Nucleosomes in serum as a marker for cell death

Area of Science:

• Biochemistry
• Clinical Chemistry
• Oncology

Background:

• Circulating nucleosomes are elevated in diseases with enhanced cell death.
• Accurate detection of nucleosomes is vital for disease monitoring.

Purpose of the Study:

• To adapt and validate the Cell Death Detection-ELISAplus (CDDE) assay for quantifying circulating nucleosomes.
• To investigate pre-analytical factors affecting nucleosome stability and detection.
• To assess the clinical utility of nucleosome detection in patients with solid tumors.

Main Methods:

• Modified the CDDE assay to include a standard curve for direct quantification.
• Assessed intra-assay and inter-assay variability (CVintraassay: 3.0-4.11%, CVinterassay: 8.6-13.5%).
• Evaluated plasma vs. serum matrices and detailed pre-analytical handling (venipuncture, centrifugation, contamination, stabilization, storage).

Main Results:

• Established a reliable method for quantifying circulating nucleosomes with good reproducibility.
• Identified critical pre-analytical factors: hemolysis, delayed processing, bacterial contamination (false positives), and improper storage (false negatives).
• Serum samples are stable for at least 6 months at -20°C when treated with 10 mM EDTA.
• Significantly higher nucleosome concentrations were found in patients with solid tumors (361 AU) compared to healthy individuals (30 AU; p=0.0001).
• Advanced tumor stages (UICC 4) showed higher nucleosome levels than early stages (UICC 1-3; p=0.0004).

Conclusions:

• The modified CDDE assay provides a robust method for quantifying circulating nucleosomes.
• Strict adherence to recommended pre-analytical procedures is essential for accurate results.
• Elevated nucleosome levels correlate with the presence and stage of solid tumors, suggesting potential as a diagnostic or prognostic biomarker.