Autologous Bone Marrow Transplantation - Blog Science Connections

Digital Image Analysis System 435
describe here a system we have developed using a microcomputer-controlled
scanning stage on a fluorescence microscope. Intensified video signals from
the microscope are analyzed by the microcomputer to permit semiautomatic
detection of fluorescent cells in bone marrow.
MATERIALS AND METHODS
The system is based on an IBM personal microcomputer (PC AT). For
data storage the system uses a 10-megabyte Bernoulli cartridge disk drive
(Iomega, Roy, CJT) formatted with an interleaf factor of 2, which increases the
speed of data storage. Software for the system was developed in Turbo
PASCAL (Borland International, Scotts Valley, CA), using serial input-output
procedures from the Turbo Asynch Tools Package (Blaise Computing,
Berkeley, CA) to control the scanning-stage MDACE 1000 motorized stage
controller (Ludl Electronics, Scarsdale, NY) via the serial interface. We have
developed a complete set of software tools to control the scanning stage,
scan various patterns on a slide, and record the locations of cells of interest
(Lee, Ludd, Reynolds, unpublished data).
Detection capability is provided by a Dage MTI intensified silicon
intensifier tube (1SIT 66) intensified video camera (Dage-MTI, Michigan City,
IN) (18-20) on a Leitz Orthoplan Fluorescence Microscope (E. Leitz,
Rockleigh, NJ). The ISIT camera provides an RS-330 video signal to a PC
Vision real-time digitizer (Imaging Technology, Woburn, MA) located in the
IBM PC AT. The PC vision board digitizes an image of 512 x 480 pixels with
256 gray levels at a rate of 30 frames per second (21). The aspect ratio of the
512 x 480 pixel image matches that of a standard video image.
Cell preparations consisted of Hoechst 33342 (H342) premarked
leukemia cells (MOLT-3, from American Type Culture Collection, Rockville,
MD) (22) seeded into human vertebral-body marrow as previously described
(23). To provide optimal detection of the H342-marked cells, the microscope
was equipped with a 100-watt mercury lamp and an ultraviolet excitation (350
nm)/blue emission (460 nm) "D" cube. Cytospins were prepared by using
centrifugal cytology buckets (CCB) (International Equipment, Needham
Heights, MA) to centrifuge marrow/leukemia mixtures onto poly-L-lysinecoated
microscope slides at 200 x g for 10 minutes (24,25). Slides were
prepared by covering one surface with greater than 100,000 molecular
weight poly-L-Iysine (Sigma Chemical, St. Louis, MO) (1 mg/ml in 0.1 M
potassium-phosphate buffer, pH 7.0) and allowing the solution to dry (26).
The CCBs were modified by placing a silicon rubber gasket between the slide
and the cell chamber, defining a cell-preparation area of 13 mm x 15 mm.
Marrow/leukemia mixtures were adjusted so that the correct number of cells
for a well (usually 2 million) was suspended in 0.4 ml of medium (Iscove's
Dulbecco's modified Eagle medium +1056 fetal bovine serum), which was
then pipetted into the well. The pipette was rinsed and the rinse (0.1 ml)