Advanced Diagnostic Hematology (0701705) Past Paper PDF

Advanced Diagnostic Hematology (0701705) 1st Semester 2024/2025 Prof Samir Awadallah Faculty of Allied Medical Sciences Department of Medical Lab Sciences Zarqa University  Textbook Clinical Hematology: Theory & Procedures 5...

Advanced Diagnostic Hematology (0701705) 1st Semester 2024/2025 Prof Samir Awadallah Faculty of Allied Medical Sciences Department of Medical Lab Sciences Zarqa University  Textbook Clinical Hematology: Theory & Procedures 5th Edition By Mary Louis Turgeon Thalassemia Chapter 13 Pages 220-233 Thalassemia Introduction Thalassemia is a group of inherited disorders caused by mutation in one or more of the globin genes of hemoglobin leading to decreased or absent synthesis of the corresponding globin chains. The defect could be mild (asymptomatic) or severe. Patients with more severe defects present with symptoms that result from one or more of the following: Decreased production of normal hemoglobin, Synthesis of abnormal hemoglobins, Unbalanced synthesis of α and non-α globin chains Ineffective erythropoiesis. Symptoms include anemia, hepatosplenomegaly, infections, gallstones, and bone deformities that alter facial features and result in pathologic fractures. Thalassemia Epidemiology Thalassemia is one of the most common genetic disorders affecting the world’s population. Approximately 1–5% of people are thought to be carriers of β-thalassemia. It is estimated that between 100,000 and 200,000 individuals worldwide are born each year with severe forms of thalassemia, and approximately 60,000 of those have β-thalassemia. In North America, about 20% of immigrants from Southeast Asia and 6–11% of African Americans have detectable α-thalassemia. Many more are silent carriers. About 6% of individuals with Mediterranean ancestry, 5% of Southeast Asians, and 0.8% of African Americans have β-thalassemia along with people from the Middle East and India. Types of Thalassemia Any of the six different normal globin genes of hemoglobin (α, β, γ, δ, ε, ζ) are likely to be affected. The two major types of classical thalassemia, α-thalassemia and β-thalassemia. When synthesis of the α-chain is impaired, the disease is α-thalassemia. When synthesis of the β chain is affected, the disease is β-thalassemia Combinations of gene deletions such as δβ-thalassemia or γδβ-thalassemia occur but are rare. A variant of β-thalassemia known as hereditary persistence of fetal hemoglobin (HPFH) is characterized by continued production of increased amounts of HbF throughout life. This disorder is characterized by a failure in the switch of γ chain production to β-chain production after birth. In homozygotes, 100% of circulating hemoglobin is HbF. Pathophysiology Normally, equal amounts of α and β chains are synthesized by the maturing erythrocyte, resulting in a β-chain to α-chain ratio of 1:1. In α- and β-thalassemia, synthesis of one of these chains is decreased or absent, resulting in an excess of the other chain. If the α-chain is affected, there is an excess of β-chain , and if the β-chain is affected, there is an excess of α-chain. This unbalanced synthesis of chains contributes substantially to the pathophysiology in thalassemia and produces several effects, all of which contribute to anemia: decrease in total erythrocyte hemoglobin production, ineffective erythropoiesis, chronic hemolysis. Pathophysiology (cont.) Excess α-chains The α-chains are highly insoluble and precipitate within the cell. The precipitates bind to the cell membrane, causing membrane damage (which leads to apoptosis) and decreased erythrocyte deformability. Macrophages in bone marrow, destroy these precipitate-filled developing erythroblasts, resulting in a large degree of ineffective erythropoiesis. Cells that enter the circulation may also contain precipitates that are removed by the spleen, causing chronic extravascular hemolysis. Excess β-chains The β-chains can combine to form hemoglobin molecules containing four β-chains, hemoglobin H (HbH, β4). This hemoglobin has a high oxygen affinity and is also unstable. Thus, it is a poor transporter of oxygen. In the fetus, when α-chains are decreased, excess γ-chains can combine to form hemoglobin molecules with four γ-chains , hemoglobin Bart’s (Hb Bart’s, γ4). This hemoglobin also has a very high oxygen affinity. Clinical and Laboratory Findings Associated with Thalassemia β-Thalassemia (Cooly’s Anemia) Described first by Thomas Cooley in Detroit in 1925 β-thalassemia is one of the most common single-gene disorders. Most mutations causing β-thalassemia are point mutations (> 200 types). These point deletions causes decreased or absent production of β-chain. The underproduction (or complete absence) of β chains contributes to: decreased hemoglobin production, ineffective erythropoiesis, chronic hemolytic process. The mutation in the β-gene may lead to: Complete absence of β-chains (β0-thalassemia) Reduced production of β-chains (β+-thalassemia) HbA2 and HbF, are increased in partial Chromosome 11 is the location of four types of compensation for the decreased HbA levels. globin genes (έ γ δ β). β-thalassemia has three main variants reflecting the clinical severity of the hemoglobinopathy. Thalassemia: The peripheral blood erythrocytes are hypochromic and microcytic and show anisocytosis, poikilocytosis, and target cells (arrows) Characteristics of β-Thalassemia Variants β-Thalassemia-Pathophysiology Symptoms of β-thalassemia major usually manifests approximately six months after birth: (hemoglobin synthesis switches from γ-chain to β-chain synthesis). In β-thalassemia major reduced synthesis, or absence of β-chain, results in an excess of free α- chains. The excess free α-chains cannot form hemoglobin tetramers, so they precipitate within the cell, damaging the cell membrane, and leading to chronic hemolysis. Marrow macrophages destroy precipitate-filled erythrocytes in the bone marrow, which results in ineffective erythropoiesis. Precipitate-filled circulating erythrocytes are destroyed prematurely in the spleen. This also contributes to the anemia of thalassemia. Additionally, accumulating α-chains contain free iron and hemichromes that generate reactive oxygen species (ROS). The ROS damage hemoglobin as well as the membrane proteins (oxidation of band 3) and lipids, decreasing membrane stability. β-Thalassemia-Pathophysiology (cont.) Overall, severe anemia is the most outstanding feature of the disorder and is responsible for many related problems. Most patients who are homozygous for β-thalassemia become dependent on transfusions and develop transfusion-related complications of iron overload, alloimmunization, and potential viral infection. Patients who have undergone splenectomy and who are transfusion-dependent, may suffer from thromboembolic complications, both venous and arterial. Abnormalities in the levels of coagulation factors and their inhibitors have been reported to result in what is defined as chronic hypercoagulable state. Abnormalities of the red cell membrane contribute to this hypercoagulability. Laboratory Findings Hb levels can be as low as 2 or 3 g/dL (20–30 g/L) in the marked microcytic and hypochromic anemia (reduced MCV, MCH and MCHC). Marked anisocytosis and poikilocytosis Basophilic stippling, polychromasia, and nRBC are noted. Reticulocytes are not increased to the degree expected for the severity of the anemia because of the high degree of ineffective erythropoiesis. Secondary leukopenia and thrombocytopenia can be produced because these components also become trapped in the enlarged spleen. Bone marrow show marked erythroid hyperplasia with an M:E ratio of 1:10 or less. Other Laboratory Findings Decreased osmotic fragility, moderately increased bilirubin, increased serum iron, and TIBC. Serum ferritin is a sensitive, accurate marker in determination of iron status. Soluble transferring receptor index is useful and is more specific than soluble transferring receptor because serum ferritin may be increased because of other pathology. Hemoglobin electrophoresis Hb electrophoresis can be performed on cord blood and provide evidence of deficient β-chain production at birth (Hb Bart’s (γ4) might be seen……..Why?). Normal cord blood contains about 50-80% HbA and 20-50% HbF. Infants with β-thalassemia major has much higher Hb F and lower HbA. In adults, hemoglobin electrophoresis shows variable results, depending on the thalassemia alleles inherited. In β0/β+- or β+/β+-thalassemia, hemoglobin electrophoresis reveals increased Hb F and decreased Hb A. In β0/β0-thalassemia: Hb A is absent (zero), Hb F 93-99%, and Hb A2 could be low, normal, or increased β-Thalassemia Minor (β0/β or β+/β) β-Thalassemia minor results from the heterozygous inheritance of either a β+ or β0 gene with one normal β- gene. About 1% of African Americans are heterozygous for β-thalassemia. The condition is usually discovered incidentally during testing for unrelated symptoms or during family study workups. Pathophysiology The normal β-gene as well as the β+ gene directs synthesis of sufficient amounts of β-chains to synthesize enough HbA for normal oxygen delivery and erythrocyte survival. Laboratory Findings Mild anemia with Hb values in the range of 9–14 g/dL (mean for women 10.9 g/dL and for men 12.9 g/dL) The RBC count is significantly increased Blood film shows features of microcytic hypochromic anemia (decreased MCV, MCH and MCHC) Blood smear also shows anisocytosis, poikilocytosis, target cells and basophilic stippling. Hemoglobin electrophoresis demonstrates an increase in HbA2 of 3.5–7.0% with a mean of 5.5%. β-Thalassemia minor frequently mistaken for a mild iron deficiency anemia on a peripheral blood smear. a-Thalassemia α-thalassemia is caused by deletions of one or more of the four α-globin genes located on chromosome 16. α-thalassemia is classified into four types on the basis of genotype and the total number of abnormal genes that result: 1. Silent carrier state (one inactive a gene) 2. a-thalassemia trait (two inactive a genes) 3. Hb H disease (three inactive a genes) 4. Hydrops fetalis with Hb Bart (four inactive α genes) The four genotypes of α-thalassemia α-Thalassemia Major (α0/α0; Hydrops Fetalis) This is the most severe form of α-thalassemia, that involves the deletion of all four α-genes (- -/- -). Both parents of the thalassemia patient must be α- thalassemia minor (2 α-genes on each parental chromosome are deleted). α-Thalassemia Major is most commonly found in Asians α-Thalassemia Major (α0/α0; Hydrops Fetalis) Pathophysiology All four α-genes are deleted in hydrops fetalis. No physiologically useful hemoglobins can be synthesized beyond the embryonic state. This disorder is incompatible with life, and infants are either stillborn or die within hours of birth. Because the α-chains are absent, erythrocytes assemble hemoglobin using the γ, δ and β chains available. Therefore, abnormal hemoglobin tetramers involving γ-chains (Hb Bart’s, γ4) are produced. Hb Bart’s has a very high oxygen affinity, that cannot supply tissues with sufficient oxygen to sustain life. The developing infant usually dies of hypoxia and congestive heart failure in utero. Hb electrophoresis shows only Hb Barts (γ4) and very small amount of Portland embryonic hemoglobin. Hemoglobin H Disease (α0/α+ or α-thal-1/α-thal-2) Hb H disease is a chronic, moderately severe hemolytic anemia that occurs most frequently in individuals from Southeast Asia. Was first described first in 1956. It occurs when three of the four α-genes are deleted (-,-/-, a). The disorder is the result of two parents, one with heterozygous α-thal-1 (- -/aa) and the other with the heterozygous α-thal-2 (- a/aa) genotype. Hemoglobin H Disease Pathophysiology The reduction in α-chain synthesis (25–30% of normal) results in the following: Decrease assembly or synthesis of HbA, HbA2, and HbF. The decrease in the α-chains leads to relative excess of β-chains, which assemble to form β- chain tetramers called HbH. The γ-chains are also are produced in excess of α-chains, especially at birth, leading to the formation of γ-chain tetramers or Hb Bart’s. HbH is unstable and tends to precipitate inside erythrocytes triggering chronic hemolytic anemia. The cells will have decreased life span because of the damage to the cell membrane by the precipitated Hb H The oxygen affinity of HbH is 10 times that of HbA, reducing oxygen delivery to the tissues. This increased oxygen affinity is reflected by the lower P50 value of HbH relative to HbA and myoglobin Hemoglobin H Disease Laboratory Results Severe anemia with hemoglobin values ranging from 3–10 g/dL (30–100 g/L) and erythrocytes that are markedly microcytic and hypochromic. Red cell indices (MCV, MCH, MCHC) are decreased. The reticulocyte count is increased from 5% to 10%. nRBC are observed on the peripheral blood smear Erythrocytes containing Hb H produce precipitated Hb H when incubated (stained) with brilliant cresyl blue. Hb Electrophoresis: At birth, 80–90% Hb Bart’s and about 10–20% Hb HbH. In adults, Hb H (5% to 30%), small amounts of Hb Bart, decreased HbA and normal or decreased amount of HbA2. Hb H inclusions Golf Ball appearance α-Thalassemia Minor The α-thalassemia minor (trait) occurs when two of the four α-genes, either on the same (cis) or opposite (trans) chromosomes, are missing. The condition is found in all geographic locations (African Americans, Southeast Asian and Mediterranean) Clinical Findings Patients with α-thalassemia trait are asymptomatic with a mild anemia and are often diagnosed incidentally or when being evaluated for family studies. Laboratory Results In newborns, Hb electrophoresis shows 5–6% Hb Bart’s. This can be helpful in diagnosing this condition. Three months after birth, Hb Bart’s become undetectable and hemoglobin electrophoresis becomes normal. The only persistent hematological abnormality thereafter is a mild microcytic, hypochromic anemia. The peripheral blood film usually demonstrates significant microcytosis with an MCV of 60–70 fL with few target cells Comparison of Hemoglobinopathies and Thalassemias Comparison between normal hemoglobins and other Hb variants seen in thalassemia Comparison between thalassemias and SCD