Diagnostic Techniques

A rapid and simplified fluorescence in situ hybridization (FISH) technique for the detection of human chromosome-specific centromeric probes is described. Using chromosomes 1-, 4-, 11and Y-specific fluorescence-labeled probes, the modified, or quick-FISH technique, was compared to two conventional FISH methods. The modified FISH technique detected human chromosomes without sacrificing sensitivity or signal quality in both fresh-frozen and paraffin-embedded tissue sections. Further studies demonstrated that this technique can be used with simultaneous application of dual-color probes. This novel technique offers the advantages of being simpler to perform and faster than conventional techniques. The quick-FISH technique can be substituted for any sensitive conventional FISH method for molecular cytogenetic analysis in fresh, fresh-frozen or paraffinembedded tissues. INTRODUCTION Chromosome-specific probes for the alpha satellite-, satellite IIIand locusspecific regions have been used in both diagnostic and experimental molecular genetic studies to detect chromosomal anomalies by fluorescence in situ hybridization (FISH) techniques (1–12). These probes have been instrumental in bringing FISH to the forefront of molecular cytogenetic studies. Using these probes, FISH techniques are routinely used for molecular cytogenetic studies in fresh-frozen, paraffin-embedded and fresh tissue samples (1–12). Although the various FISH techniques are sensitive for detecting chromosomal abnormalities, these techniques are either time-consuming or require multiple steps to perform the assay. Additionally, the previous modifications that have been described did not evaluate the techniques in multiple types of tissue (fresh-frozen, paraffin-embedded, fresh etc.). A modification of the FISH technique that uses chromosome enumeration probes (CEPs) and Hybrite (Vysis, Downers Grove, IL, USA), a very precise hybridization instrument, is described. The modified FISH technique (quick-FISH) detects specific chromosomes separately and/or simultaneously in both touch-preparation slides and paraffin-embedded tissue sections without sacrificing sensitivity or signal quality, while offering the advantages of being simpler to perform and faster than conventional techniques. The modified FISH technique was evaluated and compared to two established FISH methods (7,9) for the detection of chromosomal signals in this study. MATERIALS AND METHODS The quick-FISH method is a combination and modification of several established FISH techniques including those described by Jenkins et al. (5), Celeda et al. (1), Haar et al. (4) and Persons et al. (7). The modified quickFISH technique is detailed here. Tissue Samples and Slide Preparation Gastric mucosal biopsies were obtained from five humans with mucosa having a normal endoscopic appearance. Six biopsy specimens from each patient were fixed in 10% buffered formalin and embedded in paraffin. Histologic examination of 5-μm paraffin sections stained with hematoxylin and eosin confirmed that the mucosa was normal. Additional unstained paraffin sections were obtained for quick-FISH 826 BioTechniques Quick-FISH: A Rapid Fluorescence In Situ Hybridization Technique for Molecular Cytogenetic Analysis BioTechniques 24:826-830 (May 1998) molecular cytogenetic analysis. An additional two to four biopsy specimens from each subject were immediately frozen in liquid nitrogen and used to prepare touch-preparation slides for molecular cytogenetic analysis. A minimum of 30 touch-preparation slides were made from each gastric mucosal biopsy specimen. Slides were fixed in cold methanol/acetic acid (3:1 vol/vol) for 30 min at 4°C, air-dried at room temperature and then stored at -20°C until FISH was performed. For each probe, two touch-preparation slides from each biopsy sample were used for conventional FISH technique I (7), two touch-preparation slides were used for conventional FISH technique II (9), and five touch-preparation slides were used for quick-FISH. One slide from each specimen was used for a dual-color quick-FISH assay. Fluorescence In Situ Hybridization Directly labeled fluorescent alpha satellite DNA probes specific for the centromere of chromosomes 1, 4, 11 and Y were used in this study. The labeled probes and the chemicals for hybridization were purchased from Vysis. All probes except the chromosome 4specific probe were spectrum orangelabeled. The chromosome 4-specific probe was spectrum green-labeled. To critically evaluate the sensitivity and quality of the modified FISH technique, the quick-FISH technique described here was compared to two conventional FISH methods (7,9). The modified hybridization procedures of the quick-FISH method are what expedite and simplify the technique. Briefly, touch-preparation slides were not dehydrated during the quick-FISH method. Also, instead of heating the slides on a hot plate or in a water bath for 1 min and transferring them to an incubator overnight, quick-FISH slides were placed on the heating surface of the Hybrite programmable instrument, heated to 75°C (denaturation) for 1 min and then hybridized for only 1 min at 42°C on the same surface. Prior to placement of the slides on the heating surface of the Hybrite for denaturation and hybridization, the area of the slide containing the specimen was marked with diamond pencil marker, and 10 μL of hybridization mixture (single-color FISH: 7 μL CEP buffer provided with the probes, 0.5 μL probe and 2.5 μL water) were applied to each slide and immediately covered with a coverslip. After hybridization, slides were washed in Solution I (0.5× standard saline citrate [SSC], pH 7.4, 0.1% Nonidet BioTechniques 827 Figure 1. Examples of the high-quality centromere-specific signals obtained using the quick-FISH method are depicted. (a) Touch preparation from normal human gastric cells hybridized with a directly labeled fluorescent probe specific for the centromere of chromosome 1. (b) Normal gastric biopsy tissue touch preparation hybridized with a directly labeled fluorescent probe specific for the centromere of chromosome 11. (c) Touch preparation of normal gastric cells hybridized with a directly labeled fluorescent probe specific for the centromere of chromosome Y. (d) Paraffin-embedded gastric tissue section after hybridization with a directly labeled fluorescent probe specific for the centromere of chromosome Y. Diagnostic Techniques P-40 [NP40]) for 1 min at 37°C followed by a rinse in Solution II (2× SSC, pH 7.4, 0.1% NP40) at room temperature. Nuclei were counterstained with 10–15 μL of 4,6-diamino-2-phenylindole (DAPI) solution (1 μg/mL; Sigma Chemical, St. Louis, MO, USA) containing antifade p-phenylenediamine dihydrochloride (10 mg/mL; Sigma Chemical). To determine whether multiple probes could be applied simultaneously, dual-color hybridization was performed and evaluated. The technique was identical to that used for the single probe, except the DAPI concentration was diluted to 1/10 of the concentration used with the single probe (0.1 μg/mL), and the hybridization mixture was modified (dual-color FISH: 7 μL CEP buffer, 0.5 μL probe Y [orange], 0.5 μL probe 4 [green] and 2.0 μL water). The conventional FISH methods that were compared to quick-FISH are identical to those described by Persons et al. (7) and Qian et al. (9). In brief, slides were either dehydrated in 70%, 80% and 100% ethanol for 5 min each and then air-dried or, prior to dehydration, incubated in denaturant solution (70% formamide/2× SSC, pH 7.4) for 1 min at room temperature and then washed in the same solution for 5 min at 75°C. Following dehydration, the hybridization mixture containing CEP buffer, centromere-specific probe and water was applied to the slides under a coverslip, and the slides were incubated for 1 min at 90°C followed by overnight incubation at 42°C in a prewarmed humidified box for hybridization. Two 10-min post-hybridization washes at 45°C in 50% formamide/2× SSC (pH 7.4) were followed by two additional washes at 45°C in 2× SSC (pH 7.4) and another with 2× SSC (pH 7.4) 0.1% NP40. Fluorescence-stained touch-preparation slides prepared by both the modified and conventional techniques were examined with a fluorescence microscope (Olympus, Lake Success, NY, USA) equipped with a triple-pass filter (DAPI/Orange) for the detection of spectrum orange and DAPI. Slides were analyzed according to the criteria described by Chen et al. (2) and Qian et al. (9). A minimum of 200 interphase nuclei were examined on each slide. Overlapping and adherent nuclei were excluded from evaluation. However, overlapping and/or adherent signals from nonoverlapping nuclei were counted as one. Counts were recorded as 0, 1, 2 and >2 for the Y-specific probe. For chromosomes 1and 11specific probes, counts were recorded as 0, 1, 2, 3 and >3. The mean number of signals from each probe was calculated for each of the three techniques (conventional FISH I, conventional FISH II, quick-FISH), and the techniques were compared. Student’s t test was used to analyze and compare the two conventional FISH techniques and to compare the quick-FISH with the combined results of the two conventional FISH techniques. To determine whether this modified quick-FISH technique can be applied to molecular cytogenetic analysis of tissue embedded in paraffin, paraffin-embed828 BioTechniques Figure 2. Examples of the simultaneous application of dual-color probes specific for chromosomes Y (orange) and 4 (green) using the quick-FISH technique in normal gastric cells. (a) Cells examined with triple-pass filters exhibited three-color hybridization signals. The photograph was taken at high magnification (750×) using a traditional camera attached to a light microscope. (b) Cells examined at lower magnification (500×) with triple-pass filters exhibited three-color hybridization signals. Cell images were captured, and the photograph was obtained using Cytovision image analysis system (Applied Imaging, Santa Clara, CA, USA). Methoda Conventional FISHb Quick-FISH FISH Signal Y 1 11 ← Chromosome → Y 1 11 0 dots 0 0 0 0 0 0.2 ± 0.4 1 dot 99.1 ± 0.7 3.8 ± 1.5 4.2 ± 1.6 98.0 ± 1.5 3.8 ± 1.5 3.6 ± 1.5 2 dots 0.9 ± 0.7 94.6 ± 2.1 93.4 ± 2.0 1.2 ± 0.7 93.6 ± 1.7 93.4 ± 1.7 3 dots 0 1.6 ± 1.3 2.2 ± 0.8 0 2.6 ± 0.9 1.0 ± 1.0