Diagnostic approach for cancer cells in urine sediments by 5-aminolevulinic acid-based photodynamic detection in bladder cancer

Abstract

Bladder urothelial carcinoma is diagnosed and followed up after transurethral resection using a combination of cystoscopy, urine cytology and urine biomarkers at regular intervals. However, cystoscopy can overlook flat lesions like carcinoma in situ, and the sensitivity of urinary tests is poor in low-grade tumors. There is an emergent need for an objective and easy urinary diagnostic test for the management of bladder cancer. In this study, three different modalities for 5-aminolevulinic acid (ALA)-based photodynamic diagnostic tests were used. We developed a compact-size, desktop-type device quantifying red fluorescence in cell suspensions, named "Cellular Fluorescence Analysis Unit" (CFAU). Urine samples from 58 patients with bladder cancer were centrifuged, and urine sediments were then treated with ALA. ALA-treated sediments were subjected to three fluorescence detection assays, including the CFAU assay. The overall sensitivities of conventional cytology, BTA, NMP22, fluorescence cytology, fluorescent spectrophotometric assay and CFAU assay were 48%, 33%, 40%, 86%, 86% and 87%, respectively. Three different ALA-based assays showed high sensitivity and specificity. The ALA-based assay detected low-grade and low-stage bladder urothelial cells at shigher rate (68-80% sensitivity) than conventional urine cytology, BTA and NMP22 (8-20% sensitivity). Our findings demonstrate that the ALA-based fluorescence detection assay is promising tool for the management of bladder cancer. Development of a rapid and automated device for ALA-based photodynamic assay is necessary to avoid the variability induced by troublesome steps and low stability of specimens.

Keywords: 5-Aminolevulinic acid; bladder cancer; fluorescence; urine biomarker; urine cytology.

Figure 1

ALA-based fluorescence detection assay protocol. Urine samples were subjected to urinary tests such as urianalysis and conventional cytology. The remaining urine samples and detached T24 cells were analyzed in comparison with the untreated samples. Because photobleaching of protoporphyrin IX can be observed during light exposure, the experimental process for the 5-ALA treatment was performed in the dark. ALA, aminolevulinic acid; BTA, bladder tumor antigen; NMP22, nuclear matrix protein 22.

Figure 2

Detection of urothelial carcinoma cells by fluorescence microscopy. Cell suspension of ALA-treated urine sediments in PBS was transferred onto a microscope slide and covered using a cover slip. Cells were observed with regular light microscopy to find urothelial cells and exclude non-epithelial cells such as red blood cells. Representative photographs of T24 cells, pT1 HG bladder cancer, benign prostate hyperplasia (BPH), and chronic kidney disease (CKD) are shown. Blue arrowheads indicate red fluorescence-positive cells.

Figure 3

Quantitative detection of protoporphyrin IX using a fluorescence spectrophotometer. Fluorescent spectra of representative cases are shown. Red curves are spectra of samples treated with ALA, and blue curves are spectra of non-ALA control. Fluorescence intensities at 635 nm are compared between ALA-treated and untreated cells. The assay was run in triplicate. Both cases with bladder cancer are considered positive for the fluorescence spectrophotometric assay. a.u., arbitrary unit; LG, low grade; HG, high grade. ^†The intensity value of ALA-treated samples is higher than those of ALA-untreated controls.

Figure 4

A novel device for detecting ALA-induced intracellular protoporphyrin IX (PPIX): Cellular Fluorescence Analysis Unit (CFAU). (a) CFAU photograph: the unit consists of a sample tube rack (1), an elastic aspiration capillary (2), a fluorescence detection box (3) and a collecting tube rack (4). CFAU has a light-shielding cover to meet the need for protection against light exposure during the analysis. (b) Schematic representation of the fluorescence detection box. Numbers 1–4 correspond to those in a. The detection box contains a light-emitting diode (LED) producing stable excitation at a single wavelength of 400 nm. In this box, the cell suspension flows through a quartz capillary (100 μm diameter). The flowing cells are excited and the fluorescence is processed by the photodiode and spectrophotometer. The fluorescence intensity of each sample is measured and determined by integral value for 5 s. The spectrum of fluorescence is shown in the operating computer. (c) PPIX solution (1 ng/mL) was subjected to CFAU. Excitation light (400 nm) and PPIX-specific emission fluorescence at 635 nm are indicated by black arrows. a.u., arbitrary unit.

Figure 5

Sensitivity of Cellular Fluorescence Analysis Unit (CFAU) in the detection of urothelial carcinoma. (a) T24 cells without ALA treatment were used as simulated non-cancerous cells. The mixed cell suspension (10⁵ cells/mL) composed of untreated T24 cells and ALA-treated T24 cells at indicated ratios was prepared in PBS. Red arrows indicate the detectable fluorescent peaks emitted by ALA-treated cells. (b) T24 cells and urine samples from cases with bladder cancer were subjected to CFAU. Two representative cases are shown. One harbored a pTa/low-grade papillary-growing tumor, and the other harbored a pTis/high grade tumor. Black arrows indicate the background of urine sediments. Red arrows indicate the PPIX-specific fluorescence in comparison with non-ALA control.

Figure 6

Result comparison of conventional cytology, fluorescence cytology and fluorescent spectrophotometric assay by contingency tables. (a) Fluorescence cytology versus conventional cytology. (b) Fluorescent spectrophotometric assay versus conventional cytology. (c) Fluorescence cytology versus fluorescent spectrophotometric assay.