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Standardization of measurement of immunoglobulin-secreting cells in human peripheral circulation
S Baqar1, A A Nour El Din, D A Scott
1Department of Infectious Diseases, Naval Medical Research Institute, Bethesda, Maryland, USA.
Insights
Standardizing antibody-secreting cell (ASC) assays is crucial for vaccine immunogenicity studies. Cryopreservation of mononuclear cells (MNC) minimally impacts ASC enumeration, enabling reliable, quantitative comparisons across labs.
Area of Science:
- Immunology
- Vaccinology
- Cell Biology
Background:
- Enumerating antibody-secreting cells (ASC) in peripheral blood is a key measure of human vaccine immunogenicity.
- Current ASC assays lack standardization, hindering quantitative inter-laboratory comparisons.
- The need for standardized, practical methods for assessing vaccine responses is critical.
Purpose of the Study:
- To assess the impact of cryopreservation on isotype-specific antibody-secreting cell (ASC) enumeration.
- To compare ASC counts from fresh versus cryopreserved mononuclear cells (MNC) in healthy adults from different geographic locations (US and Egypt).
- To evaluate the effect of cryopreservation on antigen-specific ASC detection following vaccination.
Main Methods:
- Enumeration of isotype-specific ASC (IgM, IgG, IgA) from paired fresh and cryopreserved MNC.
- Comparison of ASC counts between US and Egyptian volunteer groups.
- Analysis of antigen-specific IgA and IgG ASC in volunteers vaccinated with LT(R192G) using fresh and cryopreserved MNC.
Main Results:
- Cryopreservation caused only a slight reduction (max 14%) in mean group ASC counts for IgM, IgG, and IgA.
- Results from cryopreserved cells were not significantly lower than those from fresh cells.
- No significant difference in antigen-specific IgA or IgG ASC was observed between fresh and cryopreserved MNC post-vaccination.
Conclusions:
- Cryopreservation of MNC is a viable method for ASC enumeration, with minimal impact on results.
- Standardized ASC assays using cryopreserved cells can yield reliable quantitative data for vaccine immunogenicity.
- The methodology supports the development of more practical and standardized ASC assays for global use.
Abstract:
A sensitive, and at times the most sensitive, measurement of human vaccine immunogenicity is enumeration of antibody-secreting cells (ASC) in peripheral blood. However, this assay, which is inherently capable of measurement of the absolute number of antigen-specific ASC, is not standardized. Thus, quantitative comparison of results between laboratories is not currently possible. To address this issue, isotype-specific ASC were enumerated from paired fresh and cryopreserved mononuclear cell (MNC) preparations from healthy adult volunteers resident in either the United States (US group) or Egypt (EG group). Analysis of fresh cells from US volunteers revealed mean numbers of ASC per 10(6) MNC of 617, 7,738, and 868 for immunoglobulin M (IgM), IgG, and IgA, respectively, whereas EG volunteers had 2,086, 7,580, and 1,677 ASC/10(6) MNC for the respective isotypes. Cryopreservation resulted in a slight reduction in group mean IgM, IgG, and IgA ASC (maximum reduction in group mean, 14%), but in no instance were results obtained with cryopreserved cells significantly lower than those obtained with fresh cells. To determine if cryopreservation affected the number of bacterial antigen-specific ASC detected, cells from a group of US adult volunteers who received a single oral dose of a mutated Escherichia coli heat-labile enterotoxin (LT(R192G)) were tested. There was no significant difference (P > 0.05) in the number of antigen-specific IgA or IgG ASC detected between fresh and cryopreserved MNC. The results support the views that ASC assays can be standardized to yield quantitative results and that the methodology can be changed to make the test more practical.
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