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Flow Cytometry
Flow cytometry (FCM) is invaluable in the diagnosis and classification of hematolymphoid neoplasms, and in determining prognosis and monitoring response to therapy. FCM is especially suited for immunophenotypic analysis of blood, fluids (e.g., cerebrospinal fluid [CSF], pleural fluid), and aspirations of bone marrow and lymphoid tissue. FCM is also ideal in small samples where its multiparametric nature allows the concurrent staining of cells with multiple antibodies complexed to different fluorochromes, thus maximizing data obtained from few cells. FCM can characterize surface as well as cytoplasmic protein expression. Furthermore, FCM can provide highly accurate quantitation of cellular antigens/molecules. With antibody-based therapies such as the rituximab (anti-CD20), epratuzumab (anti-CD22), gemtuzumab (anti-CD33), and blinatumomab (directed against CD19 and CD3), the use of FCM is likely to increase. FCM identification of therapeutic targets on the surface of malignant cells affects the potential utility of these forms of therapy in a given patient. Once a diagnosis is established, FCM analysis provides high sensitivity in the detection of minimal disease (on the order of 1 in 10 4 to 10 6 ), to monitor disease progression and/or the impact of prior therapy.
General Principles
In a flow cytometer, cells rapidly pass single-file through a series of finely focused lasers. The cell momentarily breaks the laser beam, scattering light at a low angle (also called forward scatter ), much like a small orb casting a shadow. This forward scatter/low-angle scatter (FSC) can be proportional to cell volume. Laser light is simultaneously scattered at high angle (side scatter) by intracellular and nuclear components. This side scatter (SSC) is proportional to the cell's complexity, which is determined by the type and amount of cytoplasmic granularity, cytoplasmic membrane irregularities (e.g., villous or hairy projections), and nuclear characteristics. Light scatter characteristics can also be used to restrict analysis to single cells (e.g., excluding doublets [two adherent cells]). These physical scatter properties accurately identify cell types and are the basis for many commercial hematology analyzers that provide automated differential cell counts.
In addition to FSC and SSC properties, cells are further characterized by staining with multiple fluorescent markers, such as antibodies conjugated to fluorochromes or DNA-binding dyes. If a cell expresses an antigen that binds to a fluorochrome-conjugated antibody, the fluorochrome will emit light at a particular wavelength that is measured by detectors. If used in combination with DNA-binding dyes, the DNA content can also be determined, yielding cell cycle data. Multiple fluorochromes (sometimes referred to as colors ), each emitting uniquely identifiable spectral characteristics, are simultaneously measured with multiple detectors. Most clinical laboratories use 6- to 8-color FCM, with some using 10 or more colors in their flow-cytometric analysis, and it is agreed that 4-color analysis is the minimal acceptable amount to ensure reliable discrimination of neoplastic cell populations in a broad range of sample types.
Initially, FCM determined the presence or absence of lineage-specific or lineage-associated antigens, but immunophenotypic interpretation has evolved from a simplistic “positive” or “negative” for a given antigen, to an assessment of the degree of expression. This approach is highly reliable in discriminating cell types and identifies characteristic FCM features and patterns unique to certain hematolymphoid neoplasias. As the antigen expression of many hematolymphoid neoplasias overlap with their normal counterparts, the ability of multiparametric FCM to highlight subtle temporal patterns and antigen intensity makes it extremely powerful in the diagnosis of neoplasia.
Technical Considerations
General
Appropriate samples for FCM include blood, bone marrow, lymph node, extranodal tissue biopsies, fine-needle aspirates (FNA), and body fluids (e.g., pleural, peritoneal, CSF). International consensus guidelines on medical indications for FCM are available and are based on patient history and presenting symptoms. Timely processing of samples is necessary to maximize cell yield, maintain cell viability and integrity, and prevent loss of abnormal cells of interest (see Stetler-Stevenson et al. for recommendations). Blood and bone marrow specimens must be collected in an appropriate anticoagulate. Lysis is the preferred approach for removing excess erythrocytes (see Stetler-Stevenson et al. for recommendations). In patients with an inaspirable marrow, or a “dry tap” (i.e., a fibrotic marrow or a marrow packed with neoplastic cells), submission of several core biopsies for FCM is appropriate. These cores are disaggregated to release cells into fluid suspension, before FCM. Portions of tissue for FCM should represent an area that is also being submitted for histology, to minimize discordance due to sampling. Intact portions of solid tissue (such as biopsies of bone marrow, lymph nodes, or other tissue masses) must be made into cell suspensions for FCM. Mechanical tissue disaggregation is fairly simple, rapid, leaves the cells relatively unaltered, and is achieved by slicing, mincing, and teasing apart the tissue with commercial devices or manual tools. Enzymatic dissociation has been used in processing fibrotic tissue; however, it can alter antigen expression and decrease viability.
Antibody-staining protocols differ according to application and specimen type. Antibody panels are designed for assessment of lineage and level of differentiation as well as subclassification, and they require an in-depth understanding of antigen-expression patterns in normal and neoplastic cells. The emission spectra of fluorochromes vary, and conjugation should be to appropriate antibodies to maximize detection (e.g., bright fluorochrome with dimly expressed antibody). Multiple antibodies are required for lineage assignment. Most antibodies are not cell lineage–specific, and neoplastic cells may lack one or more antigens of a particular lineage. Overall, the number of reagents in a panel should be sufficient to allow the recognition of all abnormal and normal cells in the sample; conversely, limiting the number of antibodies may compromise diagnostic accuracy. In general, the larger the antibody panel, the higher the sensitivity and specificity of detection and characterization. By international consensus, the number of reagents needed to adequately evaluate a specimen for potential hematologic neoplasms is dependent on the presenting symptoms. In addition, surface and intracellular markers may be of prognostic utility and should be studied.
Viability
Decreased viability is noted in solid-tissue samples and aggressive lymphomas. Nonviable cells may nonspecifically bind antibodies and interfere with accurate immunophenotyping. A low-viability sample composed entirely of neoplastic cells can yield meaningful results. Furthermore, many samples submitted for FCM are considered irreplaceable, obtained by an invasive procedure with significant trauma, and/or are difficult to impossible to re-collect. In this case, every effort must be made to obtain diagnostic information. No set cutoff exists to dictate specimen rejection for FCM, although, general guidelines suggest rejecting non-irreplaceable samples with less than75% viability. In irreplaceable specimens with poor viability, any abnormal populations should be reported. Failure to identify a neoplastic process in a sample of poor viability should not be viewed as a true negative, as subsequent testing may be informative.
Small Specimens
Diagnosis of lymphoma is frequently based on evaluation of small biopsies, FNA, and body fluids (e.g., CSF, vitreous humor, effusions). Small samples can provide sufficient cells for FCM, even when cell numbers are too low to count by conventional methods. FCM can be more sensitive than morphology, especially when neoplastic cells are admixed with normal counterparts or associated with a brisk inflammatory response, as in extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) or gastric lymphoma in endoscopic biopsies.
FCM provides increased sensitivity of detection of hematolymphoid neoplasia in FNA. Furthermore, as the WHO classification incorporates immunophenotypic criteria, FCM evaluation of FNA assists in both detection and diagnostic subclassification of lymphoma and is particularly robust in the subclassification of chronic lymphocytic leukemia, mantle cell lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma, and plasmacytoma.
Involvement of the CSF by hematopoietic malignancies may be difficult to document by morphology alone. FCM improves the detection sensitivity of non-Hodgkin's lymphoma in CSF and is vital in the diagnostic evaluation of high-grade lymphomas. In a study assessing FCM in evaluating CSF in patients at risk for having central nervous system (CNS) involvement by aggressive B-cell lymphoma, FCM was significantly more sensitive than cytology alone in disease detection and prognostication. FCM is also useful in identifying CNS leukemia and increases the detection rate over cytology alone. Thus FCM is crucial in the evaluation of CSF for hematolymphoid malignancies. It should be noted that studies have demonstrated that there is a rapid decline in CSF cell number within the first 30 minutes of sampling, and immediate stabilization with serum containing media or commercially available stabilizers is vital to preserve the specimen until it reaches the flow cytometry laboratory.
Flow Cytometry in Diagnosis and Classification of Mature B-Cell Neoplasms
FCM detection of malignant B-cell populations requires extensive knowledge of normal B-cell antigen expression and light scatter characteristics. Markers of B-cell neoplasia include light chain restriction, abnormally large B cells, abnormal levels of antigen expression, absence of normal antigens, and presence of antigens not normally present on mature B cells.
Flow-Cytometric Evaluation of Light Chain Expression
A B-cell population with monoclonal light chain expression is, with rare exception, considered a B-cell neoplasm. Monoclonal B-cell populations are infrequently demonstrated in patients with no evidence of lymphoma, though this may represent early preclinical detection of B-cell malignancy. A monotypic B-cell population is characterized by the expression of a single immunoglobulin light chain by a B-cell population, resulting in positive staining with only one light chain reagent (e.g., kappa-positive/lambda-negative population, or vice versa) ( Fig. 5-1, A ). In normal/benign lymphoid tissue, virtually every B cell expresses a single light chain immunoglobulin, and the ratio of kappa-expressing to lambda-expressing B cells is approximately 60% to 40%. Lack of surface immunoglobulin among mature B cells or a deviation from this normal ratio suggests a monoclonal B-cell population.
FCM is advantageous in that it can recognize monoclonal B cells, even in B-cell lymphopenia, owing to rapid analysis of large numbers of acquired B cells, or in a background of polyclonal B cells, owing to detection of aberrant antigens on the neoplastic cells. By examining B-cell subsets with differential CD19, CD20, or CD22 expression, an abnormal monoclonal B-cell population may be discovered (see Fig. 5-1, B and C ). Detection of a skewed kappa to lambda ratio should prompt a diligent search for an underlying monoclonal population that may be discriminated by CD19, CD20, CD22, or other antigens. For example, peripheral blood with minimal involvement by hairy cell leukemia (HCL) may, at first glance, appear to contain only polyclonal B cells; however, with specific identification of the CD20 bright + , CD22 bright + B cells, the monoclonal light chain expression of the HCL cells may be revealed (see Fig. 5-1, C ). Antibody panels can be designed to exploit the expression of disease-characterizing antigens, such as CD5 in mantle cell lymphoma or CD10 in follicular lymphoma, for detection of monoclonality. For example, the CD5 + B cells in peripheral blood with involvement by mantle cell lymphoma may be monoclonal, while the CD5 − B cells are polyclonal (see Fig. 5-1, B ). A simplistic, one-dimensional examination of cells staining with kappa, lambda, and CD5 is clearly ineffective in this case. Multiparametric analysis is essential in detecting relevant neoplastic populations.
Absence of surface immunoglobulin may also indicate a mature B-cell neoplasm, but caution is imperative when interpreting the significance of such a population. Reactive germinal center cells with dim surface immunoglobulin are increased in follicular hyperplasia and may be mistaken for neoplasm; however, germinal center cells are distinguished by higher levels of CD20, CD10 positivity, and lack of intracellular BCL-2. Kappa and lambda expression is typically dim, but can be detected when compared with immunoglobulin negative T cells within the sample. In bone marrow aspirates, plasma cells and most normal immature B cells (hematogones; benign precursor B cells) also lack surface immunoglobulin.
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