Full Karyotype Interphase Cell Analysis

Abstract

Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphases simultaneously, especially in single cells. Here, we present the selection of human probe DNA and a technique using multistep fluorescence in situ hybridization (FISH) employing four sets of six labeled FISH probes able to delineate all 24 human chromosomes in interphase cells. This full karyotype analysis approach will provide additional diagnostic potential for single cell analysis. The use of spectral imaging (SIm) has enabled the use of up to eight different fluorochrome labels simultaneously. Thus, scoring can be easily assessed by visual inspection, because SIm permits computer-assigned and distinguishable pseudo-colors to each probe during image processing. This enables full karyotype analysis by FISH of single-cell interphase nuclei.

Keywords: DNA probes; FISH; aneuploidy; chromosome enumeration; full karyotype analysis; interphase nuclei; spectral imaging.

Figure 1.

(A) FISH probe set I (see Table 2) hybridized on one metaphase spread from normal male lymphocyte and (B) one interphase nucleus. Computer-assigned pseudo-colors can be seen showing two copies each of chromosomes 13, 14, 16, 20, 21, and 22. (C) FISH probe set II (see Table 2) hybridized on one metaphase spread from normal male lymphocyte and (D) one interphase nucleus. It showed two copies each of chromosomes 15, 17, 18, 19, and one copy of chromosome X and Y. Scale bars A and C = 5 µm; B and D = 2.5 µm. Abbreviation: FISH, fluorescence in situ hybridization.

Figure 2.

(A) The emission spectra of DEAC, Spectrum Green, Spectrum Orange, Spectrum Red, Cy5, and Cy5.5 can be seen in this graph. (B) By using the distinguished emission spectra of these fluorochromes saved in a classified file, the SKY system can easily identify individual chromosomes and create a karyotype. This is an example of an abnormal female cell hybridized with probe set II showing four copies each of chromosomes 15 and 19, three copies of chromosome X, and two copies each of chromosomes 17 and 18. Abbreviations: DEAC, diethylaminocoumarin; SKY, spectral karyotyping.

Figure 3.

(A) FISH probe set III (see Table 2) hybridized on one normal male interphase nucleus (RGB colors). (B) The corresponding classified image from SKY system (pseudo-colors) showed two copies each of chromosomes 2, 3, 4, 5, and 12. The chromosome 9-Cy5.5 probe developed in-house showed weak hybridization signals and was not detected by the SKY system. (C) FISH probe set IV (see Table 2) hybridized on one normal male interphase nucleus (RGB colors). (D) The corresponding classified image from SKY system (pseudo-colors) showed two copies each of chromosomes 1, 6, 7, 8, 10, and 11. Scale bar = 2.5 µm. Abbreviations: FISH, fluorescence in situ hybridization; SKY, spectral karyotyping; RGB, red green blue.