Blood fluorescence activated cell

The various organs of the immune system such as spleen, lymph nodes, thymus and bone marrow containing the cells involved in the various immune responses offer the possibility to harvest these cells and perform in vitro assays for evaluation of effects on the immune system. When part of an in vivo animal study this may indicate a direct toxic effect of pharmaceuticals, that is, immunosuppression (Table 18.2). So, it is feasible to obtain cell suspensions for further evaluation such as determination of cellular subsets of T and B leukocytes by fluorescent activated cell sorter analysis (FACS analysis), and determination of natural killer (NK) cell activity of the spleen cell population. An advantage of this approach is that it may lead to identification of a biomarker to be used in clinical studies. In addition, in vitro stimulation of spleen cells with mitogens activating specific subsets may indicate potential effects on the functionality of splenic cell populations. Concanavalin A (Con A) and phytohemagglutinin (PHA) activate Tcells, while lipopolysaccharide (LPS) activates primarily B cell populations. Blood is collected for total white blood cell (WBC) determination and blood cell differential count. In addition, serum can be obtained for determination of serum immunoglobulins. 

The hematopoietic lineage originally was worked out by Injecting the various types of precursor cells into mice whose precursor cells had been wiped out by irradiation. By observing which blood cells were restored in these transplant experiments, researchers could infer which precursors or terminally differentiated cells (e.g., erythrocytes, monocytes) arise from a particular type of precursor. The first step in these experiments was separation of the different types of hematopoietic precursors. This is possible because each type produces unique combinations of cell-surface proteins that can serve as type-specific markers. If bone marrow extracts are treated with fluorochrome-labeled antibodies for these markers, cells with different surface markers can be separated in a fluorescence-activated cell sorter (see Figure 5-34). 

Fig. 3 B-cell recovery and SPD in all responders (pivotal trial). Median B-cell counts in peripheral blood (as measured by CD 9 positivity on Fluorescence Activated Cell Sorter (FACS) analysis) drop to zero and start recovering by the sixth month. Tumor volume (as measured by SPD) for responders continues to decrease beyond nine months despite normalization of B-cell counts [38, 39]. This figure is used by permission from the copyright holders - Grillo-Lopez A] and Idee Pharmaceuticals.

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectrophotometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anficoagulated whole blood (14,15). 

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