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Aby Joseph, Andres Guevara-Torres, Jesse B Schallek; In vivo flow cytometry measures red to white blood cell ratio in the living mouse eye. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4616. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Adaptive optics scanning light ophthalmoscopy (AOSLO) has recently provided counts of red blood cells (RBCs) in capillaries of the living mouse retina without contrast agents. However, white blood cells (WBCs) are far more sparse accounting for only 1 in every 3400 RBCs in the healthy mouse making their quantification more challenging. Here we use AOSLO to image label-free RBCs and fluorescently labeled WBCs to compute their ratio in the living mouse toward providing an in vivo complete blood count (CBC) - a physiological marker of systemic health.
Anesthetized C57BL/6J mice were imaged with an AOSLO modified to count single blood cells in capillaries as they passed a stationary 15 kHz 1-D point scan. RBCs were imaged using 796nm light backscatter in split-detector configuration (Guevara-Torres et al., 2015). A second detector simultaneously captured fluorescence of WBCs, rendered visible by IV injected acridine orange, and sodium fluorescein to label plasma (each using 488nm ex/534nm em). Capillaries were scanned for 80s and blood cells were counted with a user assisted algorithm. In vivo blood counts were compared to those from conventional hematology (HESKA HemaTrue).
AOSLO imaged sparse cells 3 µm in size flowing through capillaries (n=60 cells). Cells had a bright center and dim surround consistent with DNA labeling of the nucleus by acridine orange (figure). Automated counts of WBCs were between 0.05 to 0.19 cells/s (n=7 capillaries, 1 mouse). In the same capillaries, we measured a total blood cell count of 15 to 223 cells/s (discounting platelets). This gave a WBC:RBC ratio ranging from 1:76 to 1:2929 across capillaries (mean 1:813). The in vivo WBC:RBC ratio was consistent with ex vivo hematology performed on the same animal (1:1182) and was within an order of magnitude from values reported from the normal C57BL/6J mouse (1:3400) (Jackson Laboratory Phenome Database).
AOSLO flow cytometry provides in vivo ratios of WBCs and RBCs, an important component of the CBC - which currently requires a blood draw and ex vivo analysis. We observed a range of ratios across capillaries; likely due to WBC preferred paths in particular capillaries suggesting many capillaries need to be averaged to determine an accurate count. This work lays the foundation for future studies to identify biomarkers that differentiate WBCs from RBCs in label free strategies such as split-detection.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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