The development of multicolor panels with Quantum Dots-conjugated antibodies for diagnostics of Chronic Lymphoid Leukemia (CLL) by conventional flow cytometer

摘要

The standard flow cytometry diagnostics approach (3 colors) requires 10-15 tubes per patient and does not allow identification of small populations of tumor cells. Quantum dots (QDots) offer new possibilities in polychromatic clinical cytometry resolving organic and tandem dyes issues with stability and donor bleed-through. We used combinations of beads stained with different antibody conjugates with QDots and standard fluorochromes to calculate signal to noise ratio and stain index for QD605, QD655 and QD705-conjugated antibodies alone and included in our CLL panels. We developed 8-color panel (CD3/CD5/CD19/CD23/CD38/CD10/CD45/ Sytox Blue viability dye) panel incorporating QD605, QD655 and QD705 direct antibody conjugates (Invitrogen) for differential diagnostics of CLL. 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).