Compensation

(An informal perspective)

Prelude: You are free to copy and distribute this document at will--but please do so in its entirety, complete with figures. For information on obtaining a hardcopy for this purpose, click here. To reference this material, please include the WWW address as well as the latest modification date: May 24, 2000.

Address any questions, comments, or suggestions to Mario Roederer.

Introduction. Compensation is probably the least understood process accompanying flow cytometric analyses. Perhaps this is because it is often described with the linear algebra elements needed for its computation, and many of us have forgotten what linear algebra is, much less how to do it. However, proper compensation is absolutely crucial for some aspects of flow cytometry, one of which is antigen density measurements. Unfortunately, because compensation is often misunderstood, and because there is so much incorrect mythology about it floating around, many laboratories do not set compensation properly.

In this "perspective," I will try to address the issues surrounding compensation, attempt to dispel many of the myths, and hope that by the end, that I have not confused anyone but rather have convinced them what proper compensation is.

Please start with the quiz, and work through the perspective in order suggested. The table of contents below will allow you to go directly to any page.

Start with the Quiz!

If you are really adventurous, you can navigate through a more detailed (and more rigorous) discussion of compensation, complete with examples of the math, examples of the pitfalls of poor compensation, etc. I recommend this only after you have finished with the more informal, relaxed discussion... To start the detailed discussion, click here.

Table of contents:

Introduction (top of this page)
Quiz
Importance of compensation
Proper compensation
Errors introduced by compensation
Practical compensation
Autofluorescence
Recipe
Postscript
References

Compensation FAQ

List of Figures:

Figure 1a (quiz, part 1)
Figure 1b (quiz, part 2)
Figure 2: Compensation & cell enumeration
Figure 3: Errors introduced by compensation
Figure 4: Practical compensation
Figure 5: Compensation & sensitivity settings