Hemolytic Anemia

Jean M. Connors, MD

Brigham and Women’s Hospital

Dana Farber Cancer Institute

Harvard Medical School

Boston, MA

Conflicts of Interest

Pfizer/Bristol Meyer SquibbIndependent Review CommitteeScientific Ad BoardsConsultant

PortolaScientific Ad Boards

Unum TherapeuticsDSMB

• Overview of hemolytic anemia– Non-immune

• HS, HE• G6PD

– Immune• WAIHA• CAD

• Testing• Treatment

Outline

Question 2

• Anemia: How do you know it is hemolytic? – Increased reticulocyte count and absolute

retics– Peripheral smear: abnormal RBC

morphology– Decreased haptoglobin– Increased LDH– Increased indirect bilirubin– DAT: negative or positive?

• If positive what type of antibody

Approach

Question 2

• Classification– Intrinsic vs extrinsic to RBC– Non-immune– Autoimmune – Alloimmune

• Site of destruction– Intravascular vs extravascular

Approach

Question 2

• Inherited, with rare exceptions (PNH, acquired alpha thal)

• Mutations resulting in:– Membrane/cytoskeleton component defect– Hemoglobinopathy– Decreased enzymatic/metabolic function

Intrinsic RBC Etiology

Question 2

• Antibodies• Hypersplenism• Shear stress• Oxidants• Pathogens

– Malaria– Babesiosis– Clostridium perfringens

Extrinsic RBC Etiology

Question 1

• Most common inherited enzyme defect– Multiple mutations, track with ethnicity– can not reduce NADP or subsequently glutathione

• X-linked– males more significantly affected

• Most patients asymptomatic• Hemolytic crises linked to oxidative stress:

– Infection– Drugs– Fava beans: Mediterranean variant only

– Chemicals– Diabetic ketoacidosis

G6PD Deficiency

G6PD Deficiency

Prevalence of G6PD deficiency similar to malaria.

Glucose-6-Phosphate (G6PD) Deficiency

Figure 10-7 Heinz bodies in a patient withoxidant hemolysis.

Bunn & Lux, Chapter 10

Figure 10-6 Red cell metabolic pathways.

Question 1

• Diagnosis– Heinz bodies (denatured, pptd hgb), bite cells on smear– G6PD enzyme activity level

• Not accurate during acute crisis– Beutler fluorescent spot test– PCR for common variants (Mediterranean, A-)

• Treatment– Avoid offending drugs, foods, chemicals– Transfuse if necessary– Folic acid– Splenectomy often ineffective

G6PD Deficiency

Question 1

• Drugs– Antimalarials

• Primaquin, quinacrine– Sulfonamides– Dapsone– Phenacetin, asa– Nitrofurans– Mothballs– Rasburicasewww.g6pd.org/en/G6PDDeficiency/SafeUnsafe

G6PD Deficiency

Question 1• Mutations in cytoskeletal proteins

– Ankyrin 50-60% HS and 90% HE– Spectrin, band 3 in HS, protein 4.1 in HE

• Clinical severity ranges from mild to severe– Evidence of hemolytic anemia from birth– Exacerbations of hemolysis only when stressed

• infection

• Diagnosis– Evidence of extravascular hemolysis– Peripheral smear– Osmotic fragility test: hypotonic NaCl solutions– EMA: decreased eosin-5-maleimide binding by RBCs

assessed by flow cytometry

HS and HE

Osmotic Fragility

-HS and HE RBCs rupture with only mild change from normal tonicity.-10-20% will have normal osmotic fragility

Hereditary Spherocytosis (HS) and Hereditary Elliptocytosis (HE)

Figure 10-4 Hereditary elliptocytosis

Figure 10-3 Blood film – hereditary spherocytosis

An & Mohandas, British Journal of Haematology, 141, 367–375Bunn & Lux, Chapter 10

HS and HE

“vertical”

“horizontal”

The EndHS and HE

Sam Lux

Question 1

• Treatment: varies with severity– “typical” HS and HE: mild to moderate anemia,

increased bili, splenomegaly, erythroid hyperplasia– Severe and atypical forms rare, manifest from birth,

can have life threatening hemolytic crises • Splenectomy

– Normalizes RBC lifespan, anemia and increased biliusually resolve, spherocytes persist

– Treat with folate especially if hemolysis persists• Gallstones

– Pigmented stones common

HS and HE

Question 1

• Autoimmune

• Alloimmune– Transfusion– HSCT– Maternal/fetal: Rh, Kell

• Drug induced

Immune Hemolytic Anemia

Question 1

• Warm autoimmune hemolytic anemia (WAIHA)

• Cold agglutinin syndrome (CAS)

• Paroxsymal cold hemoglobinuria (PCH)– Donath Landsteiner antibodies

• Mixed AIHA

• Atypical AIHA (DAT negative)

• The first RCT of any treatment for any AIHA was published this year—for warm AIHA

Autoimmune Hemolytic Anemia

Complement Cascade

Extravascular hemolysis

Jandl JH: Blood. Textbook of Hematology,2nd ed. Boston: Little, Brown, 1996

www.medicinenet.com

Direct Antiglobulin Test (DAT)

Zantek 2012, Am J Hem 87:707

+ -

AHG: anti-human globulinpolyspecific mix

Anti-IgGAnti-C3

Direct Antiglobulin Test Interpretation

Ronald Blood 201

Question 1• Incidence: 1 to 2 cases/100,000 per year• F > M• Adults > children• IgG pan-agglutinins, react with all cells

– Ag target usually protein, often Rh but poorly defined– Extravascular hemolysis

• Primary (idiopathic)• Secondary

– CLL– Lymphomas and lymphoproliferative disorders– SLE

WAIHA

WAIHA: diagnosis

www.pathologystudent.com

DAT:IgG +C3d +/-

Hgb Retic LDH Bilirubin Haptoglobin

WAIHA

WAIHA: therapyOften extremely rapid onset, patients have significant

symptoms: weakness, SOB, tachycardiaTransfuse, despite difficulty with cross match

• 1st line therapy: steroids– Prednisone 1 mg/kg/d– Continue at least 2 weeks until hct clearly improved,

then slow taper– 80% respond within 3 weeks– Less than 20% have sustained response– “Failure” usually defined as requiring more than 15

mg/day of prednisone or equivalent to maintain Hgb>10; Hct >30

Primary WAIHA: Treatment

WAIHA: therapy

• 2nd line therapy: refractory or relapsedSplenectomy:

– 80% early CR, 60-70% CR at 2-3 yrs– May reduce steroid requirement– Complications: sepsis, thrombosis– Laparoscopic vs open splenectomy

Primary WAIHA: Treatment

WAIHA: therapy• 3rd line therapy: rituximab• Standard 4-wk dosing• 80% initial response• Poor data on long term outcome, but likely fewer long

term responses than in ITP• Preferred therapy in those w/ contraindications to

splenectomy

• Refractory: – MMF, csa, cytoxan, vincristine, azathioprine,

alemtuzumab

Primary WAIHA: Treatment

WAIHA: therapy• Use rituximab as 1st line with steroids?

• Meta-analysis of rituximab in AIHA – Overall response rate 70% for warm AIH– Complete response rate 42% for warm AIHA

• RCT of rituximab* vs placebo in newly diagnosed warm AIHA, in conjunction with prednisone.– 1 yr ORR 75% rituximab vs 31% placebo (p=0.032)

– 2 yrs 10/16 still with CR vs 3/16 placebo– More infections in placebo group

Primary WAIHA: TreatmentRituximab—2nd Line?

Autoimmun Rev 2015

* Two doses of 1000 mg 2 weeks apart Am. J. Hematol. 2017

• Treat WAHIA alone or underlying disorder or both?

• SLE– Steroids alone usually successful– Splenectomy not as effective, rituximab :PML risk?

• CLL– Fludarabine associated WAIHA

• NHL– Poor response to steroids, splenectomy– Treatment aimed at lymphoma

Secondary WAIHA: Treatment

Secondary WAIHA: Treatment

Lechner K , and Jäger U Blood 2010;116:1831-1838

Patients with CLL have increased risk of infection due to disease associated immunosuppression, age, treatment co-morbidity.

Primary CAD: clinical features

• Symptoms and findings

– Chronic anemia

– Intravascular hemolysis

– Hemoglobinuria, urine hemosiderin

– Plasma hemoglobin

– Acrocyanosis

Cold Agglutinin Syndromes

• IgM antibodies against carbohydrate antigens– I vs I

• Primary cold agglutinin disease (CAD)– Incidence: 1/1,000,000 F=M– Monoclonal B cell lymphoproliferative disorder usually

with IgMk against I antigen

• Secondary cold antibody-mediated AIHA– Mycoplasma pneumoniae: anti-I IgM– Infectious mononucleosis: anti-i IgM– Lymphoid neoplasms: anti-I or anti-i IgM– SLE

Cold Agglutinin Syndromes

CAS: diagnosis

DAT:IgG -C3d +

Hgb Retic LDH Bilirubin

www.pathologystudent.com Cold agglutinin titer

Cold Agglutinin Syndromes: Diagnosis

Etiology and treatment of primary cold agglutinin disease

Ulrich Jaeger Blood 2017;130:392-393

©2017 by American Society of Hematology

Primary CAD: therapy• Cold avoidance

• Steroids and splenectomy generally not effective

• Plasmapheresis– Acute management

• Chemo and immunotherapies: Goal is often PR, transfusion independence, and not CR– Rituximab ORR 57%, CR 21% Autoimmun Rev 2015

Refractory to rituximab alone– Fludarabine plus rituximab—old– Rituximab plus bendamustine--new

Primary CAD: Therapy

Question 1

• Prospective multicenter study in 45 patients – 18 previously treated with R, R+flu, other

• 4 cycles– Rituximab 375 mg/m2 day 1– Bendamustine 90 mg/m2 day1,2

• Excellent response: overall 71% • 3 relapses in 32 responders with 32 months follow up

—Q 2Rituximab and Bendamustine in CAD

Berentsen, Blood July 2017

Question 1—Q 2Complement inhibition in CAD

Question 1

• Eculizumab– Case reports – Phase II study in 13 patients: decreased hemolysis

and transfusion requirements ASH 2015

• TNT009– Inhibits C1s in the classical complement pathway– In vitro, mouse, and healthy volunteer studies

demonstrate inhibition of activation of classical complement pathway and formation of C3b

• Both require indefinite treatment or use as bridge until response from other treatments achieved

—Q 2Complement inhibition in CAD

DAT-negative WAIHA

DAT IgG molecules per RBC0 <25-120

½+ 1201+ 2002+ 300-500

3-4+ >500

Petz LD. Immune Hemolytic Anemias 2nd Ed. Philadelphia: Churchill Livingstone, 2004

DAT Negative WAHIA

Hemolytic Anemia: Unanswered questions

1-10% WAIHA cases will have negative Coombs.Reasons for a negative Coombs Test:

• Low titer antibody that is insufficient to agglutinate the cells in a Coombs Test

• Low affinity antibody that is removed with washing during the test

• Alternative isotype antibody that doesn’t react with Coombs sera –IgA: 1-2%

• Patient doesn’t have AIHA

DAT Negative WAHIA

• Enhanced DAT– Many enhanced antibody detection techniques:

• Flow cytometry, low ionic strength wash, polybrene– Detect low concentration of antibody– Detect alternative isotypes – Positive in 90% Coombs negative WAIHA– Negative test does not absolutely rule out WAIHA

– Must also consider alternative diagnosis

DAT Negative WAIHA

Autoimmune Hemolytic Anemia

• Vaccinate prior to splenectomy• Pneumococcus• Haemophilus influenza• Meningococcus• Influenza• Provide prophylactic antibiotics

• Open vs laproscopic?• Portal vein thrombosis rate estimated at 8-50%• No definitive answer for open vs laproscopic• VTE prophylaxis post-op?

• Increased risk over lifetime of thrombosis?• Increased PE, ischemic heart disease

Splenectomy

Jean M. Connors MDjconnors@bwh.harvard.edu