2x2 - The Pocket Cube: The method described on this page is called the Ortega method, which is slightly more advanced than the normal LBL (layer-by-layer) method you might have taught yourself. If you learn this, it will give you a surprising boost in speed over LBL. ![]() Abstract We describe a simple and robust flow cytometry assay for ZAP-70 and CD38 expression. The steps required to validate this assay in a clinical flow cytometry laboratory are described. Two criteria were used to characterize ZAP-70 expression into positive, negative, and indeterminate categories and applied to 111 cases of chronic lymphocytic leukemia (CLL) resulting in 29.7% positive, 56.8% negative, and 13.5% indeterminate cases. A sensitivity-specificity crossover plot between ZAP-70 and CD38 suggested a cutoff of 12.5% for defining CD38 positivity. ZAP-70+ cases were significantly more likely to be at a higher clinical stage and, together with CD38+ cases, were more likely to have unmutated IgV H. However, for individual patients, the concordance between these markers was not perfect. It may be necessary to evaluate several prognostic markers simultaneously in CLL, and availability of convenient assays for ZAP-70 and CD38 is desirable for optimal clinical decision making. Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of mature-appearing lymphocytes that typically express CD19, CD5, and CD23. Daemon tools free download for windows 7 serial number. It is the most common leukemia in the United States and Europe. CLL is a heterogeneous disease with a median survival ranging from 2 to 20 years. Based on the anticipated and observed aggressiveness of CLL in a particular patient, the treatment may vary from observation alone to multiagent chemotherapy or allogeneic bone marrow or stem cell transplantation. Until about a decade ago, the clinical stage at diagnosis was the chief prognostic factor available for CLL patients, but the correlation between clinical stage and aggressiveness of disease was not ideal. Immunoglobulin heavy chain variable region ( IgV H) mutation status was first described as an independent prognostic marker in CLL in 1998 and 1999, and it is now well established that the lack of an IgV H mutation in CLL correlates with a worse prognosis. Analysis of IgV H mutation status is a relatively expensive and time-consuming test with restricted availability. Gene expression analysis of CLL cells revealed that messenger RNA (mRNA) for ζ-associated protein-70 (ZAP-70) is overexpressed in CLL compared with normal B cells. Further work showed that expression of ZAP-70 mRNA or protein may serve as a surrogate for IgV H mutation status. Mp3 labaik allahuma labaik. By using flow cytometry, it was confirmed that leukemic cells without the IgV H mutation express detectable intracellular ZAP-70 protein, whereas those with the IgV H mutation do not. Several studies have identified CD38 expression by CLL cells as another marker of CLL prognosis. A simple, reliable, and convenient flow cytometric assay for ZAP-70 and CD38 is highly desirable. We present information regarding a single-tube assay using 4-color flow cytometry that can simultaneously examine the expression of CD38 and ZAP-70 in CLL cells. Our results from 111 patients with CLL for these prognostic parameters were compared with the results of IgV H mutation status, clinical stage at diagnosis, and cytogenetic abnormalities in a subset of the patients. ![]() Materials and Methods Samples EDTA-anticoagulated peripheral blood samples from 111 patients with B-CLL diagnosed according to the World Health Organization criteria were analyzed by flow cytometry between February 2005 and August 2008. The samples were stained and acquired within 24 hours from time of collection. Antibodies Fluorescently labeled antibodies to CD19–peridinin chlorophyll protein (PerCP)–cyanine (Cy)5.5, CD5-phycoerythrin (PE), and CD38-allophycocyanin (APC) were obtained from Becton Dickinson, San Jose, CA. ZAP-70-PE and ZAP-70− Alexa Fluor 488 (Caltag, Buckingham, England) antibodies were evaluated. The ZAP-70 antibodies were titrated using 1.25 to 10 μL of the stock solution of antibody to provide maximum delineation between normal T cells (positive staining) and normal B cells (negative staining) in control samples. ZAP-70 mouse monoclonal antibody (clone 1E7.2) conjugated with Alexa Fluor 488 (Caltag) was selected because it gave reliably positive and negative results in normal T and B cells, respectively, within a narrow range of staining intensity. Mouse IgG1 Alexa Fluor 488 (Caltag) was used as the matching isotype control.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |