11 Specimen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
163
Comments
The quality of the hybridization results depends on several parameters such
as the efficiency of the permeabilization procedure (see above). For practical reasons, it is important to consider the potential of various types of
probes on the different materials described in this chapter. The intensity
of hybridization signals clearly correlates with the size of the targeted sequences ( except when the target sequences are dispersed, as in the case of
chromosome painting probes). Formost applications, especially with regard to diagnostic settings, > BOo/o of the targets need to be labeled.
With metaphase chromosome spreads, suspended cells, and blood cells
or bone marrow smears such efficiencies can be achieved with probes of
> 10-50 kb (e.g. cosmid clones). However, in tissue sections, in particular
in paraffin-embedded material, the chromosomal target region has tobe
larger. Thus, alphoid or satellite probes recognizing tandemly repeated sequences and, therefore, resulting in strong hybridization signals are useful
tools for this application. However, although such probes are very powerful
for the diagnosis of numerical chromosomal changes, their ability to detect
structural aberrations, such as translocations, deletions, inversions and
duplications, is very limited. For this purpose local probes which result
in strong hybridization signals are needed. Such probes may be provided
bycontigs of cosmids and/oryeast artificial chromosomes (YACs) which will
become increasingly available in the course ofthe Human Genome Project.
References
Anastasi J, Le Beau MM, Yardiman JW, Fernald AA, Larson RA, Rowley JD (1992) Detection of trisomy 12 in chronic lymphocytic leukemia by flourescence in situ hybridization to interphase cells: A simple and sensitive method. Blood 79: 1796-1801
Anastasi J, Yardiman JW, Rudinsky R, Patel M, Nachman J, Rubin CM, Leßeau MM
(1991) Direct correlation of cytogenetic findings with cell morphology using in
situ hybridization: an analysis of suspicious cells in bone marrow specimens of
two patients completing therapy for acute lymphoblastic leukemia. Blood 77:
2456-2462
Arnoldus EPJ, Noordermeer IA, Peters ACB, Raap AK, van der Ploeg M (1991) Interphase cytogenetics reveals somatic pairing of chromosome 17 centromeres in normal
human brain tissue, but no trisomy 7 or sex chromosome loss. Cytogenet Cell Genet
56: 214-216
Bentz M, Döhner H, Sehröder M, Pohl S, Lichter P (1993a) Detection of chromosomal
abnormalities on previously stained peripheral blood and bone marrow smears. In:
Büchner T, Hiddemann W, Wörmann B (ed) Acute leukemias IV: Prognostic factors
and treatment strategies. Springer, Heidelberg
163
Comments
The quality of the hybridization results depends on several parameters such
as the efficiency of the permeabilization procedure (see above). For practical reasons, it is important to consider the potential of various types of
probes on the different materials described in this chapter. The intensity
of hybridization signals clearly correlates with the size of the targeted sequences ( except when the target sequences are dispersed, as in the case of
chromosome painting probes). Formost applications, especially with regard to diagnostic settings, > BOo/o of the targets need to be labeled.
With metaphase chromosome spreads, suspended cells, and blood cells
or bone marrow smears such efficiencies can be achieved with probes of
> 10-50 kb (e.g. cosmid clones). However, in tissue sections, in particular
in paraffin-embedded material, the chromosomal target region has tobe
larger. Thus, alphoid or satellite probes recognizing tandemly repeated sequences and, therefore, resulting in strong hybridization signals are useful
tools for this application. However, although such probes are very powerful
for the diagnosis of numerical chromosomal changes, their ability to detect
structural aberrations, such as translocations, deletions, inversions and
duplications, is very limited. For this purpose local probes which result
in strong hybridization signals are needed. Such probes may be provided
bycontigs of cosmids and/oryeast artificial chromosomes (YACs) which will
become increasingly available in the course ofthe Human Genome Project.
References
Anastasi J, Le Beau MM, Yardiman JW, Fernald AA, Larson RA, Rowley JD (1992) Detection of trisomy 12 in chronic lymphocytic leukemia by flourescence in situ hybridization to interphase cells: A simple and sensitive method. Blood 79: 1796-1801
Anastasi J, Yardiman JW, Rudinsky R, Patel M, Nachman J, Rubin CM, Leßeau MM
(1991) Direct correlation of cytogenetic findings with cell morphology using in
situ hybridization: an analysis of suspicious cells in bone marrow specimens of
two patients completing therapy for acute lymphoblastic leukemia. Blood 77:
2456-2462
Arnoldus EPJ, Noordermeer IA, Peters ACB, Raap AK, van der Ploeg M (1991) Interphase cytogenetics reveals somatic pairing of chromosome 17 centromeres in normal
human brain tissue, but no trisomy 7 or sex chromosome loss. Cytogenet Cell Genet
56: 214-216
Bentz M, Döhner H, Sehröder M, Pohl S, Lichter P (1993a) Detection of chromosomal
abnormalities on previously stained peripheral blood and bone marrow smears. In:
Büchner T, Hiddemann W, Wörmann B (ed) Acute leukemias IV: Prognostic factors
and treatment strategies. Springer, Heidelberg