Thalassemia.by dr narmada

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  • KleihauerBetke test. This peripheral blood from a postpartum womanwith fetomaternal hemorrhage demonstrates HbF containing fetal cells (dark red) in a background of maternal cells (ghost-like cells). it can be used to detect HbF–containing cells in β-thalassemia, hereditary persistence of hemoglobin F (some types have homogeneous distribution of HbF in the cells), sickle cell disease, δβ- thalassemia, and myelodysplastic syndrome
  • Thalassemia.by dr narmada

    1. 1. THALASSEMIA
    2. 2.  Thalassemia was defined as a clinical entity in1925 when Dr. Thomas B. Cooley and hisassociate Pearl Lee, pediatricians at the DetroitChildren’s Hospital, In the early it is called as the anaemia splenicainfantum. Whipple and Bradford proposed the namethalassemia.
    3. 3.  The alpha thalassemia is prevalent in southeastAsia, Malaysia and southern china. α +thalassemia is relatively more common in India. The beta thalassemia are seen primarily in thearea surrounding Mediterranean sea, Africa andsoutheast Asia. Carrier frequency of thalassemia in india isabout 3 % and estimated frequency ofthalassemia at birth is 1:2700.
    4. 4.  In India β thalassemia is frequent and αthalassemia is rare. β thalassemia is more common incertain communities such asSindhis, Punjabis, Bengalis, Gujratis, Parsis, Bhansalis, Jain and Lohanas. Thalassemia is prevalent in those parts ofworld where malaria has been common.
    5. 5.  Thalassemia are autosomal recessive disorders. Globin of haemoglobin A is made up of 2 alphaand 2 beta chains, synthesis of alpha chains iscontrolled by 2 gene clusters on chromosome 16and of beta chains on chromosome 11. Each globin gene has 3 exons and 2 introns.
    6. 6.  According to deficient globin chain Alpha thalassemia Beta thalassemia Delta-beta thalassemia Gamma delta beta thalassemia
    7. 7. According to clinical severityAlpha thalassemia Silent carrier Thalassemia trait HbH disease Hb Barts Hydrops foetalis syndromeBeta thalassemia Thalassemia major Thalassemia intermedia Thalassemia minor
    8. 8. αα αααα/αααα/-ααα/--Normal--/-α--/--
    9. 9.  Beta o thalassemias Complete absence of beta chain synthesis Beta + thalassemias Reduced synthesis
    10. 10.  α chains of globin are not/partlysynthesized. It is required for both HbA and HbF . Majority of α thalassemia cases result fromgene deletions.
    11. 11.  Most cases of α thalassemia result from genedeletion Other –1) Mutation which cause aberrant splicing2) Mutation of chain terminator codon3) Mutation which cause instability of α globinchain after translation.
    12. 12.  Deletion of all 4 genes. Intrauterine death of such a baby or ifborn, dies wihin first 2 hour. Baby is pale and bloated ; placenta isoedamatous ; moderate to massivehepatomegaly. Hb barts’ ( free ϒ 4 chains ) has high affinity foroxygen and therefore , oxygen does notdissociate from ϒ 4 resulting in sever tissuehypoxia and foetal death.
    13. 13.  Severe anisopoikilocytosis Microcytosis ErythroblastosisPeripheral smear
    14. 14.  --/-alpha Anemia, Hb -6-10gm/dl Reticulocyte count 4- 15 % Icterus and hepatosplenomegaly Lab findings Anisopoikilocytosis Hypochromia Microcytosis Target cells Inclusions bodies
    15. 15. Hemoglobin H disease. This blood film demonstrates microcytosis, hypochromasia, andnumerous morphologic abnormalities, including target cells, microspherocytes, andfragments. Basophilic stippling may occur.
    16. 16. Principle Hb H (b4) is an unstable hemoglobincommonly seen in a-thalassemia. Onincubation with some oxidative chemicalssuch as brilliant cresyl blue (BCB), HbH isoxidised, denatured and precipitated in theerythrocytes and seen as small, evenly-distributed, intra-erythrocytic blue dotswhich termed HbH inclusion bodies.
    17. 17.  Inclusion bodies
    18. 18.  Hb elctrophoresis demonstrates fast movingHbH band in the range of 5-35 %. HbH also demonstrate on HPLC.
    19. 19.  Α heterozygous cases 1 or 2 gene deletions.Clinically normal Hb 9-12 g/dl MCV ↓ MCH ↓ Mild microcytosis and hypochromia HbH Hb bart : not demonstrable Confirmation by DNA analysis.
    20. 20. BETA THALASSEMIA
    21. 21. 1) Mutations which affect transcription2) Mutation that affect splicing of RNA3) Mutations affecting consensus sequences4) Polyadenylation mutations5) Mutations which lead to the formation of thechain termination codon6) Frame-shift mutations7) Deletions
    22. 22.  Intron 1 position 5 (G-C) 619 base pair deletion Intron 1 position 1 (G-T) Frame shift mutation in codon 41 – 42(-CTTT) Codon 15 (G-A)
    23. 23.  Beta thalassemia major was first described by aDetroit pediatrician, Thomas Cooley, in 1925. Also known as Cooleys anemia It is the homozygous form of β 0 / β 0 or β +/β + or double heterozygous β 0 / β +. Infant are well at birth but develop moderate tosever anemia, failure tothrive, hepatosplenomegaly and bone changeswhich are prominent in face.
    24. 24.  Accumulation of free alpha chains Extravascular hemolysis Marrow and bone changes Extramedullary hemopoiesis Synthesis of HbF Iron overload
    25. 25.  AGE :1) Present within first year of life, at birthasymptomatic and after 3 month anemiadevelops.2) Infant may present with failure tothrive, intermittent infections and poor feeding. PALLOR ( progressive increase ) SPLENOMEGALY ( Hemosiderosis andhyperfunction of spleen)
    26. 26.  FACE : frontal bossing ( cranial bone thickening), overgrowth of zygomatic bone. JAUNDICE: mild BONE CHANGES : X ray demonstrates- expansionof diploe, hair on end appearance.
    27. 27. β-Thalassemia facial bone abnormalities.These changes include bossing of theskull; hypertrophy of themaxilla, exposing the upper teeth;depression of nasal bridge; andperiorbital puffinessβ-Thalassemia major. Note thepallor, short stature, massivehepatosplenomegaly,and wasted limbs in thisundertransfused case of β-thalassemia major
    28. 28. β-Thalassemia boneabnormalities. Note the “hair onend” appearance of the corticalbone caused by expansion of thebone marrow (arrows). Thesubperiosteal bone grows in radiatingstriations, which appears as“hairs.”
    29. 29.  Growth is retarded and delayed puberty. Increase susceptibility to infections. CARDIAC CHANGES : Myocardial hemosiderosisdevelops especially in transfused patients.Arrhythmias and congestive cardiac failuresupervene.
    30. 30.  ENDOCRINE SYSTEM :1) Growth hormone deficiency2) Hypothyrodism3) Hypoparathyrodism4) Diabetes mellitus
    31. 31.  Microcytic hypochromicanemia , basophilicstippling , markedanisopoikilocytosis, Target cells Reticulocytecount;mildly increased Leucocyte ;increased, Platelet ;normal Hb 3- 8 g/dl MCV= <70fl MCHC=(22to 30g/dl) MCH=(20 -28pg) S.iron(>200µg/dl), s.ferritin –markedly increased Transferrin saturationincreased, TIBC –Normal or redused
    32. 32. • Thalassemias• Smear Characteristics– Hypochromia– Microcytosis– Target Cells– Tear Drops
    33. 33. Basophilic stippling in thalassemia. Peripheral blood film demonstratingmicrocytic hypochromic RBCs and basophilic stippling (arrows). Basophilicstippling occurs inthalassemia as well as in other hematologic disorders.
    34. 34.  Hypercellular Erythroid hyperplasia is marked Erythropoisis is normoblastic M:E ratio 1:5 Dyserythropoisis Myelopoisis and megakaryopoisis are normal Bone marrow iron increased
    35. 35. bone marrow aspirate
    36. 36. bone marrow biopsy
    37. 37.  NESTROFT, a rapid, simple and cost effectivescreening test. The principle of NESTROFT isbased on the limit of hypotonicity which the redcell can withstand. In this procedure 2 ml of 0.36%buffered saline is taken in a test tube, 20ml ofwhole blood is added to it, and is allowed to standat room temperature. After 20 minutes reading istaken on a NESTROFT stand on which a thinblack line is marked. Positive test is due to thereduced osmotic fragility of red cells.Naked Eye Single Tube Red Cell Osmotic FragilityTest (NESTROFT)
    38. 38.  Hb F ↑ : the levels are higher in β zero then in βplus thalassemia. There are various methodmethod for estimation of HbF. The commonly used method is betke method :Principle : Fetal hemoglobin (HbF) is moreresistant to denaturation in alkaline solution thanadult hemoglobin (HbA). Alkali converts HbA toalkaline hematin. Alkaline hematin is insolubleand precipitates. HbF is quantitated by measuring the hemoglobinconcentration before and after denaturation.
    39. 39.  For higher level of HbF, method of Jonxis andvisser can be used. In this method rate of alkalidenaturation is measured in spectrophotometerand extraploated back to zero time to get theamount of HbF. Other method are radioimmunoassay and highperformance liquid chromatography.
    40. 40.  Principle-The term electrophoresis describesthe migration of a charged particle under theinfluence of an electric field. Differenthaemoglobin have different net charge becauseof variation in their structure. Under the influence of an electric field thesecharged particles will migrate either to thecathode or to the anode, depending on thenature of their net charge.
    41. 41.  Separation of haemoglobins withelectrophoresis at pH 8.4 (alkaline) and pH 6.2(acid). Scanning allows quantification of thehemoglobin present, bands are seen bystaining. At alkaline pH Hb C, E, A2 and O migratetogether to form a single band, Hb S, D and Galso co migrate.
    42. 42.  At acid pH Hb C separates from E and O andHb S separates from D and G. Hb E and O cannot be separated byelectrophoresis neither can Hb D and G.
    43. 43. Hemolysate preparation• Centrifuge EDTA blood at 3000-5000 rpm andremove plasma• Wash packed red cell with NSS for three timeand remove supernatant as much as possibleat the last washing round• Add DW 1.5 time the volume of PRC and mixvigorously• Add CCl4 to the half of the volume of lysedred cells and mix vigorously• Centrifuge 3000 -5000 rpm and collect theupper red portion which is “Hemolysate orHemoglobin solution)
    44. 44. Hemoglobin electrophoresis at alkali pHHb: Amphoteric molecule• Molecular net charge depends on pH of themedium.• pH > pI (Iso-electric point) : Molecular netcharge is negative.• pH < pI : Molecular net charge is positive.• pI (Iso-electric point) is the pH wheremolecular net charge of hemoglobin is zero.
    45. 45. Principle• In alkali medium, Hbs will gain negative netcharge.• Different Hbs have different molecularnegative net charge.• Being placed between cathode andanode, Hbs will move away from the anode.• The velocity of the movement depends solelyon the molecular net charge.• Pattern from cathode to anode is :A2/E, F, A, Bart’s, H
    46. 46. Reagent :Tris-EDTA-Borate (TBE)pH 8.4-8.6
    47. 47. Equipment• . Power supply for 500 V• . Electrophoretic chamber• . Cellulose acetate plate• . Sample applicator• . Stain box• . Large filter paper orblotter
    48. 48. EquipmentSample preparation well Aligning baseSample applicator
    49. 49. EquipmentBlotterCellulose acetate plate
    50. 50. EquipmentPower supplyElectrophoreticchamberCellulose acetate plate
    51. 51. Procedure• Hemolysate in wells• Serum applicator dipped and appliedon soaked cellulose acetate plate• Place cellulose acetate, face-down, inelectrophoretic chamber.• Run elctophoresis at 3 volts for 1 -2min.• Stained with Ponceau S
    52. 52.  Dip cellulose acetate plate in the stain andleave for 5 min Wash with destaining solution (5% HOAc)twice and 5 min each time or untilbackground becomes white Read Hb bands
    53. 53. Alkaline pHAcidic pH
    54. 54.  Positively charge molecules (salt andhemoglobin) bind to the carboxyl groups. Haemoglobin molecules are bound anddisplaced by increasing salt concentration. Haemoglobin variants separate out due tovariation in charge.
    55. 55. Principle Hb is amphoteric molecule and changes netcharge according to pH of medium. If pH < PI, net charge becomes positive (cation) and different Hbs have different positivecharge. HPLC separation of Hbs is based on cationexchange chromatography Stationary phase is negatively charged byfunctional group, e.g. polyaspatic acid. Mobile phase is buffer with pH lower than pIof Hbs Order of Hbs : Bart’s, H, F, A, A2/E accordingto RT
    56. 56. Normal or -thal trait -thal trait
    57. 57. Homo EHbE trait
    58. 58. Hb H disease in newborn HbE/ -thalassemia
    59. 59.  Indicated when the hemoglobinopathy notconfirmed by other methods or when theunderlying mutation important tomanagement. These are of value in predicting the severity ofdisease.. For genetic counseling defining the particularmutation or deletion is often required – this isachieved by a variety of molecular techniques.
    60. 60.  It is helpful when electrophoretic and otherusual haematological studies fail to diagnose. It demonstrate α : β ratio. Normal ratio is about1.0. It is redused in alpha thalassemia and increasedin beta thalassemia
    61. 61.  Clinical spectrum between thalassemia traitand thalassemia major. This include cases of interaction of β,α, HbE, Hb D and Hb S genes. Present in the later age ( 2-5 yr )
    62. 62.  Mild to moderate anemia Mild to moderate splenomegaly Mild skeletal and facial changes. Iron overload Recurrent leg ulcer Repeated infectionThalassemia intermedia
    63. 63.  Mild degree ofanemia Red cell count isincreased MCV<70 fl MCH<25 pg MCHC isreduced Hb 6- 9 gm/dl Reticulocyte count ( 2-5%)and S. bilirubin are slightlyraised HbF 10-30%, H bA2 < 4% Moderate degree ofanisopoikilocytosis,microcytic hypochromic,target cells,basophilic stippling
    64. 64.  Heterozygous carrier state characterized bylittle or no anemia but prominentmorphological changes of red cells
    65. 65.  Mild degree of anemia Red cell count is incrased MCV<70 fl MCH<25 pg MCHC is normal Hb >9.0 gm/dl Reticulocyte count and S. bilirubin are slightlyraised
    66. 66. MICROCYTOSISHYPOCHROMIAANISOPOIKILOCYTO-SISTEAR DROP CELLBASOPHILICSTIPPLINGTARGET CELL
    67. 67.  Bone marrow is cellular with erythroidhyperplasia. Osmotic fragility test shows resistance tohemolysis. Elevation of HbA2. HbF may be mildly increased
    68. 68. ANEMIAS ANISOCYTOSISPOIKILOCYTOSISBASOPHILICSTIPPLINGTARGET CELL DIMORPHISMIRONDEFICIENCYANEMIA1-3+ 0 ± ±ANEMIA OFCHRONICDISORDER± 0 ± ±THALASSEMIAMINORMAJOR±3+2+3+5%3+00Hb C OR E 2+ ± 50% 0
    69. 69. Serum iron decrease normal DecreaseironStoragedecrease N/increase Increase/NTIBC increase normal DecreaseOsmotic fragility decrease decrease _Bone marrow Decrease iron staining ErythriodhyperplasiaNormal morphologyelectrophoresis - HbFHbA2-IRON DEFICIENCYANEMIATHALASSEMIA ANEMIA OF CHRONICDISEASE
    70. 70.  Minor thalassemia :Alpha (Hb electrophoresis ) betadelta-beta Anemia of chronic disease (in late stagesspecially in renal disease )Anemia with normal RDW
    71. 71.  Iron deficiency anemia Beta thalassemia major & intermedia Sickle thalassemia Hb H disease Red cell Fragmentation syndromeAnemia with high RDW
    72. 72. MENTZER INDEX(M.I)= <13 SEEN IN THALASSEMIA AND >13 IN IRONDEFICIENCY ANEMIA M.I=MCVRED CELL COUNT
    73. 73. KERMAN INDEX 1:(MCV*MCH/RBC ) <250 : Minor thalassemia =>check Hb elect. 251-320: Mixed iron def. & minor thalassemia=> trial of iron & folate then check CBC & Hb elect 321-370: iron def.=> trial of iron for 1 mo. >371: normal Sensitivity =99% , Specificity=86%
    74. 74. KERMAN INDEX 2: MCV*MCH/RBC*MCHC<8 : Minor thalassemia8-10.5: Mixed iron def & minor thal.10.5-13: Iron deficiency>13: NormalNote : Sensitivity=99% , Specificity=93%
    75. 75.  Hb S – Thalassaemia Hb E – Thalassaemia Hb D – Thalassaemia HPFH – Hereditary persistence of foetalhemoglobin
    76. 76.  Double heterozygote state of Hb S and βthalassemia. Clinical feature - Mild growth retardation ,pallor and splenomegaly . Hematological feature – microcytichypochromic red cells, basophilic stippling andtarget cells are present. MCV and MCH ↓ Hb F ↑ Hb A, Hb F and Hb S are demonstrated by Hbelectrophoresis, Sickling and HPLC.
    77. 77.  Two forms Sickle cell Beta 0 thalassemia Sickle cell Beta + thalassemia
    78. 78.  There is interection of Hb D and β –thalassemia genes. Electrophoresis demonstrates Hb A, Hb F andHb D.
    79. 79.  Incrase Hb F production in adult life. Heterozygote have 20-30 % Hb F and inhomozygous 90 – 95 %.
    80. 80.  Health education Carrier screening and genetic counselling Prenatal diagnosis.Commonly employed method for screening :• Red cell indices• Single tube osmotic fragility test• Estimation of Hb A2• Haemoglobin electrophoresis at alkaline pH• Estimation of Hb F and Hb H inclusion.
    81. 81.  Red Cell Studies : CBC, One- Tube OFTest, DCIP Test Hb Studies : Electrophoresis, Microcolumnchromatography, Alkali DenaturationTest, HPLC/LPLC, ImnunologicDetection, Acid elution test DNA studies : Gene mapping, PCR, Ntsequencing, RFLP analysis
    82. 82. CLINICALFEATURET.MAJOR T.INTERMEDIA T.MINORGROWTH,DEVELOPMENTimpairedSPLENOMEGALY ++++ ++SKELETAL CHANGE,THALASSEMICFACIES++++++++++Hb <7 7-10 >10RED CELL COUNT 2-4 X 10¹² 3-4.5 X10¹² >5 x 10¹²BASOPHILICSTIPPLING++ + +TARGET CELL +++ ++ +ANISOPOIKILOCYTOSIS+++ ++ ±B.M.IRON ++++ ++ ±HbF 30-90 10-30 0-5HbA2 <4 <4 4-8MICROCYTOSIS +++ ++ +HYPOCROMIA +++ ++ +

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