June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Pre-analytical parameters have significant impacts on VEGF levels measured from human plasma
Author Affiliations & Notes
  • Johanna Madeleine Walz
    Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany
    Eye center, University of Freiburg, Freiburg, Germany
  • Daniel Boehringer
    Eye center, University of Freiburg, Freiburg, Germany
  • Heidrun L Deissler
    Department of Ophthalmology, University of Ulm, Ulm, Germany
  • Peter Heiduschka
    Department of Ophthalmology, University of Muenster, Muenster, Germany
  • Alexa Klettner
    University medical center, Department of Ophthalmology, University of Kiel, Kiel, Germany
  • Tim U. Krohne
    Department of Ophthalmology, University of Bonn, Bonn, Germany
  • Focke Ziemssen
    Department for Ophthalmology, Eberhard-Karl University Tuebingen, Tuebingen, Germany
  • Andreas Stahl
    Eye center, University of Freiburg, Freiburg, Germany
  • Footnotes
    Commercial Relationships Johanna Walz, Novartis Pharma GmbH (E), Novartis Pharma GmbH (F), Novartis Pharma GmbH (R); Daniel Boehringer, None; Heidrun Deissler, Bayer Healthcare AG (C), Novartis Pharma GmbH (C), Novartis Pharma GmbH (F), Novartis Pharma GmbH (R); Peter Heiduschka, Bayer Healthcare AG (F), Bayer Healthcare AG (R), Novartis Pharma GmbH (F), Novartis Pharma GmbH (R); Alexa Klettner, Novartis Pharma GmbH (C), Novartis Pharma GmbH (F), Novartis Pharma GmbH (R); Tim Krohne, Bayer Healthcare AG (R), Heidelberg Engineering GmbH (R), Novartis Pharma GmbH (C), Novartis Pharma GmbH (F), Novartis Pharma GmbH (R); Focke Ziemssen, Alcon Pharma GmbH (R), Alimera Sciences (C), Bayer Healthcare AG (C), Bayer Healthcare AG (R), Biogen Idec (C), Biogen Idec (R), Novartis Pharma GmbH (C), Novartis Pharma GmbH (F), Novartis Pharma GmbH (R), Pharm-Allergan GmbH (C), Pharm-Allergan GmbH (R); Andreas Stahl, Bausch&Lomb (C), Boehringer Ingelheim (C), Novartis Pharma GmbH (C), Novartis Pharma GmbH (F), Novartis Pharma GmbH (R), Zeiss (C)
  • Footnotes
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Investigative Ophthalmology & Visual Science June 2015, Vol.56, 235. doi:https://doi.org/
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      Johanna Madeleine Walz, Daniel Boehringer, Heidrun L Deissler, Peter Heiduschka, Alexa Klettner, Tim U. Krohne, Focke Ziemssen, Andreas Stahl; Pre-analytical parameters have significant impacts on VEGF levels measured from human plasma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):235. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Numerous studies include measurements of plasma VEGF concentrations in their endpoint analyses. However, if measurement parameters are not standardized, the results from different studies may not be comparable. Even within one study, measured plasma VEGF levels may vary if methods of obtaining and processing of samples are not strictly defined. In this study, we investigated which pre-analytical parameters affect plasma VEGF measurements and should therefore be carefully controlled in clinical studies.

Methods: Blood samples were obtained at three different clinical sites from six healthy volunteers at two time points using precisely defined procedures and detailed instructions for sample acquisition and processing. Several pre-analytical factors were deliberately altered to investigate their respective effects on VEGF measurements. The samples were measured at the three clinical sites (ELISA), three additional measurement centers (ELISA) and one central reading center (ELISA and Luminex). The parameters analyzed were: cannula (butterfly vs. neonatal), filling level (completely filled vs. half-filled tubes), use of anticoagulant (EDTA vs. PECT / CTAD), type of centrifuge (swing-out rotor vs. fixed angle), delay before and after centrifugation, study center and method of detection (ELISA vs. Luminex). In addition, intrapersonal fluctuations over one week and possible sex differences were investigated. A linear regression model was used for analysis.

Results: Regression analysis identified the following parameters as independent confounders of VEGF levels measured in human plasma: Study center, method of detection, sex of the proband, anticoagulation agent and type of centrifuge used. In contrast, the following parameters had no detectable independent effect on the VEGF values measured: Intrapersonal variation over one week, type of cannula, time between sampling and centrifugation, time between centrifugation and separating plasma, filling level of sampling tubes.

Conclusions: In order to achieve reliable plasma VEGF readings, type of anticoagulant, type of centrifuge and measuring method need to be standardized, ideally in one central measuring center. Failure to standardize these parameters may significantly affect results of plasma VEGF measurements in clinical trials.

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