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Updated: Jun 3, 2025
Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry
Published on: June 26, 2018
Evaluation of the effect of cell freshness on pyrogen detection using a serum-free monocyte-activation test
Katsuhiko Hayashi1, Mizuki Sano2, Toshie Kanayasu-Toyoda3
1Division of Microbiology, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan.
Abstract:
Pyrogens cause shock symptoms when released into the bloodstream. They are classified into two main categories: endotoxins (lipopolysaccharides [LPS]) and non-endotoxin pyrogens. The monocyte activation test (MAT) is an in vitro assay to detect pyrogens in human monocytes. Cells were incubated in the culture medium, and the cellular response, specifically the production of the inflammatory cytokine interleukin-6 in the culture supernatant, was analyzed using enzyme-linked immunosorbent assay (ELISA). Technical improvements, such as cell acquisition and culture media selection, will be beneficial for the popularization of MAT. The cell freshness was strictly controlled to achieve high MAT sensitivity. However, it is necessary to investigate the usability of older and stored blood samples in the MAT. This study evaluated the effect of cell freshness on MAT using peripheral blood mononuclear cells (PBMCs) isolated from 2- and 5-d-old donated whole blood samples. To mitigate the influence of serum in the culture medium, a serum-free MAT was developed using the LPS-binding protein (LBP) as an enhancer for LPS detection. PBMCs were incubated with a two-fold dilution series of LPS at 0.001-4.096 endotoxin units/mL (EU/mL). Interleukin-6 levels in the culture supernatant were quantified by ELISA in the presence and absence of LBP. In the presence of LBP, the limit of detection (LOD) for LPS was 0.001-0.008 EU/mL. However, in the absence of LBP, the LOD was 0.512 EU/mL. Peripheral PBMCs were 38.6 times more sensitive in the presence of LBP than in its absence. When utilizing the developed serum-free MAT with LBP, 5-d-old PBMCs showed LODs of 0.016-0.064 EU/mL, indicating a 3.1-fold increase in sensitivity compared with 5- to 2-d-old PBMCs. These results suggest that the sensitivity of PBMCs decreased gradually rather than sharply. The study concluded that 2-d-old PBMCs were sufficiently fresh and could be used as serum-free MAT.
Insights
This study shows that 2-day-old peripheral blood mononuclear cells (PBMCs) are suitable for a serum-free monocyte activation test (MAT) to detect pyrogens. The developed method enhances sensitivity, allowing for the use of stored blood samples in pyrogen testing.
Area of Science:
- Biotechnology
- Immunology
- Analytical Chemistry
Background:
- Pyrogens, including lipopolysaccharides (LPS), can cause severe shock symptoms when entering the bloodstream.
- The monocyte activation test (MAT) is an in vitro assay for pyrogen detection, typically using human monocytes and measuring interleukin-6 (IL-6) production.
- Optimizing cell acquisition and culture media is crucial for improving MAT sensitivity and widespread adoption.
Purpose of the Study:
- To evaluate the impact of cell freshness on the sensitivity of the monocyte activation test (MAT).
- To develop a serum-free MAT assay utilizing LPS-binding protein (LBP) for enhanced pyrogen detection.
- To determine the usability of stored peripheral blood mononuclear cells (PBMCs) in pyrogen testing.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples aged 2 and 5 days.
- A serum-free MAT was developed, incorporating LPS-binding protein (LBP) to enhance lipopolysaccharide (LPS) detection.
- Interleukin-6 (IL-6) levels were quantified using ELISA in response to varying LPS concentrations (0.001–4.096 EU/mL) with and without LBP.
Main Results:
- The limit of detection (LOD) for LPS was significantly lower in the presence of LBP (0.001–0.008 EU/mL) compared to its absence (0.512 EU/mL), indicating a 38.6-fold increase in sensitivity.
- Using the serum-free MAT with LBP, 5-day-old PBMCs showed LODs of 0.016–0.064 EU/mL, demonstrating a 3.1-fold increase in sensitivity compared to 2-day-old PBMCs.
- PBMC sensitivity decreased gradually with storage time, rather than sharply.
Conclusions:
- Two-day-old PBMCs are sufficiently fresh for use in a serum-free MAT assay.
- The developed serum-free MAT with LBP significantly enhances pyrogen detection sensitivity.
- Stored blood samples, specifically 2-day-old PBMCs, can be reliably used in MAT, facilitating broader application of pyrogen testing.
