Title: FISH 'n Chips: Automation of Fluorescent Dot Counting in Interphase Cell
Nuclei
Authors: H. Netten, I.T. Young, L.J.
van Vliet, H.J. Tanke, H. Vrolijk, and W.C.R. Sloos
in: Cytometry, vol. 28, no. 1, 1997, 1-10.
Abstract
Fluorescence in situ hybridization allows the enumeration of chromosomal
abnormalities in interphase cell nuclei. This process is called dot counting.
To estimate the distribution of chromosomes per cell a large number of
cells have to be analyzed, especially when the frequency of aberrant cells
is low. Automation of dot counting is required because manual counting
is tedious, fatiguing, and time consuming.
We have developed a completely automated fluorescence microscope system
that can examine 500 cells in approximately 15 minutes to determine the
number of labeled chromosomes (seen as dots) in each cell nucleus. This
system works with two fluorescent dyes ? one for the DNA hybridization
dots and one for the cell nucleus. After the stage has moved to a new field
the image is automatically focused, acquired by a Photometrics KAF 1400
camera, and then analyzed on a Macintosh Quadra 840AV computer. After the
required number of cells has been analyzed, the user may interact to correct
the computer by working with a gallery of the cell images.
The automated dot counter has been tested on a number of normal specimens
where DAPI was used for the nucleus counter stain and a centromeric 8 probe
was used to mark the desired chromosome. The slides contained lymphocytes
from cultured blood. We have compared the results of the dot counter with
manual counting. Manual obtained results, published in literature, has
been used as the "ground truth". For a normal specimen, 97.5% of the cells
will have two dots. Fully automated scanning of 13 slides showed that an
average of 89% of all nuclei are counted correctly. In other words, an
average of 11% has to be interactively corrected, using a monitor display.
The machine accuracies, after interactive correction, are comparable to
panels of human experts (manual). The fully automatically obtained results
are biased with respect to manual counting. An error analysis is given
and different causes are discussed. View pdf file (121
kB): Copyright
1997 Wiley-Liss Inc.
Return to the homepage
of Lucas J. van Vliet
|
