Thalhgbopathy

THALASSEMIAS AND THALASSEMIAS AND HEMOGLOBINOPATHIESHEMOGLOBINOPATHIES

Modified from an original Modified from an original presentation by Raymond L. presentation by Raymond L.

OlesinskiOlesinski

©2001 University of Kentucky©2001 University of Kentucky

Thalassemias and Hemoblobinopathies: Thalassemias and Hemoblobinopathies: Module ObjectivesModule Objectives

At the end of this module you should be able At the end of this module you should be able toto

• Explain the pathophysiology that causes Explain the pathophysiology that causes thalassemia and hemoglobinopathies.thalassemia and hemoglobinopathies.

• Explain how thalassemias are categorized.Explain how thalassemias are categorized.

Thalassemias and Hemoblobinopathies: Thalassemias and Hemoblobinopathies: Session ObjectivesSession Objectives

• Correlate the results of laboratory testing Correlate the results of laboratory testing with specific thalassemias and with specific thalassemias and hemoglobinopathies.hemoglobinopathies.


For theFor the1. Dithionite tube test1. Dithionite tube test2. Hemoglobin electrophoresis2. Hemoglobin electrophoresis3. Alkali denaturation test for fetal 3. Alkali denaturation test for fetal

hemoglobinhemoglobin

• Discuss specifics of specimen collection, Discuss specifics of specimen collection, handling, storage, and preparationhandling, storage, and preparation


• Explain the physiologic theory relevant to Explain the physiologic theory relevant to the test/procedure.the test/procedure.

• Explain the principle of the test/procedureExplain the principle of the test/procedure• Identify the disease manifestation/clinical Identify the disease manifestation/clinical

correlation.correlation.• Differentiate or resolve technical, Differentiate or resolve technical,

instrument, or physiologic causes of instrument, or physiologic causes of problems or unexpected test results.problems or unexpected test results.

Characteristics: ThalassemiaCharacteristics: Thalassemia

• Hereditary disorders that can result in Hereditary disorders that can result in moderate to severe anemiamoderate to severe anemia

• Basic defect is Basic defect is reduced productionreduced production of of selected globin chainsselected globin chains

Demographics: ThalassemiaDemographics: Thalassemia

• Found most Found most frequently in the frequently in the Mediterranean, Africa, Mediterranean, Africa, Western and Western and Southeast Asia, India Southeast Asia, India and Burmaand Burma

• Distribution parallels Distribution parallels that of Plasmodium that of Plasmodium falciparumfalciparum

Classification & TerminologyClassification & TerminologyAlphaAlpha ThalassemiaThalassemia

• TerminologyTerminology• Silent carrierSilent carrier• MinimaMinima• MinorMinor• IntermediaIntermedia• MajorMajor

SymbolismSymbolismAlpha ThalassemiaAlpha Thalassemia

• Greek letter Greek letter used to designate globin used to designate globin chain:chain:


// : Indicates division between genes : Indicates division between genes inherited from both parents:inherited from both parents:

//

• Each chromosome 16 carries 2 genes. Therefore the Each chromosome 16 carries 2 genes. Therefore the total complement of total complement of genes in an individual is 4 genes in an individual is 4


-- : Indicates a gene deletion: : Indicates a gene deletion:

--//

Classification & TerminologyClassification & TerminologyAlphaAlpha ThalassemiaThalassemia

• NormalNormal // • Silent carrierSilent carrier - - // • MinorMinor --/-/-

--/--/• Hb H diseaseHb H disease --/---/-• Barts hydrops fetalisBarts hydrops fetalis --/-- --/--

SymbolismSymbolismOther ThalassemiaOther Thalassemia

• Greek letter Greek letter used to designate globin used to designate globin chain:chain:


++: Indicates diminished, but some : Indicates diminished, but some production of globin chain by gene:production of globin chain by gene:

++


00 :Indicates no production of globin chain by :Indicates no production of globin chain by gene:gene:

00


Superscript Superscript TT denotes nonfunctioning gene: denotes nonfunctioning gene:

TT

Classification & TerminologyClassification & Terminology Beta ThalassemiaBeta Thalassemia

• NormalNormal //• MinorMinor //00

//++

• IntermediaIntermedia 00//++

• Major Major 00//00

++//++

Special CasesSpecial CasesThalassemiaThalassemia

• Hb Lepore: Hb Lepore: fusion seen in some types fusion seen in some types

of of thalassemia thalassemia• Hb Constant SpringHb Constant Spring

• chain with 31 additional amino acidschain with 31 additional amino acids• --/--/cscs

• Hereditary persistence of fetal hemoglobin Hereditary persistence of fetal hemoglobin (HPFH)(HPFH)

Special Cases: ThalassemiaSpecial Cases: Thalassemia

• Hb HHb H• 44 tetramer tetramer• Associated with --/-Associated with --/- thalassemia thalassemia

Special Cases: ThalassemiaSpecial Cases: Thalassemia

• Hb Barts & hydrops fetalis Hb Barts & hydrops fetalis • Barts is a Barts is a 44 tetramer tetramer• Associated with --/--Associated with --/--• LethalLethal• High concentrations are capable of sicklingHigh concentrations are capable of sickling

Primary Laboratory InvestigationPrimary Laboratory InvestigationThalassemiaThalassemia

Variable hemogram results proportional to Variable hemogram results proportional to the severity of the thalassemiathe severity of the thalassemia


• Severe cases present withSevere cases present with• MicrocytosisMicrocytosis• HypochromiaHypochromia• PoikilocytosisPoikilocytosis• RBC counts higher than expected for the level RBC counts higher than expected for the level

of anemiaof anemia


• Findings in severe cases can mimic those Findings in severe cases can mimic those seen in other microcytic/hypochromic seen in other microcytic/hypochromic anemiasanemias

• Results of the reticulocyte count are Results of the reticulocyte count are variablevariable

• NRBCs may be present (contrast with iron NRBCs may be present (contrast with iron deficiency anemia)deficiency anemia)

Course and TreatmentCourse and TreatmentThalassemiaThalassemia

• Time of presentationTime of presentation• Related to degree of severityRelated to degree of severity• Usually in first few years of lifeUsually in first few years of life• Untreated severe Untreated severe thalassemia thalassemia

• --/--: Prenatal or perinatal death --/--: Prenatal or perinatal death • --/---/- & --/ & --/cscs: Normal life span with chronic : Normal life span with chronic

hemolytic anemiahemolytic anemia

Course and TreatmentCourse and TreatmentThalassemiaThalassemia

• Untreated Untreated thalassemia thalassemia• Major: Death in first or second decade of life Major: Death in first or second decade of life • Intermedia: Usually normal life spanIntermedia: Usually normal life span• Minor/Minima: Normal life spanMinor/Minima: Normal life span

Characteristics: Characteristics: HemoglobinopathiesHemoglobinopathies

• Hereditary disorders that can result in Hereditary disorders that can result in moderate to severe anemiamoderate to severe anemia

• Basic defect is Basic defect is productionproduction of an of an abnormalabnormal globin chainglobin chain

DemographicsDemographicsHemoglobinopathiesHemoglobinopathies

• The demographics of hemoglobinopathies The demographics of hemoglobinopathies are varied.are varied.

Hemoglobinopathy GeneticsHemoglobinopathy Genetics

• Homozygous: Inheritance of two genes Homozygous: Inheritance of two genes from each parent coding for the same type from each parent coding for the same type of abnormal hemoglobin, e.g., Hb SSof abnormal hemoglobin, e.g., Hb SS

• Heterozygous: Inheritance of genes from Heterozygous: Inheritance of genes from each parent which code for a different type each parent which code for a different type of abnormal hemoglobin each, e.g., Hb SCof abnormal hemoglobin each, e.g., Hb SC

TerminologyTerminologyHemoglobinopathyHemoglobinopathy

Abnormal hemoglobins discovered earlier Abnormal hemoglobins discovered earlier have been given have been given letter designationsletter designations: :

Hb Hb SS

TerminologyTerminologyHemoglobinopathyHemoglobinopathy

More recently discovered hemoglobins have More recently discovered hemoglobins have been named by the been named by the city or location of city or location of discoverydiscovery::

Hb Hb C-HarlemC-Harlem

Amino Acid SubstitutionAmino Acid SubstitutionHemoglobinopathyHemoglobinopathy

Greek letterGreek letter designates affected globin chain designates affected globin chain


Superscript numberSuperscript number designates affected designates affected amino acid(s), e.g., amino acid(s), e.g.,

66


Letters and numbers in parentheses Letters and numbers in parentheses designate designate the helical segment and amino acid sequence the helical segment and amino acid sequence in that segment affected (sometimes omitted), in that segment affected (sometimes omitted), e.g., e.g.,

66(A3)(A3)


Amino acid substitutionsAmino acid substitutions are denoted by the are denoted by the three letter abbreviation for the normally three letter abbreviation for the normally occurring amino acid followed by an arrow occurring amino acid followed by an arrow followed by the three letter abbreviation for followed by the three letter abbreviation for the the substituted amino acidsubstituted amino acid::

6(A3)6(A3)Glu Glu Val Val

Classification: Classification: HemoglobinopathyHemoglobinopathy

• Functional AbnormalityFunctional Abnormality• AggregationAggregation

• PolymerizationPolymerization• CrystallizationCrystallization

• Unstable hemoglobinsUnstable hemoglobins• MethemoglobinMethemoglobin• Oxygen affinityOxygen affinity

Primary Laboratory InvestigationPrimary Laboratory InvestigationHemoglobinopathyHemoglobinopathy

• Variety of hemogram findings depending Variety of hemogram findings depending onon• TypeType• SeveritySeverity

of the specific disorderof the specific disorder• Only sickle hemoglobinopathies and Hb C Only sickle hemoglobinopathies and Hb C

will be described herewill be described here

Primary Laboratory InvestigationPrimary Laboratory InvestigationHeterozygous & Other DisordersHeterozygous & Other Disorders

• ASAS• S-ThalS-Thal• Other hemoglobinopathies, e.g., SCOther hemoglobinopathies, e.g., SC• Hb CHb C

Morphologic FindingsMorphologic Findings Hb SS vs. Hb SC vs. Hb CC Hb SS vs. Hb SC vs. Hb CC

=+

Hb S Hb C Hb SC

Morphologic FindingsMorphologic Findings Hb SS vs. Hb SC vs. Hb CC Hb SS vs. Hb SC vs. Hb CC

=+

Hb S Hb C Hb SC

+ =

Where Do Sickle Cells Come Where Do Sickle Cells Come From?From?

Sheared inmicrocirculation

IrreversibleSickle Cell

Sickle CellsSickle Cells

Secondary Laboratory InvestigationSecondary Laboratory Investigation

• Hemoglobin electrophoresisHemoglobin electrophoresis• Major test for identifying thalassemia and Major test for identifying thalassemia and

hemoglobinopathyhemoglobinopathy• TypesTypes

• Cellulose acetate: Alkaline pHCellulose acetate: Alkaline pH• Citrate agar: Acid phCitrate agar: Acid ph

Secondary Laboratory Secondary Laboratory InvestigationInvestigation

• Patterns of mobility (see handout)Patterns of mobility (see handout)


Cellulose Acetate Hb Cellulose Acetate Hb ElectrophoresisElectrophoresis - A- A22/C S F A +/C S F A +

NormalNormal


Cellulose Acetate Hb Cellulose Acetate Hb ElectrophoresisElectrophoresis - A- A22/C S F A +/C S F A +

NormalNormal

Hb SSHb SS


Cellulose Acetate Hb Cellulose Acetate Hb ElectrophoresisElectrophoresis

- A- A22/C S F A+/C S F A+

NormalNormal

Hb SSHb SS

Hb ASHb AS



- A- A22/C S F A+/C S F A+

NormalNormal

Hb SSHb SS

Hb ASHb AS

Hb SCHb SC



- A- A22/C S F A+/C S F A+

NormalNormal

Hb SSHb SS

Hb ASHb AS

Hb SCHb SC

Hb CCHb CC



- A- A22/C S F A+/C S F A+

NormalNormal

Hb SSHb SS

Hb ASHb AS

Hb SCHb SC

Hb CCHb CC

HB ADHB AD


• Solubility testing-Dithionite tube testSolubility testing-Dithionite tube test• Alkali denaturation test for quantification of Alkali denaturation test for quantification of

fetal hemoglobinfetal hemoglobin• Acid elution test for fetal hemoglobin Acid elution test for fetal hemoglobin

distributiondistribution• Unstable hemoglobin testing for Heinz Unstable hemoglobin testing for Heinz

bodiesbodies

Alkali Denaturation for Alkali Denaturation for Hemoglobin FHemoglobin F

• Recommended assay for hgb F in the Recommended assay for hgb F in the range of 2-40%range of 2-40%

• PrinciplePrinciple• Other hemoglobins are more susceptible than Other hemoglobins are more susceptible than

hgb F to denaturation at alkaline pHhgb F to denaturation at alkaline pH• Denaturation stopped by addition of Denaturation stopped by addition of

ammonium sulphateammonium sulphate• Denatured hemoglobin precipitatesDenatured hemoglobin precipitates


• Remaining hemoglobin (F) can be Remaining hemoglobin (F) can be measured spectrophotometricallymeasured spectrophotometrically

• Specimen: EDTA anticoagulated Specimen: EDTA anticoagulated whole bloodwhole blood

• QC: Normal and elevated controls QC: Normal and elevated controls should be used with each batch of should be used with each batch of specimensspecimens


Hgb F, %Hgb F, % Diff. Between Duplicates, %Diff. Between Duplicates, %

<5<5 0.50.5

5-155-15 1.01.0

>15>15 2.02.0


• Sources of errorSources of error• Too short or too long an incubation timeToo short or too long an incubation time• Filtrate turbidityFiltrate turbidity• Outdated reagentsOutdated reagents• Incorrect reagent concentrationsIncorrect reagent concentrations• Poor quality filter paperPoor quality filter paper

Acid Elution for Fetal Acid Elution for Fetal HemoglobinHemoglobin

• Indication of distribution of fetal Indication of distribution of fetal hemoglobin in a population of RBChemoglobin in a population of RBC

• Homogeneous distribution: hereditary Homogeneous distribution: hereditary persistence of fetal hemoglobinpersistence of fetal hemoglobin

• Heterogeneous distribution: thalassemiaHeterogeneous distribution: thalassemia

Course and TreatmentCourse and TreatmentSickle Cell DiseaseSickle Cell Disease

• Sickle cell diseaseSickle cell disease• Asymptomatic at birthAsymptomatic at birth• Symptoms appear as percentage of fetal Symptoms appear as percentage of fetal

hemoglobin decreases during first year of lifehemoglobin decreases during first year of life• Untreated crises increase morbidity and early Untreated crises increase morbidity and early

deathdeath

Course and TreatmentCourse and TreatmentSickle Cell DiseaseSickle Cell Disease

• Life span can be significantly increased with Life span can be significantly increased with early and effective treatmentearly and effective treatment

• Studies of natural populations reveal that Studies of natural populations reveal that individuals with sickle cell disease are individuals with sickle cell disease are capable of normal life spanscapable of normal life spans

Course and TreatmentCourse and Treatment

In both thalassemia and hemoglobinopathy In both thalassemia and hemoglobinopathy therapy is usually supportive rather than therapy is usually supportive rather than curativecurative


• Blood transfusion is used toBlood transfusion is used to• Control severe anemiaControl severe anemia• Reduce the risk of complications of sickle Reduce the risk of complications of sickle

hemoglobinopathies (cerebrovascular hemoglobinopathies (cerebrovascular accident, hypersplenism, etc.)accident, hypersplenism, etc.)


• Chronic blood transfusionChronic blood transfusion• Results in iron overload of major organs Results in iron overload of major organs

resulting in increased morbidityresulting in increased morbidity• Laboratory monitoringLaboratory monitoring• Necessitates the use of chelating agents to Necessitates the use of chelating agents to

remove excess ironremove excess iron


• Excess iron can cause the appearance of Excess iron can cause the appearance of sideroblastic conditionssideroblastic conditions

• Transfusion interferes with the typical Transfusion interferes with the typical laboratory findings for the disorderlaboratory findings for the disorder


• Alternative treatmentAlternative treatment• Activation of fetal hemoglobin genesActivation of fetal hemoglobin genes• Bone marrow transplantationBone marrow transplantation

WWW Sites of InterestWWW Sites of Interest

Joint Center for Sickle Cell and Joint Center for Sickle Cell and Thalassemic DisordersThalassemic Disorders: http://www-: http://www-rics.bwh.harvard.edu/sickle/ (Overview of rics.bwh.harvard.edu/sickle/ (Overview of sickle cell disease, thalassemia and iron sickle cell disease, thalassemia and iron kinetics)kinetics)

The Sickle Cell Information Center, Emory The Sickle Cell Information Center, Emory UniversityUniversity: : http://www.emory.edu:80/PEDS/SICKLE/ http://www.emory.edu:80/PEDS/SICKLE/ (Includes PowerPoint presentations on (Includes PowerPoint presentations on sickle cell disease)sickle cell disease)

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Thalhgbopathy