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Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells
Published on: January 29, 2014
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An optimized microplate-based method to evaluate complement-dependent hemolysis mediated by intravenous
Yonggang Wang1, Alexey Khalenkov1, Dorothy E Scott1
1Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
Summary
A new assay can measure the functional hemolytic activity of Intravenous Immunoglobulin (IVIG) products. This complement-dependent hemolysis assay (CDHA) offers a more reliable method for assessing IVIG safety and investigating adverse events.
Area of Science:
- Immunology
- Biotechnology
- Clinical Chemistry
Background:
- Intravenous Immunoglobulin (IVIG) administration can lead to hemolytic reactions due to anti-A and anti-B antibodies (isoagglutinins).
- Current regulatory limits for isoagglutinins in IVIG, measured by the direct hemagglutination test (DHAT), may not adequately predict clinical hemolysis, as reported events have increased.
- There is a need for a more sensitive and reproducible method to assess the functional hemolytic activity of IVIG products.
Purpose of the Study:
- To develop and validate a microplate-based complement-dependent hemolysis assay (CDHA) for measuring functional hemolytic activity of IVIG.
- To assess the variability of hemolytic activity across different IVIG brands and lots.
- To evaluate the correlation between the new CDHA and the existing DHAT method.
Main Methods:
- A microplate-based complement-dependent hemolysis assay (CDHA) was developed.
- An IVIG working reference reagent (NIBSC 14/160) was qualified for use as an assay control and for quantitation.
- Hemolytic activities of 36 IVIG product lots from seven brands, including six clinically hemolytic lots, were measured using the CDHA.
Main Results:
- The CDHA demonstrated reproducible measurement of functional hemolytic activity in IVIG products.
- Significant variation in hemolytic activity was observed among different IVIG brands and, to a lesser extent, between lots of the same brand.
- The correlation between the CDHA and the DHAT was not robust, suggesting limitations of the DHAT in predicting clinically relevant hemolysis.
Conclusions:
- The developed CDHA is a simple, specific, and sensitive tool for characterizing IVIG products.
- This assay can aid manufacturers, researchers, and regulatory authorities in investigating IVIG-associated hemolytic events.
- The CDHA provides a more accurate assessment of functional hemolytic potential compared to the DHAT.
