L-4 Hemolytic Anemia: Thalassemia, G6PD, and more

• Lecture 4 covers clinical and pathologic aspects of hemolytic anemia.
• This lecture will cover the pathophysiology, clinical features, diagnosis of:
• Thalassemia
• Glucose-6-phosphate deficiency (G6PD)
• Paroxysmal nocturnal hemoglobinuria (PNH)
• Spherocytosis
• Pyruvate kinase deficiency

Thalassemia

• It involves a group of inherited disorders in hemoglobin synthesis.
• It is characterized by a reduced or absent one or more chains of adult hemoglobin.
• Hemoglobin is a tetramer composed of two alpha and two beta chains.
• Thalassemia results from abnormally low production of alpha or beta-globin chains which the disease is named after.
• "+" indicates diminished, but some production still occurs (e.g., B+).
• "0" indicates complete absence of production (B°).
• It is frequently found in the Mediterranean, Africa, India, and Southeast Asia.
• Its distribution parallels that of Plasmodium falciparum.

Alpha and Beta Thalassemia

• Alpha thalassemia involves reduced formation of alpha polypeptide chains.
• Beta thalassemia involves reduced formation of beta polypeptide chains.
• Alpha Thalassemia Symptom: None or small red blood cells, hemolytic anemic anemia with pallor, fatigue, jaundice, and splenomegaly
• Beta Thalassemia Symptom: None or small red blood cells.
• Beta Thalassemia Symptom: Hemolytic anemic anemia with pallor, fatigue, jaundice, splenomegaly, and gallstones.
• Alpha thalassemia is diagnosed via small red blood cells, whereas beta thalassemia is diagnosed by hemolytic anemia with high fetal HbF.
• Alpha thalassemia is caused by a genetic mutation of the Alpha gene on chromosome 16.
• Beta thalassemia is caused by a genetic mutation of the Beta gene on chromosome 11.
• Homozygous alpha thalassemia results in Bart's Hemoglobin, hydrops fetalis, and the fetus dies in utero.
• In homozygous beta thalassemia, the fetus survives in utero with HbF.

Types of Alpha Thalassemia

• Silent alpha thalassemia carriers are asymptomatic.
• Alpha thalassemia carriers (trait) exhibit mild symptoms.
• Hemoglobin H disease presents with severe symptoms, including splenomegaly and jaundice.
• Alpha thalassemia major results in fetal death in utero.
• Silent carrier has a genotype of aa/-α with 3 genes present.
• α thalassemia trait has a genotype of -α/-α or αα/-- with 2 genes present.
• Hemoglobin H disease has a genotype of -α/-- with 1 gene.
• Hb Barts / Hydrops fetalis has a genotype of --/-- with 0 genes.

Alpha Thalassemia, Hemoglobin Bart Syndrome (Hb Barts)

• The detection of Barts hemoglobin on newborn screening indicates the presence of alpha gene deletions.
• HbH thalassemia is milder, with 3 genes absent.
• Hb Barts is a severe type, with deletion of all 4 alpha globin chains.
• Signs of Hb Bart include edema and ascites in fetal tissue, cardiomegaly, and splenomegaly.
• Hb Barts has a high oxygen affinity (left shift), hindering oxygen delivery to tissues, leading to severe hypochromic, microcytic anemia, and fetal death.
• An increased risk for the mother includes severe postpartum hemorrhage.

Beta Thalassemia

• Beta Thalassemia is inherited blood disorders caused by reduced or absent Beta globin chains.
• Symptoms range from severe anemia to asymptomatic presentation.
• Found in Southern Europeans, Greeks, Italians, Africans, and Asians.
• Normal genotype is β/β. Clinical presentation is normal
• Minor: Trait or βº heterozygous or β⁺ heterozygous results in minimal anemia; no treatment indicated.
• Intermedia with double distinct mutation results in severe heterozygote. Can be a spectrum; most often do not require chronic transfusions
• Major β⁺ homozygous(double) or βº homozygous (double) indicates Cooley's Anemia or Homozygous minor point mutation that needs careful observation and intensive treatment.

Pathogenesis of Beta Thalassemia

• Reduced β-globin synthesis leads to a relative excess of α-globin.
• Excess α-globin forms insoluble aggregates within erythroblasts, leading to ineffective erythropoiesis and cell death in the bone marrow.
• Few abnormal red cells leave the bone marrow.
• Extravascular hemolysis occurs when aggregate-containing red cells are destroyed in the spleen.
• Dietary iron absorption increases due to anemia.
• Blood transfusions introduce additional iron.
• The systemic iron overload can cause secondary hemochromatosis.
• Tissue hypoxia stimulates erythropoietin production.
• This leads to marrow expansion and skeletal deformities.

Beta Thalassemia Major

• Symptoms include severe anemia, mongoloid facial features, protrusion of upper teeth, physical growth and development delay
• Peripheral blood smear shows hypochromic, microcytic erythrocytes with extreme poikilocytosis and anisocytosis (target cells, tear drop cells, basophilic stippling and many nucleated RBCs).
• MCV ranges from 50-60 fl.
• There is a low reticulocyte count.
• Most Hemoglobin is HbF.

Beta Thalassemia Minor

• Beta Thalassemia Minor is common in people of Mediterranean and Asian descent.
• Symptoms present as jaundice and splenomegaly
• Laboratory investigation will show that Hemoglobin and MCV decreased, Fe is normal, RBC count is normal, a peripheral blood smear will show microcytosis
• Hemoglobin electrophoresis reveals increased HbA2, decreased Beta chains, and an increase in HbF.

Beta Thalassemia Hemoglobin Electrophoresis

• Beta Thalassemia Hemoglobin Electrophoresis can be used to identify sickle cell trait from sickle cell anemia. It can also be used to identify Beta Thalassaemia, this process identifies:
• Normal
• ß-Thalassaemia Trait
• ß-Thalassaemia Major
• Sickle Cell Trait
• Sickle Cell Anaemia

Glucose 6 Phosphate Dehydrogenase Deficiency (G6PD)

• G6PD is a common red blood cell enzyme deficiency.
• Approximately 400 million people have G6PD deficiency.
• G6PD deficiency affects one in four people in Africa, with males being more affected than females.
• It is an X-linked recessive disorder that results from a mutation in the G6PD gene which causes red blood cell hemolysis.
• The G6PD gene protects RBCs from reactive oxygen species (ROS).
• ROS can accumulate in damaged RBCs following infections and exposure to fava beans, aspirin, and sulfonamides.

Glucose 6 Phosphate Dehydrogenase Deficiency Presentation

• Symptoms include fatigue or muscle weakness, jaundice or pallor, dyspnea, dark urine, pain (especially abdominal), and splenomegaly.
• Diagnosis involves CBC (complete blood count) and reticulocyte count.
• Assessment of G6PD enzymatic activity.
• Molecular detection of G6PD gene mutation.

Glucose 6 Phosphate Dehydrogenase Deficiency Pathogenesis

• G6PD reduces NADP+ to NADPH while oxidizing G6PD.
• Glucose 6-phosphate dehydrogenase deficiency impairs the ability of an erythrocyte to form NADPH, resulting in hemolysis.
• Oxidant stress can be caused by certain drugs, infections, and fava beans

Glucose 6 Phosphate Dehydrogenase Deficiency Peripheral Smear

• Shows Heinz Bodies and Bite Cells

Pyruvate Kinase Deficiency

• Pyruvate kinase deficiency is an inherited disorder of the enzyme pyruvate kinase.
• It affects the survival of red blood cells.
• The main cause is a mutation in the PKLR gene.
• It has a worldwide distribution with autosomal and recessive inheritance
• It is the second most common cause of enzyme-deficient hemolytic anemia, next to G6PD deficiency.
• The symptoms range from most severe to usually detected at birth.
• Severity varies from mild to severe hemolytic anemia.
• Other clinical features include tachycardia, hemochromatosis, jaundice, icterus, splenomegaly, and leg ulcers.

Pyruvate Kinase Deficiency (PKD) Pathogenesis

• Right shift of the oxygen dissociation curve of hemoglobin occurs.
• Pyruvate kinase deficiency inhibits production of ATP and pyruvate by blocking the glycolytic pathway.
• Blockage of ATP causes all processes to stop.
• Diagnosis is made with a CBC, reticulocyte count, and direct assay of pyruvate kinase levels.
• Peripheral smear shows Burr cells.

Paroxysmal Nocturnal Hemoglobinuria (PNH)

• PNH is an acquired disease of red blood cells characterized by destruction of red blood cells which causes hemolytic anemia and blood clots.
• It also causes bone marrow failure (aplastic anemia).
• It affects young adults (35-40 years of age), with PNH affecting 2 persons per million and a median survival of about 10 years after diagnosis.
• The etiology is a mutation in the PIG (polyinnositol glycan) gene on the X chromosome in the bone marrow, which makes GPI proteins; GPI protects red blood cells from destruction.
• PNH red blood cells are more vulnerable to complement-mediated lysis.
• Diagnosis is made by flow cytometry.
• Normal flow cytogram RBCs express two PIG membrane proteins CD55 and CD59.
• PNH is deficient in CD55/59

Paroxysmal Nocturnal Hemoglobinuria (PNH)

• Many years can pass before a correct diagnosis is made.
• Symptoms include fatigue, weakness, shortness of breath, flue-like symptoms, fever, ecchymosis, and thrombosis.
• Bright red blood in the urine which looks like dark tea due to hemoglobinuria.
• Blood clots occur almost exclusively in veins as opposed to arteries, especially the hepatic vein (Budd-Chiari syndrome).
• Treatment for severe cases includes a bone marrow transplant.

Hereditary Spherocytosis

• Hereditary Spherocytosis is a Red Cell Membrane Defect
• It is an autosomal dominant inherited defect in the red blood cell membrane cytoskeletal which affects spectrin, ankyrin or band 3, leading to formation of spherocytes.
• It has Mild to moderate anemia, intermittent jaundice, and splenomegaly.
• Other signs include pigment gallstones, spherocytes, and positive osmotic fragility screening tests.

Pathogenesis of Hereditary Spherocytosis

• Inherited defects in the RBC cell membrane proteins that stabilize the lipid layer include spectrin, ankyrin, and band3.
• These mutations in the membrane result in spherocytosis.
• Splenectomy corrects the anemia, but the spherocytosis persists.
• Clinical signs include splenomegaly (500-1000 gms is affected, with 200gms being normal.)
• Signs and symptoms include anemia, splenomegaly, and jaundice

Pathogenesis of Hemolytic Anemia Hereditary Spherocytosis

• Lipid bilayer and Ankyrin are affected. Additionally Alpha & Beta spectrin can be affected.
• Red blood cell becomes a Spherocyte as the splenic macrophage destroys the red blood cell's normal shape

Elliptocytosis (ovalocytosis)

• It is a hereditary, autosomal recessive blood disorder in which large numbers of red blood cells are elliptical rather than biconcave, disc-shaped.
• Its distribution is worldwide with increased incidence in areas where malaria is endemic.
• The red blood cell's membrane is defective, which causes RBCs to become very rigid.
• Defects happen in genes for the polypeptide's alpha spectrin and beta spectrin.
• Diagnosis is made by electrophoresis and osmotic fragility tests, and a peripheral blood smear indicates elliptocytes.
• Symptoms include hemolytic anemia fatigue, dyspnea, growth failure, leg ulcers gall stones, and splenomegaly.
• Majority of patients require no treatment except splenectomy and cholecystectomy.