Flow cytometry-based validation of soluble biomarker detection

TL;DR

This study validates a flow cytometry assay for detecting four soluble immune markers with high sensitivity and accuracy, which could aid in diagnosing lung cancer.

Contribution

A validated multiplex flow cytometry assay for simultaneous detection of sCD25, sCD40L, sCD130, and sTREM-1 in serum.

Findings

The assay achieved low LOD values for sCD25, sCD40L, sCD130, and sTREM-1, showing high sensitivity.

Significant differences in soluble marker levels were observed between lung cancer patients and healthy individuals.

The method demonstrated reproducibility through intra- and inter-assay variability assessments.

Abstract

Accurate and reliable measurement of soluble markers, such as sCD25, sCD40L, sCD130, and sTREM-1, is crucial for understanding their roles in immune responses and inflammatory conditions. This study presents the validation of a multiplex flow cytometry assay using the BD FACSLyric flow cytometer, designed to simultaneously quantify these four soluble markers in serum samples.

The assay utilizes a bead-based immunoassay kit that is compatible with BD FACSLyric to enable precise detection of low concentrations of these markers. The methodological approach includes detailed sample preparation, bead conjugation, and assay optimization. Data acquisition was performed on the BD FACSLyric, with a minimum of 10 000 events recorded per sample to ensure robust data collection.

The validation results demonstrated that the assay achieved low limits of detection (LOD) for sCD25, sCD40L, sCD130, and sTREM-1. The LOD values were respectively 9.77 pg/ml, 43.95 pg/ml, 219.73 pg/ml, and 12.21 pg/ml, indicating the assay's high sensitivity. Reproducibility was confirmed through intra-assay and inter-assay variability assessments. Studies have shown that the levels of sCD25/sCD40L/sTREM-1 in the body are associated with lung cancer. Therefore, this study also used this detection method to test both lung cancer patients and healthy individuals, finding significant differences in the results, which can be used to assist in clinical diagnosis and treatment.

This assay's ability to detect multiple analytes in a single run, combined with its high sensitivity and reproducibility, makes it a valuable asset for both research and clinical applications. Future work may expand this assay to include additional markers or adapt it for other sample types, further enhancing its utility in diverse biomedical contexts.

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