1. Tabuk University Faculty of Applied Medical Sciences Department Of Medical Lab. Technology 3 rd Year – Level 5 – AY 1434-14345 1

2. Acute Lymphoblastic Leukemia (ALL) By/ Mr. Waqqas Elaas; M.Sc; MLT 2

3. Objectives Define ALL, and know the causes. Describe clinical signs and symptoms of ALL Classify ALL Explain the prognostic significance of cytogenetic abnormalities Cite methods for diagnosing AL L 3

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5. Hematopoietic stem cell Neutrophils Eosinophils Basophils Monocytes Platelets Red cells Myeloid progenitor Lymphoid progenitor B-lymphocytes T-lymphocytes Plasma cells naïve ALL AML

6. ALL lymphoblasts pancytopeniaALL: Increased production of lymphoblasts that do not mature into lymphocytes. They crowd the bone marrow resulting in pancytopenia. childhoodThe most common malignancy of childhood. Accounts for one-third of all childhood cancers. The incidence is highest at 3-7 years, falling off by 10 years with a secondary rise after the age of 40 years. The common (CDI0+) precursor B type which is most usual in children has an equal sex incidence; there is a male predominance for T-cell ALL (T-ALL). 6

7. Aetiology (causes) Unknown Genetic Predisposition : events early in life, perhaps even prenatally. –Increased incidence amongst monozygotic and dizygotic twins. –Maternal exposure to ionizing radiation. Down Syndrome. Disorder with chromosomal fragility: –Fanconi’s anemia –Bloom Syndrome Infections –HTLV1 in T cell leukemia/lymphoma –EBV in mature B cell ALL –HIV in lymphoproliferative disorders. 7

8. Classification May be on the basis of morphology or immmlological markers. The French-American-British (FAB) group subclassifies ALL into three subtypes: Subtype Morphology Occurrence (%) L1 Small round blasts 75 clumped chromatin L2Pleomorphic larger blasts 20 clefted nuclei, fine chromatin L3Large blasts, nucleoli, 5 vacuolated cytoplasm 8

9. Immunologic classification of ALL cell surface moleculesThe 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. CD molecules are utilized in cell sorting using various methods including flow cytometry. This system uses monoclonal antibodies (mAbs) generated against epitopes on the surface molecules of leucocytes. 9

10. Immunologic classification of ALL B- lineage (80%)Markers Pro-BCD19(+),Tdt(+),CD10(-),CyIg(-), CommonCD19(+),Tdt(+),CD10(+),CyIg(-), Pre-BCD19(+),Tdt(+),CD10(+),CyIg(+),SmIg(-) Mature-BCD19(+),Tdt(+),CD10(±),CyIg(±),SmIg(+) T-lineage (20%) Pre-TCD7(+), CD2(-), Tdt(+), Mature-T CD7(+), CD2(+), Tdt(+), 10 Tdt : Terminal deoxynucleotidyl transferase

11. Chromosomal/molecular abnormalities in ALL Better prognosis - normal koryotype - hyperdiploidy (having slightly more than the diploid number of chromosomes) Poor prognosis - t (8; 14) - t (4; 11) Very poor prognosis - t (9; 22); BCR/ABL (+) The most common specific abnormality in childhood ALL is the t(12; 21) translocation. 11

12. 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 ). 12

13. Laboratory diagnosis 1.CBC 1.CBC : TWBCs : may be decreased, normal or increased to 200 x 10 9 /L or more. Normocytic normochromic anemia Thrombocytopenia. Blood film examination LymphoblastsBlood film examination shows a variable numbers of Lymphoblasts. These are immature precursors, with large size, and primitive nuclei (ie the nuclei contain nucleoli). 2. Bone marrow examination 2. Bone marrow examination : hypercellular with >20% leukaemic blasts. 3. Biochemical tests 3. Biochemical tests may reveal a raised serum uric acid, serum lactate dehydrogenase (LDH) or, less commonly, hypercalcaemia. 13

14. ALL : Peripheral blood showing numerous Lymphoblasts 14

15. 15 Acute Lymphoblastic Leukemia - L1 Morphology: L1 blasts are small and homogeneous. The nuclei are round and regular with little clefting and inconspicuous nucleoli. Cytoplasm is scanty and usually without vacuoles.

16. 16 Acute Lymphoblastic Leukemia - L2 Morphology: L2 blasts are large and heterogeneous. The nuclei are irregular and often clefted. One or more, usually large nucleoli are present. The volume of cytoplasm is variable, but often abundant and may contain vacuoles.

17. 17 Acute Lymphoblastic Leukemia - L3 (Burkitt's) Morphology: L3 blasts are moderate-large in size and homogeneous. The nuclei are regular and round-oval in shape. One or more prominent nucleoli are present. The volume of cytoplasm is moderate and contains prominent vacuoles.

18. Laboratory diagnosis (cont.) 4. Cytogenetics and molecular genetics 4. Cytogenetics and molecular genetics: Cytogenetics is concerned with the study of the structure and function of the cell, especially the chromosomes. Molecular cytogenetics such as fluorescent in situ hybridization (FISH).chromosomesfluorescent in situ hybridization Cytogenetic analysis allows to profile chromosomal aberrations such as amplifications, deletions, rearrangements, point mutations & copy number changes. 18

19. Laboratory diagnosis (cont.) The most common specific abnormality in childhood ALL is the t(12; 21) translocation. Philadelphia chromosome translocation t(9; 22) increases with age. Sometimes Translocations of chromosome llq23 is found. t(15; 17) and t(8; 21) and inv(16) are favorable. 19

20. Laboratory diagnosis (cont.) 5.Cytochemistry: Blood cells contain various enzymes, fats, and other substances that can be identified by cytochemical means. ALL AML Cytochemical stains on blood and bone marrow help to distinguish the ALL from AML. myeloperoxidaseSudan black Bnonspecific esteraseacid phosphatase The most important cytochemical studies in the study of acute leukemias are myeloperoxidase (MPO), Sudan black B, nonspecific esterase (NSE), and acid phosphatase (AP). never Myeloperoxidase is an enzyme located in the granules of myeloid and monocytic cells. Myeloperoxidase is never found in lymphoid cells. If positive, is the most important marker distinguishing myeloid from lymphoid blasts. Sudan Black B is a fat-soluble dye; that stains myeloblasts but not lymphoblasts, because it stains the lipid membrane around the granules, so it Parallels MPO. 20

21. NASDA : Naphthol AS-D acetate esterase PAS : Periodic acid Schiff stain CytochemistryMorphologyDesignation PASNASDA MyeloPeroxidase Sudan black B Granul es Cytoplas m NucleoliNucleusCellsFAB ++++/--ve Scanty, moderate ly basophilic Not visible Round, homogeneous Small, uniformL1 ++++/--ve Variable One or more Irregular, nonhomogeneo us Of varying sizeL2 ++++/--ve Abundant, deeply basophilic One or more, vesicular, often prominen t Round-to-oval, homogeneous Large, uniformL3 21

22. 6. Immunophenotype/Flow cytometry 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 22

23. Home work A 69-year-old Caucasian man has an abnormal blood. White blood cell count is 75,000 with 90% mature lymphocytes and 10% neutrophils. Hemoglobin is 15.4 gms% Hematocrit 47% Platelet count 244,000/cmm Can we assume that the patient had ALL? Why? 23