Hemolytic anemia Rakesh Biswas MD, Professor, Department of Medicine, People's College of Medical Sciences, Bhanpur, Bhopal, India

Young man of 19 Complains of giddiness weakness, pallor Examination reveals a spleen mild lemon yellow sclera

How shall you investigate to find out the cause of the problem?

Laboratory investigations: Severe normochromic, normocytic anemia (hemoglobin level of 6.4 g/dL Reticulocyte count of 12.2%. Blood film:

Bilirubin level of 2.5 mg/dL, Lactate dehydrogenase (LDH) of 2140 IU/L, Haptoglobin below 7 mg/dL

Introduction Mean life span of a RBC-120days Mean life span of a RBC-120days Removed Extravascularly by- Macrophages of RE system Removed Extravascularly by- Macrophages of RE system

Hemolytic Anemia Definition: Definition: Those anemias which result from an increase in RBC destruction Those anemias which result from an increase in RBC destruction Classification: Classification: Congenital / Hereditary Congenital / Hereditary Acquired Acquired

Laboratory Evaluation of Hemolysis ExtravascularIntravascular HEMATOLOGIC Routine blood film Reticulocyte count Bone marrow examination Polychromatophilia Erythroid hyperplasia Polychromatophilia Erythroid hyperplasia PLASMA OR SERUM Bilirubin Haptoglobin Plasma hemoglobin Lactate dehydrogenase Unconjugated, Absent N/ (Variable) Unconjugated Absent (Variable) URINE Bilirubin Hemosiderin Hemoglobin  severe cases

Hemoglobinuria

Classification of Hemolytic Anemias Hereditary1. Abnormalities of RBC interior a.Enzyme defects: G-6-PD def,PK def b.Hemoglobinopathies 2. RBC membrane abnormalities a. Hereditary spherocytosis etc. b. PNH Acquiredc. Spur cell anemia 3. Extrinsic factors a. Hypersplenism b. Antibody: immune hemolysis c. Mechanical trauma: MAHA d. Infections, toxins, etc Ref : Harrison’s

Features of HEMOLYSIS BilirubinLDH Reticulocytes, n-RBC Haptoglobulins +ve Urinary hemosiderin, Urobilinogen Blood Film Spherocytes No spherocytes Fragmentation DCT +ve DCT –ve AI Hemolysis H. Sherocytosis Malaria, Clostidium Clostidium Hereditery enzymopathies Microangiopathic, Traumatic Hereditery enzymopathies Microangiopathic, Traumatic

Red Cell Membrane Defects 1.Hereditary Spherocytosis Usually inherited as AD disorder Usually inherited as AD disorder Defect: Deficiency of Beta Spectrin or Ankyrin  Loss of membrane in Spleen & RES  becomes more spherical  Destruction in Spleen Defect: Deficiency of Beta Spectrin or Ankyrin  Loss of membrane in Spleen & RES  becomes more spherical  Destruction in Spleen

RBC Membrane

C/F: C/F: Asymptomatic Asymptomatic Fluctuating hemolysis Fluctuating hemolysis Splenomegaly Splenomegaly Pigmented gall stones- 50% Pigmented gall stones- 50%

Complications Clinical course may be complicated with Crisis: Clinical course may be complicated with Crisis: : associated with infection Hemolytic Crisis: associated with infection : associated with Parvovirus infection Aplastic crisis: associated with Parvovirus infection

Inv: Inv: Test will confirm Hemolysis Test will confirm Hemolysis P Smear: Spherocytes P Smear: Spherocytes Osmotic Fragility: Increased Osmotic Fragility: Increased Screen Family members

Osmotic Fragility

Management: Management: Folic Acid 5mg weekly, prophylaxis life long Folic Acid 5mg weekly, prophylaxis life long Spleenectomy Spleenectomy Blood transfusion in Ac, severe hemolytic crisis Blood transfusion in Ac, severe hemolytic crisis

2.Hereditary Elliptocytosis Equatorial Africa, SE Asia Equatorial Africa, SE Asia AD / AR AD / AR Functional abnormality in one or more anchor proteins in RBC membrane- Alpha spectrin, Protein 4.1 Functional abnormality in one or more anchor proteins in RBC membrane- Alpha spectrin, Protein 4.1 Usually asymptomatic Usually asymptomatic Mx: Similar to H. spherocytosis Mx: Similar to H. spherocytosis Variant: Variant: 3.SE-Asian ovalocytosis: Common in Malaysia, Indonesia… Common in Malaysia, Indonesia… Asymptomatic-usually Asymptomatic-usually Cells oval, rigid,resist invasion by malarial parasites Cells oval, rigid,resist invasion by malarial parasites

Elliptocytosis

Red Cell Enzymopathies Physiology: Physiology: EM pathway: ATP production EM pathway: ATP production HMP shunt pathway: NADPH & Glutathione production HMP shunt pathway: NADPH & Glutathione production

1. Glucose-6-Phosphate Dehydrogenase ( G6PD ) Deficiency Pivotal enzyme in HMP Shunt & produces NADPH to protect RBC against oxidative stress Pivotal enzyme in HMP Shunt & produces NADPH to protect RBC against oxidative stress Most common enzymopathy -10% world’s population Most common enzymopathy -10% world’s population Protection against Malaria Protection against Malaria X-linked X-linked

(Oxidised form) (Reduced form)

Clinical Features: Clinical Features: Acute drug induced hemolysis: Acute drug induced hemolysis: Aspirin, primaquine, quinine, chloroquine, dapsone…. Aspirin, primaquine, quinine, chloroquine, dapsone…. Chronic compensated hemolysis Chronic compensated hemolysis Infection/acute illness Infection/acute illness Neonatal jaundice Neonatal jaundice Favism Favism

Inv: Inv: e/o non-spherocytic intravascular hemolyis e/o non-spherocytic intravascular hemolyis P. Smear: Bite cells, blister cells, irregular small cells, Heinz bodies, polychromasia P. Smear: Bite cells, blister cells, irregular small cells, Heinz bodies, polychromasia G-6-PD level G-6-PD level Treatment: Treatment: Stop the precipitating drug or treat the infection Stop the precipitating drug or treat the infection Acute transfusions if required Acute transfusions if required

2. Pyruvate Kinase Deficiency AR AR Deficient ATP production, Chronic hemolytic anemia Deficient ATP production, Chronic hemolytic anemia Inv; Inv; P. Smear: Prickle cells P. Smear: Prickle cells Decreased enzyme activity Decreased enzyme activity Treatment: Treatment: Transfusion may be required Transfusion may be required

Hemolobinopathies…

Autoimmune Hemolytic Anemia Result from RBC destruction due to RBC autoantibodies: Ig G, M, E, A Result from RBC destruction due to RBC autoantibodies: Ig G, M, E, A Most commonly-idiopathic Most commonly-idiopathic Classification Classification Warm AI hemolysis:Ab binds at 37degree Celsius Warm AI hemolysis:Ab binds at 37degree Celsius Cold AI Hemolysis: Ab binds at 4 degree Celsius Cold AI Hemolysis: Ab binds at 4 degree Celsius

1.Warm AI Hemolysis: Can occurs at all age groups Can occurs at all age groups F > M F > M Causes: Causes: 50% Idiopathic 50% Idiopathic Rest - secondary causes: Rest - secondary causes: 1.Lymphoid neoplasm: CLL, Lymphoma, Myeloma 2.Solid Tumors: Lung, Colon, Kidney, Ovary, Thymoma 3.CTD: SLE,RA 4.Drugs: Alpha methyl DOPA, Penicillin, Quinine, Chloroquine 5.Misc: UC, HIV

IMMUNOHEMOLYTIC ANEMIA MACROCYTE SPHEROCYTE

Direct antiglobulin test demonstrating the presence of autoantibodies (shown here) or complement on the surface of the red blood cell. complement

Inv: Inv: e/o hemolysis, MCV e/o hemolysis, MCV P Smear: Microspherocytosis, n-RBC P Smear: Microspherocytosis, n-RBC Confirmation: Coomb’s Test / Antiglobulin test Confirmation: Coomb’s Test / Antiglobulin test Treatment Treatment Correct the underlying cause Correct the underlying cause Prednisolone 1mg/kg po until Hb reaches 10mg/dl then taper slowly and stop Prednisolone 1mg/kg po until Hb reaches 10mg/dl then taper slowly and stop Transfusion: for life threatening problems Transfusion: for life threatening problems If no response to steroids  Spleenectomy or, If no response to steroids  Spleenectomy or, Immunosuppressive: Azathioprine, Cyclophosphamide Immunosuppressive: Azathioprine, Cyclophosphamide

2. Cold AI Hemolysis Usually Ig M Usually Ig M Acute or Chronic form Acute or Chronic form Chronic: Chronic: C/F: C/F: Elderly patients Elderly patients Cold, painful & often blue fingers, toes, ears, or nose ( Acrocyanosis) Cold, painful & often blue fingers, toes, ears, or nose ( Acrocyanosis) Inv: Inv: e/o hemolysis e/o hemolysis P Smear: Microspherocytosis P Smear: Microspherocytosis Ig M with specificity to I or I Ag Ig M with specificity to I or I Ag

Other causes of Cold Agglutination: Other causes of Cold Agglutination: Infection: Mycoplasma pneumonia, Infec Mononucleosis Infection: Mycoplasma pneumonia, Infec Mononucleosis PCH : Rare cause seen in children in association with cong syphilis PCH : Rare cause seen in children in association with cong syphilis

Treatment: Treatment: Treatment of the underlying cause Treatment of the underlying cause Keep extremities warm Keep extremities warm Steroids treatment Steroids treatment Blood transfusion Blood transfusion

Non-Immune Acquired Hemolytic Anemia 1. Mechanical Trauma A). Mechanical heart valves, Arterial grafts: cause shear stress damage B).March hemoglobinuria: Red cell damage in capillaries of feet C). Thermal injury: burns D). Microangiopathic hemolytic anemia (MAHA): by passage of RBC through fibrin strands deposited in small vessels  disruption of RBC eg: DIC,PIH, Malignant HTN,TTP,HUS

TRAUMATIC HEMOLYSIS

Acquired hemolysis 2.Infection F. malaria: intravascular hemolysis: severe called F. malaria: intravascular hemolysis: severe called ‘Blackwater fever’ Cl. perfringens septicemia oxidant denaturation of hemoglobin 3.Chemical/Drugs: oxidant denaturation of hemoglobin Eg: Dapsone, sulphasalazine, Arsenic gas, Cu, Nitrates & Nitrobenzene

The direct antiglobulin test was positive for complement (C3d) (++), and IgG (++-). Also was positive for agglutinins of IgM type and had a titer of 1:1024.

Serologies for human immunodeficiency virus, hepatitis B and C viruses, and Mycoplasma pneumoniae were negative. Rheumatoid factor and antinuclear antibodies were undetectable.

Prednisone therapy was started at a dose of 1 mg/kg intravenously, daily. Hemoglobin level rose to 11 g/dL, concomitantly with the improvement of hemolytic signs.

A reduction of positivity of both direct and indirect antiglobulin tests (polyvalent serum + ; C3d + ; IgG+ ), as well as a reduction of cold agglutinin titers (1:128), was observed 8 weeks after corticosteroid therapy.

Three months later, corticosteroids were tapered to a maintenance dose of 25 mg daily. Hemolysis recurred again with the fall of hemoglobin to 7 g/dL.

The direct antiglobulin test recurred positive for polyvalent serum (+++), complement (+++), and IgG (+++), while cold agglutinin titers again became strongly positive (1:256).

Immunophenotyping of bone marrow cells showed that 10% of all the cells were CD20 and CD19 positive.

CD20 is widely expressed on B- cells. CD20 could play a role in Ca2+ influx across plasma membranes, maintaining intracellular Ca2+ concentration and allowing activation of B cells.

Rituximab is a monoclonal antibody that binds to CD 20 Rituximab was started at the dose of 375 mg/mq once weekly, for a total of 4 doses

Hemoglobin value reached 13.5 g/dL just before the third dose, although biochemical signs of hemolysis remained substantially unaltered.

At the end of therapy, the hemolytic signs disappeared, the direct and indirect antiglobulin tests became negative, and cold agglutinin titers fell to 1:32 Immunophenotyping of bone marrow cells showed the absence of CD20 and CD19 B cells.

Summary of lecture Learning points