Cd4apc

Fig. 4. Peripheral blood specimen, gated on CD3+ T-cells. In this manner, the percentage of CD4+ T-cells (lower right quadrant) and CD8+ T-cells (upper left quadrant) can be quantified.

including disorders of cellular immunity (11). Lymphocyte subset analysis by flow cytometry is typically one of the first laboratory tests performed in the clinical evaluation of such patients. Moreover, lymphocyte activation, cytokine expression within specific cell subsets, and cellular cytotoxicity can all be probed using flow cytometry, although such functional assays are generally performed only in select laboratories with experience in these techniques.

2.2. CD34+ HEMATOPOIETIC STEM CELL ENUMERATION Hematopoietic stem cells are capable of reconstituting hematopoiesis; through multiple generations of proliferation and differentiation, a single pluripotent stem cell can give rise to red blood cells, white blood cells, and platelets. In patients with malignancies whose successful treatment requires extremely high doses of chemotherapy, a side effect of therapy is ablation of the bone marrow (so-called myeloablative therapy), the normal site of hematopoiesis. Without reconstitution of hematopoiesis by replacement of stem cells, either harvested from the patient prior to therapy (autologous) or from an immunologically compatible donor (allogeneic), such a potentially curative therapy would be fatal. Allogeneic stem cell transplant might also be used to cure patients with non-neoplastic, but potentially fatal genetic disorders (e.g., severe combined immunodeficiency). In either case, successful stem cell engraftment requires delivery of an adequate "dose" of stem cells to the recipient.

The expression of CD34 by hematopoietic stem cells, which can be obtained from bone marrow, peripheral blood, or umbilical cord blood samples, enables their rapid enumeration by flow cytometry (12,13). As in CD4+ T-cell enumeration, both dual-platform (requiring both flow cytometry and a conventional hematology analyzer) and single-platform (bead-based) assays are in use clinically. Several general points can be made, however, with respect to CD34+ stem cell enumeration. Because

CD34+ stem cells are generally rare (<1%), a large number of total events must be collected to enable their precise quantification. In addition, antibodies to CD34 whose binding requires recognition of carbohydrates and terminal sialic acid molecules (so-called class I antibodies) are to be avoided, as these post-translational modifications of CD34 are only variably present. Finally, it is preferable to use an antibody conjugated to the flu-orochrome phycoerythrin (PE), as its relatively high quantum yield, or brightness, enhances the sensitivity of the assay for stem cells, that are only dimly positive for CD34.

2.3. IMMUNOPHENOTYPIC ANALYSIS OF ACUTE LEUKEMIA The acute leukemias represent malignancies of lymphoid (acute lymphoblastic leukemia [ALL]) or myeloid (acute myeloid leukemia [AML]) precursors (14). Patients with acute leukemia commonly present with signs and symptoms of bone marrow failure (e.g., fatigue, bruising, anemia, thrombo-cytopenia) and, not infrequently, with an elevated white blood cell count. However, despite the many clinical similarities between ALL and AML, optimal treatment differs for these two broad categories of acute leukemia. Moreover, both ALL and AML encompass clinically and biologically distinct subsets of disease, which are often impossible to discern on the basis solely of conventional microscopic analysis. Because immuno-logic characterization of the neoplastic cells (blasts) in many cases permits more precise, therapeutically relevant diagnostic information about an acute leukemia than conventional microscopic evaluation, flow cytometry has become an important adjunct in the diagnosis of patients with acute leukemia.

2.3.1. Acute Myeloid Leukemia In AML, the neoplastic cells, or blasts, are myeloid precursor cells that might display varying degrees of differentiation to granulocytes, monocytes, or, less commonly, erythroid or megakaryocytic precursors. It is important to remember though that diagnostic specimens (typically peripheral blood or bone marrow) contain mature myeloid cells in addition to the malignant blasts. Because myeloblasts can share many of the immunophenotypic properties of these mature myeloid cells, it is important to gate selectively upon the blasts. Historically, blasts were gated on the basis only of their FSC and SSC signals. However, this approach is significantly flawed, because gates drawn solely on the basis of these intrinsic properties invariably include non-neoplastic lymphoid cells and erythroid precursors, as well as monocytes; the "population" gated using only FSC and SSC, therefore, in fact comprises a heterogeneous mixture of non-neoplastic and malignant cells. In the last decade, it has become routine to gate on blasts using the combination of dim CD45 expression and low SSC (15,16). This approach permits more precise distinction of blasts from other bone marrow elements (Fig. 5). In cases of acute leukemia, blasts gated in this fashion are substantially free of contaminating non-neoplastic cells (Fig. 6).

Once a candidate blast population has been isolated by electronic gating, it can be probed immunophenotypically using a panel of antibodies (15-17). It is important to include antibodies recognizing not only myeloid antigens but also B-lymphoid and T-lymphoid antigens, as so-called "aberrant" expression of myeloid antigens in ALL and lymphoid antigens in AML is not uncommon. In most cases, it is possible to assign unambiguously a lineage (i.e., myeloid vs T- or B-lymphoid) on the basis

CD45 PerCP

Fig. 5. CD45 expression vs SSC. Normal bone marrow (A) and bone marrow of a patient with acute leukemia (B). R2 = erythroid precursors; R3 = normal B-cell precursors (hematogones); R4 = lymphocytes; R5 = monocytes; R6 = granulocytes; R7 = blasts.

CD45 PerCP

Fig. 5. CD45 expression vs SSC. Normal bone marrow (A) and bone marrow of a patient with acute leukemia (B). R2 = erythroid precursors; R3 = normal B-cell precursors (hematogones); R4 = lymphocytes; R5 = monocytes; R6 = granulocytes; R7 = blasts.

of the surface-membrane immunophenotype of the blasts. However, in some cases, it might be useful also to evaluate cytoplasmic antigens whose expression is more specific for a given lineage. For example, detection of cytoplasmic myeloper-oxidase in a blast population would provide strong evidence for myeloid differentiation, even if that population were found to express lymphoid antigens on the cell membrane. Some of the antigens commonly evaluated to permit lineage assignment in cases of acute leukemia are summarized in Table 1.

In addition to facilitating lineage assignment, flow cytomet-ric immunophenotyping in AML can also provide more detailed information with respect to the type (e.g., granulocytic, mono-cytic, megakaryocytic, erythroid) and extent of myeloid differentiation. Perhaps more importantly, certain composite immunophenotypes have been associated with specific recurrent karyotypic abnormalities in AML, some of which require modification of the patient's treatment protocol. For example, in acute promyelocytic leukemia, absent or weak expression of HLA-DR is typical. This form of AML must be specifically recognized, because its characteristic juxtaposition of PML and RARa genes resulting from a translocation between chromosomes 15 and 17 renders the leukemic cells susceptible to alltrans retinoic acid. Another biologically distinct subset of AML, characterized karyotypically by the t(8;21) AML1-ETO is associated with a CD34+, HLA-DR+, myeloid immunophenotype, with aberrant expression of the B-cell antigen CD19 and at least partial positivity for the nuclear antigen terminal transferase (TdT). Finally, AML with abnormalities of 16q, most commonly inv(16), is associated with aberrant expression of the T-cell antigen CD2.

2.3.2. Acute Lymphoblastic Leukemia The term ALL includes precursor B-cell and precursor T-cell forms of the disease. Despite differences in clinical presentation and optimal treatment, precursor B-cell ALL and precursor T-cell ALL are morphologically indistinguishable (18). Flow cytometric immunophenotyping is, therefore, essential in establishing the diagnosis of ALL. As with AML, a panel of antibodies should be used, including B-lineage, T-lineage, and myeloid antigens (16,18-20). As noted earlier, surface-membrane myeloid antigens can be expressed aberrantly in ALL, a feature that might complicate lineage assignment of the blast population. In such instances, it is appropriate to evaluate the expression of cyto-plasmic antigens more specifically associated with B- and T-lymphoid differentiation (i.e., cytoplasmic CD22 and cytoplasmic CD3, respectively).

In addition to its role in establishing the diagnosis of precursor B-cell ALL and precursor T-cell ALL, flow cytometric immunophenotyping might provide prognostic information and predict recurrent genotypic aberrations. For example, in childhood precursor B-cell ALL, bright expression of CD20 and CD45 are adverse prognostic factors, independent of other known risk factors, whereas in childhood precursor T-cell ALL, expression of the T-cell antigen CD2 is a favorable prognostic factor. A number of recurrent balanced translocations have also been associated with specific immunophenotypic profiles. Such composite immunophenotypes have been described in precursor B-cell ALL with t(1;19) E2A-PBX, t(12;21) TEL-AML1, and t(4;11) AF4-MLL.

3. IMMUNOPHENOTYPIC ANALYSIS OF NON-HODGKIN'S LYMPHOMA AND OTHER LYMPHOPROLIFERATIVE DISORDERS

The non-Hodgkin's lymphomas (NHLs) are malignancies of B-cells, T-cells, or, rarely, natural killer cells, which present clinically with swelling of lymph nodes and/or tumoral masses in extranodal sites (14). In cases of NHL, the diagnostic specimen is often a biopsy of an affected lymph node or extranodal site. To make such a specimen suitable for flow cytometric immunophenotyping, the biopsy must be disaggregated and a single-cell suspension prepared; this is typically accomplished mechanically, although enzymatic digestion has also been used. Increasingly, tumor cells from both superficial and deep-seated lesions (the latter is performed with radiographic guidance) can

Fig. 6. Illustration of CD45 vs SSC gating in acute leukemia. In (A), a conventional mononuclear cell gate is drawn (R1) on the basis of intrinsic cellular light-scatter properties. The fluorescence signals obtained using this strategy (shown in B) are heterogeneous, as the gate includes not only blasts but also non-neoplastic erythroid precursors, T-cells, B-cells, and monocytes. Because blasts characteristically express CD45 dimly (R2 [C]), the fluorescence signals obtained using this approach (D) are essentially homogeneous. In this case, the blasts are positive for the myeloid antigen CD33 (B, D), whereas the non-neoplastic cells contaminating the R1 gate are negative for CD33 (B).

Fig. 6. Illustration of CD45 vs SSC gating in acute leukemia. In (A), a conventional mononuclear cell gate is drawn (R1) on the basis of intrinsic cellular light-scatter properties. The fluorescence signals obtained using this strategy (shown in B) are heterogeneous, as the gate includes not only blasts but also non-neoplastic erythroid precursors, T-cells, B-cells, and monocytes. Because blasts characteristically express CD45 dimly (R2 [C]), the fluorescence signals obtained using this approach (D) are essentially homogeneous. In this case, the blasts are positive for the myeloid antigen CD33 (B, D), whereas the non-neoplastic cells contaminating the R1 gate are negative for CD33 (B).

Table 1

Some Lineage-Related Antigens Commonly Evaluated in Acute Leukemia

Table 1

Some Lineage-Related Antigens Commonly Evaluated in Acute Leukemia

Myeloid

T-lymphoid

B-lymphoid

CD11b

CD1a

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