Isolation of Immune Cells

Cell Samples
For most of the diagnostic tests, unless specified otherwise, cell samples derive from human blood, which contains a rich mixture of different cell types. Various fractionation methods are typically used, as discussed below, in order to enrich for white blood cells (WBC), which comprise less than 1% of all of the cells found in the blood. Some methods are designed to isolate specific WBC subpopulations depending on the nature of the diagnostic test.


RBC Lysis
The easiest method for lysing RBC without lysing other cells is to expose whole blood briefly to 0.15 >M ammonium chloride (NH4Cl). White blood cells (WBC) can then be recovered from the lysate by pelleting them by low speed centrifugation. This treatment typically has little effect on WBC viability or function but may alter the buoyant densities of different WBC subpopulations through swelling or shrinking by osmotic imbalance.


Density Gradient Centrifugation
This is another method often used to isolate WBC and different subpopulations. Because RBC have greater average buoyant density than most WBC, these two blood cell types can easily be separated by centrifuging whole blood samples on discontinuous density gradients made with solutions of high molecular weight polymers adjusted to specific densities as illustrated in the figure at the right.

Differential WBC Subpopulation Isolation
 Again, as illustrated in the figure at the right, different WBC subpopulations can be harvested from different buoyant "layers" following centrifugation through a density gradient. Here for example, a polysaccharide solution of density = 1.077 g/ml (e.g., Ficoll-400) is shown to separate other blood cells from peripheral blood mononuclear cells (PBMC), a subpopulation with lower average density that includes most lymphocytes and monocytes. However, RBC and blood granulocytes (including neutrophils, basophils, and eosinophils) have higher average density and accumulate below the separation medium at the bottom of the tube. After 20-30 min spin in the centrifuge, the PBMC layer can easily be siphoned from the density boundary, washed, and resuspended in a solution for further analysis. Variations of this gentle and efficient technique using media adjusted to different densities allow for the bulk isolation of other WBC subpopulations from whole blood samples.



Antibody plus Complement (C')
Cell samples treated with specific antibodies plus C' can be used to eliminate unwanted cell types from a heterogeneous mixture of cells thereby enriching the sample for the remaining cell populations. Antibodies targeting specific molecules on the unwanted cell types activate the C' system of enzymes resulting in specific lysis of the antibody-coated cells (see Complement Assays). The remaining viable cells in the lysate can then be easily recovered and also concentrated by low speed centrifugation.


Fluorescent Activated Cell Sorting (FACS)
FACS is the most commonly employed method used for detecting and even isolating specific cells from a heterogeneous suspension of mixed cell types. The cell mixture is first "labeled" or "stained" with antibodies that are chemically conjugated with fluorescent dyes. Using antibodies tagged with different fluorescent light-emitting dyes (e.g., red, green, etc.) that are directed against different cell surface antigens or "markers" expressed only on certain cell subpopulations, the corresponding cell populations can be "sorted" according to the intensity and color of fluorescent light that is emitted from each cell as it passes through a laser beam. This is achieved by drawing the stained cells into a thin fluid stream that is directed to pass through a laser beam, which optically "excites" the fluorescent dyes attached to the various antibodies. The resulting fluorescence color and intensity induced from each cell are recorded electronically for further analysis (See FACS Analysis). Because the relative fluorescence intensity produced by each fluorescent tag is essentially proportional to the amount fluorescently labeled antibody bound to the cell surface, one has an indirect measure of the density of that particular cell surface marker on that particular cell. For some of the diagnostic tests, FACS is just used analytically in order to determine the various percentages of different cell types in the sample mixture that have distinct marker profiles. For other diagnostic tests, FACS is also used preparatively in order to physically isolate specific cell types according to their marker profiles so that different cell populations can be further analyzed biochemically or immunologically. For example, such cells might examined for the expression of specific mRNA or protein molecules, or they might be tested in an in vitro or in vivo immunological assay.


Cell Stimulation
This is yet another method used to enrich for specific cell types or specifically activated cells from a heterogeneous mixture of cells, frequently in combination with other methods described above. Some stimulants used in the diagnostic test results presented here lead to cell proliferation and expansion while other stimulants may simply up-regulate the expression of cell-type-specific molecules, including specific mRNA and protein molecules. Because numerous procedures (See Cell Stimulation) have been devised to stimulate or activate B and T lymphocytes specifically, this is a particularly effective method for analyzing the functions and other properties of such cells.