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be aspirated directly using a narrow-gauge needle. Such fine-needle aspiration procedures not only spare the patient potentially unnecessary surgery, but also yield single-cell specimens suitable for flow cytometric immunophenotyping with a minimum of further preparation. Flow cytometry is also frequently used in the diagnosis of other chronic lymphoproliferative disorders, which are neoplasms of mature lymphocytes and typically present in the peripheral blood and/or bone marrow, rather than lymph nodes (21-24).

3.1. B-CELL NON-HODGKIN'S LYMPHOMAS/LYMPHO-PROLIFERATIVE DISORDERS B-cell NHLs comprise the majority of NHLs in Western countries. Flow cytometric immunophenotyping is a valuable diagnostic adjunct, both in distinguishing B-cell NHLs from benign lymphoid hyperplasia and in subclassification of B-cell NHLs. Mature B-cells display an antigen receptor (antibody) that consists of heavy chains and light chains (kappa or lambda). Because the antigen receptor expressed by an individual B-cell contains either a kappa or lambda light chain, a neoplastic B-cell population representing a monoclonal B-cell proliferation should contain exclusively kappa- or lambda-bearing cells (e.g., Fig. 3B). In practice, a ratio of kappa-bearing : lambda-bearing B-cells in excess of 10:1 or less than 1 : 5 reflects the presence of a monoclonal B-cell population. For comparison, in (polyclonal) B-cell hyperplasia, the kappa : lambda ratio typically ranges between 1 : 1 and 3 : 1.

Not only does flow cytometric immunophenotyping facilitate the distinction between benign, reactive lymphoid proliferations, and monoclonal neoplasms, the extensive immunopheno-typic profiles made possible by flow cytometry enable more precise distinction among different B-cell NHLs. For example, B-cell chronic lymphocytic leukemia and mantle cell lymphoma could be confused morphologically, but their predicted clinical courses differ significantly. Although both are typically neoplasms of CD5+ B-cells, there are characteristic differences in the presence and/or intensity of expression of several molecules, including CD20, CD23, FMC-7, and immunoglobulin light chain, which facilitate distinction in most cases. Hairy cell leukemia is another B-cell lymphoproliferative disorder whose diagnosis is simplified by flow cytometry. Although neoplastic cells might not be numerous in the peripheral blood, in the appropriate clinical setting the detection of a monoclonal B-cell population with the composite immunophenotype CD11c(bright)+, CD25+, CD103+ is virtually pathognomonic of hairy cell leukemia, which requires specific therapy.

3.2. T/NK-CELL NON-HODGKIN'S LYMPHOMAS/ LYMPHOPROLIFERATIVE DISORDERS Whereas immuno-globulin light-chain restriction permits flow cytometric demonstration of B-cell monoclonality, there is no analagous immunophenotypic marker of clonality in T-cell or natural killer (NK)-cell NHLs, or lymphoproliferative disorders. Nonetheless, flow cytometry is useful in detecting immunophe-notypic aberrations that are common in T-cell neoplasia. For instance, among the so-called peripheral (i.e., nonlymphoblas-tic) T-cell lymphomas, absent or diminished expression of CD7, a ubiquitous T-cell antigen, is extremely common. Moreover, in conjunction with the clinical and morphologic findings, a detailed immunophenotypic profile as determined by flow cytometry facilitates distinction among different T-cell or NK-cell NHLs/lymphoproliferative disorders.

4. DNA CONTENT/PLOIDY ANALYSIS

Propidium iodide (PI) is a DNA-intercalating dye that binds DNA stoichiometrically; the fluorescence intensity of PI, therefore, correlates with the amount of DNA contained within the nucleus. When a population of nuclei is stained with PI or another similar DNA-binding fluorescent dye, a histogram of DNA content reflects the proportions of cells in different phases of the cell cycle. In a number of hematopoietic and solid tumors, the fraction of tumor cells in the S-phase and/or the presence of an aneuploid cell population as determined by flow cytometry has been shown to be prognostic (25). It is important to recognize that not all studies have demonstrated prognostic significance for S-phase fraction or DNA content measurement. In a few specific instances though, DNA ploidy measurements are still routinely performed for diagnostic or prognostic purposes. In gestational trophoblastic disease, for example, DNA content is commonly analyzed by flow cytometry (Fig. 7).

In these proliferative disorders of placental trophoblast, a triploid DNA content correlates pathologically with a partial hydatidi-form mole, which has no malignant potential, whereas a diploid DNA content correlates with a complete hydatidiform mole, which is associated with a markedly elevated risk for the subsequent development of choriocarcinoma, a malignancy of trophoblastic tissue (26). DNA ploidy is also typically evaluated at the time of diagnosis in patients with precursor B-cell ALL, in which a hyperdiploid karyotype is associated with a favorable prognosis (27).

5. MINIMAL RESIDUAL DISEASE ANALYSIS

Historically, it has been apparent that a significant portion of patients treated for acute leukemia who attain a complete clinical remission will, nonetheless, subsequently relapse. This observation implies the persistence of small (subclinical) amounts of disease following therapy in some patients, who are thus at elevated risk for recurrence. Patients with acute leukemia who have fewer than 5% blasts in the bone marrow following therapy are considered in morphologic remission. Although precise enumeration of blasts below this level is difficult by conventional microscopic evaluation, flow cytometric immunophenotyping is capable of detecting and precisely enumerating as few as 1 blast in 10,000 cells (i.e., 0.01% or 10-4).

Because leukemic blasts commonly display one or more "aberrant" antigens or express appropriate antigens at densities different from those seen in normal hematopoietic precursors, it is possible immunophenotypically to distinguish a minor population of leukemic blasts from normal regenerating hematopoietic precursors (28-30). In ALL, greater than 90% of cases are amenable to such immunophenotypic analysis, whereas in AML, approx 70-75% of cases can be evaluated immunophenotypically. In order to obtain sensitivity on the order of 10-4, it is necessary to analyze a large total number of cells (e.g., 500,000), a practice that is feasible using flow cytometry. Advantages of flow cytometric compared with molecular detection of minimal residual disease are rapid turnaround time and ability to quantify minimal residual disease directly. In ALL, for example, molecular detection of minimal residual disease requires sequencing of clone-specific rearrangements of immunoglobulin or T-cell receptor genes. Moreover, polymerase chain reaction-based methodologies are necessarily only semiquantitative, and several studies have shown that the percentage of residual disease, not merely its presence or absence, is significant in predicting outcome. In both AML and pediatric ALL, several groups have shown that the detection of residual leukemic blasts immediately or at different times following remission induction is associated with a higher likelihood of subsequent relapse. These results hold the potential both to identify earlier patients who will require additional therapy and to identify particularly low-risk patients who might safely be spared the toxicity of additional therapy.

6. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired clonal disorder of hematopoiesis associated clinically with hemolysis, thrombosis, and bone marrow failure

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