11

Chapter 12Chapter 12ThalassemiaThalassemia

3

ThalassemiaThalassemia

► In Chapter 12, you will be introduced In Chapter 12, you will be introduced to the thalassemias.  You will learn to the thalassemias.  You will learn about the pathophysiology, clinical about the pathophysiology, clinical signs and symptoms, laboratory test signs and symptoms, laboratory test results, and treatments for both the results, and treatments for both the alpha and beta forms of thalassemia.  alpha and beta forms of thalassemia.  Subclasses of each major form of Subclasses of each major form of thalassemia will be discussed. thalassemia will be discussed.

44

Introduction to Introduction to ThalassemiaThalassemia

5

Thalassemia Thalassemia 1 of 21 of 2

►Diverse group of disorders which manifest Diverse group of disorders which manifest as anemia of varying degrees. as anemia of varying degrees.

► Result of defective production of globin Result of defective production of globin portion of hemoglobin molecule. portion of hemoglobin molecule.

►Distribution is worldwide. Distribution is worldwide. ►May be either homozygous defect or May be either homozygous defect or

heterozygous defect. heterozygous defect. ►Defect results from abnormal rate of Defect results from abnormal rate of

synthesis in one of the globin chains.  synthesis in one of the globin chains.  ►Globin chains structurally normal (is how Globin chains structurally normal (is how

differentiated from hemoglobinopathy), but differentiated from hemoglobinopathy), but have imbalance in production of two have imbalance in production of two different types of chains.  different types of chains. 

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Thalassemia Thalassemia 2 of 22 of 2

►Results in overall decrease in amount Results in overall decrease in amount of hemoglobin produced and may of hemoglobin produced and may induce hemolysis. induce hemolysis.

►Two major types of thalassemia: Two major types of thalassemia: Alpha (Alpha (αα) - Caused by defect in rate of ) - Caused by defect in rate of

synthesis of alpha chains. synthesis of alpha chains. Beta (Beta (ββ) - Caused by defect in rate of ) - Caused by defect in rate of

synthesis in beta chains. synthesis in beta chains. ►May contribute protection against May contribute protection against

malaria. malaria.

7

Genetics of ThalassemiaGenetics of Thalassemia

►Adult hemoglobin composed two alpha Adult hemoglobin composed two alpha and two beta chains. and two beta chains.

►Alpha thalassemia usually caused by Alpha thalassemia usually caused by gene deletion;  Beta thalassemia gene deletion;  Beta thalassemia usually caused by mutation. usually caused by mutation.

►Results in microcytic, hypochromic Results in microcytic, hypochromic anemias of varying severity. anemias of varying severity.

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Beta Beta ThalassemiaThalassemia

9

Classical Syndromes of Beta Classical Syndromes of Beta ThalassemiaThalassemia

►Silent carrier stateSilent carrier state – the mildest form of – the mildest form of beta thalassemia. beta thalassemia.

►Beta thalassemia minorBeta thalassemia minor - heterozygous - heterozygous disorder resulting in mild hypochromic, disorder resulting in mild hypochromic, microcytic hemolytic anemia. microcytic hemolytic anemia.

►Beta thalassemia intermedia Beta thalassemia intermedia - Severity - Severity lies between the minor and major. lies between the minor and major.

►Beta thalassemia majorBeta thalassemia major - homozygous - homozygous disorder resulting in severe transfusion-disorder resulting in severe transfusion-dependent hemolytic anemia. dependent hemolytic anemia.

10

Silent Carrier State for Silent Carrier State for ββ ThalassemiaThalassemia

►Are various heterogenous beta Are various heterogenous beta mutations that produce only small mutations that produce only small decrease in production of beta chains. decrease in production of beta chains.

►Patients have nearly normal Patients have nearly normal beta/alpha chain ratio and beta/alpha chain ratio and nono hematologic abnormalities. hematologic abnormalities.

►Have normal levels of Hb AHave normal levels of Hb A22. .

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Beta Thalassemia Minor Beta Thalassemia Minor 1 of 21 of 2

► Caused by heterogenous mutations that Caused by heterogenous mutations that affect beta globin synthesis.  affect beta globin synthesis. 

► Usually presents as mild, asymptomatic Usually presents as mild, asymptomatic hemolytic anemia unless patient in under hemolytic anemia unless patient in under stress such as pregnancy, infection, or folic stress such as pregnancy, infection, or folic acid deficiency. acid deficiency.

►Have one normal beta gene and one Have one normal beta gene and one mutated beta gene. mutated beta gene.

►Hemoglobin level in 10-13 g/dL range with Hemoglobin level in 10-13 g/dL range with normal or slightly elevated RBC count.  normal or slightly elevated RBC count. 

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Beta Thalassemia Minor Beta Thalassemia Minor 2 of 22 of 2

► Anemia usually hypochromic and microcytic Anemia usually hypochromic and microcytic with slight aniso and poik, including target with slight aniso and poik, including target cells and elliptocytes;  May see basophilic cells and elliptocytes;  May see basophilic stippling. stippling.

► Rarely see hepatomegaly or splenomegaly. Rarely see hepatomegaly or splenomegaly. ►Have high Hb AHave high Hb A22 levels (3.5-8.0%) and levels (3.5-8.0%) and

normal to slightly elevated Hb F levels. normal to slightly elevated Hb F levels. ► Are different variations of this form Are different variations of this form

depending upon which gene has mutated. depending upon which gene has mutated. ►Normally require no treatment.  Normally require no treatment.  ►Make sure are not diagnosed with iron Make sure are not diagnosed with iron

deficiency anemia. deficiency anemia.

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Beta Thalassemia Intermedia Beta Thalassemia Intermedia 1 of 21 of 2

► Patients able to maintain minimum Patients able to maintain minimum hemoglobin (7 g/dL or greater) without hemoglobin (7 g/dL or greater) without transfusions.  transfusions. 

► Expression of disorder falls between Expression of disorder falls between thalassemia minor and thalassemia major.  thalassemia minor and thalassemia major.  May be either heterozygous for mutations May be either heterozygous for mutations causing mild decrease in beta chain causing mild decrease in beta chain production, or may be homozygous causing a production, or may be homozygous causing a more serious reduction in beta chain more serious reduction in beta chain production. production.

► See increase in both Hb ASee increase in both Hb A22 production and Hb production and Hb F production. F production.

► Peripheral blood smear picture similar to Peripheral blood smear picture similar to thalassemia minor. thalassemia minor.

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Beta Thalassemia Intermedia Beta Thalassemia Intermedia 2 of 22 of 2

► Have varying symptoms of anemia, jaundice, Have varying symptoms of anemia, jaundice, splenomegaly and hepatomegaly. splenomegaly and hepatomegaly.

► Have significant increase in bilirubin levels.  Have significant increase in bilirubin levels.  ► Anemia usually becomes worse with Anemia usually becomes worse with

infections, pregnancy, or folic acid infections, pregnancy, or folic acid deficiencies. deficiencies.

► May become transfusion dependent as adults. May become transfusion dependent as adults. ► Tend to develop iron overloads as result of Tend to develop iron overloads as result of

increased gastrointestinal absorption. increased gastrointestinal absorption. ► Usually survive into adulthood. Usually survive into adulthood.

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Beta Thalassemia Major Beta Thalassemia Major 1 of 31 of 3

► Characterized by severe microcytic, Characterized by severe microcytic, hypochromic anemia.  hypochromic anemia. 

► Detected early in childhood: Detected early in childhood: Infants fail to thrive.  Infants fail to thrive.  Have pallor, variable degree of jaundice, Have pallor, variable degree of jaundice,

abdominal enlargement, and hepatosplenomegaly. abdominal enlargement, and hepatosplenomegaly. ► Hemoglobin level between 4 and 8 gm/dL. Hemoglobin level between 4 and 8 gm/dL. ► Severe anemia causes marked bone changes Severe anemia causes marked bone changes

due to expansion of marrow space for due to expansion of marrow space for increased erythropoiesis.increased erythropoiesis.

► See characteristic changes in skull, long See characteristic changes in skull, long bones, and hand bones.  bones, and hand bones. 

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Beta Thalassemia Major Beta Thalassemia Major 2 of 32 of 3

►Have protrusion upper teeth and Mongoloid Have protrusion upper teeth and Mongoloid facial features.  facial features. 

► Physical growth and development delayed. Physical growth and development delayed. ► Peripheral blood shows markedly Peripheral blood shows markedly

hypochromic, microcytic erythrocytes with hypochromic, microcytic erythrocytes with extreme poikilocytosis, such as target cells, extreme poikilocytosis, such as target cells, teardrop cells and elliptocytes.  See marked teardrop cells and elliptocytes.  See marked basophilic stippling and numerous NRBCs.  basophilic stippling and numerous NRBCs. 

►MCV in range of 50 to 60 fL. MCV in range of 50 to 60 fL. ► Low retic count seen (2-8%). Low retic count seen (2-8%). ►Most of hemoglobin present is Hb F with Most of hemoglobin present is Hb F with

slight increase in Hb Aslight increase in Hb A22. .

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Beta Thalassemia Major Beta Thalassemia Major 3 of 33 of 3

► Regular transfusions usually begin around Regular transfusions usually begin around one year of age and continue throughout one year of age and continue throughout life.  life. 

► Excessive number of transfusions results in Excessive number of transfusions results in tranfusional hemosiderosis; Without iron tranfusional hemosiderosis; Without iron chelation, patient develops cardiac disease. chelation, patient develops cardiac disease.

►Danger in continuous tranfusion therapy: Danger in continuous tranfusion therapy: Development of iron overload. Development of iron overload. Development of alloimmunization (developing Development of alloimmunization (developing

antibodies to transfused RBCs). antibodies to transfused RBCs). Risk of transfusion-transmitted diseases. Risk of transfusion-transmitted diseases.

► Bone marrow transplants may be future Bone marrow transplants may be future treatment, along with genetic engineering treatment, along with genetic engineering and new drug therapies. and new drug therapies.

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Comparison of Beta Comparison of Beta ThalassemiasThalassemias

GENOTYPEGENOTYPE HGB AHGB A HGB AHGB A22 HGB FHGB F

NORMALNORMAL NormalNormal NormalNormal NormalNormal

SILENT SILENT CARRIERCARRIER

NormalNormal NormalNormal NormalNormal

MINORMINOR DecDec Normal to Normal to IncInc

Normal to Normal to IncInc

INTERMEDIINTERMEDIAA

DecDec Normal to Normal to IncInc

Usually IncUsually Inc

MAJORMAJOR DecDec Usually Usually IncInc

Usually IncUsually Inc

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Other Thalassemias Caused by Other Thalassemias Caused by Defects in the Beta-Cluster Defects in the Beta-Cluster

GenesGenes

►1. Delta Beta Thalassemia1. Delta Beta Thalassemia►2. Hemoglobin Lepore2. Hemoglobin Lepore►3. Hereditary Persistence of Fetal 3. Hereditary Persistence of Fetal

Hemoglobin (HPFH)Hemoglobin (HPFH)

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Delta Beta ThalassemiaDelta Beta Thalassemia

►Group of disorders due either to a gene Group of disorders due either to a gene deletion that removes or inactivates only deletion that removes or inactivates only delta and beta genes so that only alpha delta and beta genes so that only alpha and gamma chains produced. and gamma chains produced.

►Similar to beta thalassemia minor. Similar to beta thalassemia minor. ►Growth and development nearly normal.  Growth and development nearly normal. 

Splenomegaly modest.  Peripheral blood Splenomegaly modest.  Peripheral blood picture resembles beta thalassemia.picture resembles beta thalassemia.

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Hemoglobin LeporeHemoglobin Lepore

►Rare class of delta beta thalassemia. Rare class of delta beta thalassemia. ►Caused by gene crossovers between Caused by gene crossovers between

delta locus on one chromosome and delta locus on one chromosome and beta locus on second chromosome. beta locus on second chromosome.

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Hereditary Persistence of Fetal Hereditary Persistence of Fetal Hemoglobin (HPFH) Hemoglobin (HPFH) 1 of 21 of 2

►Rare condition characterized by  continued Rare condition characterized by  continued synthesis of Hemoglobin F in adult life.  synthesis of Hemoglobin F in adult life. 

►Do not have usual clinical symptoms of Do not have usual clinical symptoms of thalassemia. thalassemia.

►Little significance except when combined Little significance except when combined with other forms of thalassemia or with other forms of thalassemia or hemoglobinopathies. hemoglobinopathies.

► If combined with sickle cell anemia, If combined with sickle cell anemia, produces milder form of disease due to produces milder form of disease due to presence of Hb F. presence of Hb F.

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Hereditary Persistence of Fetal Hereditary Persistence of Fetal Hemoglobin (HPFH) Hemoglobin (HPFH) 2 of 22 of 2

►Hb F more resistant to denaturation Hb F more resistant to denaturation than Hb A. Can be demonstrated on than Hb A. Can be demonstrated on blood smears using Kleihauer Betke blood smears using Kleihauer Betke stain.  Cells containing Hb F stain. stain.  Cells containing Hb F stain.

►Classified into two groups according to Classified into two groups according to distribution of Hb F among red cells:distribution of Hb F among red cells: Pancellular HPFH - Hemoglobin F Pancellular HPFH - Hemoglobin F

uniformly distributed throughout red cells.uniformly distributed throughout red cells. Heterocellular HPFH - Hemoglobin F found Heterocellular HPFH - Hemoglobin F found

in only small number of cells.in only small number of cells.

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Beta Thalassemia with Hbg SBeta Thalassemia with Hbg S

► Inherit gene for Hb S from one parent and Inherit gene for Hb S from one parent and gene for Hb A with beta thalassemia from gene for Hb A with beta thalassemia from second parent. second parent.

►Great variety in clinical severity.   Usually Great variety in clinical severity.   Usually depend upon severity of thalassemia depend upon severity of thalassemia inherited.  Production of Hb A ranges from inherited.  Production of Hb A ranges from none produced to varying amounts.  If no Hb none produced to varying amounts.  If no Hb A produced, see true sickle cell symptoms.  If A produced, see true sickle cell symptoms.  If some Hb A produced, have lessening of sickle some Hb A produced, have lessening of sickle cell anemia symptoms.cell anemia symptoms.

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Beta Thalassemia with Hgb CBeta Thalassemia with Hgb C

►Shows great variability in clinical Shows great variability in clinical and hematologic symptoms. and hematologic symptoms.

►Symptoms directly related to which Symptoms directly related to which type thalassemia inherited. type thalassemia inherited.

►Usually asymptomatic anemiaUsually asymptomatic anemia

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Beta Thalassemia with Hgb EBeta Thalassemia with Hgb E

► Is unusual because results in more Is unusual because results in more severe disorder than homozygous E severe disorder than homozygous E disease. disease.

►Very severe anemia developing in Very severe anemia developing in childhood.  childhood. 

►Transfusion therapy required.Transfusion therapy required.

2727

Alpha Alpha ThalassemiaThalassemia

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Alpha Thalassemia Alpha Thalassemia 1 of 21 of 2

► Has wide range clinical expressions. Has wide range clinical expressions. ► Is difficult to classify alpha thalassemias due to Is difficult to classify alpha thalassemias due to

wide variety of possible genetic combinations. wide variety of possible genetic combinations. ► Absence of alpha chains will result in increase Absence of alpha chains will result in increase

of gamma chains during fetal life and excess of gamma chains during fetal life and excess beta chains later in life;  Causes molecules like beta chains later in life;  Causes molecules like Bart's Hemoglobin (Bart's Hemoglobin (γγ44) or Hemoglobin H () or Hemoglobin H (ββ44), ), which are stable molecules but physiologically which are stable molecules but physiologically useless. useless.

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Alpha Thalassemia Alpha Thalassemia 2 of 22 of 2

► Predominant cause of alpha thalassemias is Predominant cause of alpha thalassemias is large number of gene deletions in the alpha-large number of gene deletions in the alpha-globin gene. globin gene.

► Are four clinical syndromes present in alpha Are four clinical syndromes present in alpha thalassemia: thalassemia: Silent Carrier State Silent Carrier State Alpha Thalassemia Trait  (Alpha Thalassemia Alpha Thalassemia Trait  (Alpha Thalassemia

Minor) Minor) Hemoglobin H Disease Hemoglobin H Disease Bart's Hydrops Fetalis Syndrome Bart's Hydrops Fetalis Syndrome

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Silent Carrier StateSilent Carrier State

►Deletion of one alpha gene, leaving Deletion of one alpha gene, leaving three functional alpha genes. three functional alpha genes.

►Alpha/Beta chain ratio nearly normal. Alpha/Beta chain ratio nearly normal. ►No hematologic abnormalities present. No hematologic abnormalities present. ►No reliable way to diagnose silent No reliable way to diagnose silent

carriers by hematologic methods;  carriers by hematologic methods;  Must be done by genetic mapping. Must be done by genetic mapping.

►May see borderline low MCV  (78-80fL). May see borderline low MCV  (78-80fL).

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Alpha Thalassemia Trait Alpha Thalassemia Trait (Alpha Thalassemia Minor)(Alpha Thalassemia Minor)

► Also called Alpha Thalassemia Minor. Also called Alpha Thalassemia Minor. ► Caused by two missing alpha genes.  May be Caused by two missing alpha genes.  May be

homozygous (-a/-a) or heterozygous (--/aa). homozygous (-a/-a) or heterozygous (--/aa). ► Exhibits mild microcytic, hypochromic anemia. Exhibits mild microcytic, hypochromic anemia. ► MCV between 70-75 fL. MCV between 70-75 fL. ► May be confused with iron deficiency anemia. May be confused with iron deficiency anemia. ► Although some Bart's hemoglobin (Although some Bart's hemoglobin (γγ44) present at ) present at

birth, no Bart's hemoglobin present in adults. birth, no Bart's hemoglobin present in adults.

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Hemoglobin H Disease Hemoglobin H Disease 1 of 21 of 2

► Second most severe form alpha thalassemia. Second most severe form alpha thalassemia. ► Usually caused by presence of only one gene Usually caused by presence of only one gene

producing alpha chains (--/-a).  producing alpha chains (--/-a).  ► Results in accumulation of excess unpaired Results in accumulation of excess unpaired

gamma or beta chains. Born with 10-40% gamma or beta chains. Born with 10-40% Bart's hemoglobin (Bart's hemoglobin (γγ44).   Gradually replaced ).   Gradually replaced with Hemoglobin H (with Hemoglobin H (ββ44). In adult, have about ). In adult, have about 30-50% Hb H. 30-50% Hb H.

γγ44 ββ44

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Hemoglobin H Disease Hemoglobin H Disease 1 of 21 of 2

► Live normal life; however, infections, Live normal life; however, infections, pregnancy, exposure to oxidative drugs may pregnancy, exposure to oxidative drugs may trigger hemolytic crisis. trigger hemolytic crisis.

► RBCs are microcytic, hypochromic with RBCs are microcytic, hypochromic with marked poikilocytosis.  Numerous target cells. marked poikilocytosis.  Numerous target cells.

► Hb H vulnerable to oxidation.  Gradually Hb H vulnerable to oxidation.  Gradually precipitate in vivo to form Heinz-like bodies of precipitate in vivo to form Heinz-like bodies of denatured hemoglobin.  Cells been described denatured hemoglobin.  Cells been described has having "golf ball" appearance, especially has having "golf ball" appearance, especially when stained with brilliant cresyl blue. when stained with brilliant cresyl blue.

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Bart’s Hydrops Fetalis Bart’s Hydrops Fetalis SyndromeSyndrome

► Most severe form.  Incompatible with life.  Have no Most severe form.  Incompatible with life.  Have no functioning alpha chain genes (--/--). functioning alpha chain genes (--/--).

► Baby born with hydrops fetalis, which is edema and Baby born with hydrops fetalis, which is edema and ascites caused by accumulation serous fluid in fetal ascites caused by accumulation serous fluid in fetal tissues as result of severe anemia.  Also see tissues as result of severe anemia.  Also see hepatosplenomegaly and cardiomegaly. hepatosplenomegaly and cardiomegaly.

► Predominant hemoglobin is Hemoglobin Bart, along Predominant hemoglobin is Hemoglobin Bart, along with Hemoglobin Portland and traces of Hemoglobin with Hemoglobin Portland and traces of Hemoglobin H. H.

► Hemoglobin Bart's has high oxygen affinity so cannot Hemoglobin Bart's has high oxygen affinity so cannot carry oxygen to tissues.  Fetus dies in utero or carry oxygen to tissues.  Fetus dies in utero or shortly after birth. At birth, see severe hypochromic, shortly after birth. At birth, see severe hypochromic, microcytic anemia with numerous NRBCs. microcytic anemia with numerous NRBCs.

► Pregnancies dangerous to mother.  Increased risk of Pregnancies dangerous to mother.  Increased risk of toxemia and severe postpartum hemorrhage. toxemia and severe postpartum hemorrhage.

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Comparison of Alpha Comparison of Alpha ThalassemiasThalassemias

GenotypeGenotype Hb AHb A Hb BartHb Bart Hb HHb H

NormalNormal 97-98%97-98% 00 00

Silent CarrierSilent Carrier 96-98%96-98% 0-2%0-2% 00

Alpha Alpha Thalassemia Thalassemia TraitTrait

85-95%85-95% 5-10%5-10% 00

Hemoglobin H Hemoglobin H DiseaseDisease

DecDec 25-40%25-40% 2-40%2-40%

Hydrops FetalisHydrops Fetalis 00 80% (with 80% (with 20% Hgb 20% Hgb Portland)Portland)

0-20%0-20%

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Alpha Thalassemia with Hgb Alpha Thalassemia with Hgb SS

►Alpha thalassemia can occur in Alpha thalassemia can occur in combination with hemoglobin S.  Is combination with hemoglobin S.  Is fairly common combination in fairly common combination in populations of African descent. populations of African descent.

►Patient usually asymptomatic.  Have Patient usually asymptomatic.  Have less Hb S present than those with less Hb S present than those with sickle cell trait.  Have increased sickle cell trait.  Have increased presence of Hb F. presence of Hb F.

3737

Laboratory Laboratory Diagnosis of Diagnosis of ThalassemiaThalassemia

38

Laboratory Diagnosis of Laboratory Diagnosis of ThalassemiaThalassemia

►Need to start with patient's individual Need to start with patient's individual history and family history.  Ethnic history and family history.  Ethnic background important. background important.

►Perform physical examination:Perform physical examination: Pallor indicating anemia. Pallor indicating anemia. Jaundice indicating hemolysis. Jaundice indicating hemolysis. Splenomegaly due to pooling of abnormal Splenomegaly due to pooling of abnormal

cells. cells. Skeletal deformity, especially in beta Skeletal deformity, especially in beta

thalassemia major. thalassemia major.

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CBC with Differential CBC with Differential 1 of 21 of 2

►See See decrease in hemoglobin, hematocrit,decrease in hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH).  See mean corpuscular hemoglobin (MCH).  See normal to slightly decreased Mean normal to slightly decreased Mean Corpuscular Hemoglobin Concentration Corpuscular Hemoglobin Concentration (MCHC).  Will see microcytic, hypochromic (MCHC).  Will see microcytic, hypochromic pattern. pattern.

►Have Have normal or elevated RBC countnormal or elevated RBC count with a with a normal red cell volume distribution (RDW). normal red cell volume distribution (RDW).

►Decrease in MCV very noticeable when Decrease in MCV very noticeable when compared to decrease in Hb and Hct. compared to decrease in Hb and Hct.

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CBC with Differential CBC with Differential 2 of 22 of 2

►Elevated RBC count with markedly Elevated RBC count with markedly decreased MCV differentiates decreased MCV differentiates thalassemia from iron deficiency thalassemia from iron deficiency anemia. anemia.

►On differential, see microcytic, On differential, see microcytic, hypochromic RBCs (except in carrier hypochromic RBCs (except in carrier states).  See mild to moderate states).  See mild to moderate poikilocytosis.  In more severe cases, poikilocytosis.  In more severe cases, see marked number of target cells and see marked number of target cells and elliptocytes.  Will see polychromasia, elliptocytes.  Will see polychromasia, basophilic stippling, and NRBCs.basophilic stippling, and NRBCs.

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Reticulocyte CountReticulocyte Count

►Usually elevated.  Degree of Usually elevated.  Degree of elevation depends upon severity of elevation depends upon severity of thalassemia. thalassemia.

42

Osmotic FragilityOsmotic Fragility

►Have decreased osmotic fragility. Have decreased osmotic fragility. ► Is not very useful fact for Is not very useful fact for

diagnosing thalassemia.  Is an diagnosing thalassemia.  Is an inexpensive way of screening for inexpensive way of screening for carrier states.carrier states.

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Brilliant Cresyl Blue StainBrilliant Cresyl Blue Stain

► Incubation with brilliant cresyl blue stain Incubation with brilliant cresyl blue stain causes Hemoglobin H to precipitate.  causes Hemoglobin H to precipitate.  Results in characteristic appearance of Results in characteristic appearance of multiple discrete inclusions -golf ball multiple discrete inclusions -golf ball appearance of RBCs.   Inclusions smaller appearance of RBCs.   Inclusions smaller than Heinz bodies and are evenly than Heinz bodies and are evenly distributed throughout cell. distributed throughout cell.

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Acid Elution StainAcid Elution Stain

►Based on Kleihauer-Betke procedure.  Based on Kleihauer-Betke procedure.  Acid pH will dissolve Hemoglobin A from Acid pH will dissolve Hemoglobin A from red cells.  Hemoglobin F is resistant to red cells.  Hemoglobin F is resistant to denaturation and remains in cell.  Stain denaturation and remains in cell.  Stain slide with eosin.  Normal adult cells slide with eosin.  Normal adult cells appear as "ghost" cells while cells with appear as "ghost" cells while cells with Hb F stain varying shades of pink. Hb F stain varying shades of pink.

►Useful way to differentiate between Useful way to differentiate between pancellular HPFH and heterocellular pancellular HPFH and heterocellular HPFH.HPFH.

45

Hemoglobin ElectrophoresisHemoglobin Electrophoresis

► Important role in diagnosing and Important role in diagnosing and differentiating various forms of thalassemias. differentiating various forms of thalassemias.

► Can differentiate among Hb A, Hb ACan differentiate among Hb A, Hb A22, and Hb , and Hb F, as well as detect presence of abnormal F, as well as detect presence of abnormal hemoglobins such as Hemoglobin Lepore, hemoglobins such as Hemoglobin Lepore, hemoglobin Bart's, or Hemoglobin Constant hemoglobin Bart's, or Hemoglobin Constant Spring. Spring.

► Also aids in detecting combinations of Also aids in detecting combinations of thalassemia and hemoglobinopathies.thalassemia and hemoglobinopathies.

46

Hemoglobin QuantitationHemoglobin Quantitation

►Elevation of Hb AElevation of Hb A22 excellent way to excellent way to detect heterozygote carrier of beta detect heterozygote carrier of beta thalassemia.  Variations in gene thalassemia.  Variations in gene expression in thalassemias results in expression in thalassemias results in different amounts of Hb Adifferent amounts of Hb A22 being being produced. produced.

►Can also quantitate levels of Hb F.Can also quantitate levels of Hb F.

47

Routine Chemistry TestsRoutine Chemistry Tests

► Indirect bilirubin elevated in Indirect bilirubin elevated in thalassemia major and intermedia. thalassemia major and intermedia.

►Assessment of iron status, total iron Assessment of iron status, total iron binding capacity, and ferritin level binding capacity, and ferritin level important in differentiating important in differentiating thalassemia from iron deficiency thalassemia from iron deficiency anemia.anemia.

48

Other Special ProceduresOther Special Procedures

►Globin Chain Testing - determines ratio Globin Chain Testing - determines ratio of globin chains being produced. of globin chains being produced.

►DNA Analysis - Determine specific DNA Analysis - Determine specific defect at molecular DNA level.defect at molecular DNA level.

49

Differential Diagnosis of Differential Diagnosis of Microcytic, Hypochromic Microcytic, Hypochromic

AnemiasAnemiasRDWRDW Serum Serum

IronIronTIBCTIBC Serum Serum

FerritinFerritinFEPFEP

Iron Iron DeficiencyDeficiency

IncInc DecDec IncInc DecDec IncInc

Alpha ThalAlpha Thal NormNorm NormNorm NormNorm NormNorm NormNorm

Beta ThalBeta Thal NormNorm NormNorm NormNorm NormNorm NormNorm

Hgb E DiseaseHgb E Disease NormNorm NormNorm NormNorm NormNorm NormNorm

Anemia of Anemia of Chronic Chronic DiseaseDisease

NormNorm DecDec DecDec IncInc IncInc

Sideroblastic Sideroblastic AnemiaAnemia

IncInc IncInc NormNorm IncInc DecDec

Lead Lead PoisoningPoisoning

NormNorm NormNorm NormNorm NormNorm IncInc