SEARCH

SEARCH BY CITATION

PURPOSE AND APPROPRIATE SAMPLE TYPE

  1. Top of page
  2. PURPOSE AND APPROPRIATE SAMPLE TYPE
  3. BACKGROUND
  4. LITERATURE CITED
  5. Supporting Information

This panel was optimized for the evaluation of circulating endothelial cells (CECs) in peripheral blood (see Table 1). The combination of three different endothelial cell markers enables a reasonable analysis of CECs. The panel, so far tested on fresh human peripheral and cord blood, can be used to enumerate CECs in a dual platform method.

thumbnail image

Figure 1. Example of staining and gating. Cells were not gated on morphological parameters, indeed only artifacts in the upper left and lower right corner were excluded. Aggregates were gated out on a FSC-Area (FSC-A) versus FSC-High (FSC-H) dot plot. Debris, dead cells, microparticles, and platelets were excluded by gating Syto16+ cells and NiRed cells. Leucocytes were excluded by gating CD45 and CD45dim cells. CD31+ cells were then identified; CD34+ and CD146+ cells were separately gated and analyzed by boolean gating. CD106 and CD117 gates are shown as well. Live CD45CD31+CD34+CD146+ CECs are displayed in a CD106 versus SSC and a CD117 versus SSC low resolution zebra plot. Dot plots show 8,000 events each, while pseudocolor plots and low resolution zebra plots show all analyzed events. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

Table 1. Summary table for application of OMIP-011
PurposeCharacterization of CECs in peripheral blood
SpeciesHuman
Cell typesWhole blood, RBC-lysed and washed
Cross referencesn.a.
Table 2. Reagents used for OMIP-011
SpecificityFluorochrome/ReagentAb ClonePurpose
  1. APC - allophycocyanin, Cy - cyanine, ECD - energy-coupling dye, FITC - fluorescein isothiocyanate, NiRed - LIVE/DEAD fixable Near - infrared dead cells stain, PE - R-phycoerythrin.

Dead cellsNiRed Exclusion of dead cells
CD45Alexa Fluor 700HI30Exclusion of leucocytes
DNASyto 16 DNA marker
CD31Alexa Fluor 647M89D3 
CD34ECD581Endothelial cell marker
CD146PEP1H12 
CD117PE-Cy7104D2Progenitor marker
CD106PE-Cy551-10C9Activation marker

BACKGROUND

  1. Top of page
  2. PURPOSE AND APPROPRIATE SAMPLE TYPE
  3. BACKGROUND
  4. LITERATURE CITED
  5. Supporting Information

CECs as well as bone-marrow-derived endothelial precursor cells (EPCs) are very rare events in the peripheral blood that have a high potential diagnostic value in different diseases which are characterized by cardiovascular problems and/or angiogenesis, e.g., cancer, ischemia, and diabetes.

Analysis of CECs is difficult because CECs are often discriminated using a combination of antigens with low, dull, or a continuum of cell surface expression (1). Since CECs cannot be characterized by a single marker, a combination of at least two markers is necessary. Therefore, different combination of several endothelial markers (CD31, CD34, CD146, KDR, and CD144) was used in order to get a more accurate discrimination of CECs. Such a test evidenced that KDR and CD144 were very weakly expressed on the CEC cell surface and could not be reliably analyzed, whereas CD31, CD34, and CD146 were largely detected and therefore chosen for the panel (see Figure 1 and supporting information). Dead cells, microparticles, and platelets were excluded from the analysis by using a DNA stain (Syto16) and a live/dead marker (NiRed) (see Table 2). Leucocytes were excluded by gating CD45 cells (2). The addition of the progenitor marker CD117 enables to distinguish between CECs and EPCs. CD106 is expressed on endothelial cells after stimulation with cytokines and allows analysis of activated subsets of CECs.

Since the number of cells acquired influences the detection sensitivity of CECs, large numbers of cells were acquired to reliably quantify very rare CECs (40–100 cells per ml blood) (3). Four milliliters whole blood, red blood cells (RBC)-lysed and washed, were stained and about 10 million events per sample were acquired in order to ensure sufficient numbers of CECs. The use of CECs as diagnostic marker demands the enumeration of absolute numbers of CECs and EPCs per ml blood. The number of these cells per ml peripheral blood was calculated by acquiring, in a dual platform procedure, an additional tube with TruCount beads (BD) and whole blood stained with Syto16 (4). The application of a dual platform method was necessary in order to allow the analysis of rare events. It must be noted that the possible cell loss occurring in such a procedure, which involves lyse/wash steps, is a random event and does not affect CEC subsets more than other nucleated events.

Similarity to published panels

None to date.

LITERATURE CITED

  1. Top of page
  2. PURPOSE AND APPROPRIATE SAMPLE TYPE
  3. BACKGROUND
  4. LITERATURE CITED
  5. Supporting Information
  • 1
    Duda DG,Cohen KS,di Tomaso E,Au P,Klein RJ,Scadden DT,Willett CG,Jain RK. Differential CD146 Expression on circulating versus tissue endothelial cells in rectal cancer patients: Implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy. J Clin Oncol 2006; 24: 14491453.
  • 2
    Strijbos MH,Kraan J,den Bakker MA,Lambrecht BN,Sleijfer S,Gratama JW. Cells meeting our immunophenotypic criteria of endothelial cells are large platelets. Cytometry B Clin Cytom 2007; 72B: 8693.
  • 3
    Woywodt A,Blann AD,Kirsch T,Erdbruegger U,Banzet N,Haubitz M,Dignat-George F. Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: Proposal of a definition and a consensus protocol. J Thromb Haemost 2006; 4: 671677.
  • 4
    Hultin LE,Chow M,Jamieson BD,O'Gorman MR,Menendez FA,Borowski L,Denny TN,Margolick JB. Comparison of interlaboratory variation in absolute T-cell counts by single-platform and optimized dual-platform methods. Cytometry B Clin Cytom 2010; 78B: 194200.

Supporting Information

  1. Top of page
  2. PURPOSE AND APPROPRIATE SAMPLE TYPE
  3. BACKGROUND
  4. LITERATURE CITED
  5. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22071_sm_SuppFig1.tif6930KSupplemental Figure 1. Reagent titrations . Reagents were titrated on 4 ml whole peripheral blood, RBC-lysed, from healthy donors. Single FCS files were concatenated to show titrations for each reagent in a single plot. Antibodies and reagents concentrations are displayed along the x-Axis. CD146 and CD106 titrations were carried out by adding CD45 and CD34 antibodies and analyzing CD45-/CD34+ events. Concentrations used in OMIP are highlighted in red.
CYTO_22071_sm_SuppFig2.tif3177KSupplemental Figure 2. Positive controls. Reagent titrations were performed on other cell types. A CD34-ECD staining (20/100) on cord blood cells. B Staining of CD106 PE-Cy5 (2/100) on TNF-alpha-activated (16 H, 1 ng / ml) HUVEC cells (left plot) compared to control (CTRL – right plot). C Internal controls for CD34 and CD117: CD45+/CD34+/CD117+ cells represent the well-known circulating haemopoietic stem cell compartment. D Internal control for CD146 staining: CD45+/CD146+ cells are peripheral activated-T lymphocytes.
CYTO_22071_sm_SuppFig3.tif11408KSupplemental Figure 3. Gating strategy and FMO controls. A Cells were not gated on morphological parameters, only artifacts in the upper left and lower right corner were excluded. Aggregates were gated out on a FSC-Area (FSC-A) vs. FSC-High (FSC-H) dot plot. Debris, dead cells, microparticles, and platelets were excluded by gating Syto16+ cells and NiRed- cells. Leucocytes were excluded by gating CD45-, CD45dim and CD31+ cells. CD34+, CD146+, CD106+ and CD117+ cells were separately gated. B Appropriate FMO controls are shown. Dot plots show 8,000 events each, whereas pseudocolour plots and low resolution zebra plots show all analyzed events.
CYTO_22071_sm_SuppFig4.tif4919KSupplemental Figure 4. Spillover into all detectors. CompBeads were labeled individually with each of 6 fluorochromes and blood samples were stained with Syto16 or NiRed. The percentage of light from the excitation of the primary fluorochrome contaminating each of the other detectors was calculated, as suggested by Perfetto SP et al. (Nat Rev Immunol, 2004). Each bar represents the percentage of the signal subtracted in the given detector, following the compensation procedure.
CYTO_22071_sm_SuppTab1.tif3821KSupplemental Table 1. Instrument configuration. The panel was optimized for a FACSCanto II with the listed optical elements. Long pass dichroic filters were used throughout the instrument. Light is filtered to each photomultiplier tube such that only wavelengths lower than long pass filter and within the band pass are detected. DPSS – Diode Pumped Solid State, HeNe – Helium-Neon, Cy – cyanine, ECD – energy-coupling dye, LP – long pass, NiRed – LIVE/DEAD fixable near-infrared dead cells stain, PE – R-phycoerythrin.
CYTO_22071_sm_SuppTab2.tif6501KSupplemental Table 2. Commercially available reagents used in OMIP-011.
CYTO_22071_sm_SuppTab3.tif8048KSupplemental Table 3. Reagents tested, but not used. FITC – fluorescein isothiocyanate, H7 - Hilite®7, PerCP - peridinin chlorophyl protein-conjugated.
CYTO_22071_sm_SuppInfo.doc36KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.