2 To determine the distribution of ALL subtypes in these two pediatric oncology units, we reviewed the results of cases submitted for immunophenotypic analysis. 2. Patients and Methods 2.1. Patients. From October 2003, to May 2007, 279 con- secutive children (114 females, 165 males) below the age of 18 years with newly diagnosed ALL were recruited from the Onco-haematology-Pediatric service of the University Hospitals of Rabat and Casablanca. These hospitals are the main referral pediatric centers for leukemia patients in Morocco [6, 7]. The diagnosis of ALL was based on morphologic, cytochemical criteria of the French-American-British (FAB) Cooperative Working Group and ﬂow cytometric analysis. Using these criteria, 144 cases were classiﬁed as L1, 101 cases as L2, 4 cases as L3. 30 cases were not classiﬁed as FAB subtypes. 2.2. Flow Cytometry. The immunophenotyping was per- formed at the National Hygiene Institute laboratory on bone marrow aspirate or peripheral blood samples collected in EDTA according to a two-step strategy using panels of monoclonal antibodies based on the European Group for the Immunological Characterization of Leukemia (EGIL) [8] and St. Jude Children’s Research Hospital strategies [9]. Immunophenotypic characterization consisted of two consecutive steps (Table 1). According to the results obtained at the initial screening, the second level of investigation was assessed. The second panel is designed to identify stage of diﬀerentiation, prognosis features or aberrant phenotypes for monitoring minimal residual disease [10, 11]. The reactivity with ﬂuorescent conjugated monoclonal antibodies directed against lymphoid and myeloid associated antigens was evaluated on the surface of leukemic cells. The intracytoplasmic Immunoglobin (Ig), CD3, CD79a and myeloperoxidase (MPO) antigens, as well as nuclear terminal deoxynucleotidyl transferase (TdT) staining, were evaluated by ﬂuorescent conjugated monoclonal antibodies after ﬁxation and permeabilization of leukemic cell. Stained cells were analyzed by ﬂow cytometry on a three-color FACSCallibur ﬂow cytometer (Becton Dickinson Immuno- cytometer Systems) which was calibrated with a set of standardized beads (Becton Dickinson Calibrate 3). Blasts initially were gated for analysis by using CD45 versus side scatter, according to the gating strategy [12]. Leukemic samples were considered positive for a partic- ular antigen if 20% or more of leukemic cells reacted with a particular antibody. 2.3. Immunological Classiﬁcation and Nomenclature. The cases were classiﬁed in three main ALL immunological subtypes: mature B-cell ALL, precursor B-cell ALL (pB- cell ALL), and T-cell ALL [1, 13]. A case was consid- ered mature B-cell ALL if the cells expressed surface immunoglobulin (sIg) with Kappa or lambda light chains. Precursor B-cell ALLs include 3 immunological subtypes’ International Journal of Pediatrics 30 25 sse 20 a c f ro15 e b um10 N 5 0 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Age (years) PB-All T-All Figure 1: Age distribution of 275 children with acute lymphoblastic leukemia (ALL). early pre-B (CD19+, CD22+, CD79a and lack cytoplas- mic immunoglobulin μ (cμ) and surface immunoglobu- lin), pre-B (CD19+, CD22+, CD79a+ exhibit cytoplasmic immunoglobulin μ without detectable surface immunoglob- ulin) and transitional pre-B (CD19+, CD22+, CD79a and express both cytoplasmic immunoglobulin μ and surface immunoglobulin) without Kappa or Lambda light chains [14]. T-cell ALL can also be separated into 3 maturational stages, deﬁned as follows: proT (cyCD3+, CD7+, CD1a−, sCD3−) common thymocyte (cyCD3+, CD7+, CD1a+) and mature thymocyte (cyCD3+, CD7+, CD1a−, sCD3+). 2.4. Statistical Analysis. Association between the incidence of immunologic phenotype and clinical and biologic features was examined and tested by chi-square test using the Excel Stat software. 3. Results The study group comprised 279 newly diagnosed and untreated acute lymphoblastic leukemia cases (<18 years). There were 114 females and 165 males with a male/female ratio of 1.45 : 1. Immunophenotypic analysis showed that the precursor B-cell phenotype was encountered in 216 (77.42%), mature B-cell in 4 (1.43%) and T-cell in 59 (21.15%) cases. The patients’ characteristics are summarized in Table 2. The distribution of immunological ALL subtypes is clearly inﬂuenced by clinical features. Compared with pB- cell ALL, male sex (P = .008), older age (P = .048), higher leukocyte count (P < .0001) and mediastinal mass (P < .0001) seem to be the typical features of T-cell ALL subtype. The age distribution of the diﬀerent ALL subtypes (Figure 1) shows that pB-cell ALLs patients were younger with peak in incidence between 3 and 5 years. There was a trend for patients with T-cell ALL to have a more advanced age.