| Zugriffsnummer | 33689 |
| Dokumenttyp | Zeitschriftenartikel |
| Peer Review | unbekannt |
| Sprache | Englisch |
| Titel | Quantification of cells with specific phenotypes II: Determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody |
| Autor(in); Institution |
Wang, L.; Biosystems and Biomaterials Division,National Institute of Standards and Technology (NIST), Gaithersburg, USA
Stebbings, R.; Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK
Gaigalas, A.; Biosystems and Biomaterials Division,National Institute of Standards and Technology (NIST), Gaithersburg, USA
Sutherland, J.; Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK
Kammel, Martin; 8.3, Biomedizinische Optik, PTB-Berlin
John, Manuela; 8.3, Biomedizinische Optik, PTB-Berlin
Roemer, B.; Abbott GmbH & Co. KG, Wiesbaden, GERMANY
Kuhne, M.; Department of Analytical Chemistry, BAM Federal Institute for Materials Research and Testing, Berlin, GERMANY
Scheider, R.; Department of Analytical Chemistry, BAM Federal Institute for Materials Research and Testing, Berlin, GERMANY
Braun, M.; Beckman Coulter GmbH, Krefeld, GERMANY
Engel, A.; Becton Dickinson GmbH, Heidelberg, GERMANY
Dikshit, D.; Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute (CDRI), Lucknow, INDIA
Abbasi, F.; Center for Devices and Radiologic Health, Food and drug Administration (CDRH/FDA), Bethesda, USA
Marti, G.; Center for Devices and Radiologic Health, Food and drug Administration (CDRH/FDA), Bethesda, USA
Sassi, M.; Amount of Substance, Istituto Nazionale di Ricerca Metrologica (INRIM), Torino, ITALY
Revel, L.; Amount of Substance, Istituto Nazionale di Ricerca Metrologica (INRIM), Torino, ITALY
Kim, S.; Bioanalysis, Korea Research Institute of Standards and Science (KRISS), Daejeon, KOREA
Baradez, M.; Science and Innovation, LGC Limited, Teddington, UK
Lekishvili, T.; Science and Innovation, LGC Limited, Teddington, UK
Marshall, D.; Science and Innovation, LGC Limited, Teddington, UK
Whitby, L.; UK National External Quality Assessment Service (UK NEQAS) for Leucocyte Immunophenotyping, Sheffield, UK
Jing, W.; Division of Medical and Biological Measurement, National Institute of Metrology (NIM), Beijing, CHINA
Ost, V.; Partec GmbH, Muenster, GERMANY
Vonsky, M.; Department of State Standards in the Field of Physical - Chemical Measurements, D.I. Mendeleev Institute for Metrology (VNIIM), St-Petersburg, RUSSIA
Neukammer, Jörg; 8.3, Biomedizinische Optik, PTB-Berlin
|
| Quelle/Jahr | Cytometry A: 87 (2015), 3, 254 - 261 |
| ISSN | 0196-4763 (PRINT) ; 1552-4922 (ONLINE) |
| DOI | |
| Verlag | Hoboken, NJ: Wiley Blackwell |
| Freie Schlagworte | surface labelled lyophilized PBMC ; CD4 expression level ; FITC ; equivalent fluorescein fluorophore (EFF) ; quantitative flow cytometry ; calibration ; standard measurement procedure ; measurement uncertainty ; reference cell material |
| Zusammenfassung | This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants’ raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units. |
Zitierung
Wang, L., Stebbings, R., Gaigalas, A., Sutherland, J., Kammel, M., John, M., Roemer, B., Kuhne, M., Scheider, R., Braun, M., Engel, A., Dikshit, D., Abbasi, F., Marti, G., Sassi, M., Revel, L., Kim, S., Baradez, M., Lekishvili, T., Marshall, D., Whitby, L., Jing, W., Ost, V., Vonsky, M., & Neukammer, J. (2015). Quantification of cells with specific phenotypes II: Determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody. Cytometry A, 87(3), 254–261. https://doi.org/10.1002/cyto.a.22634