Flow analysis of DNA content and cell size in non-Hodgkin's lymphoma

Abstract

Cellular DNA content, Coulter volume, and light scatter were measured in cell suspensions from 30 non-Hodgkin's lymphomas in order to assess flow analysis as a quantitative and reproducible means of evaluating these diseases. Nonneoplastic control populations included 31 samples obtained from lymph nodes, spleens, tonsils, and peripheral blood. Cellular DNA and light scatter were measured on ethanol-fixed cells by flow microfluorometry using nuclei isolated from chicken erythrocytes as an internal standard. For DNA analysis, the cells were stained with propidium iodide following RNase treatment. The cellular DNA content of the human populations was expressed as a ratio between the DNA content of the human G0-G1 cells and that of the chicken erythrocyte nuclei. The mean DNA ratio for the 31 nonneoplastic samples was 2.83 +/- 0.08 (S.D.) In these samples, the coefficient of variation of the human G0-G1 peak ranged from 2.27 to 3.63% (mean 3.09 +/- 0.32%). Fifteen of 30 non-Hodgkin's lymphomas, including 7 of 15 low-grade lymphomas and 8 of 15 high-grade lymphomas, had abnormal DNA content, the majority containing hyperdiploid G0-G1 populations. In six malignant lymphomas with normal DNA content, the coefficient of variation of the human G0-G1 peak, corrected for differences in instrument setting was greater than that seen in the nonneoplastic populations. A good correlation between the percentage of cells calculated to be in the S phase of the cell cycle and the expected clinical behavior of the tumors was observed. In those lymphomas in which the S-phase percentages could be calculated, 11 of 13 low-grade lymphomas had less than 5% of the cells in S phase, and 7 of 10 high-grade lymphomas had greater than 5% of the cells in S phase. Thirteen of 21 neoplastic cases in which Coulter volume determinations were performed could be distinguished from the nonneoplastic controls on the basis of their modal volume. Although some correlation was observed between light scatter of ethanol-fixed cells and Coulter volume measurements on unfixed cells, light scatter was found to be less discriminatory. Altogether, by all three flow parameters studied, 26 of 30 (87%) of the neoplastic cases could be distinguished from nonneoplastic controls.