The Evolving Landscape of Microangiopathic Hemolytic Anemia

J. Christian Barrett, M.D.Division of Hematology, Oncology,

and Palliative Care

• Objectives– Compare and contrast the physiologic

mechanisms of the causes of microangiopathic hemolytic anemia

– Select an appropriate management strategy for a microangiopathic hemolytic anemia

The Evolving Landscape of MAHA

• 67 year old woman with history of scleroderma and Raynaud’s syndrome – 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

• 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)– Plasmapheresis plus steroids after rheumatology consultation– ACE-inhibitor– Rituximab x 2 doses– Eculizumab.

Case #1

• Intravascular hemolytic process– Reticulocytosis– Elevated LDH– Reduced haptoglobin

Fibrin and platelet-rich thrombi occluding vessel Fragmented red blood cells a.k.a. schistocytes

Microangiopathic Hemolytic Anemia

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

• Disseminated intravascular coagulopathy (DIC)• Thrombotic thrombocytopenic purpura (TTP)• Hemolytic uremic syndrome (HUS)

The G3 of MAHA

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

Endotoxins

Bacteria

Tissue Factor

Hypoperfusion shock tissue necrosis

Direct vascular injury

X

XII

VII

II (ProthrombinThrombin)

XI

IX

VIII

V

I (FibrinogenFibrin)

Tissue Factor Initiates Coagulation

XIIIStable fibrin clot

Tissue Factor

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– 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

ultralarge vWF

Ultra-large vWF

Weilberl-Palade bodies

Alpha-granules

Smaller vWF forms

ADAMTS-13(vWF cleaving enzyme)

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• 10% with CNS manifestations• 3% with Cardiac (MI)• 5% multi-organ failure• Digital ischemic gangrene

NOTE:Need not be acute onsetAnemia 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

• 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

Classic Shiga-like Toxin HUS

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

• Diacylglycerol kinase-e– Phosphorylates arachidonic acid-containing

diacylglycerol phophatidic acid

aHUS and DGKE Mutations

aHUS and DGKE Mutations

Lemeire, M, et al. Nat Genet. 2013 May;45(5):531-6.

• Other Infections– HIV– Streptococcal pneumoniae– H1N1 influenza A

• Medications– Gemcitabine– Cyclosporin– Tacrolimus (and other calcineurin inhibitors)

Other Causes of HUS

• TTP– Thrombotic microangiopathy

dominates• Renal affected

– Coagulation activated• But not consumed

– ADAMTS13 deficient

• aHUS– Thrombotic microangiopathy

dominates• Renal dominates

– Coagulation activated• But not consumed

– Complement alternative pathway abnormalities

The G3 MAHA Revisited

➤ DICo Consumptive

coagulopathy dominates

• Bleedingo Thrombotic

microangiopathy minor part of picture

• 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

Scleroderma Renal Crisis

Patient 1 Revisited

Homozygous for CFHR1-CFHR3 deletions

• 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

Case #2

• 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

Antiphospholipid Syndrome

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

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:

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:

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:– 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.

• Microangipathic Hemolytic Anemias– Disseminated intravascular coagulopathy (DIC)– Thrombotic thrombocytopenic pupura (TTP)– Hemolytic uremic syndrome (HUS)

• (classic and atypical)– 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

Schistocytes

• 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

Diagnostic Considerations

• Does all of this make any difference?• General aspects of management

– 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?

Management Strategies

• 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: TTP

• 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

Plasmaphersis: HUS

• 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

Plasmapheresis: Other MAHA

Eculizumab: Anti-C5 MoAB

www.medscape.com

• Donor considerations– Source– Donor risks

• GVHD prophylaxis• Disease relapse prophylaxis

Renal Transplantation

Pathophysiology of MAHA

aHUSAPLS

DICSTX HUSScl Renal CrisisAPLSCobalamin Disorder

TTPAPLS

Pathophysiology of MAHA

aHUSSTX HUS

DICTTPAPLSCobalamin disorder

*Not exclusive

• 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

Nomenclature in MAHA

The Evolving Landscape of MAHA

To be continued…