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February 11, 2012
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THALASSAEMIA – HEMOGLOBINOPATHIES
Presented by: Dr. Neela Ferdoushi
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February 11, 2012
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 ( or ). 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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THALASSEMIASBeta () thalassemia
The disease manifests itself when the switch from to chain synthesis occurs several months after birth
There may be a compensatory increase in and chain synthesis resulting in increased levels of hgb F and A2.
The genetic background of thalassemia is heterogenous and may be roughly divided into two types: 0 in which there is complete absence of chain
production. This is common in the Mediterranean. + in which there is a partial block in chain
synthesis. At least three different mutant genes are involved:+1 – 10% of normal chain synthesis occurs+2 – 50% of normal chain synthesis occurs+3 - > 50% of normal 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: 0/0, +1/ +1, or 0/ +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 chains precipitate causing hemolysis of RBC precursors in the bone marrow leading to ineffective erythropoiesis
In circulating RBCs, 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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THALASSEMIASThe 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 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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THALASSEMIASTherapy – transfusions plus iron chelators to
prevent hemochromatosis and tissue damage from iron overload; Gene therapy?
+2, or 3 homozygous = thalassemia intermedia Heterozygosity of 0, or + = thalassemia minor
Mild hypochromic, microcytic anemiaPatients are usually asymptomatic with symptoms
occurring under stressful conditions such as pregnancy
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 () 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 chains and later there are
excess 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 gene and occasionally to a functionally abnormal gene.
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THALASSEMIAS The normal haploid genotype is / If one gene is deleted, the haploid phenotype is
thal 2 If both genes are deleted, the haploid phenotype
is thal 1 Since one gets two genes from each parent,
there are four types of thalassemia: / thal 2 = silent carrier / thal 1, or thal 2/ thal 2 = thal trait with
mild anemia thal 1/ thal 2 = hemoglobin H disease (4 = 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 thal 1/ thal 1 = hydrops fetalis which is fatal with
stillbirth or death within hours of birth. Hemoglobin Barts (4) forms and has such a high affinity for O2 that no O2 is delivered to the tissues.
Hgb S/ thalassemia – symptomless to moderate anemia
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ALPHA THALASSEMIAS
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THALASSEMIAS
Delta/beta (/) thalassemia – both and chains are absent with no or little compensatory increase in 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 and chain synthesis which is compensated for by an increase in 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 InvestigationThalassemia
Variable hemogram results proportional to the severity of the thalassemia
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Primary Laboratory InvestigationThalassemia
• Severe cases present with• Microcytosis• Hypochromia• Poikilocytosis• RBC counts higher than expected for the level of
anemia
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Primary Laboratory InvestigationThalassemia
• 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 TreatmentThalassemia
• Time of presentation• Related to degree of severity• Usually in first few years of life• Untreated severe thalassemia
• --/--: Prenatal or perinatal death • --/- & --/cs: Normal life span with chronic hemolytic anemia
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Course and TreatmentThalassemia
• Untreated thalassemia• Major: Death in first or second decade of life • Intermedia: Usually normal life span• Minor/Minima: Normal life span
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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TerminologyHemoglobinopathy
Abnormal hemoglobins discovered earlier have been given letter designations:
Hb S
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Amino Acid SubstitutionHemoglobinopathy
Greek letter designates affected globin chain
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Amino Acid SubstitutionHemoglobinopathy
Superscript number designates affected amino acid(s), e.g.,
6
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Amino Acid SubstitutionHemoglobinopathy
Letters and numbers in parentheses designate the helical segment and amino acid sequence in that segment affected (sometimes omitted), e.g.,
6(A3)
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Amino Acid SubstitutionHemoglobinopathy
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:
6(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 carrierparents 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 InvestigationHemoglobinopathy
• 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 InvestigationHeterozygous & 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 inmicrocirculation
IrreversibleSickle 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 InvestigationCellulose Acetate Hb Electrophoresis
- A2/C S F A +
Normal
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Secondary Laboratory InvestigationCellulose Acetate Hb Electrophoresis
- A2/C S F A +
NormalHb SS
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Secondary Laboratory InvestigationCellulose Acetate Hb Electrophoresis
- A2/C S F A+
NormalHb SSHb AS
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Secondary Laboratory InvestigationCellulose Acetate Hb Electrophoresis
- A2/C S F A+
NormalHb SSHb ASHb SCHb CC
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Secondary Laboratory InvestigationCellulose Acetate Hb Electrophoresis
- A2/C S F A+
NormalHb SSHb ASHb SCHb CCHB 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 TreatmentSickle 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 TreatmentSickle 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 TreatmentSickle 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
