Transcript Thalassemia and Hemoglobinopathy

THALASSEMIAS AND
HEMOGLOBINOPATHIES
Modified from an original
presentation by Raymond L.
Olesinski
©2001 University of Kentucky
Thalassemias and Hemoblobinopathies:
Module Objectives
At the end of this module you should be able
to
• Explain the pathophysiology that causes
thalassemia and hemoglobinopathies.
• Explain how thalassemias are categorized.
Thalassemias and Hemoblobinopathies:
Session Objectives
•
Correlate the results of laboratory testing
with specific thalassemias and
hemoglobinopathies.
Thalassemias and Hemoblobinopathies:
Session Objectives
For the
1. Dithionite tube test
2. Hemoglobin electrophoresis
3. Alkali denaturation test for fetal
hemoglobin
•
Discuss specifics of specimen collection,
handling, storage, and preparation
Thalassemias and Hemoblobinopathies:
Session Objectives
•
•
•
•
Explain the physiologic theory relevant to
the test/procedure.
Explain the principle of the test/procedure
Identify the disease manifestation/clinical
correlation.
Differentiate or resolve technical,
instrument, or physiologic causes of
problems or unexpected test results.
Characteristics: Thalassemia
•
•
Hereditary disorders that can result in
moderate to severe anemia
Basic defect is reduced production of
selected globin chains
Demographics: Thalassemia
•
•
Found most
frequently in the
Mediterranean, Africa,
Western and
Southeast Asia, India
and Burma
Distribution parallels
that of Plasmodium
falciparum
Classification & Terminology
Alpha Thalassemia
•
Terminology
•
•
•
•
•
Silent carrier
Minima
Minor
Intermedia
Major
Symbolism
Alpha Thalassemia
•
Greek letter used to designate globin
chain:

Symbolism
Alpha Thalassemia
/ : Indicates division between genes
inherited from both parents:
/
•
Each chromosome 16 carries 2 genes. Therefore the
total complement of  genes in an individual is 4
Symbolism
Alpha Thalassemia
- : Indicates a gene deletion:
-/
Classification & Terminology
Alpha Thalassemia
• Normal
• Silent carrier
• Minor
• Hb H disease
• Barts hydrops fetalis
/
- /
-/-
--/
--/-
--/--
Symbolism
Other Thalassemia
•
Greek letter used to designate globin
chain:

Symbolism
Other Thalassemia
+:
Indicates diminished, but some
production of globin chain by gene:
+
Symbolism
Other Thalassemia
0
:Indicates no production of globin chain by
gene:
0
Symbolism
Other Thalassemia
Superscript T denotes nonfunctioning gene:
T
Classification & Terminology
Beta Thalassemia
• Normal
• Minor
• Intermedia
• Major
/
/0
/+
0/+
0/0
+/+
Special Cases
Thalassemia
•
•
Hb Lepore:  fusion seen in some types
of  thalassemia
Hb Constant Spring
•  chain with 31 additional amino acids
• --/cs
•
Hereditary persistence of fetal hemoglobin
(HPFH)
Special Cases: Thalassemia
•
Hb H
• 4 tetramer
• Associated with --/- thalassemia
Special Cases: Thalassemia
•
Hb Barts & hydrops fetalis
•
•
•
•
Barts is a 4 tetramer
Associated with --/-Lethal
High concentrations are capable of sickling
Primary Laboratory Investigation
Thalassemia
Variable hemogram results proportional to
the severity of the thalassemia
Primary Laboratory Investigation
Thalassemia
•
Severe cases present with
•
•
•
•
Microcytosis
Hypochromia
Poikilocytosis
RBC counts higher than expected for the level
of anemia
Primary Laboratory Investigation
Thalassemia
•
•
•
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)
Course and Treatment
Thalassemia
•
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
Course and Treatment
Thalassemia
• Untreated  thalassemia
•
•
•
Major: Death in first or second decade of life
Intermedia: Usually normal life span
Minor/Minima: Normal life span
Characteristics:
Hemoglobinopathies
•
•
Hereditary disorders that can result in
moderate to severe anemia
Basic defect is production of an abnormal
globin chain
Demographics
Hemoglobinopathies
•
The demographics of hemoglobinopathies
are varied.
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
Terminology
Hemoglobinopathy
Abnormal hemoglobins discovered earlier
have been given letter designations:
Hb S
Terminology
Hemoglobinopathy
More recently discovered hemoglobins have
been named by the city or location of
discovery:
Hb C-Harlem
Amino Acid Substitution
Hemoglobinopathy
Greek letter designates affected globin chain

Amino Acid Substitution
Hemoglobinopathy
Superscript number designates affected
amino acid(s), e.g.,
6
Amino Acid Substitution
Hemoglobinopathy
Letters and numbers in parentheses designate
the helical segment and amino acid sequence
in that segment affected (sometimes omitted),
e.g.,
6(A3)
Amino Acid Substitution
Hemoglobinopathy
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
Classification:
Hemoglobinopathy
•
Functional Abnormality
• Aggregation
•
•
Polymerization
Crystallization
• Unstable hemoglobins
• Methemoglobin
• Oxygen affinity
Primary Laboratory Investigation
Hemoglobinopathy
•
Variety of hemogram findings depending
on
• Type
• Severity
•
of the specific disorder
Only sickle hemoglobinopathies and Hb C
will be described here
Primary Laboratory Investigation
Heterozygous & Other Disorders
•
•
•
•
AS
S-Thal
Other hemoglobinopathies, e.g., SC
Hb C
Morphologic Findings
Hb SS vs. Hb SC vs. Hb CC
=
+
Hb S
Hb C
Hb SC
Morphologic Findings
Hb SS vs. Hb SC vs. Hb CC
=
+
Hb S
Hb C
+
Hb SC
=
Where Do Sickle Cells Come
From?
Sheared in
microcirculation
Irreversible
Sickle Cell
Sickle Cells
Secondary Laboratory Investigation
•
Hemoglobin electrophoresis
• Major test for identifying thalassemia and
hemoglobinopathy
• Types
•
•
Cellulose acetate: Alkaline pH
Citrate agar: Acid ph
Secondary Laboratory
Investigation
• Patterns of mobility (see handout)
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
S
F
A+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
Hb SS
S
F
A+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S
Normal
Hb SS
Hb AS
F
A+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
Hb SS
Hb AS
Hb SC
S
F
A+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C
Normal
Hb SS
Hb AS
Hb SC
Hb CC
S
F
A+
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S
Normal
Hb SS
Hb AS
Hb SC
Hb CC
HB AD
F
A+
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
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
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
Alkali Denaturation for
Hemoglobin F
Hgb F, %
Diff. Between Duplicates, %
<5
5-15
>15
0.5
1.0
2.0
Alkali Denaturation for
Hemoglobin F
•
Sources of error
• Too short or too long an incubation time
• Filtrate turbidity
• Outdated reagents
• Incorrect reagent concentrations
• Poor quality filter paper
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
Course and Treatment
Sickle 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
Course and Treatment
Sickle 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
Course and Treatment
In both thalassemia and hemoglobinopathy
therapy is usually supportive rather than
curative
Course and Treatment
•
Blood transfusion is used to
• Control severe anemia
• Reduce the risk of complications of sickle
hemoglobinopathies (cerebrovascular
accident, hypersplenism, etc.)
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
Course and Treatment
• Excess iron can cause the appearance of
sideroblastic conditions
• Transfusion interferes with the typical
laboratory findings for the disorder
Course and Treatment
•
Alternative treatment
• Activation of fetal hemoglobin genes
• Bone marrow transplantation
WWW Sites of Interest
Joint Center for Sickle Cell and
Thalassemic Disorders: http://wwwrics.bwh.harvard.edu/sickle/ (Overview of
sickle cell disease, thalassemia and iron
kinetics)
 The Sickle Cell Information Center, Emory
University:
http://www.emory.edu:80/PEDS/SICKLE/
(Includes PowerPoint presentations on
sickle cell disease)
