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Congenital hemolytic anemia - IAPP- ... Congenital hemolytic anemia Dr Rajasekar Thirugnanam Consultant hematologist and bone marrow transplant physician Kovai Medical Center and Hospital

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  • Congenital hemolytic anemia

    Dr Rajasekar Thirugnanam

    Consultant hematologist and bone marrow transplant physician

    Kovai Medical Center and Hospital


    Tamil Nadu

  • Iron

    • Iron- an essential metal for all mammalian cells

    • Serves as a mediator of enzymatic electron exchange (in cytochromes, peroxidases, ribonucleotide reductases, and catalases) and a carrier of oxygen (in hemoglobin and myoglobin).

    • However, its flexible redox state and its interactions with oxygen can also promote cellular damage if and when the reactivity of iron is not restrained by protein binding.

  • Iron, haem and globin

    Protoporphyrin ring



    Proximal histidine

    Distal histidine

  • Hemoglobin

    Within each red blood cell are some 300 million hemoglobin molecules. Each molecule contains the

    protein globin and a pigment called heme - which includes an iron atom

  • Haemoglobin

  • Hemoglobin in normal adults



    δ γ

    HbA HbF HbA2

    98% ~1%

  • Hemoglobin synthesis

    β δ γ α α

    Chromosome 16 Chromosome 11

    25% 25%

    α α β δ γ

    25% 25% 48%


    1.5% 0.5%

    1.5% 0.5%

  • Red blood cell

    Mature RBC: 7-8 μ Capillaries: 3 μ

    Slits in RE system: 2-3 μ

  • Red cell membrane

  • Red cell membrane-


  • Red Blood Cells

    • No nucleus- no cell division

    • No ribosomes- no protein synthesis

    • No mitochondria- no oxidative phosphorylation

    • Incapable of de-novo purine or pyrimidine synthesis

  • Red cell requirements of energy

    1. Maintenance of glycolysis 2. Maintenance of the electrolyte gradient between

    plasma and red cell cytoplasm through the activity of adenosine triphosphate (ATP)-driven membrane pumps

    3. Synthesis of glutathione and other metabolites 4. Maintenance of hemoglobin’s iron in its functional,

    reduced, ferrous state 5. Protection of metabolic enzymes, hemoglobin, and

    membrane proteins from oxidative denaturation 6. Preservation of membrane phospholipid asymmetry.

  • Metabolic pathways in RBCs “Energy producing” Glycolytic pathway

    ATP: Energy - membrane & metabolic reactions

    NADH: Cofactor for meth- Hb reduction

    2,3 DPG: Modulates Hb-O2 affinity

    “Protective” HMP pathway

    NADPH: cofactor in glutathione metabolism

  • Hemolytic anemia




    Increase output 6-8 times

    erythrocyte survival can be reduced to a value as low as 20 to 30 days without the

    onset of anemia Retic count > 2 %, with an absolute retic count usually greater than 100,000/microL

    Unconjugated bilirubinemia and

    increased LDH

  • Membrane


    Hemoglobin and








    hemolytic anemia


    Inherited hemolytic


  • Congenital

    hemolytic anemia


    abnormalities Hereditary sherocytosis




    abnormalities Quantitative


    α & β Thalassemia

    αβ Thalassemia


    Sickle cell disease

    Unstable hemoglobin


    abnormalities Glycolytic pathway

    HMP shunt pathway

  • HEMOGLOBIN DISORDERS Congenital hemolytic anemia

  • Hemoglobinopathy

    • An inherited mutation of the globin genes leading to a qualitative or quantitative abnormality of globin synthesis

  • disorders of haemoglobin synthesis due to

    reduced output of globin chains


  • a2b2

    a2 a1 a2 a1   

    bb --Globin Gene Cluster Chromosome 11Globin Gene Cluster Chromosome 11

    aa--Globin Gene Cluster Chromosome 16Globin Gene Cluster Chromosome 16

    b A  G b 

    Quantitative hemoglobin disorders

  • Pathophysiology of thalassaemia

    Normal (b=a)

    skeletal deformity


    marrow expansion

    precipitation of excess a

    in erythroid precursors


    erythropeoesis haemolysis


    b thalassemia (b

  • •Intramedulary hemolysis

    •Ineffective hematopoiesis

    •Reticulocytosis not pronounced

    •LDH not elevated greatly

    •Mild Indirect bilirubinemia

    •Gall stones not a feature

    •MCV- LOW


  • Pathophysiology of congenital hemolytic anemias

  • MEMBRANE ABNORMALITIES Congenital Hemolytic anemia

  • Outcome of altered membrane interactions

  • Pathophysiology of HS

  • Inheritance

    Autosomal dominant

    Xle generations of

    affected families




    Comp hetrozygous

    Severe disease

    New mutations

  • Clinical manifestations

    Typical HS Mild HS Severe

    Symptoms Asymptomatic During stress Severe

    Anemia Mild



    Absent Severe

    Spleen + +/- +

    Reticulocyte Increased N/ Increased Increased

    Excellent predictor screening test for HS- MCHC and elevated RDW.

  • Blood film

    • Typical spherocyte

    • lack central pallor, mean diameter decreased and appear intensely hemoglobinized.

    • Pincered red cells- band 3 deficiency

    • acanthocytic spherocytes - beta spectrin deficiency.

  • HS:Principle of osmotic fragility

  • Qualitative hemoglobin disorders

    • Amino acid substitution in the globin chain

    • 6th position of the β-globin chain-

    • Glutamic acid with valine- sickle hemoglobin


    • Glutamic acid with lysine- Hemoglobin C (HbC)

    • 26th position of the β-globin chain-

  • Mutation (in DNA)

    GUG CAC CUG ACU CCU GUG GAG AAG val his leu thr pro val glu lys 1 2 3 4 5 6 7 8

    Mutant mRNA

    Mutant protein

    Glutamate (glu), a negatively charged amino acid, is replaced by valine

    (val), which has no charge.

    GUG CAC CUG ACU CCU GAG GAG AAG val his leu thr pro GLU glu lys 1 2 3 4 5 6 7 8

    Normal mRNA

    Normal protein

    Sickle cell

  • Pathophysiology of Sickle Cell Disease

  • Sickle cell disease- Genotypes

    Genotype Full Name Abbreviation βs / βs Sickle cell disease- SS SCD-SS

    βs / βc Sickle cell disease- SC SCD-SC

    βs / βo thalassemia Sickle cell disease-S βo thalassemia SCD-S βo thal

    βs / β+ thalassemia Sickle cell disease-S β+ thalassemia SCD-S β+ thal

  • • Diagnosis of a sickle cell syndrome is suggested by characteristic findings on the complete blood count and peripheral smear which then require confirmation with hemoglobin electrophoresis and sickling tests

    • sickling phenomenon may be demonstrated in a thin wet film of blood (sealed with a petroleum jelly/paraffin wax mixture or with nail varnish).

    • If Hb S is present, the red cells lose their smooth, round shape and become sickled. This process may take up to 12 hours in Hb S trait, whereas changes are apparent in homozygotes and compound heterozygotes after 1 hour at 37°C.

    • These changes can be hastened by the addition of a reducing agent such as sodium dithionite as follows

  • Electrophoresis

    • Principle: When proteins applied to a membrane are exposed to a charge gradient, the components separate from each other and can be visualized by either a protein or haem stain.

    • Done on red cell concentrate so that there are no bands caused by plasma proteins

    • Electrophoresis: – Separates hemoglobins on solid support media

    – Inexpensive and quickly prepared

    – Sharp resolution of major hemoglobin bands

    – Electrophoretic variability based on charge

  • Electrophoresis

  • Normal

    Hb SS

    Hb AS

    Hb SC

    Hb CC

    Hb AD

    Hb EE

    A2/C/E/Oa S/D/G F A + -

    Electrophoresis pattern

  • HPLC Results


    Β+ thal


    Hb S



    S/β0 thal S/β+ thal

    S/α thal

    Hb 7-9 7-9 10-12 Normal

    MCV Normal 63-75 68-78 Reduce