CBER Research Projects

Project Title

Flow and Image Cytometry in B-CLL

Principal Investigator

Gerald E. Marti

Laboratory

Laboratory of Stem Cell Biology; Division of Cellular and Gene Therapy; Office of Therapeutics Research and Review

Project Summary

The Section of Flow & 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 flow cytometry laboratory consists of two FACScans and a FACStar Plus, all of which are connected with ethernet using TCP/IP. There are three other FACScan and two FACSTAR flow cytometers within CBER; they all have some level of network capability. Together these laboratories make up the CBER/FDA flow cytometry consortium. 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 as a function of time. A linkage analysis in an F1 backcross has been completed. A protocol of loss of heterozygosity, comparitive genomic hybridization and linkage analysis is being prepared to develop a genetic model.

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 selection 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.

Publications

• Am J Hum Genet 1999 Jul;65(1):265-9
  Anticipation in familial chronic lymphocytic leukemia.
  Goldin LR, Sgambati M, Marti GE, Fontaine L, Ishibe N, Caporaso N
  Pub Med

• Blood 2000 Feb 15;95(4):1413-1419
  Analysis of expressed immunoglobulin heavy chain genes in familial B-CLL.
  Sakai A, Marti GE, Caporaso N, Pittaluga S, Touchman JW, Fend F, Raffeld M
  Pub Med

• Cold Spring Harb Sym 64: 71-78 1999
  The lymphochip: A specialized cDNA microarray for the genomic-scale analysis of gene expression in normal and malignant lymphocytes.
  Alizadeh A, Eisen M, Davis RE, Ma C, Sabet H, Tran T, Powell JI, Yang L, Marti GE, Moore DT, Hudson JR, Chan WC, Greiner T, Weisenburger D, Armitage JO, Lossos I, Levy R, Botstein D, Brown PO, Staudt LM
  Pub Med

• Nature 2000 Feb 3;403(6769):503-11
  Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.
  Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti GE, Moore T, Hudson J Jr, Lu L, Lewis DB, Tibshirani R, Sherlock G, Chan WC, Greiner TC, Weisenburger DD, Armitage JO, Warnke R, Staudt LM, et al
  Pub Med

• Leuk Lymphoma 2000 Jan;36(3-4):353-65
  CD5 is A potential selecting ligand for B-cell surface immunoglobulin: a possible role in maintenance and selective expansion of normal and malignant B cells.
  Pospisil R, Silverman GJ, Marti GE, Aruffo A, Bowen MA, Mage RG
  Pub Med

• Cytometry 1998 Oct 1;33(2):188-96
  Performance of calibration standards for antigen quantitation with flow cytometry.
  Lenkei R, Gratama JW, Rothe G, Schmitz G, D'hautcourt JL, Arekrans A, Mandy F, Marti G
  Pub Med

• Cytometry 1998 Jun 15;34(3):128-42
  Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis.
  Gratama JW, Orfao A, Barnett D, Brando B, Huber A, Janossy G, Johnsen HE, Keeney M, Marti GE, Preijers F, Rothe G, Serke S, Sutherland DR, Van der Schoot CE, Schmitz G, Papa S
  Pub Med

• Cytometry 1998 Oct 1;33(2):106-14
  Standardizing flow cytometry: a classification system of fluorescence standards used for flow cytometry.
  Schwartz A, Marti GE, Poon R, Gratama JW, Fernandez-Repollet E
  Pub Med

Last Updated: 4/1/2002

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Date created: September 25, 2003

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