8 Amniotic Fluid Cells - Uncultured
117
chromosomes 13 and 21, labeled a-satellite centromere-specific DNAprobes for chromosomes 18, X and Y and labeled unique DNA probes
for chromosomes 13 (hybridizing to 13qI4) and 21 (hybridizing to 21
q22.13-21q22.2) are now commercially available as a kit (AneuVysion
EC by Vysis GmbH, 51429 Bergisch Gladbach, Germany).
The first application of these new DNA-probes was published by Eiben
et aI. (1998). In this prospective comparative study no false-negative or
false-positive results were obtained in 91 % (798/904) successfully analyzed
cases, with a minimum of 50 nuclei counted for each probe. In contrast to
Bryndorf (1997), who reported 19% of cases with less than 50 scorable
nuclei, this value dropped in the study of Eiben to 8%.
In our own study (Ulmer et aI., 2000) in 86% ofcases, 50 nuclei could be
scored for each probe. However, only 12/230 cases were classified as not
informative (less than 15 analyzable nuclei). More than 30 scorable nuclei
were obtained for the probes X,Y and 18 in 94.7% and for the probes 13
and 21 in 90% of the cases.
According to the recipe, which is included in the AneuVysion EC-Kit of
Vysis together with the experience we had with FISH in our institute, we
tried to find a procedure which allows the preparations to be done in parallel to other work in a prenatal diagnostic laboratory. It should be possible with minimal technical equipment, to accomplish this in a short time,
with a high success-rate and accurate results.
The procedure for FISH on uncultivated amniotic fluid cells will now
be described as it has been developed in our laboratory with the equipment available there. Consequently, there are several steps which can be
varied or adapted to the available conditions in other laboratories.
Outline
There are four main steps from the native amniotic fluid to the slide which
is ready for fluorescence microscopy:
1. Preparation of cells: from amniotic fluid to a slide with swollen and
well-fixed nuclei.
2. Slide pretreatment: in order to increase the accessibility of the probeDNA to the target sequences. This step normally includes a protease
and an RNase step. The RNase can be omitted; duration of the pepsintreatment depends on the number of nuclei (and thus is dependent on
the week of gestation in which the amniocentesis was done) on the
slide.
117
chromosomes 13 and 21, labeled a-satellite centromere-specific DNAprobes for chromosomes 18, X and Y and labeled unique DNA probes
for chromosomes 13 (hybridizing to 13qI4) and 21 (hybridizing to 21
q22.13-21q22.2) are now commercially available as a kit (AneuVysion
EC by Vysis GmbH, 51429 Bergisch Gladbach, Germany).
The first application of these new DNA-probes was published by Eiben
et aI. (1998). In this prospective comparative study no false-negative or
false-positive results were obtained in 91 % (798/904) successfully analyzed
cases, with a minimum of 50 nuclei counted for each probe. In contrast to
Bryndorf (1997), who reported 19% of cases with less than 50 scorable
nuclei, this value dropped in the study of Eiben to 8%.
In our own study (Ulmer et aI., 2000) in 86% ofcases, 50 nuclei could be
scored for each probe. However, only 12/230 cases were classified as not
informative (less than 15 analyzable nuclei). More than 30 scorable nuclei
were obtained for the probes X,Y and 18 in 94.7% and for the probes 13
and 21 in 90% of the cases.
According to the recipe, which is included in the AneuVysion EC-Kit of
Vysis together with the experience we had with FISH in our institute, we
tried to find a procedure which allows the preparations to be done in parallel to other work in a prenatal diagnostic laboratory. It should be possible with minimal technical equipment, to accomplish this in a short time,
with a high success-rate and accurate results.
The procedure for FISH on uncultivated amniotic fluid cells will now
be described as it has been developed in our laboratory with the equipment available there. Consequently, there are several steps which can be
varied or adapted to the available conditions in other laboratories.
Outline
There are four main steps from the native amniotic fluid to the slide which
is ready for fluorescence microscopy:
1. Preparation of cells: from amniotic fluid to a slide with swollen and
well-fixed nuclei.
2. Slide pretreatment: in order to increase the accessibility of the probeDNA to the target sequences. This step normally includes a protease
and an RNase step. The RNase can be omitted; duration of the pepsintreatment depends on the number of nuclei (and thus is dependent on
the week of gestation in which the amniocentesis was done) on the
slide.