Post on 16-Jul-2015
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.
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
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
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