# Xbeads: A Platform for Flow Cytometry Sample QC and Harmonization

> **NIH NIH R43** · CYTORUM, INC. · 2022 · $253,911

## Abstract

Summary
 Flow cytometry assays continue to expand in basic research and development, diagnostics, and
clinical studies, particularly in the fields of immuno-oncology, CAR-T cells, and antibody therapeutics for
various oncology and autoimmune diseases. The success of this platform is largely due to its unmatched
ability to measure 30 or more proteins per cell in complex primary blood cell populations at a rate of
thousands of cells per second. However, the complexity of these assay systems makes them challenging to
standardize and perform reproducibly over time and across instruments, severely hampering the types of
flow cytometry assays that can be applied in diagnostic and clinical settings. The variability associated with
flow cytometry assays arises from the difficulty in standardizing across the many instrument, reagent, and
experimental variables that multiply rapidly in more complex, high-parameter flow cytometry experiments.
While external quality control reagents and platforms such as instrument setup and compensation beads
and stabilized PBMC and whole blood controls exist, their utility is limited because they are run in parallel to
the test sample and therefore cannot capture and correct for any errors that occur during processing or
acquisition of individual test samples. Assay data can therefore vary significantly from sample to sample or
day to day, and the data acquired on different instruments is difficult to compare. Here, we propose a novel
solution to these limitations by creating an internal assay quality control system, termed Xbeads, that can be
added to each sample prior to staining and processing for flow cytometry. The beads are engineered to
assess the performance of the sample processing and data acquisition, and quantitatively measure the
activity of each antibody reagent. Importantly, any fluctuations in instrument or assay performance are
directly reflected by the fluorescent signals of the Xbeads. In this manner, samples can be normalized to the
internal Xbead signals and quantitatively compared over time, across instruments, or testing sites.
Preliminary data showed that Xbeads could be mixed, stained, and analyzed with PBMC samples, then
separated during data analysis, and each Xbead population could be clearly resolved. In this Phase I SBIR,
we will build upon these initial results by optimizing the Xbead system for reproducibility, specificity, and
long-term stability. The system will be tested by acquiring samples that have known errors in processing,
antibody performance, or instrument setup. The successful completion of the Phase I experiments will form
the foundation of a complete internal assay QC system for flow cytometry that will enable site to site, day to
day, and instrument to instrument normalization for highly complex antibody panels.

## Key facts

- **NIH application ID:** 10485014
- **Project number:** 1R43GM146557-01
- **Recipient organization:** CYTORUM, INC.
- **Principal Investigator:** Peter Krutzik
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $253,911
- **Award type:** 1
- **Project period:** 2022-03-10 → 2023-11-09

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10485014

## Citation

> US National Institutes of Health, RePORTER application 10485014, Xbeads: A Platform for Flow Cytometry Sample QC and Harmonization (1R43GM146557-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10485014. Licensed CC0.

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