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  1. 1. Leukemia(FAB/WHO)
  2. 2. FM has Leukemia
  3. 3. CANCER
  4. 4. DEFINE…Leukemia is defined as:A. Benign disease of hematopoietic tissue characterized by replacement of abnormal bone marrow elements with normal cellsB. Benign disease of hematopoietic tissue characterized by replacement of normal bone marrow elements with abnormal cellsC. Malignant disease of hematopoietic tissue characterized by replacement of abnormal bone marrow elements with normal cellsD. Malignant disease of hematopoietic tissue characterized by replacement of normal bone marrow elements with abnormal cells
  5. 5. TRUE or FALSE Leukemic cells look different than normal cells and do not function properly.
  6. 6. 4 TYPES of LEUKEMIA1. Acute Myelogenous (granulocytic) Leukemia (AML)2. Chronic Myelogenous (granulocytic) Leukemia (CML)3. Acute Lymphocytic (lymphoblastic) Leukemia (ALL)4. Chronic Lymphocytic Leukemia (CLL)
  7. 7. Acute vs Chronic Leukemia Acute ChronicAge Children & young Middle age and adults elderlyOnset Sudden insidiousDuration weeks to months yearsWBC count Variable High
  8. 8. Acute vs Chronic Leukemia Acute ChronicPlatelets Low Early: Normal/ High Late: LowAnemia High (>90%) None/ mildPredomi- Blast cells Mature cellsnant cells AML = myeloblast CML=granulocytes ALL= lymphoblast CLL=lymphocytes
  9. 9. Acute vs Chronic Leukemia Acute ChronicMarrow >20% marrow >70% marrowcellularity blasts (WHO) cellularity > 30% marrow (hypercellular); blasts (FAB) No dysplasiaDiagnosis PBS, BM exam, PBS (peripheral cytochemical blood smear) stains, surface markers, EM,chromosome
  10. 10. TRUE or FALSE Blastsare immature blood cells that cannot carry out the normal function of mature cells.
  11. 11. TRUE or FALSE Inacute leukemia, blasts are relatively more mature and they can carry out some normal functions.
  12. 12. TRUE or FALSE When leukemia affects myeloid cells, it is called lymphocytic leukemia.
  13. 13. Acute Leukemia
  14. 14. TRUE or FALSE Acute lymphocytic leukemia (ALL) is the most common type of leukemia in young children and it never affects adults.
  15. 15. AML vs CML
  16. 16. Acute vs Chronic Leukemia
  17. 17. Identify….AML or Normal bone marrow
  18. 18. ALL or Normal cells
  19. 19. Identify….CLL or Normal cells
  20. 20. Identify…. AML or CML
  21. 21. Identify… ALL or Normal cells
  22. 22. Identify… ALL or CLL
  23. 23. Acute Myelogenous Leukemia(AML) acutenonlymphocytic leukemia (ANLL) Common form of adult leukemia AML blasts do not mature Neutropenia, anemia, thrombocytopenia Variable WBC count Bone marrow blasts >20% (WHO) Bone marrow blasts > 30% (FAB)
  24. 24. TRUE or FALSE Acute myeloid leukemia (AML) is sometimes called acute nonlymphocytic leukemia (ANLL).
  25. 25. What % of blasts is recommended by the FAB classification system for a diagnosis of acute leukemia?A. 10%B. 20%C. 30%D. 50%
  26. 26. AML (+) Bb. Thomson
  27. 27. Peripheral Blood Blast Cell
  28. 28. Normal bone marrow
  29. 29. FAB vs WHO Classification French-American-British (FAB) Cx  Cellular morphology and cytochemical stain  Acute leukemia as > 30% bone marrow blasts  Widely used World Health Organization Cx  Cellular morphology and cytochemical stain  Immunologic probes of cell markers, cytogenetics, molecular abnormalities & clinical syndrome  Acute leukemia as > 20% bone marrow blasts  Standard for diagnosis
  30. 30. Acute myeloid leukemias(AML) Classification - FAB1. M0: minimally differentiated2. M1: myeloblastic leukemia without maturation3. M2: myeloblastic leukemia with maturation4. M3: hypergranular promyelocytic leukemia5. M4: myelomonocytic leukemia6. M4Eo: variant, increase in marrow eosinophils7. M5: monocytic leukemia8. M6: erythroleukemia (DiGuglielmos disease)9. M7: megakaryoblastic leukemia
  31. 31. AML classification - WHOAML not otherwise categorized1. AML minimally differentiated2. AML without maturation3. AML with maturation4. Acute myelomonocytic leukemia5. Acute monocytic leukemia6. Acute erythroid leukemia7. Acute megakaryocytic leukemia8. Acute basophilic leukemia9. Acute panmyelosis with myelofibrosis
  33. 33. AML
  34. 34. Acute Nonlymphocytic Leukemias
  35. 35. CYTOCHEMICAL STAIN Alpha napthyl acetate Periodic Acid Napthol AS-D Schiff (PAS)Myeloperoxidase Alpha napthyl Chloroacetate Sudan Black B butyrate (ALL, M5, Esterase (M1-M5) (M4, M5 ) M6,M7) (M1-M4) (M6,M7 + Napthylacetate
  36. 36. Chromosomal Leukemia Abnormalityt(8;21) M2t(15;17) M3inv, del, t(16q) M4t(9;11) M5 (M5a); M4
  37. 37. MO: Minimally differentiated Undifferentiated Blasts (No maturation) Myeloid phenotype - CD13, CD33, CD34 (-) SBB, MPO Negative: Auer rods, Esterase
  38. 38. M1 AML without maturation > 30% myeloblasts Large cells, round nucleus Nucleoli (+) scanty cytoplasm >3% MPO, SBB (+) <20% NSE (+) CD 13, 33, 117
  39. 39. M1 AML without maturation
  40. 40. Which FAB classification shows 3% (+) MPO, SBB, shows <20% (+) with nonspecific esterase and contains primarily myelobalst with distinct nucleoli?A. M1B. M2C. M3D. M4
  41. 41. M2 AML with maturation Common type >30% myeloblasts >10% granulocyte Kidney shape nucleus Nucleoli (+) (+) Auer rods Eosinophilic granules >50% MPO, SBB (+) CD 13, 33
  42. 42. M2 AML with maturation
  43. 43. Auer Rods
  44. 44. Promyelocyte constitute 10% of acute leukemia with >50% of leukemic cells positive for peroxidase and Sudan black B. What is the FAB classification?A. M1B. M2C. M3D. M4
  45. 45. M3 (hypergranular promyelocytic) Promyelocyte-predominant Large, kidney shape (+) Auer rods (faggot cells) basophilic, bilobed nuclei CD 13,33 High incidence of DIC
  46. 46. Acute myeloid leukemia with veryabnormal cells (AML M3/ t15;17)
  47. 47. M4 Acute myelomonocytic >30% myeloblast (FAB) >20% granulocyte >20% promonocytes and monocytes CD 11, 13, 33,14 (+) Auer rods common High serum lysozyme level  M4Eo = w/ eosinophilia
  48. 48. The serum lysozyme level was greatly increased in this patient. Cells are positive for CD11 and CD14 by flow cytometry. Which disease is most likely?A. Acute myeloblastic leukemiaB. Promyelocytic leukemiaC. Myelomonocytic leukemiaD. Undifferentiated leukemia
  49. 49. M5: acute monocytic leukemia1. M5a – without maturation  Monoblasts , few promonocytes1. M5b – with maturation  Blast, Promonocytes (BM), Monocytes (Blood)
  50. 50. M5a Monoblast ameboid with round to oval nuclei, prominent nucleoli, <20% promonocytes/mono Vacuolated cytoplasm
  51. 51. AML M5a
  52. 52. M5b > 20% promonocytes, monocytes Promonocytes folded, convulated nucleus Azurophilic granules
  53. 53. AML M5b
  54. 54. M6 - erythroleukemia Large, bizarre, round-to-oval cells (+) nucleoli > 50% Erythroblasts > 30 % Myeloblasts CD 45,71 Glycophorin A CD 13, 15,33 myeloblast PAS (+)
  55. 55. M6 (erythroblast)
  56. 56. M6 (erythroblast)
  57. 57. M7 – acute megakaryoblastic >30% megakaryoblasts platelet like granules on PAS stain NSE (but not BE) (+) Myeloid blasts may show SBB or MPO (+) CD 41,42,61
  58. 58. M7 (Megakaryoblast)
  59. 59. Megakaryoblast
  60. 60. ChronicMyeloproliferative Diseases (MPD)
  61. 61. Classification of chronicmyeloproliferative diseases (MPD)FAB WHO1. Chronic myelogenous CML Ph+: t(9;22)(qq34;q11), leukemia (CML) BCR/ABL Chronic neutrophilic leukemia2. Polycythemia vera (PV) Polycythemia vera (PV)3. Essential thrombocytemia (ET) Essential thrombocytemia (ET)4. Chronic Myelofibrosis Chronic idiopathic myelofibrosis
  62. 62. Myeloproliferative disease (MPD)Disorder Cell Type FeatureCML Granulocytes (Ph1), LAP/NAP, SplenomegalyPV Erythrocytes RBC, Splenomeg, Teardrop, High LAPET Platelets ThrombocytosisCMF Fibroblast NRBC, LAP, Dacrocytes
  63. 63. Chronic Myelocytic Leukemia < 5% blasts M:E ratio 10:1 Mature granulocyte all stages of maturation (+) High WBC,Splenomegaly Enlarged platelets 95% (+) Philadelphia (Ph) chromosome t(9;22) Low LAP score High LAP (leukemoid)
  64. 64. Chronic Myeloid Leukemia
  65. 65. Leukemoid CML reactionWBC High HighAnemia (-) (+)PBS Shift to the Left Shift to the left (blast) Toxic granulation Eosinophilia, Dohle bodies basophiliaLAP score High LowPhiladelphia (-) (+)chromosome
  66. 66. Chromosomal Leukemia Abnormalityt(9;22) - Philadelphia CMLchromosome
  67. 67. What is the M:E ratio in patients with chronic myelogenous leukemia?A. 1:5B. 1:10C. 10:1D. 3:1
  68. 68. What is the most common chromosomal abnormality found in chronic myelogenous leukemia?A. t(8;14)B. t(9:22)C. t(1;12)D. Trisomy 12
  69. 69. Polycythemia Vera Malignant hyperplasia of myeloid stem cell Increase erythrocytes High blood viscosity (BP)  Secondary polycythemia- High RBC (high EPO)  Normal: Plasma volume, WBC, Platelet ormal  Relative (pseudo) polycythemia  High Hgb  Normal EPO,RBC mass, WBC, platelet
  70. 70. Essential Thrombocythemia Proliferationmegakaryocytes (adults) High platelet count, giant form Platelet function abnormalities Leukocytosis Usually without hepatomegaly and splenomegaly
  71. 71. Chronic myelofibrosis Myeloid stem cell disorder Proliferation of erythroid, granuclocytic, megakaryotic precursor in marrow Marrow fibrosis Immature neutrophils, nucleated RBC 5% blasts, anemia, marked poikilocytosis, nucleated RBC Bleeding, hepatosplenomegaly
  72. 72. A peripheral blood stained smear showed 5% blasts, anemia, poikilocytosis and nucleated RBC. What condition should be suspected?A. Chronic myelogenous leukemiaB. Acute myelogenous leukemiaC. Thalassemia majorD. myelofibrosis
  73. 73. MyelodysplasticSyndromes(MDS)
  74. 74. Myelodysplastic Syndromes Clonal proliferation of hematopoietic cells, including erythroid, myeloid, and megakaryocytes Blood cytopenia (anemia) 30% blasts in bone marrow (FAB) Monocytosis, ringed sideroblast, macrocytosis
  75. 75. All of the following are characteristic of myelodysplastic syndrome EXCEPT:A. LymphocytosisB. MonocytosisC. Ringed sideroblastD. macrocytosis
  76. 76. Myelodysplastic syndromes(MDS)- FAB Classification1. Refractory Anemia (less than 5% blasts)2. Refractory Anemia with Ringed Sideroblasts (RARS) (Sideroblastic anemia)3. Refractory Anemia with Excess Blasts (RAEB) (5 to 20% blasts)4. Refractory Anemia with Excess Blasts in Transformation (RAEB-T) (20 to 30% blasts)5. Chronic Myelomonocytic Leukemia (CMML) (more than 1000 monocytes/mm3)
  77. 77. Myelodysplastic syndromes (MDS) (FAB) < 5% myeloblast > 5% myeloblast RAEB-TRA RARS RAEB ( 21-30% (6-20% myeloblast myeloblast chronic myelomonocytic leukemia monocytosis (CMML) = 20% myeloblast splenomegaly
  78. 78. RARS >15% ringed sideroblast (PB)
  79. 79. RAEB-T (vacuolated blast) >20% marrow blasts <30% peripheral blast WHO- acute leukemia instead MDS FAB - CMML
  80. 80. RAEB-Tvacuolation granular & clot like
  81. 81. Refractory Anemia Not responsive to therapy Oval macrocytes, reticulocytopenia bone marrow blast <5% peripheral blast <1%
  82. 82. Chronic MyelomonocyticLeukemia (CMML) 5-20% bone marrow blast <5% peripheral blood blast Absolute monocytosis Leukocytosis
  83. 83. Refractory Anemia with excessblasts (RAEB) 5-20% bone marrow blast <5% peripheral blood blast Common cytopenias Absolute monocytosis
  84. 84. Acute lymphocyticleukemia (children)
  85. 85. Acute Lymphocytic Leukemia French-American-British (FAB) ClassificationL1 ALL, childL2 ALL, adultL3 ALL, (Burkitt)
  86. 86. WHO classification- Cytogenetics Leukemia Chromosomal AbnormalityALL-pre B t(1;19)ALL-T t(11;14)ALL-Burkitt’s lymphoma t(8;14), t(2;8), t(8;22)
  87. 87. Immunophenotyping Types FAB TdT Tc Ag Bc Ag Precursor B L1,L2 +, - - + Precursor T L1,L2 +, + + - B-cell L3 -,- - +
  88. 88. Acute Lymphocytic LeukemiaFAB Class L1 small , uniform cells scanty cytoplasm absent nucleoli 70% c-ALL 20% T-ALL rarely B-ALL or null-ALL TdT (+) PAS (+)
  89. 89. Acute Lymphocytic LeukemiaFAB Class L2 Large, heterogenous basophilic cytoplasm Nucleoli present 50% c-ALL 30% T-ALL TdT (+) PAS (+)
  90. 90. Acute Lymphocytic LeukemiaFAB Class L3 (Burkitt’s) large varied cells deeply basophilic vacuolated cytoplasm Nucleoli (+) Usually B-ALL TdT (-) PAS (+)
  91. 91. Which of the following stain/reaction show a positive result in most patient with acute lymphocytic leukemia?A. Sudan black and peroxidaseB. Chloroacetate esteraseC. Non specific esteraseD. Terminal deoxynucleotidyl transferase
  92. 92. The leukemia commonly associated with pediatric age group is:A. Acute myeloblastic leukemiaB. Acute lymphoblastic leukemiaC. Chronic lymphoblastic leukemiaD. Chronic myelocytic leukemia
  93. 93. Chronic LymphocyticLeukemia (CLL)B cell malignancy (CD19,20) Male adult Autoimmune hemolytic anemia Lymphocytosis, homogenous, small, hyperclumped lymphocytes and smudge cells neutropenia, thrombocytopenia, Ig Small lymphocytic lymphoma Lymphadenopathy, splenomegaly, hepatomegaly
  94. 94. Chronic LymphocyticLeukemia (CLL)
  95. 95. Chronic LymphocyticLeukemia (CLL)
  96. 96. Hairy Cell Leukemia (HCL) B cell malignancy (CD 19, CD20) Marked splenomegaly Pancytopenia Cytoplasm show hair like projection (+) TRAP
  97. 97. Hairy Cell Leukemia (HCL)
  98. 98. Hairy Cell Leukemia (HCL)
  99. 99. Hairy Cell Leukemia (HCL)
  100. 100. Prolymphocytic LeukemiaB cell or T cell malignancy Marked splenomegaly Lymphocytosis, prolymphocytes Anemia, thrombocytpenia
  101. 101. Plasma Cell Dyscrasia
  102. 102. Multiple Myeloma Plasma (lymphoid)/ B lymphocyte neoplasm >30% plasma cells (BM) Skeletal system tumor of plasma cell (myeloma) Monoclonal gammopathy disease Excess IgG / IgA production electrophoresis: M spike in gamma globulin Bence Jones portein (free light); kidney damage High viscosity; rouleaux
  103. 103. Multiple myeloma
  104. 104. Normal BM / MM in BM
  105. 105. Plasma Cell Leukemia Abnormal plasma cells in blood Pancytopenia Rouleaux Monoclonal gammopathy
  106. 106. Waldenstrom’smacroglobulinemia malignancy involving excess B-lymphocytes that secrete immunoglobulins Excessive IgM (viscous) No bone tumor Lyphadenopathy, hepatosplenomegaly M spike in gamma globulin region
  107. 107. Lymphomas
  108. 108. Lymphomas Malignant cells in solid lymphatic tissue Localized to BM to blood Lyphadenopathy CD, PCR, Tissue biopsy  Hodgkin  B cell  T/NK cell (non Hodgkin)  Sezary syndrome
  109. 109. Hodgkin Lymphoma EBV associated (+) Reed Strenberg cells = large multinucleated cells with large nucleoli Mild anemia, eosinophilia, monocytosis High LAP, ESR
  111. 111. Leukemia Diagnosis1. Blood Test – CBC2. Differential blood count – immature “blast”3. Hematocrit assay- proportion of RBC blood4. Hgb assay- O2 carrying pigment5. Blood coagulation- clotting6. Blood morphology and staining – cell shape, structure, nucleus (PBS)
  112. 112. Leukemia Diagnosis7. Blood chemistry  ALP = CML diagnosis  Vitamin B12= CML  Ca, K, phosphate, uric acid = ALL
  113. 113. 1. Enzyme cytochemical staining 5 blood smear/bone marrow smear Air dried Papenheim/May Grunwald Giemsa (MGG)/ MPO / NSE
  114. 114. Cytomorphology Pappenheims MPO NSE
  115. 115. Cytochemical stain1. Myeloperoxidase (MPO) stain  (+) AML (granulocyte, monocyte, Auer rods)  (-) ALL1. Sudan Black B ( phospholipids, proteins)  (+) AML (granulocyte, monocyte, Auer rods)  (-) ALL
  116. 116. Cytochemical stain3. Esterases  Specific esterase stain (napthol AS-D chloroacetate esterase stain) = granulocyte  (+) FAB M1, M2, M3, M4  (-) FAB M5  Non specific esterase stain (alpha napthyl acetate and alpha napthyl butyrate) = monocyte  (+) FAB M5  (-) FAB M1, M2, M3, M4
  117. 117. Cytochemical stain4. Periodic Acid Schiff (PAS) (+) ALL (+) FAB M6 (erythrolekemia)5. Leukocyte Alkaline Phosphatase (LAP) Low LAP score = CML High LAP score = NLR Normal LAP score= Hodgkin
  118. 118. Cytochemical stain6. Tartrate Resistant acid Phosphatase (TRAP) (+) hairy cell leukemia7. Perl’s Prussian Blue stain(+) Siderocytes with iron inclusions (siderocytic granules/pappenheimer bodies) (+) HA, beta thallasemia major, sideroblastic Anemia(+) Sideroblasts = nRBC with iron granulesRinged sideroblasts= iron encircle nucleus(+) MDS (Refractory anemia, RARS, sideroblastic anemia)
  119. 119. A positive non specific esterase stain indicates differentiation into which cell type?A. MonocyticB. LymphoidC. MegakaryocyticD. Plasmacytoid
  120. 120. Which cytochemical stain is used to detect acute myelogenous leukemia M7?A. Terminal deoxynucleotidyl transferase (TdT)B. Myeloperoxidase (MPO)C. platelet peroxidaseD. Sudan black b (SBB)
  121. 121. 2. Immunophenotyping Principle: Highly specific Ags on cell surfaces are detected by monoclonal Abs tagged with fluorescein and the complex are determined by flow cellcytometry Examination of the proteins on cell surfaces and the antibodies
  122. 122. ImmunophenotypingTypes FAB TdT Tc Ag Bc Ag c Ag s AgPrecursor L1,L2 +, - - + -/+ -BPrecursor T L1,L2 +, + + - - -B-cell L3 -,- - + + -/+ TdT = Terminal deoxynucleotidyl transferase CAg= common Ag Sag= surface antigen
  123. 123. CD markersB cell= C19, CD20 T cell = CD2, CD3, CD5 Myeloid= CD13, CD14, CD33
  124. 124. 3. Multiparameter flow cytometry EDTA heparinized BM or blood Cell lysis/ Ficoll hypaque gradiation MFC
  125. 125. 4. CYTOGENETICS test to look for certain changes of the chromosomes (genetic material) of the lymphocytes Chromosomal analysis t(9;22) = Ph1 CML t(15;17) = M3
  126. 126. CYTOGENETICS1. Culture malignant hematopoietic tissue to obtain dividing cells in the metaphase stage where the mitotic process is stopped2. Cells are fixed3. Cells are dropped into microscope slide where chromosome fall in a random pattern4. Stained with giemsa or quinicrine5. Chromosome banding (G band or Q band)
  127. 127. Cytogenetics Metaphase after G- banding in a c-ALL with t(9;22) so called Philadelphia translocation
  128. 128. 5. Fluorescence in situhybridization cyto smears of bone marrow or peripheral blood metaphases and interphase nuclei fixation 2 h time for hybridization APL detecting PML-RARA fusion signals in interphase-FISHinterphase FISH (IP-FISH), whole chromosome painting (WCP-) FISH, 24- color FISH or comparative genomic hybridization (CGH) Hybridize overnightinterphase FISH (IP-FISH), whole chromosome painting (WCP-) FISH, 24- color FISH or comparative genomic hybridization (CGH)
  129. 129. Metaphase before and after analysis by 24-color FISH in a case with AML and complex aberrant karyotype
  130. 130. 5. Molecular Mtd- PCRFicoll Hypaque density gradient centrifugation for DNA or RNA preparationsequencing or even gene expression profiling
  131. 131. end na po…