Dr. Neela Ferdoushi presented on thalassemias and hemoglobinopathies. Thalassemias are genetic disorders caused by mutations that decrease the synthesis of the alpha or beta globin chains, resulting in imbalanced globin chain production and anemia. The clinical severity depends on the specific gene mutations inherited. Untreated homozygous forms can be fatal in childhood. Heterozygous carriers may be asymptomatic or have mild anemia. Laboratory tests can help identify abnormalities in hemoglobin type or amounts through hemograms and hemoglobin electrophoresis. Management involves blood transfusions and iron chelation therapy for severe forms.

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THALASSAEMIA –
HEMOGLOBINOPATHIES
Presented by: Dr. Neela Ferdoushi

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THALASSEMIAS
 Thalassemias are a heterogenous group of
genetic disorders
Individuals with homozygous forms are
severely affected and die early in childhood
without treatment
Heterozygous individuals exhibit varying
levels of severity
The disorders are due to mutations that
decrease the rate of synthesis of one of the two
globin chains (α or β). The genetic defect may
be the result of:

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THALASSEMIAS
 A mutation in the noncoding introns of the gene
resulting in inefficient RNA splicing to produce
mRNA, and therefore, decreased mRNA production
 The partial or total deletion of a globin gene
 A mutation in the promoter leading to decreased
expression
 A mutation at the termination site leading to
production of longer, unstable mRNA
 A nonsense mutation
Any of these defects lead to:
 An excess of the other normal globin chain
 A decrease in the normal amount of physiologic
hemoglobin made
 Development of a hypochromic, microcytic anemia

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WORLD DISTRIBUTION OF
THALASSEMIAS

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THALASSEMIAS
Beta (β) thalassemia
 The disease manifests itself when the switch from γ to
β chain synthesis occurs several months after birth
 There may be a compensatory increase in γ and δ
chain synthesis resulting in increased levels of hgb F
and A2.
 The genetic background of β thalassemia is
heterogenous and may be roughly divided into two
types:
 β0
in which there is complete absence of β chain
production. This is common in the Mediterranean.
 β+
in which there is a partial block in β chain synthesis.
At least three different mutant genes are involved:
 β+1
– 10% of normal β chain synthesis occurs
 β+2
– 50% of normal β chain synthesis occurs
 β+3
- > 50% of normal β chain synthesis occurs

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ALPHA AND BETA THALASSEMIAS

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THALASSEMIAS
 The clinical expression of the different gene
combinations (1 from mom and 1 from dad) are as
follows:
 β0
/β0
, β+1
/ β+1
, or β0
/ β+1,+2,or +3
= thalassemia major, the most
severe form of the disease.
 Imbalanced synthesis leads to decreased total RBC
hemoglobin production and a hypochromic, microcytic
anemia.
 Excess α chains precipitate causing hemolysis of RBC
precursors in the bone marrow leading to ineffective
erythropoiesis
 In circulating RBCs, α chains may also precipitate
leading to pitting in the spleen and decreased RBC
survival via a chronic hemolytic process.
 The major cause of the severe anemia is the
ineffective erythropoiesis.

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THALASSEMIAS
 The severe, chronic anemia early in life leads to
marked expansion of the marrow space and skeletal
changes due to the increased erythropoiesis.
 Untreated individuals die early, usually of cardiac
failure (due to overwork and hemochromatosis).
 Individuals may have massive splenomegaly leading to
secondary leukopoenia and thrombocytopenia. This
can lead to infections and bleeding problems.
 Lab findings include:
- hypochromic, microcytic anemia
- marked anisocytosis and poikilocytosis
- schistocytes, ovalocytes, and target cells
- basophilic stippling from α chain precipitation
- increased reticulocytes and nucleated RBCs

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THALASSEMIAS
- serum iron and ferritin are normal to increased
and there is increased saturation
- chronic hemolysis leads to increased bilirubin and
gallstones
- hemoglobin electrophoresis shows increased hgb F, variable
amounts of hgb A2, and no to very little A

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THALASSEMIA MAJOR

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THALASSEMIAS
 Therapy – transfusions plus iron chelators to prevent
hemochromatosis and tissue damage from iron
overload; Gene therapy?
 β +2, or 3
homozygous = thalassemia intermedia
 Heterozygosity of β0
, or β+
= thalassemia minor
 Mild hypochromic, microcytic anemia
 Patients are usually asymptomatic with symptoms
occurring under stressful conditions such as pregnancy
 β thalassemia may also be found in combination with any
of the hemoglobinopathies (S, C, or E) leading to a mild
to severe anemia depending upon the particular
combination.

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THALASSEMIA MINOR

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THALASSEMIAS
 Alpha (α) thalassemia
 The disease is manifested immediately at birth
 There are normally four alpha chains, so there is a great
variety in the severity of the disease.
 At birth there are excess γ chains and later there are excess
β chains. These form stable, nonfunctional tetramers that
precipitate as the RBCs age leading to decreased RBC
survival.
 The disease is usually due to deletions of the α gene and
occasionally to a functionally abnormal α gene.

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THALASSEMIAS
 The normal haploid genotype is α/ α
 If one gene is deleted, the haploid phenotype is α thal
2
 If both genes are deleted, the haploid phenotype is α
thal 1
 Since one gets two genes from each parent, there are
four types of α thalassemia:
 α/ α thal 2 = silent carrier
 α/ α thal 1, or α thal 2/ α thal 2 = α thal trait with mild
anemia
 α thal 1/ α thal 2 = hemoglobin H disease (β4 = hgb H)
Hgb H has a higher affinity for O2 and precipitates in
older cells. Anemia may be chronic to moderate to
severe.

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THALASSEMIAS
 α thal 1/ α thal 1 = hydrops fetalis which is fatal with
stillbirth or death within hours of birth. Hemoglobin
Barts (γ4) forms and has such a high affinity for O2 that no
O2 is delivered to the tissues.
 Hgb S/ α thalassemia – symptomless to moderate anemia

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ALPHA THALASSEMIAS

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THALASSEMIAS
Delta/beta (δ/β) thalassemia – both δ and β
chains are absent with no or little
compensatory increase in γ chain synthesis.
This leads to 100% hgb F and mild
hypochromic, microcytic anemia
Hereditary persistence of hgb F – are a group
of heterogenous disorders with the absence of δ
and β chain synthesis which is compensated
for by an increase in γ chain synthesis leading
to 100% hgb F. Since hgb F has an increased
affinity for O2, this results in polycythemia.

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Primary Laboratory Investigation
Thalassemia
Variable hemogram results proportional to the
severity of the thalassemia

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Primary Laboratory Investigation
Thalassemia
• Severe cases present with
• Microcytosis
• Hypochromia
• Poikilocytosis
• RBC counts higher than expected for the level of
anemia

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Primary Laboratory Investigation
Thalassemia
• Findings in severe cases can mimic those seen in
other microcytic/hypochromic anemias
• Results of the reticulocyte count are variable
• NRBCs may be present (contrast with iron
deficiency anemia)

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Course and Treatment
Thalassemia
• Time of presentation
• Related to degree of severity
• Usually in first few years of life
• Untreated severe α thalassemia
• --/--: Prenatal or perinatal death
• --/-α & --/αcs
α: Normal life span with chronic hemolytic anemia

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Course and Treatment
Thalassemia
• Untreated β thalassemia
• Major: Death in first or second decade of life
• Intermedia: Usually normal life span
• Minor/Minima: Normal life span

23. Haemoglobinopathy

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Characteristics: Hemoglobinopathies
• Hereditary disorders that can result in moderate to
severe anemia
• Basic defect is production of an abnormal globin
chain

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Definition:
The haemoglobinopathies are inherited disorders of
haemoglobin synthesis (thalassaemias) or structure (sickle
cell disorders) that are responsible for significant morbidity
and mortality allover the world.
They are seen mainly in individuals who originate from Africa,
the Middle East,, the Mediterranean, Asia and the Far East.
However, the increased mobility of the world’s population and
inter-ethnic mixing lead to prevailing of these conditions within
any region of the world.

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These disorders result in errors in oxygen-carrying capacity
of haemoglobin . Diseases linked to genetic predisposition
are not only kill prematurely, but result in long years of ill
health and disability, loss of work and income, possible
poverty, loneliness and depression.

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Sickle cell and thalassaemia are inherited disorders that are
passed on from parents to children through unusual
haemoglobin genes.
People only have these disorders if they inherit two unusual
haemoglobin genes – one from their mother, and one from their
father.
People who inherit just one unusual gene are known as ‘carriers’.
(Some people call this having a ‘trait’.) Carriers are healthy and do
not have the disorders.

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Hemoglobinopathy Genetics
• Homozygous: Inheritance of two genes from each
parent coding for the same type of abnormal
hemoglobin, e.g., Hb SS
• Heterozygous: Inheritance of genes from each
parent which code for a different type of abnormal
hemoglobin each, e.g., Hb SC

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Terminology
Hemoglobinopathy
Abnormal hemoglobins discovered earlier have been
given letter designations:
Hb S

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Amino Acid Substitution
Hemoglobinopathy
Greek letter designates affected globin chain
β

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Amino Acid Substitution
Hemoglobinopathy
Superscript number designates affected amino
acid(s), e.g.,
β6

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Amino Acid Substitution
Hemoglobinopathy
Letters and numbers in parentheses designate the helical
segment and amino acid sequence in that segment
affected (sometimes omitted), e.g.,
β6(A3)

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Amino Acid Substitution
Hemoglobinopathy
Amino acid substitutions are denoted by the three
letter abbreviation for the normally occurring amino
acid followed by an arrow followed by the three
letter abbreviation for the substituted amino acid:
β6(A3)Glu → Val

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Classification: Hemoglobinopathy
• Functional Abnormality
• Aggregation
• Polymerization
• Crystallization
• Unstable hemoglobins
• Methemoglobin
• Oxygen affinity

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Individuals with Haemoglobinopathy are:
either healthy carriers (trait ) i.e. unaware of their carrier
status unless specifically screened. If a couple both carry a
haemoglobinopathy trait there is a 1 in 4 chance with each
pregnancy that their child will inherit a clinically manifested
haemoglobinopathy.
or having clinically manifested haemoglobinopathy

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The diagram below shows the chances (for each pregnancy) of two carrier
parents having a child with a sickle cell or thalassaemia disorder.

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If the mother is anemic & the father is healthy carrier 50% of
the off springs are carriers and 50% is anaemic

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Sickle Cell is a condition that affects the normal oxygen carrying
capacity of red blood cells. When the cells are de-oxygenated and
under stress in sickle cell conditions, they can change from round
flexible disc-like cells to elongated sickle or crescent moon shape.
The effect of these changes is that the cells do not pass freely
through small capillaries and form clusters, which block the blood
vessels. This blockage prevents oxygenation of the tissues in the
affected areas resulting in tissue hypoxia and consequent pain
(known as sickle cell crisis pain) other symptoms of sickle cell
disorders include severe anaemia, susceptibility to infections and
damage to major organs.

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The term sickle-cell disease is preferred because it is more
comprehensive than sickle-cell anaemia.

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In children, sickle-shaped red blood cells often become trapped
in the spleen, leading to a serious risk of death before the age of
seven years from a sudden profound anaemia associated with
rapid splenic enlargement or because lack of splenic function
permits an infection.
Affected children may present with painful swelling of the hands
and/or feet (hand-foot syndrome).
Survivors may suffer recurrent & severe painful crises, as well
as “acute chest syndrome” (pneumonia or pulmonary infarction),
bone or joint necrosis, or renal failure.

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Primary Laboratory Investigation
Hemoglobinopathy
• Variety of hemogram findings depending on
• Type
• Severity
of the specific disorder
• Only sickle hemoglobinopathies and Hb C will be
described here

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Primary Laboratory Investigation
Heterozygous & Other Disorders
• AS
• S-Thal
• Other hemoglobinopathies, e.g., SC
• Hb C

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Morphologic Findings
Hb SS vs. Hb SC vs. Hb CC
=+
Hb S Hb C Hb SC

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Morphologic Findings
Hb SS vs. Hb SC vs. Hb CC
=+
Hb S Hb C Hb SC
+ =

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Where Do Sickle Cells Come From?
Sheared in
microcirculation
Irreversible
Sickle Cell

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Sickle Cells

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Secondary Laboratory Investigation
• Hemoglobin electrophoresis
• Major test for identifying thalassemia and
hemoglobinopathy
• Types
• Cellulose acetate: Alkaline pH
• Citrate agar: Acid ph

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Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S F A +
Normal

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Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S F A +
Normal
Hb SS

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Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S F A+
Normal
Hb SS
Hb AS

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Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S F A+
Normal
Hb SS
Hb AS
Hb SC
Hb CC

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Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S F A+
Normal
Hb SS
Hb AS
Hb SC
Hb CC
HB AD

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Secondary Laboratory Investigation
• Solubility testing-Dithionite tube test
• Alkali denaturation test for quantification of fetal
hemoglobin
• Acid elution test for fetal hemoglobin distribution
• Unstable hemoglobin testing for Heinz bodies

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Alkali Denaturation for Hemoglobin
F
• Recommended assay for hgb F in the range of 2-
40%
• Principle
• Other hemoglobins are more susceptible than hgb F to
denaturation at alkaline pH
• Denaturation stopped by addition of ammonium
sulphate
• Denatured hemoglobin precipitates

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Alkali Denaturation for Hemoglobin
F
• Remaining hemoglobin (F) can be measured
spectrophotometrically
• Specimen: EDTA anticoagulated whole blood
• QC: Normal and elevated controls should be used
with each batch of specimens

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Alkali Denaturation for Hemoglobin
F
Hgb F, % Diff. Between Duplicates, %
<5 0.5
5-15 1.0
>15 2.0

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Acid Elution for Fetal Hemoglobin
• Indication of distribution of fetal hemoglobin in a
population of RBC
• Homogeneous distribution: hereditary persistence of fetal
hemoglobin
• Heterogeneous distribution: thalassemia

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Course and Treatment
Sickle Cell Disease
• Sickle cell disease
• Asymptomatic at birth
• Symptoms appear as percentage of fetal hemoglobin
decreases during first year of life
• Untreated crises increase morbidity and early death

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Course and Treatment
Sickle Cell Disease
• Sickle cell disease
• Asymptomatic at birth
• Symptoms appear as percentage of fetal hemoglobin
decreases during first year of life
• Untreated crises increase morbidity and early death

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Course and Treatment
Sickle Cell Disease
• Life span can be significantly increased with early and
effective treatment
• Studies of natural populations reveal that individuals
with sickle cell disease are capable of normal life spans

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Course and Treatment
In both thalassemia and hemoglobinopathy therapy is
usually supportive rather than curative

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Course and Treatment
• Blood transfusion is used to
• Control severe anemia
• Reduce the risk of complications of sickle
hemoglobinopathies (cerebrovascular accident,
hypersplenism, etc.)

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Course and Treatment
• Chronic blood transfusion
• Results in iron overload of major organs resulting in
increased morbidity
• Laboratory monitoring
• Necessitates the use of chelating agents to remove
excess iron

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Course and Treatment
• Excess iron can cause the appearance of sideroblastic
conditions
• Transfusion interferes with the typical laboratory findings
for the disorder

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Course and Treatment
• Alternative treatment
• Activation of fetal hemoglobin genes
• Bone marrow transplantation