Dim expression of CD5 on human B lymphocytes has been used to delineate B1 and B2 subsets. Nevertheless, others have suggested that the molecule is an activation marker and does not predicate a subset distinction. We have used enzymatic amplification staining, a technology that enhances the resolution of flow cytometric analysis of cell surface molecules by as much as 100-fold, to determine that essentially all human B cells express CD5. Furthermore, we show that this expression is regulated during EpsteinBarr virus transformation. C D5, a 67-kDa surface glycoprotein of the scavenger receptor cysteine-rich family, appears on T lymphocytes early in their development and is abundantly expressed on all mature T cells. The expression of the molecule on B lymphocytes and its role in defining separate lineages of these cells is controversial (1, 2). It has been postulated that the low-level expression of CD5 by a subset of B lymphocytes defines a separate lineage that is associated with autoreactive antigenic specificity (2, 3). Alternatively, others have shown that CD5 expression on B cells can be enhanced by various activating agents, prompting the suggestion that CD5 is a B cell activation antigen and that B cells comprise a single lineage (4, 5).We have developed a powerful technology, enzymatic amplification staining (EAS), that significantly enhances the resolution of flow cytometric analysis of cell surface molecules (6). By using EAS, we have achieved a 100-fold enhancement in the fluorescent signal. The enhanced signal allows for the detection of molecules that could not be observed previously. Thus, we have been able to define a subpopulation of peripheral blood cells stimulated in vitro that express Fas ligand (CD178), and we have demonstrated the validity of EAS in this case by correlating the staining with functional activity (6). EAS has also been used to provide a high-resolution immunophenotype of leukemic cells from patient samples. It was shown that enzymatic amplification gives a more powerful assessment of the clonality of the leukemic cells (7).Some investigators have proposed that human B lymphocytes can be divided into B1 and B2 subsets based on the expression of CD5 (2, 3); however, others have shown that CD5 expression on B cells can be up-regulated by various activating agents, which suggests that CD5 is a B cell activation antigen (4, 5). To address these alternative models, we used EAS to assess the expression of CD5 on human B lymphocytes. We found that essentially all B cells express CD5 constitutively at low levels, and we also found that CD5 expression is regulated by Epstien-Barr virus (EBV) transformation. Flow Cytometric Analysis. For standard amplification staining (indirect staining), we incubated cells with biotinylated primary antibodies followed by an incubation with streptavidin conjugated to fluorescein isothiocyanate. For EAS, we obtained kits from Flow-Amp Systems (Cleveland), and followed the manufacturer's instructions. The incubations were all performed for 10 min at room...

Immunofluorescent staining of mammalian cells has provided a reliable approach for detection of specific antigen expression in situ. An advantage of fluorescent markers has been their applicability to automated, high-throughput cellular analysis by flow cytometry. Flow cytometry has thus become an integral component of clinical laboratory diagnostics, particularly in the areas of immunology and hematology. One of the major drawbacks of traditional immunofluorescent staining, even with flow cytometric detection, has been the difficulty in detecting low abundance cellular antigens, some of which may have clinical and scientific significance. To address these problems, staining techniques have recently been developed to increase the sensitivity of cellular antigen detection by flow cytometry. In this review we will describe a few of these techniques and focus on enzymatic amplification staining as a means to generate a highly augmented antigen-specific signal. We will also discuss practical applications of enzymatic amplification for immunostaining of clinical specimens.

KAT6A, a histone/lysine acetyltransferase is located on 8p11 and has been reported in association with a variety of partner genes like CREBBP (16p13), EP300 (22q13), LEUTX (9q13), NCOA2, NCOA3, and ASXL2. Favorable prognosis and spontaneous remission in congenital AML with t (8; 16) translocation have been reported. More recently, a single case report with spontaneous remission in congenital AML with KAT6A and another partner gene EP300 t (8; 22) has been described. Here we report an additional patient with congenital myeloid sarcoma of the skin with t (8; 22) with spontaneous remission.