2023-07-14-世界卫生组织-WHO_BS_2023.5460_WHO_3rd_International_Standard_for_Protein_S,_Plasma_32页_936kb

Summary of the Collaborative Study for Value Assignment of the 3rd International Standard for Protein S, Plasma

Core Content

This document outlines a collaborative study conducted by the Expert Committee on Biological Standardization (ECBS) to evaluate and assign values to a candidate material for the 3rd International Standard for Protein S, Plasma (NIBSC code 22/202). The study involved 21 laboratories from 8 countries, with 19 returning results. The objective was to determine if the candidate material could replace the 2nd International Standard (03/228) and to assess the relationship between the International Unit (IU) and the normal plasma unit.

Key Findings

Potency Assignments

• Sample B (22/202) was proposed as the 3rd International Standard with the following assigned potencies:
  • Functional activity: 0.71 IU/ampoule
  • Free antigen: 0.83 IU/ampoule
  • Total antigen: 0.88 IU/ampoule

Assay Performance

• Laboratories were able to perform functional, free antigen, and total antigen assays with reasonable precision.
• Intra-laboratory GCVs for functional assays ranged from 0.6% to 13.5%, with most below 5%.
• For free antigen assays, intra-laboratory GCVs ranged from 0.2% to 16.8%, again with most below 5%.
• For total antigen assays, intra-laboratory GCVs ranged from 1.3% to 14.1%, with 57% of values above 5%.
• Inter-laboratory GCVs for the candidate material (Sample B) were:
  • Functional: 4.5%
  • Free antigen: 5.2%
  • Total antigen: 3.2%
• Common plasma sample (C) had inter-laboratory GCVs of 4.3% (functional), 5.6% (free antigen), and 10.5% (total antigen).
• Local normal pooled plasma (P) had inter-laboratory GCVs of 5.6% (functional), 6.4% (free antigen), and 3.7% (total antigen).

Assay Validity

• Almost all assays fit the models used (slope-ratio or parallel-line), with only a few exceptions due to high variability or non-linearity.
• No significant outliers were found using Grubbs' Test.
• Sample C and local plasma pools showed good agreement with the 2nd IS when recalculated against the candidate material (Sample B), with no significant differences observed.

Stability of the Candidate Material

• Accelerated degradation study at various temperatures showed a predicted loss of 0.013% per year at -20°C.
• On-bench stability indicated that reconstituted material remains stable for up to 4 hours when stored on melting ice.
• Frozen and thawed aliquots of the proposed 3rd IS are not recommended for use.

Main Recommendations

• The candidate material, Sample B (22/202), should be considered the 3rd International Standard for Protein S, Plasma.
• The study confirmed the original definition of the International Unit (IU) and demonstrated the long-term stability of the current IS.
• The assigned potencies for the candidate material are consistent with the 2nd IS and are suitable for calibration purposes.

Study Design and Participants

• 21 laboratories from 8 countries participated.
• 19 laboratories returned results, including 4 clinical, 5 therapeutic, 2 regulatory, and 8 diagnostic labs.
• The candidate material (Sample B) was sourced from the UK's National Blood and Transfusion Service (NBTS) and was freeze-dried after being pooled and buffered.
• Local normal pooled plasma (P) was also collected and included in the study, with 13 laboratories contributing data.

Assay Methods and Statistical Analysis

• Laboratories used various methods for functional, free antigen, and total antigen assays.
• Functional assays were primarily clotting-based, while free and total antigen assays were mostly ELISA or immunoturbidimetric.
• Statistical analysis was conducted using geometric mean (GM) potencies and geometric coefficients of variation (GCV).
• Grubbs' Test was applied to detect outliers, and Student's T-Test was used for group comparisons.
• No subgroup analysis was feasible due to limited method diversity and sample numbers.

Participant Feedback and Expert Comments

• One laboratory raised concerns about the lack of parallelism and linearity in commercial assays, suggesting that the use of slope-ratio models should be more clearly explained.
• The ISTH SSC experts supported the proposal, acknowledging the thorough design and results.
• Some comments highlighted the low number of participants and the absence of laboratories from Asia, Australasia, and South America, but the results showed consistency with the original IU definition.
• The limited number of labs and diverse methods affected the ability to perform method comparisons, but the results were deemed acceptable for establishing the new standard.

Conclusion

The study concluded that the candidate material, Sample B (22/202), meets the criteria to be the 3rd International Standard for Protein S, Plasma. The assigned potencies are consistent with the 2nd IS and reflect the long-term stability and reliability of the current standard. The proposal was endorsed by all participants and the ISTH SSC experts.

References

• Campbell PJ. International biological standards and reference preparations. J Biol Standardisation 1974; 2: 249-267
• WHO TRS, No. 932, 2006, Annex 2. pp.114-119 (section A.7)
• Finney DJ. Statistical Method in Biological Assay. 3rd Edition