Establishing and Maintaining System Linearity

James C. S. Wood1

1 Wake Forest University School of Medicine, Winston‐Salem, North Carolina
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.4
DOI:  10.1002/0471142956.cy0104s47
Online Posting Date:  January, 2009
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A flow cytometer system transforms a received optical signal into an electrical signal whose amplitude ideally is directly proportional to the optical signal but in practice is at least related in a linear fashion. Establishment and maintenance of system linearity are prerequisites to accurate quantitative measurements. The many potential sources of offsets and nonlinearity are found in all parts of the detection, amplification, and data acquisition systems of a flow instrument. This unit aims to help investigators evaluate performance of the hardware in flow cytometer detection systems. Curr. Protocol. Cytom. 47:1.4.1‐1.4.14. © 2009 by John Wiley & Sons, Inc.

Keywords: flow cytometry; linearity; quantitation; dynamic range; proportional

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Table of Contents

  • Introduction
  • Definition of Linearity
  • Sources of Nonlinearity in a Flow Cytometer
  • Establishing System Linearity
  • Maintaining System Linearity
  • Conclusion
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
   Auer, R.E., Starling, D., Weber, B., and Wood, J.C.S. 1993. A data acquisition system for flow cytometry with wide dynamic range analog to digital conversion and digital signal processing. Cytometry 6:146A.
   Bagwell, C.B. and Adams, E.G., 1993. Fluorescence spectral overlap compensation for any number of flow cytometry parameters. Ann. N.Y. Acad. Sci. 677:167‐184.
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   Leary, J.F., Todd, P., Wood, J.C.S., and Jett, J.H. 1979. Laser flow cytometric light scatter and fluorescence pulse width and pulse rise‐time sizing of mammalian cells. J. Histochem. Cytochem. 27:315‐320.
   Muirhead, K.A., Schmitt, T.C., and Muirhead, A.R. 1983. Determination of linear fluorescence intensities from flow cytometric data accumulated with logarithmic amplifiers. Cytometry 3:251‐256.
   Parks, D.R., Bigos, M., and Moore, W.A. 1988. Logarithmic amplifier function evaluation and procedures for logamp optimization and data correction. Cytometry 2:155.
   Pinkel, D. and Steen, H.B. 1982. Simple methods to determine and compare the sensitivity of flow cytometers. Cytometry 3:220‐223.
   Roederer, M. and Murphy, R.F. 1986. Cell‐by‐cell autofluorescence correction for low signal‐to‐noise systems: Application to epidermal growth factor endocytosis by 3T3 fibroblasts. Cytometry 7:558‐565.
   Schmid, I., Schmid, P., and Giorgi, J.V. 1988. Conversion of logarithmic channel numbers into relative linear fluorescence intensity. Cytometry 9:533‐538.
   Schwarz, A., Repollet, E.F., Vogt, R., and Gratama, J.W. 1996. Standardizing flow cytometry: Construction of a standardized fluorescence calibration plot using matching spectral calibrators. Commun. Clin. Cytom. 26:22‐31.
   Steen, H.B. 1992. Noise, sensitivity, and resolution of flow cytometers. Cytometry 13:822‐830.
   Ubezio, P. and Andreoni, A. 1985. Linearity and noise sources in flow cytometry. Cytometry 6:109‐115.
   Vindelov, L. and Christensen, I.J. 1990. A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis. Cytometry 11:753‐770.
   Vindelov, L.L., Christensen, I.J., and Nissen, N.I. 1983. Standardization of high resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. Cytometry 3:328‐331.
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