Flow Cytometry–Based Cytotoxicity and Antibody Binding Assay

Mats Alheim1

1 Karolinska University Hospital, Stockholm
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.34
DOI:  10.1002/0471142956.cy0634s66
Online Posting Date:  October, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Human leukocyte antigen (HLA) antibodies with the ability to activate complement are associated with an increased risk of early antibody‐mediated graft rejection in kidney transplantation (KTx). Detection of these potentially harmful complement‐fixing HLA antibodies is commonly performed via the complement‐dependent cytotoxicity (CDC) assay according to protocols that were developed as early as 40 years ago. The read‐out for this assay is based on manual scoring by visual inspection of cells under a fluorescence microscope. CDC is often used in combination with the flow cytometry–based lymphocyte crossmatch assay (FCXM), which, with high sensitivity, detects HLA antibody binding. Here we describe a new approach wherein both cytotoxicity and antibody binding can be simultaneously assessed with flow cytometry. Two strategies are described, using either magnetic bead–enriched T and B lymphocytes or bulk peripheral blood mononuclear cells (PBMC) as donor target cells. Curr. Protoc. Cytom. 66:6.34.1‐6.34.11. © 2013 by John Wiley & Sons, Inc.

Keywords: kidney transplantation; HLA antibodies; crossmatching; flow cytometry; complement dependent cytotoxicity

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Cytotoxicity and Antibody Binding Using Magnetic Bead–Purified T and B Cells as Donor Cells
  • Basic Protocol 2: Determination of Cytotoxicity and Antibody Binding Using PBMC as Donor Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Determination of Cytotoxicity and Antibody Binding Using Magnetic Bead–Purified T and B Cells as Donor Cells

  Materials
  • Whole blood (drawn in ACD tubes) diluted 1:1 in PBS (e.g., 8 ml blood and 8 ml PBS)
  • Lymphocyte separation medium (LSM; e.g., Lymphoprep Axis‐Shield PoC AS)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS ( appendix 2A) containing 0.1% bovine serum albumin (BSA, e.g., Sigma‐Aldrich)
  • EasySep Human T Cell Enrichment Kit (StemCell Technologies, cat. no. 19051)
  • EasySep Human B Cell Enrichment Kit (StemCell Technologies, cat. no. 19054)
  • HLA antibody–positive control serum (e.g., in‐house serum or from commercial source)
  • HLA antibody–negative control serum (e.g., obtained from healthy ABO negative males or commercial source)
  • Patient (test) serum (centrifuge 10 min at 13,000 × g, 4°C, prior to use)
  • Standard rabbit complement (e.g., Cedarlane).
  • Fluorescein isothiocyanate (FITC)–conjugated goat anti‐human IgG F(ab′2) antibody (e.g., Jackson ImmunoResearch, cat. no. 096‐098)
  • 7‐aminoactinomycin D (7‐AAD) solution (e.g., Beckman Coulter, cat. no. A07704)
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Refrigerated centrifuge equipped with plate rotor
  • 12 × 75−mm flow cytometry sample tubes
  • Magnet (e.g., EasySep magnet from StemCell Technologies)
  • U‐bottom 96‐well plate
  • Flow‐cytometer tubes
  • Flow cytometer equipped with at least four fluorescence detectors (e.g., Beckman Coulter, cat. no. FC500)
  • Additional reagents and equipment for counting cells ( appendix 3A)

Basic Protocol 2: Determination of Cytotoxicity and Antibody Binding Using PBMC as Donor Cells

  Materials
  • Phycoerythrin (PE)‐conjugated anti‐human CD3 antibody (clone SK7, BD Bioscience)
  • Phycoerythrin‐Cyanin 7 (PC7) conjugated anti‐human CD19 antibody (clone J3‐119, Beckman Coulter)
  • Fluorescein isothiocyanate (FITC)–conjugated goat anti‐human IgG F(ab′2) antibody (e.g., Jackson ImmunoResearch, cat. no. 096‐098)
  • Additional reagents and equipment for determination of cytotoxicity and antibody binding using magnetic bead–purified T and B cells as donor cells ( protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Amos, D.B., Bashir, H., Boyle, W., MacQueen, M., and Tiilikainen, A. 1969. A simple micro cytotoxicity test. Transplantation 7:220‐223.
  Chen, G., Sequeira, F., and Tyan, D.B. 2011. Novel C1q assay reveals a clinically relevant subset of human leukocyte antigen antibodies independent of immunoglobulin G strength on single antigen beads. Hum. Immunol. 72:849‐858.
  Fuller, T.C., Fuller, A.A., Golden, M., and Rodey, G.E. 1997. HLA alloantibodies and the mechanism of the antiglobulin‐augmented lymphocytotoxicity procedure. Hum. Immunol. 56:94‐105.
  Garovoy, R.M., Bigos, M., Perkins, H., and Colombe B. 1983. Flow cytometry analysis: A high technology crossmatch technique facilitating transplantation. Transplant Proc. XV:1939‐1944.
  Johnson, A.H., Rossen, R.D., and Butler, W.T. 1972. Detection of alloantibodies using a sensitive antiglobulin microcytotoxicity test: Identification of low levels of pre‐formed antibodies in accelerated allograft rejection. Tissue Antigens 2:215‐226.
  Lillevang, S.T., Steinbruchel, D.A., Kristensen, T., and Kemp, E. 1992. A new flowcytometric CDC assay for detection of cytotoxic antibodies applied to hamster‐to‐rat cardiac transplantation. Transplant Proc. 24:537‐538.
  Maecker, H.T. and Trotter, J. 2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037‐1042.
  Morgan, B.P. 2000. The complement system: An overview. Methods Mol. Biol. 150:1‐13.
  Saw, C.L., Bray, R.A., and Gebel, H.M. 2008. Cytotoxicity and antibody binding by flow cytometry: A single assay to simultaneously assess two parameters. Cytometry B 74:287‐294.
  Schonemann, C., Lachmann, N., Kiesewetter, H., and Salama, A. 2004. Flow cytometric detection of complement‐activating HLA antibodies. Cytometry B 62:39‐45.
  Ta, M. and Scornik, J.C. 2002. Improved flow cytometric detection of donor‐specific HLA class II antibodies by heat inactivation. Transplantation 73:1611‐1614.
  Talbot, D., Shenton, B.K., Givan, A.L., Proud, G., and Taylor, R.M. 1987. A rapid, objective method for the detection of lymphocytotoxic antibodies using flow cytometry. J. Immunol. Methods 99:137‐140.
  Terasaki, P.I. and McClelland, J.D. 1964. Microdroplet assay of human serum cytotoxins. Nature 204:998‐1000.
  Vaidya, S., Cooper, T.Y., Avandsalehi, J., Barnes, T., Brooks, K., Hymel, P., Noor, M., Sellers, R., Thomas, A., Stewart, D., Daller, J., Fish, J.C., Gugliuzza, K.K., and Bray, R.A. 2001. Improved flow cytometric detection of HLA alloantibodies using pronase: Potential implications in renal transplantation. Transplantation 71:422‐428.
  Wetzsteon, P.J., Head, M.A., Fletcher, L.M., Lye, W.C., and Norman, D.J. 1992. Cytotoxic flow‐cytometric crossmatches (flow‐tox): A comparison with conventional cytotoxicity crossmatch techniques. Hum. Immunol. 35:93‐99.
  Won, D.I., Jeong, H.D., Kim, Y.L., and Suh, J.S. 2006. Simultaneous detection of antibody binding and cytotoxicity in flow cytometry crossmatch for renal transplantation. Cytometry B 70:82‐90.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library