The Hemoglobinopathies

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Chapter 11

• The Hemoglobinopathies

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1. Study Questions2. Homework Assignment3.
Exam for Unit 1
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The Hemoglobinopathies

• In Chapter 11 you will be introduced to the
  various hemoglobinopathies.  You will learn about
  the pathophysiology, clinical signs and symptoms,
  laboratory test results, and treatments for these
  disorders.  Be sure you learn the peripheral
  blood characteristics that make each
  hemoglobinopathy unique.  Also, remember which
  amino acid substitution creates which abnormal
  hemoglobin.

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Introduction to Hemoglobinopathies
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Introduction to Hemoglobinopathies1 of 3

• Qualitative abnormalities in globin structure,
  usually involving beta-chain.
• Most arise from single amino acid substitution. 
  Example is substitution of valine for glutamic
  acid in sixth position of beta chain producing
  Hemoglobin S (sickle cell anemia).
• Rarely see multiple substitutions.
• May or may not cause abnormal laboratory test
  results.

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Introduction to Hemoglobinopathies2 of 3

• Hemoglobin variant abnormal hemoglobin caused
  by abnormal globin chain structure.
• Hemoglobinopathy condition produced by
  hemoglobin variant. Confirmed by lab tests.
• Qualitative globin chain abnormality amino acid
  sequence wrong.
• Quantity of hemoglobin produced normal.

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Introduction to Hemoglobinopathies3 of 3

• Most hemoglobinopathies caused by single point
  change in amino acid sequence in globin chain. 
  Abnormalities caused by 
• Substitution
• Deletion
• Addition
• Hb variants follow Mendelian genetics.

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Broad Classification System for Hemoglobin
Disorders

• Qualitative
• Hemoglobins differ in sequence of amino acids
  composing globin chain. 
• Disorders called hemoglobinopathies.
• Quantitative
• Characterized by decreased production of
  hemoglobin resulting from decreased synthesis of
  one particular globin chain. 
• Called thalassemia  (Chapter 12).

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Better Classification System has Five Categories

• 1. Abnormal hemoglobins without clinical
  significance.
• 2. Aggregating hemoglobins (sickle cell anemia).
  
• 3. Unbalanced synthesis of hemoglobin
  (thalassemia).
• 4. Unstable hemoglobins.
• 5. Hemoglobins with abnormal heme function.

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Nomenclature

• Began by using letters of alphabet, soon ran out
  of letters.
• Changed to using names of places. Use
  combination of letter plus a place name. For
  Example, HbCHarlem.
• Description of variant can include chains and
  substitution. For example, homozygous Hb S is
  a2ß2S or a2ß26Val or a2ß26Glu-Val.

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Laboratory Tests and Findings

• Laboratory findings are variable.  Depends upon
  which hemoglobinopathy is being tested.
• Tests include
• CBC
• Sickle Cell Screening (Dithionite Test)
• Hemoglobin Electrophoresis
• Supravital stains for Heinz bodies
• Hb F and Hb A2 quantitation

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Dithionite Test for Sickle Cell

• Screening test for Sickle Cell Disease
• Fast
• Cheap
• Fairly accurate
• Procedure
• RBCs are lysed by saponin.
• Sodium dithionite binds and removes O2.
• HbS precipitates out.
• Solution becomes turbid. 

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Hemoglobin Electrophoresis

• Use EDTA whole blood.  Make a hemolysate by
  lysing RBCs.
• Cellulose acetate is support medium.
• Perform electrophoresis using buffer at pH 8.4.
  
• Stain is Ponseau S.
• Confirm with citrate agar electrophoresis at
  acidic pH.
• Textbook page 174

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Alkali Denaturation Test for Fetal Hemoglobin

• HbF resists denaturation by alkali.
• Spectrophotometric analysis.
• Calculated as total hemoglobin.
• Use whole blood EDTA. 
• Hemoglobin F less than 1 after 1 year of age.

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Sickle Cell Anemia and Sickle Cell Trait
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Introduction to Sickle Cell Anemia

• Is worldwide disorder.
• Autosomal co-dominant Both Hemoglobin A and
  Hemoglobin S produced.
• AS is sickle cell trait.
• SS is sickle cell disease.  Patient is homozygous
  for HbS (SS).  Results in very severe anemia.
• Probably originated in Africa.
• Hb S is point mutation for sixth amino acid in
  Beta chain. Valine substituted for glutamic
  acid. One benefit for AS persons is increased
  resistance to malaria.

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Incidence of Sickle Cell Disease

• 1 of 375 African-American live births have
  hemoglobin SS (Sickle Cell Disease) Have 85
  chance to live to age 20.
• 8-10 of American blacks carry the Hb S trait
  (heterozygous).
• Affects more than 50,000 Americans.
• In Africa, half of infants with sickle cell
  disease die in the first year of life.

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Pathophysiology of Sickle Cell Anemia 1 of 5

• SS cells may look normal when fully oxygenated
  Sickling occurs when O2 decreased.
• Other causes of sickling include decrease in pH
  and dehydration of patient.
• Cells become rigid, impeding blood flow to
  tissues. Tissue death, organ infarction, and
  pain result. 
• Sickling is reversible up to a point.
• Have both extravascular hemolysis and
  intravascular hemolysis.

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Pathophysiology of Sickle Cell Anemia 2 of 5

• Usually diagnosed early in life.
• Have all physical symptoms of anemia.
• Growth and sexual maturation slower.
• Crisis very painful. Anything that
  deoxygenates blood acts as trigger (exercise,
  illness and airplane flights). Sickle cells get
  stuck in capillaries.

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Pathophysiology of Sickle Cell Anemia 3 of 5

• Have three types of crises
• Aplastic crisis  associated with infections
  which causes temporary suppression of
  erythropoiesis.
• Hemolytic crisis  Results in exaggerated anemia.
  
• Vaso-occlusive crisis  Associated with severe
  pain.  Is hallmark symptom of sickle cell anemia.

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Pathophysiology of Sickle Cell Anemia 4 of 5

• Hands and feet swell (dactylitis), fingers grow
  at different rates.
• Joint pain in arms and legs first. May affect
  lungs.  Have chest pain and abdominal pain.
• Spleen enlarges cells become trapped. Have
  decreased blood volume (hypovolemia), and shock.
  Repeated splenic infarcts cause splenic
  dysfunction, increasing susceptibility to
  infection.

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Pathophysiology of Sickle Cell Anemia 5 of 5

• Organs Affected
• Liver Enlarges, malfunctions, jaundice,
  hyperbilirubinemia.
• Heart  Cardiomegaly, iron deposits.
• Spleen  Enlarges leading to infarction and
  fibrosis. Eventually shrivels and becomes
  nonfunctional.
• Skin Develop ulcers.
• Kidney  Hematuria and eventual failure.
• Lungs Infarction.
• Brain  Strokes.

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Peripheral Blood Findingsin Sickle Cell Anemia

• Hb 6-8 g/dL severe anemia.
• Marked aniso and poik.
• Sickle cells and Target cells.
• Ovalocytes and Schistocytes.
• NRBCs with Polychromasia.
• Increased Retics.
• Basophilic Stippling.
• Howell Jolly Bodies and Pappenheimer Bodies.
• Leukocytosis with left shift and Thrombocytosis.

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Special Hematology Tests in Sickle Cell Anemia

• Electrophoresis Hb S present.
• Sickling Test Positive.
• Osmotic Fragility Decreased.
• Sed Rate Decreased.

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Chemistry Testsin Sickle Cell Anemia

• LDH increased.
• Bilirubin Indirect and total both increased.
• Haptoglobin decreased.

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Treatment and Prognosis for Sickle Cell Anemia

• Improving.
• Prevent crisis.
• If crisis keep hydrated.   Alleviate pain.
• Treat infections with antibiotics.  Keep warm. 
  Blood transfusion if needed.
• Prophylactic penicillin treatment prevents most
  deaths due to pneumococcal infections.
• RBC exchanges are beneficial.
• Developing anti-sickling agents (hydroxyurea)
  which increases amount of Hemoglobin F in red
  cells.  Decreases amount of sickling that occurs
  during crisis.
• Bone marrow transplants in children show promise.
  
• Future treatment may include gene therapy.

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Sickle Cell Trait

• Heterozygous AS with more HbA than HbS, so
  condition is compensated for.
• Normal life, except if have severe hypoxia or
  respiratory infection.
• Patient often has normal life span.

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Laboratory Findings in Sickle Cell Trait

• Normal CBC Few target cells or sickle cells may
  be present.
• Sickle solubility test positive.
• Electrophoresis Both A and S present.

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Hemoglobin C Disease
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Introduction to Hb C Disease

• Amino acid substitution of lysine for glutamic
  acid at sixth position of Beta chain
  (a2ß26Glu-Lys).
• Is homozygous CC. 
• HbC crystals on peripheral blood.
• Second most common variant after HbS.
• Found almost exclusively in Black population.
• Chronic hemolytic anemia with associated
  splenomegaly and abdominal discomfort.

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Laboratory Findings in Hb C Disease

• Mild to moderate anemia (8-12 g/dL), splenomegaly
  and abdominal discomfort.
• Numerous target cells, few microspherocytes,
  schistocytes, and folded cells.
• May see hexagonal or rod-shaped crystals ("bar of
  gold).  Usually intracellular. Are elongated
  with blunt ends and parallel sides.
• Retic count 4-8 (slightly increased).
• Electrophoresis Most hemoglobin is HbC no HbA
  present may or may not have increase in Hb F.

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Hemoglobin C Trait (AC)

• No symptoms no anemia. 
• Target cells frequent finding.

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Treatment for Hb C

• Splenectomy may be beneficial for symptomatic CC
  homozygous persons.
• AC heterozygous persons are usually asymptomatic,
  so no treatment required.

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Hemoglobin D Disease
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Hb D Disease

• Homozygous and heterozygous forms.
• Migrates with HbS upon electrophoresis, but does
  not cause sickling of RBCs.
• Many variants All are rare forms.
• Both heterozygous and homozygous states are
  asymptomatic.
• No clinical (physical) abnormalities.

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Hemoglobin E Disease
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Hb E Disease

• ß chain variant lysine substituted for glutamic
  acid in 26th position in beta chain
  (a2ß226Glu-Lys).
• Heterozygous and homozygous forms.
• Third most common variant of hemoglobin. Occurs
  with greatest frequency in Southeast Asia.
• Frequently occurs with Beta thalassemia.
• No clinical symptoms.

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Laboratory Findings in Hb E Disease

• Similar to Hb D.
• Mild, microcytic, hypochromic hemolytic anemia.
• Many target cells.
• Electrophoresis shows E band. Normal Hb F, no Hb
  A.
• May protect against malaria.

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Hemoglobin SC Disease and Other Combinations
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Introduction to Hb SC Disease and other
Combinations

• May have combinations of abnormal hemoglobins.
• Occurs when have HbS gene from one parent and HbC
  gene from other parent (Hb SC).
• 1 in 835 American blacks have sickle C disease
  (SC).
• Patients generally less anemic and better
  prognosis than SS patients.

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Laboratory Findings in SC Disease

• Sickle cell symptoms including splenomegaly.
• Positive for anemia.  Mild if present.
• See target cells, folded, pocket-book cells, and
  rare sickle cells  Washington monument"
  crystals (fingerlike projections) may be found.
• Positive sickle solubility test.
• Electrophoresis shows equal amounts of HbC and
  HbS, no HbA. Hb F normal or elevated.

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Other Combinations of Abnormal Hemoglobins

• May find HbS in combination with other abnormal
  hemoglobins, such as Hb SD. 
• Hb SD Disease has positive solubility test. 
  Severity of disease between Hb SS and Hb SA.
• May find combination of hemoglobinopathy and
  thalassemia (Hb S/ß Thalassemia).
• Can make differentiation difficult.

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Hemoglobins with Increased Oxygen Affinity
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Hb with Increased Oxygen Affinity

• Decreased delivery of oxygen to tissues.
• Hb values from normal to 20 g/dL.
• Leukocytes and platelets normal.
• Normal life span.
• No treatment.
• Often results in polycythemia.

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Hemoglobins with Decreased Oxygen Affinity
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Hemoglobins with Decreased Oxygen Affinity

• Increased release of oxygen to tissues.
• Patient may become anemic.

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Unstable Hemoglobin Disease
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Introduction to Unstable Hemoglobin Diseases
1 of 2

• More than 180 unstable hemoglobins have been
  described, but most are not associated with
  hemolysis.
• Unstable hemoglobins are hemoglobin variants in
  which amino acid substitutions or deletions have
  weakened the binding forces that maintain the
  structure of the molecule.
• Instability may cause Hb to denature and
  precipitate in the red cells as Heinz bodies.

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Introduction to Unstable Hemoglobin Diseases
2 of 2

• Most inherited as autosomal dominant disorders.
• When anemia is present, degree of hemolysis
  varies considerably
• Most have mild compensated anemia with mild
  reticulocytosis
• Some have severe, chronic hemolysis with
  splenomegaly and jaundice.
• Hb electrophoresis usually not very helpful in
  diagnosis. Isopropanol precipitation or heat
  denaturation tests are used to detect unstable Hb.

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Special Diagnostic Tests

• Isopropanol Precipitation
• Unstable Hemoglobins precipitate out within 5
  minutes.
• Normal Hemoglobins do not precipitate.
• Heat Denaturation Test
• Washed red cells hemolyzed with H20.  Incubate. 
  Will denature hemoglobin.  See precipitation.
  Normal Hemoglobins do not precipitate.
• Heinz Body Staining
• Supravital staining. Brilliant Cresyl Blue
  Stain. Heinz bodies stain pale blue and are
  refractile. Eccentrically located.

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Methemoglobinemia (Hemoglobin M)
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Introduction to Methemoglobinemia

• Clinical condition with methemoglobin levels
  greater than 1 of total Hgb.
• HbM contains ferric iron (Fe3) Cant carry
  oxygen and results in cyanosis.
• Three causes of methemoglobinemia
• Hemoglobin M variants (dominant inheritance)
• NADH-diaphorase deficiency (recessive
  inheritance)
• Toxic substance (acquired)

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Hb M

• 5 variants of Hb M which result from single amino
  acid substitution in the globin chain that
  stabilizes iron in the ferric form.
• Patients have cyanosis, but are otherwise
  asymptomatic and no treatment is given.

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Laboratory Findings in Hb M

• Blood is chocolate brown.
• Mild hemolytic anemia.
• Heinz bodies.
• M band on electrophoresis.