thalassemias PATHOGENESIS AND PATHOLOGY PPT

THALASSEMIAS

1. Thalassemia

2. Quiz
nWhat is structure of hemoglobin A?
nWhat is the normal hemoglobin types in
normal adults?
nHemoglobin is composed of………..
and………
nThere are …. types of globin chains which
are……..
nNormally, rate of globin chain production is
equal or not equal.

3. Hemoglobin Types
Hemoglobin Type
nHgb A1—92%---------
nHgb A2—2.5%--------
nHgb F —<1%---------
nHgb H ------------------
nBart’s Hgb--------------
nHgb S--------------------
nHgb C-------------------
Globin Chains
a2b2
a2d2
a2g2
b4
g4
a2b26
gluval
a2b26
glulys

4. nThe two α chains in HbAare encoded
by an identical pair of α-globin genes
on chromosome 16,
nThe two β chains are encoded by a
single β-globin gene on chromosome
11.

6. Chromosomes

7. THALASSEMIA SYNDROMES
nThe thalassemia syndromes are a
heterogeneous group of disorders
caused by inherited mutationsthat
decrease the synthesis of adult
hemoglobin, HbA(α
2β
2).

8. Hb-A Molecule. Hb-A is the major
adult hemoglobin.

9. Thalassemia
Syndromes arising form
decreased rate or absence of
globin chain synthesis.
The resulting imbalance-globin
chain synthesis takes place,
giving rise to the excess
amount of the normally
synthesized globin chain.

10. Thalassemias

11. Hemoglobinopathies
nThe syndrome arising from the
synthesis of abnormal hemoglobin
or hemoglobin variants.
nRate of globin chain synthesis are
theoritically normal.
nAbnormal hemoglobins have
different properties from the
normal ones.

12. Hemoglobinopathies

14. Pathogenesis
Heterogenous group of disorder
Genetically determined
Reduced synthesis of one or more types of
normal haemoglobin polypeptide chain
Reduced haemoglobin involving affected chain

15. Each goblin chain have separate genetic control
α–thalassaemia affect α-chain synthesis
β–thalassaemia affect β -chain synthesis

16. How to name thalassemia?
nNamed after globin chain that is abnormally
synthesized !!!!
nReduced or absent a-globin chain : a-thalassemia
nReduced or absent b-globin chain : b-thalassemia
nReduced or absent g-globin chain : g-thalassemia
nReduced or absent d-globin chain : d-thalassemia
nReduced or absent gdb-globin chains
: gdb-thalassemia

17. Common types of thalassemia
na-thalassemia
nb-thalassemia

18. ALPHA
THALASSEMIAS

19. αThalassemia
nAbsence of αchains will result in
increase/ excess of gglobin chains
during fetal life and excess βglobin
chains later in postnatal life.
nSeverity of disease depends on number
of genes affected.

20. Classification & Terminology
AlphaThalassemia
•Normal aa/aa
•Silent carrier -a/aa
•Minor(trait) -a/-a
--/aa
•Hb H disease --/-a
•Barts hydrops fetalis--/--

21. αThalassemia
nDefects in αglobin affecting the
formation of both fetal and adult
hemoglobins, thus, producing
intrauterine as well as postnatal
disease. Unlike βthalassemia, why??

22. αThalassemia
nThe most common cause of αthalassemia is
due to αgene/s deletions.
nThe most likely mechanism for αgene deletion
is due to homologous pairing between α1 and
α2 and recombination. This results in loss of α
gene.

23. αThalassemia
a2a1 a2a1
a2
a2a1
a2a1
a2a1
a2a1
a2a1
a2a1
Normal Hb
One αgene deletion
α-Thal2
Two αgene deletions
α-Thal trait
Three αgene
deletions
Hb-H disease
Four αgene deletions
Hydrops fetalis or also called:
Erythroblastosis Fetalis.

25. Pathogenesis of α-
Thalassaemia
nIn normal individual HbA, HbA2 and HbF
need α-chain for their formation.
n4 genes of α-chain, each pair on short arm of
chromosome 16 present with genotype
α,α/α,α.
nIn α-thalassaemia, delation of α-genes
reduction or absence of synthesis of α-chain
depending on number of α-gene delation.

26. •↓α-chain synthesis free γ-chain in the fetus
& β-chain in infant of 6 months, and continue
in the rest of life.
•Complementary 4γand 4βare aggregated 
Hb Bart (4γ) and HbH (4β), respectively.

27. αThalassemia
•Predominant cause of alpha thalassemias is
large number of gene deletions in the α-globin
genes.
•There are four clinical syndromes present in
alpha thalassemia:
♫Silent Carrier State
♫Alpha Thalassemia Trait(Alpha Thalassemia Minor)
♫Hemoglobin H Disease
♫Bart's Hydrops Fetalis Syndrome

28. Silent Carrier αThalassemia
-α/αα
One alpha gene deletion, 3 intact alpha
genes.
Healthy persons.
Normal Hb and Hct
No treatment
Can only be detected by DNA studies.

29. Alpha Thalassemia Trait
•Also called Alpha Thalassemia Minor.
•Caused by two missing alpha genes.May be
homozygous (-α/-α) or heterozygous (--/αα).
•Exhibits mild microcytic, hypochromic anemia.
•MCV between 70-75 fL.
•Normal Hb electrophoresis.
•May be confused with iron deficiency anemia.
•present in adults.

30. Hemoglobin H Disease
Second most severe form alpha thalassemia.
Usually caused by presence of only one
intact αgene producing alpha chains (--/-α).
Results in accumulation of excess unpaired
gamma or beta chains. Born with 10-40%
Bart's hemoglobin (g4). Gradually replaced
with Hemoglobin H (β4). In adult, have about
5-40% HbH.
γ4 β4

31. αThalassemia: Hb-H Disease

32. 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 7-10 g/dl
Hb electrophoresis: Fast moving band
correspondent to HbH.

33. HbH 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. Hb-H preparation
Same preparation as
Retic count stain, but
with extended time of
incubation, instead of
15 minutes, 2 hours
incubation is required.

36. Hb-H inclusions

37. Blood Smear & HbH Preparation

38. Bart’s Hydrops Fetalis Syndrome
Most severe form.Incompatible with life.
Have no functioning αchain 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 we will see hepatosplenomegaly and
cardiomegaly.
Predominant Hb is Hb Bart, along with Hb
Portland and traces of HbH.
.

39. Hb Bart's has high oxygen affinity so
cannot carry oxygen to tissues.
Fetus dies in utero or shortly after
birth.
At birth, you will see severe
hypochromic, microcytic anemia
with numerous NRBCs.

40. Hydrops Fetalis
The blood film of neonate with hemoglobin Bart’s
hydrops fetalis showing anisocytosis, poikilocytosis
and numerous nucleated red blood cells (NRBC).

43. State GenotypeGenesFeatures
Normal aa/aa 4normal
Hetero a
+
Silent
carrier
aa/ –a 3Essentially normal
Hetero a°
trait
aa/ –– 2Micro / Hypo
Mild Anemia
Bart’s 2-8% (at birth)
Hb H <2%
Homo a
+
a-thal-trait
–a/ –a 2
a
+
+ a°
Hb-H
Disease
–a/ –– 1Moderate Micro/Hypo
anemia: Barts <10%, Hb
H <40%
homo a°
Hydrops
––/ –– 0HbA 0%, Bart’s 70-80%
Portland 10-20%

44. αThalassemia Syndromes

45. Hb electrophoresis at Alkaline pH mobility

46. βThalassaemia
nβThalassaemia usually results from
point mutations within the βglobin
gene cluster, βthalassaemia can be
classified according to the severity of
their symptoms into three groups:
1-βthalassaemia minor (or trait)
2-βthalassaemia major
3-βthalassaemia intermediate

47. Who is at risk?
Ethnic origin is very critical!

48. Pathogenesis
Heterogenous group of disorder
Genetically determined
Reduced synthesis of one or more types of
normal haemoglobin polypeptide chain
Reduced haemoglobin involving affected chain

49. Each goblin chain have separate genetic control
α–thalassaemia affect α-chain synthesis
β–thalassaemia affect β -chain synthesis

50. β-Thalassaemia
An absence or deficiency of β-chain synthesis of adult HbA
βChain synthesis
Hb-A
γand δchain
Hb-A = α
2β
2

52. Type of mutations that could occur
Immature mRNA
transcript
5’ 3’
Gene (DNA)
Transcription
Translation
ProteinNH2 COOH
exon1intron1exon2intron2exon3(AAA) signal
Promoter/Enhancer
5’exon1exon2exon3 AAAA…AAA 3’Mature mRNA
transcript
ATG
“start”
“stop”
codon
Processing
5’ 3’exon1 exon2 exon3AAAA…AAA
X X
X X
X
X

53. Again:
nβthalassemias are usually and mostly
due to single base pairsubstitutions
rather than deletions. Although
deletions do occur.

54. On the basis of synthetic ability β-genes are
designated as
βgene –can synthesize normal amount of β-
chain
β
+
gene –can synthesize reduced amount of
β-chain
β
0
gene –cannot synthesize β-chain

55. β-thalassaemia major
Mutation of normal β-gene β
0
-gene 
absent HbA increased HA
2and HbF
genotype –β
0
β
0
β-thalassaemia intermedia
↑HbA
2
↑HbF
↓HbA
Genotype β
+
β
+
or β
0
β

56. β-thalassaemiaminor
↑HbA
2
HbAnormal
HbFnormal

57. Pathophysiology of β-Thalassaemia
Various mutation in β-gene
Complete or partial absence of β-chain
Decreased adult HbA
α-chain synthesis remain normal
Free complementary α-chain –unstable and precipitate within
normoblasts as insoluble inclusions
Cell membrane damage & impaired DNA synthesis
apoptosis i.e. ineffective erythropoeisis

58. 70-80% marrow normoblastsundergo apoptosis
Inclusion bearing red cells undergo
sequestration & destruction in spleen

59. Reticulocytes undergo intramedullary death
Inadequate production + ineffective
erythropoiesis + haemolysis Anaemia

60. ↑Haemolysis ↑demands of phagocytic
function hyperplasia of phagocytes
Hepatosplenomegaly
To compensate anaemia extramedullary
haemopoiesis in liver, spleen & brain
Organomegaly

62. βThalassemia:The Story in Brief
•The molecular defects in βthalassemia
result in the absence or varying reduction
(according to the type of mutation) in β
chain production.
•α-Chain synthesis is unaffected and
hence there is imbalanced globin chain
production, leading to an excess of α-
chains.

63. Excess alpha chains

64. In the absence of their partners (βchains),
they are unstable and precipitate in the red
cell precursors, giving rise to large
intracellular inclusions that interfere with
red cell maturation.
Hence there is a variable degree of
intramedullary destruction of red cell
precursors, i.e. ineffective erythropoiesis.

65. The Story in Brief, continue
Those red cells which escape ineffective
erythropoiesis and mature and enter the circulation
contain α-chain inclusions that interfere with their
passage through the RES, particularly the spleen.
The degradation products of excess α-chains,
particularly heme and iron, produce deleterious
effects on red cell membrane proteins and lipids.
The end result is an extremely rigid red cell with a
shortened survival (i.e. hemolysis).

66. In brief:
The anemia is due to two main components:
Ineffective erythropoiesis (intramedullary).
Extravascular hemolysis in RES esp. Spleen
Because of two components there is increased
sequestration,
A third component that could contribute for the
severity of anemia is splenomegalythat may also
worsen the anemia because of increased plasma
volume (dilutional).

67. There is also:
•Extramedullary erythropoiesis occurs, which
also contributes for the splenomegaly, it is
worthy to note that extramedullary
erythropoiesis is not a perfect process, this is
why in thalassemias we may see tear drop
RBCs, and nucleated RBCs (NRBCs).
Although, the NRBC seen in the blood film
are from both the BM and the extramedullary
erythropoiesis.

68. The pathophysiology of β-thalassaemia

69. At what age could βThalassemia cause
its effect???
nIn contrast to αglobin, βglobin is not
necessary during fetal life (Hb-F= α2γ2),
thus the onset of βThalassemia isn’t
apparent until a few months after birth, when
HbF is switched to HbA.

70. Again: What is Thalassemia?
•A group of inherited single gene disorders
resulting in reduced or no production of one or
more globin chains
•This results in an imbalance of globin chain
production, with the normal excess chain
producing the pathological effects:
♪Damage to RBC precursors →ineffective red cell
production in BM.
♪Damage to mature red cells → hemolytic anemia
•Resulting in hypochromic, microcytic anemia

71. mRNA quantity
differs between
alpha and beta, so
there will be free
alpha chains that
will precipitate in
red cells.

73. Each one of us inherit one gene
from each parent
βGene
βGene
βGene
βGene
βGene
βGene
Homozygous: Normal
Both gene are normal
Heterozygous: one
normal and one
abnormal/mutated
Homozygous: Abnormal
Both gene are
abnormal/mutated
X
XX

74. Classical Clinical Syndromes of b
Thalassemia; bthalassemia can be
presented as:
oSilent carrier state –mildest form of b thal.
bthalassemia minor -heterozygous disorder
resulting in mild hypochromic, microcytic
hemolytic anemia.
b thalassemia intermedia -Severity lies
between the minor and major.
bthalassemia major -homozygous disorder
resulting in severe life long transfusion-
dependent hemolytic anemia.

75. Clinical SyndromesGenotype Clinical FeaturesMolecular Genetics
β-Thalassemia
major
Homozygous β-
thalassemia
(β
0
/β
0
, β
+
/β
+
,
β
0
/β
+
)
Severe; requires
blood
transfusions
Mainly point
mutations that
lead to defects in
the transcription,
splicing, or
translation of β-
globin mRNA
β-Thalassemia
intermedia
Variable (β
0
/β
+
,
β
+
/β
+
, β
0
/β, β
+
/β)
Severe but does
not require regular
blood transfusions
β-Thalassemia
minor
Heterozygous β-
thalassemia (β
0
/β,
β
+
/β)
Asymptomatic
with mild or
absent anemia;
red cell
abnormalities
seen

76. Silent Carrier State for βThalassemia
•Are various heterozygous (from one parent) β
gene mutations that produce only small
decrease in production of βglobin chains.
•Patients have nearly normal alpha/beta chain
ratio and nohematologic abnormalities.
•Have normal levels of HbA
2.

77. βThalassemia Minor (Trait)
•Caused by heterozygous (from one parent)
mutations that affect βglobin synthesis.
•βChains production and thus Hb-A production is
more reduced than the silent carrier Hb-A.
•Usually presents as mild, asymptomatic hemolytic
anemiaunless patient in under stress such as
pregnancy, infection, or folic acid deficiency.
•Have one normal βgene and one mutated βgene.
•Hemoglobin level in 10-13 g/dL range with normal
or slightly elevated RBC count (RCC).

78. βThalassemia Minor (Trait)
nAnemia usually hypochromic and microcytic with
slight aniso and poik, including target cells and
elliptocytes;also may see basophilic stippling.
nRarely see hepatomegaly or splenomegaly.
nHave high HbA
2levels (3.6-8.0%) and normal to
slightly elevated HbF levels.
nNormally require no treatment.
nYou have to make sure are not diagnosed as IDA.
nMentzer index: <13 (Why?).

79. βThalassemia Minor (Trait)
n2-6% HbF (N = < 1% after age 1 year)
n3.6 -8% HbA
2(N = 2.2-3.6%)
n87 -95% HbA (N=95-100%)

81. βthalassemia minor

82. βThalassemia Intermedia
Patients able to maintain minimum Hb (7
g/dL or greater) without transfusion
dependence.
Expression of disorder falls between
thalassemia minor and thalassemia major.
We will see increase in both HbA
2
production and HbF production.
Peripheral blood smear picture is similar to
thalassemia minor.

84. βThalassemia 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
deficiency.
May become transfusion dependent.
Tend to develop iron overloads as result of
increased gastrointestinal absorption.
Usually survive into adulthood.

85. βThalassemiaMajor
Characterized by very severe microcytic,
hypochromic anemia.
Detected early in childhood:
Hb level lies between 2 and 8 g/dL.
Severe anemia causes marked bone changes
due to expansion of marrow space for
increased erythropoiesis (Epo is increased).
See characteristic changes in skull, long
bones, and hand bones.

86. βThalassemia 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 (WHY??) and
elliptocytes.See marked basophilic stippling and
numerous NRBCs.
MCV in range of 50 to 60 fl.
Retic count seen (2-8%). But low for the degree of
anemia. RPI<2.
Most of Hemoglobin present is Hb F with slight increase
in HbA
2.

87. βThalassemia 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, liver cirrhosis, and
endocrine deficiencies.
Dangers in continuous tranfusion therapy:
Development of iron overload.
Development of alloimmunization (developing antibodies to
transfused RBCs).
Risk of transfusion-transmitted diseases (e.g. hepatitis, AIDS).
Bone marrow transplants may be future treatment,
along with genetic engineering and new drug
therapies.

88. CLINICAL MANIFESTATIONS
nThe anemia manifests 6 to 9 months after birth as
hemoglobin synthesis switches from HbF to HbA.
nIn untransfused patients, hemoglobin levels are 3
to 6 gm/dL.
nThe red cells may completely lack HbA (β
0
/β
0
genotype) or contain small amounts (β
+
/β
+
or β
0
/β
+
genotypes).
nThe major red cell hemoglobin is HbF, which is
markedly elevated.
nHbA
2levels are sometimes high but more often are
normal or low.

89. Untreated children
nThe classic presentation of children with severe
disease includes THALASSEMIC FACIES/
Mongoloid facies -maxilla hyperplasia, flat nasal
bridge, frontal bossing.
nSuffer from growth retardation and die at an
early age from the effects of anemia.
nPathologic bone fractures, and cachexia
nHepatosplenomegaly due to extramedullary
hematopoiesis is usually present.

90. nThe spleen can become so enlarged that it
causes mechanical discomfort and secondary
hypersplenism.
nThe features of ineffective erythropoiesis
include expanded medullary spaces (with
massive expansion of the marrow of the face
and skull producing the characteristic
thalassemic facies), extramedullary
hematopoiesis, and higher metabolic needs.
nPallor, hemosiderosis, and jaundice can
combine to produce a greenish brown
complexion.

91. INVESTIGATIONS
nHB: SEVER degree of anaemia
nPS: Marked variation in size –anisocytosis
and shape-
poikilocytosis,
nMicrocytosis, and hypochromia.
nTarget cells (so called because hemoglobin
collects in the center of the cell),
nBasophilic stippling,
nFragmented red cells are also common.

94. βThalassemia Major

95. βThalassemia Major
Anisopoikilocytosis, NRBC, microcytosis, hypochromia

96. βThalassemia Major
Target cells, NRBC, microcytosis, poikilocytosis

97. Cooley’s Anemia
This is another name for β
Thalassemia Major, because Cooley
was the first one to describe these
cases.

99. Thalassemic face

100. Thalassemia face

101. βThalassemia Major

102. Expansion of BM

103. βthalassemia major
Male 18 years

104. Hepatosplenomegaly

106. Hair on End Appearance

107. Dark skin due to iron overload

108. Thalassaemia major-life expectancy
•Without regular transfusion
–Less than 10 years
•With regular transfusion and no or poor
iron chelation
–Less than 25 years
•With regular transfusion and good iron
chelation
–40 years, or longer??

109. Thalassemics:
Blood
Transfusion

110. Good iron chelationusing desferoxamine(iron chelator)
prolongs the life expectancy of Cooley’s anemic patients,
otherwise cardiac failure, liver cirrhosis, and endocrine
deficiencies could occur and causing death.

111. Comparison of βThalassemias
Parameter Minor Intermedia Major
Hb 10-13 6-10 2-8
MCV (fl) 60-78 50-70 50-60
MCH (pg) 28-32 22-28 16-22
RDW NormalS. increasedIncreased
Micro/hypo Film Mild Moderate Severe
PolychromasiaV. LittleModerate Marked
Anisocytosis None Moderate Marked
PoikilocytosisNone Moderate Marked
Targetting Present Present Present

112. Comparison of βThalassemias
GENOTYPE Hb A Hb A
2 Hb F
NORMAL Normal Normal Normal
SILENT CARRIER Normal Normal Normal
βTHAL MINOR Dec N to Inc N to Inc
βTHAL INTERMEDIA Dec N to IncIncreased
βTHAL MAJOR DecUsually IncIncreased

114. Pathogenesis and Pathophysiology
nImbalanced globin chain synthesis.
nExcess of normal globin chain.
nPrecipitation of excess globin chain.
nDegradation of precipitated globin.
nRelease of oxygen free radicle.
nIneffective erythropoiesis

115. Pathogenesis and Pathophysiology(cont.)
nMembrane lipid peroxidation.
nLoss of deformability.
nLoss of lipid asymmetry
(Normally: PC/SM; out, PS/PE; in.)
nEntrapped by spleen and
destroyed (extravascular
hemolysis)

116. Clinical Symptoms
nAnemia, Jaundice.
nHepatosplenomegaly.
nBone change-->mongoloid face.
nIron overload-->growth
retardation, heart failure, DM,
dark-colored skin , etc.

117. Management
nBlood transfusion
nIron chelation: Desferroxamine,
Deferiprone (L1), Exjade
TM
nSplenectomy
nStem cell transplantation : BM
or Cord blood
nPrenatal diagnosis (PND)
nSupportive: Folic acid