B-cell Acute lymphoblastic leukemia (B-ALL) is a neoplasm of lymphoblast which are of B-cell lineage typically composed of small to medium sized blast cells, moderately condensed to dispersed chromatin with scanty cytoplasm and inconspicuous nucleoli, involving the bone marrow and/or blood.

1) FAB classification for ALL:

Subtyping of the various TYPES of ALL is done according to the French-American-British (FAB) classification, which was used for all acute leukemia’s.

・ ALL-L1: small uniform cells, with inconspicuous nucleoli.

・ ALL-L2: heterogeneous population of blasts.

・ ALL-L3: large cells with cytoplasmic vacuoles and basophilic cytoplasm.

2) WHO 2008 classification for ALL:

The recent WHO (2008) International panel on ALL recommends that the FAB classification be abandoned, since it depends on morphology with no clinical or prognostic relevance. And the WHO advocates the use of the genetics and molecular genetics with immunophenotypic classification mentioned below.

・ 1. Acute lymphoblastic leukemia/lymphoma. Synonyms: Former FAB L1/L2.

・ i. Precursor B acute lymphoblastic leukemia/lymphoma.

・ ALL with recurrent cytogenetic abnormality:

・ t (12; 21) (p12, q22) TEL/AML-1.

・ t (1; 19) (q23; p13) PBX/E2A.

・ t (9; 22) (q34; q11) ABL/BCR.

・ T (V, 11) (V; q23) V/MLL.

・ B-ALL with hyperdiploidy.

・ B-ALL with hypodiploidy.

・ B-ALL not otherwise specified [1] .

・ Immunological classification of B-ALL.

・ Immune phenotype of B-ALL:

・ The phenotype of blasts has prognostic impact in B-ALL. Common B-ALL has the best prognosis.

・ B-ALL marker.

・ CD19/cCD79a/TdT/cCD22.

・ Divided into pro-B-ALL (early pre-B-ALL), /CD10−).

・ Common ALL (CD10+).

・ Pre B-ALL (CD10+/−/cytoplasmic IgM).

・ Mature B-ALL (CD10+/rarely, CD20+, CD22+, surface IgM+)

・ Other marker used to diagnosis of B-ALL (CD34, CD45) [1] - [6] .

Childhood acute lymphoblastic leukemia comprises different biological subtypes of acute leukemia which is defined by cell morphology, immunophenotype, and genetic abnormalities, affecting the prognosis, and the treatment protocols.

1) Early pre-ALL

The leukemia cells always express CD19 and almost all cases have cytoplasmic CD22, CD79a. CD10 and TdT are expressed in 90% of cases and CD34 in more than 75% of cases. Surface CD22 expression is seen in most cases. And blasts lack expression of surface and cytoplasmic immunoglobulin, and light chains.

2) Pre-B-ALL

The pre B immunophentype is found in approximately 20% - 25% of cases and is defined by presence of cytoplasmic immunoglobulin µ heavy chains with absence of surface immunoglobulin.

3) B-Cell ALL (Mature B-ALL)

Mature B-ALL constitutes 2% - 3% of childhood cases and blasts express surface immunoglobulin µ heavy chains plus either Ķ or λ light chains [3] .

Immunoglobulins are glycoprotein molecules which function as antibodies that are produced by plasma cells in response to an immunoglobulin. All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23 kD) and two identical heavy chains (50 - 70 kD).

Flow cytometry (FC) is a powerful technique for the analysis of multiple parameters of individual cells within heterogeneous populations what will be studied simultaneously. Flow cytometers are used in a range of applications in research and diagnostic laboratories from immunophenotyping, to ploidy analysis, to cell counting and glycophorin protein GFP expression analysis. The flow cytometer performs this analysis by passing thousands of cells (which are suspended in sheath fluid) per second through a laser beam and detecting the light that emits from each cell as it passes through by detectors. The emitted light is then converted into signals by the electronics to report cellular characteristics such as size, complexity, and display them [5] .

The monoclonal antibodies used in FC are conjugated with fluoro chromes which are stimulated by laser beam in the Flowcytometer [6] .

These antibodies are against antigens found on the surface, cytoplasm or nuclei of the studies cells to identify their type.

Flow cytometric immunophenotyping is important for the distinction between acute and chronic leukemias and between ALL and AML, identification of lymphoblast lineage (B-ALL or T-ALL), and moreover the subtype of ALL (immunological classification) and assessing response to treatment, by the detection of minimal residual disease. Prognosis of ALL depends of the phenotype having significant cytogenetic and molecular abnormalities. For example, B-cell ALL withCD9+, CD10+, CD19+, CD20− or only partial, CD34− phenotype is a sensitive marker for t (1; 19) (q23; p13). B-cell ALL with a CD10−, CD15+, CD24− or partial phenotype is associated with MLL abnormality. However, there is little correlation between immunophenotyping and cytogenetic and molecular characteristics of the blasts [7] [8] .

All tubes were labeled then 100 ul were pipette into each tube from venous blood or bone marrow then washed using 1000 ul PBS in centrifuge for 5 min at 500 rpm, and the supernatant was discharged, 10 ul of Kappa-FITC/Lambda-PE (Immunostep, SL, Spain) were added and mixed gently, incubated in dark for 10 min at RT then After incubation 1000 ul of lysing solution were added, mixed gently, Incubated for 10 min in dark and finally samples were analyzed using the Flow Cytometer (4 COLOR EPICS XL-MCL BECKMAN COULTER, Miami, USA).

100 ul of whole blood or BM were added to test tube, then washed using 1000 ul phosphate buffer saline (PBS) in centrifuge for 5 min at 500 rpm, the supernatant was discharged, 100 ul of fixative solution were added, then incubated for 10 min in RT, after that 1000 ul of PBS were added and centrifuged for 5 min in 500 rpm and then supernatants were discharged s, 100 ul of permeabilization solution were incubated for 5 min at RT, 10 ul of kappa-FITC/Lambda-PE or Lambda PE were incubated for 10 min in dark, then it have been washed by PBS and re-suspended in 1000 ul PBS finally analyze were run by flow cytometer [9] - [13] .

Positivity was considered when 20% or more of the population expressed the marker as a cut off points between negative and positive scale for every marker. The percentages, mean fluorescence intensity and the parentheses were also recorded for most of the markers.