Red Blood Cell Disorders: Anemia

Sickle Cell Disease

• HbF protects against sickling, and high levels of HbF at birth are protective for the first few months of life
• Treatment with hydroxyurea increases levels of HbF

Complications of Sickle Cell Disease

• Cells continuously sickle and de-sickle while passing through the microcirculation, resulting in complications related to RBC membrane damage
• Extravascular hemolysis:
  • RBCs with damaged membranes are removed by the reticuloendothelial system, leading to anemia, jaundice, and increased risk for bilirubin gallstones
  • Massive erythroid hyperplasia ensues, resulting in:
    • Expansion of hematopoiesis into the skull and facial bones
    • Extramedullary hematopoiesis with hepatomegaly
    • Risk of aplastic crisis with parvovirus B19 infection of erythroid precursors
• Intravascular hemolysis:
  • RBCs with damaged membranes dehydrate, leading to hemolysis with decreased haptoglobin and target cells on blood smear
• Vaso-occlusion:
  • Dactylitis: swollen hands and feet due to vaso-occlusive infarcts in bones
  • Autosplenectomy: shrunken, fibrotic spleen
  • Acute chest syndrome: vaso-occlusion in pulmonary microcirculation
  • Pain crisis
  • Renal papillary necrosis: results in gross hematuria and proteinuria

Sickle Cell Trait

• Presence of one mutated and one normal chain, resulting in <50% HbS in RBCs
• Generally asymptomatic with no anemia; RBCs with <50% HbS do not sickle in vivo except in the renal medulla
• Extreme hypoxia and hypertonicity of the medulla cause sickling, leading to microinfarctions and resulting in microscopic hematuria and decreased ability to concentrate urine

Laboratory Findings

• Sickle cells and target cells are seen on blood smear in sickle cell disease, but not in sickle cell trait
• Metabisulfite screen causes cells with any amount of HbS to sickle; positive in both disease and trait
• Hb electrophoresis confirms the presence and amount of HbS
• Disease: 90% HbS, 8% HbF, 2% HbA2 (no HbA)
• Trait: 55% HbA, 43% HbS, 2% HbA2

Hemoglobin C

• Autosomal recessive mutation in the β chain of hemoglobin
• Normal glutamic acid is replaced by lysine
• Less common than sickle cell disease
• Presents with mild anemia due to extravascular hemolysis
• Characteristic HbC crystals are seen in RBCs on blood smear

Paroxysmal Nocturnal Hemoglobinuria (PNH)

• Acquired defect in myeloid stem cells resulting in absent glycosylphosphatidylinositol (GPI)
• Renders cells susceptible to destruction by complement
• Blood cells coexist with complement
• Decay accelerating factor (DAF) on the surface of blood cells protects against complement-mediated damage
• Absence of GPI leads to absence of DAF, rendering cells susceptible to complement-mediated damage
• Intravascular hemolysis occurs episodically, often at night during sleep
• Mild respiratory acidosis develops with shallow breathing during sleep and activates complement
• RBCs, WBCs, and platelets are lysed
• Intravascular hemolysis leads to hemoglobinemia and hemoglobinuria
• Sucrose test is used to screen for disease; confirmatory test is the acidified serum test or flow cytometry to detect lack of CD55 (DAF) on blood cells
• Main cause of death is thrombosis of the hepatic, portal, or cerebral veins

Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency

• X-linked recessive disorder resulting in reduced half-life of G6PD
• Renders cells susceptible to oxidative stress
• RBCs are normally exposed to oxidative stress, particularly H2O2
• Glutathione (an antioxidant) neutralizes H2O2, but becomes oxidized in the process
• NADPH, a by-product of G6PD, is needed to regenerate reduced glutathione
• ↓G6PD ↓ → NADPH ↓ → reduced glutathione → oxidative injury by H2O2 → intravascular hemolysis
• Two major variants: African and Mediterranean
• African variant: mildly reduced half-life of G6PD leading to mild intravascular hemolysis with oxidative stress
• Mediterranean variant: markedly reduced half-life of G6PD leading to marked intravascular hemolysis with oxidative stress
• High carrier frequency in both populations is likely due to protective role against falciparum malaria
• Oxidative stress precipitates Hb as Heinz bodies
• Causes of oxidative stress include infections, drugs, and fava beans
• Heinz bodies are removed from RBCs by splenic macrophages, resulting in bite cells
• Leads to predominantly intravascular hemolysis
• Presents with hemoglobinuria and back pain hours after exposure to oxidative stress
• Heinz preparation is used to screen for disease; enzyme studies confirm deficiency

Immune Hemolytic Anemia (IHA)

• Antibody-mediated (IgG or IgM) destruction of RBCs
• IgG-mediated disease usually involves extravascular hemolysis
• IgG binds RBCs in the relatively warm temperature of the central body
• Membrane of antibody-coated RBC is consumed by splenic macrophages, resulting in spherocytes
• Associated with SLE, CLL, and certain drugs
• IgM-mediated disease can lead to intravascular hemolysis
• IgM binds RBCs and fixes complement in the relatively cold temperature of the extremities
• RBCs inactivate complement, but residual C3b serves as an opsonin for splenic macrophages resulting in spherocytes
• Associated with Mycoplasma pneumoniae and infectious mononucleosis
• Coombs test is used to diagnose IHA; testing can be direct or indirect### Anemia
• Anemia is a condition characterized by a reduction in circulating red blood cell (RBC) mass, leading to signs and symptoms of hypoxia.
• Symptoms include:
  • Weakness, fatigue, and dyspnea
  • Pale conjunctiva and skin
  • Headache and light-headedness
  • Angina, especially with pre-existing coronary artery disease

Classification of Anemia

• Anemia can be classified based on mean corpuscular volume (MCV) into:
  • Microcytic anemia (MCV < 80 µm³)
  • Normocytic anemia (MCV = 80-100 µm³)
  • Macrocytic anemia (MCV > 100 µm³)

Microcytic Anemia

• Microcytic anemia is caused by decreased production of hemoglobin.
• RBC progenitor cells in the bone marrow are large and normally divide multiple times to produce smaller mature cells.
• Microcytosis occurs due to an "extra" division, which maintains hemoglobin concentration.
• Types of microcytic anemia include:
  • Iron deficiency anemia
  • Anemia of chronic disease
  • Sideroblastic anemia
  • Thalassemia

Iron Deficiency Anemia

• Iron deficiency anemia is caused by decreased levels of iron.
• Iron deficiency is the most common nutritional deficiency in the world, affecting approximately 1/3 of the population.
• Iron is consumed in heme and non-heme forms.
• Absorption of iron occurs in the duodenum, and it is transported in the blood by transferrin.
• Stored intracellular iron is bound to ferritin, which prevents iron from forming free radicals.
• Laboratory measurements of iron status include:
  • Serum iron
  • Total iron-binding capacity (TIBC)
  • % saturation
  • Serum ferritin
• Iron deficiency is usually caused by:
  • Dietary lack
  • Blood loss
  • Infants: breast-feeding (human milk is low in iron)
  • Children: poor diet
  • Adults: peptic ulcer disease, menorrhagia, or pregnancy
  • Elderly: colon polyps/carcinoma, or hookworm
• Stages of iron deficiency:
  1. Storage iron is depleted (↓ ferritin; ↑ TIBC)
  2. Serum iron is depleted (↓ serum iron; ↓ % saturation)
  3. Normocytic anemia (bone marrow makes fewer, but normal-sized, RBCs)
  4. Microcytic, hypochromic anemia (bone marrow makes smaller and fewer RBCs)
• Clinical features of iron deficiency include:
  • Anemia
  • Koilonychia
  • Pica
• Laboratory findings include:
  • Microcytic, hypochromic RBCs with ↑ red cell distribution width (RDW)
  • ↓ ferritin; ↑ TIBC; ↓ serum iron; ↓ % saturation
  • ↑ Free erythrocyte protoporphyrin (FEP)
• Treatment involves supplemental iron (ferrous sulfate).

Anemia of Chronic Disease

• Anemia of chronic disease is associated with chronic inflammation or cancer.
• Chronic disease results in production of acute phase reactants from the liver, including hepcidin.
• Hepcidin sequesters iron in storage sites, limiting iron transfer to erythroid precursors and suppressing erythropoietin (EPO) production.
• Laboratory findings include:
  • ↑ ferritin; ↓ TIBC; ↓ serum iron; ↓ % saturation
  • ↑ Free erythrocyte protoporphyrin (FEP)
• Treatment involves addressing the underlying cause.

Sideroblastic Anemia

• Sideroblastic anemia is caused by defective protoporphyrin synthesis.
• Protoporphyrin is synthesized via a series of reactions involving aminolevulinic acid synthetase (ALAS), aminolevulinic acid dehydratase (ALAD), and ferrochelatase.
• Iron is transferred to erythroid precursors and enters the mitochondria to form heme.
• If protoporphyrin is deficient, iron remains trapped in mitochondria, forming a ring around the nucleus of erythroid precursors (ringed sideroblasts).
• Laboratory findings include:
  • ↑ ferritin; ↓ TIBC; ↑ serum iron; ↑ % saturation
• Sideroblastic anemia can be congenital or acquired, due to:
  • Congenital defect in ALAS
  • Acquired causes: alcoholism, lead poisoning, vitamin B6 deficiency

Thalassemia

• Thalassemia is caused by decreased synthesis of the globin chains of hemoglobin.
• Inherited mutation; carriers are protected against Plasmodium falciparum malaria.
• Divided into α- and β-thalassemia based on decreased production of alpha or beta globin chains.
• Normal types of hemoglobin are HbF (α2γ2), HbA (α2β2), and HbA2 (α2δ2).
• α-Thalassemia is usually due to gene deletion, with:
  • One gene deleted: asymptomatic
  • Two genes deleted: mild anemia with ↑ RBC count
  • Three genes deleted: severe anemia
  • Four genes deleted: lethal in utero (hydrops fetalis)
• β-Thalassemia is usually due to gene mutations, with:
  • Two β genes are present on chromosome 11; mutations result in absent (β0) or diminished (β+) production of the β-globin chain.
  • β-Thalassemia minor (β/β+): mildest form of disease, usually asymptomatic with an increased RBC count.### β-Thalassemia Major
• Most severe form of the disease, presenting with severe anemia a few months after birth
• High HbF (α2γ2) at birth provides temporary protection
• Unpaired α chains precipitate and damage RBC membrane, leading to:
  • Ineffective erythropoiesis
  • Extravascular hemolysis (removal of circulating RBCs by the spleen)
• Consequences:
  • Massive erythroid hyperplasia
  • Expansion of hematopoiesis into the skull and facial bones
  • Extramedullary hematopoiesis with hepatosplenomegaly
  • Risk of aplastic crisis with parvovirus B19 infection
  • Chronic transfusions are often necessary, leading to risk of secondary hemochromatosis
• Blood smear shows:
  • Microcytic, hypochromic RBCs
  • Target cells
  • Nucleated red blood cells
• Electrophoresis shows:
  • HbA2 and HbF with little or no HbA

Macrocytic Anemia

• Definition: Anemia with MCV > 100 μm3
• Causes:
  • Folate deficiency
  • Vitamin B12 deficiency
  • Other causes (e.g., alcoholism, liver disease, and certain medications)
• Folate deficiency:
  • Dietary folate is obtained from green vegetables and some fruits
  • Absorbed in the jejunum
  • Deficiency develops within months due to minimal body stores
  • Causes:
    • Poor diet
    • Increased demand (e.g., pregnancy, cancer, and hemolytic anemia)
    • Folate antagonists (e.g., methotrexate)
• Vitamin B12 deficiency:
  • Dietary vitamin B12 is complexed to animal-derived proteins
  • Liberated by salivary gland enzymes and absorbed in the ileum
  • Deficiency takes years to develop due to large hepatic stores
  • Causes:
    • Pernicious anemia (autoimmune destruction of parietal cells)
    • Pancreatic insufficiency
    • Damage to the terminal ileum (e.g., Crohn disease or Diphyllobothrium latum)
• Clinical and laboratory findings:
  • Macrocytic RBCs with hypersegmented neutrophils
  • Glossitis
  • Subacute combined degeneration of the spinal cord
  • Decreased serum vitamin B12
  • Increased serum homocysteine
  • Increased methylmalonic acid

Normocytic Anemia

• Definition: Anemia with normal-sized RBCs (MCV = 80-100 μm3)
• Causes:
  • Increased peripheral destruction
  • Underproduction
• Reticulocyte count:
  • Normal reticulocyte count is 1-2%
  • Identified on blood smear as larger cells with bluish cytoplasm
  • Corrected count > 3% indicates good marrow response and suggests peripheral destruction
• Peripheral RBC destruction (hemolysis):
  • Divided into extravascular and intravascular hemolysis
  • Extravascular hemolysis:
    • Involves RBC destruction by the reticuloendothelial system
    • Clinical and laboratory findings:
      • Anemia with splenomegaly
      • Jaundice due to unconjugated bilirubin
      • Increased risk for bilirubin gallstones
  • Intravascular hemolysis:
    • Involves destruction of RBCs within vessels
    • Clinical and laboratory findings:
      • Hemoglobinemia
      • Hemoglobinuria
      • Hemosiderinuria
      • Decreased serum haptoglobin

Normocytic Anemias with Predominant Extravascular Hemolysis

• Hereditary spherocytosis:
  • Inherited defect of RBC cytoskeleton-membrane tethering proteins
  • Membrane blebs are formed and lost over time, rendering cells round (spherocytes)
  • Spherocytes are less able to maneuver through splenic sinusoids and are consumed by splenic macrophages
  • Clinical and laboratory findings:
    • Spherocytes with loss of central pallor
    • Increased RDW and MCHC
    • Splenomegaly, jaundice with unconjugated bilirubin, and increased risk for bilirubin gallstones
    • Increased risk for aplastic crisis with parvovirus B19 infection
  • Diagnosed by osmotic fragility test
  • Treatment is splenectomy
• Sickle cell anemia:
  • Autosomal recessive mutation in β chain of hemoglobin
  • Gene is carried by 10% of individuals of African descent
  • Sickle cell disease arises when two abnormal β genes are present, resulting in >90% HbS in RBCs
  • HbS polymerizes when deoxygenated, causing sickling
  • Increased risk of sickling occurs with hypoxemia, dehydration, and acidosis

Microangiopathic Hemolytic Anemia

• Intravascular hemolysis that results from vascular pathology
• RBCs are destroyed as they pass through the circulation
• Occurs with microthrombi (e.g., TTP-HUS, DIC, HELLP), prosthetic heart valves, and aortic stenosis
• Schistocytes are present on blood smear

Anemia due to Underproduction

• Decreased production of RBCs by bone marrow
• Characterized by low corrected reticulocyte count
• Etiologies:
  • Causes of microcytic and macrocytic anemia
  • Renal failure
  • Damage to bone marrow precursor cells
• Parvovirus B19:
  • Infects progenitor red cells and temporarily halts erythropoiesis
  • Leads to significant anemia in the setting of preexisting marrow stress
  • Treatment is supportive
• Aplastic anemia:
  • Damage to hematopoietic stem cells, resulting in pancytopenia
  • Etiologies:
    • Drugs or chemicals
    • Viral infections
    • Autoimmune damage
  • Biopsy reveals an empty, fatty marrow
  • Treatment:
    • Cessation of any causative drugs
    • Supportive care with transfusions and marrow-stimulating factors
    • Immunosuppression
    • Bone marrow transplantation as a last resort
• Myelophthisic process:
  • Pathologic process (e.g., metastatic cancer) that replaces bone marrow
  • Hematopoiesis is impaired, resulting in pancytopenia