Enumeration of interphase chromosomes. Comparison of visual in situ hybridization and confocal fluorescence in situ hybridization.
OBJECTIVE
To compare two visual enumeration methods to determine whether the confocal approach yielded better counts of chromosome-specific hybridization sites.


METHODS
Brightfield microscopy was used to count in situ hybridization (ISH) sites in 4-microns tissue sections. Confocal microscopy was used to collect three-dimensional (3D) data sets from fluorescence in situ hybridization (FISH) preparations made with sections of various thicknesses. Analysis of the confocal images relied on custom-built interactive visualization software.


RESULTS
The confocal method yielded higher average counts of hybridization sites per nucleus due to fewer truncated nuclei in thicker sections and to visual exclusion of the truncated nuclei that remained. Optimal section thickness was 8-12 microns. Limited penetration by FISH reagents restricted the use of thicker sections.


CONCLUSIONS
Analysis of intact nuclei visualized in three dimensions was more sensitive in demonstrating high centromere number than was brightfield ISH analysis of 4-microns sections. Improvements in semiautomated interactive software may make the confocal approach practical for accurate evaluation of chromosome number in precise histologic contexts.
Department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, DC 20306-6000, USA.
R L
RL
Becker
U V
UV
Mikel
W R
WR
Oliver
I A
IA
Sesterhenn
D002295:Carcinoma, Transitional Cell / Q000737:chemistry; D002503:Centromere / Q000737:chemistry; D002875:Chromosomes / Q000648:ultrastructure*; D002886:Chromosomes, Human, Pair 17 / Q000737:chemistry; D006652:Histological Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007399:Interphase; D018613:Microscopy, Confocal; D011419:Propidium / Q000032:analysis; D001749:Urinary Bladder Neoplasms / Q000737:chemistry
D011419:Propidium