Objective Mass cytometry (CyTOF) enables high-dimensional single-cell profiling, but especially for non-blood derived samples relies on immediate processing, limiting its application to fragile, low-yield clinical specimens such as urine. We developed a novel preservation protocol that combines viability staining with gentle fixation, enabling cryopreservation and delayed processing.

Methods Building on a previously established protocol using imidazolidinyl urea (IU) and MOPS buffer for flow cytometric applications, we enhanced the protocol for CyTOF by incorporating a one-minute pulse of cisplatin (5□µM) for live–dead discrimination. A novel quenching step with DL-methionine (5□mM) was introduced to minimize background signal without compromising antigen integrity. Samples were fixed overnight at 4□°C and cryopreserved prior to CyTOF analysis. The protocol was validated using peripheral blood mononuclear cells (PBMCs) and was applied to urine samples.

Results The preservation method maintained single-cell integrity, surface marker expression, and cisplatin staining specificity. DL-methionine efficiently quenched residual cisplatin reactivity. The protocol performed robustly across a wide range of cell inputs and maintained consistency following freeze–thaw cycles. In urine samples, immune cell subset frequencies and viability discrimination were comparable between the optimized and standard staining protocols.

Conclusion Our protocol enables delayed processing and cryopreservation of fragile clinical samples while preserving compatibility with mass cytometry workflows. By integrating cisplatin-based viability staining, DL-methionine quenching, and slow-release fixation, this method supports standardized immune profiling of low-abundance samples in clinical and translational research.

PE reoprts the following Grants or contracts from any entity: DFG, Jackstaedt Stiftung, BIH, BMBF, IBB, Vifor Consulting fees: Advisory Board Glaxo Smith Klein GSK,Bayer, AstraZeneca Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Akademie der Nieren, BDI Kickstarter, Nachlese ASN Charite, Novartis, NephroUpDate, FOMF, AstraZeneca, Alexion Patents planned, issued or pending: patent for preservation or urine cells (CHA124WO-EP-UP) Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: Spokesperson of the committee for intensive care of the German Society of Nephrology (DGfN), Vice President Deutsche Gesellschaft fuer Lupusforschung Other financial or non-financial interests: Cofounder of a Biotech Company (Kernevo; conservation of urine cells, biomarkers)

Part of this research was supported by the BIH Ad-hoc Booster grant awarded to NG.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethics committee of Charite Universitaetsmedizin Berlin gave ethical approval for this work.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors.