1. Tabuk University Faculty of Applied Medical Sciences Department Of Medical Lab. Technology 3 rd Year – Level 5 – AY 1434-1435 1

2. ACUTE MYELOID LEUKEMIA (AML) By/ Mr. Waqqas Elaas; M.Sc; MLT 2

3. Objectives Define AML and recognize the different subtypes. Recognize the common clinical presentation of a patient with AML. Identify various prognostic factors in patients with AML. Understand the different complications that are a result of the disease. Classify AML. Cite lab. methods for diagnosing AML. 3

4. What are Leukemias? Leuko = Leuco = white cells Aemia = related to blood accumulation malignant white cellsThe leukaemias are a group of disorders characterized by the accumulation of malignant white cells in the bone marrow and blood. These abnormal cells cause symptoms because of: (i) bone marrow failure (i.e. anaemia, neutropenia, thrombocytopenia) and (ii) infiltration of organs (e.g. liver, spleen, lymph nodes, meninges, brain, skin or testes). 4

5. Classification According to cell type with regard to both cell maturity and cell lineage. –Chronic More mature cells Usually occurs in adults Clinical onset is gradual Anemia and thrombocytopenia are mild WBC is increased Organomegaly is prominent 5

6. Classification –Acute Immature cells Occurs in all ages Clinical onset is sudden Anemia and thrombocytopenia are mild to severe WBC is variable Organomegaly is mild 6

7. Cell lineage Lymphoid Myeloid 7

8. Leukemias Acute Chronic AML ALL CML CLL Classification 8

9. Acute leukaemia the presence of over 20% of blast cellsAcute leukaemia is defined as the presence of over 20% of blast cells in the blood or bone marrow at clinical presentation. It can be diagnosed with even less than 20% blasts if specific leukaemia-associated cytogenetic or molecular genetic abnormalities are present. myeloblasts lymphoblasts It is further subdivided into acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) on the basis of whether the blasts are shown to be myeloblasts or lymphoblasts. 9

10. AML MyeloblastsAML is a cancer of the bone marrow characterized by the accumulation of Myeloblasts in the bone marrow and blood, usually with more than 20% of nucleated cells. AML causes bone marrow to produce too many immature white blood cells (blast cells). This Suppresses normal blood cell production.(Anemia, leucopenia, thrombocytopenia) AML occurs in all age groups. in adultsIt is the common form of acute leukemia in adults. Generally is a disease of older people and is uncommon before the age of 40. Slightly more common among men than women. 10

11. Myeloid maturation myeloblastpromyelocytemyelocytemetamyelocytebandneutrophilMATURATION Adapted and modified from U Va website

12. Aetiology (causes) Idiopathic (most). Several genotoxic agents have been linked to AML (1-2% of causes) –Ionizing radiation, benzene, atomic bomb exposure, cigarette smoking. AML secondary to prior chemotherapy (5-10%) : e.g.: –Alkylating agents –Topoisomerase II inhibitors Secondary to congenital defects (rare) : e.g.: –Down syndrome - trisomy 21 –Fanconi anemia - defect in DNA repair 12

13. Clinical features Clinical features are a result of the following: Bone marrow failure : anaemia (pallor = شحوب, lethargy = كسل); neutropenia (fever, malaise=تعب, infections of mouth, throat, skin, respiratory or others ); and thrombocytopenia (spontaneous bruises=رضوض, pupura=نزف تحت الجلد, bleeding gums and menorrhagia=دورة شهرية كثيفة). Organ infiltration : Tender bones=غضة, lymphadenopathy=تضخم الغدد اللمفاوية, moderate splenomegaly, hepatomegaly and meningeal syndrome (headache, nausea and vomiting, blurring of vision=تشويش and diplopia=رؤية مزدوجة ). 13

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15. Classification FAB (French-American-British) scheme, which divides AML into eight variants; according to morphology & cytochemistry. This is the old classification. The World Health Organization (WHO) classification attempts to be more clinically useful and to produce more meaningful prognostic information than the FAB criteria. Each of the WHO categories contains numerous descriptive sub-categories. 15

16. FAB classification of AML M0— undifferentiated blasts 3 - 30% blasts, – No azurophilic granules M1—blasts Without maturation More than 90% cells are myeloblasts - MPO+, SBB+ +8 frequently seen M2—blasts With maturation 30 - 89% are myeloblasts More than 10% maturing myeloids (promyelocyte and beyond) M3—PromyelocyticMore than 30% hypergranular promyelocytes Auer rods – DIC - t(15:17) is diagnostic M4—MyelomonocyticMore than 30% blast (myeloblasts, monoblasts and promonocytes) More than 20% monocytic elements (NSE+) More than 20% myeloid elements (MPO+, SBB+) M5 M5a—Monoblastic (more than 80% monoblasts) M5b—Monocytic (less than 80% monoblasts) More than 30% blast (myeloblasts, monoblasts and promonocytes) More than 80% NSE+ : monocytic elements Less than 20% MPO+, SBB+ : myeloid elements M6—Erythroid M6a—Erythroleukemia M6b—Pure erythroid leukemia More than 50% of nucleated marrow cells are erythroid precursors More than 30% of non erythroid elements are myeloid blasts PAS+ M7—MegakaryocyticMore than 30% blasts of megakaryocytic lineage – assoc. with fibrosis 16

17. 1. CBC 2. Blood film 3. Bone marrow 4. Biochemical tests (not diagnostic) 5. Cytochemistry 6. Immunophenotype/Flow cytometry 7. Cytogenetic analysis 17

18. Laboratory diagnosis 1.CBC : TWBCs : may be decreased, normal or increased to 200 x 10 9 /L or more. Normocytic normochromic anemia Thrombocytopenia. Blood film examination blast cells Auer Rods 2. Blood film examination shows a variable numbers of blast cells. These are immature precursors, with large size, and primitive nuclei (ie the nuclei contain nucleoli), sometimes (in M3); Auer Rods are seen. (it is difficult to differentiate under the microscope between cells of AML & ALL ) Bone marrow examination 3. Bone marrow examination : hypercellular with >20% leukaemic blasts. Biochemical tests 4. Biochemical tests may reveal a raised serum uric acid, serum lactate dehydrogenase (LDH) or, less commonly, hypercalcaemia. 18

19. AML 19

20. Auer rods are clumps of azurophilic granular material that form elongated needles seen in the cytoplasm of leukemic blasts. Auer rods result from the coalescenceالتحام/اندماج of primary granules 20

21. Auer rods in AML 21

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24. Laboratory diagnosis (cont.) 5. Cytochemistry: Non-specific esterase stain is used to identify a monocytic component in AMLs. TdT :Terminal Deoxynucleotidyl Transferase, is an enzyme marker for primitive lymphoid cells 24

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26. Laboratory diagnosis (cont.) 6. Immunophenotype/Flow cytometry Immunophenotyping is a technique used to study an Ag expressed by cells. It involves the labeling of white blood cells with antibodies directed against surface proteins on their membrane. The labelled cells are processed in a flow cytometer, a laser-based instrument capable of analyzing thousands of cells per second.flow cytometer Cluster of differentiation (CD): is a protocol used for the identification and investigation of cell surface molecules present on white blood cells, providing targets for immunophenotyping of cells. (also called cell markers). Common positive cell markers in AML are : CD13, CD33, CD34 26

27. Immunophenotype (cont.) Flow cytometry –Fast and accurate way to identify, quantify and determine lineage –Physical properties Forward scatter--cell size Side scatter--cytoplasmic granularity –Cells can be stained with fluorescently labeled antibodies that recognize cell markers CD34 –Stem cell marker CD117, CD33, CD13, MPO –Myeloid markers CD14, CD64 –Monocytic marker Glycophorin A –Erythroid marker CD41, CD61 –Megakaryocytic markers 27

28. Laboratory diagnosis (cont.) 7. Cytogenetic analysis:(analysis of chromosomal abnormalities) t(15;17)(q22,q12) t(8;21)(q22,q22) inv(16)(p13,q12) 11q23 ** Acute promyelocytic leukemia (M3) is frequently associated with disseminated intravascular coagulation (DIC = pathological activation of coagulation); and so laboratory findings of DIC may seen (including : Thrombocytopenia, prolonged PT & PTT, low fibrinogen level, high FDPs, D-dimer) 28

29. Homework What can you do to reduce the risk of leukemia? A 15 year old male had flu-like symptoms with a severe sore throat for two weeks prior to admission. On physical Exam; he showed cervical and axillary adenopathy. No other organomegaly. His CBC showed : RBCs: 3.2 x 10 12 /L, HB: 9.9 g/dL, MCV: 90.5 fL (normal), WBC :42.6 x 10 9 /L, Lymphocytes :12 %, Blast cells : 88%, PLT 22 x 10 9 /L. 1.Is it possible that the patient had an acute infection? Why? 2.If the patient had leukemia, is it ALL or AML? Why? 29