DNA ploidy and simultaneous six color immunophenotyping using FxCycle-Violet.

To standardize an easy flow cytometric DNA ploidy and simultaneous six color immunophenotyping method that can be used in small number of tumor cells identified using specific immunophenotype. This method will help in ploidy analysis in cases with low tumor cells like monoclonal gammopathy of undtermined significance or multiple myeloma with small number of plasma cells in the bone marrow sample. In partially treated B-cell acute lymphoblastic leukemia.
Conclusion:
FxCycle Violet (FCV) based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of up to seven color immunophenotyping & DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like residual disease in B-cell acute lymphoblastic leukemia, multiple myeloma (MM) and MM precursor conditions like MGUS.
Notes:
DNA ploidy is determined by calculating the DNA Index i.e. a ratio of geometric mean of G0/G1 peak of tumor cells (isolated with a specific immunophenotype) to the geometric mean of G0/G1 peak of patient's own lymphocytes present in the sample. Hence, patient's own lymphocytes represent the reference diploid cells to calculate DNA index. Gating approach: Doublets were excluded using FSC height versus area as doublets typically have disproportionately higher FSC area than FSC height. This does not affect cells in G2 phase as cells in G2 ohase typically have proportionately high FSC area and FSC height. Next, cell debris present in the sample were excluded using viability gate in SSC versus FSC dot plot. tumor cells and lymphocytes were gated using expression of surface markers. FCV unstained cells: Depending on the age of sample after collection and duration of fixation after ideal 10 minutes, FCV might not stain few cells or stain weakly. These cells might form a small trail of wealy stained cells. However, as this forms a continuous trail towards negative region, these does not interfere with the ploidy analysis. these can be easily excluded from the analysis. All daily routine quality measures for flow cytometer were performed. Fresh samples (within 12 hours of collection) were processed and were acquired at low rate to get good CV. Compensation: Generic compensation was performed using lymphocytes and tube specific or label specific was performed during analysis i.e. post acquisition compensation. Debris of dead cells were excluded using a gate based on the SSC and FSC. DI was calculated as a ratio of geometric mean of G0/G1 peak of tumor cells to G0/G1 peak of patient own lymphocytes present in the sample. Thus, this method avoids use of normal cells from other individuals.