Hemoglobin C Disease

• 2^nd most common hemoglobinopathy to be recognized
• Seen in Africa
• Lysine is substituted for glutamic acid at 6^th position (A3 helix) in β chain
• Upon deoxygenation there is reduced solubility of HbC&hence it forms intracellular crystals
• Crystals are seen after incubation of blood in 3% NaCl solution at 37 degree C
• Cells become rigid & are trapped & destroyed in the spleen
• Erythrocyte lifespan reduced to 30-55 days
• Patients are asymptomatic with mild anemia. They have mild to moderate splenomegaly. Cholelithiasis is common.
• Hemoglobin- 8-12-g/dL
• Peripheral smear
  • Small cells that appear to be folded & irregularly contracted
  • Many target cells
  • Hemoglobin C crystals can be seen if smear has been dried slowly
• Osmotic fragility – Decreased
• Hb electrophoresis
  • On citrate agar gel at acidic pH-HbC - > 90%
  • On cellulose acetate at alkaline pH, HbC migrates with HbA2, HbE&HbO – Arab
• No treatment is required. Cholecystectomy if symptomatic. No use of folic acid. Splenectomy if there is hypersplenism.

Hemoglobin D

• Glutamine is substituted for glutamic acid at 121^st position in β chain 
• Asymptomatic
• Homozygous states occasionally have increased target cells and decreased osmotic fragility
• Hb D has same electrophoretic mobility as HbS on cellulose acetate at pH 8.5
• However it is non sickling soluble hemoglobin.
• Electrophoresis on citrate agar at pH 6 allows separation of HbS&HbD. At this pH HbD migrates with HbA.
• No anemia.
• No treatment required.

Hemoglobin E

• 3^rd most common  hemoglobinopathy
• Seen in South east Asia
• Lysine is substituted for glutamic acid at 26^th position in β chain
• Has less O2 affinity (So dissociate curve shifts to right)
• Mild symptoms/ asymptomatic
• PS- Microcytic anemia with prominent target cells
• Confers protection against malaria
• Electrophoresis
  • Alkaline  - Moves with HbA2, HbC, HbO Arab
  • Acidic – Moves with HbA
• No treatment is required.

Unstable Hemoglobin Variants

(Congenital Heinz body hemolytic anemia)

• Etiopathogenesis:
  • Numerous variants (about 135 so far) of unstable Hb are discovered

Derangement of normal linkages between heme and globin on exposure to oxidant drugs and chemicals

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Partial proteolysis of chain

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Release of heme from globin binding site

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Binding of heme to non-specific sites on globin

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Formation of hemichromes (abnormal hemoglobin complex)

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Abnormal chains produce molecular instability

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Precipitation of abnormal hemoglobin (Heinz bodies) & they attach to inner surface of membrane

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Pitting by macrophages in spleen

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Rigidity, cell damage & subsequent erythrocyte hemolysis

• Homozygous conditions are incompatible with life (Autosomal dominant inheritance)
• Substitution of amino acid are seen at
  • Heme-globin link
  • α Helices
  • α – β Contacts
  • Heme pocket – Disruption of anatomy, so water is allowed inside which leads to oxidative damage to heme and its precipitation.
  • Hb-  Gun Hill- 5 amino acids are missing including heme binding site
• Clinical features
  • Congenital hemolytic anemia- Seen especially after administration of oxidant drugs – which increase the instability of hemoglobin.
  • Splenomegaly
  • Sometimes hepatomegaly, jaundice, leg ulcers
• Investigations
  • Peripheral smear
    •  Normocytic normochromic anemia
    • Basophilic stippling
    • Pitted cells (bite cells)
    • Small contracted cells
  • Retic count – Increased
  • Heinz body preparation (Incubation with brilliant cresyl blue)– Positive after splenectomy. Other conditions where they are seen are erythrocyte enzyme abnormalities, thalassemia and administration of oxidant drugs.
  • Hb electrophoresis- About 55% of unstable Hb have mobility as normal Hb
  • Heat instability test – positive
    • RBC lysate in TRIS buffer is heated to 50 – 60^o C for 60 min
    • Appearance of fine precipitate indicates positive reaction
  • Isopropanol stability test
    • Unstable hemoglobin is precipitated in 20 min by the presence of nonpolarisopropanol. (It weakens internal Hb bonds)
    • Normal Hb remains as solvent for 30-40min
• Treatment
  • Splenectomy if hemolysis is severe
  • Avoid oxidizing drugs

Hemoglobins with Altered Oxygen Affinity

• Amino acid substitutions in the globin chains close to the heme pocket
• Other substitutions include
  • Near α-β contact
  • At C terminal end of β chain
  • Near 2, 3 – DPG binding site
• Hemoglobins with decreased affinity
  • Methemoglobin
  • Hemoglobin M – Tyr substitution for Hist in heme pocket
  • Hb Kansas
• Hemoglobins with increased affinity
  • Hb Chesapeake
    • α 2 (FG4) Arg→ Leu
    • Autosomal dominant
    • Presents with polycythemia
    • Asymptomatic
    • No treatment is necessary