Thalassemia Chapter 12 PDF

17/12/2022 Thalassemia Chapter 12 Week 14 Rania Abu Seir, Ph. D. Associate Professor of Hematology...

17/12/2022 Thalassemia Chapter 12 Week 14 Rania Abu Seir, Ph. D. Associate Professor of Hematology Clinical Hematology I (0202306) Harmening DM. Clinical Hematology & Fundamentals of Hemostasis, 5th ed. Philadephia: F.A. Davis 2009. Objectives Name the hemoglobin defect of thalassemia Describe the different types of mutation involved in alpha (α) and beta (β) thalassemia Classify Thalassemias and list the cause for each List the clinical and laboratory findings in Beta and Alpha thalassemias List red cell indices that help to distinguish thalassemia from iron deficiency Describe the appearance of the peripheral smear in thalassemia 2 1  17/12/2022 Thalassemia Thalassemia was first described by Thomas Cooley & Pearl Lee (1925) called Cooley’s anemia (thalassemia major/ homozygous) Thalassemia is derived from the Greek word “thalassa” or Sea Thalassemia minor was later described Thalassemia is now widely distributed 3 World Distribution of α-Thalassemia & β-Thalassemia Found in Mediterranean populations (beta) Asian populations (alpha) African populations (alpha and beta) Figure 12-1 World distribution of alpha (a) and beta (β) thalassemia. 4 2  17/12/2022 Thalassemia Group of inherited disorders causing decreased rate of synthesis of a structurally normal globin chain (quantitative defect); characterized by microcytic/hypochromic RBCs and target cells. Reduced rate of production of one or more of the globin chains of Hgb. Interaction of different types of thalassemia, & interaction of thalassemias with Hb variants  produce a family of disorders that range in severity from death in utero to mild and symptom-less hypochromic anemia Most thalassemias are inherited in a Mendelian Recessive fashion 5 Classification of Thalassemia Thalassemias are classified depending on the type of globin chain affected into α-thalassemia, β-thalassemia, δβ-thalassemia,…etc Classified clinically according to severity into: Major: severe transfusion-dependent: either no alpha or no beta chains produced Intermediate: anemia & splenomegaly Minor: symptom-less carrier state: sufficient alpha and beta chains produced to make normal hemoglobins A, A2, and F, but may be in abnormal amounts 6 3  17/12/2022 Genetics of Hb Synthesis The α–chains have 141 aa each, while the β–like chains have 146 aa each. 7 Table 12–1 Composition of Hemoglobins Found in Normal Human Development and Abnormal Hemoglobins Found in Thalassemia State Globin Chains Hemoglobin Adult α2β2 A Α2δ2 A2 Fetus αAγ2 F αGγ2 F Embryo α2ε2 Gower 2 ζ2ε2 Gower 1 ζ2γ2 Portland α Thalassemia β4 H γ4 Bart's 8 4  17/12/2022 The Areas of Homology in the α–Globin Genes Can Lead to Mispairing Figure 12-2 The areas of homology (x, y, and z) can lead to mispairing. 9 Control of β–Globin Gene Expression 10 5  17/12/2022 Mutations in Globin Gene Causing Thalassemia More than 200 mutations are reported to cause β-thalassemia In the case of the β–globin gene, most of these mutations are point mutations and can be grouped into: Transcriptional mutants (affect promoter) Translational mutants RNA processing mutants Miscellaneous mutations In the case of the α-globin gene, most mutations are deletion mutations 11 Diagram of Globin Chain Imbalance Figure 12-4 Diagram of globin chain imbalance. Excess globin chains precipitate and damage red blood cells and their precursors. Destruction of red blood cells within the bone marrow (ineffective erythropoiesis) predominates in severe β-thalassemia. In contrast, hemoglobin H (β4) precipitates and damages circulating red blood cells (hemolysis) in severe α thalassemia. Notice the changes in hemoglobin constitution of the peripheral blood with thalassemia: hemoglobin F is increased in severe β thalassemia and hemoglobin H is detectable in severe α thalassemia. 12 6  17/12/2022 Pathophysiology of Thalassemia In α-thal: HbH (β4) and Hb Barts (γ4) ppt> shortened life span In β-thal> α2 precipitates> ineffective erythropoiesis β–thalassemia Symptoms occur at ~6 months of age, following the switch from γ-chain to β-chain synthesis Hgb A2 (α2δ2) is increased and Hgb F (α2γ2) 13 β–Thalassemia Genotypic classification β0–thalassemia haplotypes: complete absence of B-chain commonly found in the Mediterranean area (particularly in Italy, Greece, Algeria, and Saudi Arabia) β+–thalassemia haplotypes: reduced amount of B-chain that ranges from 10% to more than 50% of the normal expression level caused by a spectrum of mutations the expression of the β-chain is inversely proportional to clinical severity 14 7  17/12/2022 β–Thalassemia Clinical Expression of the Different Gene Combinations > Clinical Classification β–thalassemia major: Homozygous for B+ or B0; or compound heterozygous for B+ and B0 Markedly decreased rate of synthesis or absence of both beta chains results in an excess of alpha chains; no Hgb A can be produced; compensate with up to 90% Hgb F Excess alpha chains precipitate on the RBC membrane, form Heinz bodies, and cause rigidity; destroyed in the bone marrow or removed by the spleen Hgb is usually Clinical Classification Severe microcytic/hypochromic anemia, target cells, teardrops, many nRBCs, basophilic stippling, Howell-Jolly bodies, Pappenheimer bodies, Heinz bodies Increased serum iron increased bilirubin reflect the hemolysis The osmotic fragility test result in a patient with thalassemia major would most likely be decreased 16 8  17/12/2022 β–Thalassemia: Clinical Expression of the Different Gene Combinations β–thalassemia intermedia: Homozygous for combinations of less severe β-globin mutations Less severe clinical expression than β-thalassemia major Patients are not transfusion dependent at baseline but may need transfusion during an illness like infections 17 β–Thalassemia: Clinical Expression of the Different Gene Combinations β–thalassemia minor / trait: Heterozyg for B+ or B0 Mild form of chronic hypochromic microcytic anemia with a normal or slightly elevated RBC count, target cells, basophilic stippling Degree of anemia is variable: Hb 10.5-13.9 g/dl HbA is slightly decreased but Hgb A2 & Hgb F are mildly elevated Some patients with very mild, β++, mutations show no clinical or lab evidence of anemia (normal HbA2 level) and are called silent carriers 18 9  17/12/2022 Clinical Course & Therapy: β-Thalassemia Major Severe chronic anemia with variable degree of jaundice stunted growth & abdominal enlargement (hepatosplenomegaly) Presents within the first year of life Hb level is 4-8 g/dl Marked expansion of erythropoiesis with skeletal changes giving the typical hair-on-ends on the skull The marrow expansion of facial bones produces a characteristic facial appearance (prominent facial bones, especially the cheek and jaw) Iron overload from RBC destruction and multiple transfusions cause organ failure 19 Facial appearance of β- Skull X-ray in β-thalassemia major. thalassemia major. The skull is There is a “hair-on-ends” appearance bossed with prominent as a result of the expansion of the bone frontal & parietal bones, the marrow into cortical bone. maxilla is enlarged. 20 10  17/12/2022 Figure 12-7 Skull x-ray film of a 5- year-old child with homozygous β thalassemia. Note the dilation of the diploic space and the typical “hair-on- end” appearance caused by subperiosteal bone growth in radiating striations. 21 Figure 12-8 Face (A) and profile (B) of an 11-year- old child, with homozygous β thalassemia who is receiving hypertransfusion. The characteristic facial changes are not as prominent as those in an untransfused child, but are still present. Note the bossing of the skull, hypertrophy of the maxilla with prominent malar eminences, depression of the bridge of the nose, and mongoloid slant of the eyes. 22 11  17/12/2022 Clinical Course & Therapy: β-Thalassemia Major Therapy: blood transfusion to maintain Hb level at ~11.5 g/dL on average Hypertransfusion suppresses ineffective erythropoiesis Splenectomy reduces the blood transfusion requirement Transfusion causes severe iron overload (hemochromatosis) cause organ failure & requires iron chelation therapy using Deferoxamine, Deferiprone & ExJade Hyroxyurea increases the Hgb F levels Prevention: pre-marital screening tests 23 Clinical Course & Therapy: β-Thalassemia Intermedia Patients show variable degrees of anemia & survive without large blood transfusion requirements Usually occurs at age of 2 years or later with an Hb level of 9-10 g/dl Therapy usually consists of supportive therapy with occasional blood transfusion Serum bilirubin is more elevated than Thalassemia major They may become transfusion-dependent in case of hypersplenism Patients develop iron overload due to increased gastrointestinal absorption 24 12  17/12/2022 Clinical Course & Therapy: β-Thalassemia Minor Patients show mild microcytic hypochromic anemia with Hgb levels 10-13 g/dl Patients are usually asymptomatic except during periods of stress such as pregnancy and infection No therapy is needed It is important to differentiate them from IDA 25 Blood Transfusion in Thalassemia Concerns of blood transfusion Iron overload from RBC destruction and multiple transfusions cause organ failure Alloimmunization Transfusion-transmitted diseases 26 13  17/12/2022 Alpha (α) –Thalassemia Affects α-chain and thus affects Hgb F and Hgb A Manifested immediately after birth or in utero There is a great variety of severity because each person has 4 α-genes (αα/αα) Decreased synthesis of α-chain leads to formation of stable tetramers: Hgb Barts in the fetus (γ4) and Hgb H in adults (β4) Classification based on genotype α0–thalassemia haplotypes (α–thalassemia 1) α+–thalassemia haplotypes (α–thalassemia 2) 27 α–Thalassemia α0–thalassemia haplotypes (α–thalassemia 1): Mostly are caused by deletion of both a-genes, a1 and a2 on the same chromosome Haplotype (--/) Homozygous α0–thalassemia (--/--) α+–thalassemia haplotypes (α–thalassemia 2): Mostly are caused by deletion of a single a-gene Haplotype (-α/) It can be caused by non-deletion (mostly point mutations) & the haplotype is referred to as αTα It is also caused by unstable α-globin structural mutants 28 14  17/12/2022 α–Thalassemia Hb Constant Spring (HbCS) The α-globin variant Constant Spring of αCS is the result of a point mutation in the α2-gene and may be considered as α+-thalassemia haplotype αCS-globin chain has 31 extra αα at its tail end Heterozygotes are asymptomatic without hematologic abnormalities & produce only small amounts of Hgb CS (1%) Homozygotes show α-thalassemia minor with microcytosis & 5% Hgb CS It’s found in SEA and combines with α-thalassemia haplotypes to give α- thalassemia syndromes 29 α–Thalassemia: Clinical Expression of the Different Gene Combinations Four clinical categories depending on disease severity: Hb Barts hydrops fetalis syndrome Hb H disease 30 15  17/12/2022 α–Thalassemia: Clinical Expression of the Different Gene Combinations Hgb Barts hydrops fetalis(Major): Caused by homozygosity of α0-thalassemia haplotypes (--/--) (all four α-genes are deleted ) Produce no α-chain: no normal hemoglobin are produced Hgbs present are Hb Barts and Hb Portland 80% hemoglobin Bart’s (Gama 4) produced; cannot carry oxygen; incompatible with life; die in utero or shortly after birth No therapy is available 31 Interaction of Deletion Mutations in α-Thalassemia Figure 12-11 Simplistic look at the interactions of deletional mutations of α thalassemia. The severity of disease is predictable and depends on the number deleted α-globin genes. 32 16  17/12/2022 α–Thalassemia: Clinical Expression of the Different Gene Combinations Hb H disease Caused by compound heterozygosity of α0- and α+-thalassemia (--/α-) Three alpha genes are deleted. Decrease in alpha chains leads to beta chain excess. Hemoglobin H (β4), it is unstable hemoglobin and thermolabile, Electrophoretically, it represents a "fast" hemoglobin Heinz bodies form and rigid RBCs are destroyed in the spleen. Distinguishing characteristics include: moderate microcytic/hypochromic anemia Hgb H is up to 30% in adults, the remainder is Hgb A and small amounts of HgbA2 and Hgb Barts Infants have Hgb Barts 19-27% at birth and remainder is HgbF and HgbA 33 α–Thalassemia: Clinical Expression of the Different Gene Combinations α –thalassemia minor or α0–thalassemia trait or α–thalassemia1 trait : Caused by defects of two α-genes It’s usually the result of heterozygous for α0-thalassemia (--/aa) or homozygous for α+-thalassemia haplotypes (-a/-a) At birth, Hgb Barts is up to 6% and may be helpful in diagnosis, they disappear and is not replaced by HgbH by 3 months of age Mild microcytic/hypochromic anemia often with a high RBC count and target cells MCV is usually 70-75 fL 34 17  17/12/2022 α–Thalassemia: Clinical Expression of the Different Gene Combinations Silent carrier of α–thalassemia, also called α+–thalassemia trait or α– thalassemia 2 trait Caused by defects of one α -genes and are asymptomatic It’s usually the result of heterozygous to α+-thal (-a/aa) or (aTa/aa) At birth, Hgb Barts is up to 2%, which disappears and is not replaced by HgbH No recognizable hematologic abnormalities is present except for a borderline low MCV (78-80 fL) 35 δβ–Thalassemia & Hb Lepore Syndrome Heterozygous: Hgb A2 Normal & Hgb F increased Homozygous: absence of Hgb A & Hgb A2 Classified into two types (δβ)+-Thalassemia Hb Lepore (δβ-fusion chain) Hb anti-Lepore (βδ-fusion chain) (δβ)o-Thalassemia (δβ)o-Thalassemia (Aγδβ)o-Thalassemia δβ–thalassemia: much less common than β-thalassemia Found sporadically in Greeks, Italians, Arabs & American Blacks 36 18  17/12/2022 Hb Lepore & Related Variants: Abnormal Crossing Over Between B & D-genes Results in Hb Lepore & Anti-Lepore Figure 12-12 Hemoglobin Lepore formation. An abnormal crossing over between β and δ- globin genes gives rise to hemoglobin Lepore and to hemoglobin anti-Lepore. 37 Hereditary Persistence of Hb F (HPFH) Heterogeneous group that produces phenotypes similar to δβ- thalassemia, but the defective β-gene is almost (in some forms not completely) compensated by persistent γ-chain production Homozygous with 100 % Hgb F mild thalassemia-like picture & mildly polycythemic due to high O2 affinity of Hgb F Heterozygous have no hematological abnormality & Hgb F 20-30% In most cases, Gamma chain production equals alpha chain production 38 19  17/12/2022 Figure 12-16 Flow cytometric analysis to determine hemoglobin F distribution. The x-axis represents the amount of hemoglobin in red cells, and the y-axis represents the amount of red blood cells. A pancellular distribution of hemoglobin F in an individual with HPFH is shown in the diagram at left. A heterocellular distribution of hemoglobin in an individual with heterozygous (δβ)0 thalassemia is shown in the diagram at right. 39 Figure 12-15 Kleihauer– Betke stain of blood from a patient with hereditary persistence of fetal hemoglobin (HPFH). Note that all red cells stain red, owing to the varying amounts of hemoglobin F. 40 20  17/12/2022 Hemoglobin F (Kleihauer–Betke Method) Count dense-staining Hgb F cells and the number of ghost cells containing Hgb A to obtain percentage. 1. It is used to detect the presence of fetal cells in the maternal circulation during problem pregnancies because Hgb F in fetal cells resists acid elution. 2. It differentiates hereditary persistence of fetal hemoglobin from other conditions associated with high Hgb F levels. 3. Normal newborns have 70-90% Hgb F levels. 41 Thalassemia Associated with Hb Variants β-thalassemia/ Hgb S (β-thalassemia Sickle cell syndrome) Hgb S 65% & Hgb A 35% Severity depends on the type of β-allele affected In case of β0-allele: no Hgb A, similar to SCD In case of β+-allele: Hgb A is 15-30%, and patients have less severe sickling crisis than β0- group 42 21  17/12/2022 Thalassemia Associated with Hb Variants α-thalassemia/ Sickle cell anemia Hgb F is increased and decrease severity of the sickling process HbgF is ~16% with α0-thalassemia & ~8% with α+-thalassemia 43 Thalassemia Associated with Hb Variants β-thalassemia/ Hgb C Common in West Africa & African Americans, where β+ allele is most common Mild degree & usually asymptomatic anemia like heterozygous β-thalassemia 44 22  17/12/2022 Thalassemia Associated with Hb Variants β-thalassemia/ Hgb E Common in the Far East Present with severe anemia like homozygous β-thalassemia 45 Lab Diagnosis of Thalassemia CBC Hct, Hb, MCV MCH, are low MCHC: mild decrease RBC count: N or increased RDW: N, except in thalassemia major is increased Blood film Homozygous β-thalassemia> severe anisocytosis & poikilocytosis, target cells, elliptocytes, large numbers of NRBCs Heterozygous β-thalassemia> hypochromic microcytic, mild-moderate anisocytosis & poikilocytosis 46 23  17/12/2022 Lab Diagnosis of Thalassemia Supravital stain Retic is up to 10% in Hgb H disease and up to 5% in Homozygous β- thalassemia (low in relation to anemia) In Hgb H disease Hgb H 47 β–Thalassemia Major Figure 12-5 Peripheral smear from a patient with β-thalassemia major. Note the nucleated red cells, Howell–Jolly body in the hypochromic microcyte (arrow), numerous target cells, and moderate anisocytosis and poikilocytosis (Wright's stain). 48 24  17/12/2022 Hgb H Disease Hgb H disease (α-thalassemia): marked Hgb H disease (α-thalassemia): hypochromic microcytic cells with target Hgb H inclusions give the cell a cells and poikilocytosis “golf ball” appearance 49 Figure 12-10 Hemoglobin H inclusions (supravital stain). (From Bell, A: Hematology. In: Listen, Look and Learn. Health and Education Resources, 50 Inc., Bethesda, MD, with permission.) 25  17/12/2022 β–Thalassemia Minor / Trait Figure 12-6 Peripheral smear from a patient with thalassemia minor. Note the microcytosis and hypochromia with mild anisocytosis and poikilocytosis. A few target cells and basophilic stippling are present. (Wright's stain, magnification ×400) 51 Acid elution stain (Kleihauer & Bethke test) Useful to differentiate between heterozygous δβ-thalassemia (heterocellular Hgb F) from heterozygous pancellular HPFH Osmotic fragility Thalassemic red cells have a decreased O.F O.F is not useful for diagnosis 52 26  17/12/2022 Hb electrophoresis (see Table 12-4) Cellulose acetate/ pH 8.4 Citrate agar gel/ pH 6.2 Hgb A2 & Hgb F quantitation Elevated HbA2 is characteristic of β-thalassemia trait (3.5-7%), while Hgb F is mildly elevated (usually