Flow cytometric detection of complement-activating HLA antibodies

Area of Science:

• Immunology
• Transplantation Science
• Clinical Diagnostics

Background:

• Antibodies to Human Leukocyte Antigens (HLA) are critical factors in allograft rejection, necessitating accurate detection methods in organ transplantation.
• Flow cytometry (FC) offers potential improvements in transplantation outcomes, but existing techniques often fail to differentiate between complement-activating and non-complement-activating antibodies.
• The clinical significance of non-complement-activating antibodies remains debated, leading many labs to rely on conventional complement-dependent microlymphocytotoxicity crossmatch (CDC-XM) or combined methods.

Purpose of the Study:

• To introduce and validate a novel flow cytometric complement-dependent cytotoxicity crossmatch (FC-CDC-XM) assay.
• To demonstrate the assay's capability in specifically identifying complement-binding HLA antibodies.
• To compare the new assay's performance against conventional methods.

Main Methods:

• Development of a three-color FC-CDC-XM assay using a FACScan flow cytometer.
• Utilized fluorescence-conjugated anti-CD3 (T lymphocytes), anti-CD19 (B lymphocytes), and propidium iodide (PI) for cell viability assessment.
• Lymphocyte gating was performed using side scatter versus FL1/FL2 dot plots, with PI fluorescence measured in the FL3 channel.

Main Results:

• The FC-CDC-XM assay successfully identified all tested antibodies specific to HLA class I (n=18) and HLA class II (n=4).
• Demonstrated clear discrimination between complement-binding and non-complement-binding antibodies.

Conclusions:

• The FC-CDC-XM assay is more sensitive and objective than the standard CDC-XM.
• This novel assay is suitable for pre- and post-transplant diagnostics.
• The FC-CDC-XM assay can be applied to detect other antibody-mediated cytotoxic reactions.