Autologous Bone Marrow Transplantation - Blog Science Connections

Digital Image Analysis System 437
control of the operator, who can observe the positive cell in the microscope to
either confirm the event or exclude it as a false positive. When the operator
enters his decision on the keyboard, the computer moves to the next positive
event, allowing rapid user confirmation of the positive events. Computercontrolled
scanning of a single cytopreparation requires 24 minutes, and
semiautomatic playback for visual confirmation by the user averages 5
minutes.
In order to provide an efficient detection system, a method of placing a
large number of cells on a slide for analysis was required. We found that if we
used poly-L-lysine-coated slides, the CCBs could deposit efficiently up to 2
million cells per preparation area (the buckets provide three rectangular
cytospins per slide, 13 mm x 15 mm each) (Fig 2). Careful observation of the
supernatant from the CCBs aspirated after depositing cells failed to reveal any
cells. As a further test, H342-marked MOLT-3 cells were seeded into marrow
at a concentration of 10 cells per million, and then 0.5,1,2, and 3 million total
cells per preparation were deposited using the CCBs. We found the
deposition of the leukemia "tracer" cells to be linear until greater than 2
million cells were deposited. Thus, if one limits each preparation to 2 million
cells or less, the number of cells loaded into the CCB is effectively the number
of cells analyzed. Also, cytospins of 2 million cells or less provide a good
distribution of cells on the slide for analysis, avoiding cell stacking.
To determine the reproducibility of the system in scanning slides, a
cytopreparation of 1 million cells seeded with 100 fluorescent cells was
scanned repeatedly. As shown in Table 2, the reproducibility of the system is
MARROW
POLY—L—LYSINE
COATED SLIDE
2 MILLION
CELLS
EACH WELL
3 WELLS
PER SLIDE
EACH PORTION
OF PREP AREA
IS SCANNED
BY THE
COMPUTER
Figure 2. Use of centrifugal cytology system to prepare marrow specimen for rare-event
analysis.