Thalassemia is an inherited blood disorder in which the body makes an abnormal form of

hemoglobin.

HISTORY

In 1925, Cooley and Lee first describe four children with anemia, hepatomegaly, mild
hepatomegaly and mongoloid faces. COOLEY'S ANEMIA is a typical finding in those
characteristics and in young children with untreated beta thalassemia major.

EPIDEMIOLOGY

1.)Thalassemia and hemoglobinopathy-5% carrier

2.)Born with thalassemia major-56,000 infants

3.)Carrier frequency of beta thallassemia-0.3%-15% in South East Asia, 75%-80% in Nepal.

GENETICS OF GLOBIN SYNTHESIS

The normal Hb molecule is a tetramer of 2 alpha like synthesis with 2 beta like chains.
Combination of these chain produces six normal Hb. Three are embryonic Hb: Hb Gower 1, Hb
Gower 2, and Hb Portland. Others are fatal Hb and 2 adult Hb.

CATEGORIES OF THALLASSEMIA

Beta thalassemia-affect mainly the beta chain production and other except alpha thalassemia.
Alpha thalassemia-involve genes for the alpha 1 and 2 chains on chromosome

GENETIC DEFECTS CAUSING THALLASSEMIA

1.)Reduced or absent transcription of mRNA

2.)mRNA processing errors

3.)Translation errors

4.)Deletion of one more globin genes

PATHOPHYSIOLOGY OF THALASSEMIA:

Clinical Manifestations stem from:

1. Reduced/absent production of globin chain

2. Unequal production of alpha and beta-globin chains (Note: #2 is more significant)

 MECHANISMS OF BETA-THALASSEMIAS
unpaired, excess alpha-chains precipitate
in developing RBCs causing stress and
damage to cellular membranes.
apoptosis is triggered.
iron accumulation in RBC precursors
ineffective erythropoiesis- premature death
of RBC precursors in the bone marrow
anemia is multifactorial and results from
ineffective production and decreased
destruction
asymptomatic during fetal life because Hb
F is the predominant circulating
hemoglobin.
symptoms begin 6-24 mo. of age
in beta-thalassemia major, increase in
erythropoietin by the kidney results in
massive but ineffective erythroid dysplasia
in untreated patients, deformities occur due
to massive bone marrow expansion
MECHANISMS OF ALPHA-THALASSEMIA
decreased production of alpha-chains can
manifest in utero
decrease production in alpha-chains is
inversely proportional to y-chains Y-chains -
more stable and do no precipitate - forms
hemoglobin tetramers or Hb Bart
do not cause severe ineffective
erythropoiesis but may cause moderate
hemolytic anemia because they form
inclusion bodies when they mature.
pathologic fractures due to reduction of bone
mineral density
in children, lacy or lucent long bone
radiographs are observed.
"hair on end" radiograph appearance on
skulls because of vertical striations of bony
trabeculae Prominence of forehead,
cheekbone, and upper jaw
hepatosplenomegaly due to extramedullary
erythropoiesis
neutropenia and thrombocytopenia
increase in unconjugated bilirubin
iron accumulation in various organs
causing morbidity and mortality in adults
while growth retardation and absence of
sexual maturity in children.
may also cause cardiomyopathy, fibrosis,
and cirrhosis
suppresion of hepcidin production
Hb Bart cannot deliver oxygen to tissues
causing massive edema, heart failure, and
hepatomegaly in fetuses. This causes the
babies to die shortly after birth. BETA-
GLOBIN GENE CLUSTER
THALASSEMIAS
great heterogeneity in beta-globin
mutations lead to B thalassemia
individuals with severe beta-thalassemia
are compound heterozygote for 2 different
beta-thalassemia mutations
 Depending on the specific genotype, Hb A none or a decreased amount of beta chains
is absent of decreased, determining whether are produced.
Hb A --- produced if a beta mutation is present, ranges from 10 - 30%.

Hb F --- ranges from 70% to >90% depending on the genotype and Hb A.

RESULT OF MASSIVE DESTRUCTION OF ERYTHROID CELLS AND RELEASE OF FREE

HEMOGLOBIN:

1. Serum haptoglobin --- reduced or absent

2. Serum lactate dehydrogenase --- elevated

Transfusion therapy --- major option for patients with thalassemia major.

Transfusion regimens are termed "hypertransfusion" and are used not only to correct the anemia but to
also suppress the marked erythropoiesis.

Transfusion regimens, however, lead to an excess iron burden, which results in organ damage.

In the past, with transfusion therapy alone, thalassemic patients died in their teens, typically from
cardiac failure. Now patients undergo iron chelation therapy with the transfusion therapy.

The oral iron chelators, defarasirox and deferiprone, have been approved by the U.S. Food and Drug
Administration.

Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for thalassemia major.

Hydroxyurea therapy has benefited a few beta-thalassemia major patients, allowing them to become
transfusion-independent, but has not been beneficial in the majority of patients.

Research on the use of gene therapy to increase expression of the patient's own y-globin genes has intensified.
Ideally, in the future, gene therapy will be able to correct the genetic defect.

Beta-thalassemia major is a severe anemia leading to transfusion dependence.

BETA-THALASSEMIA INTERMEDIA

Beta-thalassemia intermedia manifests abnormalities with a severity between those of beta-thalassemia major
and minor, and does not require regular transfusion (transfusion-independent). Hemoglobin level: >7g/dL

Patients with thalassemia intermedia also have iron overload even though they do not receive transfusions.

The markedly accelerated ineffective erythropoiesis suppresses hepcidin production by the liver, which results
in more iron absorption by the intestinal enterocytes.
 Cardiac, liver, and endocrine complications, however, present 10 to 20 years later in thalassemia intermedia
patients than in patients who receive regular transfusion.

OTHER THALASSEMIA CAUSED BY DEFECTS IN THE BETA-GLOBIN GENE CLUSTER

Other thalassemias may be caused by deletion, inactivation, or fusion of a combination of genes of the beta-
globin gene cluster, such as:

1. hereditary persistence of fatal hemoglobin (HPFH)

2. delta beta-thalassemia

3. Hb Lepore thalassemia Thalassemias with Increased Levels of Fatal Hemoglobin HPFH and delta beta-
thalassemia are closely related, heterogenous conditions in which Hb F is expressed at increase levels beyond
infancy into adulthood.

These conditions have similarities but can be differentiated by the clinical presentation,
hemoglobin level, MCV, and amount of Hb F produced.
In HPFC, the beta-globin gene cluster typically contains a deletion the delta beta region that
leads to the increased production of Hb F.
The delta beta-thalassemias are also characterized by deletions in the delta and beta-globin genes
and an increase in Hb F in adult life.

CLINICAL SYNDROMES OF A-THALASSEMIA

Four clinical syndromes are present in a-thalassemia, de- pending on the gene number, cis or trans pairing, and
the amount of a chains produced.

4 syndromes:

Silent carrier state

a-thalassemia minor

Hb H disease

Hb Bart hydrops fetalis syndrome

ALPHA THALASSEMIA

Alpha thalassemia is a blood disorder that reduces the production of hemoglobin. Hemoglobin is the protein in
red blood cells that carries oxygen to cells throughout the body. Hb Bart syndrome is characterized by hydrops
fetalis, a condition in which excess fluid builds up in the body before birth.

ALPHA THALASSEMIA MINOR

Two missing or mutated genes is a condition called alpha thalassemia minor or having alpha thalassemia
trait. Children with this condition may have red blood cells that are smaller than normal (microcytosis) and
sometimes very slight anemia.
 HEMOGLOBIN H DISEASE

Hemoglobin H disease (HbH) is a form of alpha thalassemia in which moderately severe anemia develops due
to reduced formation of alpha globin chains. In this condition, as in the other forms of thalassemia, there is an
imbalance of globin chains needed to form hemoglobin.

HB BART HYDROPS FETALIS SYNDROME

Homozygous a-thalassemia ( / ) results in the absence of all a chain production and usually results
in death in utero or shortly after birth.
The fetus is severely anemic, which leads to cardiac failure and edema in the fetal subcutaneous tissues
(hydrops fetalis)
Hydropic pregnancies are hazardous to the mother, result- ing in toxemia and severe postpartum
hemorrhage.
Hydropic changes are detected in midgestation by means of ultrasound testing.
If both parents carry one a0-thalassemia haplotype ( /aa), prenatal diagnosis of homozygosity can be
made by molecular genetic testing of fetal cells from chorionic villus sampling or amniotic uid.
Absence of the a-globin genes establishes the diagnosis. Early termination of the pregnancy prevents the
serious maternal complications.

HEMOGLOBIN S-THALASSEMIA
 Sickle cell anemia (Hb SS) - a-thalassemia is a genetic abnormality due to the coinheritance of two
abnormal b-globin genes for Hb S and an a-thalassemia haplotype.
Hb S-b-thalassemia is a compound heterozygous condition that results from the inheritance of a b-
thalassemia gene from one parent and an Hb S gene from the other.
The combination of b-thalassemia and Hb S produces a phenotype similar to sickle cell anemia with a
similar incidence of stroke and a similar life expectancy.
Both conditions lack Hb A and produce severe painful crises as the predominant symptom.
Typically, the microcytosis and elevated Hb A2 level in Hb S-b0-thalassemia distinguish it from sickle
cell anemia.
HEMOGLOBIN C THALASSEMIA
Produces moderately severe hemolysis, splenomegaly, hypochromia, microcytosis, and target cells
HEMOGLOBIN E THALASSEMIA
Genetic point mutation in -globin gene
In homozygous state, clinical symptoms is similar to a mild -Thalassemia
Marked reduction of chain production when mutations are coinherited in the compound heterozygous
state

DIAGNOSIS OF THALASSEMIA
HISTORY AND PHYSICAL EXAMINATION
Finding in clinical examinations suggests thalassemia include: pallor, jaundice, splenomegaly, and
skeletal deformities
LABORATORY METHODS:
COMPLETE BLOOD COUNT WITH PERIPHERAL BLOOD FILM REVIEW
Decreased Hb, Hct, and MCHC
Increased RDW, reflecting anisocytosis (only in untreated -Thalassemia major, often normal in -
Thalassemia minor)
Microcytic and hypochromic in a Wright-stained peripheral blood film (except in silent carrier which
appears normal)
In /-Thalassemia minor and Hb H disease, cells are microcytic with target cells and slight to
moderate poikilocytosis
In -Thalassemia, poikilocytosis, target cells, and elliptocytes are present in addition to polychromasia,
basophilic stippling, Howell-Jowell bodies, Pappenheimer bodies, and nucleated RBCs
Reticulocyte Count
Elevated
In Hb H disease, reticulocyte count is 5% - 10%
In homozygous -Thalassemia, reticulocyte count is 2% - 8%
Supravital Staining
Brilliant Cresol Blue or New Methylene Blue stain is used
Hb H inclusions typically appear as small, multiple, and irregularly shaped greenish-blue bodies
In Hb disease, almost all RBCs contain Hb H inclusion
In -thalassemia minor, only few cells contain inclusions
In silent carrier -thalassemia, only a rare cell contain inclusions
Assessment of Normal and Variant Hemoglobins
Major clinical laboratory methods include: electrophoresis, cation-exchange high-performance liquid
chromatography (HPLC), and capillary zone electrophoresis (CZE) at least two of these methods is
used for confirmation of Hb variant
In Hb electrophoresis: migration and separation of Hb variants according to their charge
Pros: (1) distinguish common Hbs such as Hb A, Hb F, Hb S, and Hb C; (2) also fast moving Hbs:
Hb H and Hb Bart
Cons: (1) cannot accurately quantify Hb A2 and Hb F; (2) Hb S and Hb C by another method
because Hb D and Hb G comigrate with Hb S, Hb E, and Hb O
In HPLC: different retention time of various Hb types eluted in the column
Pros: (1) can accurately and quickly quantitate Hb A, Hb A2, and Hb F with 100% sensitivity and
90% specificity if no Hb variants are present, and (2) can also presumptively identify and quantify
Hb variants in low concentration
Cons: (1) Hb A2 and Hb E, due to same retention time are cannot be accurately quantified; (2) Hb
A2 can be overestimated if Hb S is present due to overlapping peaks; can be underestimated in
presence of Hb D
In CZE: migration of Hb fractions to the cathode at different velocities due to electro-endoosmotic flow;
absorbance of fraction is at 415 nm
Pros: can separate Hb A2 in the presence of Hb E
Cons: cannot quantify Hb A2 in the presence of Hb C
Molecular Genetic Testing
o For mutations in the HBB gene, targeted mutation analysis using polymerase chain reaction
(PCR) based methods can be initially performed for detection and quantification of the four
to six most common mutations if an individuals ethnicity is known.
o In multiethnic individuals or if the ethnicity is unknown , DNA sequencing of the HBB gene
is performed including exons, intervening sequences, splice sites and 5' and 3' untranslated
regulatory region.
o When the parents' mutation is known, analysis for the specific mutation in fetal cells can be
done on specimens from amniocentesis (15 to 18 weeks), chorionic villus sampling (10 to 12
weeks) or with preinplantation genetic diagnosis using a cell fron 3 day old embryo after in
vitro fertilization.
Other procedures
o Alkali denaturation- accurate and precise to quantify Hb F in the 0.2% to 50% range. Most
human hemoglobins are denaturated on exposure to a strong alkali, but Hb F is not.
o Automated HPLC- Used to quantify Hb F.
o Kleihauer- Betke - acid elution slide test, peripheral blood films are ethanol-fixed and
immersed in a citrate-acid buffer. Used to estimate the volume of fetal maternal hemorrhage
to determine if an increased dose of Rh immune globulin is needed for an Rh negative
mother who delivers an Rh positive baby.
o Flow cytometry- standard test to measure fetal-maternal hemorrhage quickly and accurately.
o Single tube osmotic fragility test- used to screen populations for thalassemia carriers.
Carriers have hypochromic RBC resulting in decreased osmotic fragility. This test is not
specific for thalassemia and will be positive for any condition causing hypochromia,
including deficiency anemia.
DIFFERENTIAL DIAGNOSIS OF THALASSEMIA MINOR AND IRON DEFICIENCY ANAEMIA

The RBCs in Thalassemia minor are microcytic and hypochromic, and this disease must be
differentiated from iron deficiency anaemia and other microcytic, hypochromic anaemia.
Clinical history is crucial. A family history of thalassemia raises the suspicion for this diagnosis. A
history of previously normal haemoglobin levels and RBC indices, significant bleeding, or pica leads to
diagnosis.
Pica means cravings for non-food items such as clay, dirt, or starch. And the most common pica
symptom in the U.S. is phagophagia, the craving to chew on ice.
IDA and -thalassemia minor are best differentiated using serum ferritin level, serum iron level, total
iron-binding capacity, transferrin saturation, Hb A2 level, along with a complete blood count (CBC) and
examination of a peripheral blood film.
But before evaluating Hb A2 levels for -thalassemia minor, iron deficiency should be ruled out. Low
iron levels in PTs with -thalassemia minor decrease the Hb A2 levels. The iron stores need to be
replenished before the laboratory analysis for thalassemia is undertaken.
A mild erythrocytosis (high RBC count) are found more commonly in
-thalassemia minor. In IDA, the RBC count and MCV may be normal or decreased, depending on
whether the deficiency is developing or long-standing. The RDW can be normal or increased in both -
thalassemia minor and IDA with a significant overlap of values; therefore, the RDW alone cannot
distinguish these conditions. Various discrimination indices have been proposed to distinguish
-thalassemia minor from iron deficiency anaemia using a calculation based on the RBC count, Hb
level, MCV, MCH and/or RDW
The sensitivity of these indices in discriminating -thalassemia minor and iron deficiency anaemia
ranged from 60% to 96%. The peripheral blood film may demonstrate basophilic stippling in -
thalassemia minor, which can distinguish it from iron deficiency. Because target cells can be found in
both conditions, however, their presence does not help discriminate between the two disorders.

TABLES
Reference Intervals for Normal in Adults

Beta-Thalassemia Associated with Structural Beta-Globin Varients

Genetic Designations in Thalassemia