The Open Leukemia Journal, 2010, 3, 47-54 47 1876-8164/10 2010 Bentham Open Open Access Flow Cytometry Evaluation of Minimal Residual Disease in Acute Lymphoblastic Leukaemia Type B N. Braham Jmili *,1 , M.C. Jacob 2 , S. Yacoub 1 , Y. Ben Youssef 3 , M.A. Laatiri 3 , Y. Braham 4 and M. Kottas 1 1 Laboratory of Haematology, Hospital Farhat Hached, Sousse, Tunisia 2 EFS Rhone-Alpes, Department of Cellular Immunology, Grenoble, France 3 Department of Clinical haematology, Hospital Farhat Hached, Sousse, Tunisia 4 Laboratory of Toxicology, Hospital Farhat Hached, Sousse, Tunisia Abstract: Immunophenotyping has become essential to the diagnosis and the treatment management of acute lymphoblastic leukaemia (ALL). We prospectively studied minimal residual disease (MDR) in patients with B lineage ALL who achieved mCR remission. The initial series of patients consisted on 90 cases with B ALL. Sixty-Six patients had bone marrow samples adequate for MDR studies collected on day 35 of remission induction chemotherapy. Strategy of monitoring MRD is based on flow cytometry using quadruple staining according the leukaemia associated immunophenotype found at diagnosis. Data analysis was done using an EPI XL cytometer (Coulter), acquiring 500 000 events. Of the 66 patients 40 (60, 6%) had MRD 0, 01%. B lymphoblasts of ALL may morphologically resemble to hematogones (B benign lymphocyte precursors) and their immunophentypes have similarities. Different combinations of antibodies are tested to determine which combinations are more suitable to detect B residual leukaemics cells. The results of this present study indicate that: CD10/CD38/CD19/CD45 and CD10/CD34CD19/CD45 are the more specifics and should be used to distinguish B lymphoblasts of lymphoblastic acute leukaemia from normal hematogones. Keywords: Acute lymphoblastic leukemia, lymphopoiesis B, flow cytometry, minimal residual disease. INTRODUCTION Acute lymphoblastic acute leukaemia (ALL) is a most common leukaemia in childhood with a peak incidence at 2- 5 years of age, and another peak in old age (after 50 years). The overall cure rate in children is 85%. Measurement of minimal residual disease (MRD) during clinical remission after chemotherapy has proven to be a valuable tool for predicting relapses before clinical and haematological manifestations and establishing different risk categories in patients with ALL [1]. The detection of residual leukemic cells is usually based on either molecular or immunophenotypical markers present in leukemic but not in normal cells, allowing for their specific discrimination [2]. Studies based on semiquantitative polymerase chain reaction (PCR) or immunophenotyping have demonstrated the clinical relevance of MRD investigation; both methods provide similar results [1, 3]. Evidence supporting the value of MRD investigation in acute leukaemias by immunophenotyping and/or molecular techniques has been increasingly reported over the last few years [1, 2]. Flow cytometry is nowadays the first line method for immunophenotypic identification of blast cells but is not so usual in limited resources countries [4, 5]. In Tunisia, flow cytometry (FC) is used in diagnosis of acute leukaemia and *Address correspondence to this author at the Laboratory of haematology. Hospital Farhat Hached, 4000 Sousse, Tunisia; Tel: 00 216 73 22 33 11; Fax: 00 216 73 22 67 02; E-mails: jmilinejia@yahoo.fr, brahamyoussef@yahoo.fr we think that it is the more suitable method to measure MRD than molecular methods which are not done systematically and are more expensive. Flow cytometry is a practical tool for monitoring MRD in patients with acute lymphoblastic acute leukaemia (ALL). This approach is based on the identification of immunophenotypes expressed by leukemic cells but not by normal lympho-haematopoietic cells in bone marrow and peripheral blood [6]. Flow cytometry measurement of MDR in B ALL present some particularities and difficulties to distinguish in the bone marrow neoplastic lymphoblasts of ALL from benign B-lymphocyte precursors known as hematogones [7, 8]. The aim of this study was analytical: to describe flow cytometric conditions for immunophenotypic analysis at diagnosis of acute leukaemia and to establish a protocol for the measurement of minimal residual disease MRD assayed by four flow cytometry at the end of chemotherapy induction in patients with B ALL in complete morphologic remission using the optimal combination of antibodies. An important question was to determine whether panel of antibodies provided informations of residual cells. PATIENTS, MATERIALS AND METHODS Patients Criteria for entering the study were: Unequivocal diagnosis of B ALL based on morphologic, cytochemical and immunophenotypical criteria [9-11] phenotypically