Addition of formaldehyde releaser imidazolidinyl urea and MOPS buffer to urine samples enables delayed processing for flow cytometric analysis of urinary cells: A simple, two step conservation method of urinary cells for flow cytometry

Abstract

IntroductionKidney 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.MethodsThe protocol utilizes a combination of the formaldehyde releasing agent imidazolidinyl urea (IU) and MOPS buffer, leading to gentle fixation of urinary cells.ResultsThe 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.OutlookThe 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 CD4+ T Cells Predict Renal Relapse in ANCA-Associated Vasculitis

“…Spontaneously voided mid-stream urine samples (median volume 60 ml) were obtained at baseline, followed by immediate dipstick analysis and on-site fixation using our previously reported urine cup preservation set with 3-( N -morpholino)propanesulfonic acid (MOPS, Carl Roth GmbH+ Co. KG) buffer and imidazolidinyl urea (IU, Sigma-Aldrich), enabling storage at 4°C for a maximum of 7 days. 29 For conservation of urine samples, we combined liquid MOPS buffer (1 mol/L) and powdered IU (20 g/L of total volume) in a ratio of 1:3 relative to the total volume. The MOPS buffer was prepared as follows: 20.95 g MOPS, 0.82 g of sodium acetate (Carl Roth), and 1.85 g of ethylenediamine tetraacetic acid (Sigma-Aldrich) were dissolved in 100 ml of deionized water.…”

Issue highlights—November 2023

“…Thus, preservation of urine samples for delayed analysis of urine cells, currently remains an unmet need. In this issue of Cytometry B, Freund et al (2023) developed a simple, two‐step, preservation procedure for urine samples, based on gentle fixation of urinary cells in imidazolidinyl urea as fixative and MOPS to prevent precipitate formation. The method proved suitable for further flow cytometric analyses of urinary cells for up to 6 days, in the absence of significant numerical and phenotypic changes (Freund et al, 2023).…”

“…In this issue of Cytometry B, Freund et al (2023) developed a simple, two‐step, preservation procedure for urine samples, based on gentle fixation of urinary cells in imidazolidinyl urea as fixative and MOPS to prevent precipitate formation. The method proved suitable for further flow cytometric analyses of urinary cells for up to 6 days, in the absence of significant numerical and phenotypic changes (Freund et al, 2023). Thereby, the proposed preservation method paves the way for further investigations that would explore more in‐depth the potential utility of flow cytometry in both kidney and urinary tract diseases, not only in individual laboratories where a flow cytometer is available, but also in multicentric studies, as it might also facilitate shipment of samples from smaller centers to reference flow cytometry laboratories.…”

“…This new issue of Cytometry B (Clinical Cytometry) consists of five main manuscripts which contain original research in the field of clinical cytometry. A manuscript describing a simple (new) method for preservation of urinary cells for subsequent flow cytometric analyses (Freund et al, 2023) opens this issue of the journal. It is followed by three papers related to the application of flow cytometry in the field of acute leukemias.…”

“…vation of urinary cells for subsequent flow cytometric analyses (Freund et al, 2023) opens this issue of the journal. It is followed by three papers related to the application of flow cytometry in the field of acute leukemias.…”