Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD‐1 Expression Levels and PD‐1 Receptor Occupancy by Nivolumab and Pembrolizumab

Dick Pluim, Willeke Ros, IHC Miedema, Jos H. Beijnen, J. H.M.S. Schellens

Research output: Contribution to journalArticleAcademicpeer-review

13 Citations (Scopus)


We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4 + and CD8 +-T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1 + cells. Samples were stable for at least 153 days of storage at −80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1 + immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8 +-T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle.

Original languageEnglish
Pages (from-to)1053-1065
Number of pages13
JournalCytometry Part A
Issue number10
Publication statusPublished - 1 Oct 2019
Externally publishedYes


  • PD-1 receptor occupancy
  • immune cell subsets
  • immunotherapy
  • nivolumab
  • pembrolizumab
  • pharmacodynamic

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