Flow Cytometry
A powerful tool for defining, characterizing and enumerating cells, lymphocytes, is the flow cytometer, which detects and counts individual cells passing in a stream through a laser beam. A flow cytometer quipped to separate the identified cells is call a fluorescence-activated cell sorter (FACS). These instruments can study the properties of cell subsets identified using monoclonal antibodies to cell-surface proteins. Individual cells within a mixed population are first tagged by treatment with specific antibodies labeled with fluorescent dyes, or by specific antibodies followed by labeled anti-Ig. The mixture of labeled cells is then forced with a much larger volume of saline through a nozzle, creating a fine stream of liquid containing cells spaced singly at intervals. As each cell passes through a laser beam, it scatters the light and any dye molecules bound to the cell will be excited and will fluoresce. Sensitive photomultiplier tubes detect both the scattered light, which gives information on the size and granularity of the cell, and the fluorescence emissions, which give information on the binding of the labeled monoclonal antibodies and therefore, on the expression of cell-surface proteins by each cell. In addition, flow cytometry can be modified to sort and sequence subsets of cell populations. These are called cell sorters, and they do this by passing a flowing stream over a piezoelectric crystal, which vibrates at high frequency. (1)
Flow cytometry is widely utilized in cell trafficking studies. For lymphocyte trafficking, extraction of blood derived, or lymph derived leukocytes, fluorescently labeling and reinfusing the cells into an animal, flow cytometry can determine subsequently where, in which compartments, these cells will eventually reside. Samples are extracted at several times after injection with the labeled cells, in order to properly assess lymphocyte distribution. Random distribution between compartments, depicts trafficking. (1)
Due to the property that certain cells can naturally autofluorensce, negative and positive controls must be applied when examining labeled cells. A negative contrl is a set of unlabeled cells, which can give an accurate measure of autofluoresce. Cells to which a known amount of fluorochrome has been given, would serve as a suitable positive control. (2)
SNARF is utilized for intracellular pH measurements, and emits differently at different pHs. To label cells, used as an example to show two flow cytometry (to give an idea of double staining - 2 flow graph). Can assess any decreased function by comparing SNARF (dye stain everything) to other dyes and comparing the level of lymphocytes migrating to other compartments, like the lymph. SNARF labeled cells in vitro. It can be used to label cells in vivo. Cell trackers could be used to assess proliferative properties, through cell tracking studies using intravital microscopy (IVM). IVM, through visualization through green fluorescent protein GFP expression on a CD45 e.g., promoter (transgenic), would allow us to see all cells with this receptor expressed, and we could compare levels between before and after antigen stimulation experiments, through counting lymphocytes and extrapolating. (1,6,7,8)
The inherent overlap of emission spectra of antibody fluorescent labels makes compensation necessary. Compensation is the process by which each fluorescent PMT can substract a percentage of the signal from other PMT's (PMT detectors). Significant spill-over of fluorescent light may occur, The best way for determining the fluorescence compensation requires running samples that are individually stained with the fluorochrome components of your multi-color samples (5). Compensation controls: In experiments, which require simultaneous staining using two or more fluorochromes, it is necessary to prepare controls that have been stained with each dye separately. These controls, termed compensation controls, are required for adjusting cross-over signals between dyes or detectors. This is critical for multicolor immunophenotyping. Statistically, you set the position of regions so that you separate your positives from your negatives. Look at the Mean peak position corresponding to the appropriate region for the parameter you are compensating. They should be the same (4,5). For the autofluorescence correction, an unstained control sample is used to measure the autofluorescence signals within each fluorescence channel. The natural fluorescence of each cell cluster in an unstained control sample has to be subtracted from the observed fluorescence of the respective cell cluster in the stained sample.
The purpose of fluidics system is to transport particles in a fluid stream for laser beam interrogation, with only one cell or particle moving through the laser beam at any given moment. The sample is therefore, injected into a stream of sheath fluid within the flow chamber. The design of the flow chamber causes the sample core to be focused in the center of the sheath fluid. The flow of sheath fluid accelerates the particles and restricts them to the center of the sample core, in a process of hydrodynamic focusing. The calibration of flow cytometers in the clinical laboratory is of primordial importance for the interlaboratory comparison of quantitative and classification results. Brightly fluorescent alignment beads are used to control the overall optical and electronic performance of a flow cytometer. The measurement of a mixture of typically four fluorescent beads with knwon amounts of antibody binding sites or bound antibodies permits the clibration of the fluorescence scale of a flow cytometer in "bound fluorescent antibody molecules". A non fluorescent bead characterizes the assay noise level of the flow cytometer. When a filter is placed at a 45 degree angle to a light source, light which would have been trasmitted by that filter is still transmitted but light that would have been blocked is reflected (at a 90 degree angle). Used this way, a filter is called a dichroic filter. (1,2,5,6,7,8)
A dot plot is a two parameter data graph used for acquisition of data and also for analysis. Each dot on the display represents one event that the flow cytometer analyzed. Dot plots can also quantitate percentages of cells with various properties. In addition, dot plots can show intensities. Stokes shift in luminescent spectroscopy, is a displacement of spectral lines or bands toward longer wavelengths (lower frequencies) than those of the corresponding absorption lines or bands (difference between 2 lines). Physical constraints imply that to maintain focusing for particles of cellular dimensions the stream requires a velocity of several metres per second. At this speed a typical cell would traverse its own diameter in a few microseconds which necessitates a rapid analytical system. This usually employs a highly focused laser beam for excitation and sensitive photomultiplier tubes (PMTs) for detection. Receive fluorescence signals.
The fluorescence-activated cell sorter (FACS) is a powerful instrument used in flow cytometry to physically separate and identify types of cells from heterogeneous populations. (1,5,6,7,8) Flow cytometry can be used in various types of surface analysis and immunophenotyping, including platelet associated immunogloublining, studying platelets. Once labeled, the cells are forced to flow rapidly and single-file through the FACS instrument where they are excited by light from a focused laser beam. Each cell scatters some light and the labeled cells generate fluorescence signals from the dye. These two parameters are gauged by photodetectors, and the FACS computer uses them to make a decision to apply an electrostatic charge to cells with positive fluorescent intensities. The now charged single drops containing the cells of interest emerge from a nozzle and pass between two high voltage deflection plates and are thereby separated into a collection tube from the other unmarked cells. Up to 30,000 cells per second can be discriminated by the computer in modern sorters (1800000 cells per minute - over two directions, left and right)(changed through magnetism incorporation - cell sorting). In ad DNA histogram cells are stained with a DNA-binding dye, such as propidium iodide. The DNA histogram can yield estimates for the number of cells in G1/G0, S and G2/M phases of the cell cycle. Cell turnover is the time for a cell
to leave (for example, dying) and be further replaced in a given period. Cell half-life is half the time required for (plural = half cells to disapper) a cell to disappear (from total). Cell lifespan is the time between cell creation and cell death. Human T cells probably live for 3-12 months (average) (different for memory, much longer and activated T cells, anergy, etc...). The lifespan of a T cell in a child is the same as in an octagenarian. However, an octagenarian would have more memory cells and therefore, have more longer living cells than a child (greater, longer time of exposure to antigens). (1,5,6,7)
Blood curves of labeled lymphocytes in AIDS patients, would have labeled lymphocytes at a higher concentration % of population, as there are less cells due to death, and infection. The same pattern would be evident, however at higher labeled cells than normally. In patients with chronic lymphocytic leukemia, the opposite would occur. The same pattern would be evident, however, there would be far less labeled cells % of total than normal, due to an increase of lymphocytic cells. In addition, labeling of the leukemic cells, would create a distortion of pattern (migration) if these cells are specific for a given compartment and bios their movement, for example to the lymph, thereby localizing highly in certain compartments. In a cardiac allograft recipients, who is on anti-immune drugs, the labeled cells would be at a higher % of population, but the pattern of movement would remain normal (movement of cells out of blood). To obtain information in pathological tissues in addition to blood data, adding pathological tissue would also benefit.
References
1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA
2. Delves, P. and Roitt, I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA
3. 1994. Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA
4. Cruse, J. and Lewis R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA
5. http://biology.berkeley.edu/crl/flowcytometry.shtml
6. Lodish, H., Baltimore, D., Berk, A., Zipursky, Matsudaira, and Darnell, J. 2000. Molecular Cell Biology. American Scientific Books, USA
7. www.spectracell.com/jana.html
8. Andrade, W., Johnston, M. and Hay, J.B. 1998. The relationship of blood lymphocytes to the recirclating lymphocyte pool. Blood 91: 1653-1661.