[Flow cytometry detection of erythrocyte antigens and antibodies. Technical aspects and new clinical applications].

Abstract

Background: Although hemagglutination techniques have proved worthwhile since many years in immunohematology, they also have several disadvantages. They are manual and subjective visual methods, which make it difficult to quantitate red cell antibodies or surface antigens. Flow cytometric analysis overcomes these limitations because of its ability to analyze individual populations of cells by sensitive, reproducible, and objective methods.

Materials and methods: Washed red cells from regular blood donors and patients were analyzed natively and after treatment with enzymes (sialidase, protease) or pneumococcal polysaccharides, using monoclonal Rh antibodies, human 7s-immunoglobulin, and FITC-labeled anti-human IgG or FITC anti-T lectin. The fluorescence intensity of single red cells was determined in the Ortho Cytoron Absolute flow cytometer.

Results: We determined the optimal test conditions and normal values by investigation of 50 blood donors. The fluorescence intensity of untreated red cells proved to be constant and therefore was used to adjust the instrument. Furthermore, the data of experimental adsorptions of red cells with pneumococcal antigens, sialidase (Vibrio cholerae) and protease (papain) as well as data from patients suffering from chronic HBV infection and autoimmune hemolytic anemia and acute pancreatitis are presented. A special software program was developed for statistical analysis and graphical presentation of the raw data. The computer program permits to analyze results from different experiments or from different dates and depicts them comparatively in overlay histograms, which may be useful in a serial study of changes of the antibody concentration or antigen expression.

Conclusion: The flow cytometric analysis of red cells proves to be a simple, rapid, reproducible, and objective method for antigen and antibody quantitation. Furthermore, this technique may be a useful new tool for the investigation of acute, infection-associated hemolytic anemia.