Using the Process of Compensation to Prevent False Positive Data Caused by Fluorescence Spillover: A Practical Example

  • Christopher Hammerbeck
  • Christine Goetz
  • Li Jen PengEmail author
Part of the Techniques in Life Science and Biomedicine for the Non-Expert book series (TLSBNE)


When fluorochromes are excited by a laser, they emit photons of light at a range of wavelengths in what is known as that fluorochromes emission spectrum. A portion of this emission spectrum is detected or is “seen” by the flow cytometer. However, when the emission spectrums of multiple fluorochromes overlap, fluorescence spillover can occur. This may negatively impact flow cytometry data, resulting in false positives. Compensation must be performed to eliminate spectral overlap between closely related fluorochromes; otherwise, cells may appear to express surface markers that they do not actually express.

This chapter combines the theoretical concepts discussed in the previous chapters with a simple, real-world example demonstrating how fluorescence spillover can impact flow cytometry results, how compensation is performed, as well as considerations to reduce the impact of fluorescence spillover and compensation when designing flow cytometry experiments.


Compensation Spillover value AutoCompensation Manual compensation Spectral overlap Fluorescence spillover Bandpass filter Emission spectrum Compensation bead Fluorochrome PMT Spillover matrix table Spectrum viewer Single-stain controls 

Supplementary material

Supplementary Video 1

(MP4 309381 kb)


  1. 1.
    Murphy K. Janeway’s immunobiology. In: Garland science. 8th ed. New York: Taylor & Francis Group, LLC; 2012.Google Scholar
  2. 2.
    Compensation; an informal perspective. Mario Roederer.

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Christopher Hammerbeck
    • 1
  • Christine Goetz
    • 1
  • Li Jen Peng
    • 1
    Email author
  1. 1.Department of Antibody DevelopmentR&D Systems/Bio-TechneMinneapolisUSA

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