Statistical methods in interphase cytogenetics: An experimental approach

Abstract

In situ hybridization (ISH) techniques on interphase cells, or interphase cytogenetics, have powerful potential clinical and biological applications, such as detection of minimal residual disease, early relapse, and the study of clonal evolution and expansion in neoplasia. Much attention has been paid to issues related to ISH data acquisition, i.e., the numbers, colors, intensities, and spatial relationships of hybridization signals. The methodology concerning data analysis, which is of prime importance for clinical applications, however, is less well investigated. We have studied the latter for the detection of small monosomic and trisomic cell populations using various mixtures of human female and male cells. With a chromosome X specific probe, the male cells simulated monosomic subpopulations of 0, 1, 5, 10, 50, 90, 95, 99, and 100%. Analogously, when a (7 + Y) specific probe combination was used, containing a mixture of chromosome No. 7 and Y-specific DNA, the male cells simulated trisomic cell populations. Probes specific for chromosomes Nos. 1 , 7 , 8 , and 9 were used for estimation of ISH artiIn situ hybridisation (ISH) techniques are now widely applied in cytogenetic research and clinical diagnosis. Important features of the molecular ISH approach distinguishing it from morphological banding cytogenetic techniques are: (1) the possibility of interphase cell analysis or "interphase cytogenetics" (8); (2) unambiguous identification of marker chromosomes facts. Three statistical tests, the Kolmogorov-Smirnov test, the multiple-proportion test, and the z'-max test, were applied to the empirical data using the control data as a reference for ISH artifacts. The Kolmogorov-Smirnov test was found to be inferior for discrimination of small monosomic or trisomic cell populations. The other two tests showed that when 400 cells were evaluated, and using selected control probes, monosomy X could be detected at a frequency of 5%

Sensitive detection of tumour cells in effusions by combining cytology and fluorescence in situ hybridisation (FISH)

“…Signal counting was performed by two investigators, and intraobserver and interobserver counting variations were evaluated repeatedly. In order to evaluate the frequencies of aneusomic effusion cells with statistical reliability, centromeric signals of 100 -1000 nuclei were scored, with highnumber cell counting in samples with a low frequency of aneuploidy (Kibbelaar et al, 1993). When necessary, we used a two-tiered scoring procedure: (1) in all 358 effusions, scoring of nuclei in single-colour FISH evaluation was performed, and if aneusomy above cutoff for any of the tested chromosomes (Table 2) was present, malignancy could be documented; otherwise, in step (2), scoring of selected, namely hyperdisomic, nuclei was performed in dual-colour FISH evaluation, which allowed for the detection of rare FISH-aneuploid cells (Fiegl et al, 1999).…”

State‐of‐the‐art FISHing: Automated analysis of cytogenetic aberrations in interphase nuclei

“…For example, more than 400 cells have to be analyzed to detect a minor clone of 10% with 99% confidence if the analytical sensitivity is 95% (10). To detect minimal residual disease, circulating tumor cells or microchimerism, the number of analyzed nuclei must be raised even more to reach adequate statistical reliability (11)(12)(13), which consequently leads to a prolonged time of analysis. However, it is important to note that FISH probes inevitably lead to false positivity (FP) that may greatly influence the limit of detection.…”

Interphase fluorescence in situ hybridization improves the detection of malignant cells in effusions from breast cancer patients

“…effusion sample 22), the percentage of counted nuclei with three or more signals was always more than 1%, which is considered as the lower detection limit of FISH analysis (Kibbelaar et al, 1993). To validate specificity, all effusions with an aneuploidy rate below 5% were reviewed using two-colour FISH.…”