Addition of formaldehyde releaser imidazolidinyl urea and MOPS buffer to urine samples enables delayed processing for flow cytometric analysis of urinary cells

Abstract

Kidney diseases are a major health concern worldwide. Currently there is a large unmet need for novel biomarkers to non-invasively diagnose and monitor kidney diseases. Urinary cells are promising biomarkers and their analysis by flow cytometry has demonstrated its utility in diverse clinical settings. However, up to date this methodology depends on fresh samples, as cellular event counts and the signal-to-noise-ratio deter over time. Here we developed an easy-to-use two-step preservation method for conservation of urine samples for subsequent flow cytometry. The protocol utilizes a combination of the formaldehyde releasing agent imidazolidinyl urea (IU) and MOPS buffer, leading to gentle fixation of urinary cells. The preservation method increases acceptable storing time of urine samples from several hours to up to 6 days. Cellular event counts and staining properties of cells remain comparable to fresh untreated samples. The hereby presented preservation method facilitates future investigations on flow cytometry of urinary cells as potential biomarkers and may enable broad implementation in clinical practice.

Urinary CD8+HLA-DR+ T Cell Abundance Non-invasively Predicts Kidney Transplant Rejection

“…We used spontaneously voided urine. We developed a urine-cup-based fixation system with imidazolidinyl urea (IU, Sigma-Aldrich) and 3-(N-morpholino)propanesulfonic acid (MOPS, Carl Roth GmbH + Co. KG) to preserve urine samples (23). Specimen were stored at 4 • C for up to 7 days, centrifuged (600 g, 6 min) and frozen in 90% fetal calve serum (FCS) and 10% dimethylsulfoxide (DMSO) (cohort 1 and 3).…”