A novel multiparametric approach for analysis of cytoplasmic immunoglobulin light chains by flow cytometry

We describe a novel flow cytometric approach using a two-step acquisition technique to determine the cytoplasmic immunoglobulin light chains (LC) expression. Samples were prepared by a lysed-whole-blood technique and incubated with CD38-PE (phycoerythrin) and CD45-FITC (fluorescein isothiocyanate). The cells were fixed and acquired on an FACSCalibur flow cytometer (first acquisition). The cells were then permeabilized, incubated with either kappa-FITC or lambda-FITC and reacquired (second acquisition). Analysis of the data was performed by gating on the differing intensities of CD38 and evaluating them for the presence of a shifting FITC-positive population from the first acquisition to the second acquisition. The FITC fluorescence intensity of the second acquisition was equal to the sum of surface CD45 expression obtained during the first acquisition and the cytoplasmic LC expression obtained during the second acquisition. Thus, the shifting (increase) of FITC fluorescence intensity during the second acquisition is specifically due to the cytoplasmic expression of either the kappa or lambda LC. We studied 15 multiple myeloma (MM) patients and 10 controls (samples from patients without plasma cell dyscrasias). None of the controls showed evidence of any clonal populations. Thirteen of 15 MM patients exhibited clonal plasma cells (CD38 bright), ranging from 0.01% to 34% of total events collected. In addition, we identified another minute clonal population of lymphocytes (CD38 dim, CD45 bright, low forward and side scatter) in 12 of 13 MM patients with clonal plasma cells. This population, ranging from 0.01% to 0.6% of total events collected, had the same LC restriction as the clonal plasma cells. Patients with a ratio of minor clonal population to clonal plasma cells less than 0.07 tended to remain in partial or complete remission than those with a ratio > or =0.07 (4/5 versus 1/4, P <.1, chi(2)). We conclude that this method is highly sensitive and permits us to identify the minute clonal population of lymphocytes in MM patients. Our preliminary observations with a small cohort of patients imply that this minute clonal population may have important prognostic significance. The prognostic significance should be confirmed by further studies.