976 (H)
LDH -269 (H)LDH -269 (H)
Haptoglobin 64 (N)Haptoglobin 64 (N)
COOMBS test and RH positiveCOOMBS test and RH positive
Iron studies: normalIron studies: normal
Vit B12/folate were normalVit B12/folate were normal
Peripheral smear: no schystocytesPeripheral smear: no schystocytes

32. TCH work-upTCH work-up
Liver tests were normalLiver tests were normal
TSH -0.23 (L)TSH -0.23 (L)
Fecal occult test -NEGATIVEFecal occult test -NEGATIVE

33. TCH work-upTCH work-up
Fibrinogen – 307 (range 175-425)Fibrinogen – 307 (range 175-425)
INR -1.3 (0.9-1.1)INR -1.3 (0.9-1.1)
PT – 16.1 (11.9-14.8)PT – 16.1 (11.9-14.8)
aPTT -23.5 (23.6-33.5)aPTT -23.5 (23.6-33.5)

34. Serum protein electrophoresisSerum protein electrophoresis
– Alpha1,2; beta normalAlpha1,2; beta normal
– Gamma globulin 3.9 (H)Gamma globulin 3.9 (H)
– Globulin 4 (H)Globulin 4 (H)
– Albumin (3.2)Albumin (3.2)
Kappa/lambda ratio –normalKappa/lambda ratio –normal
– Kappa-12.5 (3.3-14)Kappa-12.5 (3.3-14)
– Lambda -14.10 (5.7-26.3)Lambda -14.10 (5.7-26.3)
– Ratio – 0.89 (0.26-1.65)Ratio – 0.89 (0.26-1.65)

35. Parvovirus B 19 IgG positive, no IgMParvovirus B 19 IgG positive, no IgM
Consult to hematologyConsult to hematology

36. Bone Marrow biopsyBone Marrow biopsy
Cellularity is 85%.Cellularity is 85%.
Hyperplasia of the erythroid, as well as theHyperplasia of the erythroid, as well as the
megakaryocyte series with normalmegakaryocyte series with normal
morphologic features.morphologic features.
Dysplasia is not evident.Dysplasia is not evident.
Findings most compatible with peripheralFindings most compatible with peripheral
platelet destruction.platelet destruction.

37. Bone marrow biopsyBone marrow biopsy
Special STAINS:Special STAINS:
Storage iron decreased.Storage iron decreased.
Reticulin stain shows no increase inReticulin stain shows no increase in
reticulin fibrosisreticulin fibrosis
PAS stain highlights the megakaryocytePAS stain highlights the megakaryocyte
populationpopulation

38. ITP diagnosis and therapyITP diagnosis and therapy
IVIG
WinRho
Rituximab 1st
Rituximab 2nd
Splenectomy
Methyprednisolone, f/b PDN taper
1st
2nd

39. QuestionQuestion
Why was this patient refractory toWhy was this patient refractory to
platelet transfusions therapy ?platelet transfusions therapy ?

40. PlateletsPlatelets
Glycoproteins (GP)Glycoproteins (GP)
GP Ib/IX =receptor forGP Ib/IX =receptor for
VWFVWF
GP IIb/IIIa = receptor forGP IIb/IIIa = receptor for
fibrinogenfibrinogen
GP Ia/IIa = collagenGP Ia/IIa = collagen
receptor.receptor.
HLA class I: A, BHLA class I: A, B
The cell surface is a jungle!!
Gebel and Bray,
Transplantation 2004

41. 0
100
200
300
400
500
600
A B C DRB1
1990
1997
2001
2002
2004 N > 2400
HLA -A and B are important inHLA -A and B are important in
platelet transfusionplatelet transfusion

42. Refractoriness to plateletRefractoriness to platelet
transfusiontransfusion
ELISA-positive for:ELISA-positive for:
platelet-specific antibodies (1a, 1b, 3a, 3b,platelet-specific antibodies (1a, 1b, 3a, 3b,
4a, 5a, 5b) and4a, 5a, 5b) and
HLA-specific antibodiesHLA-specific antibodies

43. Broad IgG reactivity towards HLABroad IgG reactivity towards HLA
antigens determined by Luminexantigens determined by Luminex
single-antigen beadssingle-antigen beads
Calculated PRA = 82%
SELF HLA -NEGATIVE

44. The high (82%) allo-reactivity wasThe high (82%) allo-reactivity was
explained by only two specificities:explained by only two specificities:
anti-HLA A3 (private epitope) andanti-HLA A3 (private epitope) and
anti-HLA w4 (public epitope)anti-HLA w4 (public epitope)
A3
W4 epitope

45. The w4 epitope (78-82 RENLR) is known asThe w4 epitope (78-82 RENLR) is known as
a very immunogenic public epitopea very immunogenic public epitope
Magenta – Heavy Chain (Alpha chain)
Blue – Light chain (Beta-2 microglobulin)
Brown – Peptide
Yellow – Bw4 epitope

46. Patient had no previous transplantsPatient had no previous transplants
and/or transfusions;and/or transfusions;
Three pregnancies;Three pregnancies;
Patient and husband HLA typingPatient and husband HLA typing
Husband’s mismatched HLA class I:
A3
B57 (bearing the Bw4 epitope)

47. Antibodies towards HLA class IIAntibodies towards HLA class II
were also explained by pregnancywere also explained by pregnancy
sensitizationsensitization
Husband’s mismatched HLA: DR7, DR17, DQ2, DQ9

48. The IgG subtype analysis demonstratedThe IgG subtype analysis demonstrated
IgG1, which is a strong complement binderIgG1, which is a strong complement binder

49. IMMUNEIMMUNE
TROMBOCYTOPENIATROMBOCYTOPENIA
(ITP)(ITP)

50. Immune thrombocytopenia vs ITPImmune thrombocytopenia vs ITP
can no longer be considered idiopathiccan no longer be considered idiopathic
DefinitionDefinition
– Platelet count < 100 x 10Platelet count < 100 x 1099
/L/L
– No other cause of thrombocytopeniaNo other cause of thrombocytopenia
– NoNo clinically evidentclinically evident secondary formsecondary form
Rodeghiero F, et al. Blood. 2009;113:2386-2393

51. Timing in ITPTiming in ITP
NewlyNewly diagnosed < 3 modiagnosed < 3 mo
Persistent ITPPersistent ITP – 3 -12 mo and patients not– 3 -12 mo and patients not
reaching/maintaining remission off therapyreaching/maintaining remission off therapy
Chronic ITP: >Chronic ITP: > 12 mo12 mo

52. Primary ITP – mechanismsPrimary ITP – mechanisms
–molecular mimicrymolecular mimicry
–epitope spreadepitope spread
Primary vs Secondary ITPPrimary vs Secondary ITP

53. Molecular mimicryMolecular mimicry
HIV HCV H. pylori

54. ITP Post-infection:ITP Post-infection:
Molecular Mimicry GPIIIa (aa 49-66)Molecular Mimicry GPIIIa (aa 49-66)
Nardi MA, et al. Proc Natl Acad Sci U S A. 1997;94:7589-7594. 2. Zhang W, et al.
Blood. 2009;113:4086-4093. 3. Takahashi T, et al. Br J Haematol. 2004;124:91-96.

55. EPITOPE SPREADEPITOPE SPREAD
Cines DB,Cines DB,
NEJM 2002;NEJM 2002;
346:995-1008346:995-1008

56. Alloantibodies determineAlloantibodies determine
–reduced platelet survivalreduced platelet survival
–impaired platelet productionimpaired platelet production
(megakaryocytes)(megakaryocytes)

57. Prevalence ofPrevalence of SecondarySecondary ITPITP
SLE 5%
APS 2%
CVID 1%
CLL 2%
Evan’s 2%
ALPS, post-tx 1%
HIV 1%
Hep C 2%
H. pylori 1%
Postvaccine 1%
Misc systemic infection 2%
Primary
80%
Cines DB, et al. Blood. 2009;113:6511-6521.

58. Initial Evaluation for ITP 2010Initial Evaluation for ITP 2010
ASH: CBC, blood smear, BM (age > 60ASH: CBC, blood smear, BM (age > 60
yrs), r/o other causesyrs), r/o other causes
Infection: HIV, HCV, HBV, H. pylori, PPDInfection: HIV, HCV, HBV, H. pylori, PPD
Autoimmune: ANA, APLA, ATA/TSHAutoimmune: ANA, APLA, ATA/TSH
Secondary: DAT, reticulocytes, LFTs, CTSecondary: DAT, reticulocytes, LFTs, CT
scan/echoscan/echo
Bone marrow: reticulin at baseline?Bone marrow: reticulin at baseline?
Provan D, et al. Blood. 2010 115: 168-186

59. BM> 60 yrsBM> 60 yrs to exclude MDS/ CLLto exclude MDS/ CLL
Quantitative IG before patients receive IVIG (CVIDQuantitative IG before patients receive IVIG (CVID
or IgA deficiency)or IgA deficiency)
Direct antiglobulin testDirect antiglobulin test--
22% pt have DAT+22% pt have DAT+
Blood group Rh(D) typingBlood group Rh(D) typing::
if treatment with anti-if treatment with anti-
RhD immunoglobulin is being consideredRhD immunoglobulin is being considered
1. Provan D, et al. Blood. 2010. 2. Cines DB, et al. Blood. 2009;113:6511-6521.

60. Additional TestsAdditional Tests
Thyroglobulin antibodies and/or TSHThyroglobulin antibodies and/or TSH -8% to 14% ITP-8% to 14% ITP
can develop hyperthyroidism; the presence ofcan develop hyperthyroidism; the presence of
hyper/hypothyroidism result in resistance to therapyhyper/hypothyroidism result in resistance to therapy
ANAANA is predictive of chronicity in childhood ITPis predictive of chronicity in childhood ITP
H/o fetal loss/ thrombosis-H/o fetal loss/ thrombosis- antiphospholipid antibodiesantiphospholipid antibodies
15% have ITP15% have ITP
1. Provan D, et al. Blood. 2009;[Epub ahead of print]. 2. Liebman H. Semin Hematol. 2007;44(4 suppl 5):
S24-S34. 3. Altintas A, et al. J Thromb Thrombolysis. 2007;24:163-168.

61. Treatment in ITPTreatment in ITP

62. Timeline: introduction of modernTimeline: introduction of modern
day ITP drug treatmentday ITP drug treatment
Splenectomy
Corticosteroids
IVIg
Anti-D
Rituximab
TPO mimetics
1900 1920 1960 20001940 1980

63. ITPITP
Who and When to TreatWho and When to Treat
Only platelet # < 20-30 x 10Only platelet # < 20-30 x 1099
/L or/L or
symptomaticsymptomatic
Bleeding risk > 60 yrsBleeding risk > 60 yrs
Post-splenectomyPost-splenectomy
Bleeding and infection contribute equallyBleeding and infection contribute equally
to mortalityto mortality
Provan D, et al. Blood. 2010 115: 168-186

64. Relevant factors:Relevant factors:
– extent of bleedingextent of bleeding
– comorbidities predisposing to bleedingcomorbidities predisposing to bleeding
– complications of specific therapiescomplications of specific therapies
– activity and lifestyleactivity and lifestyle
– potential interventions that may causepotential interventions that may cause
bleedingbleeding
– patient expectations, anxietypatient expectations, anxiety

65. CorticosteroidsCorticosteroids
IVIg and Anti-D (Winrho)IVIg and Anti-D (Winrho)
Rituximab (anti CD20 mAb)Rituximab (anti CD20 mAb)
Splenectomy –removes B and plasmaSplenectomy –removes B and plasma
cellscells
Anti-plasma cell drugs (proteasomeAnti-plasma cell drugs (proteasome
inhibitors)inhibitors)
TPO mimmeticsTPO mimmetics
ITPITP
How to TreatHow to Treat

66. Rajan Lakhia, MD
Suzanne Partridge, MD
Hoxworth Blood Center

67. Treatment
Strategy
Response
Rate
Time to
Response
Response
Duration
Cortico-
steroids
Prednisone 70-80% Several days
to weeks
Uncertain
Me-Pred ≤ 95% Few days 23% have >
50x109
/L at 39
months
Immune
Globulin
IVIg ≥ 80% 1-2 days 3-4 weeks, months
in some
Anti-D: 50-80%
(dose
dependent)
1-5 days (dose
dependent)
Typically 3–4
weeks, months in
some
Provan D, et al. Blood. 2009;[
Frontline Therapies -Adult ITPFrontline Therapies -Adult ITP

68. IV Immune globulin (IVIg)IV Immune globulin (IVIg)
Mechanism of actionMechanism of action
– Effects on humoral and cellular immune functionEffects on humoral and cellular immune function11
– Impairs of clearance of opsonized plateletsImpairs of clearance of opsonized platelets11
Effect mediated through FcγRIIb receptor (murineEffect mediated through FcγRIIb receptor (murine
model)model)22
– Increases clearance of antiplatelet antibodies viaIncreases clearance of antiplatelet antibodies via
saturation of the FcRn (rodent model)saturation of the FcRn (rodent model)33
1. Sandler and Tutuncuoglu. Expert Opin Pharmacother. 2004;5:2515-2527. 2. Samuelsson et al.
Science. 2001;291:484-486. 3. Hansen and Balthasar. Blood. 2002;100:2087-2093.
4. George J, et al. Blood. 1996;88:3-40.

69. RituximabRituximab
Anti-CD20 monoclonal antibodyAnti-CD20 monoclonal antibody
Hypothesis: B-cell depletion: ?Hypothesis: B-cell depletion: ? ↓ antiplatelet↓ antiplatelet
antibodiesantibodies
No RCT studies availableNo RCT studies available
Effective dose not known, not FDA-approvedEffective dose not known, not FDA-approved
Before splenectomy, when the surgicalBefore splenectomy, when the surgical
option is not well accepted/ high risk ofoption is not well accepted/ high risk of
complicationscomplications

70. SplenectomySplenectomy
Mechanism of actionMechanism of action
– Removes a primary site of platelet destructionRemoves a primary site of platelet destruction
by macrophagesby macrophages
– Possible site of autoantibody productionPossible site of autoantibody production
Sandler SG, et al. Expert Opin Pharmacother. 2004;5:2515-2527.

71. Splenectomy: complete responseSplenectomy: complete response
vs timevs time
47 case series, adults only,
f/u median: 29 mos (range:
1-153)
Kojouri K, et al. Blood. 2004;104:2623-2634.
0
20
40
60
80
100
0 40 160
Follow-up (Mos)
%CompleteRemission
20 8060 100 140120
r = -0.1031, P = .490

72. Kosugi S, et al. Br J Haematol. 1996;93:704-706. Copyright © 1996. Reproduced with permission of John
Wiley & Sons, Inc.
Patient Group
Anaplastic
anemia
(n = 12)
Now vs thenNow vs then
30
ITP
(n = 43)
Normal
(n = 21)
TPO(femtomoles/mL)
25
15
20
10
5
0
Plasma TPO Levels in ITP
1) healthy: platelet # is high
and serum thrombopoietin is
low
2) ITP: both the platelet #
and serum thrombopoietin
levels are low
3)AA: platelet # low, but
serum thrombopoietin levels
are high

73. %Control
MegakaryocytesSuppression of MegakaryocyteSuppression of Megakaryocyte
Production by ITP PlasmaProduction by ITP Plasma
100
75
50
25
0
ITP-1 ITP-2 ITP-3 ITP-4 ITP-5 ITP-6 ITP-7 ITP-8 ITP-9 ITP-10 ITP-11 ITP-12
McMillan R, et al. Blood. 2004;103:1364-1369.