Detection of Mature T-Cell Leukemias by Flow Cytometry
Detection of Mature T-Cell Leukemias by Flow Cytometry
A broad array of antibodies directed against the variable (V) region of the T-cell receptor (TCR) β (Vβ) chain has become available in a directly conjugated multicolor format that permits assessment of 19 of 25 Vβ families, covering 70% of the normal circulating T-cell repertoire. These antibodies were used to detect expanded T-cell populations in 43 peripheral blood samples submitted for suspected T-cell malignancy. Of 43 samples, 27 were diagnosed as follows: T-cell large granular lymphocyte leukemia, 14 samples; Sézary syndrome, 4 samples; T-cell prolymphocytic leukemia, 5 samples; or T-cell non-Hodgkin lymphoma or T-cell lymphoproliferative disorder not otherwise specified, 4 samples. The remaining 16 samples were determined to be nonneoplastic. All samples were diagnosed before assessment with anti-Vβ flow cytometry.
Evaluation for T-cell monoclonality often is necessary for the diagnosis of T-cell malignancy. The presence of T-cell monoclonality in clinical specimens is assessed routinely by molecular methods that examine the rearrangement of T-cell receptor (TCR) genes. Southern blot analysis for rearrangement of the TCRβ chain has been used frequently in clinical laboratories for the assessment of T-cell monoclonality. Because all T cells undergo γ chain gene rearrangement during ontogeny, it also is possible to study rearrangement of the γ chain gene for analysis of T-cell monoclonality. The smaller number of TCRγ genes has been convenient for the development of polymerase chain reaction (PCR) assays for gene rearrangement. PCR assays can be more sensitive than TCRβ Southern blot analysis, are less labor-intensive, and do not require high-molecular-weight DNA, permitting paraffin-embedded tissue samples to be used as a source of DNA for retrospective studies or when fresh or frozen tissue samples are not available.
Flow cytometric analysis using antibodies directed against the V region of the TCRβ chain (Vβ) permits direct detection of expanded T-cell populations. The human Vβ locus consists of 65 genes, 46 of which are functional, divided into subfamilies based on sequence homology. The predominance of a single Vβ family within a T-cell population (Vβ restriction) is highly suggestive of an expanded T-cell clone. Recent studies have used Vβ flow cytometric analysis to evaluate T-cell expansions in a variety of mature T-cell populations. Although single anti-TCR Vβ antibodies have existed for years,7 their practical use in clinical diagnosis has been feasible only following the recent development of large numbers of Vβ family-specific antibodies that can detect the majority of normal circulating T cells. Compared with molecular studies of TCR gene rearrangement, flow cytometric evaluation of Vβ chain use permits direct quantitation and immunophenotypic characterization of the Vβ family of the malignant clone. After characterization of the malignant clone, Vβ flow cytometric analysis also can be used to follow up the treatment and clinical course in a particular patient. In the present study, we demonstrated the usefulness of a multicolor panel of directly conjugated Vβ antibodies for rapid detection of T-cell malignancy in peripheral blood samples from patients with T-cell lymphoproliferative disorders.
A broad array of antibodies directed against the variable (V) region of the T-cell receptor (TCR) β (Vβ) chain has become available in a directly conjugated multicolor format that permits assessment of 19 of 25 Vβ families, covering 70% of the normal circulating T-cell repertoire. These antibodies were used to detect expanded T-cell populations in 43 peripheral blood samples submitted for suspected T-cell malignancy. Of 43 samples, 27 were diagnosed as follows: T-cell large granular lymphocyte leukemia, 14 samples; Sézary syndrome, 4 samples; T-cell prolymphocytic leukemia, 5 samples; or T-cell non-Hodgkin lymphoma or T-cell lymphoproliferative disorder not otherwise specified, 4 samples. The remaining 16 samples were determined to be nonneoplastic. All samples were diagnosed before assessment with anti-Vβ flow cytometry.
Evaluation for T-cell monoclonality often is necessary for the diagnosis of T-cell malignancy. The presence of T-cell monoclonality in clinical specimens is assessed routinely by molecular methods that examine the rearrangement of T-cell receptor (TCR) genes. Southern blot analysis for rearrangement of the TCRβ chain has been used frequently in clinical laboratories for the assessment of T-cell monoclonality. Because all T cells undergo γ chain gene rearrangement during ontogeny, it also is possible to study rearrangement of the γ chain gene for analysis of T-cell monoclonality. The smaller number of TCRγ genes has been convenient for the development of polymerase chain reaction (PCR) assays for gene rearrangement. PCR assays can be more sensitive than TCRβ Southern blot analysis, are less labor-intensive, and do not require high-molecular-weight DNA, permitting paraffin-embedded tissue samples to be used as a source of DNA for retrospective studies or when fresh or frozen tissue samples are not available.
Flow cytometric analysis using antibodies directed against the V region of the TCRβ chain (Vβ) permits direct detection of expanded T-cell populations. The human Vβ locus consists of 65 genes, 46 of which are functional, divided into subfamilies based on sequence homology. The predominance of a single Vβ family within a T-cell population (Vβ restriction) is highly suggestive of an expanded T-cell clone. Recent studies have used Vβ flow cytometric analysis to evaluate T-cell expansions in a variety of mature T-cell populations. Although single anti-TCR Vβ antibodies have existed for years,7 their practical use in clinical diagnosis has been feasible only following the recent development of large numbers of Vβ family-specific antibodies that can detect the majority of normal circulating T cells. Compared with molecular studies of TCR gene rearrangement, flow cytometric evaluation of Vβ chain use permits direct quantitation and immunophenotypic characterization of the Vβ family of the malignant clone. After characterization of the malignant clone, Vβ flow cytometric analysis also can be used to follow up the treatment and clinical course in a particular patient. In the present study, we demonstrated the usefulness of a multicolor panel of directly conjugated Vβ antibodies for rapid detection of T-cell malignancy in peripheral blood samples from patients with T-cell lymphoproliferative disorders.
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