Objective Methods To Detect B Cell Clones in Flow Cytometry Data
SA Fitzgerald, HJ Meyerson, W Wang, P Fu. University Hospitals Case Medical Center, Cleveland, OH; Case Western Reserve University, Cleveland, OH
Background: Objective statistics-based methods to evaluate flow cytometry data for B cell clones, including those not readily apparent on flow cytometry dot plots, would aid in identifying patients with clonal B cell populations.
Design: Three different objective approaches were taken. All methods were based on aligning data points from flow cytometry data and scanning for a statistically significant string of consecutive kappa or lambda events. Raw values of FSC, SSC, CD19-APC, CD45-PerCP, kappa-FITC and lambda-PE from CD19+ cells were used. Each cell was classified as either kappa or lambda based on the FITC/PE absolute fluorescent ratio greater or less than 1 for the cell. The first method was a one dimensional scan of CD19 (CD19 scan). CD19 events were aligned from dim to bright CD19 and scanned sequentially while scoring each data point kappa or lambda. The second method was two dimensional using CD45 and CD19 (CD45/19 scan). Cells were first aligned based on expression of CD45 (dim to bright) and secondarily aligned by CD19 expression prior to scanning. The third approach was multidimensional incorporating FSC, SSC, CD19 and CD45 (multi-D scan). The Euclidean distance of all cells relative to a single cell was calculated using FSC, SSC, CD45 and CD19. The cells were then aligned from nearest to furthest from the index cell and the number of consecutive closest neighbors with a single light chain was determined. If this number reached a statistically significant level a clone was considered present. The analysis was repeated for each cell. The results from all methods were compared with flow cytometry diagnoses previously rendered using standard visual inspection of dot plots.
Results: 84 cases were analyzed. CD19 scan was positive in 24/29 and CD45/19 scan in 28/29 cases positive by standard flow. CD19 scan scored positive in 6 and CD45/19 scan in 4 of 56 samples negative by standard methods. Both methods identified a clone in one sample not observed by standard flow cytometry. The multi-D scan was applied to 43 cases. 12 were positive by standard flow cytometry and all were positive by the multi-D scan. 9 cases were positive by multi-D scan and negative by standard flow cytometry.
Conclusions: Objective statistics-based methods to evaluate flow cytometry data for B cell clones are feasible and may be more sensitive than standard visual inspection of dot plots. Multi-dimensional approaches may be most effective.
Wednesday, March 24, 2010 1:00 PM
Poster Session VI # 202, Wednesday Afternoon