Flow cytometers are instruments that can quantify fluorescence intensity data and provide unique information about cell populations. Fluorescence intensity calibration allows the establishment of a comparable window of analysis across different times and laboratories. Significant advances have been made
From: Methods in Molecular Biology vol. 378: Monoclonal Antibodies: Methods and Protocols Edited by: M. Albitar © Humana Press Inc., Totowa, NJ
in terms of calibration reagents, standardized sample preparation, and data analysis to ensure interlaboratory comparability and reproducibility. The design and use of calibration beads labeled with predefined amounts of dye allows instrument-independent expression of fluorescence intensity in units of molecules of equivalent soluble fluorochrome (1). This method was refined by the combined use of such standards with monoclonal antibodies (MAb) conjugated 1/1 with phycoerythrin (PE), allowing translation of fluorescence intensity into numbers of antibodies bound per cell (ABC) (2). Commercially available 1:1 labeled MAb have been used in patient diagnostics for several years now. In HIV infection, for example, antigen density changes in CD38 expression of CD8-positive T-cells may be an important indicator of disease progression (3).
In an earlier study, we quantitatively determined the simultaneous expression of HLA-DR and aminopeptidase N/CD13 on peripheral blood monocytes of patients suffering major trauma (4). Trauma patients are known to have an early onset depression of the overall cellular immune response, which is associated with a high rate of infection and mortality. Several investigators have shown that the expression of monocyte HLA-DR as a marker for their antigen-presenting capacity is severely impaired and correlates with the outcome of patients (5,6). The mechanisms involved in the regulation of HLA-DR include shedding of HLA-DR from the cell surface and regulation of HLA-DR gene transcription (7). We described that the recovery of the attenuated monocytic HLA-DR expression after trauma was accompanied by a strong increase in the membrane enzyme aminopeptidase N/CD13 on monocytes (4). Fourteen days after trauma, the monocytic expression of CD13 was still much higher than in normal volunteers used as a control group. Membrane peptidases are multifunctional molecules. Not only do these enzymes hydrolyse small peptide mediators, resulting in activation or inactivation; they also function as receptors and as molecules participating in cell motility and in adhesion to extracellular matrix (8). Therefore, the expression of these antigens on leukocytes could be useful for characterizing the function of the immune system. We investigated the expression of two membrane peptidases on granulocytes in patients undergoing open heart surgery. Using quantitative immunofluorescence and flow cytometry, we showed a similar time-course both of the expression of HLA-DR on monocytes and of the membrane peptidases Aminopeptidase N/CD13 and neprilysin/CD10 on granulocytes.
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