What is Thalassemia

How is inherited
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Hemoglobinopathies
Hemoglobinopathies are inherited single-gene disorders that affect Hb production and
function. It is estimated that around 7% of the world population carries a globin-gene
mutation; and in the majority of cases, it is inherited as an autosomal recessive trait.
Hemoglobinopathies can be classified broadly as qualitative and quantitative disorders.
Qualitative hemoglobinopathies result from α- or β-globin gene mutations that cause
structural alterations in the Hb molecule. Quantitative hemoglobinopathies, or
thalassemias, arise from mutations that cause decreased synthesis of otherwise normal α-
or β-globin chains, resulting in imbalance of the subunits.

The hemoglobinopathies are inherited disorders

of abnormal globin chain production that could
be quantitative or qualitative in nature.

Thalassemias
Thalassemia is an inherited disorder of hemoglobin synthesis that results in the reduction
of the total hemoglobin in the body leading therefore to anemia. The thalassemias are a
heterogeneous group of hereditary disorders of hemoglobin synthesis found worldwide,
in many countries around the world and particularly in persons of Mediterranean, African
and Asian ancestry.
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Thalassemia is derived from the words:
“Thalassa” which in Greek means the sea.
“emia” which is a disease of the blood.

Thalassemia means disease of the blood related to the sea.

It was previously thought that this type of disease was confined to the Mediterranean Sea
including Lebanon. However, we now know that this group of anemias, although
prevalent in the Mediterranean basin, is present in many other areas and particularly in
Southeast Asia.

Thalassemias

Anemias can be due to difficulties (among other things) in the formation of Heme (Iron
deficiency anemia) or of globin chain synthesis (Hemoglobinopathies).
Thalassemias for instance are a group of disorders resulting from diminished production
or complete absence of globin chain production. These are classified according to which
globin chain is synthesized at a reduced rate. The main types which have been defined
with certainty are the beta and alpha thalassemias.

In ß thalassemia, synthesis of the ß chain is defective. In α thalassemia, it is synthesis of

the chain that is defective. Finally, with δ-β thalassemia, both δ and β chains are reduced.
Both the alpha and beta chains are structurally normal, the defect is quantitative.

Normal Hemoglobins

Normally after the age of six months, we have the following normal hemoglobins:
HbA 2 2 ~ 95 % of the total adult Hb
Hb F 2 2 < 1 % of the total adult Hb
Hb A2 2 2 < 3.1 % of the total adult Hb

N.B.: Hb F or Fetal Hb is normally present in high concentrations during fetal life but
the concentration of this Hb falls rapidly after birth and assumes the normal adult level
of 1% or less after the age of six months.
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How is thalassemia passed on from parents to their children?

Genes control every characteristic of the body. One gene for each characteristic comes
from the mother and the other from the father. Therefore, among many other genes, two
genes control how β globin is made inside the RBC and four α genes control how α
globin is made inside the RBC.

Remember, the globin chain structural genes are located on chromosomes 16 and 11.
There are two copies of the hemoglobin alpha gene on each chromosome 16 or four α
genes per cell. In contrast, there is only one ß gene on each chromosome 11 or two ß
genes per cell.

Normally all four α genes and both ß genes are active in the production of globin chains.

In thalassemia, globin chain synthesis is absent or reduced because of mutation or

deletion of the genes that code for the globin chains.

Deletions of α genes tend to underlie most cases of α-thalassemia. The severity of

symptoms depends on how many of these genes are lost. Loss of one or two genes is
usually asymptomatic, whereas deletion of all four genes is fatal to the unborn child.
In contrast, over 100 types of mutations affect the ß genes, and deletion mutations are
rare. Some mutations tend to cause a reduction, rather than a complete absence, of β-
globin chains and so result in milder disease. Other mutations tend to cause total absence
of β-globin chains leading to severe disease.
Currently, severe thalassemia is treated by blood transfusions, and a minority of patients
are cured by bone marrow transplantation. Mouse models are proving to be useful in
assessing the potential of gene therapy.
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Beta thalassemia:
In β thalassemia for instance, there is partial or complete absence of beta chain
production, therefore there is decreased or total absence of HbA.
 Normal people are normal because they have two normal β genes for Hb.
 Healthy carriers of beta thalassemia trait have one normal β gene for Hb and one
altered gene.
 People with beta thalassemia major have two altered β genes for Hb.

Types of beta thalassemia:

β-thalassemia is characterized by mutations in the β-globin gene, which reduce or
completely stop gene expression. There are approximately 100 genetic mutations that
have been described that cause beta thalassemia, designated as beta 0 or beta + mutations.
Mutations that eliminate expression are labeled β-zero, while those that reduce expression
are labeled β-plus. Severity of this condition is dependent on the extent of β-globin chain
under expression.

Beta zero (o) type: the beta globin chain is completely absent and therefore no HbA is
produced at all.

Beta plus ((+) type: the beta globin chain is formed but in reduced amounts which
allows a small amount of adult Hb to be made.

Clinical Aspects
At present, thalassemia diseases are classified into transfusion-dependent thalassemia
(thalassemia major) and non-transfusion-dependent thalassemia (thalassemia intermedia).
This classification is based on the clinical severity of patients determining whether they
do require regular blood transfusions to survive (transfusion-dependent thalassemia) or
not (non-transfusion-dependent thalassemia). Definitive diagnosis of thalassemia and
hemoglobinopathies requires a comprehensive workup from complete blood count,
hemoglobin analysis, and molecular studies to identify mutations of globin genes.
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I. Homozygous beta thalassemia

A- Beta zero (o) thalassemia (thalassemia major)
In the beta zero (o) type, there is absolutely no beta chain synthesis, so there is a
complete absence of Hb A. However, there is a normal alpha chain synthesis,
therefore there is excess alpha chains. In compensation to this, some other chain
has to be formed, otherwise the person will not survive, and this other chain is the
same chain that is formed in the embryo (gamma chain), so the alpha chains will
be used with the gamma chains to form Hb F. As a result, we will get some
increase in HbF but this increase is not enough to meet the needs of the body and
therefore these children suffer from severe anemia that we call beta thalassemia
major.

Causes of anemia in beta zero ( o) thalassemia

1. Since beta chain is not produced and gamma chain is slightly increased,
there is a diminished total Hb synthesis.

2. The excess alpha chains precipitate in the RBC and make the RBC
membrane rigid so that it can no more deform, therefore this RBC cannot
pass through the sinusoids of the BM and is therefore broken down there
and we get as a result, what is known as ineffective erythropoiesis.

3. What escapes is trapped in the sinusoidal lining of the spleen, so we get a

shortened RBC survival and a hemolytic component as a result.
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What happens to iron in beta zero ( o) thalassemia?

1. These alpha chain precipitates destroy the RBC(s) all over the body so we
get accumulation of iron in the stores and serum ferritin increases as a
result.

2. The destruction of RBC(s) initiates outpouring of erythropoietin that

increases iron absorption from the intestine and ultimately results in an
increased level of iron in the body.

3. Finally, regular transfusions will cause iron overload that will lead to
hemochromatosis and multiple organ damage. Iron damages the liver
causing diabetes mellitus, the endocrine organs leading to failure of
growth, delayed or absent puberty, hypothyroidism, and
hypoparathyroidism and lastly the myocardium, causing significant heart
problems that are most usually the cause of death in these patients.

What happens to the bones in beta thalassemia major?

Since there is a marked proliferation of the erythroid series in the BM of these
patients, the BM becomes so loaded with cells of the erythroid series at an early
age that these cells slowly expand the bone, which is still soft in children. These
children develop bone deformities as a consequence, with frontal bossing, cheek
bone and jaw protrusions, distortions of ribs and vertebrae and pathologic
fractures of the long bones.

Diagnosis of beta thalassemia major

The hematological profile consists of measurements of the RBC parameters and

includes Hb concentration, hematocrit, RBC number as well as RBC indices
including mean cell volume (MCV), mean corpuscular Hb (MCH), and red-cell
distribution width (RDW, an indicator of RBC size variation).
Quantitative hemoglobinopathies may have an impact on the red-cell indices, and
accordingly these indices are critical to the differential diagnosis of thalassemia.
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The general classification of thalassemia is hypochromic and microcytic anemia;
therefore, the MCV can be considered as a key diagnostic indicator.
Thalassemic individuals have a reduced MCV, and an MCV of 70 femtoliter or
less, is maximally sensitive and specific for presumptive diagnosis of thalassemia
syndromes.
The RBC count is also useful as a differential tool, because thalassemias produce
a microcytic hypochromic anemia with an associated increase in the RBC
number. Other microcytic hypochromic anemias, including iron deficiency and
possibly anemia of chronic disease, are more typically associated with a decrease
in the RBC number that is proportional to the degree of decrease in Hb
concentration.
The Hb concentration can provide complementary information since it is typically
decreased in thalassemia. The thalassemia-minor conditions produce minimal
decrements in the Hb concentration, whereas thalassemia intermedia and
thalassemia major may be associated with moderate to severe decreases in Hb
concentration.

A. ABC:

1. Hb , Hct 
2. The RBC indices indicate a microcytic hypochromic anemia (MCV,
MCH).
3. RDW ↑

B. Peripheral blood film

1. As is the case with many anemias, anisopoikilositosis is detected.

2. Because of a marked decrease in hemoglobin synthesis,

 microcytic, hypochromic RBC(s)

3. Because of the decrease in hemoglobin synthesis,

 target cells

4. Because of increased erythropoiesis

 immature RBC(s), including polychromatic and stippled RBCs,
along with nucleated RBC(s)
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N.B.
The reticulocyte count increases depending on the severity of the
anemia but this increase is less severe than the erythroid
hyperplasia, due to the ineffective erythropoiesis.

4. Because of RBC destruction when passing through the sinusoidal

lining  fragments of RBC(s) or shistocytes.

5. Because of alpha chain precipitate over the cytoplasm and the

nucleus  chromosomal breakage and Howell Jolly bodies which
are only seen following splenectomy. Cabot rings may also be seen
as a result of chromosomal breakage.

C. Bone marrow picture

,

The BM shows increase in cellularity due mainly to erythroid cells.

D. Hemoglobin electrophoresis

Because CBC and peripheral smear findings are non-specific, hemoglobin

electrophoresis or high-performance liquid chromatography (HPLC) are
necessary to assess the different types of hemoglobin found in the sample and
confirm a diagnosis of β-thalassemia. These assays will reveal abnormal
percentages of normal hemoglobin types (HbA, HbA2, and HbF).

Here we separate the different hemoglobins by the electric current on cellulose

acetate at pH of 8.6. Hemoglobins, which are negatively charged proteins, will
move to the positive pole (anode) when we pass the electric current. The
separated hemoglobins are quantified by staining and measuring the intensity of
the color formed:
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Normally, the slowest Hb is HbA2 and the fastest is HbA.

Hemoglobin pattern in beta thalassemia major

1. Complete absence of HbA
2. Variable amount of HbA2 (which could be low, normal or high) so
its level is unimportant.
3. All the rest is HbF (95%).

Additional Diagnostic Tests

Supplemental tests for β-thalassemia may include:
 Serum ferritin level: Serum ferritin may be used as an indicator of
iron storage and predictor of cardiac problems
 Molecular analysis: Commonly occurring mutations of the β-
globin gene can be detected by PCR-based procedures or sequence
analysis of the β-globin gene

B- Beta plus (+) thalassemia

This is a very varied group:
If the beta chain formation is minimal  same as beta zero type
(beta thalassemia major).

If the beta chain formation is high enough  the child will have only
moderate anemia compatible with life (beta thalassemia intermedia).

Diagnosis of beta plus thalassemia

Decrease in Hb, Hct, MCV and MCH

Peripheral blood film

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1. Marked anisopoikilocytosis
2. Microcytic hypochromic RBC(s)
3. Target cells
4. Schistocytes
5. Some polychromasia and stippled RBC(s)

N.B: Note that the reticulocyte count is slightly increased

Peripheral blood smear from thalassemia intermedia patient, showing

anisopoikilocytosis (variation of cell size and shape),a nucleated red cell,
and basophilic stippling. © 2019 StatPearls Publishing, LLC.6

Bone marrow picture

The BM picture is similar to that of beta zero thalassemia: It shows

increase in cellularity due mainly to erythroid cells.

Hemoglobin electrophoresis

1. HbA: decreased amounts (10-20%)

2. HbF: high (70-80%)
3. HbA2: variable (2-7%)

OR

1. HbA: significant production (70-80%)

2. HbF: (10-30%)
3. HbA2: variable (2-7%)
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II. Heterozygous beta thalassemia

Heterozygous inheritance of a β-thalassemia mutation results in a trait
condition, sometimes termed β-thalassemia minor (here only one gene is
involved).

Depending on whether we have beta zero or beta plus, the individuals can
be very mildly to moderately anemic.

People with  thalassemia trait are perfectly healthy themselves, so they

carry  thalassemia but they are not ill. Most people with  thalassemia
trait do not know that they have it, but screening is essential to diagnose
those carriers who might pass on the gene to their children. If their partner
also has  thalassemia trait, there is a 25% chance that their child will
have  thalassemia major.

Diagnosis of the heterozygous state

A. ABC:
The ABC shows:
1. A slight decrease in Hb & Hct that may not be significant at times.
2. A significant decrease in MCV and MCH.
3. The RDW can be used to differentiate between microcytic anemias,
most notably iron deficiency (increase in RDW), and the thalassemia
trait condition, which — in contrast — tends to produce a uniform
microcytic red-cell population without a concomitant increase in
RDW. RDW may provide complementary information but is not
useful as a lone indicator.
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B. Peripheral blood film

1. Microcytic hypochromic RBC(s)
2. Target cells
3. Cigar shaped RBC(s)
4. Polychromatic and stippled RBC(s)

C. Hemoglobin electrophoresis
Hemoglobin electrophoresis shows a predominance of HbA, an invariable
increase in HbA2, with or without an increase in Hb F:

1. Hb A predominant
2. The great majority show an elevated HbA2 (> 3.5%) and this is
very significant (4-7%).
However, this increase in A2 may be masked by the coexistence of
Fe deficiency anemia.

Series of blood counts of beta thalassemia minor,

all with high Hb A2

4. Elevation of HbF in around 50% of carriers, but still below 7%.

A notable confounding factor in hemoglobin electrophoresis is

concomitant iron deficiency, which can conceal underlying thalassemia
minor and make the HbA2 pattern appear normal.
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Differential Diagnostic Tests

Most people with thalassemia minor are diagnosed when their complete blood count
(CBC) reveals mild microcytic anemia (anemia characterized by small, pale RBCs).
Additional tests are needed for differential diagnosis, since microcytic anemia can also be
caused by iron deficiency, lead poisoning, sideroblastic anemia, or anemia of chronic
disease.

Iron deficiency may be easily ruled out as a cause by performing reference range iron
studies (serum iron, total iron binding capacity, percent transferrin saturation).

In children, the Mentzer index (the ratio of MCV to RBC count) may help differentiate
iron deficiency from thalassemia:
Ratio >13 in iron deficiency
Ratio <13 in thalassemia
Indices such as MCV, RBC volume distribution width (RDW), and an individual's
medical history can rule out between these conditions as well as other conditions.

In patients who are β-thalassemia carriers, the RDW will be normal or slightly increased.
This finding can help differentiate thalassemia minor from other microcytic hypochromic
anemias such as iron deficiency anemia characterized by an increase in RDW or
sideroblastic anemia, in which RDW values are typically very high.

Consequently, a finding of microcytic anemia in a person with a normal RDW will

commonly be caused by thalassemia, whereas microcytic anemia in a person with an
elevated RDW does not suggest thalassemia but warrants further testing.
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CASE STUDIES

Case 1

Healthy Caucasian adult.

Blood count:

Hb : 12.5 g/dl
RBC : 6.2 1012 / l
MCV : 61 fl
RDW :13.4 %

Note :

Near normal hemoglobin level.

High red cell count
Low MCV
Normal RDW

Hb electrophoresis :

Cellulose acetate pH 8.4

Hb A2 ---5.5 %( normal range 1.5-3.5%)

Hb F-----2.5% (normal range < 1%)

Diagnosis : Typical beta thalassemia minor

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Case 2.

An underdeveloped 4-year-old boy of Italian origin with splenomegaly and severe

anemia that required blood transfusions to maintain life (and to maintain hemoglobin
above 10g/dl).

Blood smear

Note: bizarre fragmented red cells variable in size and shape; occasional nucleated RBC;
polychromes. Note that the normal red cells are probably transfused.

Diagnosis
Beta thalassemia major (Homozygous o thalassemia).

Comment:

o = beta gene producing no beta chains. Therefore the patient is unable to produce any
beta chains and hence no Hb A. When untreated he has about 95% Hb F (remainder
being Hb A2) But 95% of very little! The switch from gamma chain production to
(attempted) beta chain production has occurred normally. This clinical and hematologic
picture is typical of Mediterranean beta thalassemia major.

Treatment

 Transfusion to maintain hemoglobin level at least 10g/dl.

 Chelation therapy to remove excess iron present due to hemolysis and
transfusions.

Bone marrow transplantation is treatment of choice where available.

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Management of Thalassemia

Thalassemia major is usually a fatal disease. Actually, patients with β thalassemia major
do depend on regular transfusions to live. In other words, the condition is not compatible
with life without the transfusions, and as such, we call it transfusion dependant anemia.

The child with untreated thalassemia has a very severe anemia, first detected in early
childhood and characterized by increasing hepatosplenomegaly, slight jaundice and
marked bone changes due to an expanded BM cavity from massive erythroid hyperplasia.
Atypical facies results, with prominence of the forehead, cheek bones and upper jaw.
Physical growth and development may be impaired. Finally thinning of the bony cortex
may result in pathologic fractures.

The Hb level falls to very low levels in the patient who has not been given transfusions.

The solution to all these problems should be:

1. Regular blood transfusions every 4-6 weeks.
2. Iron chelation therapy which is combined with hyper transfusion, to
prevent iron overload.

Desferal (Desferrioxamine) is given by subcutaneous infusion over 8-12

hours, 5-7 days weekly. It is started in infants after 10-15 units of blood
have been transfused or when the serum ferritin level exceeds 1000-1500
ng/ml (Normal: 10-250 ng/ml). Chelated iron is largely excreted in urine
and partly in the stools (1/3 of chelated iron).

Clinical trials with oral chelation agents are now in use. L1 has been found
as effective as desferrioxamine in increasing urinary iron excretion.

3. Vitamin C increases excretion of iron produced by desferrioxamine  ~

200 mg is administered daily.

4. Splenectomy may be needed to reduce blood requirements. This is

delayed until the patient is over 6 years because of the high risk of
dangerous infections post-splenectomy earlier in life.
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5. A different sort of treatment called “bone marrow transplantation” is now

possible for some children with  thalassemia major. When it is
successful, the person does not need any blood transfusion. However, it is
not a simple solution and it is very expensive. In each case, there is a risk
that the BM transplantation may not work or the patient may die or have
serious complications. We hope that these problems will be gradually
overcome in the future.

Prevention remains the best solution to these problems!