Enrichment of Circulating Endothelial Cells by CD34 Microbeads Followed by Enumeration Using Flow Cytometry

Abstract

Background Circulating endothelial cells (CECs) are a potential biomarker of angiogenesis. CECs increase in numbers after vessel injury. Higher CEC numbers are reported in cancer patients. Most methods for CEC detection and enumeration rely on flow cytometry (FCM); however, there is no agreement on CEC phenotype and the detection method to be used. This leads to uncertainty about the clinical applicability and variation between studies on CEC numbers reported. Objective To develop a selective and accurate method for CEC enumeration in peripheral blood by enrichment, followed by FCM in healthy volunteers (HV) and cancer patients. Methods Samples were enriched using CD34 microbeads, stained with nuclear dye and anti-CD14, CD15, CD45, CD34 and CD146 antibodies. Putative CECs were examined for Weibel–Palade bodies (WPBs) using anti–von Willebrand factor (vWF) antibody and fluorescence microscopy. Linear range of detection (R 2), recovery and precision (coefficient of variation percentage [CV%]) were defined in three experiments by spiking a known number (range 12–12,800 CECs/4 mL) of surrogate endothelial cells in peripheral blood. Sample storage was determined at –80°C for up to 2 months. Results Sorted CECs showed vWF in the WPBs. The relationship between spiked and detected surrogate cells was R 2 = 1.0, recovery of 94.0 to 101.4% and CV% of 1.0 to 18.4%. Recovery ± standard deviation (within-run days 1, 2 and 3) were, respectively, 102.5% ± 8.2, 97.8% ± 4.6, 99.1% ± 7.7, and after 2 months 94.3% ± 15.3. The median CECs/mL in patients was 24.1 versus 14.4 in HVs. Conclusion This method for selective, sensitive and reliable CEC analysis by FCM allows for investigation of CECs as a biomarker in clinical research.

A Phase 1 Dose‐Escalation Study of Low‐Dose Metronomic Treatment With Novel Oral Paclitaxel Formulations in Combination With Ritonavir in Patients With Advanced Solid Tumors

“…Total levels of plasma TSP-1 were measured in duplicate in platelet-rich and platelet-poor plasma with an enzyme-linked immunosorbent assay using commercially available kits (Quantikine, R&D systems, Minneapolis, Minnesota). 48 Blood from the 2 CTAD tubes for CEC enumeration was prepared according to the method developed by Burylo et al 49 The mean TSP-1 (in ng/mL/10 6 platelets) and CEC levels (in CECs per milliliter) were calculated from the plasma values per time point. The fold change from baseline of the mean levels, with the baseline level set at 0, and standard deviation of each time point was calculated per patient.…”