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Quantitative Fluorescence Measurements with Multicolor Flow Cytometry

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Flow Cytometry Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1678))

Abstract

Multicolor flow cytometer assays are routinely used in clinical laboratories for immunophenotyping, monitoring disease and treatment, and determining prognostic factors. However, existing methods for quantitative measurements have not yet produced satisfactory results independent of flow cytometers used. This chapter details a procedure for quantifying surface and intracellular protein biomarkers by calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure includes the following critical steps: (a) quality control (QC) and performance characterization of the multicolor flow cytometer, (b) fluorescence calibration using hard dyed microspheres assigned with fluorescence intensity values in equivalent number of reference fluorophores (ERF), (c) compensation for correction of fluorescence spillover, and (d) application of a biological reference standard for translating the ERF scale to the ABC scale. The chapter also points out current efforts for implementing quantification of biomarkers in a manner which is independent of instrument platforms and reagent differences.

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References

  1. Fluorescence calibration and quantitative measurements of fluorescence instensity (2004) NCCLS (I/LA24-A)

    Google Scholar 

  2. Certificate of analysis, standard reference material 1932, fluorescein solution (2012) National Institute of Standards and Technology https://www-s.nist.gov/srmors/view_cert.cfm?srm=1932

  3. Wang L, Gaigalas AK, Marti G, Abbasi F, Hoffman RA (2008) Toward quantitative fluorescence measurements with multicolor flow cytometry. Cytometry A 73(4):279–288. doi:10.1002/cyto.a.20507

    Article  Google Scholar 

  4. Certificate of analysis, standard reference material 1934, fluorescent dyes for quantitative flow cytometry (visible spectral range) (2016). National Institute of Standards and Technology. https://www-s.nist.gov/srmors/view_cert.cfm?srm=1934

  5. Perfetto SP, Chattopadhyay PK, Wood J, Nguyen R, Ambrozak D, Hill JP, Roederer M (2014) Q and B values are critical measurements required for inter-instrument standardization and development of multicolor flow cytometry staining panels. Cytometry A 85(12):1037–1048. doi:10.1002/cyto.a.22579

    Article  Google Scholar 

  6. Davis KA, Abrams B, Iyer SB, Hoffman RA, Bishop JE (1998) Determination of CD4 antigen density on cells: role of antibody valency, avidity, clones, and conjugation. Cytometry 33(2):197–205

    Article  CAS  Google Scholar 

  7. Wang L, Abbasi F, Ornatsky O, Cole KD, Misakian M, Gaigalas AK, He HJ, Marti GE, Tanner S, Stebbings R (2012) Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry. Cytometry A 81(7):567–575. doi:10.1002/cyto.a.22060

    Article  Google Scholar 

  8. Wang M, Misakian M, He HJ, Bajcsy P, Abbasi F, Davis JM, Cole KD, Turko IV, Wang L (2014) Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry. Clin Proteomics 11(1):43. doi:10.1186/1559-0275-11-43

    Article  CAS  Google Scholar 

  9. Wang L, Degheidy H, Abbasi F, Mostowski H, Marti G, Bauer S, Hoffman RA, Gaigalas AK (2016) 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

    Article  Google Scholar 

  10. Roederer M (2002) Compensation in flow cytometry. Curr Protoc Cytom 22:1.14.1–1.14.20. doi:10.1002/0471142956.cy0114s22

    Google Scholar 

  11. Stewart CC, Stewart SJ (2001) Titering antibodies. Curr Protoc Cytom 4:1.1–1.13. doi:10.1002/0471142956.cy0401s14

    Google Scholar 

  12. Chase ES, Hoffman RA (1998) Resolution of dimly fluorescent particles: a practical measure of fluorescence sensitivity. Cytometry 33(2):267–279

    Article  CAS  Google Scholar 

  13. Hoffman RA, Wood JC (2007) Characterization of flow cytometer instrument sensitivity. Curr Protoc Cytom 40:1.20.1–1.20.18. doi:10.1002/0471142956.cy0120s40

    Google Scholar 

  14. The quantiFlash™ User Manual (December 2015)

    Google Scholar 

  15. Wood JC (1998) Fundamental flow cytometer properties governing sensitivity and resolution. Cytometry 33(2):260–266

    Article  CAS  Google Scholar 

  16. Degheidy H, Abbasi F, Mostowski H, Gaigalas AK, Marti G, Bauer S, Wang L (2015) Consistent, multi-instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference. Cytometry B Clin Cytom 90:159–167. doi:10.1002/cyto.b.21253

    Article  Google Scholar 

  17. Wang L, Abbasi F, Gaigalas AK, Vogt RF, Marti GE (2006) Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B-cells. Cytometry B Clin Cytom 70(6):410–415. doi:10.1002/cyto.b.20140

    Article  Google Scholar 

  18. Degheidy H, Bauer S, Marti GE, Wang L (2014) Flow cytometer performance characterization, standardization and calibration against CD4 on T lymphocytes enables quantification of biomarker expression for immunological applications. J Biomed Sci Eng 7:756–768

    Article  CAS  Google Scholar 

  19. Schiel JE, Mire-Sluis A, Davis D (2014) Monoclonal antibody therapeutics: the need for biopharmaceutical reference materials. In: Schiel JE, Davis DL, Borisov OV (eds) Monoclonal antibody therapeutics: structure, function, and regulatory space, vol 1. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization, ACS Symposium Series. doi:10.1021/bk-2014-1176.ch001

    Google Scholar 

  20. Wang L, Abbasi F, Gaigalas AK, Hoffman RA, Flagler D, Marti GE (2007) Discrepancy in measuring CD4 expression on T-lymphocytes using fluorescein conjugates in comparison with unimolar CD4-phycoerythrin conjugates. Cytometry B Clin Cytom 72(6):442–449. doi:10.1002/cyto.b.20354

    Article  Google Scholar 

  21. Wang L, Gaigalas AK, Abbasi F, Marti GE, Vogt RF, Schwartz A (2002) Quantitating fluorescence intensity from Fluorophores: practical use of MESF values. J Res Natl Inst Stand Technol 107:339–353. doi:10.6028/jres.107.027

    Article  CAS  Google Scholar 

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Acknowledgments

We are indebted to Steve Perfetto, Vaccine Research Center of NIH for his help with the troubleshooting the use of the quantiFlash™ LED pulser for the instrument sensitivity characterization. Authors are also thankful for Heba Degheidy, Fatima Abbasi, Howard Mostowski, Gerald Marti, and Steven Bauer all from FDA, and Robert Hoffman at Cytometry and Biophotonics Consulting, CA for their collaborative effort on quantitative flow cytometer measurements in the unit of antibodies bound per cell.

Disclaimer Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose. One Author, James Wood declares a financial interest with A·P·E Angewandte Physik & Elektronik GmbH, Germany.

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Authors and Affiliations

  1. National Institute of Standards and Technology (NIST), 100 Bureau Drive, Stop 8312, Gaithersburg, MD, 20899, USA

    Lili Wang & Adolfas K. Gaigalas

  2. Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA

    James Wood

Authors
  1. Lili Wang
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  2. Adolfas K. Gaigalas
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  3. James Wood
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Corresponding author

Correspondence to Lili Wang .

Editor information

Editors and Affiliations

  1. Flow Cytometry Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Teresa S. Hawley

  2. Department of Anatomy and Regenerative Biology, School of Medicine and Health Sciences, George Washington University, Washington, District of Columbia, USA

    Robert G. Hawley

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Wang, L., Gaigalas, A.K., Wood, J. (2018). Quantitative Fluorescence Measurements with Multicolor Flow Cytometry. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 1678. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7346-0_6

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  • DOI: https://doi.org/10.1007/978-1-4939-7346-0_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7344-6

  • Online ISBN: 978-1-4939-7346-0

  • eBook Packages: Springer Protocols

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