Hemolytic Anemia2007

OVERVIEW HEMOLYTIC ANEMIA Normal RBC life span is 110-120 days LIU YANFANG MD Ph D Professor Division of Hematology Department of Medicine

OVERVIEW

Definition • Hemolysis – Accelerated rate of red cell destruction (<120 days)

With age the red blood cell has :

• May be well compensated by increased red cell production by the bone marrow

– Decreased membrane lipid component – Spherocytic shape – Less pliability and deformability

– Elevated reticulcoyte count

• Hemolytic Anemia These changes lead to the aged RBC being trapped and removed by the RE system

– Accelerated rate of red cell destruction beyond the ability of the bone marrow to fully compensate

CLINICAL PRESENTATION: More commonly associated with hemolytic anemia

CLINICAL PRESENTATION Patients may present with a variety of symptoms due to anemia (not necessarily hemolytic anemia) – Related to tissue hypoxia

•

Jaundice

• Scleral icterus • Gallstones – bilirubinate

• Fatigue, weakness, lightheadedness, CP, SOB

– Related to cardiovascular compensation • Tachycardia, palpitations

– Related to underlying etiology

• Splenomegaly – LUQ pain, early satiety • Leg ulcers • Acute crisis – ex. Aplastic crisis

LABORATORY EVALUATION: Suspected Hemolytic Anemia Initial • CBCP with MCV • Reticuloctye count (retic. count) • Review peripheral blood smear – Nucleated red cells, reticulocytosis

• Bilirubin-direct and indirect • Lactate Dehydrogenase (LDH) • Consider haptoglobin, urinalysis

LABORATORY EVALUATION Additional / Directed studies • Coombs’ test • Underlying disorders • Osmotic fragility test • Hgb electrophoresis • G-6PD assays • Enzyme assays • Bone marrow aspirate / biopsy – Reversed M:E ratio

Increased RBC Destruction Š ↑ BM Erythroid Production Š Peripheral Blood RBCs - Anemia - ↑ Reticulocytes - Poikilocytosis (Abnormal Shape) Š Body Retains RBC Products - Iron (Hemochromatosis) - Bilirubin (Jaundice, Gallstones)

EXTRAVASCULAR HEMOLYSIS

Site of Hemolysis • 90% of cases

Extravascular vs. Intravascular

• Inappropriate removal of RBC’s by the RE system • Exaggeration of the normal removal of RBC’s

Extravascular Hemolysis (Spleen)

Extravascular Pathway for RBC Destruction (Liver, Bone marrow, & Spleen) Phagocytosis & Lysis

Hemoglobin

Globin

Heme

Amino acids

Fe2+

Amino acid pool

INTRAVASCULAR HEMOLYSIS

Bilirubin

Excreted

INTRAVASCULAR HEMOLYSIS

• 10% of all cases • Red cells are disrupted in the general circulation • Free hemoglobin is released into the plasma

INTRAVASCULAR HEMOLYSIS By products of intravascular hemolysis include: Hemoglobinemia Hemoglobinuria Hemosiderinuria Low haptoglobin

Hemolytic Anemias Intrinsic RBC Abnormalities: Hereditary Š Membrane Proteins - Spherocytosis Š Enzymes - G6PD Deficiency Š Hemoglobin Synthesis Sickle Cell Disorders, Thalassemia Acquired Š Membrane Defect - PNH

Hemolytic Anemias

Extrinsic (Extracorpuscular) Abnormalities Acquired: Š Antibody Mediated Š Mechanical Trauma to RBC’s Š Infections (Malaria)

Hereditary Spherocytosis 1. Autosomal dominant inheritance; incidence 1/5000. 2. Defective "band 4.1" protein causing decreased spectrin in red cell membrane. 3. Clinical manifestations: chronic anemia, splenomegaly, gallstones, aplastic crisis. 4. Lab tests: Spherocytes on blood smear, increased osmotic fragility. 5. Treatment: Splenectomy is essentially curative.

Hereditary Spherocytosis

Hereditary Spherocytosis Mutation of Ankyrin Gene (Most Common Defect)

Shear forces in circulation

Abnormal Ankyrin Protein

↓ membrane stability

Deficiency of Spectrin Assembly

Chronic hemolytic anemia

membrane loss MΦ

splenomegaly

spherocyte

Hereditary Spherocytosis Laboratory Findings Š PBS - Moderate Anemia - Spherocytes - Reticulocytes Š BM - Erythroid Hyperplasia Š Coomb’s Test - Negative Š Osmotic Fragility Test - Sensitivity to Lysis in Hypotonic Solution Family History

Spherocytes

Enzyme Deficiency Glucose-6-Phosphate Dehydrogenase: Š Deficiency (>400 Variants) Š X Linked, Males > Females - African Americans (Males 10%) - Mediterranean Groups (↑ Severity) Š No Symptoms Unless Oxidative Stress - Therapeutic Drugs, Fava Beans Š Denatured Hemoglobin Precipitates → RBCs Removed by Spleen

Hemoglobin Precipitates (Special Stain)

G6PD Deficiency

*GSH

O-

glutathione +

hydrogen peroxide Hemoglobin precipitates (Heinz bodies)

- plucked out by spleen - bite cells (RBCs phagocytized in spleen)

Bite Cell - G6PD Deficiency

Hemoglobin Synthesis Adult hemoglobin - 96% HgA (a2B2) a2

B2

a2

B2

Clinically significant variant hemoglobins - usually B abnormalities

Hemoglobin S Disorders Hemoglobinopathies Abnormal Hgb Structure (Qualitative) zHereditary Disorders z>300 Types Abnormal Hemoglobin zHemoglobin S Disorders - Most Prevalent - Sickle Cell Disease - Homozygous (SS) - Sickle Cell Trait - Heterozygous 40% Hgb S, 60% Hgb A (Anemia Rare)

Š Point Mutation of B Globin Gene GLU

HgA

6th HgS

VAL

Š Hgb S Gene - 8% in American Blacks - 30% in Some African Populations - Confers Resistance to plasmodium falciparum Malaria

Sickle Cells

Sickle Cell Disease Sickle cell Deoxyg enation (or ↓H 0 2 , ↓pH)

O2

↑ Ca ↓ K, O2

Polymerization of Hgb S

Sickle Cell Disease Š Irreversibly Sickled Cells are Hemolyzed in the Spleen (Children - Splenomegaly) Š Microvascular Occlusion - Tissue Infarcts and Pain - Autosplenectomy (Adults) Š ↑ Infections - Salmonella Osteomyelitis Š Aplastic Crisis - Usually Parvovirus

Sickledex Screen Sickle Cell Disorders

Normal RBCs

Sickled RBCs

Diagnosis Š Hemoglobin Electrophoresis - Identify Hgb S Š Sickledex - In Vitro Sickling After Adding Reducing Agent Š DNA Analysis - Prenatal Testing

Thalassemia

Hemoglobin Electrophoresis

Š Absent or È Synthesis of Globin Chains (Quantitative) Š Most Frequent in Mediterranean, African, or Asian Populations Š β - Thalassemia - È β Chain Synthesis (Gene Mutations) Š α - Thalassemia - È α Chain Synthesis (1-3 of 4 Genes Deleted) (SE Asians)

* Sickle Cell Trait Heterozygous

β - Thalassemia Minor β β

βo β

È β chain synthesis

β - Thalassemia Major β β

βo βo

No β Chain Synthesis

β+ β+

È β Chain Synthesis

Chromosome 11 β β

β+ β

Mild or no anemia

È β chain synthesis

β β

Severe Anemia

β - Thalassemia Major Š Peripheral Blood Smear - Severe Hypochromic Microcytic Anemia (↓ Hgb A → ↓ MCHC) Š Bone Marrow - Erythroid Hyperplasia - Skeletal Deformities Š Extramedullary Hematopoiesis - Splenomegaly and Hepatomegaly

α - Thalassemia

β - Thalassemia Major Complications: Š ↑ α Chains - Hemolytic Anemia, Ineffective Erythropoiesis Š Growth Retardation Š Systemic Iron Overload Chronic Blood Transfusions ⇓ Cirrhosis, Cardiomyopathy (Cardiac Failure), Death (2nd-3rd Decade)

Paroxysmal Nocturnal Hemoglobinuria z Acquired Disorder z 25% Paroxysmal and Nocturnal z Mutation of Stem Cells - No Anchor Protein (Chronic Hemolysis)

C

C

C

ComplementInduced Lysis (Intravascular - Hgb in Urine)

Chromosome 16

α α α

α α

α α α - thalassemia trait (+/- anemia)

Silent carrier HbH disease

α

(severe anemia)

Hb Bart Hydrops fetalis (lethal in utero)

Hemolytic Anemias Extrinsic Abnormalities - Acquired: Š Antibody Mediated (Spherocytes) Š Mechanical Trauma (Schistocytes) - Heart Valves, Microthrombin Fibrin Strands in Vessels (DIC, TTP, HUS) Š Infections - Malaria - Organisms Destroy RBCs

Immune Hemolytic Anemias Antibody Mediated: Š RBC Destruction Caused By Antibody to RBC Surface Antigen Š Phagocytosis in Spleen Š More Common with Aging Š 2 Types - Warm and Cold Autoimmune Hemolytic Anemias

Immune Hemolytic Anemias Warm Antibody Type (IgG, 37o C): Š IgG Reacts with RBC Surface Antigens Š Primary (Iidiopathic, 60%) Š Secondary: Leukemia, Lymphoma, SLE, Drugs Š Spherocytes - Spleen Removes Membrane Protein from Ab Coated RBCs Š Positive Direct Coomb’s Test

Spherocytes

Autoimmune Hemolytic Anemia 1. Autoantibodies to own red cells. Associated with underlying infection or tumor. 2. Two forms exist: Warm AIHA: IgG antibodies cause acute hemolysis. Associated with lupus, leukemia, or lymphoma. Cold AIHA: IgM antibodies cause insidious hemolysis. Associated with mycoplasma, viruses, or lymphoma. 3. Lab tests: Direct antiglobulin test, followed by antibody identification. 4. Treatment: Steroids, treat underlying disease.

Direct Coombs Test Coombs Test Detects Antibodies Human Globulin

Agglutination Ab

Ab

Antibodies to Human Globulins

+

Ab

Ab

Patient RBC

Immune Hemolytic Anemias Cold Antibody Type (IgM, <30o C) Š Usually Not Clinically Significant Š Acute - mycoplasma pneumoniae - Infectious Mononucleosis (Mild Transient Anemia) Š Chronic - Idiopathic, Lymphoma

Mechanical Trauma - Schistocytes

Malaria Š Most Common Acquired Hemolytic Anemia Worldwide Š Tropical Distribution with Variety of Species Š Parasites Destroy RBCs Š Cyclical Hemolysis Produces Fever and Chills Š Splenomegaly - ↑ Mononuclear Cells

Malaria in RBCs

NORMAL BILIRUBIN METABOLISM

• Uptake of bilirubin by the liver is mediated by a carrier protein (receptor) • Uptake may be competitively inhibited by other organic anions • On the smooth ER, bilirubin is conjugated with glucoronic acid, xylose, or ribose • Glucoronic acid is the major conjugate - catalyzed by UDP glucuronyl tranferase •“Conjugated” bilirubin is water soluble and is secreted by the hepatocytes into the biliary canaliculi • Converted to stercobilinogen (urobilinogen) (colorless) by bacteria in the gut • Oxidized to stercobilin which is colored • Excreted in feces • Some stercobilin may be re-adsorbed by the gut and re-excreted by either the liver or kidney

Prehepatic (hemolytic) jaundice • Results from excess production of bilirubin (beyond the livers ability to conjugate it) following hemolysis • Excess RBC lysis is commonly the result of autoimmune disease; hemolytic disease of the newborn (Rh- or ABO- incompatibility); structurally abnormal RBCs (Sickle cell disease); or breakdown of extravasated blood • High plasma concentrations of unconjugated bilirubin (normal concentration ~0.5 mg/dL)

Intrahepatic jaundice • Impaired uptake, conjugation, or secretion of bilirubin • Reflects a generalized liver (hepatocyte) dysfunction • In this case, hyperbilirubinemia is usually accompanied by other abnormalities in biochemical markers of liver function

Posthepatic jaundice • Caused by an obstruction of the biliary tree • Plasma bilirubin is conjugated, and other biliary metabolites, such as bile acids accumulate in the plasma • Characterized by pale colored stools (absence of fecal bilirubin or urobilin), and dark urine (increased conjugated bilirubin) • In a complete obstruction, urobilin is absent from the urine