Machine learning assisted microfluidics dual fluorescence flow cytometry for detecting bladder tumor cells based on morphological characteristic parameters

Bladder cancer (BC) is the most common malignant tumor and has become a major public health problem, leading the causes of death worldwide. The detection of BC cells is of great significance for clinical diagnosis and disease treatment. Urinary cytology based liquid biopsy remains high specificity for early diagnosis of BC, however, it still requires microscopy examination which heavily relies on manual operations. It is imperative to investigate the potential of automated and indiscriminate cell differentiation technology to enhance the sensitivity and efficiency of urine cytology. Here, we developed a machine learning algorithm empowered dual-fluorescence flow cytometry platform (μ-FCM) for urinary cytology analysis. A phenotype characteristic parameter (CP) which correlated with the size of the cell and nucleus was defined to achieve the differentiation of the BC cells and uroepithelial cells with high throughput and high accuracy. Based on CP analysis, SV-HUC-1 cells were almost differentiated from EJ cells and effectively reduced the overlap with 5637 cells. To further differentiate SV-HUC-1 cells and 5637 cells, support vector machine (SVM) machine learning algorithm was optimized to assist data analysis with the highest accuracies of 84.7 % for cell differentiation including the specificity of 91.0 % and the sensitivity of 75.0 %. Furthermore, the false positive rate (FPR) compensation enabled the detection rates of rare BC cells predicted by the well-trained SVM model were close to the true proportions with the recognition error in 0.4 % for the tumor cells. As a proof of concept, the developed μ-FCM system successfully demonstrates the capacity to identify the distribution of exfoliated cells in real urine samples. This system underscores the significance of integrating AI with microfluidics to perform high-throughput phenotyping of exfoliated cells, offering a pathway toward scalable, efficient, and automatic microfluidic systems in the fields of both biosensing and in vitro diagnosis of BC.