Internationale Zeitschrift für Herz-Kreislauf-Forschung

Test-Retest Reliability of Flow-Cytometric Quantification of Circulating Endothelial Cells and Endothelial Progenitor Cells in Cardiovascular Diseases

Josephine Wagner1, Fabian Weisrock1, Max Fritschka2, Sebastian Beckmann1, Simon Litmeier1, Elvis Tahirovic1, Sara Radenovic3, Andreas Busjahn4, Thomas Krahn5, Wilfried Dinh6* and Hans-Dirk Düngen1*#

Circulating Endothelial Cells (CECs) and Endothelial Progenitor Cells (EPCs) are gaining importance as quantifiable surrogate biomarkers of Endothelial Dysfunction (ED). Lack of a common definition and, consequently, a standardized quantification method, has limited clinical applicability of these biomarkers. A reliable, reproducible, and practicable method is required to achieve the full potential of these parameters. The objective of this study was to evaluate the test- retest reliability over a short period of time (~7 days) of flow cytometric quantification of CECs and EPCs in human whole blood from patients with cardiovascular disease. 100 patients (mean age, 65 ± 10 years, 30 women) were enrolled into a prospective study consisting of 4 patients groups: heart failure with reduced ejection fraction (HFrEF; n=25), heart failure with preserved ejection fraction (HFpEF; n=26), diabetic nephropathy (DN; n=25), and hypertension (HTN; n=24). In addition, 11 healthy volunteers were included as a control group. At 2 study visits, a blood sample was drawn, which underwent an identical sequence of preparation and analysis. CECs (DNA+, CD45dim, CD31+, and CD146+) and EPCs (CD45dim, CD34br, CD133+, and CD31+, FSClow– medium, SSClow) were counted via flow cytometry. To assess short-term test-retest reliability, correlation (intraclass correlation) and agreement (Bland-Altman plot) of the measurements obtained at the 2 study visits were evaluated. Across all patients, median CECs/mL and EPCs/mL were 12 (5th/95th percentile: 6/22) and 679 (447/1281) at visit 1 and were 11 (6/24) and 736 (510/1105) at visit 2, respectively; intraclass correlation (ICC) was poor for CEC count (0.106; ICC- 95% CI -0.08–0.29) and good for EPC count (0.9; 0.86–0.93). In patients with HFpEF, ICC was poor for CEC count (0.294; 95% CI -0.08–0.6) and moderate in strength for EPC count (0.694; 0.43–0.85). In patients with HFrEF, ICC was poor for CEC count (0.076; -0.32–0.45) and excellent in strength for EPC count (0.946; 0.88–0.98). In patients with DN, ICC was poor for CEC count (-0.031; -0.44– 0.37) and excellent in strength for EPC count (0.946; 0.88–0.98). In patients with HTN, ICC was poor for CEC count (0.143; -0.27–0.51) and moderate in strength for EPC count (0.668; 0.37–0.84). In healthy controls, ICC was poor for CEC count (0.378; -0.26–0.78) and good in strength for EPC count (0.846; 0.59–0.96). A Bland-Altman plot showed a positive correlation of variations of differences and increasing median CEC counts; there were no distinct trends for median EPC counts. Our analyses indicate that flow cytometric quantification of EPC concentrations is reliable in patients with HFpEF, HFrEF, DN, and HTN. Quantification of CEC concentrations showed poor test-retest reliability across all patient groups. Further research is necessary to elucidate the nature of this finding, which could be due to higher biological variability in patients with severe ED. Clinical Trial Registration Identifier: NCT02299960.