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The Thalassemias

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    The Thalassemias The Thalassemias Presentation Transcript

    • Hemoglobinopathies
    • Hemoglobin structure Hgb A tetramer  globin  globin  globin  globin
    •  
    • Globin chain synthesis  cluster - chromosome 16  cluster - chromosome 11  Gower 1  Portland Embryonic  Gower II  F Fetal <1%  A2 1.5-3.5% Adult  A >95% Globin chain component Hgb name Development period % of adult Hgb     G    A 
    • HAEMOGLOBINOPATHIES Qualitative (structural) Combined (Quantitative & Qualitative) Quantitative
    • Thalassemia
      • Heterogenous group of disorders due to an imbalance of  and  globin chain synthesis
        •  thalssemia:  -globin chain production decreased
        •  thalassemia:  globin chain production decreased
      • The globin chains that are produced are normal
      • Quantitative deficiency:
        •  o thalassemia: No  -globin chain is made
        •  + thalassemia: decreased  -globin chain is made
      • With 4  genes and 2  genes there is wide phenotypic variation
    • Types of Thalassemia
      •  thal : excess of  globins , leading to formation of  globin tetramers (  4 ) that accumulate in the erythroblast , leading to ineffective erythropoiesis . Two types of mutations, the β0 in which no β globin chains are produced and β+, in which some β chains are produced but at a reduced rate.
      •  thal : excess of  globins , leading to the formation of  globin tetramers (  4 ) called hemoglobin H. Results in hemolysis, generally shortening the life span of the red cell. Hemoglobin H-Constant Spring disease is a more severe form of this hemolytic disorder. Most severe form is  thalassemia major, in which fetus produces no  globins, which is generally incompatible with life.
    • Demographics: Thalassemia
      • Found most frequently in the Mediterranean, Africa, Western and Southeast Asia, India and Burma
      • Distribution parallels that of Plasmodium falciparum
    • Genetics of Thalassemia
    • Alpha Thalassemia
      • Inadequate production of alpha chains
      • Hemoglobin analysis normal; can be detected by  globin gene analysis
      • Absence of 1-2 alpha chains
        • Common
        • Asymptomatic
        • Does not require therapy
      • Absence of 3 alpha chains
        • Microcytic anemia (Hgb 7-10)
        • Splenomegaly
      • Absence of 4 alpha chains
        • Hydrops fetalis (non-viable)
    •  
    • Laboratory Findings in Alpha Thalassemia   chains Hgb (g/dl) MCV (fl) RDW  /  Normal Normal Normal  /-  12-14 75-85 Normal  -/  - or - -/  11-13 70-75 - -/-  7-10 50-60 - -/- - - - -
      • Minor (Trait)  /  + or  /  ° 10-13
      • Intermedia  + /  + 7-10
      • Major  + /  ° or  °/  ° < 7
      Beta Thalassemia Clinical Syndrome Genotype Hemoglobin (g/dl) Inadequate production of  chains
      • Minor (Trait)  /  + or  /  ° 90-94 3.5-8 1-10
      • Intermedia  + /  + 5-60 2-8 20-80%
      • Major  + /  ° 2-10 1-6 >85
      •  °/  ° 0 1-6 >94
      Beta Thalassemia - Hgb analysis Clinical Syndrome Genotype A A2 F Hemoglobin analysis: Increased levels of Hgb A2 and Hgb F
    •  
    • Signs and symptoms
      • Thalassemia carriers (trait):
      • Usually no signs or symptoms are apparent, except for a mild anemia.
      • Carriers are usually initially detected through screening, or when performing routine CBC (complete blood count). Later it can be confirmed using hemoglobin electrophoresis.
    • Signs and symptoms- Cont
      • Thalassemia major:
      • Signs such as paleness and growth retardation, are readily detectable since the first year of life. Those are mainly due to severe anemia. Later bone deformities and hepato-splenomegaly develops .
    •  
    • Laboratory diagnosis
      • Thalassemia minor:
      • -Blood smear shows hypochromia and microcytosis (similar to Iron Deficiency Anemia).
      • -Blood indices: MCV< 75 fl, Hb usually> 10, Hematocrit> 30%, RDW < 14%.
      • -Hemoglobin A2 often elevated > 3%, sometimes reaching 7-8%.
    • Laboratory diagnosis- Cont
      • Thalassemia major:
      • -Blood smear shows profound microcytic anemia, with extreme hypochromia, tear drop, target cells and nucleated RBCs.
      • -Hemoglobin may be very low at 3-4 g/dl.
    • Primary Laboratory Investigation Thalassemia
      • Severe cases present with
        • Microcytosis
        • Hypochromia
        • Poikilocytosis
        • RBC counts higher than expected for the level of anemia
    • Thalassemia major
    • Prenatal diagnosis
      • Early prenatal diagnosis can be done using first fetal blood sampling, and later chorion villus biopsy and direct analysis of the globin genes.
      • The error rate in experienced centers is now well under 1%.
    • Beta thalassemia major treatment
      • Transfusion
      • Iron chelation
      • stem cell transplant
    • Management and treatment
      • Thalassemia minor (trait) :
      • No need for any treatment, since the carriers are usually symptomless.
      • Thalassemia major:
      • The severe life-threatening anemia, requires regular life long blood transfusion, to compensate for damaged red blood cells.
    • Management and treatment- Cont
      • Thalassemia Major:
      • The continuous blood transfusion will eventually lead to iron overload, which must be treated with chelation therapy to avoid organ failure.
    • Source: Cooley’s Anemia Foundation
    • Management and treatment- Cont
      • Thalassemia Major -Continued:
      • Other novel treatments like bone-marrow transplantation are very costly.
      • New treatments includes the use of oral chelators, to replace the chelation treatment using Desferal delivered by infusion under the skin through a battery-operated pump.
      • Gene therapy is also an option still researched
    • Prevention efforts
      • Pre marital screening to make sure that the couple are not both carriers.
      • Provision of counseling and health education for the thalassemics, their families and the public .
      • Provision of prenatal testing for thalassemia.
      • Reduction of marriages between relatives.
    • Approach to Beta Thalassemia
      • Screening/counseling
      • RBC transfusion therapy
      • Agents to increase hemoglobin F (Hydroxyurea)
      • Bone marrow transplantation
    • Hemoglobinopathy-antenatal diagnosis
      • Test partners of heterozygous or affected individuals
      • Antenatal diagnosis from DNA obtained by chorionic villus sampling, or by amniocentesis
    • Clinical Presentations of Abnormal Hemoglobins
      • Sickling disorder
      • Thalassemia or microcytic anemia
      • Cyanosis
      • Erythrocytosis
      • Hemolytic anemia
      • Asymptomatic (screening or family study)
    • Structural Haemoglobin Variants
      • Mostly: single AA substitution.
      • Over 300 variants.
      • Many:
      • Harmless, Accidently discovered (screening).
      • May alter Hb stability clinical disorders.
    • The Sickling Disorders
      • Definition
      • A group of disorders characterised by sickling of RBC on deoxygenation (unstable Hb).
      • Include:
      • Heterozygous state of Hb S (sickle cell trait , genotype A S ).
      • Homozygous state of Hb S (sickle disease, genotype S S).
      • Compound heterozygous state of Hb S with Hb SC, SD, SE and other structural variants.
      • Inheritance of sickle cell together with thalassemia.
    • Pathogenesis
      • Substitution of valine for Glutamic acid at position 6 in the B chain (B 6 Glut valine) = Hb S.
      • Gene defect:
        • Replacement of adenine by thymine in the DNA code (GAG GTG).
    •  
    • Patho-physiology of sikle cell disease
    • Clinical picture
      • In infancy:
      • Higher levels of Hb F in neonates protective 8 – 20 weeks.
      • Chronic hemolytic anaemia + Jaundice.
      • Growth & developmental retardation (skeletal deformities): Frontal bossing, short stature, inequalities between upper & lower segments, deformities ………
      • Anaemia & jaundice (ch. Haemolysis).
      • Dactylitis.
      • Splenomegaly resolves by infarcts (auto spelenectomy).
      • Splenic sequestration.
      • Fulminant pnuemococcal septicemia.
      • Chronic leg ulcers.
    • Paediatric emergencies
      • High fever ? Septicaemia.
      • Acute splenic sequestration.
      • Aplastic crisis.
      • Stroke.
      • Acute chest syndrome.
      • Hand-foot syndrome.
    • Pneumococcal septicaemia Prophylactic penicillin works but pneumococcal vaccine does not work well when it is most needed. Penicillin is given as monthly IM injections to ensure compliance. Given 4 months to 4 years when last injection is given with 23V vaccine. Protocol will need modifying with rapid emergence of penicillin resistance and conjugate vaccine.
    • Definition Sudden increase in spleen size, fall in Hb > 2g/dl, and reticulocytosis. The spleen size decreases spontaneously or post transfusion. Accounted for 24% deaths in first 10 years of study. Acute Splenic Sequestration
    • Aplastic crisis Human parvovirus B19 infection accounts for virtually all clinically defined episodes. High secondary infection rate so that siblings of affected cases should be closely monitored. Immunity appears to be life long with no recurrent episodes. Most cases can be safely treated by outpatient transfusion.
      • Pathology
      • Mostly ischaemic in childhood associated with blockage of major cerebral vessels.
      • Mostly haemorrhagic in adults often associated with berry aneurysms at base of brain.
      Stroke
      • Prevention of first or recurrent strokes
        • Chronic transfusion programmes
        • Hydroxyurea
        • Bone marrow transplantation
      Stroke
      • Chronic transfusion programmes (problems)
        • Regular supply of blood
        • Transfusion-acquired infections
        • Red cell alloimmunization
        • Transfusion reactions
        • Iron accumulation necessitating chelation
        • Venous access
      Stroke
      • Bone marrow transplantation
      • Cost and expertise involved
      • Availability of HLA matched marrow (16%)
      • Mortality of 5-10% within 3 months
      • Long term effects of marrow ablation
      • Graft versus host disease
      • Long term effect on growth and fertility
      Stroke
    • Acute chest syndrome
        • complex pathology resulting from infection, infarction, fat embolism, acute pulmonary sequestration.
        • major cause of death at all ages after 2 years
        • monitor closely with
        • pulse oximetry
        • exchange transfusion
        • may be life-saving
    • Acute chest syndrome Radiological change in 3d with acute pulmonary sequestration following gallbladder surgery
    • Hand-foot Syndrome
    • Hand-foot Syndrome Note avascular necrosis of bone marrow of metacarpal (left) compared with normal marrow in proximal phalanx (right)
    • Painful Crisis
      • Hospitilization.
      • Resustation: IV fluid, Oxygen, Antibiotics.
      • Analgesia:
        • Given on regular basis (not on need basis).
        • Early in starting crisis.
        • Prefered smaller, frequent than larger over longer periods (swings of euphoria& pain).
        • Inadequate doses= inadequate control.
        • Haemolysis
        • Daily Hb, Retics TRANSFUSION
        • Aplasia
    • Laboratory Diagnosis sickle Cell Disease
      • 1- Haemogram:
      • Variable anaemia (Hb 6 – 8 gm/dl).
      • TLC & Platalet: N, .
      • retics (10 – 20%).
      • Smear:
        • Sickled erythrocytes, normoblasts, fragmented RBCs, target cells (SC), basophilic stippling, Howell-Jolly & pappenheimer bodies (hyposplensim).
      • 2- ESR:
      • Low as sickle cells fail to form rouleaux.
    • 3- Screening Tests
      • a) Sickling test:
        • In vitro deoxygenation of Hb to induce sickling.
        • So. Metabisultite + cell suspension and immediately covered cause sickling.
      • b) Solubility test:
      • Solubility testing-Dithionite tube test
      • Modification of sickling test:
        • Lysing RBCs release Hb then add metabisulfite to Hb solution + DW form Dark pptate indicates Hb S.
    • Screening for Sickle Cell Trait and Disease
      • RBC lysate with concentrated phosphate buffer and sodium hydrosulfite
      • Incubate 10-20 min
    • 4- Hb Electrophoresis
      • On alk pH or with agar strach gel.
      • The electrophoretic pattern of normal & abnormal Hb depends on the amount of –ve charges that they carry.
      • The net charge varies due to the gain of –ve charges in AA substitution.
      • Hb A > Hb S (B Glu  val ) > Hb C (B Glu  lys )
      • -ve mobility.
      • So, Hb S migerates slower than Hb A.
      • Several ab Hb may overlap.
      • e.g. Hb S & D, Hb C with E.
      • Hb SC & Hb SO Arab: the clinical data may be the only clue.
      5- HPLC & PCR Amplication
    • Hemoglobin electrophoresis: Identification of abnormal hemoglobins
    • HPLC: Sickle cell anemia (Hgb SS)
    • 6- Prenatal diagnosis
      • When parents are both sickle trait to determine fetal homozygosity.
      • prenatal measuring of the B s / B a synthetic ratio in the fetal blood.
      • May be made from:
        • 1 st trimester chronic villus sampling (10 wk gestation).
        • 2 nd trimester fetal RBCs from umbilical cord or trophoblast.
        • 3 rd trimester DNA fetal fibroblast (amniotic fluid).
      • Restriction endonuclease:
      • To detect sickle mutation in the fetal B globin gene.
      • Olignucleotide probes specific for globin point mutation.
      • PCR amplification followed by restriction enzyme digestion of amplified DNA.
      • ARMS (Amplification Refractory Mutation System) PCR.
    • Sickle Cell Trait (Hb A – S)
      • The HETEROZYGOUS from Hb A – s.
      • Sickling does not occur under normal conditions.
      • CLINICAL FEATURES:
      • No clinical disability unless extreme anoxia.
      • May suffer vaso-occlusive crisis (hypoxia, anasthesia, pregnancy, or flying in unpressurized aricrafts).
      • Renal papillary necrosis cause haematuria, hypothenuria (dil urine).
      • Racial background.
      • Sickling test if +ve during anasthesia
      adequate O 2 Avoid postop. dehyd.
    • Management of Sickle Cell Disease
      • Maintenance Therapy
      • Prenatal diagnosis programs.
      • Avoid: Infection, Hypoxia, Dehyration, Exhaustion, Psychological stress.
      • Prophylactic:
        • Oral penicillin infection & crisis.
        • Vaccination: pneumococcal, meningococcal & H. influenza.
        • Malaria (if endomic).
    • For Patients:
      • Regular blood transfusion is not required.
      • Adaptation to anaemia is successful (Hb S has low O 2 affinity).
      • Early & prompt treatment of infection.
      • Folic acid supplements.
      • Major surgical emergencies or recurrent crisis: HYPERTREANSFUSION OR EXCHANGE.
      • Pregnancy:
        • Good antenatal, folic acid.
        • If Hb or crisis regular transfusion.
      • Oral contraceptive ? Veno-occlusive crisis (contraindicated).
      • 8- Occular (Retinopathy): LASER viterous Hage.
      • 9- Haematuria Warning sign
      • 10- CRF: as other causes, renal transplant.
      • 11- Hip pain (aspectic necrosis): Hip replacement.
      • 12- Splenomegaly with hypersplenism, sequestration crisis: splenectomy.
    • Priapism
      • Stilbosterol 5 mg daily.
      • Conservative (1 st 24hs.: Hydration, Analgesia, Exchange transfusion).
      • Surgery: Cavernosus-Spongiosum shunt.
      • Anticoagulants: little value.
      • Leg Ulcer
      • Unsatisfactory, bed rest, debridment (often relapse).
      • Skin graft limited success.
      • Transfusion dose not speed healing.
    • New Therapies
      • I- Antisikling Agents:
      • Agents that increase Hb F.
      • a) Hydroxyurea:
      • Painful crisis by:
      • Hb F, cell volume, alter cell hydration, WBCs.
      • Only in adults (child leukomogenic).
      • b) rhu EPO:
      • Additive effect with hydroxyurea.
      • c) 5-Azacytidine:
          • Prevents methylation of the Y glob gene Hb F.
          • Risk of 2ry malignancy.
      • d) Short chain fatty acids:
      • Butyrate Hb F.
      • II- Bone Marrow Transplantant:
      • Controversial.
      • III- Gene Therapy:
      • Potentially curative.
      • Gene transfer attempted with variable results.
      • Expression exogenous gene too low to be of benefit.