Chapter 6
Epithelial Cells from Buccal Smears and Urine
ANITA RAUCH
Introduction
Aneuploidy is usually investigated by karyotyping lymphocytes from peripheral blood. However, in several aneuploidy syndromes, tissue mosaicism is frequently found. In Pallister-Killian syndrome, for example, the
supernumerary isochromosome 12p is usually not seen in blood lymphocytes, although it occurs in up to 100% of fibroblasts. Therefore it is necessary to karyotype other tissues than blood. Traditionally, fibroblasts
from a skin biopsy were used to analyze suspected mosaic karyotypes.
However, this rather invasive procedure is often refused by patients.
Therefore, interphase FISH on cells from buccal smears (Ohashi et al.
1993) or urinary sediment provides a useful noninvasive alternative method to analyze numerical chromosomal aberrations, if tissue mosaicism is
suspected.
In order to achieve a high hybridization efficiency, which also allows
accurate analysis even of low percentage mosaicism by FISH with single
copy probes, we recommend pretreatment of cells with hypotonic solution, RNase and pepsin. For hypotonic treatment we are using a solution
containing dithioerythritol, which was used by Otto to investigate Chinese
hamster cell lines by flow karyotyping (Otto 1988). In addition, application
of directly labeled probes reduces background fluorescence, which is especially disturbing if caused by bacteria in a signal imitating manner.
~ Anita Rauch, Institut fUr Humangenetik, Schwabachanlage 10, Erlangen, 91054,
Germany (phone +49-9131-8522318; fax +49-9131-209297;
e-mail arauch@humgenet.uni-erlangen.de)
PROTOCOL
Epithelial Cells from Buccal Smears and Urine
ANITA RAUCH
Introduction
Aneuploidy is usually investigated by karyotyping lymphocytes from peripheral blood. However, in several aneuploidy syndromes, tissue mosaicism is frequently found. In Pallister-Killian syndrome, for example, the
supernumerary isochromosome 12p is usually not seen in blood lymphocytes, although it occurs in up to 100% of fibroblasts. Therefore it is necessary to karyotype other tissues than blood. Traditionally, fibroblasts
from a skin biopsy were used to analyze suspected mosaic karyotypes.
However, this rather invasive procedure is often refused by patients.
Therefore, interphase FISH on cells from buccal smears (Ohashi et al.
1993) or urinary sediment provides a useful noninvasive alternative method to analyze numerical chromosomal aberrations, if tissue mosaicism is
suspected.
In order to achieve a high hybridization efficiency, which also allows
accurate analysis even of low percentage mosaicism by FISH with single
copy probes, we recommend pretreatment of cells with hypotonic solution, RNase and pepsin. For hypotonic treatment we are using a solution
containing dithioerythritol, which was used by Otto to investigate Chinese
hamster cell lines by flow karyotyping (Otto 1988). In addition, application
of directly labeled probes reduces background fluorescence, which is especially disturbing if caused by bacteria in a signal imitating manner.
~ Anita Rauch, Institut fUr Humangenetik, Schwabachanlage 10, Erlangen, 91054,
Germany (phone +49-9131-8522318; fax +49-9131-209297;
e-mail arauch@humgenet.uni-erlangen.de)
PROTOCOL