Autologous Bone Marrow Transplantation - Blog Science Connections

438 Digital Image Analysis System
Table 2. Reproducibility of Detecting Cells
With Digital Image Analysis System
T r
'al
Cells Detected
1 91
2 89
3 90
4 88
quite good; we attribute the decrease in detected cells in later scans to
quenching of the H342 stain owing to repeated analysis.
To determine the sensitivity of the system, we seeded human bone
marrow with H342-marked leukemia cells at concentrations of 50,10, and 1
target cell per million marrow cells. Figure 3 shows the number of H342-
stained leukemia cells detected in marrow at various seeding concentrations;
each bar represents analysis of one cytopreparation (for one target cell per
million, 2 million cells per preparation were scanned). Thus, detection of
seeded cells with the system was reliable down to one target cell per million
marrow cells.
DISCUSSION
Now that relatively specific antibodies for detecting malignant cells in
bone marrow are becoming available, assay systems that maximize the ability
of the antibodies to detect a given target cell are needed. Sensitive detection
of infrequent leukemia or tumor cells in blood or marrow using flow
cytometry has been reported (8,11-13). However, flow cytometry is less than
ideal for detecting infrequent malignant cells, because target cells found in
clumps (especially common in solid tumors) are excluded from analysis
owing to the narrow aperture of the flow chamber. Slide-based detection
assays (which do not exclude tumor clumps) of cells stained for tumorassociated
antigens using immunofluorescence or immunoperoxidase have
demonstrated sensitivities from 1/10,000 to 1/100,000, but require laborintensive
manual scanning of the slides (Table 1). To overcome this
limitation of slide-based assays, we have developed a semiautomated system
for scanning slide preparations for infrequent cells. To use the system
efficiently, we employ a modified centrifugal cytology method that allows
highly efficient depositing of 2 million cells into a preparation area ideally
suited for "rare event" scanning.
The ability of the system to "replay" positive events for user confirmation
can be exploited in a variety of ways. In our model-system studies, we used the
replay to exclude false fluorescent signals based on the morphology of the
H342-stained cells. In using the system with immunostained cell preparations,
one could also use a second color of fluorescence or differing antigen
locations (cell surface vs. nuclear or cytoplasmic). Simultaneous two-color