Hexaminolevulinate (HAL) induced
Protoporphyrin IX (
PpIX) fluorescence is commonly used to differentiate
cancer cells from normal cells in vivo, as for instance in blue light cystoscopy for
bladder cancer diagnosis. A detailed approach is here provided to use this diagnostic principle ex vivo in an immunosensor device, towards enabling non-invasive
cancer diagnostic from body fluids, such as urine. Several factors susceptible to affect the applicability of HAL-assisted diagnosis in body fluids were tested. These included the cell viability and its impact on
PpIX fluorescence, the storage condition and shelf life of HAL premix
reagent, light exposure (360-450 nm wavelengths) and its corresponding effect on both intensity and bleaching of the
PpIX fluorescence as a function of the microscopy imaging conditions. There was no significant decrease in the viability of
bladder cancer cells after 6 h at 4 °C (student's t-test: p > 0.05). The cellular
PpIX fluorescence decreased in a time-dependent manner when
cancer cells were kept at 4 °C for extended period of time, though this didn't significantly reduce the fluorescence intensity contrast between
cancer and non-
cancer cells kept in the same condition for 6 h. HAL premix
reagent kept in long term storage at 4 °C induced stronger
PpIX fluorescence than
reagent kept in the - 20 °C freezer. The
PpIX fluorescence was negatively affected by repeated light exposure but increased with illumination intensity and exposure time. Though this applied to both healthy and
cancer cell lines, and therefore did not statistically improved the differentiation between cell types. This study revealed important experimental settings that need to be carefully considered to benefit from the analytical potential of HAL induced fluorescence when used in technologies for the diagnosis of
cancer from body fluids.