Enzyme-labeled liquid-based cytology (ELLBC): a new noninvasive diagnostic method for bladder cancers

Fifty patients who were initially diagnosed with suspected bladder cancers, based on symptoms such as hematuria or bladder irritation and urinary ultrasound suggestive of bladder mass, were selected from the Second Affiliated Hospital of Anhui Medical University (Anhui, China) between January 2022 and December 2022 (Patients with a combination of other urologic tumors and a combination of severe infectious diseases were excluded). Non-morning urine samples were collected from the patients, and ELLBC and CC tests were performed. Before participating in the study, all patients signed a consent form that allowed clinicians to access their medical records for clinical information. The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (Ethics Approval No. YX2022-064). The process is illustrated in Fig. 1.

After admission, 100 mL of fresh urine (non-morning urine) was collected from the patients using enzyme-free EP tubes for examination by ELLBC and CC, respectively. Only one urine sample is required for ELLBC and three samples are required on three consecutive days for CC. The CC slide preparation involved double centrifugation of the sediment obtained from the urine samples, followed by Papanicolaou staining. The slides were then sent to the pathologist for microscopic reading after completion of the smear and transparency operations. For ELLBC, 100 mL of collected fresh urine was centrifuged at 2000 rpm for 10 min, washed with enzyme-labeled liquid-based cytological preservation solution, and prepared until the cells were partially dry. The partially dry cell smear was then fixed in a fixing solution at room temperature for 4 min to remove red blood cells, rinsed with tap water for 2 min, placed in a staining solution of acid phosphatase at 37 °C for 5–10 min, rinsed with running water for 2 min, dyed with nuclear redyeing solution for 5 min, rinsed again with running water for 2 min, and finally sealed with neutral gum after thorough air drying. The cytology reports were completed using the Paris system and the results were categorized as positive or negative. Positive results indicated confidence or suspicion of malignancy, while negative results indicated findings other than malignancy, such as inflammation or benign conditions. The final diagnosis of the patient was based on the histopathological findings. The dyeing process of ELLBC is shown in Figure S1, and the composition of the acid phosphatase stain solution is described in Table S1. The cytology reports were completed using the Paris system, with results categorized as positive or negative. Positive results indicated confidence or suspicion of malignancy, while negative results indicated findings other than malignancy, such as inflammatory or benign conditions. In all cases, the diagnosis was confirmed by the same pathologist, and the final diagnosis of the patient was based on the histopathological findings, which served as the gold standard. Figure 2 shows a representative image of the staining.

The study's findings revealed that in the urine cytology of patients with chronic haematuria, the CC preparation's background was more muddy (Fig. 5).