Fig. 1

Key elements of the Accellix cartridge based HLA-DR flow cytometry assay. a Cartridge. The sample is added to the cartridge, which is then placed in the reader instrument. Reagents are added to the sample by crushing the 1st blister on the cartridge that releases the reagents and simultaneously moves the sample to a first mixing chamber. Additional mixing is performed by moving the composition of the sample and reagents from one mixing chamber to the other through a tortuous path by compressing and releasing a polymer bellows in order to alternately apply pressure and vacuum to the sample. After the incubation period, emission level reference beads are added by crushing the 3rd blister to release the beads into the 2nd mixing chamber. The resulting composition is again moved back and forth between the two mixing chambers in order to obtain a uniform particle suspension. At the end of this mixing process, the composition is in the first mixing chamber. The reading bellows, which had been previously compressed, is now released in order to apply vacuum to draw the specimen from the first mixing chamber through the reading cuvette. b Optical detection. Fluorescence emission detection is performed by dispersing the emission using an optical grating and collecting the dispersed emission using a PMT array. c Dot plot histograms. Results from the automatic classification algorithm are shown on the 2 plots. First, junk is excluded based on the absence of any fluorescent signal. Reference beads are identified because they do not express pan WBC CD45 marker. PMNs used as a negative control are identified based on the levels of CD45, CD14, and forward scatter. Finally, lymphocytes used as a positive control are separated from monocytes based on CD14 expression. Importantly, it is to note that these histograms are not accessible to Accellix users and are only provided here as an illustration of cell identification process