Flow Cytometry and Fluorescence

The fluorescent antibody techniques described are extremely valuable qualitative tools, but they do not give quantitative data. This shortcoming was remedied by development of the flow cytometer, which was designed to automate the analysis and separation of cells stained with fluorescent antibody. The flow cytometer uses a laser beam and light detector to count single intact cells in suspension (Figure 6-15). Every time a cell passes the laser beam, light is deflected from the detector, and this interruption of the laser signal is recorded. Those cells having a fluorescently tagged antibody bound to their cell surface antigens are excited by the laser and emit light that is recorded by a second detector system located at a right angle to the laser beam. The simplest form of the instrument counts each cell as it passes the laser beam and records the level of fluorescence the cell emits; an attached computer generates plots of the number of cells as the ordinate and their fluorescence intensity as the abscissa. More sophisticated versions of the instrument are capable of sorting populations of cells into different containers according to their fluorescence profile. Use of the instrument to determine which and how many members of a cell population bind fluorescently labeled antibodies is called analysis; use of the instrument to place cells having different patterns of reactivity into different containers is called cell sorting.

The flow cytometer has multiple applications to clinical and research problems. A common clinical use is to determine the kind and number of white blood cells in blood samples. By treating appropriately processed blood samples with a fluorescently labeled antibody and performing flow cytometric analysis, one can obtain the following information:

■ How many cells express the target antigen as an absolute number and also as a percentage of cells passing the beam. For example, if one uses a fluorescent antibody specific for an antigen present on all T cells, it would be possible to determine the percentage of T cells in the total white blood cell population. Then, using the cell-sorting capabilities of the flow cytometer, it would be possible to isolate the T-cell fraction of the leukocyte population.

■ The distribution of cells in a sample population according to antigen densities as determined by fluorescence intensity. It is thus possible to obtain a measure of the distribution of antigen density within the population of cells that possess the antigen. This is a powerful feature of the instrument, since the same type of cell may express different levels of antigen depending upon its developmental or physiological state.

■ The size of cells. This information is derived from analysis of the light-scattering properties of members of the cell population under examination.

Flow cytometry also makes it possible to analyze cell populations that have been labeled with two or even three different fluorescent antibodies. For example, if a blood sample is reacted with a fluorescein-tagged antibody specific for T cells, and also with a phycoerythrin-tagged antibody specific for B cells, the percentages of B and T cells may be determined simultaneously with a single analysis. Numerous variations of such "two-color" analyses are routinely carried out, and "three-color" experiments are common. Aided by appropriate software, highly sophisticated versions of the flow cy-tometer can even perform "five-color" analyses.

Flow cytometry now occupies a key position in immunology and cell biology, and it has become an indispensable clinical tool as well. In many medical centers, the flow cytometer is one of the essential tools for the detection and classification of leukemias (see the Clinical Focus). The choice of treatment for leukemia depends heavily on the cell types involved, making precise identification of the neoplastic cells an essential part of clinical practice. Likewise, the rapid measurement of T-cell subpopulations, an important prognostic indicator in AIDS, is routinely done by flow-cytometric analysis. In this procedure, labeled monoclonal antibodies against the major T-cell subtypes bearing the CD4 and CD8 antigens are used to determine their ratios in the patient's blood. When the number of CD4 T cells falls below a certain level, the patient is at high risk for opportunistic infections.

Ultrasonic nozzle vibrator

Deflection plates

A- B-cells

Cells stained with:

Anti-A + Anti-B antibody Anti-A antibody Anti-B antibody Unstained

Ultrasonic nozzle vibrator

Cells stained with:

Deflection plates

Blood Plate
o

A- B-cells

Anti-A antibody fluorescence

Computer screen

A- B+ cells .

A+ B+ cells

A- B- cells

A+ B- cells . •

Anti-A antibody fluorescence

FIGURE 6-15

Separation of fluorochrome-labeled cells with the flow cytometer. In the example shown, a mixed cell population is stained with two antibodies, one specific for surface antigen A and the other specific for surface antigen B. The anti-A antibodies are labeled with fluorescein (green) and the anti-B antibodies with rhodamine (red). The stained cells are loaded into the sample chamber of the cytometer. The cells are expelled, one at a time, from a small vibrating nozzle that generates microdroplets, each containing no more than a single cell. As it leaves the nozzle, each droplet receives a small electrical charge, and the computer that controls the flow cytometer can detect exactly when a drop generated by the nozzle passes through the beam of laser light that excites the fluo-rochrome. The intensity of the fluorescence emitted by each droplet that contains a cell is monitored by a detector and displayed on a computer screen. Because the computer tracks the position of each droplet, it is possible to determine when a partic ular droplet will arrive between the deflection plates. By applying a momentary charge to the deflection plates when a droplet is passing between them, it is possible to deflect the path of a particular droplet into one or another collecting vessel. This allows the sorting of a population of cells into subpopulations having different profiles of surface markers.

In the computer display, each dot represents a cell. Cells that fall into the lower left-hand panel have background levels of fluorescence and are judged not to have reacted with either antibody anti-A or anti-B. Those that appear in the upper left panel reacted with anti-B but not anti-A, and those in the lower right panel reacted with anti-A but not anti-B. The upper right panel contains cells that react with both anti-A and anti-B. In the example shown here, the A~B~—and the A+B+— subpopulations have each been sorted into a separate tube. Staining with anti-A and anti-B fluorescent antibodies allows four subpopulations to be distinguished: A~B~, A+B+, A~B+, and A+B~.

CLINICAL FOCUS

CLINICAL FOCUS

Flow Cytometry and Leukemia Typing

Leukemia s the unchecked proliferation of an abnormal clone of hematopoietic cells. Typically, leukemic cells respond poorly or inappropriately to regulatory signals, display aberrant patterns of differentiation, or even fail to differentiate. Furthermore, they sometimes suppress the growth of normal lymphoid and myeloid cells. Leukemia can arise at any maturational stage of any one of the hematopoietic lineages. Lymphocytic leukemias display many characteristics of cells of the lymphoid lineage; another broad group, myelogenous leukemias, have attributes of members of the myeloid lineage. Aside from lineage, many leukemias can be classified as acute or chronic. Some examples are acute lymphocytic leukemia (ALL), the most common childhood leukemia; acute myelogenous leukemia (AML), found more often in adults than in children; and chronic lym-phocytic leukemia (CLL), which is rarely seen in children but is the most common form of adult leukemia in the Western world. A fourth type, chronic myelogenous leukemia (CML), occurs much more often in older adults than in children.

The diagnosis of leukemia is made on the basis of two findings. One is the detection of abnormal cells in the bloodstream, and the other is observation of abnormal cells in the bone marrow. Clinical experience has shown that designing the most appropriate therapy for the patient requires knowing which type of leukemia is present. In this regard, two of the important questions are: (1) What is the lineage of the abnormal cells and (2) What is their maturational stage? A variety of approaches, including cytologic examination of cell morphology and staining characteristics, immuno-

phenotyping, and, in some cases, an analysis of gene rearrangements, are useful in answering these questions. One of the most powerful of these approaches is immunophenotyping, the determination of the profile of selected cell-surface markers displayed by the leukemic cell. Although leukemia-specific antigens have not yet been found, profiles of expressed surface antigens often can establish cell lineage, and they are frequently helpful in determining the maturational stages present in leukemic cell populations. For example, an abnormal cell that displays surface immuno-globulin would be assigned to the B-cell lineage and its maturational stage would be that of a mature B cell. On the other hand, a cell that had cytoplasmic ^ heavy chains, but no surface immuno-globulin, would be a B-lineage leukemic cell but at the maturational stage of a pre-B cell. The most efficient and precise technology for immunophenotyping uses flow cytometry and monoclonal antibodies. The availability of monoclonal antibodies specific for each of the scores of antigens found on various types and subtypes of hematopoietic cells has made it possible to identify patterns of antigen

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