Cd10 apc

PBMC were isolated by density gradient centrifugation on Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) as described previously [5] (link). Frequencies of lymphocyte subsets were estimated in PBMC by means of flow cytometry using mAb against CD19-PE.Cy7, CD4-APC, CD27-FITC, CD10-APC, IgD-PE, IgM-PE.Cy5, IgG-APC and CD138-APC (BD Pharmingen, San Jose, CA, USA). Non-specific fluorescence was established with APC-mouse IgG1κ, PE-Cy7-mouse IgG1κ, FITC-mouse IgG1κ, PE-mouse IgG2aκ, PE-mouse IgG1κ isotype controls. At least 200,000 events per sample were collected in a FACSAria II flow cytometer (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Lymphocytes were identified by forward and side-angle light scatter characteristics. At least 80,000 lymphocyte-gated cells were analyzed for each sample. Absolute numbers were estimated based on the frequencies of the cells in flow cytometry and lymphocytes in the differential leukocyte count (Sysmex K-800 Automated Hematology Analyzer, Sysmex America, Inc., Mundelein, IL, USA), and expressed as the number of cells per microliter of blood.

Phenotypic analysis of cell suspensions was performed by flow cytometry. Cells were stained with a panel of seven 8-colour antibody combinations (Table 1). The following 6 monoclonal antibodies were used as a “backbone” in all combinations: CD38-PerCP5.5, CD10-APC, CD19-APC-H7, CD5-V450, CD45-V500 (BD Biosciences) and CD27-PeCy7 (Beckman Coulter). All FITC- or PE-labelled antibodies were specific of a particular combination, i.e. CD20-FITC, CD44-FITC, CD24-PE, CD40-PE, (Beckman Coulter); CD43-FITC, CD81-FITC, CD86-FITC, CD21-PE, CD22-PE, CD23-PE, CD268 (BAFF-R)-PE, IgD-PE, (BD Biosciences) IgM-FITC (Dako). Clone and isotype specificity of these antibodies are detailed in S1 Table; the antibodies were used at the dilution recommended by the manufacturers.
Samples containing 10⁶ cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH₄Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 10⁴ cells were acquired in the CD19⁺ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).

MPFC was performed with a panel of antibodies designed for B‐ALL. The diagnostic panel for B‐ALL contained CD45, CD7, CD19, CD13, CD33, CD34, CD117, HLA‐DR, CD10, cMPO, cCD3 and cCD79a and its extended panel included CD66c, CD22, CD20, CD58, CD38, CD123, CD45, cytoplasmic heavy chain of immunoglobulin M(cu) and cytoplasmic TDT (BD Bioscience; San Jose, CA; Beckman‐Counter; Indianapolis, IN).
¹⁵ (link) FACS Aria II (BD Biosciences) with Diva program was used to acquire and analyze the data. MRD panel included CD58‐FITC, KORSA‐PE, CD34‐Percp, CD20‐PE‐cy7, CD10‐APC, CD19‐APC‐H7, CD38‐V450 and CD45‐V500 (BD Bioscience and Beckman‐Counter).
Negative MRD by MPFC was defined as <10⁻⁴ blasts (0.01%) in bone marrow samples and Flow‐MRD positivity was defined as >10⁻⁴ blasts (0.01%) in bone marrow. In our study, patients with sustained undetectable MRD were defined according to negative MRD results observed at the end of consolidation and any of the previous time points.