Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches
The validity of endothelial progenitor cells as biomarkers and their therapeutic potential depend on the accuracy of techniques used for enumeration. This study assessed the agreement between 6 flow cytometric methods and a CFU assay used for EPC quantification. Two blood samples were obtained from...
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| Veröffentlicht in: | Journal of immunological methods 2008-03, Vol.332 (1), p.31-40 |
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| container_title | Journal of immunological methods |
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| creator | Van Craenenbroeck, Emeline M.F. Conraads, Viviane M.A. Van Bockstaele, Dirk R. Haine, Steven E. Vermeulen, Katrien Van Tendeloo, Viggo F. Vrints, Christiaan J. Hoymans, Vicky Y. |
| description | The validity of endothelial progenitor cells as biomarkers and their therapeutic potential depend on the accuracy of techniques used for enumeration. This study assessed the agreement between 6 flow cytometric methods and a CFU assay used for EPC quantification.
Two blood samples were obtained from 30 healthy volunteers (60 samples). CD34+/VEGFR2+ cells were analyzed with flow cytometry, starting from whole blood (
A–C) or PBMC (
D–F), using different gating strategies:
A: lymphocyte gating;
B and D: exclusion of autofluorescent cells (CD3 negative selection);
C and E: exclusion of autofluorescence and cell aggregates (pulse shape analysis by FSCarea/FSCpeak);
F: exclusion of autofluorescence, cell aggregates and non-nucleated cells (Draq 5). PBMC were cultured under endothelial cell conditions to assess CFU numbers.
Moderate agreement was found between methods B–C and D–E (ICC 0.647 and 0.530). Comparison of methods B–D and C–E showed poor agreement (ICC 0.178 and 0.249). This was also the case for techniques that considerably differed with regard to gating strategies (A–B, A–F, B–F). CFU numbers did not correlate with flow cytometric quantification (all
p
>
0.05).
Agreement between methods for EPC quantification is moderate to poor, which may explain apparent controversies in literature. Although each protocol is highly reproducible, this study cautions against comparing study results gathered with different enumeration techniques. |
| doi_str_mv | 10.1016/j.jim.2007.12.006 |
| format | Article |
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Two blood samples were obtained from 30 healthy volunteers (60 samples). CD34+/VEGFR2+ cells were analyzed with flow cytometry, starting from whole blood (
A–C) or PBMC (
D–F), using different gating strategies:
A: lymphocyte gating;
B and D: exclusion of autofluorescent cells (CD3 negative selection);
C and E: exclusion of autofluorescence and cell aggregates (pulse shape analysis by FSCarea/FSCpeak);
F: exclusion of autofluorescence, cell aggregates and non-nucleated cells (Draq 5). PBMC were cultured under endothelial cell conditions to assess CFU numbers.
Moderate agreement was found between methods B–C and D–E (ICC 0.647 and 0.530). Comparison of methods B–D and C–E showed poor agreement (ICC 0.178 and 0.249). This was also the case for techniques that considerably differed with regard to gating strategies (A–B, A–F, B–F). CFU numbers did not correlate with flow cytometric quantification (all
p
>
0.05).
Agreement between methods for EPC quantification is moderate to poor, which may explain apparent controversies in literature. Although each protocol is highly reproducible, this study cautions against comparing study results gathered with different enumeration techniques.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/j.jim.2007.12.006</identifier><identifier>PMID: 18255093</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Adult ; Biological and medical sciences ; Cell Count ; Circulating endothelial progenitor cells ; Colony forming unit assay ; Colony-Forming Units Assay - methods ; Endothelial Cells - cytology ; Female ; Flow cytometric quantification ; Flow Cytometry - methods ; Fundamental and applied biological sciences. Psychology ; Fundamental immunology ; Humans ; Male ; Molecular immunology ; Rare event analysis ; Reproducibility of Results ; Stem Cells - cytology ; Techniques</subject><ispartof>Journal of immunological methods, 2008-03, Vol.332 (1), p.31-40</ispartof><rights>2007 Elsevier B.V.</rights><rights>2008 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c478t-974e812a550356629a4a1d3c2f174a4268b3e4d7d9880e7461e95d70fe87e9d23</citedby><cites>FETCH-LOGICAL-c478t-974e812a550356629a4a1d3c2f174a4268b3e4d7d9880e7461e95d70fe87e9d23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jim.2007.12.006$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20259147$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18255093$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Van Craenenbroeck, Emeline M.F.</creatorcontrib><creatorcontrib>Conraads, Viviane M.A.</creatorcontrib><creatorcontrib>Van Bockstaele, Dirk R.</creatorcontrib><creatorcontrib>Haine, Steven E.</creatorcontrib><creatorcontrib>Vermeulen, Katrien</creatorcontrib><creatorcontrib>Van Tendeloo, Viggo F.</creatorcontrib><creatorcontrib>Vrints, Christiaan J.</creatorcontrib><creatorcontrib>Hoymans, Vicky Y.</creatorcontrib><title>Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches</title><title>Journal of immunological methods</title><addtitle>J Immunol Methods</addtitle><description>The validity of endothelial progenitor cells as biomarkers and their therapeutic potential depend on the accuracy of techniques used for enumeration. This study assessed the agreement between 6 flow cytometric methods and a CFU assay used for EPC quantification.
Two blood samples were obtained from 30 healthy volunteers (60 samples). CD34+/VEGFR2+ cells were analyzed with flow cytometry, starting from whole blood (
A–C) or PBMC (
D–F), using different gating strategies:
A: lymphocyte gating;
B and D: exclusion of autofluorescent cells (CD3 negative selection);
C and E: exclusion of autofluorescence and cell aggregates (pulse shape analysis by FSCarea/FSCpeak);
F: exclusion of autofluorescence, cell aggregates and non-nucleated cells (Draq 5). PBMC were cultured under endothelial cell conditions to assess CFU numbers.
Moderate agreement was found between methods B–C and D–E (ICC 0.647 and 0.530). Comparison of methods B–D and C–E showed poor agreement (ICC 0.178 and 0.249). This was also the case for techniques that considerably differed with regard to gating strategies (A–B, A–F, B–F). CFU numbers did not correlate with flow cytometric quantification (all
p
>
0.05).
Agreement between methods for EPC quantification is moderate to poor, which may explain apparent controversies in literature. Although each protocol is highly reproducible, this study cautions against comparing study results gathered with different enumeration techniques.</description><subject>Adult</subject><subject>Biological and medical sciences</subject><subject>Cell Count</subject><subject>Circulating endothelial progenitor cells</subject><subject>Colony forming unit assay</subject><subject>Colony-Forming Units Assay - methods</subject><subject>Endothelial Cells - cytology</subject><subject>Female</subject><subject>Flow cytometric quantification</subject><subject>Flow Cytometry - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fundamental immunology</subject><subject>Humans</subject><subject>Male</subject><subject>Molecular immunology</subject><subject>Rare event analysis</subject><subject>Reproducibility of Results</subject><subject>Stem Cells - cytology</subject><subject>Techniques</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU2LFDEQhoMo7rj6A7xILnrrtpJOOh09LYtfsCCCnkM2qZ7J0N0Zk7Q6_94MM-hNT0Xgqacq9RLynEHLgPWv9-0-zC0HUC3jLUD_gGzYoHijNMiHZAPAecOU1FfkSc57AGDQw2NyxQYuJehuQ45fVruUMAZnS4gLjSN1Ibl1qs9lS3HxsexwCnaihxS3uIQSE3U4TfkNvaEzll30cYrbKpioi_PBppDPohx-0XGKP6k7lljJFBy1h6qxbof5KXk02injs0u9Jt_ev_t6-7G5-_zh0-3NXeOEGkqjlcCBcVv37WTfc22FZb5zfGRKWMH74b5D4ZXXwwCoRM9QS69gxEGh9ry7Jq_O3jr4-4q5mDnk0wfsgnHNRoEAIVX3X5CDUkpLVkF2Bl2KOScczSGF2aajYWBOwZi9qcGYUzCGcVODqT0vLvL1fkb_t-OSRAVeXgCb6ynHZBcX8h-OA5eaCVW5t2cO681-BEwmu4CLQx8SumJ8DP9Y4zeVPqzT</recordid><startdate>20080320</startdate><enddate>20080320</enddate><creator>Van Craenenbroeck, Emeline M.F.</creator><creator>Conraads, Viviane M.A.</creator><creator>Van Bockstaele, Dirk R.</creator><creator>Haine, Steven E.</creator><creator>Vermeulen, Katrien</creator><creator>Van Tendeloo, Viggo F.</creator><creator>Vrints, Christiaan J.</creator><creator>Hoymans, Vicky Y.</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T5</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20080320</creationdate><title>Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches</title><author>Van Craenenbroeck, Emeline M.F. ; Conraads, Viviane M.A. ; Van Bockstaele, Dirk R. ; Haine, Steven E. ; Vermeulen, Katrien ; Van Tendeloo, Viggo F. ; Vrints, Christiaan J. ; Hoymans, Vicky Y.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c478t-974e812a550356629a4a1d3c2f174a4268b3e4d7d9880e7461e95d70fe87e9d23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Adult</topic><topic>Biological and medical sciences</topic><topic>Cell Count</topic><topic>Circulating endothelial progenitor cells</topic><topic>Colony forming unit assay</topic><topic>Colony-Forming Units Assay - methods</topic><topic>Endothelial Cells - cytology</topic><topic>Female</topic><topic>Flow cytometric quantification</topic><topic>Flow Cytometry - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>Humans</topic><topic>Male</topic><topic>Molecular immunology</topic><topic>Rare event analysis</topic><topic>Reproducibility of Results</topic><topic>Stem Cells - cytology</topic><topic>Techniques</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Van Craenenbroeck, Emeline M.F.</creatorcontrib><creatorcontrib>Conraads, Viviane M.A.</creatorcontrib><creatorcontrib>Van Bockstaele, Dirk R.</creatorcontrib><creatorcontrib>Haine, Steven E.</creatorcontrib><creatorcontrib>Vermeulen, Katrien</creatorcontrib><creatorcontrib>Van Tendeloo, Viggo F.</creatorcontrib><creatorcontrib>Vrints, Christiaan J.</creatorcontrib><creatorcontrib>Hoymans, Vicky Y.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Van Craenenbroeck, Emeline M.F.</au><au>Conraads, Viviane M.A.</au><au>Van Bockstaele, Dirk R.</au><au>Haine, Steven E.</au><au>Vermeulen, Katrien</au><au>Van Tendeloo, Viggo F.</au><au>Vrints, Christiaan J.</au><au>Hoymans, Vicky Y.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>2008-03-20</date><risdate>2008</risdate><volume>332</volume><issue>1</issue><spage>31</spage><epage>40</epage><pages>31-40</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>The validity of endothelial progenitor cells as biomarkers and their therapeutic potential depend on the accuracy of techniques used for enumeration. This study assessed the agreement between 6 flow cytometric methods and a CFU assay used for EPC quantification.
Two blood samples were obtained from 30 healthy volunteers (60 samples). CD34+/VEGFR2+ cells were analyzed with flow cytometry, starting from whole blood (
A–C) or PBMC (
D–F), using different gating strategies:
A: lymphocyte gating;
B and D: exclusion of autofluorescent cells (CD3 negative selection);
C and E: exclusion of autofluorescence and cell aggregates (pulse shape analysis by FSCarea/FSCpeak);
F: exclusion of autofluorescence, cell aggregates and non-nucleated cells (Draq 5). PBMC were cultured under endothelial cell conditions to assess CFU numbers.
Moderate agreement was found between methods B–C and D–E (ICC 0.647 and 0.530). Comparison of methods B–D and C–E showed poor agreement (ICC 0.178 and 0.249). This was also the case for techniques that considerably differed with regard to gating strategies (A–B, A–F, B–F). CFU numbers did not correlate with flow cytometric quantification (all
p
>
0.05).
Agreement between methods for EPC quantification is moderate to poor, which may explain apparent controversies in literature. Although each protocol is highly reproducible, this study cautions against comparing study results gathered with different enumeration techniques.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>18255093</pmid><doi>10.1016/j.jim.2007.12.006</doi><tpages>10</tpages></addata></record> |
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| ispartof | Journal of immunological methods, 2008-03, Vol.332 (1), p.31-40 |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Adult Biological and medical sciences Cell Count Circulating endothelial progenitor cells Colony forming unit assay Colony-Forming Units Assay - methods Endothelial Cells - cytology Female Flow cytometric quantification Flow Cytometry - methods Fundamental and applied biological sciences. Psychology Fundamental immunology Humans Male Molecular immunology Rare event analysis Reproducibility of Results Stem Cells - cytology Techniques |
| title | Quantification of circulating endothelial progenitor cells: A methodological comparison of six flow cytometric approaches |
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