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% <3.5%

α

β α α

α α γ β

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<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

•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

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

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 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

Enzymopathies

Red cell enzymopathies associated with

haemolytic anemia

• Enzymopathies of Glutathione metabolism (HMP pathway)

• Enzymopathies of the glycolytic pathway

Disorders of HMP shunt and glutathione metabolism

Hb-O2

interaction

HMP shunt and glutathione metabolismHMP shunt and glutathione metabolism

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

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)

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

Drug’s and G6PD deficiency

Unstable hemoglobin

Structural abnormalities

Substitutions in primary sequence

Alterations in tertiary or quarternery

structure

unstable globin polypeptide chain

or Hb tetramer

precipitate

inside the red cell

• 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.

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

Clinical manifestations

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

Lab diagnosis

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

Examination

• Anemia

• Icterus

• Splenomegaly

• Thalassemic features

• Features of cholelithiasis or cholecystitis

Investigations- preliminary

• Complete hemogram

• Reticulocyte count (correct for anemia) or absolute reticulocyte count

• Bilirubin estimations

• LDH

• Peripheral smear examination

• X Ray

• U/S abdomen

Peripheral smear findings

HS

HE

HPP

SS

SC

Thal Maj

G6PD def

PK def

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

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

Therapy

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

HS-Indications for splenectomy

• Growth retardation

• skeletal changes

• symptomatic hemolytic disease

• anemia induced compromise of

vital organs

• development of leg ulcers

• Extramedullary hematopoietic

tumors.

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.

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.

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.

Thank you