Immunomonitoring in bladder cancer: a short report on stability of leukocytes and proteins in refrigerated urine samples.

BACKGROUND

Urine biomarkers are crucial for monitoring patient responses in treating urological pathologies, including non-muscle invasive bladder cancer (NMIBC). Yet, analysing urine biomarkers poses several challenges, including ensuring specimen stability during transportation and analytical processing. This prospective feasibility study aimed to investigate how urinary leukocytes and proteins are impacted by storing and refrigerating urine.

METHODS

Stability of leukocytes from four healthy donors (HD) spiked into urine supernatants was analyzed for up to 72 h at 4°C. Urine samples from five NMIBC patients undergoing BCG treatment were divided into two portions, followed by either immediate processing or overnight refrigeration. Urinary cell content and soluble factors were analyzed by multiparameter flow cytometry and Luminex®, respectively.

RESULTS

We confirmed the stability of healthy donor peripheral blood leukocytes spiked into cell-free urine supernatants from healthy donors or untreated bladder cancer patients for up to 72 h under refrigeration at + 5℃. Additionally, we conducted immune cell and proteomic analysis from urine samples obtained from five NMIBC patients receiving Bacillus Calmette-Guérin (BCG) therapy either processed immediately or after overnight refrigeration. We successfully demonstrated that leukocyte and protein composition remain stable in refrigerated urine from BCG-treated NMIBC for 24 h. This included granulocytes, monocytes, and T cells, as well as total protein, creatinine and 46 additional individual immune-related mediators.

CONCLUSIONS

This work demonstrates the compatibility of refrigerated urine shipment from the collection sites to analytical laboratories with both immunophenotyping and proteomic analysis and establishes clear logistical benefits for numerous clinical settings focused on monitoring patient immune responses in the urine matrix.