RAPID COMMUNICATION |
Correspondence to: Klaus Hermann Wiedorn, Institut für Pathologie, Klinikum Stuttgart, Kriegsbergstr. 60, 70174 Stuttgart, Germany. E-mail: k.wiedorn@katharinenhospital.de
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Summary |
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Seventy paraffin-embedded cervical biopsy specimens and condylomata were tested for the presence of human papillomavirus (HPV) by conventional in situ hybridization (ISH) and ISH with subsequent signal amplification. Signal amplification was performed either by a commercial biotinyltyramide-based detection system [GenPoint (GP)] or by the novel two-layer dextran polymer visualization system EnVision+ (EV), in which both EVhorseradish peroxidase (EVHRP) and EValkaline phosphatase (EVAP) were applied. We could demonstrate for the first time, that EV in combination with preceding ISH results in a considerable increase in signal intensity and sensitivity without loss of specificity compared to conventional ISH. Compared to GP, EV revealed a somewhat lower sensitivity, as measured by determination of the integrated optical density (IOD) of the positively stained cells. However, EV is easier to perform, requires a shorter assay time, and does not raise the background problems that may be encountered with biotinyltyramide-based amplification systems. (J Histochem Cytochem 49:10671071, 2001)
Key Words: EnVision+, dextran polymer conjugate, biotinyltyramide, GenPoint+, in situ signal amplification, ISH
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Introduction |
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ISH has become an integral part of molecular pathology as a main molecular tool for both diagnostics and research. ISH enables the detection and precise localization of nucleic acid targets in cells and tissues without destruction of morphology. Originally radioactive probes were used for ISH. However, non-radioactive probes, particularly biotin or digoxigenin, are now often favored because they are less hazardous to work with, can be much more quickly developed, allow a much better spatial resolution, and are least as sensitive as radioactively labeled probes. Whereas the kind of non-isotopic label overall appears to be almost irrelevant with respect to the sensitivity of ISH, the detection system may enhance signal intensity. Thus, the combination of alkaline phosphatase and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) reveals stronger signals than peroxidase and diaminobenzidine (DAB). Signal intensity can be further improved by using triple-step biotin detection methods with anti-biotin/anti-mouse/APAAP or anti-biotin/APanti-mouse/APAAP.
Despite these improvements in sensitivity ISH usually detects only medium to high copy number nucleic acid targets. In contrast to ISH, in situ PCR (IS-PCR) allows the detection even of low copy number nucleic acids, but this method is much more cumbersome and is often hampered by poor reproducibility and morphology, especially when applied to paraffin-embedded tissue. Recently, a new system for signal enhancement in enzyme immunoassays (EIA) and immuno-cytochemistry (IHC) using the water-soluble dextran polymer conjugate EV has been described (
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Materials and Methods |
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Sample processing from fixation up to permeabilization was performed as described by
Permeabilization was done either by incubation with 250 µg/ml Proteinase K (Roche Diagnostic; Mannheim, Germany) for 15 min at 37C or by incubation in Target Retrieval Solution (TRS) (S1700; Dako) at 95C for 30 min. Thereafter sections were incubated at room temperature (RT) twice for 5 min in 0.1 M Tris-NaCl, pH 7.5.
Quenching
If peroxidase/AEC was used as a detection system, inactivation of endogenous peroxidase was performed by incubation in peroxidase blocking reagent (S2001; Dako) for 30 min at 37C followed by a 1-min incubation each in 50%, 70%, and 90% ETOH at RT. Slides were dried for 5 min at 37C.
ISH
ISH was performed on a MISHA (Shandon; Frankfurt, Germany) in situ thermocycler using either a commercial DNA probe (PathoGene) according to the recommendations of the manufacturer or a cocktail of 5'-DIG-labeled oligonucleotides as described previously (
Evaporation control was performed as described elsewhere (
Posthybridization washes were performed according to the recommendations of the manufacturer for the commercial DNA probe or once in 2 x SSC, 0.1% SDS at RT for 10 min, followed by two washes in 1 x SSC, 0.1% SDS at 55C for 15 min.
Detection
Unless explicitly stated, all incubations were performed at ambient temperature. For probes handled with EV the procedure was as follows: three washes in 1 x TBST (S3306; Dako) for 1 min; 30-min incubation in protein block (X909; Dako) at 37C; 30-min incubation in primary anti-biotin antibody diluted in antibody diluent (S0809; Dako); either an unconjugated monoclonal mouse antibody (M0743; Dako) diluted 1:20 or an enzyme-conjugated polyclonal rabbit antibody diluted 1:30 (HRP conjugate P5106, AP conjugate D5105; both from Dako) was used; 3 incubations in 1 x TBST for 1 min; 30-min incubation with the EV conjugate at 37C. The primary antibody was detected using either EVanti-mouseHRP (K4001; Dako) or EVanti-mouserabbitAP (K4018; Dako), three incubations in 1 x TBST for 1 min. Staining was completed by incubation in NBT/BCIP, Fast Red, New Fuchsin for AP detection or AEC+ (K3469; Dako) for HRP detection, and counterstaining with eosin and hemalum, respectively, for 30 sec. Probe detection with GP was performed with one cycle of biotinyltyramide incubation as described previously (
Slides hybridized with the PathoGene probe were treated according to the recommendations of the manufacturer if not processed with EV. Slides hybridized with the DIG-labeled oligonucleotide cocktail were treated with anti-DIGAP or anti-DIGHRP (Roche Diagnostic) diluted 1:100 for 30 min at 37C after to the blocking reaction.
Controls
Several controls, including solution-phase PCR (
Determination of Staining Intensities
Staining intensities of the chromogen were determined by measuring the IOD using the image analysis software ImagePro Plus3.01 (Media Cybernetics; Silver Spring, MD), which was adopted for cell detection by applying the integrated macro programming and VisualBasic 5.0 (Microsoft Corporation; Munich, Germany) (
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Results |
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To ensure an optimal comparison between conventional ISH and ISH with subsequent signal enhancement by EV, in all experiments the same enzyme-conjugated primary antibody was used as the first layer. EVHRP revealed a dramatic increase in signal intensity (Fig 1), exhibiting strong distinct signals without background staining. However, with EVAP no specific staining was generated (data not shown). This phenomenon was also reproducible with HRP- or AP-conjugated antibodies from other manufacturers.
Only the application of unconjugated antibodies resulted in a significant increase in signal intensity using EVAP, although the degree of amplification was less than that observed with EVHRP (Fig 2) for all chromogens tested. In contrast to EVHRP, EVAP often generates a filamentous background staining even after short incubation periods with the chromogen. This background staining could be reduced by a 30-min pre-incubation with a protein block at 37C. This additional step was not required for EVHRP.
Conditions initially optimized for ISH without signal enhancement could be maintained for EVHRP detection. However, for EVAP detection, either a prolonged incubation with proteinase K or a two- to threefold higher concentration of proteinase K was required for optimal results. For example, after doubling of the proteinase K concentration during permeabilization, EVAP exhibited an increased signal, whereas signal intensity with EVHRP decreased, as could be expected after overdigestion (data not shown). Nevertheless, even after optimization of the permeabilization step, EVAP detection showed a lower sensitivity than EVHRP (Fig 2) and often resulted in an inferior morphology due to the extended and more aggressive protease treatment.
When EVHRP and EVAP are compared, the former shows significantly shorter incubation periods for the chromogen to give equivalent signal intensities. However, prolonged incubation with the chromogen intensifies the diffusion artifact that is especially inherent to the slow NBT/BCIP reaction and therefore impairs spatial resolution (Fig 2). However, EVAP in combination with NBT/BCIP exhibits stronger staining than Fast Red or New Fuchsin (data not shown).
The signal amplification power of EV was estimated to be approximately 50- to 100-fold. Forty percent of samples that were negative with conventional ISH showed a positive signal with an optimized procedure using EVHRP. All positive results after signal enhancement could be verified by conventional solution-phase PCR as true positives (data not shown).
When the signal intensity achieved by EVHRP was compared to that of the biotinyltyramide-based amplification system, GP exhibited an IOD of two- to 10-fold higher as determined by the Image Analysis Program IPP3.01 (Fig 3).
Comparing the commercial probe with the oligonucleotides, they showed the same results, with the exception that the latter usually exhibited a lower signal intensity (data not shown).
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Discussion |
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Our study clearly demonstrates for the first time that the novel dextran polymer-based detection system EV, which up until now has only been reported to enhance sensitivity in immunoassays and immunohistochemistry, is also a potent tool for signal amplification in combination with ISH, thus dramatically increasing sensitivity and shortening assay times.
Whereas
However, in contrast to GenPoint, EV exhibited only minor unspecific background staining, especially when proteinase K was used for permeabilization, which did not interfere with correct interpretation as confirmed by solution-phase PCR. In this case, GenPoint may yield high background levels, which can be reduced by applying TRS for permeabilization, although this will simultaneously decrease sensitivity. This result is in agreement with the findings of
Especially when used with HRP and AEC+ as chromogen, EV shows a distinct staining pattern of individual cells without loss of spatial resolution. However, this problem will likely arise in applying EVAP, preferably when used in combination with NBT/BCIP, as a diffusion artifact that is inherent to the relatively slow NBT/BCIP reaction (
In two other studies (
In conclusion, EVHRP in combination with AEC+ appears to be the method of choice with regard to handling, spatial resolution, and background staining. EVHRP is also suitable for signal enhancement after ISPCR using the same protocol. The use of EV will enable the application of ISH even for the detection of low-copy nucleic acids and therefore confine the requirement for the application of the much more cumbersome method of IS-PCR. It also enables shorter assay times compared to conventional ISH- or tyramide-based amplification systems and easier handling (with respect to GP), although not showing the same amplification power as GP, which will yield a two- to tenfold higher IOD.
Received for publication May 2, 2001; accepted May 16, 2001.
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Literature Cited |
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