Fig. 1
Extended leukocyte sample quality window defined for up to 72h in refrigerated urine matrix. a Experimental protocol of the healthy donor leukocytes spiking experiments. Blood leukocytes from four healthy donors were spiked at three concentrations (103, 104 or 105 cells/mL) into cell-free urine supernatants obtained from either healthy donors (n = 2) or untreated NMIBC patients (n = 2) and analysed by flow cytometry either fresh or after incubation for 24h, 48h or 72h at 4°C. This figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. b Gating strategy from urine spiked with different numbers of leukocytes (103, 104 or 105 cells): after debris, doublets and dead cells exclusion, frequencies of monocytes (CD45+CD14+CD3−), T cells (CD45+CD3+CD14−) and granulocytes (CD45+CD15+CD14−) were determined. Since no difference in stability was observed between different concentrations of spiked leukocytes, these were therefore treated as technical replicates. c Frequency of granulocytes (CD15+), monocytes (CD14+) and T cells (CD3+) from spiked leukocytes after 0h, 24h, 48h or 72h at 4℃. Comparisons were made using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (with no statistically significant differences). Graphs include n = 6 independent leukocyte/urine matrix conditions per timepoint. d Correlations between the frequencies of immune cell subsets determined in fresh samples (0h) and samples refrigerated for 24h, 48h or 72h. A Spearman r test with two-tailed P-value was performed