Comparative analysis of circulating endothelial progenitor cells in age-related macular degeneration patients using automated rare cell analysis (ARCA) and fluorescence activated cell sorting (FACS)

PLoS One. 2013;8(1):e55079. doi: 10.1371/journal.pone.0055079. Epub 2013 Jan 24.


Background: Patients with age-related macular degeneration (ARMD) begin with non-neovascular (NNV) phenotypes usually associated with good vision. Approximately 20% of NNV-ARMD patients will convert to vision debilitating neovascular (NV) ARMD, but precise timing of this event is unknown. Developing a clinical test predicting impending conversion to NV-ARMD is necessary to prevent vision loss. Endothelial progenitor cells (EPCs), defined as CD34(+)VEGR2(+) using traditional fluorescence activated cell sorting (FACS), are rare cell populations known to be elevated in patients with NV-ARMD compared to NNV-ARMD. FACS has high inter-observer variability and subjectivity when measuring rare cell populations precluding development into a diagnostic test. We hypothesized that automated rare cell analysis (ARCA), a validated and FDA-approved technology for reproducible rare cell identification, can enumerate EPCs in ARMD patients more reliably. This pilot study serves as the first step in developing methods for reproducibly predicting ARMD phenotype conversion.

Methods: We obtained peripheral venous blood samples in 23 subjects with NNV-ARMD or treatment naïve NV-ARMD. Strict criteria were used to exclude subjects with known angiogenic diseases to minimize confounding results. Blood samples were analyzed in masked fashion in two separate laboratories. EPCs were independently enumerated using ARCA and FACS within 24 hours of blood sample collection, and p<0.2 was considered indicative of a trend for this proof of concept study, while statistical significance was established at 0.05.

Results: We measured levels of CD34(+)VEGFR2(+) EPCs suggestive of a trend with higher values in patients with NV compared to NNV-ARMD (p = 0.17) using ARCA. Interestingly, CD34(+)VEGR2(+) EPC analysis using FACS did not produce similar results (p = 0.94).

Conclusions: CD34(+)VEGR2(+) may have predictive value for EPC enumeration in future ARCA studies. EPC measurements in a small sample size were suggestive of a trend in ARMD using ARCA but not FACS. ARCA could be a helpful tool for developing a predictive test for ARMD phenotype conversion.

Publication types

  • Comparative Study

MeSH terms

  • Aged
  • Aged, 80 and over
  • Automation*
  • Cell Separation
  • Endothelial Cells / cytology
  • Female
  • Flow Cytometry
  • Fluorescence
  • Humans
  • Macular Degeneration / blood*
  • Male
  • Middle Aged
  • Stem Cells / cytology*

Grant support

SHC would like to acknowledge the Adler Foundation, Hope for Vision Career Development Award, and Research to Prevent Blindness for unrestricted grant support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.