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)