2023-01-29-世界卫生组织-WHO_BS_2023.2445_WHO_RR_for_Anti-HLA_run_controls_106页_2mb

Summary of WHO Collaborative Study for Establishing Anti-HLA Reference Reagents

Background

The presence of donor-specific anti-HLA antibodies in transplant recipients can lead to hyperacute rejection, making their detection crucial for successful transplantation outcomes. Flow cytometry crossmatch (FCXM) and Luminex-based assays (bead-based single antigen bead, SAB) are sensitive methods for detecting anti-HLA antibodies, but they require standardized reference reagents to validate and harmonize testing. This international collaborative study evaluated four anti-HLA reference reagents (RR) developed by the National Institute for Biological Standards and Control (NIBSC) to determine their suitability for WHO International Reference Reagent status.

Study Design

• Participants: 21 laboratories from eight countries evaluated the reference materials using their in-house procedures for FCXM and Luminex assays.
• Materials: Four reference reagents were assessed:
  • 10/142: Negative plasma for anti-HLA.
  • 17/212: Negative serum for anti-HLA.
  • 17/238: Strong positive plasma for anti-HLA.
  • 21/378: Weak positive plasma for anti-HLA.
• Assays: Both FCXM and Luminex SAB assays were used to test the reagents, with performance evaluated based on fluorescence intensity values, percentage reactive antibody (% PRA), and qualitative categorization (negative, weak positive, strong positive).
• Stability: Accelerated thermal degradation and real-time stability monitoring confirmed the reagents' stability over time.

Key Findings

• Performance in FCXM Assays:

  • Negative controls (10/142 and 17/212) were consistently identified as negative by 96-96.4% of participants.
  • Strong positive control (17/238) was identified as strong positive by 94-100% of participants.
  • Weak positive control (21/378) showed variable recognition due to differences in laboratory-specific cut-off criteria and assay sensitivity, but was categorized as weak positive by 8-67% of labs, depending on the cell type (T or B cells).
  • Variability in results highlighted the need for standardized thresholds and assay harmonization.

• Performance in Luminex Assays:

  • Negative controls (10/142 and 17/212) were classified as negative in 71-73% of cases.
  • Strong positive control (17/238) was universally identified as strong positive.
  • Weak positive control (21/378) showed lower consensus positivity (19.3% for class I, 4.6% for class II), indicating intermediate reactivity.
  • Methodological differences, such as pre-treatment and software usage, contributed to variability in % PRA values.

• Stability and Suitability:

  • All reagents demonstrated stability under standard storage conditions (4°C and -20°C) based on real-time and accelerated degradation studies.
  • The reagents performed reliably across different assays and laboratories, with no significant deviations in functionality.

Proposal

Based on the collaborative study results, the Expert Committee on Biological Standardization (ECBS) is proposed to endorse the establishment of the following four anti-HLA reference reagents as WHO International standards:

1. 10/142: WHO International reference reagent—Negative plasma for anti-HLA.
2. 17/212: WHO International reference reagent—Negative serum for anti-HLA.
3. 17/238: WHO International reference reagent—Strong positive plasma for anti-HLA.
4. 21/378: WHO International reference reagent—Weak positive plasma for anti-HLA.

These reference reagents will serve as qualitative intra-assay variability controls, enabling trend monitoring and harmonization of FCXM and Luminex assays globally.

Conclusion

The study demonstrates that these anti-HLA reference reagents are fit for purpose and can standardize antibody detection assays, improving reliability in transplantation compatibility testing. Their WHO International recognition will enhance global accessibility and utility, particularly for laboratories outside the European Economic Area.