Hemoglobinopathies: Qualitative Defects

Hemoglobin % Sickle Cell Disease

A 0 A2 N F 5-20 S 80-95 C 0

Hemoglobin % Sickle Cell Trait

A 50-65% A2 N F N S 35-45 C 0

Hemoglobin % Normal Adult

A 97 A2 3 F <1 S 0 C 0

Hemoglobinopathy

Abnormality Qualitative abnormality. Abnormality in amino acid sequence of globin chain, not in amount of globin produced Examples Sickle Cell Anemia and trait Hemoglobin C disease and trait

Which of the following best describes a hemoglobinopathy? A) Any problem involving hemoglobin destruction. B) Any problem associated with hemoglobin production. C) A deletion of the loci of one or more hemoglobin chains. D) A substitution of an amino acid in the hemoglobin chain.

D) A substitution of an amino acid in the hemoglobin chain. A hemoglobinopathy is a substitution of an amino acid in the hemoglobin chain. When an amino acid substitution occurs, a new protein is formed. Hemoglobin destruction or problems with hemoglobin production are not exclusive to hemoglobinopathies. Other anemias that are not considered hemoglobinopathies may also involve increased hemoglobin destruction or problems with hemoglobin production. However, other anemias demonstrating hemoglobin production problems do not form an abnormal hemoglobin protein. Deletion of a hemoglobin chain loci results in decreased production of that chain (thalassemia), and not production of a different hemoglobin protein.

The gene loci for the alpha globin chains are adjacent to the locus for which other globin chain? A) Beta B) Delta C) Epsilon D) Zeta

D) Zeta The order of globin gene loci on chromosome 16 is Zeta, Alpha 2, and Alpha 1. Beta globin gene, delta globin gene, and epsilon globin genes are found on chromosome 11.

HbS

Peripheral Blood Homozygotes Normocytic, normochromic anemia; reticulocytosis, poikilocytosis with sickled and boat-shaped cells Hb Present on Electrophoresis Homozygous: HbS, F, A2 Heterozygous: HbA, S, F, A2 Mutation β6(A3) Glu--->Val Geographic Distribution Tropical Africa and Mediterranean areas, Middle East, India, Nepal

What is Sickle Cell Anemia due to?

due to the production of a mutant beta globin prone to sickling

What is Basophilic Stippling?

Description -Round or irregularly shaped granules of variable number and size, distributed throughout the RBC -Composed of aggregates of ribosomes (RNA) -Stain bluish-black with Wright's stain Associated Disease States -Lead Poisioning -Anemias associated with Abnormal hemoglobin synthesis -Thalassemia

(- -/ -α) α^0/ α^+

Genotypic Description Heterozygous α-thalassemia-1/α-thal-2 Phenyotype Hemoglobin H disease Labs Moderate to marked Anemia Target Cells Basophilic Stippling Microcytic Hypochromic Severity Chronic, moderately severe hemolytic anemia Hemoglobins Present Birth = Hb Bart Adult= HbH

What inclusion would result from mothball poisoning?

Heinz Body

A 4-year-old girl is brought to her pediatrician for a check up. The child's skin is slightly jaundiced, and she has mild splenomegaly. Her Hemoglobin and Hematocrit are reduced. Her mean corpuscular volume is 90um^3, and her reticulocyte count is 7%. A Coombs test is performed and is negative. A hemoglobin electrophoresis shows an abnormal component, constituting less than 25% of the total. A blood smear shows inclusion bodies within the RBCs. Which of the following is the most likely diagnosis? a) Beta thalassemia b) Heinz body anemia c) Hereditary spherocytosis d) Pernicious anemia e) Sickle cell anemia

b) Heinz body anemia Heinz body anemia is an autosomal dominant disease that causes an abnormal hemoglobin variant. The mutant hemoglobin precipitates within the RBC, forming an inclusion, or Heinz body. The RBC is then either phagocytosed or removed by the spleen. Patients present with hemolytic anemia, jaundice, splenomegaly, and dark colored urine. The MCV is normal, and electrophoresis usually reveals the abnormal component as a small percentage of the total. The reticulocyte count is increased as the body tries to make new blood to compensate. If the Coombs test were positive, this would indicated the problem was extracorpuscular

The most appropriate screening test for hemoglobin S is: a) Kleihauer-Betke b) dithionite solubility c) heat instability d) fluorescent spot

b) dithionite solubility The solubility test is a rapid test for Hb S. The presence of Hb S is confirmed by hemoglobin electrophoresis

A patient has the following blood values: TEST RESULT RBC 6.5X10^6/uL Hgb 13.0g/dL HCT 39.0% MCV 65fL MCH 21.5pg MCHC 33% These results are compatible with: a. Iron deficiency b. Pregnancy c. Thalassemia minor d. Beta thalassemia major

c

This hemoglobinopathy results from a fusion product of the delta and beta gene: a) Hgb D b) Hgb G c) Hgb Lepore d) Hgb Constant Spring

c) Hgb Lepore In Hgb Lepore, a non-alpha chain is a delta/beta globin hybrid in which the N-terminal end of a delta chain is fused to the C-terminal end of a beta chain

The hemoglobin variant which is seen frequently in the South East Asian population, demonstrates a microcytic blood smear, and migrates with HgbC at pH 8.6 is: a) HgbBarts b) HgbF c) HgbE d) HgbH

c) HgbE On alkaline electrophoresis, HgbE migrates with HgbA2, HgbC and HgbOarab. HgbE is seen commonly in Southeast Asia and presents with a microcytic, hypochromic anemia

Which of the following is characteristic of Hemoglobin H? a) it is a tetramer of gamma chains b) it is relatively stable c) electrophoretically, it represents a "fast" hemoglobin d) it has a lower oxygen affinity than hemoglobin A

c) electrophoretically, it represents a "fast" hemoglobin HgbH represents a fast migrating hemoglobin

A characteristic morphologic feature in hemoglobin C disease is: A) Macrocytosis B) Spherocytosis C) Rouleaux formation D) Target cells

D

True/False In Sickle Cell Disease there is No HbA on hemoglobin electrophoresis.

True

Sickle Cell Anemia pathophysiology

-caused by a mutation (GAG--->GTG conversion) in the HBB gene (hemoglobin Beta gene) -results in the substitution of nonpolar valine for polar glutamic acid -AA subsitution on surface results in a net decrease in negative charge -decreases the molecules electrophoretic mobility -Solubility of HbS in deoxygenated state is markedly reduced -tendency for deoxyhemoglobin S molecules to polymerize into rigid aggregates

Hb H Disease

3 gene deletions ---high 02 affinity and lack of heme-heme interaction 4 Beta chains Severe hemolytic anemia --due to presence of excess Beta-chain inclusions --Macrophage destruction of RBCs w/ inclusions --Signal for apoptosis of RBC Hgb: 8-10g/dL Chronic hemolysis w/ variable jaundice and cholethiasis (bilirubin stones) Hb Barts 25% HbA: Decreased HbA2: Decreased HbF: Decreased

What is Hb Barts?

4 gamma chains

Which of the following is consistent with the diagnosis of heterozygous beta-thalassemia? A) Increased Red blood cell count B) High MCV C) Decreased HbA2 D) Decreased Iron stores

A) Increased Red blood cell count The RBC count is increased in heterozygous beta thalassemia more than expected for the hemoglobin concentration

Why are symptoms of Beta-thalassemia major delayed until approximately 6th month of life?

Beta-thalassemia major is characterized by ABSENCE of synthesis of Beta-genes ---Although synthesis of Beta-gene begins at approx 3rd trimester of fetal development, quantities sufficient to assemble significant amounts of HbA do not occur until 6 months of age ---The lack of production of Beta-chains and resultant lack of HbA does not manifest until 6 months of age when HbA synthesis is expected

The osmotic fragility test would be most useful for the diagnosis of which of the following disorders? A) Alpha thalassemia minor B) Beta thalassemia major C) Hereditary spherocytosis D) Sickle cell anemia E) Sickle cell trait

C) Hereditary spherocytosis Hereditary spherocytosis is due to an intrinsic abnormality in the membrane support structure of the red cell. A distinctive feature of this disorder is marked susceptibility of the red cells to lysis in hypotonic solutions. This feature is the basis of the osmotic fragility test, which is quite specific for spherocytosis

In a patient with an increased red cell mass into the 99th percentile and serum erythropoietin level below reference range for normal, which of the following criteria confirms a diagnosis of polycythemia vera? A) bone marrow panmyelosis B) inv(16) mutation C) JAK2 V617F mutation D) BCR-ABL 1 translocation

C) JAK2 V617F mutation Polycythemia vera diagnostic criteria include the presence of the JAK2 V617 mutation as major criteria

Which of the following genotypes is associated with the hydrops fetalis form of thalassemia? Beta Chain Genes Alpha-Chain Genes A) Four abnormal Two normal B) Two abnormal Four normal C) Two normal Four abnormal D) Two normal Two normal

C) Two normal Four abnormal Hydrops fetalis is a form of Alpha-thalassemia, which results from deletion of the four genes responsible for Alpha-chain synthesis. Deletion of one gene produces a silent carrier. Deletion of two genes causes alpha-thalassemia trait (with a mild hypochromic anemia; choice D) Deletion of three genes results in HbH disease (with hemolysis), and deletion of four genes leads to the condition known as hydrops fetalis and death in utero. Beta-thalassemia is due to a genetic abnormality at a single site that results in defects in mRNA processing. Since a person recieves genetic material from both parents, the patient can have 0,1 or 2 abnormal genes at this site but not four(choice A) Homozygotes for beta-thalassemia (choice B) have beta-thalassemia major Heterozygotes have beta-thalassemia minor.

HbH Disease Electrophoresis

Elevated None Decreased HbA HbF HbA2 Abnormal Hb Bart's HbH

Target Cell in 3 disorders

Thalassemia Hemoglobinopathy IDA

What is Hydrops fetalis?

"generalized edema of the fetus" 4 gene deletions Gamma chains of fetal hemoglobin will form tetramers called Hb Barts ----very high O2 affinity ----no O2 released to perfuse the tissues Intrauterine death occurs

HbH pathophysiology

-Symptomatic but nonfatal -Dramatic reduction in α-chain synthesis -----results in decrease HbA, HbA2, HbF -excess Beta chains -----Beta tetramers===HbH -Thermolabile, unstable, precipitate inside RBC -Reduce deliver O2 to tissue -High O2 Affinity attributed to lack fo heme-heme interaction and absence of bohr effect

Which of the three normal adult hemoglobins would be affected in hydrops fetalis?

Hydrops fetalis occurs when there is a deletion in all four Alpha genes; therefore, all three normal adult hemoglobins, HbA, HbA2, and HbF are affected because each contains an Alpha chain.

A child's parents both have sickle cell trait. The physician orders a hemoglobin electrophoresis on the child. Results of electro- phoresis on cellulose acetate at pH 8.4 show 65% HbS, 30% HbA, 3% HbF, 2% HbA2. Explain these results, and suggest further testing that could help in diagnosis:

In sickle cell trait, the amount of HbS is usually less than 50%, whereas in sickle cell anemia, it is typically >90%. The presence of some HbA suggests sickle cell trait in this patient. The patient could have inherited another abnormal hemoglobin gene such as thalassemia. Electrophoresis at acid pH should be performed to eliminate the possibility of hemoglobins with similar mobilities at alkaline pH. DNA testing can provide evidence of thalassemia. A microcytic hypochromic anemia is also a clue to the presence of thalassemia.

α-Thalassemia trait Labs

Increased RBC Count Decreased HbA HbA2 HbF

Hemoglobin A2

Molecular Structure 2 alpha 2 delta Adult Reference Value 1.5%-3.7%

Hemoglobin F

Molecular Structure 2 alpha 2 gamma chains Adult Reference Value <2%

Hemoglobin A

Molecular Structure 2 alpha chains 2 Beta chains Adult Reference Value >95%

Hemoglobin S

Molecular Structure Valine subsituted for glutamic acid in 6th position of Beta chain Adult Reference Value 0

Target Cells are typically observed in what 3 disorders?

Thalassemia Hemoglobinopathy Iron-Deficiency Anemia

Differentiate the etiology of thalassemias and hemoglobinopa- thies.

Thalassemia results from deletional or nondeletional mutations in one or more globin genes that reduce or eliminate synthesis of the corresponding globin chain(s). Hemoglobinopathy is a qualitative hemoglobin disorder caused by a point mutation in a globin gene, usually the b@gene that results in an amino acid substitution in the corresponding globin chain and alters hemoglobin stability and function.

Why do newborns with sickle cell anemia not experience epi- sodes of vaso-occlusive crisis?

The predominant hemoglobin in newborns is HbF, which does not sickle. HbS does not become the predominant hemoglobin until after the first 6 months of life.

Thalassemias are characterized by: a) structural abnormalities in the hemoglobin molecule b) absence of iron in hemoglobin c) decreased rate of heme synthesis d) decreased rate of globin synthesis

d

The presence of hemoglobin H may be demonstrated by: a) Prussian Blue stain b) Wright Stain c) Giemsa stain d) Brilliant cresyl blue

d) Brilliant cresyl blue HgbH inclusions are seen after incubation of blood with brilliant cresyl blue and give the RBC the appearance of a golf ball. [Mckenzie 2015, p 295]

What is beta-thalassemia due to?

due to the production of inadequate levels of beta globin

Hb H treatment

long term transfusion therapy and splenectomy Iron chelation therapy avoids iron overload and toxicity or Bone Marrow transplant