The development of multicolor panels with Quantum Dots-conjugated antibodies for diagnostics of Chronic Lymphoid Leukemia (CLL) by conventional flow cytometer
N. Barteneva, I. Vorobjev
Harvard Medical School, US
Keywords: quantum dots, antibodies, chronic lymphoid leukemia, flow cytometry
Abstract:Diagnostics of leukemia and lymphomas by flow cytometry emerged in 90-th years of last century when several consensus panels had been introduced and based on three-four color cytometry. The standard flow diagnostics approach requires 10-15 tubes per patient and does not allow identification of small populations of tumor cells. The major obstacles for development of multicolor diagnostics are 1) problem of multicolor compensation; 2) problem of different brightness of existing fluorophores. Quantum dots (QDots) offer new possibilities in polychromatic clinical cytometry resolving organic and tandem dyes issues with stability and donor bleed-through. We developed 8-color panels incorporating QD605, QD655 and QD705 direct antibody conjugates (Invitrogen) for differential diagnostics of CLL lymphocytes (CD3/CD5/CD19/CD23/CD38/CD10/CD45/viability dye). Fluorescent staining of CLL patients and normal controls was analyzed with 3-laser FACSCanto2 (BDBiosciences) cytometer. We used combinations of beads stained with different antibody conjugates with QDots and standard fluorochromes to calculate signal to noise and stain index for QD605, QD655 and QD705 alone and included in our CLL panels. Using this panel we were able to define the population of CLL cells that was as small as 1% of the overall PBMC population – it was determined in dilution experiments (adding mixture of ~0.2% CLL to the normal blood.