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Related Experiment Videos

T and B cell crossmatching using three-colour flow cytometry

A Robson1, S Martin

  • 1Tissue Typing Laboratory, St Mary's Hospital, Manchester, UK.

Transplant Immunology
|September 1, 1996
PubMed
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A new three-colour flow cytometry method accurately crossmatches T and B cells for alloantibodies. This sensitive technique improves detection of antibodies against donor cells compared to traditional methods.

Area of Science:

  • Immunology
  • Transplantation immunology
  • Clinical diagnostics

Background:

  • Accurate crossmatching is crucial for successful organ transplantation.
  • Detecting patient alloantibodies against donor T and B cells is essential to prevent rejection.
  • Existing methods may lack sensitivity or specificity in identifying antibody targets.

Purpose of the Study:

  • To develop and validate a highly sensitive three-colour flow cytometry method for simultaneous T and B cell crossmatching.
  • To compare the sensitivity and accuracy of this new method against conventional crossmatching techniques and complement-dependent cytotoxicity assays.
  • To determine the utility of three-colour flow cytometry in identifying specific antibody targets against donor T and/or B cells.

Main Methods:

Related Experiment Videos

  • Development of a three-colour flow cytometry assay utilizing fluorescently conjugated monoclonal antibodies (anti-CD19 for B cells, anti-CD3 for T cells) and a detection antibody for bound IgG.
  • Incubation of donor lymphocytes with patient serum followed by analysis of bound IgG on T and B cell populations.
  • Comparative analysis of results from three-colour flow cytometry, conventional crossmatching, and complement-dependent cytotoxicity assays using test sera and donor-recipient pairs.

    • Main Results:

    • The three-colour flow cytometry method demonstrated high sensitivity in detecting bound patient IgG alloantibodies on both T and B cells.
    • No significant difference was observed in bound IgG detection between three-colour flow cytometry and conventional crossmatching methods across various samples.
    • The three-colour flow cytometry assay showed increased sensitivity compared to complement-dependent cytotoxicity assay in 8 out of 72 crossmatches, aiding in antibody target identification.

    Conclusions:

    • Three-colour flow cytometry provides a sensitive and simultaneous method for crossmatching T and B cells.
    • This technique enhances the ability to identify specific antibody targets against donor T and/or B cells.
    • The method offers improved sensitivity over complement-dependent cytotoxicity assays, potentially improving transplant outcomes.