Potential Diagnostic Application of the Quantitative Assessment of Red Blood Cell Membrane Protein Expression

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RelevantRBC membrane proteins are selected based on the literature and these data are compared to Mass Spectrometry (MS) measurements and the data in the erythrocyte database. For Flow Cytometry (FACS) measurements, RBC ghosts (hemoglobin depleted erythrocyte membranes) are prepared from a small drop of blood and these ghosts are labeled by a fluorescent lectin derivative (e.g. wheat germ agglutinin-AlexaFluor647, WGA-A647) to allow selective gating. Thereafter, the ghosts are labeled with specific monoclonal antibodies, then with suitable fluorescent secondary antibodies (for details see. The measurements are carried out in a flow cytometer, preferably equipped with automatic high throughput plate sampler.

Inorder to obtain quantitative results in the FACS assay, each membrane protein and each selective first antibody has to be carefully titrated to give maximum level interactions. Still, the results of the FACS measurements do not provide an actual number of the given membrane protein per RBC, and data have to be evaluated based on the protein levels obtained in a certain population or under specific disease conditions.