Optical Imaging of Leukemia Figure 6. (A) Representative flow cytometric analysis of bone marrow of a mouse transplanted with leukemic cells from patient AML sample treated or not with anti-hCD45 mAb. The bone marrow is overrun by CD45+ cells in the two cases. (B) Liver and spleen CD45 immunostaining at x20. doi:10.1371/journal.pone.0030690.g006 mAb is described as a mAb that does not mediate antibody- dependent cellular cytotoxicity or complement-mediated cyto- toxicity [23]. Therefore it appears possible to use this anti- hCD45 mAb as a diagnostic tool to detect human leukemia foci in mouse experimental models. Nevertheless, considering that CD45 is a protein tyrosine phosphatase implicated in the regulation of CXCR4 mediated chemotaxis and MAP kinase activation; further tests may be required to document potential impact of the labeled mAb on the sensitivity towards therapeu- tical agents in oncopharmacology studies. Furthermore, it will be useful to examine any potential impact of anti-hCD45 mAb on disease development in different human leukemia models with varying CD45 expression and determine the imaging sensitivity with low expression levels. These observations could be only performed at the time various human leukemia models will be established. The FRI performed on mice engrafted with leukemic cells from patient samples showed several leukemic foci in the bones (sternum, tibia, and femur), the liver and the spleen. These results support a previous study performed with several human AML samples [24]. Furthermore, a good correlation between the fluorescent foci detected by the fluorescent anti-hCD45 mAb and the presence of human CD45+ cells (showed by immunohisto- chemical and flow cytometry analyses) was observed. This demonstrated a specific fixation of the fluorescent mAb on the leukemic foci. The fact that a fluorescent signal can be detected in the liver and the spleen, which have a high absorption coefficient, suggests that the Alexa Fluor 750 was suited to this application. So, NIR fluorescent anti-hCD45 mAb appears to be an efficient diagnostic tool for human leukemia engraftment in mice but in addition, it could be considered also for the staging of the disease and to assess the efficiency of a therapeutic agent. However for such applications, quantitative fluores- cence imaging is required and such a goal can be achieved only using fluorescence tomography and further validation studies. In conclusion, we demonstrated that the detection of human leukemic foci in mice with a low dose of mAb labeled with a near- infrared fluorochrome is possible but it would be necessary to further determine the limit of in vivo detection as compared to reference methods to assess the evolution of the pathology. Of course only CD45+ leukemia cells can be detected with this antibody which is the case of 90% of AML [8]. Author Contributions Conceived and designed the experiments: SP ALP SL AK AP. Performed the experiments: SP AP. Analyzed the data: SP. Contributed reagents/ materials/analysis tools: LC CR CD. Wrote the paper: SP AK NG. PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30690