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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
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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.
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Iron, haem and globin
Protoporphyrin ring
Nitrogen
Fe++
Proximal histidine
Distal histidine
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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
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Haemoglobin
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Hemoglobin in normal adults
α
δ
δ γ
HbA HbF HbA2
98% ~1% <3.5%
α
β α α
α α γ β
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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%
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Red blood cell
Mature RBC: 7-8 μ Capillaries: 3 μ
Slits in RE system: 2-3 μ
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Red cell membrane
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Red cell membrane-
interactions
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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
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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.
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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
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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
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Membrane
abnormalities
Hemoglobin and
Enzyme
abnormalities
Infections
Mechanical
Drugs
Hypersplenism
Congenital
hemolytic anemia
or
Inherited hemolytic
anemia
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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
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HEMOGLOBIN DISORDERS Congenital hemolytic anemia
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Hemoglobinopathy
• An inherited mutation of the globin genes leading to a qualitative or quantitative abnormality of globin synthesis
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disorders of haemoglobin synthesis due to
reduced output of globin chains
Thalassemias
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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
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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<a)
b0
b a b a
a0
()4
Hb Bart’s
Intrauteral death
a+
a thalassemia (a<b)
(b)4
HbH
mild anemia
b a b a
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•Intramedulary hemolysis
•Ineffective hematopoiesis
•Reticulocytosis not pronounced
•LDH not elevated greatly
•Mild Indirect bilirubinemia
•Gall stones not a feature
•MCV- LOW
Thalassemia
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Pathophysiology of congenital hemolytic anemias
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MEMBRANE ABNORMALITIES Congenital Hemolytic anemia
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Outcome of altered membrane interactions
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Pathophysiology of HS
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Inheritance
Autosomal dominant
Xle generations of
affected families
Autosomal
recessive
Homozygous/
Comp hetrozygous
Severe disease
New mutations
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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.
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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.
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HS:Principle of osmotic fragility
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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-
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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
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Pathophysiology of Sickle Cell Disease
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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
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• 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
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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
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Electrophoresis
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Normal
Hb SS
Hb AS
Hb SC
Hb CC
Hb AD
Hb EE
A2/C/E/Oa S/D/G F A + -
Electrophoresis pattern
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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 Reduced
MCH Normal Reduced Reduced Reduced
HbA2 Normal Raised Raised Normal
HbF Varies 4-7 5-21 Normal
HbS Major
band
Remaining
Major band
Remaining
Major band
25-30
HbA Absent Absent 5-15 Major band
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Enzymopathies
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Red cell enzymopathies associated with
haemolytic anemia
• Enzymopathies of Glutathione metabolism (HMP pathway)
• Enzymopathies of the glycolytic pathway
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Disorders of HMP shunt and glutathione metabolism
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Hb-O2
interaction
HMP shunt and glutathione metabolismHMP shunt and glutathione metabolism
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Normal or wild type enzyme is G6PD B
Most whites and Asians and majority of blacks
Common variant G6PD A+ (Class IV)
20-30% of Blacks
G6PD A - : responsible for primaquine sensitivity in blacks
Class III- unstable enzyme (normal catalytic activity)
G6PD Mediterranean: Abn variant seen in whites
Same electrophoretic mobility as G6PD B but synthesized
at a reduced rate and results in severe hemolysis (Class II)
http://www.rubic.rdg.ac.uk/g6pd/ Mutations listed in G6PD database
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Pathophysiology
• As red cells age activity of G6PD declines exponentially
• G6PD: in-vivo half life of 62 days • Normal old RBC’s have sufficient G6PD activity to
generate NADPH and thereby sustain GSH levels in the face of oxidant stress
• G6PD variants associated with hemolysis: unstable and have much shorter half lives
• G6PD A- activity normal in reticulocytes but half life only 13 days
• G6PD Med: greater instability (1/2 life in hours)
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G6PD deficient RBC’s
GSH depletion
Oxidants
Infections
Drugs
Fava
Oxidation of
other RBC SH
containing proteins
Oxidation of
SH groups on Hb-
Denatured Hb- Heinz bodies
Oxidation of membrane
SH groups – membrane
polypeptide aggregates- rigid
Non-deformable RBC’s –
extravascular hemolysis
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Drug’s and G6PD deficiency
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Unstable hemoglobin
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Structural abnormalities
Substitutions in primary sequence
Alterations in tertiary or quarternery
structure
unstable globin polypeptide chain
or Hb tetramer
precipitate
inside the red cell
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• Hb mutants with substitutions that alter the structure and solubility of the molecule
• Unstable Hb precipitates as intracellular inclusions (dark globular aggregates – Heinz bodies)
• Reduced life expectancy hemolytic syndrome HEINZ BODY HEMOLYTIC SYNDROME.
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Heinz body stain
• Heinz bodies: late sign of oxidative damage, represents denatured hemoglobin within a cell.
• Causes: 1. Unstable Hb 2. Drugs & chemicals causing
oxidant damage 3. Congenital enzyme defects
affecting glutathione metabolism (G6PD, Glutathione reductase, synthetase, peroxidase deficiency).
Principle: Red cells exposed to oxidant stress induced by a chemical to induce injury, even to normal red cells. Cells with any of the above will have increased numbers of Heinz bodies.
G6PD enzyme level estimation
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Clinical manifestations
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Clinical manifestations Thalassemia
Intramedulary hemolysis
Ineffective hematopoiesis
Reticulocytosis not pronounced
LDH not elevated greatly
Mild Indirect bilirubinemia
Gall stones not a feature
Rest of the congenital hemolytic anemias
Extramedulary hemolysis (Extravascular)
Effective hematopoiesis
Reticulocytosis pronounced
LDH elevated significantly
Indirect bilitubinemia significant
Gall stones -frequent
Common to non thalassemic cong HA Crisis- Aplastic (due to parvo virus B19 infection) Hemolytic crisis Megaloblastic crisis Leg ulcers
G6PD deficiency
Episodic exacerbations of anemia
secondary to drugs, infections,
DKA, fava bean ingestion
Sickle cell disease Painful crisis Acute chest syndrome Priapism Splenic sequestration syndrome Infectious complications (functional asplenia): Strep.pneumonia sepsis Salmonella/ Staph Osteomyelitis
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Lab diagnosis
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Approach to diagnosis • History
– Anemia- severity
– Recurrent jaundice
– Precipitating factors (Eg: Medications)
– Family history • Consanguinity
– Blood transfusion history • Age of commencement
• Frequency
• Response to transfusion
• Gall stones
• Crisis symptoms
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Examination
• Anemia
• Icterus
• Splenomegaly
• Thalassemic features
• Features of cholelithiasis or cholecystitis
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Investigations- preliminary
• Complete hemogram
• Reticulocyte count (correct for anemia) or absolute reticulocyte count
• Bilirubin estimations
• LDH
• Peripheral smear examination
• X Ray
• U/S abdomen
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Peripheral smear findings
HS
HE
HPP
SS
SC
Thal Maj
G6PD def
PK def
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Establish that anemia present
RBC indices
Microcytic Normocytic Macrocytic
Retics
Normal/low Increased
ESTABLISH THAT HEMOLYSIS IS PRESENT
Increased red cell destruction
Indirect bilirubinemia
+ intravascular hemolysis
•Increased LDH
•Hemoglobinemia
•Hemoglobinuria
•Hemosiderinuria
Increased red cell production
Increased reticulocyte count
+ Nucleated red cells
Bone marrow erythroid hyperplasia
Hemoglobin synthesis
Defect
Iron def or poor use
Globin synthesis def
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Hemolytic anemia established
Coomb’s test
Positive Negative
Auto (AIHA) Examine PS
Immune HA
Allo IHA
Non Immune HA
Rule out
extrinsic
non immune causes
Consider Intrinsic causes
Blister cells
Prickle cells
Red cell
inclusions
Enzyme test
Heinz bodies
HPLC/RDB
Sickle cells
Target cells
SC Prep
Hb Electro
HPLC/RDB
Spherocytes
OF
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Therapy
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Sickle cell disease
Deoxygenation HbS concentration
Acidosis Polymerization
O2 Increase HbF- Hydrea
Decrease HbS- Exchange Tx Stem cell Tx
RBC adhesion & rigidity
Hemolysis
Endothelial damage Vaso-occlusion
? Aspirin, analgesics Hydration, Tx
Anemia
Transfusion Folate supplement
Decreased NO
Pulmonary hypertension
Sildenafil
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HS-Indications for splenectomy
• Growth retardation
• skeletal changes
• symptomatic hemolytic disease
• anemia induced compromise of
vital organs
• development of leg ulcers
• Extramedullary hematopoietic
tumors.
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Splenectomy
• Splenectomy prior to the formation of bilirubin gallstones can eliminate the need for cholecystectomy later in life.
• Where possible,- laparoscopic splenectomy rather than operative splenectomy.
• Where possible- splenectomy be deferred until at least age six, rather than at an earlier age - due to the higher risk of overwhelming sepsis in young children who undergo splenectomy.
• All patients who have been splenectomized are at risk for sepsis with encapsulated organisms.
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Immunizations
• appropriately timed preoperative immunization against S. pneumoniae, meningococcus, and H. influenzae type b.
• Timing of immunizations – Vaccines should be administered at least
14 days prior to scheduled splenectomy. If this is not possible, these immunizations can be given after the 14th postoperative day.
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Antibiotic prophylaxis
• Daily antibiotic prophylaxis – Oral penicillin or amoxicillin till age 5 for at least 1 yr post splenectomy
• Empiric antibiotic therapy for fever – Amoxicillin-clavulanate —
– Cefuroxime axetil —
– Extended-spectrum fluoroquinolones — In adults or adolescents only:
After taking the first dose, the patient should proceed
without delay to the nearest urgent care facility for
evaluation and further therapy.
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Thank you