Optimization of the fluorescence in situ hybridization (FISH) technique for high detection efficiency of very small proportions of target interphase nuclei

Abstract

Using commercially available fluorochrome-labeled probes specific for chromosomes X, Y, 13, 18, and 21, we optimized the technical protocols for fluorescence in situ hybridization (FISH) so that the highest sensitivity and specificity were achieved. Also, we compared the optical properties of different types of fluorescent labels in an effort to develop the most efficient FISH protocol, including the determination of which types of labels are the easiest to count accurately. The lymphocytes were purified from blood of normal male and female newborns, normal male and female adults, and a trisomy 21 male adult. Male and female lymphocytes were mixed in five different combinations. For each combination, the male lymphocytes either from newborns or from adults were diluted with female lymphocytes in seven different proportions. For each of these 35 different cell mixtures, 100,000 nuclei were analyzed and scored in a blind fashion. Among the different fluorochrome-labeled probes, the highest sensitivity and specificity were achieved when SpectrumAqua CEP-Y/SpectrumOrange CEP X probe mixture, SpectrumAqua CEP-18, SpectrumOrange LSI-13, and SpectrumOrange LSI-21 were hybridized. The hybridization sensitivity and specificity were higher than 99% for the identification of chromosomes X, Y, 13, and 18, and higher than 98% for the detection of trisomy 21. The proportion of false-positive signals was under 0.005% for XY detection and lower than 0.14% for autosome detection. With these high hybridization sensitivities and specificities, the optimized FISH protocol developed in our laboratory has the potential to detect very rare events, e.g., when the proportion of cells being sought is lower than 0.01%. In other words, our protocol allows the specific detection of one male cell sunken among 10,000 female cells.

The use of non-physiological conditions to isolate fetal cells from maternal blood

“…Analysis was carried out without knowing in advance fetal sex. The mean percentage of cells which showed one X and one Y signal or two X signals was N98% using this protocol [23]. This hybridization efficiency was comparable to the controls.…”

“…Hybridization was performed according to the procedure described by Yan et al [23]. To detect fetal cells with trisomy 21 and trisomy 18, slides were processed with TriGen LSI 21 SpectrumOrange and CEP 18 (a satellite) SpectrumOrange hybridizing centromere of human chromosome 18 (18p11.1-q11.1 Vysis).…”

Efficiency of Manual Scanning in Recovering Rare Cellular Events Identified by FluorescenceIn SituHybridization: Simulation of the Detection of Fetal Cells in Maternal Blood

“…Diffused signals can be explained by chromatin extension forming chromatin fibers, which links two or more condensed domains of chromatin. These fibers usually show a very weak signal, which fades faster than a normal one [19]. The nature of defective signals resulting from overdecondensed chromatin points to the importance of prehybridization steps in the FISH technique.…”

“…A robust system allowing detection of one male fetal cell or one trisomic 21 cell among 10,000 to 100,000 maternal cells would be extremely useful. Such system would obviate the need or at least significantly reduce the required level of enrichment of fetal cells and facilitate screening large number of slides, making prenatal diagnosis more easily achievable [19]. Evaluation of the efficiency of these automatic slide-scanning devices is mandatory before clinical implementation.…”

“…However, the accuracy of the manual scanning, which is considered the gold standard, in the detection of rare cellular events had never been validated. Other studies measured the detection efficiency by using prediluted artificial sample mixtures (spiked samples) [1, 19, 20, 22–24]. Dilutions of target cells within a whole cell population are reliable within certain limits of dilution.…”