Download Chapter 1

MLAB 1415: Hematology
Keri Brophy-Martinez
Chapter 11:
Thalassemia
1
Introduction to
Thalassemia
2
Thalassemia

Diverse group of congenital disorders which
manifest as anemia of varying degrees.

Result of quantitative defective production of
one or more globin portion(s) of hemoglobin
molecule.

Distribution is worldwide.

Defect results from abnormal rate of synthesis
in one or more of the globin chains.
3
Thalassemia
Results in overall decrease in amount of
hemoglobin produced and may induce
hemolysis.
 Two major types of thalassemia:

◦ Alpha (α) - Caused by defect in rate of
synthesis of alpha chains.
◦ Beta (β) - Caused by defect in rate of synthesis
in beta chains.

May contribute protection against malaria.
4
Genetics of Thalassemia
May be either homozygous defect or
heterozygous defect.
 Adult hemoglobin composed two alpha and
two beta chains.
 Genetic defects usually falls into one of below
categories

◦
◦
◦
◦
◦
Gene deletion
Promoter deletion
Nonsence mutation
Mutated termination
Splice site mutation
5
Genetics of Thalassemia
Alpha thalassemia usually caused by gene
deletion
 Beta thalassemia usually caused by
mutation.

6
Review of Hgb Structure

Normal globin genes
◦ Alpha, beta, delta, gamma
 Form hgb A (97%), hgb A2, hgb F
◦ Epsilon, zeta: in utero
◦ Gamma: 3rd trimester until birth
Ratio of β-chain to α-chain is 1:0
 Thalassemia causes an excess of one of
these chains

7
Pathophysiology

α-chain excess
unstable
 Precipitates within the cell, binding to the cell membrane
causes damage
 Macrophages destroy the damaged RBCs in the bone
marrow, leads to ineffective erythropoiesis
 Spleen also removes damaged RBCs, leads to chronic
extravascular hemolysis


β-chain excess
◦ Unstable
◦ Combines to form hgb molecules with 4 β-chains
( hemoglobin H)
 Infants: excess gamma chains combine with hgb molecules
(hemoglobin Bart’s)
◦ High oxygen affinity, poor transporter of oxygen
8
General Clinical Findings
Anemia
 Hypoxia
 Splenomegaly
 Gallstones
 Skeletal abnormalites
 Iron toxicity
 Fractures

9
Comparison of Hemoglobinopathies
and Thalassemias
Disease
Hemoglobinopathy
RBC
count
Indices
RBC Morph
Abnormal
Hb
Ancestry
Normocytic
Target cells,
sickle cells
(HbS),
Crystals
(HbC)
HbS,HbC,
HbE etc
African
Mediterranean
Middle Eastern
Asian
Target cells,
basophilic
stippling
HbH
Hb Bart’s
African
Mediterranean
Asian
Normochromi
c
Thalassemia
Microcytic
Hypochromic
Retic
Count
Thalassemia: globin chains structurally normal
Hemoglobinopathies: globin chain is abnormal
10
Beta
Thalassemia
11
Classical Syndromes of Beta
Thalassemia




Beta thalassemia minima/ Silent
carrier state – the mildest form of beta
thalassemia.
Beta thalassemia minor - heterozygous
disorder resulting in mild hypochromic,
microcytic hemolytic anemia.
Beta thalassemia intermedia - Severity
lies between the minor and major.
Beta thalassemia major - homozygous
disorder resulting in severe transfusiondependent hemolytic anemia.
12
Beta thalassemia minima/ Silent
Carrier State for β Thalassemia
Are various heterogenous beta mutations
that produce only small decrease in
production of beta chains.
 Patients have nearly normal beta/alpha
chain ratio and no hematologic
abnormalities.
 Have normal levels of Hb A2.

13
Beta Thalassemia Minor
Caused by heterogenous mutations that affect
beta globin synthesis.
 Usually presents as mild, asymptomatic
hemolytic anemia unless patient in under stress
such as pregnancy, infection, or folic acid
deficiency.
 Have one normal beta gene and one mutated
beta gene.
 Hemoglobin level in 9-14 g/dL range with
normal or slightly elevated RBC count.

14
Beta Thalassemia Minor






Anemia usually hypochromic and microcytic
with slight aniso and poik, including target cells
and elliptocytes; May see basophilic stippling.
Rarely see hepatomegaly or splenomegaly.
Have high Hb A2 levels (3.5-8.0%) and normal to
slightly elevated Hb F levels.
Are different variations of this form depending
upon which gene has mutated.
Normally require no treatment.
Make sure are not diagnosed with iron
deficiency anemia.
15
Beta Thalassemia Intermedia
Patients able to maintain minimum hemoglobin
(7 g/dL or greater) without transfusions.
 Expression of disorder falls between thalassemia
minor and thalassemia major. May be either
heterozygous for mutations causing mild
decrease in beta chain production, or may be
homozygous causing a more serious reduction in
beta chain production.
 See increase in both Hb A2 production and Hb F
production.
 Peripheral blood smear picture similar to
thalassemia minor.

16
Beta Thalassemia Intermedia






Have varying symptoms of anemia, jaundice,
splenomegaly and hepatomegaly.
Have significant increase in bilirubin levels.
Anemia usually becomes worse with infections,
pregnancy, or folic acid deficiencies.
May become transfusion dependent as adults.
Tend to develop iron overloads as result of
increased gastrointestinal absorption.
Usually survive into adulthood.
17
Beta Thalassemia Major/ Cooley’s
anemia
Characterized by severe microcytic,
hypochromic anemia.
 Detected early in childhood:

◦ Infants fail to thrive.
◦ Have pallor, variable degree of jaundice, abdominal
enlargement, and hepatosplenomegaly.
Hemoglobin level between very low
 Severe anemia causes marked bone changes due
to expansion of marrow space for increased
erythropoiesis.
 See characteristic changes in skull, long bones,
and hand bones.

18
Beta Thalassemia Major






Have protrusion upper teeth and Mongoloid
facial features.
Physical growth and development delayed.
Peripheral blood shows markedly hypochromic,
microcytic erythrocytes with extreme
poikilocytosis, such as target cells, teardrop cells
and elliptocytes. See marked basophilic stippling
and numerous NRBCs.
MCV in range of 50 to 60 fL.
Low retic count seen (2-8%).
Most of hemoglobin present is Hb F with slight
increase in Hb A2.
19
Beta Thalassemia Major
Regular transfusions usually begin around one
year of age and continue throughout life.
 Excessive number of transfusions results in
tranfusional hemosiderosis; Without iron
chelation, patient develops cardiac disease.
 Danger in continuous tranfusion therapy:

◦ Development of iron overload.
◦ Development of alloimmunization (developing
antibodies to transfused RBCs).
◦ Risk of transfusion-transmitted diseases.

Bone marrow transplants may be future
treatment, along with genetic engineering and
new drug therapies.
20
Comparison of Beta Thalassemias
GENOTYPE
HGB A
HGB A2
HGB F
NORMAL
Normal
Normal
Normal
MINIMA
Normal
Normal
Normal
MINOR
Dec
Normal to Inc
Normal to Inc
INTERMEDIA
Dec
Normal to Inc
Usually Inc
MAJOR
Dec
Usually Inc
Usually Inc
21
Other Thalassemias Caused by Defects
in the Beta-Cluster Genes
1. Delta Beta Thalassemia
 2. Hemoglobin Lepore
 3. Hereditary Persistence of Fetal
Hemoglobin (HPFH)

22
Delta Beta Thalassemia
Group of disorders due either to a gene
deletion that removes or inactivates only
delta and beta genes so that only alpha
and gamma chains produced.
 Similar to beta thalassemia minor.
 Growth and development nearly
normal. Splenomegaly modest. Peripheral
blood picture resembles beta thalassemia.

23
Hemoglobin Lepore
Rare class of delta beta thalassemia.
 Caused by gene crossovers between delta
locus on one chromosome and beta locus
on second chromosome.

24
Hereditary Persistence of Fetal
Hemoglobin (HPFH)
Rare condition characterized by continued
synthesis of Hemoglobin F in adult life.
 Do not have usual clinical symptoms of
thalassemia.
 Little significance except when combined with
other forms of thalassemia or
hemoglobinopathies.
 If combined with sickle cell anemia, produces
milder form of disease due to presence of Hb
F.

25
Hereditary Persistence of Fetal
Hemoglobin (HPFH)


Hb F more resistant to denaturation than Hb A. Can be
demonstrated on blood smears using Kleihauer Betke
stain. Cells containing Hb F stain.
Classified into two groups according to distribution of Hb
F among red cells:
◦ Pancellular HPFH - Hemoglobin F uniformly distributed
throughout red cells.
◦ Heterocellular HPFH - Hemoglobin F found in only
small number of cells.
26
Beta Thalassemia with Hgb S
Inherit gene for Hb S from one parent and gene
for Hb A with beta thalassemia from second
parent.
 Great variety in clinical severity. Usually depend
upon severity of thalassemia
inherited. Production of Hb A ranges from none
produced to varying amounts. If no Hb A
produced, see true sickle cell symptoms. If some
Hb A produced, have lessening of sickle cell
anemia symptoms.

27
Beta Thalassemia with Hgb C
Shows great variability in clinical and
hematologic symptoms.
 Symptoms directly related to which type
thalassemia inherited.
 Usually asymptomatic anemia

28
Beta Thalassemia with Hgb E
Is unusual because results in more severe
disorder than homozygous E disease.
 Very severe anemia developing in
childhood.
 Transfusion therapy required.

29
Alpha
Thalassemia
30
Alpha Thalassemia
Predominant cause of alpha thalassemias is large
number of gene deletions in the alpha-globin
gene.
 Four clinical syndromes present in alpha
thalassemia:

◦
◦
◦
◦
Silent Carrier State
Alpha Thalassemia Trait (Alpha Thalassemia Minor)
Hemoglobin H Disease
Bart's Hydrops Fetalis Syndrome
31
Silent Carrier State





Deletion of one alpha gene, leaving three functional alpha
genes.
The 3 remaining genes direct synthesis of adequate
numbers of α-chains for normal hgb synthesis.
No hematologic abnormalities present.
Alpha/Beta chain ratio nearly normal.
No reliable way to diagnose silent carriers by
hematologic methods; Must be done by globin gene
analysis mapping.
May see borderline low MCV (78-80fL).
32
Alpha Thalassemia Trait
(Alpha Thalassemia Minor)






Also called Alpha Thalassemia Minor.
Occurs when two of the four alpha genes are
missing.The unaffected globin chains can
compensate for the missing genes.
Exhibits mild microcytic, hypochromic anemia.
MCV between 70-75 fL.
May be confused with iron deficiency anemia.
Although some Bart's hemoglobin (γ4) present at
birth, no Bart's hemoglobin present in adults.
33
Hemoglobin H Disease



Second most severe form alpha thalassemia.
Only one alpha gene out of four is functional
Results in accumulation of excess unpaired
gamma or beta chains.
◦ The excess chains pair up to form tetrads
 Beta: hemoglobin H (adults)
 Gamma: hemoglobin Bart’s (infants)



Unstable
Precipitates within RBCs triggers hemolysis
High affinity for oxygen which reduces oxygen
delivery to the tissues
34
Hemoglobin H Disease
Live normal life; however, infections, pregnancy,
exposure to oxidative drugs may trigger
hemolytic crisis.
 RBCs are microcytic, hypochromic with marked
poikilocytosis. Numerous target cells.
 Hb H vulnerable to oxidation. Gradually
precipitate in vivo to form Heinz-like bodies of
denatured hemoglobin. Cells been described has
having "golf ball" appearance, especially when
stained with brilliant cresyl blue.

35
Bart’s Hydrops Fetalis Syndrome or
α-Thalassemia major





Most severe form. Incompatible with life. Have no
functioning alpha chain genes
Baby born with hydrops fetalis, which is edema and ascites
caused by accumulation serous fluid in fetal tissues as
result of severe anemia. Also see hepatosplenomegaly and
cardiomegaly.
Predominant hemoglobin is Hemoglobin Bart, along with
Hemoglobin Portland and traces of Hemoglobin H.
Hemoglobin Bart's has high oxygen affinity so cannot
carry oxygen to tissues. Fetus dies in utero or shortly
after birth. At birth, see severe hypochromic, microcytic
anemia with numerous NRBCs.
Pregnancies dangerous to mother. Increased risk of
toxemia and severe postpartum hemorrhage.
36
Comparison of Alpha Thalassemias
Genotype
Hb A
Hb Bart
Hb H
Normal
97-98%
0
0
Silent Carrier
96-98%
0-2%
0
Alpha
Thalassemia
Trait
85-95%
5-10%
0
Dec
25-40%
2-40%
0
80% (with
20% Hgb
Portland)
0-20%
Hemoglobin H
Disease
Hydrops Fetalis
37
Laboratory
Diagnosis of
Thalassemia
38
Laboratory Diagnosis of Thalassemia
Need to start with patient's individual
history and family history. Ethnic
background important.
 Perform physical examination:

◦
◦
◦
◦
Pallor indicating anemia.
Jaundice indicating hemolysis.
Splenomegaly due to pooling of abnormal cells.
Skeletal deformity, especially in beta thalassemia
major.
39
CBC with Differential



See decrease in hemoglobin, hematocrit, mean corpuscular
volume (MCV), and mean corpuscular hemoglobin
(MCH). See normal to slightly decreased Mean
Corpuscular Hemoglobin Concentration (MCHC). Will
see microcytic, hypochromic pattern.
Have normal or elevated RBC count with a normal red
cell volume distribution (RDW).
Decrease in MCV very noticeable when compared to
decrease in Hb and Hct.
40
CBC with Differential


Elevated RBC count with markedly decreased MCV
differentiates thalassemia from iron deficiency anemia.
On differential, see microcytic, hypochromic RBCs (except
in carrier states). See mild to moderate poikilocytosis. In
more severe cases, see marked number of target cells and
elliptocytes. Will see polychromasia, basophilic stippling,
and NRBCs.
41
Reticulocyte Count

Usually elevated. Degree of elevation
depends upon severity of thalassemia.
42
Osmotic Fragility
Have decreased osmotic fragility.
 Is not very useful fact for diagnosing
thalassemia. Is an inexpensive way of
screening for carrier states.

43
Brilliant Cresyl Blue Stain

Incubation with brilliant
cresyl blue stain causes
Hemoglobin H to
precipitate. Results in
characteristic
appearance of multiple
discrete inclusions -golf
ball appearance of
RBCs. Inclusions
smaller than Heinz
bodies and are evenly
distributed throughout
cell.
44
Acid Elution Stain

Based on Kleihauer-Betke
procedure. Acid pH will
dissolve Hemoglobin A from
red cells. Hemoglobin F is
resistant to denaturation and
remains in cell. Stain slide
with eosin. Normal adult
cells appear as "ghost" cells
while cells with Hb F stain
varying shades of pink.
45
Hemoglobin Electrophoresis
Important role in diagnosing and differentiating
various forms of thalassemias.
 Can differentiate among Hb A, Hb A2, and Hb F, as
well as detect presence of abnormal hemoglobins
such as Hemoglobin Lepore, hemoglobin Bart's,
or Hemoglobin Constant Spring.
 Also aids in detecting combinations of
thalassemia and hemoglobinopathies.

46
Hemoglobin Quantitation
Elevation of Hb A2 excellent way to detect
heterozygote carrier of beta
thalassemia. Variations in gene expression
in thalassemias results in different amounts
of Hb A2 being produced.
 Can also quantitate levels of Hb F.

47
Routine Chemistry Tests
Indirect bilirubin elevated in thalassemia
major and intermedia.
 Assessment of iron status, total iron
binding capacity, and ferritin level important
in differentiating thalassemia from iron
deficiency anemia.

48
Other Special Procedures
Globin Chain Testing - determines ratio of
globin chains being produced.
 DNA Analysis - Determine specific defect
at molecular DNA level.

49
Differential Diagnosis of Microcytic,
Hypochromic Anemias
RDW
Serum
Iron
TIBC
Serum
Ferritin
FEP
Inc
Dec
Inc
Dec
Inc
Alpha Thal
Norm
Norm
Norm
Norm
Norm
Beta Thal
Norm
Norm
Norm
Norm
Norm
Hgb E Disease
Norm
Norm
Norm
Norm
Norm
Anemia of
Chronic Disease
Norm
Dec
Dec
Inc
Inc
Inc
Inc
Norm
Inc
Dec
Norm
Norm
Norm
Norm
Inc
Iron Deficiency
Sideroblastic
Anemia
Lead Poisoning
50