Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology

Abstract

Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, <em>In Vivo</em> Tissue Analysis

“…Other inks, such as proflavine or fluorescein, can be used instead of pyranine ink with appropriate approval. [4][5][6][7]11 Any probe feature may be modified from this design. For the high-resolution fluorescence microendoscopy modality, the 1 mm diameter image fiber consisted of 50,000 individual core fibers with 4.5 µm spacing, resulting in a constant sub-cellular spatial resolution of 4.5 µm.…”

“…Finally, filters and excitation light source may be modified depending on the excitation/emission spectra of fluorescent dyes. [4][5][6][7] In addition to modifying the probe and high-resolution fluorescence microendoscopy instrumentation, the sub-diffuse reflectance spectroscopy instrumentation can be modified.…”

“…[1][2][3] One such imaging technique, high-resolution fluorescence microendoscopy, can image apical tissue micro-architecture with sub-cellular resolution in a small, microscale field-of-view, using a topical contrast agent such as proflavine, fluorescein, or pyranine ink. 1,[3][4][5][6][7][8][9][10][11] This imaging modality has shown promising clinical performance in qualitatively differentiating diseased and healthy epithelial tissue in realtime with low inter-observer variability. 8 Occasionally, investigators will use high-resolution fluorescence microscopy data to extract quantitative features such as cell and nuclear size or gland area, but this remains a primarily qualitative technique targeted towards visualizing tissue morphology.…”

Comparative Study Regarding the Properties of Methylene Blue and Proflavine and Their Optimal Concentrations for In Vitro and In Vivo Applications

“…This phenomenon is due to the fact that human erythrocytes have low DNA content, thus it determined a low fluorescence signal from these cells. While the erythrocytes are not visible with PRO staining, the monolobar and multilobar structures within leukocytes are clearly distinguishable [73].…”

Fluorescein as a topical fluorescent contrast agent for quantitative microendoscopic inspection of colorectal epithelium

“…Therefore, proflavine predominantly highlights cellular nuclei in cytology specimens [18], as well as squamous bulk tissue such as oral and esophageal [4,7]. Proflavine is an amphipathic dye comprised of a three carbon ring structure, and capable of intercalation within the minor groove of the DNA helix [19].…”