11 Specimen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
155
With fresh blood smears (up to two weeks) startat step 3. For older blood Blood smears
smears, extensive rehydration (steps 1 and 2) is required.
Procedure
1. Incubate stained blood smear in PBS overnight at 37°C.
2. Repeat step 1 using TE pH 7.5.
3. Incubate in methanol/acetic acid (19:1) for 10 min at room temperature
(first fixation step ).
4. Repeat with methanol/acetic acid (3:1) for 10 min at room temperature
(second fixation step).
5. Dehydrate in a series of 70o/o, 90o/o, 100o/o ethanol, 5 min each, at room
temperature.
6. Proceed with fluorescence in situ hybridization protocol (see Chap. 12).
Fig. 1. Fluorescence in situ hybridization on blood smears. Photomicrographs ofhybridization signals obtained with an alphoid probe specific for chromosome 8 (A-D, F, G); E dual
colour hybridization. A Hybridization of a chromosome 8 specific alphoid probe to a metaphase spread of a leukemia patient with trisomy 8: three signals are seenbothin the metaphase
andin the interphase nucleus; B Hybridization to a blood smear of a pro band: note the weil
preserved morphological features of the segmented cells. The hybridization signals are indicated by arrows; C Hybridization to a blood smear of a leukemia patient with trisomy
8: three signals are seen in all nuclei; D Nuclei on a blood smear of a patient with acute myeloid
leukemia with trisomy 8 and a subclone with tetrasomy 8 after in situ hybridization: all cells
belong to the subclone with tetrasomy 8; E Metaphase of a patient with myeloid leukemia, who
had a small percentage of trisomy 8 by interphase cytogenetics and a marker chromosome.
This experimentwas performed to address the question, if the marker chromosome contains
material of chromosome 8. Hybridization was done with a chromosome 8 specific plasmid
library (detected via rhodamine, red signals) and a chromesome 17 specific alphoid probe
(detected with FITC). Only the two #8 homologs are stained by the DNA library; F, G Consecutive morphological (F) and cytogenetic (G) assessment of a blood smear of a patient with
myeloid leukemia and trisomy 8: in all myeloid cells, three signals can be detected. In the
lymphocyte, only two hybridization signals were seen, which are in a different focal plain.
(Reprinted from Benzet al. 1993, Macmillan Press Ltd.)
155
With fresh blood smears (up to two weeks) startat step 3. For older blood Blood smears
smears, extensive rehydration (steps 1 and 2) is required.
Procedure
1. Incubate stained blood smear in PBS overnight at 37°C.
2. Repeat step 1 using TE pH 7.5.
3. Incubate in methanol/acetic acid (19:1) for 10 min at room temperature
(first fixation step ).
4. Repeat with methanol/acetic acid (3:1) for 10 min at room temperature
(second fixation step).
5. Dehydrate in a series of 70o/o, 90o/o, 100o/o ethanol, 5 min each, at room
temperature.
6. Proceed with fluorescence in situ hybridization protocol (see Chap. 12).
Fig. 1. Fluorescence in situ hybridization on blood smears. Photomicrographs ofhybridization signals obtained with an alphoid probe specific for chromosome 8 (A-D, F, G); E dual
colour hybridization. A Hybridization of a chromosome 8 specific alphoid probe to a metaphase spread of a leukemia patient with trisomy 8: three signals are seenbothin the metaphase
andin the interphase nucleus; B Hybridization to a blood smear of a pro band: note the weil
preserved morphological features of the segmented cells. The hybridization signals are indicated by arrows; C Hybridization to a blood smear of a leukemia patient with trisomy
8: three signals are seen in all nuclei; D Nuclei on a blood smear of a patient with acute myeloid
leukemia with trisomy 8 and a subclone with tetrasomy 8 after in situ hybridization: all cells
belong to the subclone with tetrasomy 8; E Metaphase of a patient with myeloid leukemia, who
had a small percentage of trisomy 8 by interphase cytogenetics and a marker chromosome.
This experimentwas performed to address the question, if the marker chromosome contains
material of chromosome 8. Hybridization was done with a chromosome 8 specific plasmid
library (detected via rhodamine, red signals) and a chromesome 17 specific alphoid probe
(detected with FITC). Only the two #8 homologs are stained by the DNA library; F, G Consecutive morphological (F) and cytogenetic (G) assessment of a blood smear of a patient with
myeloid leukemia and trisomy 8: in all myeloid cells, three signals can be detected. In the
lymphocyte, only two hybridization signals were seen, which are in a different focal plain.
(Reprinted from Benzet al. 1993, Macmillan Press Ltd.)