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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

    Coimbatore

    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

    Nitrogen

    Fe++

    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

    α

    δ

    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%

    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-

    interactions

  • 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

    Reduced

    erythrocyte

    lifespan

    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

    abnormalities

    Hemoglobin and

    Enzyme

    abnormalities

    Infections

    Mechanical

    Drugs

    Hypersplenism

    Congenital

    hemolytic anemia

    or

    Inherited hemolytic

    anemia

  • Congenital

    hemolytic anemia

    Membrane

    abnormalities Hereditary sherocytosis

    Elliptocytosis

    Stomatocytosis

    Hemoglobin

    abnormalities Quantitative

    Thalassemias

    α & β Thalassemia

    αβ Thalassemia

    Qualitative

    Sickle cell disease

    Unstable hemoglobin

    Enzyme

    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

    Thalassemias

  • 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

    anemia

    marrow expansion

    precipitation of excess a

    in erythroid precursors

    Ineffective

    erythropeoesis haemolysis

    b+

    b thalassemia (b

  • •Intramedulary hemolysis

    •Ineffective hematopoiesis

    •Reticulocytosis not pronounced

    •LDH not elevated greatly

    •Mild Indirect bilirubinemia

    •Gall stones not a feature

    •MCV- LOW

    Thalassemia

  • 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

    Autosomal

    recessive

    Homozygous/

    Comp hetrozygous

    Severe disease

    New mutations

  • Clinical manifestations

    Typical HS Mild HS Severe

    Symptoms Asymptomatic During stress Severe

    Anemia Mild

    to

    moderate

    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

    http://stonechurchclinic.ca/Rh-prevention-program/rbc/image_preview

  • Qualitative hemoglobin disorders

    • Amino acid substitution in the globin chain

    • 6th position of the β-globin chain-

    • Glutamic acid with valine- sickle hemoglobin

    (HbS)

    • 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

    http://content.nejm.org/content/vol342/issue25/images/large/10f1.jpeg

  • 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

    HbS

    Β+ thal

    Homo

    Hb S

    Hom

    HbS

    S/β0 thal S/β+ thal

    S/α thal

    Hb 7-9 7-9 10-12 Normal

    MCV Normal 63-75 68-78 Reduce