FLOW CYTOMETRIC IMMUNOFLUORESCENCE AND DNA ANALYSIS - USING A 1.5 MW HELIUM-NEON LASER (544 NM)

We evaluate a 1.5 mW HeNe laser (544 nm) for use on an EPICS(R) Elite with a 76 mu m Sortsense flow cell. The two applications chosen were i mmunofluorescence and DNA analysis. We measured the fluorescence thres hhold of phycoerytherin calibration beads to be approximately 336 MESF . Cell analysis with a HeNe laser and Argon laser correlated well for the 1CD4(PE), CD56(PE) CD19(PE) conjugates, with correlation coefficie nts of 0.98, 0.99, 0.94, respectively. The % positive and mean channel fluorescence were comparable to the results obtained with a 15 mW Arg on laser. In addition, a three-color configuration yielded excellent r esults. Cell analysis of CD4(PE), CD3(ECD) and CD19(Cy-Chrome) with th e HeNe laser and Argon laser correlated well with correlation coeffici ents of 0.96, 0.95, and 0.92, respectively. The histograms showed good separation between the negative cells, the dimly staining cells and t he brightly staining cells. Propidium Iodide was chosen for DNA analys is. Full CV values for whole blood DNA fluorescence using the green la ser were good at 2.6%. These data indicate the low power 544 nm laser is sufficient to do immunophenotyping and DNA analysis. Results may be explained by higher quantum efficiency and lower background fluoresce nce. The wavelength of the 544 nm laser is much closer to the excitati on peaks of PI, PE, and the tandem dyes ECD and Cy-Chrome. Also, the R aman scattering of water for the 544 nm laser has a longer wavelength maximum than the emission peaks of PI, PE, and ECD. The major advantag es of this laser for the research laboratory are small size, no coolin g fan, low power requirements and low cost. (C) 1995 Wiley-Liss, Inc.