Interphase or metaphase nuclei can be accessed in molecular cytogenetic analyses. Metaphase spreads are routinely studied by fluorescence in situ hybridization (FISH) to answer clinical genetic questions. Even though metaphase quality is essential for FISH studies, there is limited ability in clinical cases to improve the quality of cytogenetic preparations. However, the quality of preps is important for the exact localization of FISH signals, which is necessary to identify individual chromosomes and chromosomal sub-regions using inverted DAPI banding. Here we present an efficient and easy-to-perform variant of standard slide pretreatment before a normal FISH procedure. This method reproducibly leads to solid, “steel,” nonfuzzy, and well-DAPI-banded metaphases. This protocol works in blood lymphocyte and amniotic fluid-derived fibroblasts. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol : Slide pretreatment for high-quality metaphases for molecular cytogenetics.

The method presented here is helpful in molecular cytogenetics, specifically in preparing high-quality metaphase spreads to be evaluated after fluorescence in situ hybridization (FISH). The area of molecular cytogenetics developed from banding cytogenetics in the 1980s and belongs to the concert of cytogenetic approaches being applied in research and diagnostics (known as chromosomics) (reviewed in Liehr, 2021). Chromosomics aims to uncover the underlying principles of genetics and genomics to achieve a general understanding of life itself (Claussen, 2005). Chromosomics began with the microscopic visualization of chromosomes by Walther Flemming in 1879 (in 1888, Heinrich Wilhelm Waldeyer invented the designation chromosome = stained body) and continued with Walter Sutton's and Theodor Boveri's chromosome theory of inheritance in 1902/03. This was followed up in 1910 with the general proof of Thomas Hunt Morgan that genes are aligned like pearls on a necklace along the chromosome (reviewed in Cremer and Cremer, 2006). However, human cytogenetics as we know it began with the publication of the correct modal human chromosome number as 46 in 1956 (Tjio and Levan, 1956). Even today, GTG-banding (G-bands by Trypsin using Giemsa), based on Lore Zech's groundbreaking discovery that human chromosomes show reproducibly inducible banding patterns (Schlegelberger, 2013), is still the gold standard and best starting point for human genetic diagnostics for many clinical questions (Liehr, 2021).

FISH is well-established, and multiple probe sets have been developed to address diagnostic problems in tumor and clinical genetics, the latter including prenatal and postnatal cases (Liehr, 2021). When FISH-probe sets are being applied to metaphase nuclei, it is desirable to obtain clear signals on well-spread chromosomes, which optimally should be identifiable by an inverted DAPI-banding pattern. Inverted DAPI banding is almost identical to the GTG-banding pattern (Henegariu et al., 2001; Liehr, 2022). Quality DAPI banding makes it possible to verify (i) that the correct FISH probe has been used and (ii) that the probe is located on the expected chromosome or was relocated to another chromosome due to a rearrangement (Coullin, Philippe, Ravise, and Bernheim, 1999). However, in routine FISH-diagnostic testing, patient material is often limited, and cytogenetic preparations are of variably quality. The purpose of this protocol is to provide a useful pretreatment to aid in obtaining the most accurate and informative results possible for the sake of the patient (OECD, 2007).

Chromosomal pretreatment is a standard practice in most laboratories that perform FISH. This pretreatment helps to improve chromosome quality using pepsin and formaldehyde (Liehr, 2017). Pepsin digestion removes proteins distributed around the chromosomes on the metaphase spread, enabling better access for DNA probes to the chromosomal target DNA, leading to higher signal intensities. The use of formaldehyde after pepsin treatment allows binding of the remaining protein and stabilizes the chromosomal structure by cross-linking. Both treatments prepare chromosomes to exhibit a banding pattern when counterstained with DAPI (Liehr, 2017). Still, they are insufficient to uniformly produce the desired quality of metaphase spreads in all cases (Fig. 1).

The protocol described here was originally developed to obtain high-quality, blood lymphocyte-derived metaphases for comparative genomic hybridization (CGH). In utilizing this method, we were able to obtain “steel” (fixed and clearly defined chromosomes with clear banding pattern), regardless of the variation in the quality of the blood lymphocyte preparation (Liehr, Heller, Starke, and Claussen, 2002). We have determined that pretreatment is also well-suited for standard FISH procedures, as reported here for the first time. As a negative control, amnion and non-pretreated blood cells (Figs. 1A and 1B) were processed with 3-color FISH, using a commercially available probe set, and compared to pretreated preparations (Figs. 1C to 1H). Pretreated preparations showed a clear difference from their untreated counterparts, further indicating the appropriateness of this method.

The protocol outlined here is a simple but highly effective change of the standard slide pretreatment protocol: the two-step protocol (pepsin followed by formaldehyde) (Figs. 1C and 1D). We have modified this to a three-step protocol (formaldehyde, followed by pepsin and formaldehyde again) (Figs. 1E and 1F). This simple modification led to a much more uniform, high quality, “steel,” well-inverted DAPI-banded chromosomal preparations with at least as strong a FISH signal as in the standard protocol (Fig. 1). Accordingly, evaluation of FISH results became much more straightforward, as chromosome identification became much less time-consuming. As shown in Figure 1H, a simple switch to the order of the standard two-step pretreatment protocol (now treating with formaldehyde followed by pepsin) did not yield equal or better results. Thus, we propose including an additional formaldehyde pretreatment before the standard pepsin and formaldehyde pretreatment to obtain the best results.

CAUTION : All work with formaldehyde solutions must be performed in a suitable fume hood with efficient ventilation.

This protocol is a broadening of a standard slide pretreatment protocol. It can be easily integrated into any FISH slide pretreatment protocol variant, encompassing a pepsin and formaldehyde step. The main point of the protocol is the inclusion of an initial formaldehyde treatment before the standard pepsin followed by formaldehyde pretreatment. Interestingly, formaldehyde fixation does not impair the capability of pepsin to remove superfluous proteins.

Materials

• DAPI (Merck, Darmstadt, Germany, #124653), 1 mg/100 ml pure water (Ampuwa, Fresenius Kabi, Bad Hersfeld, Germany, #06605508)

• Formaldehyde (37%, Sigma-Aldrich, Hamburg, Germany, #818708), 3 ml in 97 ml PBS/MgCl₂

• FISH probe (SHOX, CytoCell, Cambridge, UK, #LPU 025)

• Glass slides (76 × 26 mm) (Thermo Scientific, Waltham, USA, #MENZAB00000102E012)

• PBS/MgCl₂ (190 ml 1× PBS, Biochrom, Berlin, Germany, # L1825, and 10 ml of 1 M MgCI₂, Merck, Darmstadt, Germany, #2189.2)

• Pepsin solution (50 µl of 10% pepsin, Sigma-Aldrich, Hamburg, Germany, # P7012, in 100 ml distilled water with 1 ml of 1 M HCI, Sigma-Aldrich, Hamburg, Germany, #1090571000)

• Patient sample (Cytogenetic preparation of blood or amnion cells in Carnoy's fixative, 3:1 methanol/acetic acid, Merck, Darmstadt, Germany, # CAS 67-56-1 and #64-19-7)

• 2× SSC solution (from 20× SSC, GIBCO BRL, Karlsruhe, Germany, #15557-036, in distilled water)

• Coplin jar (100 ml)

• Fume hood with efficient ventilation

• Phase-contrast microscope (Zeiss or equivalent)

• Micropipettes (e.g., Eppendorf 200 and 20 µl)

1.Put clean glass slide(s) into a Coplin jar with purified water. Place in the refrigerator at 4°C.

2.Place suspension with interphases and metaphases on a slide using a 20-µl micropipette and let air dry for at least 1 hr. Let slide(s) dry overnight at room temperature for best results.

3.Transfer glass slide(s) into the formaldehyde solution for 10 min at room temperature.

4.Rinse the slide(s) in 2× SSC solution for 5 min in a Coplin jar at room temperature.

5.Transfer the slide(s) to freshly made pepsin solution for 3 min at 37°C in a Coplin jar.

6.Rinse slide(s) in PBS/MgCl₂ at room temperature for 5 min.

7.Transfer slide(s) into formaldehyde solution again for 10 min at room temperature.

8.Rinse the slide(s) in PBS/Mg₂ for 5 min at room temperature.

9.Immerse slide(s) in 70% ethanol for 5 min; then remove slides and place in 90% ethanol for 5 min, followed by 100% ethanol for 5 min, all at room temperature.

10.Air-dry the slides for 20 min and store them at −20° or use them immediately in FISH.

Background Information

Formaldehyde is extensively used to fix tissues, including chromosomal structures, which consist mainly of protein. Each kind of chromosomal banding other than FISH-based banding is protein-mediated (Claussen et al., 2002). Thus, the use of formaldehyde as a critical component of FISH was introduced some decades ago (Coullin et al., 1999; Henegariu et al., 2001). The advantage of its use was obvious when examining FISH results with and without formaldehyde fixation (Fig. 1). Even still, the concept of treating samples twice with formaldehyde with an intervening pepsin treatment has not been introduced before now. It is possible that precaution was taken to avoid disrupting the effectiveness of the pepsin treatment, but we have demonstrated that the additional formaldehyde treatment has the opposite effect and improves the final product.

Critical Parameters

The protocol is very robust. Apart from standard precautions to be observed when working with cytogenetic material on slides (like paying attention to proper labeling of slides and mark which side is up), no specific cautions or concerns accompany the use of this protocol.

Troubleshooting

None.

Understanding Results

The results of seven FISH experiments are presented in Figure 1, performed on samples with karyotype 46, XY (amnion shown in Figures 1A, C, and E; blood in Figures 1B, D, F, and H). This test was performed with 3 male and 3 female probes, each for amnion and blood-derived chromosome preparations. Typical results for one male probe are presented in Figure 1.

Both pretreatments enhanced signal quality and intensity in amnion-derived metaphases (Figures 1A, C, and E). In terms of shape, Chromosome quality did not substantially differ between untreated (Fig. 1A) and treated chromosomes (Figs. 1C and E). However, the inverted DAPI-banding pattern was best visualized after using the three-step protocol (Fig. 1E).

Compared to amnion-derived preparations, blood-derived metaphase spreads benefitted much more from each kind of pretreatment (Figs. 1B, D, F, and H). The pretreated blood-derived chromosomes also show much more intense FISH signals. Additionally, the effect of pretreatment (Figs. 1D, F, and H) was clearly visible compared to a non-pretreated, extremely low-quality preparation (Fig. 1B). While both of the two-step pretreatment protocols already led to a strikingly positive effect on chromosome shape and visibility (Figs. 1D and 1H), the three-step treatment yielded an almost perfect, GTG-like banding pattern (Fig. 1F).

Time Considerations

The described protocol can be completed in about 1 hr, about 15 min more than the standard pretreatment.

Acknowledgments

Open access funding enabled and organized by Projekt DEAL.

Author Contributions

Thomas Liehr : Conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, supervision, validation, original draft, review, and editing; Anja Weise : Resources, review, and editing; Luisa Person : Methodology, validation, visualization, review, and editing; Niklas Padutsch : Methodology, visualization, review, and editing; Stefanie Kankel : Formal analysis, investigation, methodology, validation, visualization, review and editing.

Conflict of Interest

The authors have no conflicts of interest to declare.

Data Availability Statement

The data that supports the findings of this study are available in this article.

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