Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays

Area of Science:

• Immunology
• Transplantation Science

Background:

• Human leukocyte antigen (HLA) compatibility is critical for solid organ transplantation success.
• Donor-specific anti-HLA antibodies (DSAs) are a major barrier to transplantation and require accurate detection.
• Existing cell-based and HLA bead-based assays for DSA identification have limitations, including the prozone effect, which can lead to false-negative results.

Purpose of the Study:

• To investigate the impact of the prozone phenomenon on cell-based assays (flow cytometric cross-match, FCXM) and HLA bead-based assays (panel reactive antibodies, PRA).
• To compare the utility of these assays in histocompatibility testing under prozone conditions.

Main Methods:

• Four experiments were conducted using known positive samples, pooled sera, and commercially available positive control sera.
• Flow cytometric cross-match (FCXM) and HLA bead-based panel reactive antibody (PRA) assays were performed.
• Serial dilutions of pooled sera and positive concoctions were analyzed to assess the prozone effect.

Main Results:

• Pooled sera showed unexpected median fluorescence intensity (MFI) in FCXM, while PRA indicated high positivity.
• The prozone effect caused declining MFI in FCXM with positive concoctions, whereas PRA values remained stable.
• Diluting samples revealed a sudden increase in FCXM MFI, peaking at 1:4 dilution, while PRA values remained consistently high.

Conclusions:

• Cell-based assays, specifically FCXM, are more susceptible to the prozone effect, leading to potential false-negative results.
• Solid-phase assays, such as flow PRA, demonstrate resistance to the prozone effect, offering greater reliability in DSA detection.
• These findings highlight the differential impact of the prozone phenomenon on various assay types used in histocompatibility testing.