Standardization, Calibration, and Control in Flow Cytometry

Lili Wang1, Robert A. Hoffman2

1 Biosystems and Biomaterials Division, NIST, Gaithersburg, Maryland, 2 Independent Consultant, Livermore, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.3
DOI:  10.1002/cpcy.14
Online Posting Date:  January, 2017
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Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions and cautions are provided for common terms to alert the reader to critical distinctions in meaning. This unit presents extensive background on particle types and cautions and describes practical aspects of methods to standardize and calibrate instruments. Procedures are provided to characterize performance in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity. Finally, suggestions follow for analyzing particles used for calibration. © 2017 by John Wiley & Sons, Inc.

Keywords: standardization; calibration; control; particles; MESF (molecules of equivalent soluble fluorochrome); ERF (equivalent number of reference fluorophores); sensitivity; resolution

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Table of Contents

  • Introduction
  • Standards, Standardization, and Jargon
  • Definitions
  • Overview of Standardization and Performance Characterization in Flow Cytometry
  • Instrument Performance Characterization, Standardization and Calibration
  • Characterizing Particles for Calibration and Control of a Flow Cytometer
  • Concluding Remarks
  • Disclaimer
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
  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. doi: 10.1111/j.1749‐6632.1993.tb38775.x.
  Bauer, K.D. 1993. Quality control issues in DNA content flow cytometry. Ann. N.Y. Acad. Sci. 677:59‐77. doi: 10.1111/j.1749‐6632.1993.tb38765.x.
  Baumgarth, N. and Roederer, M. 2000. A practical approach to multicolor flow cytometry for immunophenotyping. J. Immunol. Methods 243:77‐97. doi: 10.1016/S0022‐1759(00)00229‐5.
  Bevington, P. 1969. Data Reduction and Error Analysis for the Physical Sciences. McGraw‐Hill, New York.
  Bikoue, A., George, F., Poncelet, P., Mutin, M., Janossy, G., and Sampol, J. 1996. Quantitative analysis of leukocyte membrane antigen expression: Normal adult values. Cytometry 26:137‐147. doi: 10.1002/(SICI)1097‐0320(19960615)26:2%3c137::AID‐CYTO7%3e3.0.CO;2‐D.
  Bohmer, R.M., Papaioannou, J., and Ashcroft, R.G. 1985. Flow‐cytometric determination of fluorescence ratios between differently stained particles is dependent on excitation intensity. J. Histochem. Cytochem. 33:974‐976. doi: 10.1177/33.9.2410484.
  Brown, M.C., Hoffman, R.A., and Kirchanski, S. 1986. Controls for flow cytometers in hematology and cellular immunology. Ann. N.Y. Acad. Sci. 468:93‐103. doi: 10.1111/j.1749‐6632.1986.tb42032.x.
  Chase, E.S. and Hoffman, R.A. 1998. Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity. Cytometry 33:267‐279. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c267::AID‐CYTO24%3e3.0.CO;2‐R.
  CLSI. 2008. Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping of Peripheral Blood Lymphocytes; Approved Guideline H42‐A2. Clinical and Laboratory Standards Institute, Wayne, Pa.
  Darzynkiewicz, Z. 1993. The cell cycle: Application of flow cytometry in studies of cell reproduction. In Clinical Flow Cytometry: Principles and Application (K.D. Bauer, R.E. Duque, and T.V. Shankey, eds.) pp. 13‐40. Williams and Wilkins, Baltimore.
  Darzynkiewicz, Z. 2011. Critical aspects in analysis of cellular DNA content. Curr. Protoc. Cytom. 56:7.2.1‐7.2.8.
  Davis, K.A., Abrams, B., Iyer, S.B., Hoffman, R.A., and Bishop, J.E. 1998. Determination of CD4 antigen density on cells: Role of antibody valency, avidity, clones and conjugation. Cytometry 33:197‐205. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c197::AID‐CYTO14%3e3.0.CO;2‐P.
  Doornbos, R.M., Deurloo, K.E., De Grooth, B.G., Sloot, P.M., and Greve, J. 1994. Lissajous‐like patterns in scatter plots of calibration beads. Cytometry 16:236‐242. doi: 10.1002/cyto.990160307.
  Dressler, L.G. 1990. Controls, standards, and histogram interpretation in DNA flow cytometry. Methods Cell Biol. 33:157‐171. doi: 10.1016/S0091‐679X(08)60522‐1.
  Eisert, W.G. and Nezel, M. 1978. Internal calibration to absolute values in flow‐through particle size analysis. Rev. Sci. Instrum. 49:1617‐1621. doi: 10.1063/1.1135357.
  Fulwyler, M.J. 1979. Standards for flow cytometry. In Flow Cytometry and Sorting (M.R. Melamed, P.F. Mullaney, and M.L. Mendelsohn, eds.) pp. 351‐358. John Wiley & Sons, New York.
  Fulwyler, M.J., Perrings, J.D., and Cram, L.S. 1973. Production of uniform microspheres. Rev. Sci. Instrum. 44:204‐206. doi: 10.1063/1.1686082.
  Gaigalas, A.K., Li, L., Henderson, O., Vogt, R., Barr J., Marti, G., Weaver, J., and Schwartz, A. 2001. The development of fluorescence intensity standards. J. Res. Nat. Inst. Stand. Technol. 106:381‐389. doi: 10.6028/jres.106.015.
  Gaucher, J.C., Grunwald, D., and Frelat, G. 1988. Fluorescence response and sensitivity determination for ATC 3000 flow cytometer. Cytometry 9:557‐565. doi: 10.1002/cyto.990090608.
  Halter, M., Bier, E., DeRose, P.C., Cooksey, G.A., Choquette, S.J., Plant, A.L., and Elliott J.T. 2014. An automated protocol for performance benchmarking a widefield fluorescence microscope. Cytometry A 85:978‐985. doi: 10.1002/cyto.a.22519.
  Henderson, L.O., Marti, G.E., Gaigalas, A., Hannon, W.H., and Vogt, R.F. 1998. Terminology and nomenclature for standardization in quantitative cytometry. Cytometry 33:97‐105. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c97::AID‐CYTO3%3e3.0.CO;2‐H.
  Hoffman, R.A. 2009. Pulse width for particle sizing. Curr. Protoc. Cytom. 50:1.23.1‐1.23.17.
  Hoffman, R.A. and Wood, J.C. 2007. Characterization of flow cytometer instrument sensitivity. Curr. Protoc. Cytom. 40:1.20.1‐1.20.18.
  Hoffman, R.A., Wang, L., Bigos M., and Nolan, J.P. 2012. NIST/ISAC standardization study: Variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads. Cytometry A 81A:785‐796. doi: 10.1002/cyto.a.22086.
  Horan, P.K. and Loken, M.R. 1985. A practical guide for the use of flow systems. In Flow Cytometry: Instrumentation and Data Analysis (M.A. Van Dilla, P.N. Dean, O.D. Laerum, and M.R. Melamed, eds.) pp. 259‐280. Academic Press, New York.
  Horan, P.K., Muirhead, K.A., and Slezak, S.E. 1990. Standards and controls in flow cytometry. In Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 397‐414. Wiley‐Liss, New York.
  Hultin, L.E., Matud, J.L., and Giorgi, J.V. 1998. Quantitation of CD38 activation antigen expression on CD8+ T cells in HIV‐1 infection using CD4 expression on CD4+ T lymphocytes as a biological calibrator. Cytometry 33:123‐132. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c123::AID‐CYTO6%3e3.0.CO;2‐K.
  Hurley, A.A. 1997a. Principles of quality control. Curr. Protoc. Cytom. 00:3.1.1‐3.1.2.
  Hurley, A.A. 1997b. Components of quality control. Curr. Protoc. Cytom. 00:3.2.1‐3.2.4.
  Hurley, A.A. 1997c. Quality control in phenotypic analysis by flow cytometry. Curr. Protoc. Cytom. 00:6.1.1‐6.1.4.
  Islam, D., Lindberg, A.A., and Christensson, B. 1995. Peripheral blood cell preparation influences the level of expression of leukocyte cell surface markers as assessed with quantitative multicolor flow cytometry. Cytometry 22:128‐134. doi: 10.1002/cyto.990220208.
  Iyer, S.J., Hultin, L.E., Zawadzki, J.A., Davis, K.A., and Giorgi, J.V. 1998. Quantitation of CD38 expression using QuantiBRITE beads. Cytometry 33:206‐212. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c206::AID‐CYTO15%3e3.0.CO;2‐Y.
  Kachel, V. 1990. Electrical resistance pulse sizing: Coulter sizing. In Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 45‐80. Wiley‐Liss, New York.
  Kantor, A.B., Moore W.A., Meehan, S., and Parks, D.R. 2016. A quantitative method for comparing the brightness of antibody‐dye reagents and estimating antibodies bound per cell. Curr. Protoc. Cytom. 77:1.30.1‐1.30.23. doi: 10.1002/cpcy.6.
  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. doi: 10.1177/27.1.374594.
  Lenkei, R. and Andersson, B. 1995. Determination of the antibody binding capacity of lymphocyte membrane antigens by flow cytometry in 58 blood donors. J. Immunol. Methods 183:267‐277. doi: 10.1016/0022‐1759(95)00064‐H.
  Lenkei, R., Gratama, J.W., Rothe, G., Schmitz, G., D'hautcourt, J.L., Årekrans, A., Mandy, F., and Marti, G. 1998. Performance of calibration standards for antigen quantitation with flow cytometry. Cytometry 33:188‐196. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c188::AID‐CYTO13%3e3.0.CO;2‐Q.
  Muirhead, K.A. 1993a. Quality control for clinical flow cytometry. In Clinical Flow Cytometry: Principles and Application (K.D. Bauer, R.E. Duque, and T.V. Shankey, eds.) pp. 177‐199. Williams and Wilkins, Baltimore.
  Muirhead, K.A. 1993b. Establishment of quality control procedures in clinical flow cytometry. Ann. N.Y. Acad. Sci. 677:1‐20. doi: 10.1111/j.1749‐6632.1993.tb38758.x.
  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. doi: 10.1002/cyto.990030404.
  NCCLS. 1998a. Terminology and definitions for use in NCCLS documents: Approved Standard NRSCL8‐A. Clinical and Laboratory Standards Institute. Wayne, Pa.
  NCCLS. 1998b. Clinical applications of flow cytometry: Quality assurance and immunophenotyping of peripheral blood lymphocytes; Approved Guideline H42‐A. Clinical and Laboratory Standards Institute. Wayne, Pa.
  Owens, M.A. and Loken, M.R. 1995. Flow Cytometry for Clinical Laboratory Practice: Quality Assurance for Quantitative Immunophenotyping. Wiley‐Liss, New York.
  Owens, M.A., Vall, H.G., Hurley, A.A., and Wormsley, S.B. 2000. Validation and quality control of immunophenotyping in clinical flow cytometry. J. Immunol. Methods 243:33‐50. doi: 10.1016/S0022‐1759(00)00226‐X.
  Poncelet, P. and Carayon, P. 1985. Cytofluorometric quantification of cell‐surface antigens by indirect immunofluorescence using monoclonal antibodies. J. Immunol. Methods 85:65‐74. doi: 10.1016/0022‐1759(85)90274‐1.
  Poncelet, P., Bikoue, A., Lavabre, T., Poinas, G., Parant, M., Duperray, O., and Sampol, J. 1991. Quantitative expression of human lymphocyte membrane antigens: Definition of normal densities measured in immunocytometry with the QIFI assay. Cytometry 5:82‐83.
  Rembaum, A. 1979. Microspheres as immunoreagents for cell identification. In Flow Cytometry and Sorting (M.R. Melamed, P.F. Mullaney, and M.L. Mendelsohn, eds.) pp. 335. John Wiley & Sons, New York.
  Roederer, M. 2001. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45:194‐205. doi: 10.1002/1097‐0320(20011101)45:3%3c194::AID‐CYTO1163%3e3.0.CO;2‐C.
  Salzman, G.C., Singham, S.B., Johnston, R.G., and Bohren, C.F. 1990. Light scattering and cytometry. In Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 81‐107. Wiley‐Liss, New York.
  Schmid, I., Schmid, P., and Giorgi, J. 1988. Conversion of logarithmic channel numbers into relative linear fluorescence intensity. Cytometry 9:533‐538. doi: 10.1002/cyto.990090605.
  Schwartz, A. and Fernandez‐Repollet, E. 1993. Development of clinical standards for flow cytometry. Ann. N.Y. Acad. Sci. 677:28‐39. doi: 10.1111/j.1749‐6632.1993.tb38760.x.
  Schwartz, A., Sugg, H., Ritter, T.W., and Fernandez‐Repollet, E. 1983. Direct determination of cell diameter, surface area, and volume with an electronic volume sensing flow cytometer. Cytometry 3:456‐458. doi: 10.1002/cyto.990030613.
  Schwartz, A., Fernandez‐Repollet, E., Vogt, R., and Gratama, J. 1996. Standardizing flow cytometry: Construction of a standardized fluorescence calibration plot using matching spectral calibrators. Cytometry 26:22‐31. doi: 10.1002/(SICI)1097‐0320(19960315)26:1%3c22::AID‐CYTO4%3e3.0.CO;2‐I.
  Schwartz, A., Marti, G.E., Poon, R., Gratama, J.W., and Fernández‐Repollet, E. 1998. Standardizing flow cytometry: A classification system of fluorescence standards used for flow cytometry. Cytometry 33:106‐114. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c106::AID‐CYTO4%3e3.0.CO;2‐H.
  Schwartz, A., Wang, L., Early, E., Gaigalas, A., Zhang, Y., Marti, G.E., and Vogt, R.F. 2002. Quantitating fluorescence intensity from fluorophore: The definition of MESF assignment. J. Res. Nat. Inst. Stand. Technol. 107:83‐91. doi: 10.6028/jres.107.009.
  Serke, S., van Lessen A., and Huhn, D. 1998. Quantitative fluorescence flow cytometry: A comparison of the three techniques for direct and indirect immunofluorescence. Cytometry 33:179‐187. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c179::AID‐CYTO12%3e3.0.CO;2‐R.
  Shapiro, H. 2003. Practical Flow Cytometry, 4th ed. John Wiley & Sons, Hoboken, N.J.
  Sharpless, T.K. and Melamed, M.R. 1976. Estimation of cell size from pulse shape in flow cytofluorometry. J. Histochem. Cytochem. 24:257‐264. doi: 10.1177/24.1.1254921.
  Sharpless, T.K., Bartholdi, M., and Melamed, M.R. 1977. Size and refractive index dependence of simple forward‐angle scattering measurements in a flow system using sharply focused illumination. J. Histochem. Cytochem. 24:257‐264. doi: 10.1177/24.1.1254921.
  Stebbings, R., Wang, L., Sutherland, J., Kammel, M., Gaigalas, A.K., John, M., Roemer, B., Kuhne, M., Schneider, R.J., Braun, M., Leclere, N., Dikshit, D., Abbasi, F., Marti, G.E., Porcedda, P., Sassi, M., Revel, L., Kim, S.K., Marshall, D., Whitby, L., Jing, W., Ost, V., Vonski, M., and Neukammer, J. 2015. Determination of CD4+ cell count per µl in reconstituted lyophilized human PBMC pre‐labelled with anti‐CD4 FITC antibody. Cytometry 87A:244‐253. doi: 10.1002/cyto.a.22614.
  Steen, H.B. 1992. Noise, sensitivity, and resolution of flow cytometers. Cytometry 13:822‐830. doi: 10.1002/cyto.990130804.
  Stewart, C.C. and Steinkamp, J.A. 2005. Quantitation of cell concentration using the flow cytometer. Cytometry 2:238‐243. doi: 10.1002/cyto.990020407.
  Tanqri, S., Vall, H., Kaplan, D., Hoffman, B., Purvis, N., Porwit, A., Hunsberger, B., Shankey, T.V., and on behalf of ICSH/ICCS working group. 2013. Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS—Part III: Analytical Issues. Cytometry B 84B:291‐308. doi: 10.1002/cyto.b.21106.
  Wang, L., Gaigalas, A.K., Abbasi, F., Marti, G.E., Vogt, R.F., and Schwartz, A. 2002. Quantitating fluorescence intensity from fluorophores: Practical use of MESF values. J. Res. Nat. Ins. Stand. Technol. 107:339‐353. doi: 10.6028/jres.107.027.
  Wang, L. and Gaigalas, A.K. 2011. Development of multicolor flow cytometry calibration standards: Assignment of equivalent reference fluorophores (ERF) unit. J. Res. Nat. Ins. Stand. Technol. 116:671‐683. doi: 10.6028/jres.116.012.
  Wang, L., DeRose, P., and Gaigalas, A.K. 2016a. Assignment of the number of equivalent reference fluorophores to dyed microspheres. J. Res. Nat. Ins. Stand. Technol. 121:269‐286. doi: 10.6028/jres.121.012.
  Wang, L., Degheidy, H., Abbasi, F., Mostowski, H., Marti, G., Bauer, S., Hoffman, R.A., and Gaigalas, A.K. 2016b. Quantitative flow cytometry measurements in antibodies bound per cell based on a CD4 reference. Curr. Protoc. Cytom. 75:1.29.1‐1.29.14. doi: 10.1002/0471142956.cy0129s75.
  Wheeless, L.L. 1993. The clinical utility of DNA cytometry. Ann. N.Y. Acad. Sci. 677:82‐85. doi: 10.1111/j.1749‐6632.1993.tb38767.x.
  Wood, J.C.S. 1993. Clinical flow cytometry instrumentation. In Clinical Flow Cytometry: Principles and Application (K.D. Bauer, R.E. Duque, and T.V. Shankey, eds.) pp. 71‐92. Williams and Wilkins, Baltimore.
  Wood, J.C.S. 1998. Fundamental flow cytometer properties governing sensitivity and resolution. Cytometry 33:260‐266. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c260::AID‐CYTO23%3e3.0.CO;2‐R.
  Wood, J.C.S. 2009. Establishing and maintaining system linearity. Curr. Protoc. Cytom. 47:1.4.1‐1.4.14.
  Wood, J.C.S. and Hoffman, R.A. 1998. Evaluating fluorescence sensitivity on flow cytometers: An overview. Cytometry 33:256‐259. doi: 10.1002/(SICI)1097‐0320(19981001)33:2%3c256::AID‐CYTO22%3e3.0.CO;2‐S.
  Wood, B., Jevremovic, D., Bene M.C., Yan, M., Jacobs, P., Litwin, V., and on behalf of ICSH/ICCS working group. 2013. Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS—Part V: Assay performance criteria. Cytometry B 84B:315‐323. doi: 10.1002/cyto.b.21108.
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