Hemolytic Anemias

Hereditary Anemias

Hemolytic Anemias Definition

  • Premature destruction of red blood cells (RBCs) either in the blood vessels (intravascular hemolysis) or elsewhere in the human body (extravascular).
  • Most commonly occurs within the spleen but can also occur in the macrophagocyte (reticuloendothelial system).

Symptoms of Hemolytic Anemia

  • General Symptoms of anemia: Pallor, fatigue, shortness of breath, palpitation, dizziness, headache.
  • Symptoms related to hemolysis: Chills, jaundice, dark urine, enlarged spleen.

Causes (Classification) of Hemolytic Anemia

  • Corpuscular (Hereditary): The cause is related to RBC itself.
  • Extra-corpuscular: The cause is external to RBC.

Corpuscular Causes

  • I- INHERITED causes can be due to:
    • A- Defects of red cell membrane (as hereditary spherocytosis and hereditary elliptocytosis).
    • B- Defects in hemoglobin production (thalassemia, sickle-cell disease).
    • C- Defective red cell metabolism (glucose-6-phosphate dehydrogenase deficiency, pyruvate kinase deficiency).
  • II- ACQUIRED:
    • Paroxysmal nocturnal hemoglobinuria (PNH), characterized by complement-induced intravascular hemolytic anemia.

Hereditary Hemolytic Anemias Associated with Membrane Abnormalities

Erythrocyte Membrane

  • The erythrocyte membrane plays a critical role in the maintenance of the biconcave shape and integrity of the red cell.
  • It consists of a lipid bilayer with embedded trans-membrane proteins and an underlying membrane protein skeleton that is attached to the bilayer via linker proteins.

Clinical Phenotypes of Hereditary Membrane Disorders

  • Mutations in the genes that control the proteins of the membrane and their interaction mainly produce changes in the shape of red cells, which is characteristic in any individual.
  • Many of the conditions are inherited as autosomal dominant disorders.
  • The mutations affecting the red cell membrane are many and heterogeneous, but the effect on the phenotype can be classified in five main categories:
    1. Hereditary Spherocytosis
    2. Hereditary Elliptocytosis
    3. Hereditary Pyropoikilocytosis
    4. Southeast Asian Ovalocytosis
    5. Hereditary Acanthocytosis
    6. Hereditary Stomatocytosis

HEREDITARY SPHEROCYTOSIS

  • Inheritance: HS is typically inherited in an autosomal dominant fashion. In approximately 25% of cases, HS is due to autosomal recessive inheritance or de novo mutations.
  • Pathophysiology:
    • Characterized by osmotically fragile, spherical erythrocytes.
    • HS is typically caused by a red cell membrane protein deficiency.
      • About 60% of HS cases result from a defect in the ankyrin–spectrin complex.
      • A further 25% involve deficiency in band 3.
      • In the remainder of the dominantly inherited HS families, there is a deficiency of protein 4.2, or no abnormality has yet been identified.
    • The membrane protein deficiency destabilizes the lipid bilayer, causing microvesicles to bud off from weakened areas, which leads to spherocyte formation.
    • Spherocytes exhibit a decreased surface area-to-volume ratio and are dehydrated, which decreases their deformability.
    • The passage of spherocytes through the spleen is impeded, and during erythrostasis, they are engulfed by splenic macrophages and destroyed.

Clinical Feature

  • The typical clinical picture of HS combines evidence of hemolysis with spherocytosis and positive family history.
  • Other features of hemolytic anemia may be present, e.g., splenomegaly, gallstones, mild jaundice.
  • Occasional aplastic crises occur, e.g., with parvovirus B19 infection.
  • The majority of HS patients have a moderate degree of hemolytic anemia.
  • Severe cases may be diagnosed in infancy or childhood, but mild cases may escape detection until adulthood or may remain undetected.
  • 10% of HS patients have severe disease in infancy. A small number of these, typically with autosomal recessive HS, present with life-threatening, transfusion-dependent anemia.
  • An asymptomatic carrier state has been suggested in the case of clinically asymptomatic parents whose children present with typical HS.

Investigations

  1. Blood film: Spherocytes are the hallmark of the disease and are characterized by a smaller diameter, darker staining, and a decreased or absent central pallor, compared to normal red cells.
  2. Erythrocyte indices reflect: a mild to moderate decrease in hemoglobin in most patients and an increased MCHC in approximately 50% of cases.

Osmotic Fragility Test

  • The test measures the sensitivity of red cells to lysis in vitro to swelling caused by incubation in increasingly hypotonic saline solutions.
    • Test: RBCs incubated in saline at various concentrations, this results in cell expansion and eventually rupture.
    • Results:
      • Normal RBCs can withstand greater volume increases than spherocytic RBCs.
      • Positive result (i.e., confirms HS) when RBCs lyse in saline at near to isotonic concentration, i.e., 0.6–0.8g/dL (whereas normal RBCs will simply show swelling with little lysis).
      • Osmotic fragility is more marked in patients who have not undergone splenectomy, and if the RBCs are incubated at 37°C for 24h before performing the test.
      • Note: a normal result does not exclude HS and may occur in 10–20% cases.

Therapy and Prognosis

  1. Patients with well-compensated haemolysis and no transfusion requirements need no treatment other than reassurance and folic acid.
  2. Radiolucent gallstones, if detected by chance on ultrasound, are common and need no treatment unless complications arise. Recurrent cholecystitis or obstruction would be an indication for cholecystectomy, which would also be an indication for splenectomy.
  3. Splenectomy restores the lifespan of the red cells to normal and hence cures the haemolysis and jaundice in the majority of patients because splenic sequestration is the primary determinant of erythrocyte survival in HS.
    • Indications for splenectomy:
      1. Patients with marked haemolysis producing symptoms or requiring transfusion should be splenectomized.
      2. Recurrent aplastic crises are also an indication.
      3. Attacks of cholecystitis or biliary colic warrant cholecystectomy and splenectomy. Laparoscopic splenectomy has become the method of choice in centers with surgeons experienced in the technique. Occasionally, splenectomy may not correct the anemia, usually due to an accessory spleen.
  4. Transfusion in patients with aplastic crises or severe hemolysis.

HEREDITARY ELLIPTOCYTOSIS

  • Pathophysiology: Spectrin defects and a deficiency of protein 4.1 are the most common underlying causes of HE.
  • Hereditary elliptocytosis (HE) is a heterogeneous disease characterized by the presence of elliptical or oval erythrocytes on the blood film.
  • Inheritance: HE is typically inherited as an autosomal dominant disorder, and de novo mutations are rare.

Laboratory Features

  1. Blood film:
    • The hallmark of HE is the presence of normochromic, normocytic elliptocytes.
    • Poikilocytes may be present in severe HE.
    • The degree of hemolysis does not correlate with the number of elliptocytes.
    • The reticulocyte count generally is less than 5% but may be higher when hemolysis is severe.

Hereditary Hemolytic Anemia Associated With Red Cell Enzyme Deficiency

Hexose Monophosphate Shunt (Pentose Phosphate Pathway)

  • The HMP shunt catabolizes approximately 10% of the glucose, which is essential for maintaining adequate concentrations of reduced glutathione (GSH).
  • GSH levels are maintained by conversion of NADPH to NADP; NADP is reduced back to NADPH in a reaction catalyzed by glucose 6- phosphate dehydrogenase (G6PD).
  • GSH protects the erythrocyte from oxidant damage; it maintains hemoglobin in the reduced functional state and preserves vital cellular enzymes from oxidant damage.
    • If enzymes in the HMP shunt are deficient:
      • The cell’s reducing power is compromised, and oxidized hemoglobin accumulates.
      • Subsequently, denaturation in the form of Heinz bodies, which aggregate at the cell membrane, causing membrane damage.
      • As the cells pass through the spleen, the macrophages attempt to remove the Heinz bodies, leading to premature extravascular hemolysis (bite cells).
  • The most common enzyme deficiency of the HMP shunt is G6PD deficiency.

Enzyme Deficiencies in the Hexose Monophosphate Shunt

  • G6PD deficiency is the most common erythrocyte enzyme disorder.
  • Inheritance: Sex-linked disorder carried by a gene on Xq28
  • Females are homozygous for the mutation, while heterozygote females appear to have two populations of cells, one deficient in G6PD and one normal.

G6PD Variants

  • More than 400 variants (isoenzymes) of the G6PD enzyme have been identified.
  • The mutant enzymes have been categorized according to the degree of deficiency and hemolysis.
  • The World Health Organization classifies G6PD genetic variants into five classes (the first three of which are deficiency states):
    • Class I: Severe deficiency (<10% activity) with chronic (nonspherocytic) hemolytic anemia
    • Class II: Severe deficiency (<10% activity), with intermittent hemolysis
    • Class III: Moderate deficiency (10-60% activity), hemolysis with stressors only
    • Class IV: Non-deficient variant, no clinical sequelae
    • Class V: Increased enzyme activity, no clinical sequelae

Pathophysiology of G6PD Deficiency

  • Normally, G6PD activity is highest in young cells and decreases as the cell ages. Thus, even older cells normally retain enough G6PD activity to maintain adequate GSH levels.
  • The reticulocytes released from the bone marrow in response to the hemolytic episode also have enough enzyme activity to maintain metabolic activity even under oxidant stress.
  • Normal erythrocytes have been shown to use only 0.1% of their maximum G6PD enzyme capacity. This also explains why even G6PD-deficient cells can maintain normal function and hemolysis is sporadic.
  • In excessive oxidant stress, cells that are most deficient undergo oxidant damage and are rapidly removed from circulation. This accounts for the sporadic hemolysis that accompanies oxidant stress in G6PD deficiency.
  • In most G6PD variants, hemolysis is self-limited (referring to the fact that hemolysis stops after a time even if the oxidant stress continues). It occurs because the older, most G6PD-deficient erythrocytes initially are destroyed, but the younger cells remain because they have sufficient enzyme activity to avoid hemolysis.
  • G6PD deficiency causes the formation of Hienz bodies. They are removed from the erythrocytes by splenic macrophages, producing “bite” cells and blister cells.
  • With progressive membrane loss, spherocytes can be formed, which are less deformable than normal and become trapped and hemolyzed in the spleen (extravascular hemolysis).
  • Cell membrane damage can be severe enough for the cell to hemolyze in the circulation.
  • This intravascular hemolysis can be acute and accompanied by hemoglobinemia and hemoglobinurea.

Clinical Features of G6PD Deficiency

  1. Most persons with G6PD deficiency have no clinical symptoms, and they are not anemic.
  2. Acute haemolytic anaemia in response to oxidant stress
  3. Neonatal jaundice.
  4. Congenital non‐spherocytic haemolytic anaemia (rare)

Acute Hemolytic Anemia

  • Diagnosis usually occurs during or after infectious illnesses or following exposure to certain drugs precipitating a hemolytic attack.
  • Hemolysis is variable and depends on the degree of oxidant stress, the G6PD isoenzyme, and sex of the patient.
  • The symptoms are those of acute intravascular hemolytic anemia.
  • Sudden anemia develops with a 3–4 g/dL drop in hemoglobin.
  • Jaundice is not prominent. The patient may experience abdominal and low back pain, as well as dark or black urine due to hemoglobinuria.
  • Complications: Renal failure
  • Favism: Sudden severe hemolytic episode that develops in some individuals with G6PD deficiency after the ingestion of fava beans. This is associated with severe G6PD deficiency, especially the G6PD Mediterranean variant.
    • It is the main form of the disease in Egypt
    • Severe favism usually affects children between the ages of 2 and 5 years.
    • The most likely components of the bean responsible for the sensitivity are isouramil and divicine.
    • The first signs of favism are malaise, severe lethargy, nausea, vomiting, abdominal pain, chills, tremor, and fever, and usually hemoglobinuria occurs a few hours after ingestion of the beans.

Laboratory Investigations for G6PD Deficiency

  • CBC with blood smear:
    • During or immediately following a hemolytic attack, polychromasia, occasional spherocytes, RBCs fragments, and bite cells.
    • Bite cells: with one or more semicircular portions removed from the cell margin, are frequently thought to be typical of G6PD deficiency.
    • Reticulocytosis is characteristic following a hemolytic episode.
    • Leukocytes can increase during hemolytic attacks.
    • Platelets are usually normal
  • Chemistry:
    • Unconjugated bilirubin and serum LDH can be increased.
    • Haptoglobin commonly decreases during the acute hemolytic phase
    • Urinary hemosiderin.

Enzyme Activity

  • Definitive diagnosis depends on the demonstration of a decrease in erythrocyte G6PD enzyme activity.
  • In affected individuals, the enzyme activity can appear normal during and for a time after a hemolytic attack because older cells with less G6PD are preferentially destroyed during the attack and reticulocytes have normal activity.
  • For this reason, assays for G6PD should be performed 2 to 3 months after a hemolytic episode.

Enzyme Deficiencies in the Glycolytic Pathway

  • Pyruvate kinase deficiency is the most common enzyme deficiency in the glycolytic pathway.
  • It is the second most common erythrocyte enzyme deficiency.
  • Many pyruvate kinase enzyme mutations coincide with the disorder’s variety of clinical manifestations.
  • The more severe types are noted in infancy, whereas the milder types may not be detected until adulthood.
  • Inheritance is autosomal recessive.

Pathophysiology of Pyruvate Kinase Deficiency

  • More than 180 different mutations in the PK gene (PKLR gene on chromosome 1q21) affecting the enzyme activity have been identified.
  • Pyruvate kinase is the enzyme that catalyzes the final step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP.
  • In PK deficiency, this energy-producing reaction is prevented, resulting in a loss of two ATP molecules per molecule of glucose catabolized.
  • The cell’s inability to maintain normal ATP levels results in alterations of the erythrocyte membrane; failure of the cation pumps causing potassium loss as well as sodium and calcium gain; and dehydration (echinocytes).
  • The decrease in echinocyte deformability enhances erythrocyte sequestration in splenic cords and phagocytosis by macrophages.
  • Individuals tolerate the anemia well because of the increase in 2,3-BPG that accompanies the distal block in glycolysis.
  • The two to three times normal increase in 2,3-BPG enhances the release of oxygen to the tissues.

Clinical Findings of Pyruvate Kinase Deficiency

  • Clinical symptoms vary depending on the degree of anemia, which is mild to severe.
  • Jaundice can occur with intermittent hemoglobinuria.
  • Gallstones are a common complication.
  • PK deficiency can be life threatening in neonates.
  • When anemia is present at birth, PK deficiency should be considered with and differentiated from other etiologies associated with anemia in this population (ABO/Rh incompatibility, thalassemia, G6PD deficiency, and hereditary spherocytosis).
  • Neonates with a severe phenotype usually have severe jaundice that may require exchange transfusions.
  • Splenectomy can result in stabilization of the hemoglobin and decrease the need for transfusions.
  • Patients with milder forms of PK deficiency are commonly diagnosed in early adulthood although they could have had neonatal jaundice.

Laboratory Investigations for Pyruvate Kinase Deficiency

  • Patients with PK deficiency have a normocytic, normochromic anemia with hemoglobin levels of 6–12 g/dL.
  • Reticulocytosis ranges from 2 to 15% and increases more after splenectomy, often above 40%.
  • Serum unconjugated indirect bilirubin and LDH are increased, and haptoglobin is decreased or absent.
  • Enzyme activity: In performing enzyme tests for PK, the erythrocytes are separated from leukocytes because leukocytes contain more PK than erythrocytes.
  • Thus, in erythrocyte PK deficiency (mutations of the second locus), only the erythrocytes are deficient; leukocytes are normal.
  • Treatment
    • There is no specific therapy for PK deficiency.
    • Transfusions help maintain the hemoglobin above 8–10 g/dL.
    • Splenectomy can improve the hemoglobin level and decrease the need for transfusions in some affected individuals; however, hemolysis continues.

HEMOGLOBINOPATHIES

Structure, Genetic Control, and Synthesis of Hemoglobin

  • The embryonic haemoglobins include Hb Portland (ζ<em>2γ</em>2\zeta<em>2\gamma</em>2), Hb Gower 1 (ζ<em>2ϵ</em>2\zeta<em>2\epsilon</em>2), and Hb Gower 2 (α<em>2ϵ</em>2\alpha<em>2\epsilon</em>2).
  • In the fetus, HbF(α<em>2γ</em>2\alpha<em>2\gamma</em>2) predominates, Fetal haemoglobin (HbF) exhibits a higher oxygen affinity than adult haemoglobins in vivo; the higher oxygen affinity of HbF relative to adult haemoglobin facilitates the transfer of oxygen across the placenta from the maternal to the fetal circulation.
  • In adults, HbA (α<em>2β</em>2\alpha<em>2\beta</em>2) comprises over 95% of the total haemoglobin, with a minor component of HbA2 (α<em>2δ</em>2\alpha<em>2\delta</em>2) in the red blood cells.

Types of Hemoglobinopathy

  • NB. all are genetically inherited
    • Qualitative
      • Affecting the structure and therefore maybe the function of the globin molecule. Eg. HbS, HbC, HbD Punjab, HbE, hundreds of others
    • Quantitative
      • Affecting the amount of globin molecules produced
        • Either alpha chain defects - alpha thalassaemias
          • 4 genes, 2 from each parent,
        • Or beta chain defects - beta thalassaemias
          • 2 genes, 1 from each parent

The Thalassaemias And Related Disorders

  • The term ‘thalassaemia’ is derived from the Greek word (meaning ‘the sea’) since many of the early cases came from the Mediterranean region.
  • However, it is now clear that the disorder is not just limited to the Mediterranean region but occurs throughout the world, prevalent in the tropical and subtropical regions including the Middle East, parts of Africa, Indian subcontinent, and Southeast Asia.
  • It appears that heterozygotes for thalassaemia are protected from the severe effects of malaria and natural selection has increased and maintained their gene frequencies in these malarious regions.
  • Mutation decreases the synthesis of one globin β, it produces imbalanced production of α- and β-globins results in damage to precursors of RBCs in the bone marrow. This damage occurs largely because the excess unpaired globin is unstable, and it precipitates within early RBC precursors in the bone marrow and oxidatively damages the cellular membrane.
  • For example, if β-globin synthesis is diminished by a mutation, there will be a relative excess of α-globins.

Beta Thalassemia

  • The β thalassaemias pose by far the most important public health problems because they are common and usually produce severe anaemia in their homozygous and compound heterozygous states.
  • Genetic basis of disease: β-thalassemia is extremely heterogeneous at the molecular level.
  • The β thalassaemias are considered to be autosomal recessive disorders since individuals who have inherited one abnormal β gene (carrier) are asymptomatic and the inheritance of two abnormal β globin genes is required to produce a clinically detectable phenotype.
  • More than 300 different mutations have been found in association with the β-thalassemia phenotype. They fall into:
    • Non deletional: The vast majority of β thalassaemia mutations are point mutations (i.e. single-base substitutions) and small insertions or deletions of one to two bases. These may involve any step in globin chain production: transcription, translation or post-translational stability of the globin gene product.

Beta Thalassemia Variants

  • Beta thalassemia is caused by variants (mutations) in the hemoglobin beta locus (beta globin gene) that lead to impaired production of beta globin chains:
    • The clinical phenotype depends on which an individual carries.
      • Beta0 variants are those that abolish beta globin production, and
      • Beta+ variants can produce some beta globin but far less than the normal amount.
    • Homozygous or compound heterozygous for beta0 thalassemia mutations are more likely to have transfusion-dependent beta thalassemia (previously called beta thalassemia major)
    • Beta+ thalassemia mutation combined with a beta0 thalassemia mutation typically have transfusion-dependent beta thalassemia.
    • Two beta+ thalassemia mutations typically have transfusion-independent beta thalassemia, although some may be transfusion dependent or may become transfusion dependent later in life.
    • Heterozygous for beta+ thalassemia inherit a beta thalassemia allele from one parent and a normal beta globin allele from the other parent. These individuals have profound microcytosis but mild or minimal anemia; they are largely asymptomatic and are often identified incidentally (by family testing or on a complete blood count [CBC] done for other reasons). Anemia can become more significant during pregnancy.

Clinical Phenotype of Beta-Thalassemia

The clinical phenotype of patients with β-thalassemia is heterogeneous and is determined primarily by:

  1. The globin chain imbalance due to the number and severity of the abnormal alleles inherited.
  2. Coinheritance of other abnormalities affecting α- or γ-globin synthesis or structural abnormalities of hemoglobin (eg, HbC, HbE) also affects the chain imbalance and hence the clinical phenotype.

Pathophysiology Of Beta Thalassemia

  • The molecular defects in β thalassaemia result in absent or reduced β-chain production while α-chain synthesis is unaffected.
  • The imbalance in globin chain production leads to an excess of α-chains>>highly unstable and precipitate in red cell precursors>> intracellular inclusions that interfere with red cell maturation
  • RBCs destruction occurs due to:
    1. Mechanical damage (α-chain inclusions) interfering with their passage through the spleen
    2. Degradation products of excess α-chains, particularly haem and iron >>marked abnormalities of electrolyte homeostasis and membrane deformability.
  • The end result is an extremely rigid red cell with a shortened survival
  • Thus, the anaemia of β-thalassaemia results from a combination of ineffective erythropoiesis and haemolysis.
  • RBC membrane damage with increased surface expression of anionic phospholipids, platelet activation, and changes in hemostatic regulatory proteins contribute to a hypercoagulable state in thalassemia, which is worsened after splenectomy.
  • Anemia >>erythropoietin production>>BM expansion and skeletal deformities.
  • Because the spleen is being constantly bombarded with abnormal red cells, it hypertrophies.
  • These F cells come under selection in the marrow and peripheral blood and thus individuals with β thalassaemia have variable increases in HbF due to selective survival of these F cells.

Alpha -THALASSEMIA

  • Distribution: There is a high prevalence of α-thalassemia in areas of the Old World where malaria was once endemic, including Africa, the Mediterranean region, Southeast Asia, and, to a lesser extent, the Middle East.
  • Molecular basis: Normal individuals have four α - globin genes on chromosome 16, the normal α genotype being represented as αα/αα. The α thalassemias can be classified as:
    • α0 thalassaemia, in which no α- chains are produced from the linked pair
    • α+ thalassaemia, in which production of α - chain from the affected chromosome is reduced, due to deletion of one of the linked genes, –α/αα, or impairment due to a point mutation, designated αTα/αα.
    • α+-thalassemia, the deletion type is due to unequal crossover (recombination) of the linked genes, whereas the nondeletion type includes mutations resulting in abnormal transcription or translation or the production of unstable α-globin.

The Pathophysiology of Alpha Thalassaemia

  • A deficiency of α - chains leads to the production of excess γ or β chains, which form Hb Bart’s (γ<em>4\gamma<em>4) and HbH (β</em>4\beta</em>4), respectively. These soluble tetramers do not precipitate extensively in the bone marrow and hence erythropoiesis is more effective than in β thalassaemia.
  • HbH is unstable and precipitates in red cells as they age.
  • The inclusion bodies cause red cell membrane damage and obstruction in the spleen leading to shortened red cell survival.
  • Furthermore, both Hb Bart’s and HbH have a very high oxygen affinity and their oxygen dissociation curves resemble myoglobin. Thus, the severe forms of α-thalassaemia are due to defective haemoglobin production, the synthesis of homotetramers that are physiologically useless and a haemolytic component.

Alpha-Thalassemia Genotypes and Clinical Classification

  • Below is a table summarizing the different genotypes of alpha-thalassemia and their corresponding clinical classifications:
    • 4 genes (αα/αα ): Normal
    • 3 genes (αα/-α): Silent carrier
    • 2 genes (-α/-α or αα/--): Alpha thalassemia trait
    • 1 gene (-α/--): Hb H Disease
    • 0 genes (--/--): Hb Barts/Hydrops fetalis

Alpha-Thalassemia Trait (Minor)

  • Heterozygotes for α+- thalassemia (–α/αα), so-called silent carriers, are clinically normal with minimal to no anemia, or morphologic abnormalities of RBCs and normal electrophoresis.
  • Thalassemia trait (2-gene deletion α-thalassemia) occurs in 2 forms: α0 - thalassemia trait (– –/αα) or homozygosity for α+-thalassemia (–α/–α). Individuals with thalassemia trait have a lifelong mild microcytic anemia.
  • No specific treatment in these groups (–α/αα, – –/αα, –α/–α) except for ethnic groups genetic counseling ( risk of HbH or hydrops in the offspring)

Beta-Thalassemia Trait (Minor)

  • β-Thalassemia trait (minor) is asymptomatic and is characterized by mild microcytic anemia. Most commonly, it arises from heterozygous β-thalassemia (β-thalassemia trait).
  • Patients with β-thalassemia trait may have a hemoglobin ranging from 9 g/dL to a normal value.
  • Peripheral smear shows microcytic, hypochromic RBCs, poikilocytes, and target cells. Basophilic stippling is variable.
  • RBC survival is normal, with minimal ineffective erythropoiesis. Individuals with β-thalassemia trait are asymptomatic and do not require therapy.
  • They should be identified to reduce the risk of inappropriate iron supplementation.

Transfusion-dependent Thalassemia (TDT) vs. Non-transfusion-dependent thalassemia (NTDT)

  • Patients with TDT require regular blood transfusions for survival
  • NTDT includes a wide spectrum of clinical phenotypes, ranging from mild to moderately severe anemia.
  • Patients with NTDT do not require regular blood transfusions for survival, but intermittent transfusions may be required in acutely worsening anemia due to infection or acute illness, or to allow for normal growth and development in childhood.
  • Some patients with NTDT may require regular transfusions later on in life, often in adulthood, due to complications of the disease including worsening anemia and splenomegaly.

Clinical Picture & Complications of Thalassemia

  1. Anemia
    • Individuals with severe β-thalassemia have presented with expansion of the bone marrow spaces secondary to erythroid hyperplasia, hepatosplenomegaly, and extramedullary hematopoiesis in the chest and abdomen.
    • The external appearance is characterized by pallor and slight jaundice, frontal bossing, and other abnormalities of the facies secondary to marrow expansion, and abdominal enlargement due to hepatosplenomegaly.
    • Usually, these manifestations are absent or minimally present in patients with thalassemia major if transfusion therapy is initiated early during the first year of life provided that the hemoglobin levels are maintained at 9–10 g/dL.
  2. Osteoporosis
    • Reduced bone mineral density and consequent susceptibility to fractures, occurring in up to 90% of individuals and tends to be more common in NTDT patients.
  3. Cholelithiasis
    • Gallstones, depending on the transfusion regimens, inefficient erythropoiesis, and hemolysis.

Other Complications of Thalassemia

  • Growth impairment
  • Secondary Gout
  • Endocrine and metabolic abnormalities
    • Hypogonadism
    • Hypothyroidism
    • Insulin resistance and diabetes
  • Chronic skin ulceration is more commonly seen in NTDT due to reduced tissue oxygenation and increases with increasing age and iron burden.
  • Pulmonary hypertension is the major cardiovascular complication in NTDT patients.

Laboratory Testing for Thalassemia

  1. CBC and routine laboratory studies
    • Profound hypochromic, microcytic anemia accompanied by bizarre red cell morphology is a hallmark of beta thalassemia major and HbH disease
    • Mild microcytosis or anemia may be seen in thalassemia minor
    • The reticulocyte count is elevated but less than expected for the degree of anaemia due to ineffective erythropoiesis.
  2. Iron profile:
    • Serum iron level is elevated, with saturation as high as 80%.
    • Ferritin is also raised.
  3. Erythropoietin levels will be high.
  4. Bone marrow aspirate
    • BMA is not essential to make the diagnosis, but if performed shows very marked erythroid hyperplasia, with dyserythropoiesis.
    • Many of the erythroid precursors show inclusions after incubation with methyl violet; similar inclusions are found in the peripheral red cells after splenectomy.
  5. Liver function tests show elevation of bilirubin, AST and LDH, with a normal ALT.
  6. Haptoglobin low
  7. Coombs negative
  8. Hb electrophoresis: Four methods may be used to quality and quantity of hemoglobin:
    • Hemoglobin electrophoresis acidic and alkaline
    • Iso-electric focusing
    • Capillary Electrophoresis
    • High-performance liquid chromatography (HPLC)

Hemoglobin Electrophoresis

  • A. Hemoglobin electrophoresis at acidic and alkaline medium:
    • (i) Haemoglobin Electrophoresis at Alkaline pH 8.4–8.6 (EDTA): At alkaline pH, haemoglobin is a negatively charged protein, and when subjected to electrophoresis will migrate toward the anode (+). Electric current is applied till adequate separation is achieved. The cellulose acetate strips are removed from the chamber, stained and dried.
      • The test sample should be compared with a control sample containing known normal and abnormal hemoglobins.
      • Usually, a control sample known to contain hemoglobins A, F, S, and C is always included in every electrophoresis and is applied to each strip next to the test sample.
  1. Imaging
    • Skeletal surveys show classical changes to the bones but only in patients who are not regularly transfused.
    • They result from expansion of marrow spaces and usually disappear when marrow activity is reduced by regular transfusions.
    • Plain skull X-ray shows the classical 'hair on end' appearance.

MANAGEMENT of Thalassemia

Anemia Management in Thalassemia

  • For children with beta thalassemia major, chronic transfusion is initiated in early childhood to maintain a hemoglobin level higher than 9.5 g/dL.
  • For individuals with thalassemia intermedia who have anemia that is severe enough to require transfusion, decisions must be made regarding whether to initiate a chronic transfusion regimen in order to suppress ineffective erythropoiesis or to provide periodic transfusions for symptomatic relief and/or during periods of increased stress.
  • For individuals with thalassemia minor, transfusions are not required; anemia is very mild or absent.
  • Chronic transfusion program, aim:
    1. Correcting anaemia improves oxygen delivery to the tissues and facilitates normal growth and development.
    2. Suppression of abnormal erythroid hyperplasia limits damage to bones, reduces excessive iron absorption and reduces extramedullary haemopoiesis. Ideally transfusions are with packed red cells & leucodepleted blood reduces the risk of transfusion reactions and cytomegalovirus infection.

Assessment of Iron Stores and Chelation Therapy

  • Iron overload inevitably complicates regular blood transfusions.
  • Each unit of transfused blood contains about 200–250 mg iron.
  • Ferritin and transferrin saturation should be regularly checked in thalassemia patients to detect iron overload.
  • To prevent hemosiderosis, iron needs to be chelated and excreted.
  • Currently, three drugs are used for iron chelation: desferrioxamine, deferiprone, and deferasirox. Iron chelation is generally initiated in one or more of the following settings:
    1. At the same time a chronic transfusion program is started
    2. After the serum ferritin exceeds 1000 ng/mL (1000 mcg/L)
    3. After the liver iron concentration exceeds 3 mg iron per g of dry weight or cardiac MRI T2 < 20 millisec
    4. After transfusion of approximately 20 to 25 units of RBCs

Other Treatments for Thalassemia

  • Vitamin supplement: Vitamins and trace minerals represent key buffers against the oxidative stress due to iron overload.
    • Ex: Folate supplementation is advised for patients with a hyperactive bone marrow (1-2 mg /day), vitamin D is given to prevent osteoporosis.
  • Luspatercept: is a subcutaneous agent that improves red blood cell (RBC) maturation through differentiation of late stage erythroid precursors.
  • Splenectomy: In cases of hypersplenism
  • Bone marrow transplantation: is generally seen as the treatment of choice if there is an HLA - identical sibling and the child is transfusion dependent. The prognosis for HSCT from an HLA-matched sibling donor is excellent. The success of stem cell transplantation is generally reduced as children get older, iron overload increases and iron-related organ damage increases.

Fetal Hemoglobin Inducers

  • Butyrate analogues such as L-carnitine (fatty acid oxidation)
  • Hydroxyurea (HU)

Gene therapy: is a future prospect for treatment.

Molecular Basis of Sickling

  • Homozygosity for a unique hemoglobin gene mutation (HBB glu7val, GAG —> GTG, rs334, sickle hemoglobin, HbS), located on chromosome 11 (