Unlabeled Competitor Antibody to Reduce Nonlinear Signal Spillover in Mass Cytometry

Palak Sekhri, Miriam Y. Kim, Gregory K. Behbehani

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Mass cytometry (MCM; CyTOF) utilizes isotopically purified metal-tagged antibodies for single-cell analysis and can analyze more than 40 parameters simultaneously with minimum signal spillover to other mass channels as compared to fluorescent flow cytometry. In spite of this improvement, various factors such as metal oxidation, abundance sensitivity related spillover, and metal impurities can cause measurable amounts of spillover in MCM that can potentially lead to misinterpretation of data. Linear spillover can be corrected by applying compensation; however, we demonstrate that at high signal intensities, MCM channel spillovers are frequently nonlinear. This report describes a simple method to correct for nonlinear signal spillover (due to abundance sensitivity, isotopic contamination, or oxide formation) that can occur at high signal intensity through the use of unlabeled competitor antibodies to the specific metal-tagged antibodies causing spillover. This method significantly decreased high signal intensity and nonlinear spillover to other mass channels while maintaining saturating antibody concentrations, thereby facilitating accurate staining and compensation. In contrast, the common method of using under-titrated antibodies to overcome spillover lead to staining intensity that varied with cell numbers and antigen abundance. We demonstrate that this technique reduces total signal without significantly altering immunophenotypic or functional measurement of relative antigen levels and could be used to enable improved linear compensation of signal spillovers from high abundance antigens. Statement of Significance: Mass cytometry is becoming a well-established technology for comprehensive analysis of complex biological samples, due to its ability to enable measurement of more than 40 simultaneous parameters. Due to the use of isotopically pure metal-tagged antibodies, measurement channel spillover in mass cytometry is drastically lower than in fluorescent cytometry but can still occur due to metal oxidation, isotopic impurities, or abundance sensitivity when mass signals have high intensity. We show in this report that high abundance antigens with high signal intensity exhibit non-linear mass channel spillovers that cannot be easily compensated. We also demonstrate a simple method for the use of unlabeled competitor antibody to decrease antigen signal intensity while maintaining antigen abundance to allow for more accurate linear compensation. This method performs more consistently than the commonly used approach of using under-titrated antibodies. We believe that this report has immediate practical utility for researchers using mass cytometry and can be broadly utilized to enable compensation of mass cytometry data when needed. We thus feel that this article merits publication as a Brief Report in Cytometry Part A.

Original languageEnglish
Pages (from-to)898-909
Number of pages12
JournalCytometry Part A
Issue number8
StatePublished - 2019


  • compensation
  • mass cytometry
  • spillover
  • unlabeled antibodies


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