00019606-200803000-00009ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 by Lippincott Williams & Wilkins.17March 2008
p 59-63Looking for Ferns:
Optimization of Digestion
Pretreatment in Fluorescence In Situ Hybridization (
FISH) Technique on
Paraffin-embedded TissuesOriginal ArticlesTojo, Marta BSc*; Perez-Becerra, Raquel BSc*; Vazquez-Boquete, Angel BSc*; García-Rivero, Arancha BSc†; García-Caballero, Tomas MD, PhD*; Forteza, Jeronimo MD, PhD*; Fraga, Maximo PhD**Departamento de Anatomía Patológica y Ciencias Forenses and Servicio de Anatomía Patológica, Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain†Servicio de Genética, Centro Oncológico, A Coruña, SpainThis work was supported by a grant from Instituto de Salud Carlos III (FIS PI 061902).Reprints: Maximo Fraga, PhD, Departamento de Anatomía Patológica y C. Forenses and Servicio de Anatomía Patológica, Complejo Hospitalario Universitario de Santiago, Spain (e-mail:
[email protected]).AbstractFluorescence in situ hybridization (
FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on
paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of
enzymatic digestion. To establish optimal digestion time,
enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24
paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent
FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for
FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of
FISH technique on
paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.Fluorescence in situ hybridization (
FISH) is a useful technique for the study of numerical and structural genetic abnormalities in metaphase and interphase cells. It has important advantages over other genetic testing methods, such as polymerase chain reaction (PCR), reverse transcription-PCR, and conventional cytogenetics.1–3 It is widely used on cell suspensions, imprints, and frozen tissues. However, its routine application on
paraffin-embedded tissues, which represent the major source of diagnostic material in anatomic pathology laboratories, is hampered by technical problems.4–7 The most common include weakness or inconsistency of hybridization signals and the presence of high background fluorescence, which can render results uninterpretable. In addition, the interpretation of
FISH on tissue sections may be more complex by truncated and/or overlapping nuclei (resulting in lack and/or overlap of hybridization signals).8 To overcome these difficulties, some authors have advocated the use of isolated nuclei from thick tissue sections8,9; however, this method is limited because morphologic correlation cannot be made.4 In our opinion, the ideal technique continues to be
FISH on whole thin section, as the overall structure is preserved and comparison can be made with hematoxylin and eosin and/or immunostainings performed in serial sections.The technical problems with
FISH on
paraffin-embedded tissue can be divided into 2 related groups. First, there are factors inherent to tissue conditions, namely initial tissue handling, fixation conditions, paraffin-embedding process, and even specimen age.7,10 Second, there are factors involving the technical steps of
FISH designed to minimize the impact of previous tissue conditions, that is enzymatic
pretreatment, hybridization conditions, and posthybridization washing conditions.5 A particularly important factor is fixation, because it influences the efficiency of hybridization and, unfortunately, may be highly variable between specimens. For most Anatomic Pathology laboratories, formalin is the standard fixative. Formalin results in the formation of methylene bridges between amino groups in the DNA and/or proteins, which helps to preserve the cellular composition and morphology of the tissue but reduces the penetration of acid nuclei probes.11 Therefore,
pretreatment of sections, usually in the form of
enzymatic digestion, is a key step in
FISH technique on formalin-fixed tissues.An extended fixation time is known to substantially reduce the number of hybridization signals.5 There is general agreement that the longer the fixation, the stronger should be the
pretreatment of tissue sections,12 which is similar to other techniques, such as immunohistochemistry.13 As fixation may be quite variable between specimens, the use of a standard
pretreatment may result in failure of
FISH technique. Consequently, if initial results are unsatisfactory, a “trial and error” approach is usually performed; that is, analysis is repeated with increased
pretreatment.14 However, this may be time consuming. Another option is to perform
FISH simultaneously on several sections from the same specimen with different
pretreatment intensities. However, this requires tissue sections that could be employed for other purposes.There are considerable technical variations in
FISH on
paraffin-embedded samples, especially regarding factors such as section thickness and dilution as well as time and type of enzyme. Pepsin and proteinase K are widely used in
enzymatic digestion. With these proteases, digestion usually takes between 10 and 30 minutes, which is obviously influenced by tissue-section thickness and enzyme concentration.1,8,15,16We present a simple method that improves hybridization in
FISH on
paraffin-embedded samples by monitoring the time of
enzymatic digestion under microscopic control and also saves time and reactives by reducing repetition and increasing reproducibility.MATERIALS AND METHODSTissue SamplesTo perform this study, we analyzed 65 formalin-fixed
paraffin-embedded tissue samples from the Hospital Clínico-Universitario de Santiago: 12 normal tonsils as negative control for translocation for each probe set, 15 follicular lymphomas, 15 mantle cell lymphomas, 15 Burkitt lymphomas, and 8 anaplastic cell lymphomas. Three-micron thick sections were cut from each case and placed in pretreated slides (ChemMate Capillary Gap Microscope Slides, DakoCytomation, Glostrup, Denmark).ProbesThe detection of chromosome translocations was carried out using 4 probe sets from Vysis, Inc (Downers Grove, IL): (1) LSI IGH SpectrumGreen/LSI BCL2 SpectrumOrange for t(14;18) detection, (2) LSI IGH SpectrumGreen/LSI CCND1 SpectrumOrange for t(11;14) detection, (3) LSI IGH SpectrumGreen/MYC SpectrumOrange/CEP8 SpectrumAqua for t(8;14) detection, and (4) LSI ALK Dual Color Probe SpectrumGreen/SpectrumOrange for translocations involving band 2p23.
Pretreatment ProtocolSlides were dried overnight at 55°C or at 60°C for 20 minutes. Deparaffination and rehydratation was carried out with 3 xylene washes, 2 absolute-alcohol washes, two 96°C alcohol washes, and 1 distilled-water wash, each for a period of 3 minutes.After deparaffination and rehydratation, slides were placed in SSC2× (saline sodium citrate) wash for 3 minutes in water bath. To avoid or mitigate tissue autofluorescence, slides were immersed in HCl (acid chlorhydric) 0.1 M for 20 minutes (in our experience, this step is not essential).
Paraffin-embedded sample
pretreatment was carried out with a NaSCN (sodium thiocyanate) 1 M, at 80°C for 30 minutes (some samples were pretreated with citric acid yielding similar results). Afterwards, slides were placed in distilled water for 1 minute and in SSC2× for 5 minutes.Enzymatic DigestionTo perform the
enzymatic digestion protocol, we used 0.05 mg/mL pepsin solution in HCl 0.01 N or 0.01 mg/mL proteinase K solution in buffer TE (Tris 10 mM, ethylene diaminetetra acetic acid 1 mM pH 8). Initially, to find optimal digestion time, series sections were made for 24 cases. Enzyme activity for the various sections taken from each specimen was stopped at different times (3-min intervals for biopsies and 10-min intervals for autopsy samples). Stop process consisted in slide immersion in SSC2× for 1 minute and tissue drying in thermal plate at 45°C; this wash step proved to be indispensable for discerning morphologic tissue modifications, as we will describe later. Tissue appearance was then observed under light microscopy with 10× and 40× lenses with slightly closed diaphragm. After that, the
FISH technique was continued to evaluate the efficiency of each digestion time. From these initial tests, it became evident that optimal tissue digestion was achieved when tissue showed fernlike formations under light microscopy.This method for optimizing digestion was then applied to the remaining routine pathologic diagnostic samples (41 more cases).After detecting fernlike formations, the slides were placed in 2 baths of SSC2× for 3 minutes and dehydrated in increasing alcohol solutions of OH70, OH96, and OH100°C for 2 minutes each. Then the slides were air dried at room temperature for 15 minutes.Probe Preparation and HybridizationProbe mix was carried out according to the manufacturer's specifications. We avoided using formamide because of its health risks. Hybridization was performed either on a thermal plate or on the DakoCytomation Hybridizer (Dako Co, Denmark), first at 84°C for 5 minutes and finally at 37°C overnight.Stringent Wash and Preparation MountingFor posthybridization washes, we used SSC0.4×with 0.3% NP-40 (Nonidet P-40) at 73°C for 3 minutes. Then the slides were placed in SSC2× with 0.1% NP-40 at room temperature. Afterwards, the slides were air dried for 20 minutes and mounted in DAPI II (4′-6′-diamidino-2-feniloide) counterstain from Vysis, Inc (Downers Grove, IL).Image AnalysisImage analysis was performed and photographed using an Eclipse E400 Nikon fluorescence microscope (Nikon, Tokyo, Japan) equipped with DAPI (nuclei) and SpectrumGreen/SpectrumOrange dual bandpass filter sets.RESULTSAs previously described, we initially tried to optimize the digestion step in 24
paraffin-embedded samples by stopping
enzymatic digestion at 3/10-minute intervals. Every stop was followed by observation of tissue appearance under light microscopy. After that, the
FISH technique was continued to evaluate the effectiveness of different digestion times.We found that optimal tissue digestion was achieved when tissue showed fernlike formations under light microscopy, regardless of whether pepsin or proteinase K was used. When fernlike formations were not observed, either by shorter or longer digestion, the hybridization signals were lacking or very weak. It must be stressed that the only stop solutions with which we found fernlike arrangement was SSC2×.This method for optimizing digestion was then applied to the remaining routine pathologic diagnostic samples (41 additional cases). Accordingly, digestion time was highly variable and related to the type of sample. The extreme values were 9 and 120 minutes; the longest value was extraordinary and occurred in only one autopsy sample (Fig. 1). The usual digestion times were 9 to 12 minutes for endoscopic biopsies, 15 to 30 minutes for surgical biopsies, and 25 to 40 minutes for autopsies.JOURNAL/dimp/04.03/00019606-200803000-00009/figure1-9/v/2021-02-17T195944Z/r/image-jpeg
An outstanding example of the influence of digestion time in tissue morphology and hybridization efficiency (autopsy sample): as digestion time increases (A, B), 10 minutes; (C, D), 40 minutes; (E, F), 80 minutes; (G, H), 120 minutes fernlike image (left side) is more clearly displayed and hybridization signals are improved (right side). BCL2/IgH probe set from Vysis, Inc (Downers Grove, IL) (green signals, IgH; red signals, BCL2; no nuclear counterstaining).With this microscopic monitoring of digestion time procedure, an adequate hybridization signal was obtained in every case.DISCUSSIONFISH is a powerful technique for the detection of numerical and structural chromosomal aberrations. In this respect, it has well-known advantages over classic cytogenetics and molecular techniques such as Southern blot and PCR; for example, the ability to study interphase cells, and its very high sensitivity. The availability of commercial probes for
FISH detection of chromosomal abnormalities has facilitated its incorporation into the routine diagnostic armamentarium, best exemplified in hematopathology.17,18FISH is a well-established technique that is easy to perform in fresh cell suspensions or lymph node imprints that have been fixed with methanol and glacial acetic acid. However, its application on
paraffin-embedded tissues is hampered by several technical problems, mainly the inability to obtain an optimal signal from probes. One reason for this is that fixatives and paraffin interfere with the hybridization step that can give rise to nonconclusive results.5 Nevertheless, efforts must be made to solve such problems, because
paraffin-embedded samples are a common source of diagnostic material in pathology laboratories and are essential for carrying out retrospective studies.11,19A variety of pretreatments, such as microwaving and/or protease digestion,6,15,16 has been proposed for obtaining good results with
FISH on paraffin sections. With respect to protease digestion, both proteinase K and pepsin have been used, but there is considerable heterogeneity among studies concerning their concentration and digestion time. Although relatively good results have been reported using a common digestion time for all biopsies in paraffin sections (usually ranging from 10 to 30 min),1,10,15,16 individual
optimization is often recommended to obtain best results and greater reproducibility.20In our opinion, empirical
optimization is a time-consuming and reactive-consuming approach, owing to ample variations in biopsy fixation times. This is particularly evident in samples of consultation cases from other centers, it may be even more dramatic in autopsy samples,21 where fixation is usually intense. An extreme example was one autopsy case in which digestion
pretreatment was as long as 120 minutes.When we began method testing (24 initial cases), we checked the morphologic appearance of the tissue in 3/10-minute intervals (see section Materials and Methods). Afterwards, when additional cases were being evaluated, we realized that monitoring under the microscope could be more spaced if the morphology did not change after the first visual control, and it could be adapted to the type of sample, thus avoiding unnecessary morphologic checking: in general, endoscopic samples required a total digestion time of about 10 minutes, routine biopsies 15 to 30 minutes, and autopsy samples 20 to 40 minutes. On the other hand, if the fernlike pattern begins to appear, usually in the edge of the section, the digestion should be stopped shortly thereafter.Required digestion time is influenced not only by fixation and enzyme concentration, but also by thickness of paraffin sections. In our experience, 3-μm thick sections were most suitable for digestion control and interpretation of the results (data not shown). Thicker sections need longer digestion and are more prone to overlapping of nuclei.8,22The fernlike pattern was observed regardless of whether pepsin or proteinase K were used. Proteinase K has been reported to leave highly fluorescent speckles within the cells.11 Other authors mention the influence of enzymatic
pretreatment on the efficiency of hybridization, and they note that the best results were obtained with pepsin.5 However, these observations are not supported by our experience. The advantage of using pepsin is that it is dependent on pH and ceases to act without the need for a stop reactive when pH is changed.As we have done for
FISH, some authors have also stressed the appropriateness of other PCR techniques for the morphologic evaluation of pretreated samples. For in situ PCR, Bagasra and Harris23 recommend stopping digestion when small round bubbles appear on the cytoplasmic membrane of the cells-of-interest.In conclusion, the method here proposed, controlling digestion time until visualization of fernlike structures on the sample under light microscopy, is highly useful insofar as optimizing the results of hybridization is concerned. Repeated attempts are not necessary for a successful technique, thus saving time and reactives and it is particularly useful when dealing with consultation cases or autopsy samples.ACKNOWLEDGMENTThe authors are indebted to Luisa Anllo for her expert technical assistance.REFERENCES1. Einerson RR, Kurtin PJ, Dayharsh GA, et al.
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FISH;
pretreatment;
optimization;
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An outstanding example of the influence of digestion time in tissue morphology and hybridization efficiency (autopsy sample): as digestion time increases (A, B), 10 minutes; (C, D), 40 minutes; (E, F), 80 minutes; (G, H), 120 minutes fernlike image (left side) is more clearly displayed and hybridization signals are improved (right side). BCL2/IgH probe set from Vysis, Inc (Downers Grove, IL) (green signals, IgH; red signals, BCL2; no nuclear counterstaining).Looking for Ferns:
Optimization of Digestion
Pretreatment in Fluorescence In Situ Hybridization (
FISH) Technique on
Paraffin-embedded TissuesTojo Marta BSc; Perez-Becerra, Raquel BSc; Vazquez-Boquete, Angel BSc; García-Rivero, Arancha BSc; García-Caballero, Tomas MD, PhD; Forteza, Jeronimo MD, PhD; Fraga, Maximo PhDOriginal ArticlesOriginal Articles117p 59-63
