The Section of Flow and Image Cytometry is concerned with quantitative flow cytometry as it relates to our regulatory needs. An interagency working group (FDA, NIH, CDC, NIST) has been formed and is working on a flouresence intensity standards initiative. The next area of data analysis in flow cytometry will consist of a comparison of cluster analysis programs. The major program concerns the etiology and pathogenesis of common and familial B cell lymphocytic leukemia (B-CLL). This has consisted of an evaluation of two and three color surface immunofluorescence, cell cycle analysis and sorting of B cells for PCR analysis of monoclonality. This latter activity is being undertaken as part of a linkage analysis study in familial B-CLL. It is designed to detect early preclinical CLL in third and fourth generation family members to facilitate the formal genetic linkage analysis. Presently, we use magnetic beads and/or sorting to enrich B cells. Oligomeric primers for V-region families (VH1 through VH6) are used for clonal evaluation. An NZB mouse colony has been established to evaluate a murine model of B-CLL. Image analysis is being carried out to examine the morphological heterogeneity of lymphocytes seen on conventionally prepared blood films using albumin. A linkage analysis in an F1 backcross has been completed. Our work in flow cytometry has permitted us to develop an assay for the earliest detection of BCLL and we have called this a B cell monoclonal lymphocytosis (BCML). It appears to be increased in the non affected members of families with BCLL. In addition we have been able to split BCLL lyphocytes into a diploid GoG1 and aneuploid peak using DAPI and an UV based flow cytometer. In collaboration with Akira Sakai in Mark Raffeld's laboratory we have also examined the Ig gene sequence of affected family members with BCLL. They are equally devided between germ line and somatically mutated forms. The germline configuration may be more often associated with the leukemic form while the somatically mutated forms may have a more lymphomatous clinical course. The level of CD5 expression seemed to also correlate with the leukemic form. In collaboration with Richaard Popisil in Rose Mage's laboratory we have been able to show that one of the ligands for CD5 may be the heavy chain of Ig and binding is mediated by the VH framework region. Such an interaction is believe to be able to regulate selction and expansion of a transformed B cell. Finally we have have collaborated with Louis Staudt and colleagues in the flow cytometric sorting of tonsil B cells for the formation of a cDNA libray used to make a cDNA microarray. We are now trying to apply the single cell pcr methodolgy to an analysis of BCML.
