title: Quantification of circulating endothelial and progenitor cells: comparison of quantitative PCR and four-channel flow cytometry.
abstract: BACKGROUND
  Circulating endothelial cells (CEC) and endothelial precursor cells (CEP) have been suggested as markers for angiogenesis in cancer. However, CEC/CEP represent a tiny and heterogeneous cell population, rendering a standardized monitoring in peripheral blood difficult. Thus, we investigated whether a PCR-based detection method of CEC/CEP might overcome the limitations of rare-event flow cytometry.
  
  
  RESULTS
  To test the sensitivity of both assays endothelial colony forming cell clones (ECFC) and cord blood derived CD45- CD34+ progenitor cells were spiked into peripheral blood mononuclear cells (PBMNC) of healthy volunteers. Samples were analyzed for the expression of CD45, CD31, CD34, KDR or CD133 by 4-color flow cytometry and for the expression of CD34, CD133, KDR and CD144 by qPCR. Applying flow cytometry, spiked ECFC and progenitor cells were detectable at frequencies >/= 0.01%, whereas by qPCR a detection limit of 0.001% was achievable. Furthermore, PBMNC from healthy controls (n = 30), patients with locally advanced rectal cancer (n = 20) and metastatic non-small cell lung cancer (NSCLC, n = 25) were analyzed. No increase of CEC/CEP was detectable by flow cytometry. Furthermore, only CD34 and KDR gene expression was significantly elevated in patients with metastatic NSCLC. However, both markers are not specific for endothelial cells.
  
  
  CONCLUSIONS
  QPCR is more sensitive, but less specific than 4-channel flow cytometry for the detection of CEC/CEP cell types. However, both methods failed to reliably detect an increase of CEC/CEP in tumor patients. Thus, more specific CEC/CEP markers are needed to validate and improve the detection of these rare cell types by PCR-based assays.
authors.affiliation: Tumor Biology and Angiogenesis Laboratory, Division of Hematology and Oncology, Innrain 66, Innsbruck Medical University, 6020 Innsbruck, Austria. michael.steurer@i-med.ac.at
authors.forename: Michael
authors.initials: M
authors.lastname: Steurer
authors.forename: Johann
authors.initials: J
authors.lastname: Kern
authors.forename: Matthias
authors.initials: M
authors.lastname: Zitt
authors.forename: Albert
authors.initials: A
authors.lastname: Amberger
authors.forename: Monika
authors.initials: M
authors.lastname: Bauer
authors.forename: Günther
authors.initials: G
authors.lastname: Gastl
authors.forename: Gerold
authors.initials: G
authors.lastname: Untergasser
authors.forename: Eberhard
authors.initials: E
authors.lastname: Gunsilius