Sequential versus Simultaneous Quantitative Analysis of Biomarkers in Individual Cells by ICP-MS and Mass Cytometry: A Focus on Immunotherapy

Quantification strategies to establish the content of different elements in individual cells are crucial for elucidating any kind of biological conclusion. Technically, this process is hampered when using inductively coupled plasma mass spectrometry (ICP-MS) with nonsimultaneous mass analyzers. This work aims to establish under which set of conditions the sequential and simultaneous detection of different elements in individual cells provides comparable results. Thus, endogenous elements (P and Fe) and Ir as an exogenous cell marker are sequentially measured and quantified using ICP-MS revealing that cell storage and dilution conditions are more critical than cell deposition within the sample introduction device. Under optimized conditions, the interaction of an immunotherapeutic agent (Rituximab) that directs against the B-lymphocyte antigen called cluster of differentiation 20 (CD20) on a B-cell chronic lymphocytic leukemia cell line (MEC-1) is quantified using both single-cell analysis by ICP-MS (SC-ICP-MS) and mass cytometry (CyTOF) after labeling with Lu. Although the obtained values are in good agreement, a general trend is observed with a slight bias toward higher results in SC-ICP-MS versus CyTOF probably ascribed to the automated data treatment system used in CyTOF, discarding double cell events. Both analytical approaches (SC-ICP-MS and CyTOF) showed comparable levels of CD20 expression around 1 × 10⁴ receptors/cell in good agreement with the literature, proving the suitability of a similar calibration strategy for both instruments. The application of CyTOF quantification strategies has permitted the analysis of CD20 in healthy subjects and patient samples as proof of concept for the applicability of the proposed work.