A NEW STRATEGY FOR VALIDATION OF MONOCLONAL ANTIBODY IN FLOW CYTOMETRY

Abstract

Objectives: Clinical flow cytometric analysis of neoplastic hematolymphoid cells relies on evaluation of surface membrane and intracellular antigens expression patterns, which are related to normal cells of a particular lineage and maturational stage. A complete characterization of the studied population includes information about presence, absence and level of expression of a series of relevant antigens recognized by highly specific monoclonal antibodies (MAbs). Usually, the fluorescence intensity is considered to be directly proportional to the amount of antigen expressed in a specific population, as assessed by flow cytometry using the mean peak of channel fluorescence (MPCF), the proper use of MAbs is essential for this condition to be valid. Data show that laboratories use a wide variety of MAb dilutions, even those obtained from the same manufacturer. Ideally, the titre value should be determined for every MAb and is defined as the amount of antibody that is required to satura te the maximum number of antigen binding sites on a selected cell population, ensuring that antibodies are used efficiently yet still remain in excess. Our objective is to report a new statistical criteria adopted by our laboratory to establish optimal MAb dilution for flow cytometry analysis based on the discrimination of unstained and positively stained cells using MPCF. Methods: We have tested in our laboratory 57 MAbs from different manufacturers, from May/2010 until August/2011 (27 conjugated to FITC, 11 to PE, 4 to PerCP/PEDY647/CyQ or RPECy5 and 5 to APC). The tests were performed using bone marrow or peripheral blood samples which presented any positive population (normal or neoplastic) for the studied MAbs. The staining procedure followed manufacturers ́ protocols, with different volumes of Mabs (recommended by manufacturer followed by titrations 1:2, 1:4 and >1:4). At least 10,000 events were acquired for each sample using the flow cytometer BD FACSCalliburTM, and data was analyzed by CellQuestTM, obtaining the MPCF values for unstained and positively stained cells. The percentage difference between the MPCF (PD-MPCF) values was calculated applying this formula: (MPCF from positively stained population–MPCF from unstained population) x 100/MPCF from positively stained population. An adequate PD-MPCF must be 95% or more. Results: Among MAbs tested, 42/57 (73,68%) were likely to be titrated, 18 being titrated to 1:2, 22 to 1:4 and 2 to >1:4. Analysis according to the fluorochrome conjugated to MAb demonstrated more efficient titration for APC (3 MAbs in 5), while those conjugated with FITC showed difficulty in titration (9/27 have not shown good performance at lower concentrations than those indicated by the manufacturer and titers >1:4 were not appropriate for any samples). Six MAbs had titration disapproved by the criterion used (PD-MPCF) but were also disapproved in the concentration recommended by the manufacturer because the expected fluorescence level was dim for the population analyzed. Conclusions: We conclude that PD-MPCF is an efficient and objective instrument as quality control for validation and titration of MAbs, and that the cutoff of 95% ensures proper separation between positive and negative stained populations showing good correlation with the performance in daily routine.