In vitro evaluation of the multiplexing imaging strategy in AML cell lines. (A) Combined flow cytometry results of mAb multiplexing in 3 cell lines (HL-60, NB4, and MOLM-13) using all possible combinations of the mAbs CD33/45/13/HLA ABC as 1 (n = 12), 2 (n = 18), 3 (n = 12), or 4 mAbs (n = 3). Mean fluorescence intensity (MFI) values show means ± SEM. (B) Pellets of 1 × 10⁶ NB4^GFP cells prestained with 1-4 Alexa Fluor 680–labeled mAbs and immersed into a liposyn solution containing 3% whole blood were imaged at depths of 1-9 mm by time-domain optical imaging. Imaging with 3 multiplexed mAbs results in greater depth detection and increased fluorescence over 1 and 2 mAbs, respectively (mean ± SEM, n = 3). (C) Similarly, cell pellets (1 × 10⁶ cells) implanted at defined locations (brain, subcutaneous [s.c.], intramuscular [i.m.], and thorax) in a postmortum mouse illustrates (D) significantly increased fluorescence intensity with increased multiplexing of mAbs (n = 4). Fluorescence images from each panel (eg, brain) have the same scale. *P < .05; **P < .01; ***P < .001.