The 10-color panel consisting of 15 monoclonal antibodies (mAbs) is developed to detect leukemia and lymphoma cells in small cell samples [hypoplastic bone marrow (BM), fine needle aspirates, or cerebral spinal fluids (CSFs)]. MAbs conjugates were selected to identify populations of distinct cell lineages and to determine stages of differentiation based on specific antigen expression patterns. As such, conjugates containing the same fluorochrome could be combined. This panel is tested on peripheral blood (PB), BM, and CSF and provides a strong improvement of diagnostic potential.1, 21
Table 1. Summary for OMIP-010
Improvement of phenotyping of leukemia and lymphoma cells in small cell samples by a panel consisting of 10 colors and 15 mAb
Most immunophenotypic analyses are performed using up to five-color panels and are usually adequate in samples containing high cell numbers. Samples that contain few cells and samples with minimal residual disease need high-level multicolor analyses to gather sufficient data for diagnostic purposes (1). However, the commercial availability of fluorochrome-labeled mAbs can hinder the formation of multicolor panels. Using CD45-Krome Orange (2), we defined a 10-color panel with 15 mAbs for the detection of aberrant cells in small samples. The other 14 mAbs were selected based on their sensitivity of recognition for different cell lineages and developmental stages. MAbs to low-density antigen were conjugated to bright dyes. In some cases, two mAbs were conjugated to the same fluorochromes and combined where each mAb was independently directed against antigens expressed exclusively on different cell populations.
Conjugates were titrated to find the adequate concentration, thereby maximizing the signal-to-noise ratio. Subsequently, PMT settings and compensation of spectral overlap were performed on each single conjugate. All incubations were performed at room temperature in the dark. This panel enables a reliable clinically diagnostic immunophenotyping of small cell samples with more accurate results and, as such, improves patient care.
SIMILARITY TO PUBLISHED OMIPs
None to date.
The authors thank Marij Leenders for panel development experiments; Beckman Coulter, Marseille (Dr. F. Montero) for providing the CD45-KO, (Dr. L. Nieto-Gligorovski and Dr. E. Gautherot) for conjugation of mAbs, and Dr. T. Matt Holl for correcting the manuscript.