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The Evolving Landscape of
Microangiopathic Hemolytic Anemia
J. Christian Barrett, M.D.
Division of Hematology, Oncology,
and Palliative Care
The Evolving Landscape of MAHA
• Objectives
– Compare and contrast the physiologic
mechanisms of the causes of microangiopathic
hemolytic anemia
– Select an appropriate management strategy for a
microangiopathic hemolytic anemia
Case #1
•
67 year old woman with history of scleroderma and Raynaud’s syndrome
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Chronic abdominal pains and bloating
Fell out of her chair with significant tonic seizures.
BP 188/74 during seizure in ED  136/71 minutes later
Platelet count 36,000 with creatinine of 3.8
Peripheral smear schistocytes with Haptoglobin <8 with LDH 663
CT head negative  MRI/MRA with minor stenosis bilateral ICA
Additional pre-therapy labs returning over fist several days
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ADAMTS13 level 56%
PT/PTT and fibrinogen normal
normal C3, C4, and CH50 complement levels
Antiphospholipid antibody panel was negative
ANA 1:2560 centromeric
Therapy (over the span of 3 weeks)
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Plasmapheresis plus steroids after rheumatology consultation
ACE-inhibitor
Rituximab x 2 doses
Eculizumab.
Microangiopathic Hemolytic Anemia
• Intravascular hemolytic process

– Reticulocytosis
– Elevated LDH
– Reduced haptoglobin
Fibrin and platelet-rich thrombi occluding vessel

Fragmented red blood cells a.k.a. schistocytes
Microangiopathic Hemolysis:
Fragmented RBCs
Microangiopathic Hemolytic Anemia
• Pathology General Findings
– Arteriolar and capillary wall thickening
– Subendothelial protein and cellular debri
– Fibrin and platelet rich thrombi occluding vessel
The G3 of MAHA
• Disseminated intravascular coagulopathy (DIC)
• Thrombotic thrombocytopenic purpura (TTP)
• Hemolytic uremic syndrome (HUS)
Disseminated Intravascular
Coagulopathy (DIC)
• Rapid and massive activation of coagulation
– fibrin deposition
• Consumption of platelets and factors
• Generation of fibrin split products
• Results in hemorrhage and thrombosis
– Balance differs acute vs. chronic
• End-organ damage
Sepsis and DIC
Direct vascular injury
Endotoxins
Tissue Factor
Bacteria
Hypoperfusion
shock  tissue
necrosis
Tissue Factor Initiates Coagulation
XII
Tissue Factor
XI
IX
VII
VIII
X
V
II (ProthrombinThrombin)
I (FibrinogenFibrin)
XIII
Stable fibrin clot
Acute DIC
• Laboratory Testing
– Prolonged aPTT, PT, TT
– Low fibrinogen
– Increased fibrin split products (D-dimers)
– Thrombocytopenia
– Microangiopathic hemolytic anemia
• MINOR AND VARIABLE COMPONENT
Thrombotic Thrombocytopenic Purpura
(TTP)
• Classic Pentad
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Microangiopathic Hemolytic Anemia
Thrombocytopenia
CNS involvement
Renal Involvement
Fever
NOTE:
Only need the first two criteria as
entry to consideration
Thrombotic Thrombocytopenic Purpura
(TTP)
• >90% mortality if untreated
• Treatment is plasmapheresis
• Deficiency of von Willebrand cleaving enzyme
ADAMTS 13 and VWF
WeilberlPalade bodies
Alphagranules
ultralarge
vWF
ADAMTS-13
(vWF cleaving
enzyme)
Ultra-large
vWF
Smaller vWF
forms
Figure 4. Scheme depicting the role of shear stress and ADAMTS13 in regulating the
interaction between VWF and platelets.
Tsai H JASN 2003;14:1072-1081
©2003 by American Society of Nephrology
Thrombotic Thrombocytopenic Purpura
(TTP)
• Pathogenesis
– Deficiency of von Willebrand cleaving enzyme
• Ultralarge molecular weight vWF multimers (ULVWF)
– Not cleaved as normally would be
– ULVWF promote platelet aggregation
– Result is microthrombi  hemolysis and platelet consumption

Inciting event “second hit”????
Hemolytic Uremic Syndrome (HUS)
• Hemolytic anemia with schistocytes
• Thrombocytopenia
• Renal impairment (elevated creatinine)
– However, any organ can be affected
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10% with CNS manifestations
3% with Cardiac (MI)
5% multi-organ failure
Digital ischemic gangrene
NOTE:
Need not be acute onset
Anemia and thrombocytopenia not absolute
Classic HUS
(aka “Post-Diarrheal”* HUS)
• Usually seen in children or pregnant women
• Can occur in epidemic forms
– Shiga-like toxin-producing E. coli O157:H7
• Clinically looks like TTP involving only the kidney
– No CNS symptoms
– Marked renal impairment
– Low mortality (virtually none if treated)
*Use caution with D+ versus D- designations
Classic Shiga-like Toxin HUS
• Shiga-like toxin E.coli
– 92% of cases are seasonal May-November
• Overall hospitalization rate 17%
• HUS occurs ~7-10 days after infection
Classic Shiga-like Toxin HUS
Strain variation
– E.coli O157:H7
• HUS in 5-15% of cases
• Children especially susceptible to HUS
– elderly to dying
• Antibiotics potentially harmful and NOT helpful
– STX gene within antibiotic-inducible prophages
– E.coli O104:H4
• HUS in 22%
• 88% of HUS cases in young women
• Antibiotics do help decrease STX production
Classic Shiga-like Toxin HUS
• STX structure
– A subunit = enzymatic subunit
– B subunit = cell-binding subunit
– Organized in pentamers
• B-subunit binds…
– Globotriaosylceramide GB3 membrane receptors
• Glomerular endothelium
• Tubular epithelium
Classic Shiga-like Toxin HUS
• Pathogenesis
– Internalized
– Transport to endoplasmic reticulum
– Enzymatic A subunit has N-glycosidase activity
• Removes an adenosine from 28S ribosomal RNA
• Inhibits cellular protein synthesis
Classic Shiga-like Toxin HUS
• Pathogenesis
– Cellular apoptosis and necrosis
• Inflammation with cellular adhesion
• Tissue factor release
• Decreased thrombomodulin expression
– Thrombotic microangiopathy
Atypical HUS
• Familial
• Onset at any age
– Slight childhood predominance (60% of cases)
• 70% of childhood cases occur before the age of 2 years
• Inciting event
– Viral illness (URI or gastroenteritis)
– Pregnancy (esp. post-partum period)
Noris, M. et al. (2012) STEC-HUS,
atypical HUS and TTP are all
diseases of complement activation
Nat. Rev. Nephrol..2012.195
Activation and inactivation of the alternative complement pathway.
Atkinson J P , and Goodship T H J Exp Med
2007;204:1245-1248
© 2007 Rockefeller University Press
Factor H Mutations
Atypical HUS Pathogenesis
• Quantitative or qualitative deficiencies of the
alternative complement pathway
– Fluid phase proteins
• CFI, CFH
• C3 and CFB (qualitative defects only with these)
– Membrane bound proteins
• MCP and Thrombomodulin
www.inkling.com
www.kidneypathology.com.ar/01.htm
Why the Kidneys?
• Fenestrated monolayer of endothelium
• Lower DAF and CD59 expression
• Reduced thrombomodulin/tissue factor ratio
aHUS and DGKE Mutations
• Diacylglycerol kinase-e
– Phosphorylates arachidonic acid-containing
diacylglycerol  phophatidic acid
aHUS and DGKE Mutations
Lemeire, M, et al. Nat Genet. 2013 May;45(5):531-6.
Other Causes of HUS
• Other Infections
– HIV
– Streptococcal pneumoniae
– H1N1 influenza A
• Medications
– Gemcitabine
– Cyclosporin
– Tacrolimus (and other calcineurin inhibitors)
The G3 MAHA Revisited
• aHUS
➤ DIC
– Thrombotic microangiopathy
dominates
• Renal dominates
o Consumptive
– Coagulation activated
coagulopathy
dominates
• Bleeding
o Thrombotic
microangiopathy
minor part of picture
– Complement alternative
pathway abnormalities
• But not consumed
• TTP
– Thrombotic microangiopathy
dominates
• Renal affected
– Coagulation activated
• But not consumed
– ADAMTS13 deficient
Scleroderma Renal Crisis
• Endothelial damage
– Unknown mechanism – Immune-mediated?
• Increased in patients with corticosteroid exposure?
• Reduced in patients with ACE-I therapy?
– Platelet and coagulation activation
– Fibrinoblastic and non-fibroblastic connective
tissue proliferation  proliferative
endarteropathy
• “onion-skin” vascular injury
– Small vessel injury >>> glomerular injury
Patient 1 Revisited
Homozygous for CFHR1-CFHR3 deletions
Case #2
• 65 year old woman
– TTP diagnosed 4 years prior with ADAMTS13 86%
– AMI 3 years prior
– DVT and PTE 1 year prior
– Weakness with falls
– Chronic intermittent headaches
Antiphospholipid Syndrome
• A Clinical-pathologic Diagnosis
– At least one test positive for an antiphospholipid
• Repeated at least 12 weeks later
• Less than 5 years before the clinical event
• Moderate to High Titer
– Presence of Disease Manifestation
• Arterial thrombosis
• Venous Thrombosis
• Pregancy morbidity
– Three+ losses <10 weeks gestation otherwise not explained
– One loss >10 weeks gestation (morphologically normal fetus)
– One+ premature birth <34weeks due to pre-eclampsia, eclampsia,
placental insufficiency
Antiphospholipid Syndrome
• Anti-B2GPI/B2GPI complex
– Binds receptors on endothelial cells and platelets
• Reduced nitric oxide production
• Increases tissue factor expression
• Platelet activation and aggregation
– Inhibition of anticoagulation
• Disruption of the thrombomodulin-Protein C system on
endothelial surface
– Inhibition of fibrinolysis
• Blocks B2GPI from acting as a cofactor for tPA
– Activation of complement
• Immune complexes activate classical pathway (C1q)
– Tissue factor expression
– Platelet storage granule and microparticle release
Case #3
 History:
 50 year-old AAM consulted to see for possible TTP/HUS
 2 weeks of progressive DOE and fatigue
 URI symptoms about 2 weeks ago that improved with supportive care, but
DOE progressed even as his URI symptoms improved.
 10-lbs unintentional weight loss in past month or so.
 PMH: HTN
 Lisinopril 20 mg daily
 HCTZ 25 mg daily
 Physical Examination:
 VS: T36.3, HR 106, BP 132/52, SpO2 97% on RA
 Eyes + Conjunctival pallor. No scleral icterus.
Case #3
 Labs:
 WBC 3.8, Hgb 4.1, HCT 11.0, Plts 89K, MCV 108.3
 Differential: Neu 48.2% Lym 48.1%
 BMP within normal limits (Cr 0.76).
 Liver panel
 Total bili 3.1 Direct bili 0.5
 ALP 77 ALT 63 AST 268
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PTT 26 PT 11.0 INR 1.1
Urine: small blood, 8 RBCs/HPF
Retic count 10.2 10e9/L, Ret % 1.0 % L
Haptoglobin <8
LDH 9496
DAT negative
Transferrin sat 45 %, ferritin 264 ng/mL
Vitamin B12 223, folate 7.6
Case #3
 4 units FFP infused plus prednisone 1 mg/kg started
 Next AM Labs
 WBC 3.3, Hgb 6.0 (decreased), Plts 75 (decreased)
 Smear with Multiple fragmented red blood cells;
 LDH 6976 (decreased), ALT 72 AST 234 (decreased)
 Transfusion medicine consulted for plasma exchange.
After 5 Days of Plasmapheresis
 Lab trends during plasmapheresis
 LDH 6976-->4805-->3400--> 2653-->1,145
 Plt: 75-->62-->48-->54-->59-->48-->47-->56
 ADAMTS13 activity: 80% (ref> = 67)*
 Plasmapheresis was discontinued
*Collected after initial FFP had been infused
8 Days After Started Therapy
 Blood counts at discharge
 Hgb 8.2 HCT 23.5 WBC 8.4 MCV 97.1 PLT 96 LDH 658
 Discharged to home
 Discharged on prednisone 60 mg daily
 Following discharge additional lab data returned
 Methylmalonic acid 39,423
Outpatient Follow-up One Month
Later
 One month later
 Tapering steroids
 Labs:
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WBC 9.9, Hgb 14.6 ,Plt 238, MCV 91.4,
Total bili 1.6, Direct bili 0.3,
AST 15, ALT 16, LDH 339,
haptoglobin <8
 Three months later
 Off steroid.
 Labs:
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WBC 4.2, Hgb 10.6, platelets 160, MCV 92.9,
Total bili 1.9, Direct bili 0.4
AST 91, ALT 44, LDH 1815
Haptoglobin <8. No schistocytes on smear.
Readmitted One Month Later
• Admission laboratories:
– WBC 2.6 Hgb 5.7 HCT 15.9 MCV 104.2 PLT 109
• Smear showed fragmented RBCs
– LDH 10,766
– Haptoglobin <8
– DAT IgG: Positive
• Additional labs ordered including:
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Vitamin B12 99
Methylmalonic acid 6038
Homocysteine (03/24/12): 44.9
Anti-intrinsic factor Ab (03/24/12): positive
Anti-cardiolipin antibodies (03/24/12): negative for IgG and IgM
Lupus anticoagulant (03/24/12): negative
B2-glycoprotein 1 antibodies (03/24/12) negative for IgA, IgG, and IgM
vWF protease activity (03/24/12): 99%
Pernicious Anemia
• 10% of the patients had life threatening hematological
manifestations:
– symptomatic pancytopenia (5%)
– "pseudo" thrombotic microangiopathy(2.5%)
– hemolytic anemia (1.5%)
Hemolysis and B12 Deficiency
• Maturation arrest of nucleated precursors results in
intramedullary hemolysis
• A positive direct Coombs test is a common finding in
untreated pernicious anemia.
Vitamin B12: Role in DNA synthesis and
Cell Division
Hemolysis and Homocysteine
• Homocysteine decreases cellular production of
glutathione peroxidase-1, an antioxidant enzyme
 leads to hemoglobin precipitates within the
RBC***
• Homocysteine can cause endothelial dysfunction
via oxidative injury to the membrane lipid and
protein components
***J Biol Chem. 2005 Apr 22;280(16):15518-25. Epub 2005 Feb 25.
Homocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasing translation.
Schistocytes
• Microangipathic Hemolytic Anemias
– Disseminated intravascular coagulopathy (DIC)
– Thrombotic thrombocytopenic pupura (TTP)
– Hemolytic uremic syndrome (HUS)
• (classic and atypical)
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MAHA, NOS (not otherwise specified)
HIV infection
Vasculitis (i.e. SLE)
Antiphospholipid Syndrome
Scleroderma crisis
Malignant hypertension
Eclampsia and HELLP
Transplantation
Medications
B12 and folate deficiency or metabolic disorders
• Valvular and Aortic disease hemolysis
Diagnostic Considerations
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DIC profile (PT/PTT/Fibrinogen/D-Dimer)
ADAMTS13 activity and inhibitor
Stool culture for STEC or PCR for Stx
ANA, LAC, APL Antibodies
Pregnancy test and LFT
HIV serologies
C3, C4, CFH, CHI, CFB, and anti-CFH Antibodies
– Genetic mutation analysis CFH, CHI, CFB, C3, MCP
• MCP expression on leukocytes
• DGKE mutations in pediatric cases
• Cobalamin metabolism
– Homocysteine, methionine, urine MMA, and MMACHC mutation analysis in all
children
– B12, homocysteine, and MMA in selected adults
Management Strategies
• Does all of this make any difference?
• General aspects of management
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Avoid platelets unless absolutely necessary
Central access (keep in mind long-term needs)
Hemodialysis support if necessary
Hypertension should be controlled
• Treat specific cause as best identified
– B12 supplementation for a cobalamin metabolism
deficiency or defect
• Plasmapheresis?
Plasmaphersis: TTP
• Plasmapheresis is absolutely beneficial
– FFP 10-20 mL/kg if not volume overloaded or HTN
– Laboratory testing timing
• Why helpful?
– Congenital deficiency of ADAMTS13
• Replaces ADAMTS13
– Acquired deficiency of ADAMTS13
• Removes inhibiting antibody
• Replaces ADAMTS13
Plasmaphersis: HUS
• Plasmapheresis may be beneficial
– CFH, CFI, CFB and C3 mutations
• Replaces the factor which is missing or mutated
– CFH antibodies
• Removes the inhibiting antibody
• Replaces CFH
• But, NOT ALWAYS
– MCP and Thrombomodulin
• Cellular membrane-bound proteins
• Plasmapheresis provides no potential benefit
– STX-mediated HUS
• Toxin internalized early thus not removed
– Drug-induced HUS
Plasmapheresis: Other MAHA
• Plasmapheresis may help antibody-mediated
diseases via immunoglobulin removal
– Antiphospholipid syndrome
– Lupus vasculitis
• Plasmapheresis will not help others
– DIC
– Cobalamin metabolism disorders
– Hypertensive crisis
– Valvular or aortic disease
Eculizumab: Anti-C5 MoAB
www.medscape.com
Renal Transplantation
• Donor considerations
– Source
– Donor risks
• GVHD prophylaxis
• Disease relapse prophylaxis
Pathophysiology of MAHA
DIC
STX HUS
Scl Renal Crisis
APLS
Cobalamin Disorder
Endothelial Injury
TTP
APLS
aHUS
APLS
Activation of
Platelets and
Coagulation
Activation of
Complement
Pathophysiology of MAHA
DIC
TTP
APLS
Cobalamin disorder
Systemic
Renal
(dominant)*
aHUS
STX HUS
*Not exclusive
Nomenclature in MAHA
• TTP = ADAMTS13 deficiency
• Classic HUS = Shiga-like toxin HUS
• Atypical HUS = Complement pathway HUS?
– Will we in the future further delineate?
• aHUS-complement
• aHUS-DGKE
• Secondary HUS
• MAHA, NOS
– 10-25% of “TTP” cases
– 30% of “HUS” cases
The Evolving Landscape of MAHA
To be continued…