THALASSEMIAS Muhammad Asif Zeb

HEMOGLOBIN DISORDERS

Genetic disorders of hemoglobin are the most

common genetic disorders worldwide

Mutations in the globin genes are the most

prevalent monogenic disorders worldwide &

affect approx. 7% of the world population

Α AND Β THALASSEMIA

• The α gene is duplicated: 2 genes per chromosome, therefore 4 α genes in a normal adult. • The β gene is single therefore the normal adult will have two β genes

α and β globin gene clusters Chromosomes 11 and 16

NORMAL HUMAN HEMOGLOBIN

Embryonic Hemoglobin

• Hgb Gower 1 (ζ2,ε2)

• Hgh portland (ζ2, γ2)

• Hgb Gower 2 (α2, ε2)

Fetal Hemoglobin

• Hgb F (α2,γ2)

Adult Hemoglobin

• Hgb A (α2,β2) 98%

• Hgb A2 (α2,δ2) 1.5- 3.2%

• Hgb F (α2,γ2) <1%

Early embryogenesis Yolk sac erythroblasts

Major Hemoglobin of Intra-uterine life

Adult Hemoglobin

THALASSEMIA

Are characterized by a reduced rate of

production of normal hemoglobin due to

absent or decreased synthesis of one or

more types of globin polypeptide chain

TYPES OF THALASSEMIA

• Inherited anemia characterized by a decrease in synthesis one or more of the globin chains. The defect may affect the

α chains

β chains

γ chains

δ chains

• The clinical syndromes arise from a combination of

Inadequate production of hemoglobin

Unbalanced accumulation of globin chains

Α-THALASSEMIA SYNDROMES

• Group of disorders characterized by decreased

synthesis of α-chains usually due to gene

deletions

• The mutation is a functionally abnormal α

gene

• The clinical severity can be classified according

to the number of genes that are missing or

inactive

Α-THALASSEMIA SYNDROMES

Four possible α thalassemia syndromes:

1. α-thalassemia (silent carrier): One of the four α-

globin gene fails to function.

2. α-thalssemia-trait (α Thal trait minor): Two of the

four α-globin gene fails to functions.

3. Hgb H disease: Three of the four α-globin gene

fails to functions.

4. Bart’s Hydops Fetalis: Four of the four α-globin

gene fails to functions.

CLASSIFICATION

Syndrome # alpha genes

deleted

Newborn Hb

Barts (γ4) %

Clinical Picture

Silent Carrier 1 1-2 Silent

Alpha

thalassemia trait 2 3-10

Mild hypochromic,

microcytic anemia

Hb H Disease 3 25 Hb H (β4) mild

hemolytic anemia

Hydrops fetalis 4 80-100 Death in utero or

shortly after birth

α Thalassemia

Α-THALASSEMIA MAJOR

• --/--( α0 -thal) (Hydrops fetalis)

• Most severe form of α-thalassemia, involving the deletion of all 4 genes resulting the absence of α chains.

• In the absence of α chains, erythrocytes assemble hemoglobin using the δ,ϒ & β chains available.

• Therefore abnormal hemoglobin tetramer involving gamma chains (Hb Bart’s, ϒ4) & beta chains(HbH, β4 )is produced.

Α-THALASSEMIA MAJOR

Hb Bart’s has high affinity for oxygen so this

Hb can’t supply tissue with sufficient O2 to

sustain life & developing infant dies of

hypoxia & congestive heart failure.

CLINICAL FEATURES

• Infants that survive until birth exhibit significant physical changes upon routine exam

• The babies are

- Underweight, edematous with distend

abdomen,

- Hepatosplenomegaly due to extramedulary

hematopoiesis

- Massive bone marrow hyperplasia

• Hemolysis is severe, as there is extensive deposition of hemosiderin

LAB DIAGNOSIS

• There is severe anemia

• hemoglobin 3-10 g/dl

• Microcytic hypochromic RBCs

• marked anisocytosis & poikilocytosis

• Increased NRBCs

• Hb electrophoresis on cellulose acetate membrane at alkaline PH shows:

Hb Bart’s 80-90%

Hb Portland 10-20%

Hb H sometimes detectable

Hb A, HbA2, Hb F absent

HEMOGLOBIN H DISEASE

--/-α (HETEROZYGOUS)

Occurs when 3 of 4 α genes are deleted

This disorder usually results when 2

heterozygous parents, one with --/αα & the

other expressing –α/αα genotype, bear

children.

PATHOPHYSIOLOGY

• The reduction of α-chain synthesis results in the decrease in the assembly of HbA, HbA2, and HbF.

• A decrease in α-chain creates a relative excess

of beta chain which unite to form a tetrad of 4

beta chains called HbH.

• HbH is unstable & tend to

precipitate inside erythrocytes trigering chronic

hemolytic anemia.

• It has an O2 affinity 10 times that of

HbA, which reduces oxygen delivery to tissues

causes tissue hypoxia.

LAB DIAGNOSIS

• Hemoglobin level 8-10 g/dl

• Reticulocyte count moderately raised ranging

5-10%

• Microcytic hypochromic red cells

• Marked poikilocytosis

• Target cells

• Basophilic stippling

• NRBCs

Peripheral Blood Smear: stained With brilliant cresyl blue, showing Precipitated Hgb H

LAB DIAGNOSIS

• Hemoglobin electrophoresis of affected neonates shows

- Hb Bart’s 25% with decreased levels of

HbA, HbA2,HbF

• After birth β chains begin to replace gamma chains & Hb H replaces the Hb Bart’s.

- Hb H in Adults 2-40%

- HbA2 Decreased (1.5%)

- HbF Normal

DEMONSTRATION OF HB H INCLUSION BODIES

“In alpha thalssemia, red cell containing Hb H

are incubated with a solution of a redox

dye(brilliant cresyl blue), HbH which is relatively

unstable, precipitates & red cells are pitted by

numerous inclusions, an appearance likened to

the surface of a golf ball”

THALASSEMIA TRAIT

--/αα and -α/-α

Also called Thalassemia minor, occurs when

two of the four α gene, either on same or

opposite chromosomes, are missing

There is a measureable decrease in the

production of α- containing hemoglobins, the

unaffected hemoglobin genes are able to direct

synthesis of globin

THALASSEMIA TRAIT

chains faster than normal & compensate

for effected genes

• Patients with α-thalassemia trait are

asymptomatic with mild anemia and are

often diagnosed incidentally or when being

evaluated for family studies

• These patients have normal life span & do

not need medical intervention for their

thalassemia

LAB DIAGNOSIS

MCV 60-70 fl

Slight anemia

In peripheral blood film, red cells with

- Hypocromia & Microcytosis

- Poikilocytosis

- Target Cells

- Basophilic Stippling

In Hb electrophoresis, Hb Bart’s 5-6%

BETA THALASSEMIA

• occurs when one or both of the two genes needed for making the beta globin chain of hemoglobin are variant.

• The severity of illness depends on whether one or both genes are affected and the nature of the abnormality.

• If both genes are affected, anemia can range from moderate to severe.

• The severe form of beta thalassemia is also known as Cooley’s anemia.

THALASSEMIA

Greek letter used to designate globin chain:

+: Indicates diminished, but some production of globin chain by gene:

+

0 :Indicates no production of globin chain by gene:

0

CLASSIFICATION

Genotype Genetic

description Phenotype

βο/βο Homozygous Major

βο/β+ Heterozygous Major or

Intermedia

β+/β+ Homozygous Major or

Intermedia

βο/β Heterozygous Intermedia or

minor

β+/β Heterozygous Minor

β /β Homozygous Normal

Β-THALASSEMIA MAJOR

• βο/βο, βο/ β+, β+/ β+

• Is a severe, transfusion dependent, inherited

anemia

• Also referred to as COOLEY’S ANEMIA

• Caused by a homozygous or double

heterozygous inheritance of abnormal β gene

resulting in marked reduction or absence in β-

chain synthesis.

• Presents early in life

PATHOPHYSIOLOGY

• The molecular defects in - β thalassemia result in absent or reduced β-chain production. α-Chain synthesis is unaffected leading to an excess of α-chains

• Lack of beta chain production can be classified into four categories

– Reduced HbA

– Compensatory production of abnormal Hb

– Ineffective erythropoiesis

– Erythroid hyperplasia

PATHOPHYSIOLOGY

CLINICAL FEATURES

• Symptoms 1st observed in infants are;

- Irritability, pallor,

- Diarrhoea, fever, enlarged abdomen

- Growth retardation

- Brown pigmentation of skin

- Chronic hemolysis often produces

gallstones

- Gout & icterus

CLINICAL FEATURES

- Bone changes accompany the hyperplastic marrow, marrow cavities enlarge in every bone,expanding the bone & producing characteristic bossing of the skull

- Facial deformities & “hair-on-end” appearance of the skull on X-ray

- Massive splenomegaly

• Severe infections, septicemia,pericarditis

• Iron deposition leads to organ dysfunction, diabetes & cirrhosis

Marked bony changes from bone marrow proliferation

Bone marrow hyper expansion gives the classic “hair on end” appearance on X-ray of the skull

LAB DIAGNOSIS

On CBC TLC Normal

- Hb low (2-3 g/dl)

- MCV, MCH, low

PLT: Normal to Decrease

- Reticulocyte count increases to the degree

expected for the severity of anemia

because of the increase degree of

ineffective erythropoises

• On peripheral blood picture

- Marked anemia

- Marked microcytosis & hypochromia

- Anisocytosis & poikilocytosis

- Target cells, tear drop cells, fragmented RBC, polychromasia

- NRBCs

- Basophilic stippling

• Osmotic fragility test reveals increased resistance to hemolysis

• Bone Marrow:

- is not usually necessary for diagnosis

but, when performed, show marked

erythroid hyperplasia

• Bone Marrow iron stores increased

BIOCHEMISTRY

- Serum iron raised

- TIBC saturated

- Serum transferrin receptor variable

- Serum ferritin raised

- Serum unconjugated bilirubin raised

- Serum uric acid raised

- Urine dipyrrols (hB degradation products)

are present

serum & red cell folate reduced

HB ELECTROPHORESIS

In adults, the absence of HbA, 90% HbF, & low,

normal, or increase HbA2 is characteristic of

βο/βο thalassemia

Β-THALASSEMIA MINOR

βο/ β or β+/ β

Results from the heterozygous inheritance of

either a β+ or βο gene with one normal β gene

The normal β gene directs synthesis of

sufficient amount β-chains to synthesize

enough HbA for near normal oxygen delivrey &

erythrocyte survival

Β-THALASSEMIA MINOR

In case of a heterozygous β+ patient, the

thalassemic gene can also contribute to β-

chain production

They are asymptomatic except in periods of

stress as can occur during pregnancy,

infections & folic acid deficiency

Under these conditions,a moderate microcytic

anemia develops

LAB DIAGNOSIS

Erythrocyte count is doubled (>5×10/L) for what is expected at the given Hb conc.

MCV 55-70fl

MCH less than 22pg

MCHC 29-33 g/dl

Mild anemia with hemoglobin ranges 9-14 g/dl

Reticulocyte count mild elevated

LAB DIAGNOSIS

• Peripheral blood picture shows mild anemia, Anisocytosis & poikilocytosis with

- Target cells

- Basophilic stippling

- NRBCs are not usually found

• On Hb electrophoresis

- HbF normal

- HbA > 3.5%

Β-THALASSEMIA INTERMEDIA

• βο/ β+, β+/ β+ or βο/ β+

• All three patterns of inheritance, homozygous, heterozygous, double heterozygous can produce β-Thalassemia intermedia

• The homozygous & double heterozygous forms represent a mutation in both β alleles resulting in a moderate degree of β chain synthesis

• Symptoms intense during pregnancy & infection

LAB DIAGNOSIS

CBC reflects a moderate microcytic

hypochromic anemia with Hb value 6-10 g/dl

Erythrocyte count raised

Target cells are prominent poikilocytes

observed, basophilic stippling & NRBCs are

present

HbA2 5-10%

HbF 30-75%

SECOND LINE IDENTIFICATION OF THALASSEMIA

• Second line identification tests based on results of Hb electrophoresis on cellulose acetate membrane are;

- Estimation of HbA2

- Estimation HbF by

– Alkali denaturation by modified betke method

– Acid elution method

OTHER TESTS

PCR for identification of mutation

Hb Electrophoresis

DIFFERENTIAL DIAGNOSIS

• Iron deficiency anemia

• Anemia of chronic disease

• Sideroblastic anemia

THANX