Tabuk University

Faculty of Applied Medical SciencesDepartment Of Medical Lab.

Technology

3rd Year – Level 5 – AY 1434-14345

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HEMATOLOGY – 2, MLT 307

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Acute Lymphoblastic Leukemia (ALL)

By/Mr. Waqqas Elaas;

M.Sc; MLT

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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 ALL

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Hematopoieticstem cell

Neutrophils

Eosinophils

Basophils

Monocytes

Platelets

Red cells

Myeloidprogenitor

Lymphoidprogenitor

B-lymphocytes

T-lymphocytes

Plasmacells

naïve

ALL

AML

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ALL• ALL: Increased production of lymphoblasts that do not mature

into lymphocytes. They crowd the bone marrow resulting in pancytopenia.

• The 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).

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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.

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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 75clumped chromatin

L2 Pleomorphic larger blasts 20clefted nuclei, fine chromatin

L3 Large blasts, nucleoli, 5vacuolated cytoplasm

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Immunologic classification of ALL

• The 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.

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Immunologic classification of ALL

B- lineage (80%) MarkersPro-B CD19(+),Tdt(+),CD10(-),CyIg(-),Common CD19(+),Tdt(+),CD10(+),CyIg(-),Pre-B CD19(+),Tdt(+),CD10(+),CyIg(+),SmIg(-)

Mature-B CD19(+),Tdt(+),CD10(±),CyIg(±),SmIg(+)

T-lineage (20%) Pre-T CD7(+), CD2(-), Tdt(+),Mature-T CD7(+), CD2(+), Tdt(+),

Tdt : Terminal deoxynucleotidyl transferase

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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.

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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

الجلد =bleeding gums and menorrhagia ,تحت كثيفة شهرية .(دورة• Organ infiltration : Tender bones=غضة, lymphadenopathy= تضخم

اللمفاوية moderate splenomegaly, hepatomegaly and ,الغددmeningeal syndrome (headache, nausea and vomiting, blurring of vision=تشويش and diplopia= مزدوجة .(رؤية

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Laboratory diagnosis1. CBC :• TWBCs : may be decreased, normal or increased to 200 x 109/L

or more.• Normocytic normochromic anemia• Thrombocytopenia.• Blood 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 : hypercellular with >20% leukaemic blasts.

3. Biochemical tests may reveal a raised serum uric acid, serum lactate dehydrogenase (LDH) or, less commonly, hypercalcaemia.

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ALL : Peripheral blood showing numerous Lymphoblasts

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Acute Lymphoblastic Leukemia - L1Morphology: 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.

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Acute Lymphoblastic Leukemia - L2Morphology: 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.

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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.

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Laboratory diagnosis (cont.)

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).

Cytogenetic analysis allows to profile chromosomal aberrations such as amplifications, deletions, rearrangements, point mutations & copy number changes.

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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.

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Laboratory diagnosis (cont.)5. Cytochemistry: Blood cells contain various enzymes, fats, and other substances that can be identified by cytochemical means. Cytochemical stains on blood and bone marrow help to distinguish the ALL from AML. The most important cytochemical studies in the study of acute leukemias are myeloperoxidase (MPO), Sudan black B, nonspecific esterase (NSE), and acid phosphatase (AP). 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.

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NASDA : Naphthol AS-D acetate esterase PAS : Periodic acid Schiff stain

Cytochemistry Morphology DesignationPAS NASDA MyeloPeroxidase

Sudan black BGranul

esCytoplas

mNucleoli Nucleus Cells FAB

+++ +/- -ve -ve Scanty,moderate

ly basophilic

Not visible

Round,homogeneous

Small, uniform L1

+++ +/- -ve -ve Variable One or more

Irregular,nonhomogeneo

usOf varying size L2

+++ +/- -ve -ve Abundant,

deeply basophilic

One or more,

vesicular,often

prominent

Round-to-oval,homogeneous

Large, uniform L3

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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

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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?