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Rare-Event Analysis and Sorting

Molecular Cytometry Unit
Division of Infectious Diseases
Department of Internal Medicine
University of Texas Medical Branch
Galveston, Texas 77555-08412 

Director: James F. Leary, Ph.D.

1. Facilities: The Molecular Cytometry Unit consists of 6 laboratories in an 11-room complex on the seventh floor of the Sealy & Smith Professional Building on the University of Texas Medical Branch campus in Galveston, Texas: (1) Human Gene Probe/Gene Transfer Laboratory for the production and analysis of human sequence-specific DNA and RNA probes, containing Perkin Elmer In-Situ PCR System 1000 and System 2400 thermal cyclers for PCR (polymerase chain reaction), and FISH (fluorescence in-situ hybridization) preparation areas; (2) Cell Culture/Micromanipulation Laboratory, containing tissue culture facilities, a Nikon Diaphot inverted fluorescence microscope with a Narashige micromanipulator/microinjection system for manipulation of single cells, chromosome microdissection, and gene transfers; (3) Software Development/Biocomputing Laboratory, where flow and image cytometry software is written in C/C++ object-oriented programming under Windows and NT environments and a variety of molecular biology software is available; (4) Instrument Prototyping/CAD (Computer-Aided Design) Laboratory, for designing, building, and testing new instrumentation; (5) Fluorescence Microscopy/Image Analysis Laboratory, including a Nikon upright Optiphot with cameras, and with image analysis hardware and software under construction; and (6) Flow Cytometry and Cell Sorting Laboratory, containing the high-speed cell sorter and associated computers for basic and clinical research applications including (a) human breast cancer, (b) AIDS research, (c) human stem cells, and (d) prena

2. Instrumentation: The flow cytometer/cell sorter consists of a high-speed, multiparameter home-built instrument and system of Pentium and 80486 personal computers (PC) with extensive software. Laser excitations presently consist of a dual argon-ion laser/dye laser system (a 5W UV-enhanced, Spectra/Physics Model 2025 argon-ion laser and a 300mW air-cooled Ion Laser Technology Model 5500AWC argon-ion laser). Special capabilities include time-of-flight sizing systems capable of sizing cells as small as 0.3 microns in length or diameter, and a high-speed "rare-event" system capable of analyzing cells at rates in excess of 100,000 cells per second, allowing analysis of rare cell subpopulations as small as 10-7 frequency. A sophisticated Windows data acquisition system on a PC allows for acquisition of 8-parameter, 12-bit FCS-standard listmode data at rates in excess of 10,000 cells per second. The HiReCS system (High Resolution Cell Sorter) also allows for visualization, analysis, and sorting on the basis of up to 6 colors of fluorescence and 3 principal components. Correlations between flow cytometric parameters can be explored through the use of principal component/biplot analyses. Unique patented "flexible sorting" strategies can be used to optimize yield or purity. The file-server running Windows NT is a 100 Mhz Pentium with data analysis system capable of time-sharing multiple users, with 32 Mb memory, 2 Gb disk storage, and 250 Mb (QIC-80) tape backup, and runs a sophisticated package of home-developed multiparameter data analysis software. It also ru statistical packages with conversion of FCS-standard data to/from "S-Plus" for general statistical analyses, and "CART (Classification and Regression Tree)" binary-tree analysis of flow cytometric and patient data. Commercial software includes WINLIST, MODFIT, and ISOCONTOUR (Verity Software House), cluster analysis and display of complex multiparameter data by two PC-based programs, CLUSTER, and BIVARPLT (Software Resource Company). Any 3 parameters including raw and principal components parameters of multidimensional data can be visualized using a special PC-based autostereoscopic display terminal and the facility's own unique 3-dimensional software, PCSTEREO, which can also produce stereo-pairs on a conventional display system. A 3-D mouse is used to address regions of this data space for direct reprocessing of listmode files or as "seed points" for data clusters for "guided" cluster analysis. This system is also being used to provide 3-dimensional visualization and spatial mapping, using the 3-D mouse and special home-built software, of confocal microscopic images of cells and chromosomes labeled by FISH. Another in-house program, CELTAG, allows modeling of FCS-standard listmode data mixtures for predicting the limits of cell-sorting strategies for rare cells. Additional resources within the Division in the neighboring laboratory of Dr. Norman Roberts are a Beckman Model J-6M centrifugal elutriator for cell subpopulation enrichment, and a Becton Dickinson FACSort flow cytometer/cell sorter and associated MacIntosh Quadra 650 running CellQuest.

3. Specialization Areas: This facility has for the past 15 years specialized in all aspects of cell preparation, immunocytochemistry, advanced instrumentation and data analysis software for detection, analysis and sorting, and molecular characterization of rare cell subpopulations.

4. Special Opportunities: A number of training opportunities exist. Data files in FCS format can be downloaded to UTMB from interested researchers for analysis. If the special instrumentation at UTMB is needed, arrangements can be made to bring experiments to Galveston. More general training in rare events analysis and sorting techniques will be provided for small groups.

5. Special Courses Offered: There will be specialized courses in different areas of rare cell detection, analysis, and isolation which will be advertised to the community.

6. Graduate Program: Through the Pathology Department Dr. Leary offers a full graduate-level semester course in molecular cytometry.

7. Local Accommodations: A number of hotels are located within a 10 minute drive of the University campus, including some on the beach and others in the historical district. Marchi Travel Agency (409-762-2612) can assist.

8. Transportation: Houston Hobby Airport is 41 miles northwest of Galveston Island; Houston Intercontinental Airport is 68 miles northwest of Galveston. Interstate 45 goes from Houston two miles out into the Gulf of Mexico on causeways to Galveston Island. Route 45 then becomes Broadway and if taken to 6th Street leads directly to the Sealy & Smith Professional Building, where parking is always available.

9. Cost of Internship: Cost of internships is variable depending on the need for resources and time. Group course costs will be stated in the particular course announcements.

10. Communication:

James F. Leary, Ph.D., Professor of Internal Medicine
Director, Molecular Cytometry Unit - Route 0841
Sealy & Smith Professional Bldg, Suite 724
University of Texas Medical Branch
Galveston, Texas 77555-0841

Phone: (409) 747-1930
FAX: (409) 772-6527
E-mail: jleary@beach.utmb.edu