Flow Cytometric Assessment of HLA Alloantibodies

Robert A. Bray1, Howard M. Gebel1, Thomas M. Ellis2

1 Emory University, Atlanta, Georgia, 2 The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.16
DOI:  10.1002/0471142956.cy0616s27
Online Posting Date:  February, 2004
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Abstract

Antibodies against HLA molecules are formed in response to exposure to foreign HLA molecules, which can occur as a result of blood transfusion, pregnancy, or transplant. Blood components, particularly those containing cellular elements, are the most common cause of HLA antibodies. This unit describes technical aspects of the flow cytometric crossmatch (FCXM), flow cytometric microparticle assays, and cellā€based flow cytometric screening assays. The collective goal for these assays is to clearly identify the presence of HLA antibody, determine the titer of antibody, and elucidate the specificities (i.e., HLA antigens) to which they will react. Knowledge of this information is critical for organ allocation and accurate assessment of the immunological risk for a patient at the time of transplantation. In addition, the identification of HLA antibodies in blood components may be useful in planning appropriate transfusion support strategies for selected patients.

Keywords: alloantibody; flow cytometry crossmatch; HLA antibody; transplantation

     
 
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Table of Contents

  • Basic Protocol 1: Detection of HLA Antibodies by Flow Cytometric Crossmatch
  • Alternate Protocol 1: Detection of HLA Antibodies Using A Microparticle Screening Assay
  • Basic Protocol 2: Determination of HLA Specificity (Class I and Class II) Using Flow Microparticles
  • Basic Protocol 3: Cell‐Based Flow Cytometric Panel Reactive Antibody (FC‐PRA) Assay
  • Support Protocol 1: Treatment of Cell Preparation Containing Lympho‐Kwik or Percoll
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of HLA Antibodies by Flow Cytometric Crossmatch

  Materials
  • Donor sample: freshly purified lymphocytes or mononuclear cells with >80% viability, isolated from lymph node, spleen, or peripheral blood; or thawed cryopreserved mononuclear cells
  • Patient sample: serum from potential recipient(s)
  • Negative control: normal human or pooled human sera from non‐sensitized individuals (NHS)
  • Positive control: pooled positive serum from sensitized patients with high anti‐HLA antibody titer (PPS)
  • Flow wash buffer (FWB; see recipe), ice cold
  • FITC‐conjugated goat anti‐human IgG [F(ab′) 2 fragment, Fcγ‐specific; Jackson Laboratories; PE conjugate also available]
  • Anti‐human CD3‐PerCP (or other conjugated fluorochrome with excitation at 488 nm and emission >650 nm)
  • Anti‐human CD19‐PE (or CD20‐PE)
  • 1% paraformaldehyde in PBS, pH 7.2 ± 0.2, ice cold; store protected from light up to 2 weeks at 4°C
  • 6 × 50–mm glass tubes (Kimble), 1.5‐ml conical plastic tubes, or 96‐well microtiter plates
  • Tabletop centrifuge
  • Vacuum aspirator
  • Flow cytometer with 488‐nm excitation and filters for collection of green (525 ± 15 nm), orange (575 ± 15 nm), and red (>650 nm) fluorescence
  • Analysis software
  • Additional reagents and equipment for pronase treatment (Vaidya et al., ), and cell counting ( appendix 3A)

Alternate Protocol 1: Detection of HLA Antibodies Using A Microparticle Screening Assay

  Materials
  • Sera to be tested
  • Negative control: normal human or pooled human sera from non‐sensitized individuals (NHS; One Lamda or Pel‐Freez)
  • Positive control: pooled positive serum from sensitized patients with high antibody titer (PPS; One Lamda or Pel‐Freez)
  • Class I and class II FlowPRA beads (a combined kit or separate products from One Lambda).
  • 1:10 (v/v) diluted flow bead wash buffer (provided with the kit from One Lambda) in water; use at room temperature
  • FITC‐conjugated goat anti‐human IgG [F(ab′) 2 fragment, Fcγ specific; Jackson Laboratories; PE conjugate also available]
  • Tabletop centrifuge (e.g., Fisher or Beckman)
  • 6 × 50–mm glass tubes, 5‐ml plastic conical tubes, or 96‐well tissue culture plates
  • Repeat pipettor (e.g., Gilson Pipetman) with DNA gel‐loading tips
  • Vacuum aspirator
  • Flow cytometer with 488‐nm excitation and filters for collection of green fluorescence

Basic Protocol 2: Determination of HLA Specificity (Class I and Class II) Using Flow Microparticles

  Materials
  • Patient serum
  • Negative control sera
  • Positive control sera
  • Liquid nitrogen
  • FlowPRA specific class I beads and/or flowPRA specific class II beads (One Lambda); provided in 4 groups of 8 beads/group
  • 1:10 (v/v) diluted flow bead wash buffer (included in specificity bead kits from One Lambda) in water; use at room temperature
  • FITC‐conjugated goat anti‐human IgG [F(ab′) 2 Fc specific] or FITC‐conjugated goat anti‐human IgM [F(ab′) 2 Fc specific]
  • Tabletop centrifuge (e.g., Fisher or Beckman)
  • Polyethylene microultracentrifuge tubes
  • 6 × 50–mm glass tubes or 96‐well tissue culture plate
  • Vortexer
  • 5‐µl single‐shooter syringe (e.g., Hamilton)
  • Vacuum aspirator
  • Repeat pipettor (e.g., Eppendorf) with a multi‐channel adapter
  • Flow cytometer with 488‐nm excitation and filter set for detection of green and orange fluorescence

Basic Protocol 3: Cell‐Based Flow Cytometric Panel Reactive Antibody (FC‐PRA) Assay

  Materials
  • Sera to be tested
  • Negative controls: normal human and pooled human sera (NHS)
  • Positive control: pooled positive serum titered for the sensitivity of the flow (PPS)
  • Frozen cell pools, frozen using techniques that preserve maximum viability; appropriate concentration is ∼8 × 106/ml
  • RPMI containing 20% FBS
  • Flow wash buffer (FWB, see recipe), ice cold
  • FITC‐conjugated goat anti‐human IgG [F(ab′) 2, Fc specific; Jackson Laboratories]
  • Phycoerythrin (PE)‐conjugated anti‐human CD3 monoclonal antibody (Becton‐Dickinson), diluted according to manufacturer's specification
  • 1% paraformaldehyde in PBS, pH 7.2 ± 0.2
  • Airfuge (e.g., Beckman) with microultracentrifuge tubes and protective caps
  • 15‐ml conical tubes
  • Colored tape
  • Benchtop centrifuge (e.g., Beckman GP)
  • 6‐ml polypropylene tubes (e.g., Falcon)
  • 96‐well U‐bottom cell culture plate (Corning‐Costar)
  • Repeat pipettor (Eppendorf) and 8‐channel attachment with appropriate tips
  • Transfer pipet (e.g., Brinkman Transferpette‐12) with appropriate tips
  • Absorbent pad
  • Flow cytometer with 488‐nm excitation and filters for collection of green and orange fluorescence
  • 6 × 50–mm glass tubes (Baxter diSPo culture tubes)
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 1: Treatment of Cell Preparation Containing Lympho‐Kwik or Percoll

  Materials
  • Cell preparation treated with Percoll or Lympho‐Kwik
  • RPMI medium with 10% FBS
  • Flow wash buffer (FWB, see recipe)
  • 37°C incubator or water bath
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Figures

Videos

Literature Cited

Literature Cited
   Bray, R.A. 1994. Flow cytometric crossmatching in solid organ transplantation. In Methods in Cell Biology: Flow Cytometry, Vol. 41 (Z. Darzynkiewicz, H.A. Crissman, and, J.P. Robinson, eds.) pp. 103‐119. Academic Press, New York.
   Bray, R.A. and Gebel, H.M. 2000. Transplantation immunophenotyping. In Immunophenotyping: Cytometric and Cellular Analysis (C. Stewart, and, J. Nicholson, eds.) pp. 321‐332. Academic Press, New York.
   Bray, R.A., Lebeck, L.L., and Gebel, H.M. 1989. The flow cytometric crossmatch: Dual‐color analysis of T and B cells. Transplantation 48:834‐840.
   Bray, R.A., Cook, D.J., and Gebel, H.M. 1997. Flow cytometric detection of HLA alloantibodies using class I coated microparticles. Hum. Immunol. 55:36.
   Bray, R.A, Sinclair, D.A, Wilmoth‐Hosey, L., Lyons, C., Chapman, P., and Holcomb, J. 1998. Significance of the flow cytometric PRA (FC‐PRA) in the evaluation of patients awaiting renal transplantation. Hum. Immunol. 59:121.
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