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Clinical flow cytometry: milestones along the pathway of progress. (Cover Story).

Introduction

Cytometry is the art and science of measuring cells. Flow cytometry is that branch which studies cells suspended in fluid during analysis.

Flow cytometry (FCM) is a powerful technology capable of rapidly measuring multiparameter characteristics of individual particles. This review will focus on intact eukaryotic cells or nuclear masses isolated from them. Prokaryotic microorganisms, such as yeasts, which are 200 times smaller than a human diploid cell, as well as bacteria, protozoa, algae, and molds, might also be measured by flow cytometry. (1) The feasibility of detection of these smaller cells has been documented; however, practical applications remain to be developed and accepted. Detection and measurement of viral products or DNA is currently below the detection level of most commercially available systems at the time of this writing. Methods to enhance detection of intracellular viral products -- in particular, those associated with HIV -- are currently in development, but not yet available for clinical application.

Historical overview of flow cytometry

The concept of automatically counting particles suspended in fluid is not new, first being described by Moldavan in 1934. (2) Wallace Coulter filed a patent application for automated counting of particles in 1949 and presented the concept of simultaneous counting and sizing of particles 46 years ago. (3,4) This led to the introduction of automated blood cell counting to the clinical lab. The concept of combining differences in electrical conductivity with radio-frequency differences was described in 1966, but it was not introduced until the 1990s. The various models of Coulter Counters gradually replaced manual methods of cell counting. The adaptation of cell sizing with a second parameter measured by spectrophotometry was soon to follow, the Technicon series of systems being an outstanding example. Today, most automated differential systems generate five- and six-part differential analysis and have had this capability for more than 20 years. (5) Oddly, most laboratorians do not think of today's automated hem atology systems as flow cytometers, although they most certainly are. Moreover, they may play important roles in the quality control and quality assurance of immunologic assays performed on laser flow cytometers.

Kamentsky and Melamed introduced two concepts that significantly extended flow-cytometric applications -- the use of spectrophotometry to quantitate specific cellular components and the classification of cells using a combination of multiple simultaneous measurements. (6)

The introduction of a laser as the light source for flow-based immunoassays came from Van Dilla and the Los Alamos group in 1969. (7) This group developed the first instrument having orthogonal axes of flow, illumination; and detection using an argon-ion laser. The passage of a cell through the precisely focused laser beam scatters light in 360-degrees of arc. Strategically positioned photodiodes measure this scattered light, revealing information on cell size and structure. Weaker signals from fluorescent probes are detected and amplified by photomultiplier (PMT) tubes. (8)

Flow cytometry has the capability of detecting and measuring features of whole cells, subcellular organelles, and intracellar products. Flow cytometer systems may also be designed to physically remove defined cell populations through the process known as cell sorting. (9) These isolated, purified populations may then be studied by other techniques to assess normalcy versus malignancy, as well as functional capability and status. Cell sorting is not commonly employed in the clinical laboratory, but it remains primarily a research-based investigative tool.

The birth of clinical flow cytometry

Serendipity in science: The laser-based flow cytometry and monoclonal antibodies made possible the immigration of flow cytometry from the research lab to the clinical laboratory. This would not have occurred without the marriage of laser flow cytometry and monoclonal antibodies tagged with various fluorescent probes.

In the early 1980s, research-based biologic applications developed clinical significance; however, acceptance of these new assays was not rapid, and many doubted the existence and the relevance of these T- and B-cell populations.

Kohler and Milstein, with their creation of the hybridoma, opened the door to almost unlimited immunologic testing possibilities. (10) The hybridoma, a fused cell line involving a plasma cell and a carefully selected lymphocyte, continuously secretes predetermined antibodies of one specific type known as monoclonal antibodies (MoAb). A monoclonal antibody recognizes and binds to one specific epitope or site. Characterization of cell populations by these monoclonal antibodies has become popularly known as "immunophenotyping." Given the proposed vast diversity of the human antibody epitope repertoire ([10.sup.9] to [10.sup.17]), to these site- or epitope-specific antibodies theoretically offered the possibility of generating an immunology agent against essentially any immunogenic substance.

The new morphology

Before immunophenotyping, the ability to distinguish one cell type from another was dependent on differences in the cell's morphologic or functional features. Peripheral blood mononuclear leukocytes (PBMCs), lymphocytes, and monocytes, microscopically examined after Romanowsky staining, commonly generated a great deal of subjective morphologic classification. The routine staining process of dead, dried blood cells on smears yielded even less information about the varied functional potential or capabilities of the cells. Specific cell types were identified through pattern recognition based on physical morphology: cell size, nuclear shape, chromatin pattern, and various cytoplasmic features, such as granules.

As automated hematology systems evolved sophisticated differential methodology, in the 1980s the resultant two-parameter cytograms based on cell size and a coupled parameter created distinguishing clusters of signals (dots) (Figure 1). This ushered in the beginning of a new form of morphologic pattern recognition based on signal cluster analysis. (11) The most successful operators were those who could review the computer-generated clusters and mentally associate them with the more traditional microscopic blood cell morphology. Because the beginning of data acquisition in most flow cytometry assays begins with identifying the clusters of interest as generated by light-scatter signals, individuals with expertise in hematology pattern recognition commonly make the best flow operators. In general, the greater the depth of understanding of cell biology, morphology, and function, the better the hematology system or flow operator is at establishing the desired regions in both the acquisition and analysis phases of a ssays.

The marriage of immunology and hematology

From the onset, flow cytometry and fluorescent probes had obvious applications in cellular immunology. It was hardly surprising that among the first monoclonal antibodies created were those to epitopes on molecules expressed on the surface membranes of lymphocytes. Reinherz et al. were among the first to create and use monoclonal antibodies in the identification of lymphocytes according to differences in expression or frequency of certain cell membrane surface antigens. (12) The T-cell family was the first to be identified and characterized. (13) Monoclonal antibodies then were focused on the task of detecting and defining the three major lymphocyte families (T, B, and NK) and their functional subsets -- for example, T-helper/inducer (CD3+CD4+) and T-suppressor/cytotoxic (CD3+CD8+) cells.

Early studies were performed on isolated, peripheral blood mononuclear cells (PBMCs), not whole blood. PBMCs may be obtained by a variety of techniques, including density gradient separation, antibody-selective microcolumns, or antibody-coated magnetic bead selection. Density gradient selection using Ficoll Hypaque (FH)-based gradients remains the most common approach. These FH preparations, correctly performed in a timely fashion, within a specific gravity range of 1.077 to 1.080, yield specimens dominated by Lymphocytes and monocytes, with some basophils. (14) From the beginning, it was known that not all lymphocytes were isolated in the interphase layer of these gradients and fell through with the granulocytes, thereby escaping analysis. Establishing the appropriate analytic regions on the basis of two-parameter light-scatter characteristics is reasonably straightforward with normal specimens (Figure 2).

For a brief period, it was assumed that many detected surface membrane antigen epitopes might be unique to lymphocytes. (15) In part, this assumption arose as a result of working with isolated PBMCs and single-color immunofluoresence. This was also a period during which surface receptors could be detected and quanittated; however, what functional purposes they served were unknown. With the passage of time and the delineation of receptor function, co-expression on multiple cell lines became more ological. (16) The ligands that bound to various areas of the surface receptor molecules were identified, functions were described, and the pieces of the immune network puzzle began to come together. Techniques for whole blood lysis and multicolor immunophenotyping were yet to come. These developments would be of significant value in further definition of the various surface membrane receptors.

The development of reliable techniques of lysing blood specimens to remove erythrocytes and platelets without destroying the leukocytes bypassed the labor-intensive procedure of gradient separation and permitted analysis of the entire leukocyte population. With correct identification of the lymphocyte in the analysis process.

The analysis of a heterogenous blood cell population for the presence of a certain cell type, such as T-cells, might be considered qualitative, while the frequency of a population is quantitative. The three lymphocyte families -- T, B, and NK and their major subsets -- are currently the most frequently assayed cells in the clinical laboratory. (18) Most flow cytometric immunophenotypic asssays are both qualitative and quantitative, and they generate reports that contain relative frequency expressed as a percentage of a population and absolute number based on leukocyte differential information. In selected laboratories, the creation of individual patient files provides for longitudinal tracking to facilitate detection of trends. (19) Summation of information in this form assists the clinician by eliminating the necessity of paging through chart data to detect the needed information.

From single-color to rainbow staining

The excitation and emission wavelengths of fluorescein isothiocyanate (FITC) fit well with the spectral characteristics of the argon laser spectrum. This made FITC the first widely employed tag of monoclonal antibodies. Marking cells with a single color probe mandated separate tubes for each mark. Analyzing the stained lymphocytes one tube at a time was not only labor-intensive, but also involved speculation as to which cells shared surface epitopes.

By 1984, the use of two-color immunofluorescence assay, which permitted simultaneous assay of two different epitopes at the same time, was available. As the decade ended, simultaneous three-color assays were performed in the cutting edge clinical labs. As the number and type of fluorochromes increased, a second laser, usually helium-neon, was added to the standard argon laser on flow systems to achieve the required excitation wavelengths and desired separation of emitted excitation signals. With the simultaneous application of multiple monoclonal antibody probes, the complexity and diversity of the immune network began to emerge. Today, four-color simultaneous staining is common, and five-color clinical cytometry systems are being released.

Separating lymphocytes and monocytes

The availability of simultaneous detection of epitopes has been very valuable in resolving a long-standing problem in blood cell identification. Distinguishing between lymphocytes and monocytes has always been a challenge in mononuclear classification. The creation of monoclonal antibodies, which identified leukocyte epitopes such as CD45, added a very useful tool for accomplishing this task. CD45 is a molecule expressed on all leukocytes, but which is expressed on the surface of most lymphocytes at a higher frequency. Consequently, lymphocytes bind a greater amount of tagged anti-CD45 and generate a more intense fluorescent signal, which separates the lymphocyte cluster from the other leukocytes. Monocytes and lymphocytes frequently display overlapping signal cluster patterns by light scatter, and to some degree, CD45. (20) The use of anti-CDl4, which is expressed by monocytes but not lymphocytes, improved the setting of analytical gates for both populations. The combination of CD45 and CDl4 in routine lymph ocyte panels is now common. Comparison of cytograms from hematology systems with the those generated by the flow cytometer assists in assuring that larger reactive lymphocytes, particularly those with granules, are included in the correct analytic gate. In addition, it alerts the flow operator to the presence of an abnormal specimen that may contain nucleated red blood cells, blasts, or other abnormalities. The automated five- or six-part differential commonly provided the absolute values used in the generation of the final flow analysis report.

Cost versus time versus information

The use of monoclonal cocktails made of three or four different antibodies all tagged with distinctive fluorochromes and coupled with whole blood lysis methodology permits a high throughput with minimal labor intensity. Commercially prepared monoclonal cocktails for these common combinations are available from several commercial sources. The manufacturer performs the titration process and provides recommendations for use -- that is, the amount of cocktail to be used with a set concentration of cells. A basic lymphocyte (immune) screening panel typically consists of detection and quantitation of CD3, CD4, CD8, CDl9, and CDl6/56. Anti-CD45/CD14 are included to assist in distinguishing lymphocytes from monocytes; however, these values are not typically reported. This panel reveals the frequency of T-cells (CD3+), B-cells (CDl9+), and natural killer cells (CD3-CD16+CD56+) It also provides the frequency of T-helper-inducer cells (CD3+CD4+) and T-suppressor/cytotoxic cells (CD3+CD8+). It does not provide informatio n of cell activation or signaling pathway receptors, frequency of T subsets such as the Th1 or Th2, stem or blast cells, or B-lymphocytes such as immunoblasts or plasma cells, nor nonlymphoid elements. Detection and characterization of these cells requires additional monoclonal antibodies.

When less common multi-antibody cocktails are required for specific protocols or clinical disorders, then the process of antibody combination becomes more complicated. Dependent on the protocol, these combinations are less likely to be commercially available, except by custom order. Consequently, selection of which fluorescent label to use for a particular antibody, the combination of antibodies, and their titration for optimal staining become in-house exercises.

Nonetheless, simultaneous quantitation of expressed epitopes on both intracellular and surface membrane molecules makes assessment of lineage, subset, activation, and functional capabilities of cells now practical. In fact, so much information can be provided that analysis and interpretation become challenging and sometimes ambiguous. This new clinical tool, flow cytometry, can be both art and science at their frustrating best.

References

(1.) Steen HB. Flow cytometric studies of microorganisms. In: Melamed M, Lindomo T, Mendelsohn M, eds. Flow Cytometry and Cell Sorting. 2nd ed. New York: Wiley-Liss; 1990:605-622.

(2.) Moldavan A. Photo-electric technique for the counting of microscopical cells. Science. 1934;180:188-189.

(3.) Coulter WH. Means for counting particles suspended in a fluid. U.S. patent 2,66,508. October 20, 1953.

(4.) Coulter WH. High speed automatic blood cell counter and cell size analyzer. Proc. Natl Electron Conf. 1956;12:1034-1042

(5.) Ornstein L, Ansley HR. Spectral matching of classical cytochemistry to automated cytology. J Histochem Cytochem. 1974;22:453-469.

(6.) Kamentsky LA, Melamed MR. Instrumentation for automated examination of cellular specimens IEEE. 1969;57:2007-2016.

(7.) Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR. Cell microfluorometry: A method for rapid fluorescence measurement. Science. 1969;163:1213-1214.

(8.) Patrick CW. Principles of flow cytometric analysis. Am Soc Clin Pathol Press. 1982;10:1-15.

(9.) Parks D, Herzenberg L Fluorescence-activated cell sorting: Theory, experimental optimization, and applications in lymphoid cell biology. Methods Enzymol. 1984;108:197-241.

(10.) Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature Lon. 1975;256:495-497.

(11.) Smart Y, Cox J, Murphy B, Enno A, Burton R. Flow cytometric enumeration of absolute lymphocyte number in peripheral blood using two parameters of light scatter. Cytometry. 1985;6:172-174.

(12.) Reinherz EL, Kung PC, Goldstein G, Schlossman SF. Separation of functional subsets of human T cells by a monoclonal antibody. Proc Natl Acad Sci USA. 1979;76:4061-4065.

(13.) Reinherz EL. Kung PC, Goldstein G, Schlossman SF. A monoclonal antibody with selective reactivity with functionally mature thymocytes and all peripheral human T cells. J Immunol. 1979;123:1312-1317.

(14.) Patrick CW, Swartz SJ, Harrison KA, Keller RH. Collection and preparation of hematopoietic cells for cell marker analysis. Lab Med. 1984;15(10):659-665.

(15.) Patrick CW, Keller RH, Milson TJ, Janicek, KM. Monoclonal antibodies: Clinical utility and the misunderstood epitope. Lab Med. 1984;15(12):795-802.

(16.) Lovett EJ, Schnitzer B, Keren OF, Flint A, Hudson J, McClateley DD. Application of flow cytometry to diagnostic pathology. Lab Invest. 1984;50(2):115-140.

(17.) Mosmann TR, Cherwinski H, Bond BW, Gieldlin MA, Coffman RL. Two types of murine helper T cell clone: Definition according to profiles of lymphokines activities and secreted proteins. J Immunol. 1906;136:2343-57.

(18.) Gratama JW, Kraan J, Van den Beemd R, Hooibrink B, Van Ockstaele DR, Hooijkaas HH. Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study. Cvtometry. 1993;30:166-177.

(19.) Mandy FF, Bergeron M, Minkus T. Evolution of leukocyte immunophenotyping as influenced by the HIV/AIDS pandemic: A short history of the development of gating strategies for CD4 T-cell enumeration. Cytometry (Communications in Clinical Cytometry). 1977;30:157-165.

(20.) Owens MA, Loken MR. Quantitative immunophenotyping in HIV infection. Row Cytometry Principles for Clinical Laboratory Practice. New York: Wiley-Liss; 1995:72-110.

Dr. Catherine Patrick is currently serving on the Board of Directors for a private biotech company in South Florida and is executive director of CCT Ltd. of Ft. Lauderdale. FL.
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Author:Patrick, Catherine W.
Publication:Medical Laboratory Observer
Article Type:Cover Story
Date:Sep 1, 2002
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