Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference

Lili Wang1, Heba Degheidy2, Fatima Abbasi2, Howard Mostowski2, Gerald Marti3, Steven Bauer2, Robert A. Hoffman4, Adolfas K. Gaigalas1

1 National Institute of Standards and Technology (NIST), Gaithersburg, Maryland, 2 Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, 3 Center for Devices and Radiological Health, U.S. Food and Drug Administration, Silver Spring, Maryland, 4 Consultant, Livermore, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.29
DOI:  10.1002/0471142956.cy0129s75
Online Posting Date:  January, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument‚Äźindependent across all fluorescence channels. This unit details a procedure for quantifying surface and intracellular biomarkers by calibrating the output of a multicolor flow cytometer in units of antibody bound per cell (ABC). The procedure includes (1) quality control of the flow cytometer, (2) fluorescence intensity calibration using hard dyed microspheres assigned with fluorescence intensity values, (3) compensation for fluorescence spillover between adjacent fluorescence channels, and (4) application of a biological reference calibrator to establish an ABC scale. The unit also points out current efforts for quantifying biomarkers in a manner that is independent of instrument platforms and reagent differences. ¬© 2016 by John Wiley & Sons, Inc.

Keywords: multicolor flow cytometry; fluorescence calibration; equivalent number of reference fluorophores (ERF); CD4+ lymphocytes; antibody bound per cell (ABC)

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Staining Cells for Quantification of CD20
  • Basic Protocol 2: Quality Control, Calibration, and Instrument Compensation
  • Basic Protocol 3: Quantification of CD20 in Units of ABC
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Staining Cells for Quantification of CD20

  • Freshly drawn whole blood from a normal healthy individual
  • 1× phosphate‐buffered saline (PBS), pH 7.4 (Life Technologies, cat. no. 10010‐023)
  • Fetal bovine serum (FBS; Sigma‐Aldrich)
  • Fluorescently labeled monoclonal antibodies (BD Biosciences):
    • CD4 APC (SK3 clone, cat. no. 340443)
    • CD4 PerCP‐Cy5.5 (SK3 clone, cat. no. 341654)
    • CD4 FITC (SK3 clone, cat. no. 340133)
    • CD4 PE‐Cy7 (SK3 clone, cat. no. 348789)
    • CD4 V450 (SK3 clone, cat. no. 651850)
    • CD20 APC (L27 clone, cat. no. 340941)
    • CD20 PerCP‐Cy5.5 (L27 clone, cat. no. 340955)
    • CD45 FITC (cat. no. 555482)
    • CD3 V450 (cat. no. 560365)
    • CD19 PE‐Cy7 (cat. no. 560728)
  • Lysing solution, fixative‐free (Life Technologies, cat. no. HYL‐250)
  • 1% (w/v) paraformaldehyde (Electron Microscopy Sciences) in 1× PBS
  • Cyto‐Trol control cell kit (Beckman Coulter), including reconstitution buffer
  • Disposable 12 × 75−mm polystyrene Falcon tubes (BD Biosciences)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Boonstra, J.G., van Lom, K., Langerak, A.W., Graveland, W.J., Valk, P.J., Kraan, J., van ‘t Veer, M.B., and Gratama, J.W. 2006. CD38 as a prognostic factor in B cell chronic lymphocytic leukaemia (B‐CLL): Comparison of three approaches to analyse its expression. Cytometry B Clin Cytom. 70:136‐141. doi: 10.1002/cyto.b.20106.
  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)33:23.0.CO;2-R.
  Davis, K.A., Abrams, B., Iyer, S.B., Hoffman, R.A., and 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)33:23.0.CO;2-P.
  de Vos van Steenwijk, P.J., Heusinkveld, M., Ramwadhdoeb, T.H., Löwik, M.J., van der Hulst, J.M., Goedemans, R., Piersma, S.J., Kenter, G.G., and van der Burg, S.H. 2010. An unexpectedly large polyclonal repertoire of HPV‐specific T cells is poised for action in patients with cervical cancer. Cancer Res. 70:2707‐2717. doi: 10.1158/0008-5472.CAN-09-4299.
  Degheidy, H., Bauer, S., Marti, G.E., and 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. doi: 10.4236/jbise.2014.79074.
  Degheidy, H., Abbasi, F., Mostowski, H., Gaigalas, A.K., Marti, G., Bauer, S., and Wang, L. 2015. Consistent, multi‐instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference. Cytometry B Clin Cytom. doi: 10.1002/cyto.b.21253.
  Eisaman, M.D., Fan, J., Migdall, A., and Polyakov, S.V. 2011. Invited review article: Single‐photon sources and detectors. Rev. Sci. Instrum. 82:071101. doi: 10.1063/1.3610677.
  Estess, P., Salmon, S.L., Winberg, M., Oi, V.T., and Buck, D. 1990. Molecular mapping of immunogenic determinants of human CD4 using chimeric interspecies molecules and anti‐CD4 antibodies. In Current Research in Protein Chemistry: Techniques, Structure, and Function (J. Villafranca, ed.), pp. 499‐508. Academic Press, San Diego.
  Hoffman, R.A. 2001. Standardization and quantitation in flow cytometry. In Methods in Cell Biology: Cytometry (J.P. Robinson and Z. Darzynkiewicz, eds.), pp 299‐340. Academic Press, San Diego.
  Hoffman, R.A., and Wood, J.C.S. 2007. Characterization of flow cytometer instrument sensitivity. Curr. Protoc. Cytom. 40:1.20.1‐1.20.18.
  Hoffman, R.A., Recktenwald, D.J., and Vogt, R. 1993. Cell‐associated receptor quantitation. In Clinical Flow Cytometry: Principles and Application (K.D. Bauer, R.E. Duque, and T.V. Shankey, eds.), pp 469‐477. Williams & Wilkins, Baltimore.
  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 81A:785‐796. doi: 10.1002/cyto.a.22086.
  Hultin, L., Matud, J., and Giorgi, J.V. 1998. Quantitation of CD38 activation antigen expression on CD81 T‐cells in HIV‐1 infection using CD4 expression on CD41 T‐lymphocytes as a biological calibrator. Cytometry 33:123‐132. doi: 10.1002/(SICI)1097-0320(19981001)33:23.0.CO;2-K.
  Iyer, S., 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)33:23.0.CO;2-Y.
  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. In press.
  National Institute of Standards and Technology (NIST). 2007. Certificate of analysis, standard reference material 1932, fluorescein solution. (http://ts.nist.gov/measurementservices/referencematerials/index.cfm).
  Perfetto, S.P., Chattopadhyay, P.K., Wood, J., Nguyen, R., Ambrozak, D., Hill, J.P., and Roederer, M. 2014. Q and B values are critical measurements required for inter‐instrument standardization and development of multicolor flow cytometry staining panels. Cytometry 85A:1037‐1048. doi: 10.1002/cyto.a.22579.
  Richards, J.O., Treisman, J., Garlie, N., Hanson, J.P., and Oaks, M.K. 2012. Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures. Cytometry 81A:374‐381. doi: 10.1002/cyto.a.22047.
  Roederer, M. 2002. Compensation in flow cytometry. Curr. Protoc. Cytom. 22:1.14.1‐1.14.20.
  Stebbings, R., Wang, L., Sutherland, J., Kammel, M., Gaigalas, A.K., John, M., Roemer, B., Kuhne, M., Schneider, R.J., Braun, M., Engel, A., Dikshit, D., Abbasi, F., Marti, G.E., Sassi, M., Revel, R., Kim, S.K., Baradez, M., Lekishvili, T., Marshall, D., Whitby, L., Jing, J., Ost, V., Vonski, M., and Neukammer, J. 2015. Quantification of cells with specific phenotypes I: Determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti‐CD4 FITC antibody. Cytometry 87A:244‐253. doi: 10.1002/cyto.a.22614.
  Tembhare, P.R., Marti, G.E., Wiestner, A., Degheidy, H., Farooqui, M., Kreitman, R.J., Jasper, G.A., Yuan, C.M., Liewehr, D., Venzon, D., and Stetler‐Stevenson, M. 2013. Quantification of expression of antigens targeted by antibody‐based therapy in chronic lymphocytic leukemia. Am .J. Clin. Pathol. 140:813‐818. doi: 10.1309/AJCPYFQ4XMGJD6TI.
  Voet, D. and Voet, J.G. 1990. Biochemistry, p. 1153. John Wiley & Sons, New York.
  Wang, L., Abbasi, F., Gaigalas, A.K., Vogt, R.F., and Marti, G.E. 2006. Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells. Cytometry B Clin Cytom. 70B:410‐415. doi: 10.1002/cyto.b.20140.
  Wang, L., Galgalas, A.K., Marti, G.E., Abbasi, F., and Hoffman, R.A. 2008. Toward quantitative fluorescence measurements with multicolor flow cytometry. Cytometry 73A:279‐288. doi: 10.1002/cyto.a.20507.
  Wang, L., Gaigalas, A.K., and Yan, M. 2011. Quantitative fluorescence measurements with multicolor flow cytometry. In Flow Cytometry Protocols (T.S. Hawley and R.G. Hawley, eds.), pp. 53‐65. Humana Press/Springer, New York. doi: 10.1007/978-1-61737-950-5_3.
  Wang, L., Abbasi, F,. Ornatsky, O., Cole, K.D., Misakian, M., Gaigalas, A.K., He, H.J., Marti, G.E., Tanner, S., and Stebbings, R. 2012. Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry. Cytometry 81A:567‐575. doi: 10.1002/cyto.a.22060.
  Wang, M., Misakian, M., He, Hua‐Jun, Abbasi, F., Davis, J.M., Cole, K.D., Turko, I.V., and Wang, L. 2014. Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry. Clin Proteomics 11:43. doi: 10.1186/1559-0275-11-43.
  Wang, L., Stebbings, R., Gaigalas, A.K., Sutherland, J., Kammel, M., John, M., Roemer, B., Kuhne, M., Schneider, R.J., Braun, M., Engel, A., Dikshit, D., Abbasi, F., Marti, G.E., Sassi, M., Revel, R., Kim, S.K., Baradez, M., Lekishvili, T., Marshall, D., Whitby, L., Jing, J., Ost, V., Vonski, M., and Neukammer, J. 2015. Quantification of cells with specific phenotypes II: Determination of CD4 expression level on reconstituted lyophilized human PBMC labeled with anti‐CD4 FITC antibody. Cytometry 87A:254‐261. doi: 10.1002/cyto.a.22634.
  Wood, J.C.S. 2009. Establishing and maintaining system linearity. Curr. Protoc. Cytom. 47:1.4.1‐1.4.14.
PDF or HTML at Wiley Online Library