# Application of spectral flow cytometry for comprehensive detection of immune metabolism in patient-derived microsamples

## TL;DR

A new spectral flow cytometry platform enables detailed analysis of immune cell metabolism using small blood samples, revealing metabolic changes in heart failure patients.

## Contribution

A high-dimensional spectral flow cytometry platform for immunometabolic profiling using minimal clinical microsamples.

## Key findings

- The platform allows 20-parameter analysis from just 100 μL of whole blood.
- Heart failure patients show reduced naive T cells and NK-like T cells with a shift to glucose-dependent metabolism.
- The method captures metabolic interactions not accessible with traditional sequential detection.

## Abstract

Single-cell metabolic characteristics are powerful indicators of cellular physiological and pathological states. Flow cytometry enables high-throughput single-cell metabolic profiling and immunophenotyping; however, spectral overlap impedes simultaneous detection of multi-parametric features. This limitation imposes larger sample requirements and increased variabilities due to metabolic dynamics—constraints acutely magnified in studies utilizing clinical microsamples. To overcome these shortcomings, we developed a spectral flow cytometry platform for immunometabolic features, using 13 dual-probe combinations and 11 fluorophore probes. Requiring only 100 μL of whole blood per assay, this platform enables concurrent detection of 4 metabolic biomarkers and 16 immune markers. Application to patients with heart failure revealed heterogeneous metabolic landscapes across 20 immune subpopulations, showing reduced frequencies of naive T cells and NK-like T cells, with a metabolic shift from fatty acid dependence to glucose avidity. Our framework captures metabolic interactions previously inaccessible by sequential detection and will help to enable precision immunometabolism research.

•A spectral flow cytometry platform for single-cell immunometabolic profiling•The platform enables 20-parameter analysis from 100 μL peripheral blood•We identify metabolic shifts in lymphocytes from patients with heart failure

A spectral flow cytometry platform for single-cell immunometabolic profiling

The platform enables 20-parameter analysis from 100 μL peripheral blood

We identify metabolic shifts in lymphocytes from patients with heart failure

Immunometabolism underpins a wide range of disease states. Spectral flow cytometry has advanced immunophenotyping, but its potential for assaying single-cell, multi-parametric metabolic characteristics has not been fully unlocked. Here, we leverage the multiplexing capacity of spectral flow cytometry to establish a platform for simultaneous assessment of multiple metabolic and immune features with limited sample consumption.

Bai et al. develop a spectral flow cytometry platform for high-dimensional analysis of single-cell immunometabolic signatures using small amounts of peripheral blood. Application to samples from patients with heart failure suggests a metabolic shift toward glucose use in T cell subsets, demonstrating the method’s potential for precision studies of immunometabolism in disease.