Hemolytic Anemias:

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Chapter 10

• Hemolytic Anemias
• Enzyme Deficiencies

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1. Study Questions2. Homework Assignment3.
Exam for Unit IV
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Hereditary Enzyme Deficiencies

• In Chapter 10, you will learn about the hemolytic
  anemias caused by enzyme deficiencies in the red
  blood cells. The two most important enzyme
  deficiencies are Glucose-6-Phosphate
  Dehydrogenase (G6PD) deficiency and Pyruvate
  Kinase (PK) deficiency.  You will study the
  pathogenesis and etiologies of these disorders.
  Laboratory results and treatment will be
  discussed.

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History of Hereditary Enzyme Deficiencies 1 of 2

• First connected with anti-malarial drug
  8-aminoguinoline. Patients developed hemolysis,
  cyanosis, and methemoglobinemia.
• 1953 - Evaluated cases of congenital hemolytic
  anemias that had three common characteristics
• 1) No detectable abnormal hemoglobin.
• 2) Negative DAT. 
• 3) Normal osmotic fragility.
• Coined term "hereditary non-spherocytic hemolytic
  anemia (HNSHA)". Later found to be associated
  with red cell enzyme abnormalities.

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History of Hereditary Enzyme Deficiencies 2 of 2

• Most common anemia of this group caused by
  deficiency of glucose-6-phosphate dehydrogenase
  (G6PD), an enzyme in hexose monophosphate
  pathway.
• Second most common enzyme deficiency pyuvate
  kinase (PK), an essential enzyme in
  Embden-Meyerhof pathway.
• Are deficiencies in other enzymes.

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Glucose-6-Phosphate Dehydrogenase
Deficiency(G6PD)
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G6PD

• Glucose-6-Phosphate Dehydrogenase
• One of more common enzyme deficiencies. 
• Occurs worldwide.
• Isolated more than 400 variants of G6PD enzyme.
  Almost all of variants resulted from point
  mutations that produced structurally abnormal or
  functionally defective enzymes.

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G6PD Mode of Inheritance

• Transmitted by mutant gene located on X
  chromosome.
• Disorder fully expressed in males Only
  expressed in homozygous females.
• Heterozygous females have enzymatically dimorphic
  red cell population (have both normal G6PD
  activity and decreased G6PD activity in red
  cells). Expression of G6PD activity varies
  widely in heterozygotes.

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G6PD Distribution

• Distribution worldwide.
• Highest incidence in darkly pigmented racial and
  ethnic groups - African Americans, Mediterranean
  ancestry and Asians. 
• Whites tend to have Mediterranean form of G6PD
  deficiency (Gd Med), which is associated with
  favism.

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Pathogenesis 1 of 4

• G6PD catalyzes the first step in the hexose
  monophosphate shunt of the aerobic glycolytic
  pathway.
• G6PD is required for the reduction of NADP to
  NADPH, which is required for the reduction of
  glutathione. Reduced glutathione helps protect
  hemoglobin from oxidative denaturation.
• Activity of G6PD highest in young erythrocytes
  and decreases with aging.

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Pathogenesis 2 of 4

• Under normal conditions, individual with G6PD
  deficiency compensates for shortened erythrocyte
  life span by reticulocytosis.
• Oxidative stress, however, can lead to mild to
  severe hemolytic episode.
• More than 50 chemical agents known to induce
  hemolysis in G6PD deficient erythrocytes.
• Hemolytic episodes result when G6PD erythrocytes
  fail to maintain adequate levels of reduced
  glutathione, resulting in oxidation of hemoglobin
  which leads to progressive precipitation of
  irreversibly denatured hemoglobin (Heinz Bodies).

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Pathogenesis 3 of 4

• Cells lack normal deformability and consequently
  have difficulties in passing through
  microcirculation.
• Cells are more easily lysed.
• Have intravascular hemolysis or early
  sequestration in liver and spleen.
• Early destruction of erythrocytes may be seen on
  peripheral blood smear as bite cells or helmet
  cells.

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Pathogenesis 4 of 4

• Certain individuals exhibit sensitivity to fava
  beans (favism) After eating fava beans or after
  inhaling fava bean pollen, may have a severe
  hemolytic episode.
• Favism found in some individuals with G6PD
  deficiency of Mediterranean and Canton types.

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Clinical Manifestations of G6PD 1 of 2

• Majority of G6PD deficient individuals
  asymptomatic most of the time May never be
  aware of trait.
• G6PD activity 20 of normal sufficient for normal
  red cell function and survival under ordinary
  conditions.
• However, newborns, individuals taking certain
  drugs or who get infections may have varying
  degrees of hemolysis.
• Symptoms related to severity of hemolysis.

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Clinical Manifestations of G6PD 2 of 2

• If anemia does result, is normochromic and
  normocytic. Have decreased RBC count and
  decreased HH. Retic count is elevated. May have
  hemoglobinuria or jaundice.
• Hemolysis associated with G6PD Med more easily
  induced than with the other types of deficiency. 
  Usually more severe and may require blood
  transfusions.

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Laboratory Testing for G6PD

• G6PD deficiency should be suspected after
  hemolytic episode following administration of
  chemical or therapeutic agent known to cause
  reaction in patients with G6PD deficiency.
• Nonspecific test results include
• Hemoglobinuria
• Decreased HH
• Presence of Heinz bodies
• Hemolysis in serum
• Elevated bilirubin level
• Markedly decreased haptoglobin level
• Investigate when evidence (family history, drug
  sensitivity or both) present.

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Laboratory Testing for G6PD

• Several screening procedures available
• Can induce oxidation of hemoglobin to produce
  Heinz bodies. Use supravital stain crystal
  violet to see them Heinz bodies NOT seen on
  Wrights stain. NOTE Heinz bodies NOT seen
  with pyruvate kinase deficiency.
• Methemoglobin reduction test
• Ascorbate-cyanide test
• Fluorescent spot test (specific for G6PD
  deficiency)
• Specific G6PD assay (positive only in G6PD
  deficiency)

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Pyruvate Kinase (PK) Deficiency
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History of PK

• First discussed when De Gruchy reported that some
  patients with hereditary non-spherocytic
  hemolytic anemia had elevated concentrations of
  2,3-DPG. Suggested block in anaerobic glycolysis
  further down pathway.
• Enzyme identified in 1961 as pyuvate kinase.
• Lack of PK means lack of capacity to generate
  ATP.  Results in red cell membrane fragility and
  a hemolytic anemia.
• PK deficiency most common enzymatic disease
  involving anaerobic glycolysis.

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PK Mode of Inheritance

• Is autosomal recessive trait, but true
  homozygotes rare and restricted to children of
  consanguineous parents.
• 20 mutant variants of PK known.
• Both sexes equally affected.
• Probably found worldwide - Most known cases in
  Northern Europe, United States and Japan.

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Pathogenesis

• PK deficiency results in decreased capacity to
  generate ATP, and the lack of ATP results in the
  membrane pumps failing.
• Results in water loss with cell shrinkage,
  distortion of cell shape, and increased membrane
  rigidity.
• Results in premature RBC destruction in spleen
  and liver with consequent anemia.

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Clinical Manifestations

• Severity of disease varies from mild to severe
  depending upon properties of PK enzyme.
• True homozygotes are anemic and jaundice at birth
  and may require repeated transfusions throughout
  life.
• Less severely affected patients may be discovered
  later in life because of anemia, jaundice and/or
  splenomegaly. Hemolytic anemia is more pronounced
  during infections or periods of stress.
  Increased incidence of gallstones.
• See increase of 2,3-DPG, which allows for
  decreased oxygen affinity by hemoglobin so that
  oxygen released more readily to tissues (right
  shift in oxygen dissociation curve).
• Removal of spleen may be beneficial in some
  patients.

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Laboratory Testing 1 of 2

• Peripheral blood shows normochromic, normocytic
  anemia with varying degrees of reticulocytosis.
• May see increased poly, aniso, poik and NRBCs.
• HH is decreased (Hb 6-12 g/dL).
• Unconjugated bilirubin is increased.
• Haptoglobin level is decreased or absent.

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Laboratory Testing 2 of 2

• Screening tests are available to detect PK
  deficiencies, but tests are nonspecific.
• Screening tests include
• Osmotic Fragility Normal on fresh blood
  increased after incubation
• Autohemolysis Increased
• DAT Negative
• Red Cell Survival Tests decreased 
• Fluorescent screening test (simple and sensitive)
• If screening test positive, must confirm with a
  quantitative PK enzyme assay.
• Most PK deficient patients have 5-25 of normal
  activity.

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Methemoglobin Reductase Deficiency
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Methemoglobin Reductase Deficiency 1 of 2

• Hemoglobin that is oxidized from ferrous to
  ferric state called methemoglobin. Usually have
  about 1 methemoglobin in circulation.
• Balance between methemoglobin formation and
  reduction maintained by NADH-methemoglobin
  reductase pathway.
• Methemoglobin may occur either when decreased
  enzyme activity occurs or when production of
  methemoglobin exceeds reducing capacity of enzyme
  system.
• Hereditary deficiency of the enzyme
  NADH-methemoglobin reductase is an autosomal
  recessive trait  Heterozygotes usually
  asymptomatic unless challenged by certain drugs.

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Methemoglobin Reductase Deficiency 2 of 2

• Major clinical feature is cyanosis because
  methemoglobin cannot carry O2, may exhibit
  symptoms of anemia.
• Some patients may develop mild compensatory
  polycythemia.
• Disorder usually benign. In severe cases,
  administration of methylene blue may be given to
  activate NADH-methemoglobin reductase system. 
• Methemoglobinemia may also be caused by
  Hemoglobin M disease or by acute reactions to
  various drugs or toxins.

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Differentiation of Methemoglobinemias
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Other Enzyme Deficiencies

• Other enzyme deficiencies very rare.
• Most labs usually screen for enzyme deficient
  hemolytic anemia with the following tests
• DAT
• Erythrocyte survival
• Autohemolysis
• Osmotic fragility
• Heinz body tests
• Confirmation of the specific enzyme deficiency is
  usually done at reference labs.

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Summary of RBC Enzyme Deficiencies

• G6PD Deficiency
• Pyruvate Kinase (PK) Deficiency
• Methemoglobin Reductase Deficiency