Stable Circulating Histone Complexes for Non-Invasive Liquid Biopsy Diagnostics

Dataset Description

This dataset comprises prospectively and repeatedly collected peripheral blood samples obtained from four healthy adult volunteers (two males and two females) in Brno, Czech Republic, between 2023 and 2025. The dataset contains quantitative and imaging-based measurements of histones and histone complexes in human serum and plasma under controlled experimental conditions.

Type of Data
The dataset includes:

·       Multispectral imaging flow cytometry data (ImageStreamX MkII)

·       Quantitative measurements of histone abundance and histone complex formation

·       Processed and gated cytometry outputs

Sample Collection and Processing
Peripheral blood samples were collected from healthy donors using two standardized protocols to obtain serum and plasma:

·       Serum samples: Blood was drawn into serum-separating tubes, allowed to clot at room temperature for 30 minutes, and centrifuged at 1000 g, 2000 g, 4000 g, and 8000 g for 10 minutes at 4 °C.

·       Plasma samples: Blood was collected into EDTA-coated tubes and centrifuged under identical conditions.

Following processing, all samples were immediately stored at −80 °C to preserve molecular integrity for downstream analyses.

Temporal Design
Samples were collected repeatedly over time from the same individuals to enable longitudinal analysis. The dataset also includes measurements assessing:

·       Effects of long-term storage

·       Effects of repeated freeze–thaw cycles

·       Temporal variation in histone levels

Experimental Measurements
Histone levels and histone complexes were quantified using multispectral imaging flow cytometry (ImageStreamX MkII, Luminex Corporation). Samples were immunostained with predefined panels of primary and fluorescent secondary antibodies.

For each measurement:

·       25 μL of serum or plasma was incubated overnight at 4 °C with primary antibodies

·       Secondary antibody staining was performed for 2 hours at room temperature

·       A total of 20,000 events per sample were acquired using multiple laser excitation channels

Data Processing and Analysis
Raw image data were processed using the IDEAS® 6.2 software package. A standardized three-step gating strategy was applied:

1.     Focus selection based on image quality metrics

2.     Exclusion of aggregates and artifacts

3.     Identification of fluorescence-positive objects corresponding to histones and their complexes

Histone levels and complex abundances were quantified as a percentage of the total gated population.

Statistical Analysis
Statistical evaluation of the dataset was performed using GraphPad Prism (version 10.01). The analysis included:

·       Averaging of histone abundances across repeated experiments

·       Parametric Welch’s t-test for normally distributed data

·       Non-parametric Mann–Whitney U test for non-normal distributions

·       Wilcoxon test for paired comparisons (e.g., long-term storage effects)

·       Mixed-effects matched-pairs analysis for repeated measurements (e.g., time-course and freeze–thaw effects)

Data are reported as mean ± standard deviation (SD). Statistical significance thresholds were defined as:
p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****).

The provided dataset contains primary, minimally processed data only; therefore, the accompanying files do not include any statistical analyses or derived statistical outputs.