Hemolytic Anemia: The Hemoglobinopathies PDF

26/11/2022 Hemolytic Anemia: The Hemoglobinopathies Chapter 11 Rania Abu Seir, Ph. D. Associate Professor of Hematology...

26/11/2022 Hemolytic Anemia: The Hemoglobinopathies Chapter 11 Rania Abu Seir, Ph. D. Associate Professor of Hematology Clinical Hematology I (0202306) Harmening DM. Clinical Hematology & Fundamentals of Hemostasis, 5th ed. Philadephia: F.A. Davis 2009. Objectives List examples of Hemoglobinpsthies and their nomenclature Describe the cause of Sickle Cell Anemia and list the clinical and laboratory findings List findings of hemoglobin C disease and SC disease List characteristics for hemoglobin D, E, and other variants 1  26/11/2022 Composition of Normal Physiologic Hemoglobin Hemoglobinopathies: Definition A group of qualitative or quantitative inherited abnormalities in the structure of Hb resulting from alterations in the DNA genetic code for one or more globin chains; changes in RBC deformability and electrophoretic mobility can occur. Inheritance: inherited according to Mendelian genetics or arise from new genetic mutations There are >900 naturally occurring genetically determined variants of human Hgb, many are harmless and only about 200 of these variants are clinically significant Hgb-pathies are inherited as co-dominant traits Hemoglobinopaties show geographic distribution 2  26/11/2022 Hemoglobinopathies: Definition Homozygous/disease conditions (both globin chains affected) are more serious than heterozygous Heterozygous/Trait conditions (only one globin chain affected). Target cells are associated with the hemoglobinopathies. Hemoglobin electrophoresis, isoelectric focusing and/or DNA (PCR) analysis may be used to confirm the diagnosis. amino acid substitution causing formation of Hgb S is the most common, Hgb C is the second most common, and Hgb E is the third most common. Figure 11-1 Inheritance of abnormal hemoglobins. A. With one parent heterozygous for an abnormal hemoglobin, the offspring have a one-in-two chance of carrying the trait. B. With one parent homozygous for an abnormal hemoglobin, all offspring will carry the trait because that parent can contribute only an abnormal gene. C. With both parents heterozygous for the abnormality, the chances are one in four for normal, two in four for heterozygous, and one in four for homozygous. D. With both parents carrying the same abnormal hemoglobin—one homozygous and one heterozygous—the offspring have a 50– 50 chance of being either homozygous or heterozygous. E. With parents carrying two different abnormal hemoglobins, offspring have a one-in-four chance of not inheriting an abnormality, a one-in-two chance of carrying the trait for one or the other abnormality, and a one-in-four chance of carrying both abnormalities in codominance. 3  26/11/2022 The Globin Gene Clusters on Chromosomes 16 & 11 The α–chains have 141 aa each, while the β–like chains have 146 aa each. Review of Normal Hb Structure 4  26/11/2022 Nomenclature First Hb discovered was HbS (Crescent or Sickle cell) Alphabetical letters according to their electrophoretic mobility beginning with letter C; e.g, Hb C Geographic place, e.g, Hb. Koeln Letter + geographic place for Hbs with identical mobility on electrophoresis, HbC Harlem, HbC Georgtown, HbO Arab, HbO Indonesia The letter M was given to Hbs that tend to form MetHb Hemoglobinopathies: Classification Three main broad groups: 1. Structural variants, with altered structure of the globin chain  (qualitative, “Hemoglobinopathies”) Aggregating Hbs Unstable Hbs Hbs with abnormal heme function (O2 transport) 2. Disorders with reduced rate of synthesis of one or more globin chain, > (quantitative, “Thalassemia”) 3. HPFH (Hereditary Persistence of Fetal Hb), failure to complete the normal neonatal switch from fetal (HbF) to adult Hb (HbA) 4. Abnormal Hbs without clinical significance !! 5  26/11/2022 Hemoglobinopathies The majority of Hb variants result from β–chain defects Defects of α-, γ- or δ–chains do occur, but most of them are benign Molecular defects that result in Hb variants Most Hb variants (>90%) result from a single nucleotide/ aa substitution Example: HbS (Glu6Val) or HbC (Glu6Lys) Multiple nucleotide substitution Long or short globin chains Fusion globin chains Table 11–2 Classification of Hemoglobinopathies Abnormal hemoglobins without clinical significance Aggregating hemoglobins (structural abnormalities with amino acid substitution away from the crevice of the heme, i.e., HbS, HbC) Unbalanced synthesis of hemoglobin (thalassemia) Unstable hemoglobins Hemoglobins with abnormal heme function (structural abnormalities with amino acid substitutions near the crevice of the heme) 6  26/11/2022 Nomenclature Abnormal Hbs are now assigned both a common name and a scientific designation HbS is designated as: α2β2S, or α2β26Val, or α2β26Glu-Val, or α2β2 6(A3)Glu-Val HbG Philadelphia is designated as: α2GPhilβ2, or α268Lysβ2, or α268Asn-Lysβ2, or α268(E17)Asn-Lysβ2 Nomenclature Hb S: Valine substitutes Glutamic acid at 4.Hb OArab: β121Glu→Lys( mild dis.) the 6th position of the β chain ; 5.Hb G Philadelphia: α268Asn→Lys α 2 β2 6Glu→Val or α2 β2 6val or α2β2S 6. Hb Punjab:α2 β2 121Glu→Gln 7. Hb Memphis: α223Glu→Gln 2. Hb C:Lysine substitutes glutamic acid at the 6th position of the β chain ; α2β26Glu→Lys If Homozygous→ The two chains are (HbC dis.) involved If Heterozygous → one chain is involved 3.Hb E: β26Glu→Lys 7  26/11/2022 Sickle Cell Anemia The most common type of severe Hb-pathy ~ 8% of American blacks are heterozygous for HbS In Africa the gene frequency approaches 30% Protective effect of HbS against Plasmodium falciparum malaria !! HbS is common in Mediterranean, South & Central America, India. Hb S: Valine substitutes Glutamic acid at the 6th position of the β chain ; α 2 β2 6Glu→Val or α2 β2 6val or α2β2S 8  26/11/2022 Sickle Cell Anemia The term “Sickle cell disease” is used generically to describe a group of genetic disorders characterized by production of abnormal HbS Sickle cell disease include: HbSS disease, HbSC disease, HbS/β-thalassemia Homozygotes (sickle cell anemia or HbSS disease): all HgbA is replaced by HgbS (NO HgbA) , and approximately 80% Hgb S and 20% Hgb F (the compensatory hemoglobin) are seen. Hgb A2 is variable. Symptomatic Heterozygotes (sickle cell trait): only about half of HbA is replaced by HbS, asymptomatic Geographical Distribution of Hb Disorders 9  26/11/2022 Sickle Cell Anemia: Pathophysiology On deoxygenation, HbS undergo polymerization This change RBCs to elongated crescentic, or sickle, shape Low O2, low pH & dehydration promote sickling Sickling of RBCs is initially reversible by oxygenation membrane damage occurs with each episode of sickling, and eventually the cells accumulate Ca++, lose K+ and water, and become irreversibly sickled, despite oxygenation 10  26/11/2022 Sickle Cell Anemia: Pathophysiology Rigid sickled cells, Increase blood viscosity slows circulation hypoxia in small vasculature, causes lack of oxygen to the tissues, resulting in tissue necrosis. Sickled cells may plug small blood vessels low pH, PO2  increase # of sickle cells acute & chronic tissue damage  painful crisis & infarction of organs Presence of HbA & HbF in cells with HbS modify the degree of sickling Pathophysiology & Morphologic Consequences of Sickle Cell Anemia 1 2 11  26/11/2022 Factors Enhancing Sickling 1. Degree of oxygenation: HbAS sickle at 15 mm Hg, while Hb SS at 40 mm Hg. 2. Low pH: right shift, dec. affinity, inc. deoxy form 3. Low temp. →low blood flow inc. sickling. 4. Dehydration:→↓pH, (fever, vomiting diarrhea) 5. Infections: Pneumonia, → hypoxia. Factors Enhancing Sickling Cont. 6. Vascular stasis sickle cell inc. blood viscosity slows circulation, inc. time of exposure to hypoxic environment, promotes further sickling, infarction in spleen occur, leg ulcers & renal lesions 7. Osmolarity: Sickle cells in hypertonic medium, lose water, cell dehydrates, inc. intracellular Hb conc., inc. sickling. 8. Amount of Hb S in RBCs: Hb AS contain < 40% HbS & 60%HbA, but Hb SS has 80-100% Hb S. 9. An irreversible sickle cell has: ↑Hb conc., ↑Ca, ↓K, ↓ATP → short survival intravascularly. 12 26/11/2022 13  26/11/2022 Sickle Cell Anemia: Clinical Findings 3 types of “Sickle crises” Aplastic, associated with infection like parvovirus, resolve in 5-10 days. Indicator: low Retic Hemolytic, reflect acute exacerbation of anemia with a fall in Hb, Hct & increased Retic, jaundice. Usually caused by acute splenic sequestration. Indicators: sudden weakness, rapid pulse, pallor & enlarged spleen. Painful (vaso-occlusive), Caused by occlusion of small blood vessels mediated by adhesion of sickled cells to endothelium. Usually lasts 4-6 days but may last for weeks. It’s triggered by infection, fever, acidosis, dehydration SCD: Clinical Findings Organs at greatest risk: spleen, kidney & Bone Marrow where blood flow is slow, (kidney failure being a common outcome) hyposplenism and joint swelling also occur. Vaso-occlusive crisis occurs with increased bone marrow response to the hemolytic anemia. Crisis can be initiated by many physiological factors, including surgery, trauma, pregnancy, high altitudes, etc. Sickle Cell Anemia (HbSS disease) is diagnosed early in life & presents with severe chronic HA, Hb level of 6-8 g/dl, jaundice 14  26/11/2022 SCD: Sickle cells, target cells. Table 11–5 Clinical Manifestations of Sickle Cell Anemia Cutaneous manifestations (leg ulcers) Cardiac enlargement Joint and skeletal problems Arthritis Renal complications (renal papillary necrosis) Bone marrow infarctions Conjunctival vascular abnormalities Gastrointestinal symptoms Hepatomegaly Autosplenectomy Cholelithiasis Priapism (persistent, painful penile erection) 15  26/11/2022 Hematologic Nonhematologic Aplastic crisis Abnormal growth Hemolytic crisis Bone and joint abnormalities Vaso-occlusive crisis Pain Table 11–6 Salmonella infection Clinical Features Hand–foot dactylitis Genitourinary of Sickle Cell Renal papillary necrosis Anemia by Priapism Spleen and liver Category Autosplenectomy Hepatomegaly Jaundice Cardiopulmonary Enlarged heart Heart murmurs Pulmonary infarction Eye Retinal hemorrhage Central nervous system Leg ulcers Risky pregnancy Figure 11-6 Asthenic physique with mild jaundice. 16  26/11/2022 Figure 11-8 Hand–foot syndrome in a patient with sickle cell anemia. 17  26/11/2022 Table 11–7 Organisms Implicated in Causing Infections in Patients with Sickle Cell Anemia Bacterial Viral Fungal Parasitic Streptococcus pneumoniae Rubeola Coccidioides immitis Plasmodium Haemophilus influenzae Cytomegalovirus Histoplasma spp. Neisseria meningitidis capsulatum Mycoplasma pneumoniae Staphylococcus aureus Streptococcus pyogenes Mycobacterium tuberculosis Escherichia coli Salmonella spp Sickle Cell Trait Sickle cell trait is caused when valine replaces glutamic acid at position 6 on one beta chain Heterozygote form HbAS: One normal beta chain can produce some Hgb A approximately 40% Hgb S and 60% Hgb A are produced, with normal amounts of Hgbs A2 and F. Usually asymptomatic but occasionally episodes of hematuria & hyposthenuria, Anemia is rare but, if present, will be normochromic/normocytic, and sickling can occur during rare crisis states (same as in Hgb SS). severe respiratory infection, excessive exercise Drastic lowering of pH & O2 can precipitate a crisis This heterozygous trait is the most common hemoglobinopathy in the United States. Apparent immunity to Plasmodium falciparum 18  26/11/2022 Figure 11-9 Sickle trait (peripheral blood). Note the normal appearing smear. Diagnosis Diagnosis is made after 6 months of age (time of beta-gamma globin chain switch), Life expectancy of 50 years with proper treatment. Death usually results from infection or congestive heart failure. 19  26/11/2022 Lab Diagnosis: Tests for Diagnosis of Sickle Cell Anemia CBC Retic count PB smear Hb electrophoresis (alkaline/ cellulose acetate; acid/citrate agar) HbA2 and HbF measurement Lab Diagnosis: Tests for Diagnosis of Sickle Cell Anemia Hb 6-8 g/dl RBC indices: usually normochromic normocytic PB smear: numerous target cells, fragmented cells, polychromasia(resulting from premature release of reticulocytes), NRBCs, suckled cells. Siderotic granules, Pappenheimer bodies, and Howell-Jolly bodies. 20  26/11/2022 Lab Diagnosis: Tests for Diagnosis of Sickle Cell Anemia Retic count: 5-20% There may be neutrophilic leukocytosis with a shift to left & thrombocytosis BM: erythroid hyperplasia, except aplastic crisis SC trait: sickle cells are not present in PB smear, but may occur during a crisis episode Bone marrow erythroid hyperplasia (M:E ratio decreases) Increased bilirubin and decreased haptoglobin are characteristic due to hemolysis. SCD: Sickle cells, target cells. 21  26/11/2022 Where Do Sickle Cells Come From? Sheared in microcirculation Irreversible Sickle Cell 22  26/11/2022 Sickle Cells Lab Screening for Sickle Cell Anemia Screening of newborns for HbS has been established in many developed countries Cellulose acetate electrophoresis (alkaline pH), followed if necessary by citrate agar electrophoresis (acid pH) SC Anemia: HgbS> 80% & HgbF 1-20%, HgbA2 is slightly elevated with a mean of 3.4% (No Hgb A) SC trait: HgbA 60%, HgbS 40%, HgbA2 is usually elevated (3.6%). Hgb S migrates with hemoglobins D and G on alkaline hemoglobin electrophoresis can differentiate using acid electrophoresis. Isoelectric focusing (IEF) 23  26/11/2022 Lab Screening for SCD HPLC (DNA analysis) Sickling tests and HbS solubility test> are not suitable for newborns because of low HbS in newborns. The most appropriate screening test for hemoglobin S is Dithionite solubility (Insoluble). The most appropriate screening test for detecting hemoglobin F is Kleihauer-Betke Hb Electrophoresis 24  26/11/2022 Hb Electrophoresis Cellulose Acetate (pH 8.6) Hemoglobin Citrate Agar (pH 6.2) Hemoglobin Electrophoresis Electrophoresis HbS Sickling Test http://www.academic.marist.edu/~jzmz/topics/inclusions/inclusions33.html 25  26/11/2022 HbS Solubility Test Hgb SS( sickle cell anemia or sickle cell disease ) Positive hemoglobin solubility screening test Hgb AS (sickle cell trait) Positive hemoglobin solubility screening test Hemoglobin S is insoluble when combined with a reducing agent (sodium dithionite). Hgb S will crystallize and give a turbid appearance to the solution. The test will not differentiate homozygous from heterozygous conditions containing Hgb S. Follow up a positive solubility test with Rodak BF. Hematology: clinical principles and applications. Philadelphia: hemoglobin electrophoresis Saunders; 2002. Figure 11-11 Tube solubility screening test for sickle cell anemia. 26  26/11/2022 Treatment Exchange Blood transfusion for acute situations for prevention of certain complications like stroke Hyroxyurea Bone marrow transplantation Gene therapy Table 11–11 Treatment of Sickle Cell Anemia: Goals of Therapeutic Approaches Increase the production of fetal hemoglobin in the adult. Decrease microvascular entrapment of sickled cells. Modify the oxygen affinity or solubility of sickle hemoglobin. Change the volume of the sickle erythrocyte. Alter expression of the abnormal, sickle gene. Prevent endothelial damage. Counter oxidant-induced injury. 27  26/11/2022 HbC Disease & Trait Hgb C disease is caused when lysine replaces glutamic acid at position 6 on both beta chains. Defect is inherited from both parents HbC > α2β26Glu-Lys HbC is seen most frequently in Africa with incidence reaching up to 17- 28% in Ghana Clinical symptoms: mild chronic hemolytic anemia with splenomegaly RBC morphology: typically normocytic & normo- or hyperchromic, numerous target cells (50-90%); characterized by intracellular rodlike C crystals, occasionally microspherocytes and fragmented cells and folded cells HbC Disease & Trait Retic is slightly increased Hb electrophoresis/alkaline: – HbC disease: HgbC 90% plus 2% A2, HbF retinal hemorrhage, renal papillary necrosis Seen in African, Mediterranean, and Middle Eastern populations HbS with Other Abnormal Hbs: HbSC Disease Hgb SC disease Laboratory: Moderate to severe normocytic/normochromic anemia with target cells; characterized by SC crystals; may see rare sickle cells or C crystals; Positive hemoglobin solubility screening test Hb electrophoresis: HbS and C are separated at alkaline pH, HbF is 1% Causes: – Hb M variants (dominant inheritance) – NADH-methemoglobin reductase deficiency (recessive inheritance) – Toxic substance (acquired) 37  26/11/2022 Assessment Questions Assessment-Q1 Clinical manifestations of a homozygous mutation involving the beta-globin gene will most likely appear: A. During embryonic development B. In the neonate at birth C. No later than 3 weeks after birth D. By 6 months of age 76 38  26/11/2022 Assessment-Q1 Clinical manifestations of a homozygous mutation involving the beta-globin gene will most likely appear: A. During embryonic development B. In the neonate at birth C. No later than 3 weeks after birth D. By 6 months of age 77 Assessment-Q2 Which of the following statements about sickle cell syndromes is false? A. Asplenism may result from repeated sickling crises in the homozygous state. B. Heterozygous persons may be partly protected from infection by falciparum malaria. C. Hemoglobin S is more soluble in dithionite than is normal hemoglobin. D. Trait conditions are generally asymptomatic with no sickle cell formation. 78 39  26/11/2022 Assessment-Q2 Which of the following statements about sickle cell syndromes is false? A. Asplenism may result from repeated sickling crises in the homozygous state. B. Heterozygous persons may be partly protected from infection by falciparum malaria. C. Hemoglobin S is more soluble in dithionite than is normal hemoglobin. D. Trait conditions are generally asymptomatic with no sickle cell formation. 79 Assessment-Q3 Which of the following electrophoretic results is consistent with a diagnosis of sickle cell trait? A. Hgb A: 40% Hgb S: 35% Hgb F: 5% B. Hgb A: 60% Hgb S: 40% Hgb A2: 2% C. Hgb A: 0% Hgb A2: 5% Hgb F: 95% D. Hgb A: 80% Hgb S: 10% Hgb A2: 10% 80 40  26/11/2022 Assessment-Q3 Which of the following electrophoretic results is consistent with a diagnosis of sickle cell trait? A. Hgb A: 40% Hgb S: 35% Hgb F: 5% B. Hgb A: 60% Hgb S: 40% Hgb A2: 2% C. Hgb A: 0% Hgb A2: 5% Hgb F: 95% D. Hgb A: 80% Hgb S: 10% Hgb A2: 10% 81 Assessment-Q4 A cellulose acetate electrophoresis revealed a large band of hemoglobin in the hemoglobin S position. This band quantified at 95%. The peripheral smear revealed 70% target cells, and the solubility test was negative. Based on this information, what is the hemoglobin?L A. Hemoglobin C B. Hemoglobin D C. Hemoglobin E D. Hemoglobin S 82 41  26/11/2022 Assessment-Q4 A cellulose acetate electrophoresis revealed a large band of hemoglobin in the hemoglobin S position. This band quantified at 95%. The peripheral smear revealed 70% target cells, and the solubility test was negative. Based on this information, what is the hemoglobin?L A. Hemoglobin C B. Hemoglobin D C. Hemoglobin E D. Hemoglobin S 83 Assessment-Q4 Hereditary pyropoikilocytosis (HP) is a red cell membrane defect characterized by: A. Increased pencil-shaped cells B. Increased oval macrocytes C. Misshapen budding D. Bite cells 84 42  26/11/2022 Assessment-Q5 A patient with suspected sickle cell trait has negative solubility test results, but hemoglobin electrophoresis at pH 8.6 shows an apparent A-S pattern. What is the most likely explanation? A. Patient has hemoglobin AS, and the solubility test is incorrect. B. Patient has hemoglobin AA, and the electrophoresis is incorrect. C. Patient has hemoglobin AD or AG, and both procedures are correct. D. Tests need to be repeated; impossible to determine which procedure is correct. 85 Assessment-Q5 A patient with suspected sickle cell trait has negative solubility test results, but hemoglobin electrophoresis at pH 8.6 shows an apparent A-S pattern. What is the most likely explanation? A. Patient has hemoglobin AS, and the solubility test is incorrect. B. Patient has hemoglobin AA, and the electrophoresis is incorrect. C. Patient has hemoglobin AD or AG, and both procedures are correct. D. Tests need to be repeated; impossible to determine which procedure is correct. 86 43  26/11/2022 Assessment-Q6 A native of Thailand has a normal hemoglobin level. Hemoglobin electrophoresis on cellulose acetate shows 70% hemoglobin A and approximately 30% of a hemoglobin with the mobility of hemoglobin A2. This is most consistent with hemoglobin: A. C trait B. E trait C. O trait D. D trait 87 Assessment-Q6 A native of Thailand has a normal hemoglobin level. Hemoglobin electrophoresis on cellulose acetate shows 70% hemoglobin A and approximately 30% of a hemoglobin with the mobility of hemoglobin A2. This is most consistent with hemoglobin: A. C trait B. E trait C. O trait D. D trait 88 44  26/11/2022 End 45