Hemoglobinopathies and Thalassemia Handouts PDF 2022-2023

HEMATOLOGY 1 HEMOGLOBINOPATHIES AND THALASSEMIAS HEMOGLOBINOPATHIES These are a group of inherited disorders causing globin chain in the synthesis due to the amino acids’ substation (qualitative defect); changes in RBC deformability and electrophor...

HEMATOLOGY 1 HEMOGLOBINOPATHIES AND THALASSEMIAS HEMOGLOBINOPATHIES These are a group of inherited disorders causing globin chain in the synthesis due to the amino acids’ substation (qualitative defect); changes in RBC deformability and electrophoretic mobility can occur. Homozygous/disease condition (both globin chains affected) is more serious than; Heterozygous/traitcondition (only one globin chain affected). Target cells are associated with the hemoglobinopathies. Hgb electrophoresis, isoelectric focusing and/or DNA (PCR) analysis may be used to confirm the diagnosis. The amino acid substitution causing formation of Hgb S is the most common; Hgb C is the 2nd most common and the Hgb E the 3rd most common. Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023 Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023 SICKLE CELL DISEASE (HgB SS)  Sickle cell disease is caused when valine replaces glutamic acid atposition 6 on both beta chains. It results in a decrease in Hgb solubility and function. Defects are inherited from both parents.  Occurs most commonly in: o Sub-Saharan Africa = 64.4% o Arab-India = 22.7% o The Americas = 7.4% o Eurasia = 5.4% o SoutheastAsian = 0.1%  Highest frequency in sub-Saharan Africa where approximately 230,000 babies are born with sickle cell disease.  No Hgb A is produced and approximately 80% Hgb S and 20%Hgb F (the compensatory Hgb) is seen,Hgb A2 is variable.  Hgb insolubility results when deoxyhemoglobin is formed. Hgb crystallizes in erythrocytes. It is characterized by the classic sickled shape erythrocyte. Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023  HEMOGLOBINSOLUBILITY TEST Testtube in the left= NEGATIVE(Clearsolutionwithvisible background) Testtube in right= POSITIVE (Turbidsolution) CLINICAL FINDINGS LABORATORYFINDINGS >Erythrocytesbecomerigidandtrappedin >Severe normo/normohemolytic anemia capillaries; bloodflowrestrictioncauses lack withpolychromasia resultingfrom of O2to the tissues, resultingin tissue prematurerelease ofreticulocytes. necrosis. >BMerythroidhyperplasia (M: ERatio >All organs areaffected, with kidney failure decreases) beinga commonoutcome; hyposplenism >Sicklecells, target cells, nucleatedRBCs, andjoint swellingoccur. Pappenheimerbodies, andHowell-Jolly >Vaso-occlusive crisisoccurswith increase bodies areseen. BMresponse tothehemolytic anemia.Crisis >Increasedbilirubin anddecrease can be initiatedby many physiologicfactors, haptoglobin are characteristicsdue to includingsurgery, trauma, pregnancy, high hemolysis. altitudes, etc. >(+) Hgbsolubility test. >Apparent immunity to >Hgb Smigrateswith HgbDandG on alkaline Hgbelectrophoresis;can differentiateusingacidelectrophoresis. SICKLE CELL TRAIT (HgbAS) Sickle cell disease is caused when valine replaces glutamic acid atposition 6 on one beta chain. Defect is inherited from one parent. One normal beta chain can produce some Hgb A. Approximately 60% Hgb A and 40% Hgb S are produced, with normal amounts of Hgb A2 and F. This heterozygous traitis the most common hemoglobinopathy in the USA. Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023  CLINICALFINDINGS /LABORATORYFINDINGS Sickle cell traitgenerally producesnoclinicalsymptoms.Anemia israre but ifpresent, will be normo/normoandsicklingcan occur duringrare crisis states (sameas inHgbSS) (+) Hgbsolubility test. Apparent immunity to P. falciparum. HEMOGLOBIN C DISEASE / Hgb CC Hgb C disease is cause when lysine replaces glutamic acid atposition 6 on both beta chains. Defectis inherited from both parents. Occurs in the African-American (2%-3%) and WestAfrican (17%-28%) populations. No Hgb A is produced; approximately 90%Hgb C, 2% Hgb A2 and 7% Hgb F are produced. Mild anemia may be present. The heterozygous Hgb C trait patientis asymptomatic, with no anemia, the one normal beta chain is able to produce approximately 60% Hgb A and 30%-40%Hgb C, with normal amounts of HgbA2 and Hgb F. CLINICAL FINDINGS / LABORATORY FINDINGS Mildnormo/normoanemia withtarget cells;characterizedby intracellular rodlike Ccrystals. Slight to Moderate increase inreticulocytesandnRBC. (-) Hemoglobin Solubility Test HgbCmigrateswithHgbA2, E, andOon alkalineHgb electrophoresis:candifferentiateHgbusingacidelectrophoresis. HEMOGLOBIN C-HARLEM (HEMOGLOBIN C-GEORGETOWN) Hgb C – Harlem has a double substitution on the beta chain. Valine forglutamic acid at position 6 and aspartic acid for asparagine atposition 73. Heterozygous are asymptomatic, but patients with compound heterozygosity for Hb S and Hb C-Harlem have crisis similar to Hgb SS disease. (+) Hemoglobin Solubility Test HEMOGLOBIN SC DISEASE Hgb SC disease is a double heterozygous condition where an abnormal sickle gene from one parent and an abnormal C gene from the other parentare inherited. At position 6, glutamic acid is replaced by valine in one beta chain and by lysine on other beta chain. Seen in WestAfrican (25% prevalence), Mediterranean and Middle Eastern populations. No Hgb A is produced; approximately 50%Hgb S, and 50%Hgb C are produced. Compensatory Hgb F may be elevated up to 7%. Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023  CLINICAL FINDINGS / LABORATORY FINDINGS Symptoms: less severe than sickle cell anemia butmore severe than Hgb C disease. Laboratory: Moderate to severe normo/normo anemia with target cells; Characterized by SC crystals; May be see rare sickle cell or C crystals. (+) Hgb solubility screening test. HEMOGLOBIN E (Hb EE) Caused when lysine replaces glutamic acid at position 26 on the beta chain. Found more commonly in Southeast Asian (30%), African,and African-American populations. Homozygous condition results in: Mild anemia with microcytes and target cells. Shortened RBC life-span. It resembles thalassemia trait. Heterozygotes are asymptomatic. Hgb E migrates with Hgb A2, C and O on alkaline Hgb electrophoresis. LABORATORYFINDINGS (-) Solubility Test Electrophoresis: o Homozygous = > 90% HbE o Heterozygous = 30 – 40% HbE Low MCV (55 – 65 fL) Normal Reticulocyte Count HEMOGLOBIN D Group of at least 16 Beta chain variants. Caused when glycine replaces glutamic acid atposition 121 on the beta chain. Found more commonly on Middle Eastern and Indian populations. Mostvariants are named for the place they were discovered. o Hb D – Punjab (affects about 3% of the population in Northwestern India) o Hb D – Los Angeles (affects < 2% of African-Americans) Both homozygous and heterozygous conditions are asymptomatic. Hgb D migrates with Hgb S and Hgb G on alkaline Hgb electrophoresis. HEMOGLOBIN G Hb G – Philadelphia o A variant of Hb G where lysine replaces asparagine at position 68 in alpha chain. o Mostcommon G variant in African-American. o It is also found in Ghana. HEMOGLOBIN M Caused by a variety of mutations in the alpha, beta and gamma globin genes,all of which are resultin the production of methemoglobin (hence Hb M designation). Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023  Hemoglobin carries Ferric state iron which makes it incapable of carrying oxygen. HEMOGLOBIN O - ARAB Caused when lysine substitutes glutamic acid at position 121. It is found in Kenya, Israel, Egypt, and Bulgaria with 0.4% prevalence in African-American. No clinical symptoms are exhibited exceptfora mild splenomegaly in homozygotes. However, this variant with Hb S presents severe clinical conditions similar to those in HbSS. CLINICALANDLABORATORYFINDINGS o (-) Solubility Test o Mild hemolytic anemia with target cells ELECTROPHORESI ELECTROPHORESI S S Cellulose Acetate HPLC CitrateAgar (Alkaline pH @8.4) (AcidpH @6.0 – 6.2) Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023 THALASSEMIAS Group of inherited disorders causing decrease rate of synthesis of a structurally normal globin chain (quantitative defect); characterized by micro/hypo RBC and target cells. Classified according to the globin chain affected. Found in Mediterranean (beta); Asian (alpha); and African (alpha and beta) populations. Severity varies from no clinical abnormalities to transfusion-dependent to fatal. Thalassemia major: severe anemia; either no alpha or no bête chains produced. Thalassemia minor/trait: Mild anemia; sufficient alpha and beta chains to produce to make normal Hgb. A1,A2 and F but may be abnormal in amounts. BETA-THALASSEMIA Major/Homozygous (Cooley anemia) 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, from Heinz bodies and cause rigidity; destroyed in the BM or removed by the spleen. Patients are transfusion dependent (major therapeutic option). Typically, 10 – 15 mL / kg of RBC are transfused every 2 – 5 weeks. Hematopoietic Stem Cell Transfusion (HSCT) is the only curative therapy for thalassemia major. CLINICAL SYMPTOMS LABORATORY FINDINGS Severemicro/hypo anemia Target cells Teardropcells ManynRBC Basophilicstippling Howell-Jolly bodies Pappenheimer bodies Heinz bodies Increase serumiron and increasedbilirubin reflect the hemolysis β-Thalassemia Intermedia This is the term used to describe anemia that is more severe than β thalassemia minor but does notrequire regular blood transfusion to maintain hemoglobin level and quality of life (transfusion-independent). Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023 Minor/Trait (Heterozygous) Decreased rate of synthesis of one of the beta chains; the other beta chain is normal. LABORATORYFINDINGS Mild or asymptomatic micro/hypo anemia with a normal or slightly elevated RBC count Target cells Basophilic stippling. Hgb A is slightly decreased, butHgbA2 is slightly increased to compensate. Silent Carrier state of Beta Thalassemia  The designation βsilent includes the various heterogenous β-globin gene mutation that produces only a small decrease in production of β chains.  The silent carrier state is nearly normal α-β chain ratios and no hematologic abnormalities.  ALPHA-THALASSEMIA Major (Hydrops Fetalis) All four alpha genes are deleted, no normal Hgb are produced. 80% Hgb Bart’ s (γ4) produced; cannot carry O2 Incompatible with life; Baby dies in utero or shortly after birth. Hemoglobin H Disease 3 alpha genes are deleted. Decrease in alpha chains leads to beta chain excess. Hgb H (β4), an unstable Hgb is produced. Heinz bodies form and rigid RBCs are destroyed in spleen. Distinguishing characteristic include: Moderate micro/hypo anemia Up to 30% Hgb H; the restis Hgb A. Minor/Trait  2 alpha genes are deleted. Patients are usually asymptomatic and discovered accidentally. Up to 6% Hgb Bart’ s in newborns may be helpful in diagnosis. Absent by 3 months of age.  Mild micro/hypo anemia with often with a high RBC count and target cells. Silent Carrier  One alpha gene is deleted. Patients are asymptomatic and are often not diagnosed unless gene analysis is done.  Borderlow MCV may be the only sign. THALASSEMIA AND HEMOGLOBIN INTERACTIONS  Caused by inheritance of a thalassemia gene from one parent and a Hgb variantgene.  Severity and symptoms depend on the specific interactions.  Common interactions include: o Hgb S/Alpha-thalassemia Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023  o Hgb S/Beta thalassemia o Hgb C/Beta-thalassemia o Hgb E/Beta thalassemia Mild Anemia Hgb S /Alpha-Thalassemia African Ancestry High Hgb Levels Low Reticulocyte Count Africa Mild hemolytic Anemia with Hgb S / Beta-Thalassemia Middle East Splenomegaly India Microcytosis Moderately severe hemolysis, splenomegaly, Hgb C /Beta-Thalassemia hypochromia, microcytosis and target cells Southeast Asia Symptoms are similar to β Hgb E / Beta-Thalassemia thalassemia major or Eastern India intermedia LABORATORY METHODS I. Complete Blood Count with Peripheral Blood film Review INCREASED DECREASED Hemoglobin Hematocrit RBC Count MCV RBC Distribution Width (RDW) MCH MCHC Thalassemia Minor Microcytes (α and β) Target Cells Hb H Disease Slight to Moderate Poikilocytosis Extreme poikilocytosis Homozygous and Compound Target Cells Heterozygous β-Thalassemia Elliptocytes Polychromasia Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023  Basophilic Stippling Howell-Jolly Bodies Pappenheimer Bodies Nucleated RBC : Usually micro / hypo cells except for silent carrier phenotypes. II. Reticulocyte Count o Elevated Reticulocyte Count indicating that the bone marrow is responding to hemolytic process. o In Hb H disease reticulocytes can reach 5 to 10%. o In homozygous β-Thalassemia 2 – 8%. III. Assessment Of Normal and Variant Hemoglobin IV. Molecular Genetic Testing V. Alkali Denaturation Test – accurate and precise to quantify Hb F in the 0.2 – 50% range. VI. Kleihauer-Betke Acid Elution Test  Peripheral blood films are ethanol fixed and immersed in citrate-acid buffer (pH 3.3).  Adult hemoglobin is eluted from the RBC whereas Hb F resists elution and remains in the cell.  When the RBCs are stained, RBCs with Hb F will take up the stain meanwhile, RBCs with adult hemoglobin will appear as “ghost RBC”. Preparedby:LOUIS JOHNR.BEJO, RMT, MSPH A.Y. 2022- 2023