Faculty Collaboration Database

Immunoglobulin isotype identification in red cell antibodies using flow cytometry. Transfusion 1999 Jul;39(7):756-62

Date

07/21/1999

Pubmed ID

10413285

DOI

10.1046/j.1537-2995.1999.39070756.x

Scopus ID

2-s2.0-0032776381 (requires institutional sign-in at Scopus site)   4 Citations

Abstract

BACKGROUND: Identifying the isotype of an immunoglobulin (IgM vs. IgG) detected in a patient sample is especially important in anticipating the risk of hemolytic disease of the newborn. Currently, 2-mercaptoethanol (2-ME) treatment of a sample is used in the authors' laboratory to degrade IgM, and this is followed by retesting. This method has multiple drawbacks. The purpose of this study was to develop a flow cytometry (FC) assay that would replace the 2-ME treatment protocol (2-ME treatment).

STUDY DESIGN AND METHODS: A preliminary FC assay was developed, modified, and refined through the use of stock antibodies. Then, 10 samples containing antibodies were tested in parallel by the FC assay and 2-ME treatment.

RESULTS: When a 10-unit mean channel fluorescence change was used as an index of a positive result, the FC assay detected all isotypes identified by 2-ME treatment. The FC assay was also able to identify mixtures of isotypes. One antibody that had not reacted in conventional agglutination testing was detected by the FC assay. The amount of fluorescence and the agglutinating strength of the antibody did not parallel each other. In one case, this discrepancy may have reflected an antibody that was primarily IgA.

CONCLUSIONS: The FC assay appears to be as accurate as 2-ME treatment in differentiating IgG from IgM. The FC assay produces a positive endpoint for both isotypes, will identify IgA, requires less sample, and has no odor.

Author List

Price WR, Johnson ST, Curtis BR

Author

Brian Curtis PhD Director in the Platelet & Neutrophil Immunology Laboratory department at BloodCenter of Wisconsin

MESH terms used to index this publication - Major topics in bold

Antibodies, Anti-Idiotypic
Erythrocytes
Flow Cytometry
Fluorescence
Humans
Immunoglobulin G
Immunoglobulin Isotypes
Immunoglobulin M
Isoantibodies
Mercaptoethanol
Reproducibility of Results