RESUMO
Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. This unit describes a technique to measure ZAP-70 protein expression in whole-blood specimens from CLL samples. The protocols presented include an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP-70 protein with significantly improved signal-to-noise ratio, an optimized combination of antibodies-fluorophores to maximize ZAP-70 expression levels, standardized methodology for instrument setup, including compensation, to improve inter- and intra-laboratory reproducibility, and a method to index ZAP-70 protein expression levels to internal positive and negative cell populations. Residual normal T and B cells function as internal positive and negative controls. These are used to index ZAP-70 protein expression levels in the CLL population.
Assuntos
Leucemia Linfocítica Crônica de Células B/sangue , Proteína-Tirosina Quinase ZAP-70/sangue , Proteína-Tirosina Quinase ZAP-70/genética , Antígenos CD/sangue , Linfócitos B/imunologia , Antígenos CD5/sangue , Permeabilidade da Membrana Celular , Citometria de Fluxo , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Antígenos CD15/sangue , Valores de Referência , Linfócitos T/imunologiaRESUMO
BACKGROUND: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. METHODS: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. RESULTS: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. CONCLUSIONS: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory.
Assuntos
Linfócitos B/química , Citometria de Fluxo/métodos , Células Matadoras Naturais/química , Leucemia Linfocítica Crônica de Células B/diagnóstico , Linfócitos T/química , Fixação de Tecidos/métodos , Proteína-Tirosina Quinase ZAP-70/análise , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Linfócitos B/imunologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/imunologia , Permeabilidade da Membrana Celular , Humanos , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Reprodutibilidade dos Testes , Coloração e Rotulagem , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/biossíntese , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
Lethal autoimmunity associated with IL-2Rbeta-deficient mice is prevented after thymic transgenic expression of wild-type IL-2Rbeta in IL-2Rbeta(-/-) mice (Tg -/- mice). Here, we show that CD4(+)CD25(+) regulatory T cells were not readily detected in IL-2Rbeta(-/-) mice, but the production of functional CD4(+)CD25(+) T cells was reconstituted in Tg -/- mice. Adoptive transfer of normal CD4(+)CD25(+) T cells into neonatal IL-2Rbeta-deficient mice prevented this lethal autoimmune syndrome. The CD4(+)CD25(+) T cells in disease-free adult IL-2Rbeta-deficient recipient mice were present at a near normal frequency, were solely donor-derived, and depended on IL-2 for expansion. These observations indicate that the essential function of the IL-2/IL-2R system primarily lies at the level of the production of CD4(+)CD25(+) regulatory T cells.
