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 & TerminologyAlpha 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 & TerminologyAlpha 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 Thalassemia0
:Indicates no production of globin chain by gene:
0
Symbolism Other ThalassemiaSuperscript 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 ThalassemiaVariable 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 HemoglobinopathyAbnormal hemoglobins discovered
earlier have been given letter designations:
Hb S
Terminology HemoglobinopathyMore recently discovered hemoglobins
have been named by the city or location of discovery:
Hb C-Harlem
Amino Acid Substitution HemoglobinopathyGreek letter designates
affected globin chain
Amino Acid Substitution HemoglobinopathySuperscript number
designates affected amino acid(s), e.g.,
6
Amino Acid Substitution HemoglobinopathyLetters 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 HemoglobinopathyAmino 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 S F A+
Normal
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis - A2/C S F A+
Normal Hb SS
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis - A2/C S F A+
Normal Hb SS Hb AS
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis - A2/C S F A+
Normal Hb SS Hb AS Hb SC
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis - A2/C S F A+
Normal Hb SS Hb AS Hb SC Hb CC
Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis - A2/C S F A+
Normal Hb SS Hb AS Hb SC Hb CC HB AD
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 FHgb F, % Diff. Between
Duplicates, %
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 TreatmentIn 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 InterestJoint 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)