Flow Cytometric Screening for the HLA‐B27 Antigen on Peripheral Blood Lymphocytes

Wilfried H.B.M. Levering1, Kees Sintnicolaas1, Henk Wind2, Herbert Hooijkaas2, Jan W. Gratama2

1 Laboratory for Histocompatibility and Immunogenetics, Sanquin Blood, Bank South West Region, Rotterdam, 2 Erasmus MC, Rotterdam
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.22
DOI:  10.1002/0471142956.cy0622s33
Online Posting Date:  August, 2005
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HLA‐B27 is common to the entire group of seronegative spondyloarthropathies, and assessment of HLA‐B27 is of diagnostic importance if any of these diseases are considered. Among the alternatives to traditional typing by the complement‐dependent cytotoxicity assay, flow cytometric HLA‐B27 screening is most widely used in general diagnostic laboratories, as it is simple, rapid, and cost effective. This unit describes screening for HLA‐B27 on peripheral blood lymphocytes using more than one HLA‐B27 monoclonal antibody to detect possible cross‐reactivity with non‐HLA‐B27 antigens. Screening is hampered by the lack of true monospecific anti‐HLA‐B27 monoclonal antibodies. Cross‐reactivities of anti‐HLA‐B27 with other HLA‐B antigens have been reported. The authors recommend use of GS145.2 and FD705 antibodies, as this combination avoids most false‐positive conclusions. Lyse‐and‐wash sample processing is employed. A multiparameter flow methodology is applied. Three to four parameters–forward scatter, side scatter, HLA‐B27 expression, and, in some cases CD3 or B7 expression–are acquired.

Keywords: HLA‐B27; flow cytometry; cross‐reactivity; HLA‐typing; monoclonal antibody

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Patient blood samples to be screened for HLA‐B27, anticoagulated with acid citrate dextrose or EDTA
  • Positive control: freshly obtained blood sample with known HLA‐B27‐positive HLA‐B typing result
  • Negative control: freshly obtained blood sample with known HLA‐B27‐negative HLA‐B typing result and no cross‐reactivity with the HLA‐B27 MAb used (see )
  • HLA‐B27 Kit (BD Biosciences; optional) containing:
    • Anti‐HLA‐B27‐FITC/CD3‐PE (clones anti‐GS145.2 and SK7, respectively)
    • 10× BD FACS lysing solution
    • Calibration beads
    • HLA‐B27 Kit software (optional)
  • 10× ammonium chloride lysing solution ( appendix 2A) or BD FACS lysing solution (BD Biosciences)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.2% BSA (see recipe)
  • PBS containing 1% (w/v) paraformaldehyde
  • FITC‐conjugated anti‐HLA‐B27 monoclonal antibodies: GS145.2 (BD Biosciences), and FD705 (One Lambda; http://www.onelambda.com); combined use of GS145.2 and FD705 is recommended (see above)
  • FITC‐conjugated mouse IgG2b isotype control MAb (One Lambda)
  • 12 × 75–mm polypropylene tubes
  • Tabletop centrifuge
  • Flow cytometer equipped to detect forward scatter (FS) and side scatter (SS), with a laser emitting at 488 nm and at least two fluorescence detectors, i.e., for fluorescein isothiocyanate (FITC) and phycoerythrin (PE)
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Literature Cited

Literature Cited
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