Introduction
Since the early ‘50s, flow cytometry has become a highly refined technology for a quick and precise exploration of cell surfaces. By means of flow cytometry, it is now possible to collect information concerning cell surface molecules and their expression during cell differentiation; to study the cell cycle by quantitatively assessing DNA modifications; to develop functional studies of a variety of cell parameters and to obtain enriched cell populations for further studies. It can be rightly said that, in this forty-year period, flow cytometry has become an essential tool to biologists, pathologists and clinicians. It would be impossible to detail all flow cytometry’s applications in the study of structural elements and of the functions of the immune system, in a brief chapter. This chapter will try to summarize, in a concise and easy to grasp manner, the development of flow cytometry techniques and their most frequent applications in the study of immune processes. The reader should refer to the many books on this topic, which offer a more detailed description of the techniques and applications. He should also look up the vast and constantly increasing literature that describes the innovative uses of flow cytometry to investigate the diverse aspects of the immune system and inflammation in health and sickness.

Background
Early attempts to analyse cells quickly and precisely date back to the ‘50s. In fact, the early works on flow cytometry were directed at assessing this technique’s potential to determine the DNA content of cells and to distinguish between normal and malignant cells. The finding that two distinct populations of lymphocytes, T and B, interacted in the immune response, increased interest in the study of these two populations, directed at characterizing them and understanding their functions and interactions. In the first place methodologies were developed to identify lymphocytes T and B through fluorescence microscopy with anti-Ig (lymphocyte B) and through the formation of the ovine erythrocyte rosette (rosette E, rosette T). The ability to produce monoclonal antibodies and the simplification of flow cytometry have revolutionized, and still do, our skill to conduct precise, objective and fast cell analyses in a variety of clinical and research situations.

Monoclonal antibodies
The very discovery of a simple method of producing monoclonal antibodies triggered attempts at conducting studies directed at producing monospecific reagents to identify cell surface molecules expressed in the diverse stages of differentiation, and to be used together with flow cytometry. Right from the early stages, monoclonal antibodies that recognize similar antigens on immune and inflammatory cells and their products were quickly introduced by investigators and commercial suppliers of laboratory reagents. And, as time passed, all monoclonal reagents’ suppliers established a name to identify each monoclonal antibody. This lead to issues concerning the specificity of every new monoclonal antibody and to considerable confusion in recording the data obtained through flow cytometry and other methods that used monoclonal antibodies, as the reagents of different suppliers lead to different designations. There were two important requirements: 1) to certify the specificity of every monoclonal antibody, and 2) to establish a nomenclature system to exchange information in a common language. Hence, a Committee of experts was organized to periodically assess the results of specificity tests for every new monoclonal antibody and give each of them, once approved, a CD code, or a wCD code if the antibody was still being assessed. These designations reflect the antigen group towards which a particular monoclonal antibody will be specific. A careful examination of the current list of monoclonal antibodies reveals the explosion of activity in this field, to the advantage of an increasingly deeper understanding of cells and molecules involved in immune and inflammatory responses. Hence the combination of monoclonal antibodies easily available with the development of flow cytometry techniques has obviously contributed towards the birth of a new paradigm for a quick, precise and in many cases quantitative analysis of the cells.

Basic principles of flow cytometry
The hemicytometer consists of three main components: a fluidic system that controls cell detecting and cell flow; an optical system that “questions” the cells while they cross a laser beam; and an electronic system that controls the equipment and collects, depicts and analyses the data. The cells are introduced as a single cell suspension into a flow of isotonic chlorinated solution and they travel like “a flow inside a flow”, forming a line and passing through a spout in single file. While they travel and are at last expelled or recovered (in other words they are separated by specific cell types), they cross a ray of light that “questions” every cell with regard to the characteristics required. The sources of light most frequently used are gas lasers, preferably the argon one as it emits an excitation light at 488 nm, with the most common fluorochromes currently used. The light signal emitted by a cell at the questioning site in a hemicytometer’s electronic component is converted into an electric impulse (it will generally be an analogue signal). Analogue signals are then converted into digital signals that are represented on a screen by a cathode ray tube. The advent of increasingly powerful computers has enabled the introduction of computerized systems to control hemicytometer operations and to collect, memorize and analyse data. Single cells are required for flow cytometry measurements and this is a limiting factor that prevents studies on tissues that cannot be easily scattered in a single cell suspension. The samples are usually suspended in a buffered chlorinated solution with the phosphate, 0.2% bovine serum albumin and 0.01% sodium azide (PBS+). Following exposure to the specific monoclonal antibody for 30 minutes in the dark, to avoid the reduction of fluorescent radiation, the samples are washed, suspended again in PBS+ and studied in the hemicytometer. It may be useful to recall certain basic principles concerning the manipulation of samples. In clinical practice, the most common samples are peripheral blood, bone marrow aspirates and monodispersed lymph node cell suspensions. In research situations, the sample can be of any type of cell suspended in a monodispersed state. For peripheral blood and bone marrow it is necessary to remove the erythrocytes by means of a lysing agent (generally ammonium chloride) that will not influence mononuclear cells subject to the examination (Fig. 1).

 

Una cellula (oggetto più scuro e ovale) portando antigeni superficiali è esposta ad un anticorpo monoclonale e gli eritrociti sono lisati prima che il campione venga introdotto nella camere a flusso

The cells flow in single file in the flow chamber and when one of them intersects the laser beam (question point) the light that strikes the cell is scattered in all directions. The light scattered forward (indicating the cell size) and the light scattered sideways (indicating the cell’s inside structure) are collected by the photomultiplying tubes that amplify the weak light signal. The light signals are then processed as described above and represented as dots on the screen - each will represent an event as established by the cell’s light dispersion properties. A distribution will thus be obtained by the groups of dots that represent distinct cell populations, with various dimensions and inside structures (a cytogram or “bit map”). At this stage, it is necessary to outline the cell population that is about to be further examined to determine the characteristics studied by means of the fluorescent signals emitted by the monoclonal antibodies bound to cell surface antigens. This is performed by placing an electronic window (“gate”) around the group that interests us (Fig. 2).

 

Schema di un citogramma che mostra tre popolazioni cellulari distinte come delineate per mezzo delle proprietà che spargono la luce. Dimensione = dispersione in avanti e complessità = dispersione ai lati

While portions of “stained” cells with different monoclonal antibodies are consecutively introduced into the flow cytometer, the cells examined will be those present in the “gate” established. The data will then be represented in the form of a histogram, the number of events that lead to the relative fluorescent intensity, according to a logarithm-logarithm scale (Fig. 3).

Schema di un istogramma raffigurante l'intensità della fluorescenza verso il numero di cellule contate. La freccia indica il punto nel quale scompare la fluorescenza di fondo come determinato da un controllo isotopico

 

Besides, it is possible to analyse the samples stained with combinations of monoclonal antibodies, each one labelled with a unique and different fluorochrome. Figure 4 shows a sample that was “double-stained” to detect at the same time cells that express two distinct surface antigens.

Schema di un istogramma da un'analisi a due colori di linfociti T. Doppio-colorate, le cellule CD3/CD4+ nel quadrante 2 rappresentano linfociti T aiutanti

 

In the same manner it is possible to study cells labelled with three or more monoclonal antibodies thanks to computers, which help in representing data deriving from these complex pictures. Fig. 5 describes an example of a four-colour analysis.

Stampa di emocitometro a flusso da un campione di linfociti da un donatore normale. Pannello 1: la distribuzione cellulare secondo la dispersione di luce. Pannello 2 "Gating" (o raggruppamento?) secondo la dispersione ai lati (complessità o struttura cellulare) verso CD45 (pan-antigene leucocito, presente su tutti i leucociti). Pannello 3 e pannello 4: linfociti CD3/CD4+ e linfociti CD3/CD8+

In order to establish the lymphocyte gate by means of a “side-scatter” towards CD45, the lymphocytes in the gate are examined for CD3+/CD4+ cells and for CD3+/CD8+ cells. The use of different fluorochromes specific for the four monoclonal antibodies used enables to perform this test on a single cell sample. This example stresses the power of flow cytometry to perform cell analyses in a more precise and easy manner, examining multi-parameters on a single sample, a very useful tool when there are few cells available for the analysis. In short, the ability to interpret the data of the multi-stain analysis lies in the ability to represent the same data, so that every cell population, as detected by only one different antigen, is clearly outlined. It is equally important to compensate for the projection of the various emission bands from the various fluorochromes. Computerized data analysis is of the greatest importance in these studies on multi-stain fluorescence.

Data analisys
Hemicytometer manufacturers offer a large variety of software to elaborate data. In recent years there has been a lot of progress in the production of software that is increasingly better and easier to use, and that enables the operator to observe and modify data representations while the samples are being processed. The data acquired can also be transferred to another computer to continue the analysis at a distance from the hemicytometer. Considering that the range of software and computers is really wide, the reader should refer to the more detailed descriptions of hardware and software available (see bibliography).

Immunophenotyping of leukaemia and limphomas
During the last 20 years considerable progress has been made in the use of flow cytometry in phenotyping and monitoring lymphoproliferative and myeloproliferative diseases. The production of increasingly larger quantities of monoclonal antibodies directed towards cell surface antigens expressed during cell differentiation has contributed to this progress. Besides, this has enabled to continue a detailed study of the differentiation stages of lymphoid and myeloid cells, thus contributing to a better understanding of the mechanism underlying immune and inflammatory phenomena. The samples presented for analysis are lymph nodes, bone marrow aspirates or peripheral blood. It is extremely important to ascertain that coagulation is complete and that the samples do not contain microclots, because these would block the flow chamber and hence the cell flow. During the analysis, portions containing at least one million cells are perfectly exposed to monoclonal antibodies in special plates for cell immunophenotyping in each of the various lymphoproliferative and myeloproliferative diseases. To save time, cells and material, and to obtain a more precise typing of cells and detect more surface antigens at the same time, it is now a common practice to perform multi-staining procedures for analyses with 2, 3 or 4 dyes. The plates used in our laboratory are listed in Table 1. The samples are exposed to monoclonal antibodies for 30 minutes in the dark at four degrees, then a lysing solution is added to every tube to lyse the erythrocytes, and the samples are centrifuged, suspended again in a chlorinated solution and washed, and again suspended in a chlorinated solution to be read. In the interpretation of data, it is important to determine the presence or absence of abnormal cells, a sign of a malignant transformation. In the case of B cell tumours, it suffices to prove that the majority of cells are monoclonal to establish the presence of a malignant clone. Alternatively, if the cells do not express their light chains, kappa and lambda, a cell population which expresses the CD19 and CD20 antigens of the B cells can be interpreted as a sign of a B cell’s malignant clone. In the case of T lymphocytes, it is virtually impossible to establish a clonal transformation. It is important to establish the presence of an abnormal phenotype that is compatible with a T lymphocyte during the earliest differentiation stage of peripheral and mature lymphocytes. In myeloproliferative diseases, the discovery of cells that are less phenotypically differentiated and are not usually present in the peripheral compartment would be a sign of a malignant transformation. A large number of monoclonal antibodies is now available to accurately immunophenotype cells and establish the cell differentiation stage reached. It is thus possible, through the construction of suitable plates of monoclonal antibodies, to examine cell samples with great precision and to establish their immunophenotypes. Besides, the finding that malignant cells could carry certain combinations of antigens, usually present in cells in the main compartment and not in the peripheral one, is of further help to establish an abnormal immunophenotype. It must be stressed that the results of flow cytometry immunophenotyping must be correlated with the morphological and histochemical examination of the blood, lymph nodes and aspirates and bone marrow biopsies. Even the cytogenetic analysis, besides the abovementioned ones, is an invaluable help to ascertain a diagnosis and propose a cure.

Monitoring HIV infections
The evidence of a progressive drop in the number of CD4+ lymphocytes in HIV infections has opened a new chapter in the use of flow cytometry to monitor diseases. The last decade has seen an increase in work focussed on perfecting the methodologies for the exact count of CD4+ lymphocytes in the blood of individuals infected with HIV. This movement towards increasing precision has been motivated partly by economic considerations, as the detection of 200 lymphocytes CD4+/ul was one of the criteria (and probably the most important one) to determine eligibility to financial aid. Obviously flow cytometry, as all other technologies, is subject to variability, both with regard to the operators and the instruments, hence the methodology to count CD4+ lymphocytes continues to be monitored and perfected. Figure 6 presents the best method currently accepted to count CD4+ cells - two-colour fluorescence, though three and four-colour fluorescence is used when possible.

Procedura di colorazione per i campioni dai pazienti HIV+, secondo le migliori tecniche di controllo della qualità attualmente accettate

It is important to receive the blood placed in ethylenediaminetetraacetate as anticoagulant, to store the samples at ambient temperature till they undergo the procedure, to analyse them within 24 hours, and to assure that there are no microclots in the samples. A basic fluorescence must be established with isotypic controls, to ensure that there is no aspecific bond formed by a special aspecific immunoglobulin of the same isotype as the monoclonal antibody used. CD14/CD45 double staining enables the “gating” of all CD14/CD45+ cells (that is, leukocytes that are not monocytes). The steps that follow count the CD4+ and CD8+ cells, on the total number of CD3+ (T cells). The strict quality control of instruments, reagents and processes inside the laboratory is of the greatest importance in all the stages of this procedure, besides the frequent monitoring of the results to assure reproducibility in the various geographical areas. A yet controversial issue concerns the translation of flow cytometric data, obtained as percentages of a cell population, into absolute data, that is cells/ul. Tools that record the results as absolute numbers have been introduced in an attempt to avoid the need to calculate the absolute number by using the results of the blood cell counts obtained with blood analysers and the percentage of CD4+ cells obtained by means of flow cytometry. The purpose of this chapter does not involve a discussion on the worth and defects of these instruments. We advice the reader to refer to the abundant literature concerning these instruments and their features, besides the many issues being discussed with regard to the absolute count of CD4+ cells.

Other metHodologies
There continues to be interest in developing new applications for clinical and research purposes.

Reticulocyte count
Flow cytometry immediately appeared useful towards a more objective and precise reticulocyte count. An easy method to this end, it is now available and its use is growing fast because it offers an objective methodology that does not require visual observation with a microscope and also involves less effort. This technology uses fluorescent dyes that bind with the residue RNA, thus identifying the reticulocytes. Dyes that have proved useful combine the RNA-specific bond with a good emission of a light signal, and this enables to discriminate the dye bound to the cells from the ones in the background. Auromine O and thiazole orange are currently the choice dyes in the analysis of reticulocytes.

Anti-platelet antibodies
The ability to detect anti-platelet antibodies can be useful in thrombocytopenia cases. The antibodies can be against the platelet antigens or against the immunoglobulin bound to the platelet and it is useful to detect them in thrombocytopenias of diverse origin. Flow cytometry supplies a very objective and precise method to detect these antibodies, and it is preferable to other methods so far available. A greater advantage appears obvious in the presence of a low platelet count, as flow citometry can be conducted on small samples.

Immunological monitoring in transplants
The OKT3 monoclonal antibody targeted against the T cell pan-antigen CD3 has become an important part of immunosuppressive treatment in the cure of patients with transplants of organs. Its use is based on its antagonism towards T-lymphocytes, the first cells involved in a rejection, thus avoiding rejection crises by means of a mechanism that has not been still completely understood. OKT3 must be administered in doses that maintain the amount of T-cells at a non-detectable level. The flow cytometric measurement of T cells / ul has become normal in monitoring patients that are receiving OKT3 following an organ transplant. It is of the greatest importance in these measuring procedures to obtain highly precise and reproducible results in sequential measurements. Another recently introduced method is the assessment of the effect of cyclosporines on target cells, to guide therapeutic dosage. The measurement of intracellular IL2 in peripheral blood lymphocytes stimulated with “PMA-ionomycin” can detect a suppression of the response to the stimulation, expressed in the amount of intracellular IL2, in lymphocytes of patients treated with increasing blood levels of Cyclosporine A. This method can help discriminate between toxic and therapeutic doses and also develop an optimal dose.

Resistance to multi-drug treatment
It has become obvious that drug resistance is a cause of therapeutic failure in the chemiotherapy of cancer, and that a variety of mechanisms can be responsible for this phenomenon. A mechanism that has been explained involves the outflow of therapeutic drugs from the cells, mediated by a glycoprotein-P (Pgp). A monoclonal antibody type against Pgp is available to conduct studies on the Pgp activity in drug resistance, a useful methodology in assessing multi-drug treatments.

Intracellular measuring
Cell membrane molecules, exposed on the cell surface, are easily detected, as these molecules are immediately available for monoclonal antibodies. A more complex problem was that of binding monoclonal antibodies to antigens localized inside the cell - it required the monoclonal antibody to be introduced into the cell preserving at the same time the integrity of the cell membrane so that the cell type could be established by identifying the surface antigens. The solution to this problem came with the demonstration that monoclonal antibodies could be introduced into the cell by modifying the membrane’s integrity with light fission that does not significantly destroy surface antigens. The development of this technique has enabled the study of intracellular molecules, such as cytokines, in various cells, thus contributing to our understanding of the cellular functions through the sequential measurement of molecules that are expressed while the cell is either differentiating or metabolizing. Other intracellular measurements have involved a study of calcium transport by means of dyes that are carried together with calcium. A useful example of this method is the possibility of measuring the intracellular flow of calcium, an early event in cell activation.

The ‘grain’ technology
A particularly burning issue was the possibility of analysing cell products in a solution, rather than inside the cells. In recent years a method requiring the use of plastic grains or pearls coated with a monoclonal antibody targeted at the product to be detected was proposed as an answer to this problem. The grains are then treated with the same monoclonal antibody labelled with a fluorochrome and studied in a hemicytometer. The fluorescence present on the grains shows that the product to be detected by the grain has been bound and it also shows the following bond between the fluorescent monoclonal antibody and the product bound to the grain. It is now possible, by means of measuring charts, to quantitatively detect the cellular products in fluids up to a pg level. It is interesting to note that the combination of grain detection of an extracellular, soluble product, with the detection of the same product inside the cell, can supply precious data on the accumulation and expression of the product and its secretion, thus enabling kinetic studies on activated cells.

DNA analisis
Even if it is not directly pertinent with the topic discussed in this chapter, the DNA content analysis used to establish the cell cycle and cell ploidy requires a few words. The earliest use of flow citometry was directed at analysing DNA by using dyes that would form a bond with it and then measuring the fluorescent intensity emitted by a certain dye. A resting cell would contain a normal amount of DNA, while a cell at the end of the DNA duplication process, and in every mitotic stage, would contain twice the amount of DNA. During DNA synthesis, there would be a different DNA content in every cell. The use of dyes that form stoichiometrical bonds with DNA allows the quantitative detection of the DNA content. The perfecting of the DNA analysis method has made use of DNA detection to assess ploidy and DNA synthesis in malignant cells, in order to relate this data with the degree of malignancy and the prognosis. Literature in this field has marked an exponential growth, as there has been an increasing effort to confirm the use of DNA analysis as an important aid in the diagnosis and cure of neoplastic diseases. There is still some controversy with regard to the role played by DNA analysis in supplying the information required, beyond those acquired by other conventional and established methods. While more parallel studies continue to add information to those already existent, it will become clear how and when DNA analysis can be a reliable diagnostic tool.

Conclusions
I have tried in this chapter to present a clear summary of certain basic aspects of flow citometry, in particular with regard to its use in immunology. It is my hope that this brief summary will encourage readers to study in detail those aspects of flow citometry that could help them in their work, both clinical and investigative. And I would also like to encourage those who are interested, but did not find in this context the information that can be applied to their needs, to search new ways of using flow citometry. It is still a young technology, and many applications have still not been developed. Hence, to close: enjoy your work and good hunting! Translated by interpres sas

Mariano Francis La Via
Department of Pathology and Laboratory Medicine
University of South Carolina - Charleston SC - USA