Polyclonal and monoclonal antibodies are used in a wide range of in vitro assays in many different formats. The formats of the technology range from sophisticated flow cytometry (FCM) to simple enzyme-linked immunosorbent assay (ELISA). Data from antibody-facilitated assays are often used to support a tentative clinical diagnosis, evaluate disease progress, and provide prognostic information. In vitro assays are also used to determine the presence of bacteria, viruses, and antigens in biologic fluids. Increased hormone levels characteristic of disease states or pregnancy are determined by antibody-facilitated assays.

Flow Cytometry

A flow cytometer consists of four components: (1) a fluidics system, (2) single or dual lasers, (3) optical detectors, and (4) an on-board computer system that collects and analyzes data. As part of FCM, cells are placed in a fluidic system that contains sheath fluid or saline. Using hemodynamic focusing, cells are concentrated at a nozzle and passed in a single file through a laser (the interrogation point) at a rate of several thousand cells per second. Lasers are usually of high-power, water-cooled argon or krypton. The computer records each cell as a dot on a linear or logarithmic “x–y” graph. As the cell is passed through the interrogation point, light is scattered in all directions. One optical detector collects forward, or low-level, light scatter. Forward scatter is laser light that passes around the cells and is roughly proportional to cell size. An optical detector converts the light passing around the cell to voltage impulses, which are stored in the computer. In essence, small cells generate small voltages, and large cells generate large voltages. Another detector collects light scattered from the laser at a 90-degree angle, which is called side scatter. Side scatter is a measure of the internal complexity or granularity of a cell. Since peripheral blood cells vary in both size and granularity, a linear plot of side scatter versus forward scatter is used to separate peripheral blood cells into lymphocyte, monocyte, and granulocyte populations (Figure 14-1).

Figure 14-1 A schematic showing the flow cytometric segregation of peripheral blood cells based on size and granularity.

Enumeration of Lymphocyte Subpopulations

Lymphocyte phenotyping (lymphocyte subset enumeration) is a routine test used to screen for immunodeficiencies, evaluate disease progress, support a tentative diagnosis, and provide prognostic information. In patients with acquired immuno deficiency syndrome (AIDS), FCM is used to monitor CD4 cell numbers.

An inverse correlation exists between CD4 numbers and the risk for specific bacterial and fungal infections. Clinicians use CD4 data to determine when and what type of prophylactic antibiotic therapy should be implemented. FCM is also used to differentiate between acute B cell leukemia (ABCL) and myelogenous leukemia, to diagnose chronic lymphocytic leukemia (CLL), and to differentiate between hairy cell leukemia and mantle cell lymphoma.

Proper identification and classification of hematopoietic neoplasms are critical because treatment of leukemia and lymphoma are radically different. For example, CLL is usually treated with nucleoside analogues and alemtuzumab directed at CD52 expressed by leukemic cells. First-line therapy for hairy cell leukemia is chlorodeoxyadenosine (2-CdA). Conversely, mantle cell lymphoma is treated with hyperfractionated cyclophosphamide, doxorubicin, vinblastine, and dexamethasone, with or without rituximab.

Dual staining of malignant cells has some prognostic value. For example, patients with ABCL have a poor prognosis if the malignant cells express CD10, CD34, and CD13. Conversely, the prognosis is excellent and so is response to chemotherapy when ABCL cells express CD45, CD135, and CD34.

In the enumeration of lymphocytes, size and granularity limits are used to draw an electronic “gate” around the lymphocyte population. Any subsequent data are derived solely from the “gated” area. The presence of lymphocytes in the gated area is verified by using fluorochrome-labeled monoclonal antibodies. Fluorochromes have high intensity and minimal overlapping spectra. As labeled antibodies pass the interrogation point, the fluorochrome absorbs light (excitation) at a specific wavelength. When the excited fluorochrome returns to a ground state, light is emitted at a different wavelength. Using mirrors and filters, the emitted light is delivered to photomultiplier detectors. In the detectors, fluorescence is converted to voltage intensity and stored in the computer as a dot on an x–y graph. Fluorescence yields information on the number of cells with fluorochrome, and the fluorochrome intensity is an index of the number of fluorescent markers per cell.

Although numerous fluorochromes can be used in FCM, fluorescein isothiocyanate (FITC) and phycoerythrin (PE) are commonly used in laboratory studies. FITC is a fluorescein derivative that is easily conjugated to antibodies via primary amines. In most conjugations, three to six FITC molecules are covalently linked to a single antibody. FITC is excited at 488 nanometer (nm) by an argon laser, and light is emitted at 530 nm. PE is a large-molecular-weight protein commonly found in cyanobacteria and red-green algae. Only one molecule of PE can be conjugated to an antibody. It is maximally excited at 533 nm and emits a very bright light at 570 nm.

To verify the percent of lymphocytes in the “gated” population, cells are labeled with two different antibodies—usually CD45-PE and CD3-FITC. The former is a pan-leukocyte marker, and the latter reacts only with T cells. Using a histogram of FITC versus PE immunofluorescence, cells are electronically placed into four quadrants: (1) Cells lacking both markers are placed in the lower left quadrant. (2) Cells expressing only the CD45 marker are placed in the upper left quadrant. (3) CD3-positive cells are shown in the lower right quadrant. (4) Cells expressing both markers are placed in the upper right quadrant. Generally, 95% of lymphocytes are found in the lower right quadrant (Figure 14-2).

Figure 14-2 Flow cytometric analyses using dual labeling with CD3-FITC (fluorescein isothiocyanate) and CD45-PE (phycoerythrin). Cells staining with CD3-FITC are shown in the lower right quadrant. Each dot in the quadrant represents a single lymphocyte. Since there are no cells staining with CD45-PE (*upper left quadrant*), the lymphocyte population is essentially pure.

After the lymphocyte population is verified, total T cell, CD4, and B cell populations can be enumerated using dual staining techniques, as shown in Table 14-1. Dual-color immunofluorescence enumerations of the peripheral blood CD4 subset are shown in Figure 14-3. Cells are incubated with two monoclonal antibodies conjugated to two different fluorochromes. FITC-conjugated antibodies are directed at CD3, and PE is linked to an anti-CD4 antibody. Dual-labeled CD4 helper/amplifier cells are shown in the upper right quadrant. The study is repeated using CD3–CD8 dual staining for T cytotoxic cells and CD16–CD56 staining for natural killer (NK) cells. B cells are identified by staining with anti-CD19.

Table 14-1

Minimum Monoclonal Panel Used to Determine Lymphocyte Subsets

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