Journal of Immunological Methods 335 (2008) 21–27

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Journal of Immunological Methods

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Research paper

High-reproducible flow cytometric endothelial progenitor cell determinationin human peripheral blood as CD34+/CD144+/CD3−lymphocyte sub-population

Santiago Redondo a, Mihail Hristov b,c, Antonio A. Gordillo-Moscoso a, Emilio Ruiz a,Christian Weber b, Teresa Tejerina a,⁎a Department of Pharmacology, School of Medicine, Universidad Complutense, Madrid, Spainb Institute for Molecular cardiovascular Research (IMCAR), RWTH Aachen University Hospital, Aachen, Germanyc Interdisciplinary Center for Clinical research “BIOMAT”, RWTH Aachen University Hospital, Aachen, Germany

a r t i c l e i n f o

Abbreviations: DAPI4′, 6-diamidino-2-phenylindon3,3,3′,3′-tetramethylindocarbocyanine perchlorate;genitor cells; EDTAethylenediamine tetraacetate; APCafluorescein isothiocyanate; PEphycoerythrin.⁎ Corresponding author.

E-mail address: teje@med.ucm.es (T. Tejerina).

0022-1759/$ – see front matter © 2008 Elsevier B.V.doi:10.1016/j.jim.2008.02.011

a b s t r a c t

Article history:Received 17 July 2007Received in revised form 1 January 2008Accepted 12 February 2008Available online 19 March 2008

Although determination of circulating endothelial progenitor cell (EPC) in peripheral blood byflow cytometry is an emerging marker for cardiovascular medicine, a common standardizedprotocol is still not available, due to the low numbers achieved in peripheral blood. In thepresent paper we describe a novel technique for EPC quantification as CD34+/CD144+/CD3−cells within the lymphocyte gate, which increases the percentages of EPC positivity describedbefore and also offers high intra-assay reproducibility. These improvements are based on agating strategy for big-sized lymphocytes, smooth fixation and cytometric clearance of CD3+lymphocytes (T-cells). This last procedure is able to increase intra-assay Pearson's correlationfrom 0.8517 to 0.8908. Therefore, the technical setting described here offers a high-performance and clinically oriented EPC determination strategy in human peripheral blood.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Flow cytometryEndothelial progenitor cellsMarkers

1. Introduction

One of the most outstanding medical breakthroughswithin the last years has been the identification, isolationand determination of the potential clinical importance ofendothelial progenitor cells (EPCs). Some reports relate theblood amount (Vasa et al., 2001a,b) and in vitro function (Hillet al., 2003) of these precursors and the clinical cardiovascularoutcome, expressed as Framingham's risk score (Vasa et al.,2001a,b). Moreover, their number in peripheral blood hasbeen related to future cardiovascular events (Schmidt-Luckeet al., 2005) angiographically defined coronary obstruction(Kunz et al., 2006), or endothelial function measured asbrachial dilation flow (Hill et al., 2003).

e; Dil1,1′-dioctadecyl-EPCsendothelial pro-llophycocyanin; FITC-

All rights reserved.

However, there is still no standardized protocol for EPCdetermination, a problem originated by the rarity of thesecells in the peripheral circulation (Khan et al., 2005). Sincepioneering reports of EPCs positivity from total lymphocytesyielded small differences in percentage (Schmidt-Lucke et al.,2005), current efforts are being made in order to enrich theEPC positivity and therefore enhance the technical strength,in order to establish a common protocol. State-of-the-arttechnical approaches are based on a smooth fixationprocedure (Rustemeyer et al., 2006) and cytometric clearanceof non-analysed populations (Khan et al., 2005).

The majority of blood calculations of EPCs as a pharma-cological target determine this cell type as the CD34+/KDR+lymphocyte sub-population (Vasa et al., 2001a,b; Schmidt-Lucke et al., 2005; Hristov et al., 2007). Nevertheless, recentreports have remarked the importance of mature CD34+/CD144+ cells as a marker of EPC differentiation in responseto statins, which may explain the transient increase andfurther decrease of CD34+/KDR+ EPCs in response to long-term treatment with statins (Deschaseaux et al., 2007;Hristov et al., 2007; Vasa et al., 2001a,b).

Table 1Demographic and clinical pattern of our described group of healthy subjects

Parameter Value

n 23Male/female 13/10Age 45.87±2.079Glucose (mg/dl) 83.04±2.408Creatinine (mg/dl) 0.929±0.025Triglycerides (mg/dl) 127.4±15.17Total cholesterol (mg/dl) 217.6±8.380HDL-cholesterol (mg/dl) 55.35±2.436LDL-cholesterol (mg/dl) 136.7±7.382Hemoglobin (g/dl) 14.75±0.273Platelets (cells/μl) 229400±12020White cells (cells/μl) 7600±522.5Eosinophils (cells/μl) 187.6±17.24Basophiles (cells/μl) 226.6±176.1Lymphocytes (cells/μl) 2616±178.0Monocytes (cells/μl) 504.2±55.07Neutrophils (cells/μl) 4246±353.6CD34+/CD144+ lymphocytes (%) 0.09504±0.01652CD34+/CD144+/CD3− lymphocytes (%) 0.1863±0.03853CD3 lymphocytes (%) 54.45±2.601Total CD34+/CD144+ (cells/μl) 1.848±0.2689

Themeasurement of EPCswas performed as described in Materials andmethodsand the table shows the mean value of duplicated measures from each subject.

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The aim of the current paper is to define and test theinternal reproducibility of a novel cytometric EPC determina-tion as CD34+/CD144+/CD3− cells from the lymphocyte gate,as a potential clinically-driven target for vascular pharmacol-ogy. The novelty of the method described here lies in the highintra-assay reproducibility and the significant increase of EPCpercentage in peripheral blood, due to three major technicalshifts: gating strategy for large lymphocytes, smooth fixationand cytometric clearance of T-cells.

2. Materials and methods

2.1. Population description

A cohort of 23 healthy subjects (13 males and 10 females,mean age 46±2 years) was recruited from workers of theUniversidad Complutense, Madrid, Spain. No acute or chronicdiseases or medications were present. Their blood wascollected after overnight harvest and used for preventivemedicine determinations, whereas the remaining blood waskept for this study (1–2 ml). Principles of the Declaration ofHelsinki were respected. This group of subjects volunteeredfor the current analysis and the blood extraction was notrequired from the Company. Moreover, according to theSpanish Sensitive Data Protection Act, their names weremaintained secret. The clinical and laboratory features ofanalyzed population are shown in Table 1.

2.2. Blood management

The blood was collected in EDTA-coated Vacutainer®tubes (Becton Dickinson, Franklin Lakes, USA) and kept onice. EPC staining was performed within next 2 h after bloodcollection. Prior to it, blood from every single tube wasseparated into five 100 μl aliquots in citrate-coatedEppendorf vials and noted as listed below: Allophycocyanin

(APC)-IgG1, Fluorescein Isothiocyanate (FITC)-IgG1, Phycoer-ythrin PE-IgG2B, and CD34/CD144/CD3 (last one induplicate).

2.3. Blood staining for mature EPC markers

Our main novelty was to perform routine EPC staining inperipheral blood and, additionally, to remove T-cells, markedas CD3+ lymphocytes, in order to increase the percentage oflymphocyte population which expresses EPC markers, giventhat the T-cell fraction includes the majority of lymphocytepopulation in healthy human peripheral blood.

Antibody staining was made according to a methodpreviously described (Hristov et al., 2007). Briefly, 10 μl ofrespective antibodies were added to each 100 μl of aliquotedblood: mouse IgG1K isotype control allophycocyanin con-jugated (555751, Becton Dickinson), mouse IgG1k isotypecontrol isothiocyanate fluorescein conjugated (555748, Bec-ton Dickinson) mouse IgG2B isotype control phycoerythrinconjugated (IC0041P, R&D Systems, Minneapolis, USA),mouse IgG1K anti-CD3 allophycocyanin conjugated (555335,Becton Dickinson), mouse IgG1k anti-CD34 isothiocyanatefluorescein conjugated (555821, Becton Dickinson), mouseIgG2B anti-CD144 (VE-cadherin) phycoerythrin conjugated(FAB9381P, R&D Systems). Then, the blood was gently mixedand incubated for 30 min on ice protected from light. Afterthat time, red blood cells were lysed by a commercial lysissolution (347691-MSDS-A, Becton Dickinson) for 10 min atroom temperature. Final paraformaldehyde concentrationwas 1%. Then, vials were centrifuged at 1000 rpm for 7 minand the pellet was resuspended in PBS. After two washingsteps, the vials (200 μl suspension) were kept at 4 °C,protected from the light, and cytometric analysis was madewithin the next 24 h.

2.4. Cytometric analysis

A FACSCalibur flow cytometer (Serial Number E2295,Becton Dickinson, San Jose, CA, USA) was used from theFlow Cytometry Service, Universidad Complutense deMadrid.Cell suspension was driven and the lymphocyte populationwas gated. Notably, we included large lymphocyte populationin our gating strategy (Fig. 1, panel A). Using appropriatefluorescence compensation, gated events were analysed forPE and FITC positivity, according to the threshold establishedin the same determination (CellQuestPro, Becton Dickinson).In order to increase the statistical n for an accurate cytometricanalysis, all the gated events of each entire sample (19,790±2586 events, expressed as mean±SEM of all analysed vials)were assessed.

Double FITC+/PE+ events were then considered as CD34+/CD144+ events, respectively (Fig. 1, panel E). At the samedetermination, we separated APC+ events as CD3+ T-cells,given that these lymphocytes are considered as the mostabundant lymphocyte population and their exclusion wouldrelatively increase the EPC population at the highest extent.Thus, the percentage of CD34+/CD144+ and CD34+/CD144+/CD3− lymphocytes were obtained in duplicate from everysingle subject. As an added advantage of this approach, thepercentage of CD3+ lymphocytes (T-cells) was obtained at thesame time.

Fig. 1. Panel A: Gating strategy for cytometric determinations. Human peripheral blood was stained and lysed as described in Materials and methods. Then, thelymphocyte populationwas gated, including big lymphocytes, as shown in the profile. Panel B, C and D: Cytometric profiles for control immunoglobulins: APC-IgG1,FITC-IgG1 and PE-IgG2B (respectively) were preformed as explained in Materials and methods. Panel E: Double positive FITC+/PE+ gated events were registered asdouble CD34+/CD144+ events, (upper right quadrant).

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2.5. FACS sorting and in vitro culture assays

In order to assess whether the procedure of CD3− T-cellexclusion can be reproduced in vitro, we performed a two-way high pressure aseptic sorting with yield precision mode(FACSAria, Becton Dickinson) to separate CD3+ versus CD3−lymphocyte sub-sets. Briefly, peripheral blood mononuclearcells (PBMCs) from buffy coat preparations of healthy donorswere separated by density gradient centrifugation withBiocoll separating solution (Biochrom AG, Berlin, Germany).After two washing steps, the PBMCs were incubated with FcRblocking reagent (Miltenyi Biote, Bergisch-Gladbach, Ger-many), 5 min on ice. They were then incubated with anti-human CD3−APC antibody (300311, Bio Legend, CA, USA),30 min on ice. After washing, labeled cells (5×107/ml) wereresuspended in HEPES-buffered Hank's Balanced Salt Solutionwith 0.5% BSA and 2 mmol/l EDTA, filtered trough a 35 μmnylonmesh and subjected to FACS sorting. Collected CD3+ and

CD3− lymphocytes (1×106) were plated on fibronectin-coated4-chamber slides (BD Falcon) or 6-well plastic plates andcultured in microvascular endothelial growth medium with5% FBS and supplements (MV-2; PromoCell, Heidelberg,Germany) as previously described (Hristov et al., 2007).After 7–10 days in culture, adherent cells were investigatedby fluorescence microscopy for uptake of Dil-Ac-LDL (CellSystems, St. Katharinen, Germany) and further stained for VE-cadherin (sc-6458, goat polyclonal antibody, Santa CruzBiotechnology, CA, USA). A secondary FITC-conjugated anti-goat antibody (Sigma-Aldrich, Taufkirchen, Germany) wasused for fluorescence detection. Finally, cells weremounted inVectashield with DAPI (Vector Laboratories Inc., CA, USA).Images were recorded using an Olympus IX71 inversemicroscope with CellM Imaging Software (Olympus, Ham-burg, Germany) and a Leica DMLB epifluorescence micro-scope (Leica Microsystems GmbH, Wetzlar, Germany) withDiskus Software (Hilgers, Königswinter, Germany).

Table 3Three different correlation coefficients (Pearson's intra-class correlationcoefficient or ICC and Lin's coefficient) are shown for CD3-exccludedpercentage of CD34+/CD144+ EPCs

Test Estimated IC 95%

Pearson 0.8908 0.7563–0.953ICC 0.85 0.68–0.930Lin 0.8051 0.643–0.8982

Notably, PearsonTs correlation increases up to 0.89 for duplicated measures.

Fig. 2. Percentages of CD34+/CD144+ lymphocytes for raw analyses (left bar,white) and when CD3+ cells are excluded (right bar, black). Of note, positivepopulation turns enriched in a significant manner. Each measurement wasperformed in duplicate.

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2.6. Statistics

Repeatability was analyzed by four different statisticalmethods, Pearson's correlation, ICC (Intra-class correlation),Lin's correlation coefficient, and Bland–Altman's limits ofconcordance. These statistical tests were done using free-access R software.

In addition, comparison for cell percentages was done by aStudent's t test (GraphPadPrism program, version 3.0).pb0.05 was considered as statistical significant.

3. Results

3.1. Role of T-cell clearance on the percentage of double-positivecells

As shown in Fig. 2, T-cell clearance using a CD3-APCantibody is able to increase the percentage of double-positiveCD34+/CD144+ EPCs within the lymphocyte population in asignificant manner.

3.2. Correlation coefficients

Correlation coefficients include the accepted threshold(Shrout and Fleiss, 1979; Lin, 1989; Kramer and Feinstein,1981; Bland and Altman,1986; Bland,1995) for three differentstatistical tests: Pearson's correlation, intra-class correlation(ICC) and Lin's correlation coefficient, both for raw (Table 2)and T cell-cleared (Table 3) CD34+/CD144+ lymphocytedetermination. Interestingly, Pearson's correlation isincreased when T-cells are excluded and therefore the finalpercentage is increased.

Table 2Three different correlation coefficients (Pearson's intra-class correlationcoefficient or ICC and Lin's coefficient) are shown for raw percentage of CD34+/CD144+ EPCs

Test Estimated IC 95%

Pearson 0.8517 0.6773–0.9355ICC 0.85 0.68–0.930Lin 0.8165 0.6287–0.9143

3.3. Dispersion graphs

Fig. 3 shows the dispersion of the points when thedifferences between the first and second measurementwere represented, both for raw (Panel A) and T cell-excluded

Fig. 3. Graph plot shows the data concerning Linn's repeatability coefficientfor raw (panel A, left) and CD3-excluded (panel B, right) EPCs measured asCD34+/CD144+ lymphocytes, as referred in Materials and methods. In bothcases, line passes near zero, although this is achieved in panel B in a deepermanner.

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(Panel B) CD34+/CD144+ lymphocytes. In panel B, the line islocated more closely to the zero segments.

In order to visualize the dispersion by anothermethod, theBland and Altman's limits of confidence were also repre-sented in Fig. 4, for raw (panel A) and Tcell-excluded (panel B)CD34+/CD144+ lymphocytes. Scatter points are located nearerthan the zero segments in panel B.

3.4. Results from the in vitro assay

Analysis of human PBMCs by flow cytometry revealed 67.4±4.5% CD3+ (P3) and 28.4±5.4% CD3− (P2) cells within thelymphocyte population P1, respectively (n=5; Fig. 5, panel A).Moreover, staining for CD34 and CD144 was intensivelypresent on the CD3− in contrast to the CD3+ sub-populationwithin the lymphocyte gate. Accordingly, populations P2 and

Fig. 4. Bland and Altman's concordance limits for raw (panel A, left) and CD3-exccluded (panel B, right) EPCs measured as CD34+/CD144+ lymphocytes, asexplained in Materials and methods. Although the majority of duplicatedevents are included within the limits, the scatter has a more concentratedappearance in panel B (right scheme), for CD3+ excluded EPCs.

P3were used for aseptic FACS sorting.While sorted CD3+ cellsdid substantially adhere neither on fibronectin-coated glassslides, nor on tissue culture plastic plates (data not shown), asmall fraction of the CD3− sub-population adhered on thefibronectin matrix. Interestingly, these cells acquired a round,cobblestone and spindle-shaped phenotype when furthermaintained in endothelial culture medium for 7–10 days(Fig. 5, panels B–D). Notably, the adherent CD3− cells showedpositivity for VE-cadherin (Fig. 5, panels E, F) and intensivelytook-up acetylated-LDL (Fig. 5, panel G), a phenotype andfunctional feature corresponding to endothelial cells. Thus, aCD3− lymphocyte sub-set from humanperipheral blood startsto differentiate into endothelial-like cells in vitro.

4. Discussion

First clinical studies with endothelial progenitors werebased on the culture of these cells, obtained from 15–20 ml ofpatient's peripheral blood, 5–7 days of culture and counting ofcolony forming units (Hill et al., 2003; Honold et al., 2006).The problems to be faced were related to the clinicaldisposability of this amount of blood from patients, as wellas the reproducibility of observations and the impossibility ofmassive analyses, due to the effort and the time-consumingnature of the technique. In contrast, direct cytometricdeterminations of vascular progenitors in blood offer theadvantage of a rapid, cheap and accessible technique, using asmall blood volume which can be collected from patients invirtually all clinical conditions (Vasa et al., 2001a,b; Schmidt-Lucke et al., 2005; Kunz et al., 2006; Hristov et al., 2007).

Studies about quantitative EPC determination in periph-eral blood and its relation to clinical parameters haveexperienced an exponential increase in recent years. Most ofthem regard these cells as an antiatherogenic factor, inverselycorrelated to Framingham's risk score (Hill et al., 2003; Vasaet al., 2001a,b), coronary obstruction (Kunz et al., 2006) andthe coming of future cardiovascular events (Schmidt-Luckeet al., 2005). However, others consider them as proathero-genic (George et al., 2005). A major difficulty for the inter-pretation of this apparent contradiction is the lack of astandard for EPC quantification in blood (Khan et al., 2005).Originated from CD34+ stem cells at the bone marrow level,these cells lose CD133+ and gain KDR+ in their process ofdifferentiation when they reach the peripheral blood, wherethey become CD31+, vonWillebrand factor+ and CD144+ priorto denuded vessel adhesion (Hristov andWeber, 2004). Due toits advanced position in the continuous process of EPCdifferentiation, CD144+ (or VE-cadherin) has gained impor-tance as a pharmacological target for statin treatment(Deschaseaux et al., 2007). In addition, CD34+ would avoidany potential overlapping between the EPC population andCECs, passively detached from disrupted vessels (Boos et al.,2007). Research on the EPC differentiation pathway has madetwo important distinctions according to theirmaturation statein vitro. Early-outgrowth EPCs are derived from bone marrowCD34+ or circulating CD14+ precursors and possess highcapacity of cytokine secretion (Asahara et al., 1997; Rehmanet al., 2003; Schober et al., 2005). However, their functionalcapacity to form vessel-like structures in vitro is relativelypoor. In contrast, late-outgrowth EPCs are more able toproliferate and to form capillary-like structures (Lin et al.,

Fig. 5. Panel A: Flow cytometry assessment of CD3− (P2, green dots) versus CD3+ (P3, blue dots,) lymphocyte sub-populations which have been further used foraseptic sorting. Staining for CD34 and CD144 on the gated CD3+ versus CD3− sub-populations within the lymphocyte gate (population P1). Representative dot plotswere shown. Panels B–D: Representative photomicrographs of adherent sorted CD3− cells. Panels E–G: Representative immunofluorescence staining for VE-cadherin (green; panels E, F) and Dil-Ac-LDL (red; panel G) of CD3− cells. Nuclei were stained with DAPI, respectively (blue; panels F, G). Magnification ×100 (panelsB, E) and ×200 (panels C, D, F, G).

26 S. Redondo et al. / Journal of Immunological Methods 335 (2008) 21–27

2000; Mukai et al., 2007), and possess highly advancedmarkers of endothelial differentiation, such as CD144 (VE-cadherin), Tie-2 and the endothelial nitric oxide synthase(Gulati et al., 2003). Notably, both cell clusters seem tosynergize in an animal experimental model of revasculariza-tion after limb ischemia (Yoon et al., 2005).

The vast majority of translational reports for EPC numberin blood are based on flow cytometry from lysed peripheralblood (Vasa et al., 2001a,b; Schmidt-Lucke et al., 2005; Kunzet al., 2006; Hristov et al., 2007). However, despite theadvantage of the broad clinical disposability of this technique,some concerns have been raised about the extremely lowfrequency of the EPC population in peripheral blood, whichmay often fall below the minimal resolution capacity of mostflow cytometry devices and therefore would avoid a reliabledetermination by using this method, even if control immu-noglobulins are used in these studies (Khan et al., 2005).

An important issue for EPC enrichment is a meditatedgating strategy and the clearance of other populations. In thepresent study, we have focused on the lymphocyte gate, aspreviously described (Grisar et al., 2005; Hristov et al., 2007;Vasa et al., 2001a,b). Nevertheless, the light scatter populationof big lymphocytes was included, given the large size and

complexity that EPCs acquire in culture. According to thisgating strategy, clearance of CD3+ T-cells was performed,since the CD3+ sub-population contains the majority oflymphocytes (above 70%) in peripheral blood and the stainingfor CD34 and CD144 was intensively present only on the CD3−sub-population. An additional advantage of this method is thesimultaneous calculation of T-cell number, an emerginginterest for immunological evaluation of atherosclerosis(Robertson and Hansson, 2006). Moreover, our in vitro resultsrevealed that sorted CD3− sub-sets possess adhesion poten-tial, develop distinct morphology in culture and initiatedifferentiation towards the endothelial lineage.

In the present study, key flow cytometry technical featuresin order to increase EPCmarker positivity have been taken intoconsideration. These features include a smooth lysis andfixation procedure and the avoidance of an extended storage.In this manner, Rustemeyer et al. (2006) were able to report anincrease of CD34+ from 1.3 (formaldehyde at 2% solution) to2.9% (formaldehyde at0.2% solution).Our resultswere obtainedusing a smooth fixation procedure (fixation at the time of lysis,10 min with a final formaldehyde concentration of 1%).

Taken together, the present study reports a reproducibleand clinically oriented method for CD34+/CD144+/CD3−

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lymphocyte quantification in human peripheral blood, acellular sub-population of late EPCs. This novel method fordetection of putative differentiated EPCs may prove anemerging target for current cardiovascular pharmacology.Nevertheless, the present technical setting offers the limita-tion of the asymptomatic status of the clinical cohort. Furtherlarger clinical studies will assess whether current observa-tions may be extrapolated to diseased populations andwhether quantitative measurements performed here aresuitable from a clinical point of view.

Acknowledgments

Our group receives funding from FISS PI041018 (HealthResearch Fund from the Spanish Ministry of Health) and fromRECAVA (Cardiovascular network of the Spanish Ministry ofHealth). The authors would like to thank the Servicio deMedicina del Trabajo and Centro de Microscopía y Citometría(Both of them fromUniversidad Complutense, Madrid, Spain),for their excellent technical assistance.

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