Lecture 7-Chapters 24 PDF

LECTURE 7: CHAPTERS 24 HEMOGLOBINOPATHIES Jahan Abdi |Clinical Science |Fall 2024  LEARNING OBJECTIVES. What laboratory test would differentiate between the hemoglobinopathies?. What is the most prevalent hemoglobinopathy?. What is the diffe...

LECTURE 7: CHAPTERS 24 HEMOGLOBINOPATHIES Jahan Abdi |Clinical Science |Fall 2024  LEARNING OBJECTIVES. What laboratory test would differentiate between the hemoglobinopathies?. What is the most prevalent hemoglobinopathy?. What is the difference between a disease and a trait in the hemoglobinopathies?. Discuss the genetic variations in the hemoglobinopathies.  HEMOGLOBIN DEVELOPMENT Composition of human HgB Globin chains and HgB in embryo Globin chains and HgB in fetus Loading… Globin chains and HgB at birth Globin chains and HgB in adults  HEMOGLOBINOPATHIES PATHOPHYSIOLOGY Definition: abnormality in Hb structure and function due to a mutation in one or more globin genes Factors affecting Hb function – Type of amino acid substituted homozygous has disease that is clincally apparent – Location in globin chain heterozygous is normal – Number of genes mutated pattern of inheritance Impact of zygosity: if the patient is homozygous (aka disease), clinical picture is more severe than when they are heterozygous (aka trait). SS is sickle cell disease, AS is sickle cell trait both parents are carriers = risk for patient and children might inherit both (homozygous which is a disease) if one parent is normal and other is carrier = heterozygous  HEMOGLOBINOPATHIES CLASSIFICATION Homozygous (most common variants): sickle cell disease Hb S: α2β26Val (severe hemolytic anemia; sickling) Hb C: α2β26Lys (mild hemolytic anemia) Hb D-Punjab: α2β2121Gln (no anemia) beta chain Hb E: α2β226Lys (mild microcytic anemia) Heterozygous: Hb variants causing functional aberrations or hemolytic anemia in the heterozygous state Loading… Hemoglobins associated with methemoglobinemia and cyanosis: Hb M-Saskatoon (α2β263Tyr) Hemoglobins associated with increased oxygen affinity: Hb Bethesda: α2β2145His Hemoglobins associated with decreased oxygen affinity: Hb Kansas: α2β2102Thr chronic anemia / bm makes enough Unstable hemoglobins: RBC but are lysed bc of hg dysfunction Hb precipitates as Heinze bodies (Hb Hammersmith, Hb Zurich, Hb Ann Arbor) Tetramer of normal chains (only in alpha thalassemia): HbH (β4), Hb Bart (γ4)  HEMOGLOBIN S Etiology – Structural formula: α2β26Glu→Val Trait: AS Disease: SS – Frequency in population Globally the sickle cell gene occurs at the highest frequency in five geographic areas: sub-Saharan Africa, Arab-India, the Americas, Eurasia, and Southeast Asia Incidence with malaria – Malaria species involved: SCT (AS) gives protection against Plasmodium falciparum – Mechanism: Selective destruction of RBCs containing parasites decreases the number of malarial organisms and increases the time for immunity to develop HEMOGLOBIN S two varientes of hg when two diff varieties are in both parents Pathophysiology HEMOGLOBIN S Shape of red blood cells (RBCs) Oxygenated Deoxygenated Process of sickling Homozygous state (disease): when oxygen saturation decreases to less than 85% Heterozygous state (trait): sickling does not occur unless the oxygen saturation of hemoglobin is reduced to less than 40% Reversible sickling: Hb S-containing RBCs that change shape in response to oxygen tension, responsible for vasoocclusive crises Irreversible sickle cells do not change their shape regardless of the change in oxygen tension or degree of hemoglobin polymerization, seen on PBS as actual sickle cells  HEMOGLOBIN S Clinical features – Age when symptoms begin: 6 months after birth – Types of crises: vasoocclusive or “painful,” splenic sequestration (dropping of Hb to below 6g/dL, mostly in infants and young adults, could lead to autosplenectomy), chronic hemolytic, megaloblastic (due to folate depletion), and aplastic (most common life-threatening hematologic complications and are usually associated with parvovirus B19 infection) – Risk factors for initiation of vasoocclusion: polymerization, decreased deformability, sickle cell-endothelial cell adherence, WBC and platelet activation. Triggered by acidosis, hypoxia, dehydration, infection and fever, and exposure to extreme cold  HEMOGLOBIN S Laboratory diagnosis – Type of hemolytic anemia – Blood smear evaluation – Reticulocyte count (usually high, if low, signal of aplastic crisis) – Mean corpuscular volume (MCV) and RBC distribution width (RDW) indices – Cell counts: leukocytosis (neutrophilia), thrombocytosis – Bone marrow studies HEMOGLOBIN S Loading… oat shape that will eventually move to sickle cell a transitional shape  HEMOGLOBIN S Laboratory diagnosis – Tube solubility test Principle: deoxygenated Hb S less soluble in solution generating add blood and hold card behind turbidity (reducing agent sodium dithionite) and if you can't see through = + = turbid False negatives: infants younger than 6 months and in low HCT False positives: hyperlipidemia, too much blood added to the tube – Hb electrophoresis Principle: separation of hemoglobin molecules in an electric field primarily as a result of differences in total molecular charge Alkaline (pH 8.4) electrophoresis: hemoglobin molecules assume a negative charge and migrate toward the anode (on cellulose acetate or recently agarose) Acid (pH 6.2) electrophoresis: some hemoglobins assume a negative charge and migrate toward the anode, whereas others are positively charged and migrate toward the cathode. HEMOGLOBIN S A is fastest S is a bit slower  most hg will migrate together A follows F negative positive  VERY IMPORTANT HB S ELECTROPHORESIS Hb S migrates with Hb D and Hb G on alkaline electrophoresis but separates from Hb D and Hb G on acid which will migrate together and which electrophoresis. will separate due to acidic electrophoresis fastest Hb D and Hb G are further differentiated S + D + G all migrate all at once = retest A2 for alpha thalasemia minor from Hb S in that they produce a negative result on the hemoglobin solubility test. Similarly, Hb C migrates with Hb A2, Hb E and Hb O on alkaline do acidic electrophoresis to help migrate electrophoresis but separates on acid clump electrophoresis HB S ELECTROPHORESIS FIGURE 24.8. Electrophoretic separation of Hb at Alkaline pH. 1: Normal adult make only hg A 2 and 3: 17-year-old patient with sickle cell anemia (Hb SS) only hg S for 2 + 3 = SS sickle cell 5 and 6: patient with sickle cell anemia, recently transfused (note the presence of Hb A from the 5 + 6 has AS transfused red blood cells) 4 and 7, Hbs A/F/S/C standard. SICKLE CELL TRAIT (SCT) The term sickle cell trait (SCT) refers to the heterozygous state (Hb AS) and describes a benign condition that generally does not affect mortality or morbidity except under conditions of extreme exertion PBS: Normal RBC morphology, with the exception of a few target cells. No abnormalities in the leukocytes and thrombocytes are seen. The hemoglobin solubility screening test yields positive results, and SCT is diagnosed by detecting the presence of Hb S and Hb A on hemoglobin electrophoresis or HPLC. In individuals with SCT, electrophoresis reveals approximately 40% or less Hb S and approximately 60% or more Hb A, Hb A2 level is normal or slightly increased, and Hb F level is within the reference interval.  HEMOGLOBIN C also called bar of gold It is the most common non-sickling variant encountered in the United States and the third most common in the world. α2β26Glu→Lys Unlike HbS the structure of Hb C polymers differ and they form under high oxygen tension. Hb S polymers are long and thin, whereas the polymers in Hb C form a short, thick crystal within the RBCs. Homozygous hemoglobin C disease (Hb CC) manifests as a milder disease compared with SCD. Mild splenomegaly and hemolysis may be present. Vasoocclusive crises do not occur. Heterozygous hemoglobin C trait (Hb AC) is asymptomatic.  HEMOGLOBIN C Laboratory diagnosis – Type of hemolytic anemia: mild to moderate, normocytic normochromic in homozygous Hb C disease – Blood smear evaluation: occasional micro/hypo, marked increase in target cells, nRBC – Reticulocyte count: slightly to moderately high (2-3%) – Crystals (“bar of gold”) – HgB electrophoresis: – No Hb A is present in Hb CC disease: Hb C > 90%, Hb F at less than 7% and Hb A2 at approximately 2%. – In Hb AC trait, about 60% Hb A and 30% Hb C are present. – On alkaline hemoglobin electrophoresis Hb C migrates in the same position as Hb A2, Hb E, and Hb O-Arab  HEMOGLOBIN E Features of HgB – Mostly in southeast Asia – Amino acid substitution: α2β226Glu→Lys Clinical features – Trait versus disease: EE mild anemia with microcytes and target cells, needs to be differentiated from IDA, beta-thal minor, trait (AE) asymptomatic – Malaria: because the highest incidence of the Hb E gene is in areas of Thailand where malaria is most prevalent, it is thought that P. falciparum multiplies more slowly in Hb EE RBCs than in Hb AE or Hb AA RBCs and that the mutation may give some protection against malaria.  HEMOGLOBIN E Negative hemoglobin solubility test, must be confirmed using electrophoresis or HPLC. Hb E disease (homozygous): >90% Hb E, mild anemia (hemoglobin between 11.0 to 13.0 g/dL), a very low MCV (55 to 65 fL), few to many target cells, and a normal reticulocyte count. The heterozygous state (AE) has normal hemoglobin levels, a mean MCV of 65 fL, slight erythrocytosis, target cells, and approximately 25% to 30% Hb E. On alkaline hemoglobin electrophoresis, Hb E migrates with Hb C, Hb O, and Hb A2. On citrate agar electrophoresis at an acid pH, Hb E can be separated from Hb C, but it comigrates with Hb A and Hb O  DOUBLE HETEROZYGOSITY WITH HEMOGLOBIN S Hb SC: most common compound heterozygous syndrome – Hb features Amino acid substitutions Ethnic distribution: 25% in West Africa, 1 in 833 births per year in the U.S. – Clinical features: milder than SS, may cause vasooclusion, – Laboratory features CBC: a mild normocytic, normochromic anemia with many features associated with sickle cell anemia. The hemoglobin level is usually 11 to 13 g/dL, and the reticulocyte count is 3% to 5% Blood smear: few sickle cells, target cells, and intraerythrocytic SC crystals Hb solubility test is positive Electrophoresis results: Hb C and Hb S migrate in almost equal amounts (45%) on alkaline electrophoresis, and Hb F is normal