ARTICLE |
Correspondence to: Harold M.J. Kerstens, Dept. of Pathology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: h.kerstens@pathol.azn.nl
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Summary |
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In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer. (J Histochem Cytochem 48:709718, 2000)
Key Words: cell block processing, cytology, molecular diagnosis, uterine cervix, immunocytochemistry, in situ hybridization, PCR, image cytometry
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Introduction |
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In diagnostic cytology of the uterine cervix, cytomorphological evaluation of Papanicolaou-stained smear preparations (Pap smears;
To improve the reliability of diagnostic cytology, molecular techniques can be used to detect non-morphological markers as an adjunct to the conventional cytomorphology. Potential molecular biomarkers of cervical intraepithelial lesions are now under investigation and await implementation in routine diagnostic procedures. Candidate ancillary tests include monitoring for Ki-67 and p53 expression with immunocytochemistry (
Additional molecular techniques can be applied only if a sufficient number of preparations are made from a single cell sample. In present day practice, however, only a single smear is made for routine examination. Valuable diagnostic cells present on the brush are discarded but might be used for the preparation of extra microscopic specimens (
The aim of this study was to develop a cell block processing procedure (AgarCyto) for brush material of the uterine cervix that would enable us to assess cytomorphology and to perform multiple ancillary molecular techniques using standard laboratory protocols. Here we describe the optimization of fixation and cell embedding concerning cytomorphology and signal-to-noise ratio of immunocytochemical staining and in situ hybridization techniques. Furthermore, we describe the use of AgarCyto sections for HPV typing by line probe assay (LiPA)-PCR and an additional step in the protocol for DNA index measurements by image cytometry.
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Materials and Methods |
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Cell Culture and Cytological Specimens
For the development of the AgarCyto cell block procedure and the protocols for ancillary molecular techniques, five different cell lines were used. Human cervical carcinoma cell lines HeLa, SiHa, and CaSki, human lymphoblast leukemia cell line Molt-4, and human vulval carcinoma cell line A431 were used for optimization of immunocytochemistry (ICC), in situ hybridization (ISH), and PCR. A431 and Molt-4 were also used for optimization of DNA image cytometry. All cell lines were obtained from the American Tissue and Cell Collection (ATCC; Manassas, VA) and were cultured according to the supplier's instructions. For validation of the techniques on clinical samples, 50 cervical scrapings were collected at the Department of Obstetrics & Gynecology (University Hospital Nijmegen, The Netherlands).
Fixations
To optimize for a good balance between cytomorphology and signal-to-noise ratios of ICC and ISH, chessboard experiments were performed in which fixation types and times were varied (Table 1). Subconfluently grown cell cultures were trypsinized and fixed in suspension for 2, 16, and 64 hr in one of the following fixatives: (a) 50% ethanol/2% carbowax, a standard collection fluid in cytology (
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Clinical material was obtained using a Cervex brush (Rovers; Oss, The Netherlands), and a routine cervical smear was made. The brush with remnant material was put into a storage vial with primary fixative and transported (Fig 1A). Within 16 hr, cervical cells were transferred from the transport vial into 50-ml tubes ( Fig 1B), spun down at 2000 rpm for 10 min, secondarily fixed in suspension for up to 64 hr, and processed into AgarCytos.
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AgarCyto Cell Block Preparation
The AgarCyto cell block preparation is based on earlier protocols described by
Immunocytochemistry
ICC detection for Ki-67 and p53 in AgarCytos of tumor cell lines and cervical scrapings was demonstrated by standard immunocytochemical procedures. Four-µm-thick sections were dewaxed in xylol and methanol and endogenous peroxidase was blocked for 15 min in 1% H2O 2 in methanol. Antigens were unmasked by microwave heating in buffered 0.01 M citrate solution, pH 6.0, for 3 min at 850 W and 10 min at 150 W, and cooled to room temperature (RT) in the same solution for 30 min. Preparations were rinsed in PBS for 20 min. All primary antibodies were diluted in PBS and incubated overnight at 4C, and all following antibodies were diluted in PBS/1% bovine serum albumin (BSA; Sigma, St Louis, MO) and incubated for 30 min at RT. All washings between the incubations were performed in PBS. For p53 immunodetection, slides were preincubated with 10% normal horse serum in PBS and then incubated with DO-7 monoclonal antibody (MAb) against wild-type and mutant p53 (1:50; Novocastra, Newcastle, UK), biotinylated horse anti-mouse (1:200; Vector Laboratories, Burlingame, CA), and horseradish peroxidase avidinbiotin complex (ABC Elite; Vector Laboratories). Ki-67 was detected, without preincubation, by MAb Mib-1 (1:50; Dianova, Hamburg, Germany), followed by horseradish peroxidase-conjugated rabbit anti-mouse (1:100; DAKO, Glostrup, Denmark). Peroxidase was visualized with 0.05% diaminobenzidine (DAB; Sigma)/0.15% H2O2 in PBS for 5 min at RT. Specimens were counterstained with hematoxylin and mounted in Permount (Fisher Scientific; Fair Lawn, NJ).
In Situ Hybridization
DNA probe pUC1.77 for the centromere region of chromosome 1 (cen 1;
For detection of HPV 16 DNA, the catalyzed reporter deposition (CARD) signal amplification method was used (
Microscopic Evaluation
To assess the quality of the experimental immunostaining, the results were correlated to Ki-67 and p53 immunoexpression levels of these cell lines as known from the literature. Ki-67 antigen is expressed in proliferating cells and is therefore present in more than 50% of cells of a subconfluent cell culture (
Microscopic evaluation of AgarCytos was performed on a Leica Dialux transmission microscope (Leica; Wetzlar, Germany) equipped for digital image capture with a 3-CCD color video camera (DXC-950P; Sony, Tokyo, Japan) and a computer-interfaced frame grabber (Intellicam; Matrox Electronic Systems, Dorval, QC, Canada). Composite figures were produced using Adobe Photoshop 5.0.
Line Probe AssayPolymerase Chain Reaction
LiPA-PCR for simultaneous detection of 25 HPV types was performed as described (
DNA Image Cytometry (ICM)
DNA ICM requires a microscopic preparation technique that preserves nuclear integrity. Therefore, to include DNA image cytometry in a multiparameter analysis of a cervical scraping, a 200-µl sample has to be taken from the primary fixed cell suspension for cytospin preparation before AgarCyto embedding. We used Molt-4 and A431 cell lines for optimization of the sample preparation. The cell suspensions were primarily fixed for 16 hr in (a) 50% ethanol/2% carbowax, the standard cell fixative for DNA image cytometry (
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Results |
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AgarCyto Cell Block Preparation
We optimized the cell block preparation for exfoliate cell material with respect to cell yield by (a) pelleting the cell suspension in agar in a 1.5-ml Eppendorf tube (Fig 1A1C) as described by
Cytomorphology
AgarCyto cell block sections of differently fixed cell lines with known morphological features were H&E-stained for morphological evaluation ( Table 1). The ethanol-containing fixatives (ethanol/carbowax, ethanol/carbowax/formaldehyde, and CytoRich) gave good cell morphology if the fixation lasted no longer than 16 hr. The cell shape was maintained and a normal nucleocytoplasmic ratio was observed. Longer fixation times compromised morphology. A fair morphology was observed after fixation in ethanol/carbowax or CytoRich for 16 hr or in ethanol/carbowax/formaldehyde for 64 hr. Neutral-buffered formaldehyde resulted in shrinkage of the cells and therefore overall poor morphology. In contrast, Unifix, a modified formaldehyde fixative, gave good morphology even when the cells were fixed for 64 hr. Cytomorphology of cervical scrapings after fixation in Unifix and AgarCyto processing was well preserved (see Fig 3A), and dysplastic groups were easily recognized. The morphological features used for the classification of Pap smears also apply to AgarCytos, with minor modifications. A comprehensive comparison between cytomorphology of Pap smears and AgarCytos is pending and will be published elsewhere.
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Immunocytochemistry
The influence of different fixatives and fixation times on immunocytochemical staining for Ki-67 and p53 using standard laboratory protocols was monitored with regard to signal intensity, background staining, and staining efficiency Table 1. The cell morphology was not altered by the immunostaining procedure. Using the ethanol-based fixatives, Ki-67 immunodetection resulted in weak to moderate staining of the nuclei in only a small fraction of the cells, and the cells showed a high background staining. Prolonged fixation in ethanol-based fixatives generally decreased the intensity of immunostaining. Cells fixed in neutral-buffered formaldehyde gave strong, specific nuclear staining for Ki-67 in a representative fraction of the cells, but also showed some background. Best results were obtained with Unifix-fixed cells, which gave strong Ki-67 immunostaining without any background in a representative fraction of the cells.
P53 immunostaining of the positive control cell line A431 was strongly positive with all fixation protocols. Antigen retrieval steps were required to detect low levels of wild-type and mutant p53 in cervical tumor cell lines and Molt-4, respectively. Using different fixation protocols, we observed differences in the staining efficiency and signal-to-noise ratio for the detection of low-level p53. Immunostaining for p53 was weak in ethanol/carbowax and negative in CytoRich. In contrast, fixation with ethanol/carbowax/formaldehyde gave moderate p53 immunostaining, with a lower but still considerable background. Longer fixation times decreased the intensity of immunostaining. Neutral-buffered formaldehyde and Unifix gave clear, specific nuclear staining for p53 in a representative fraction of the cells. Best results were obtained with fixation in Unifix for 64 hr, because the signals were clearly observed without any background staining. Using the Unifix fixation protocol, AgarCyto preparations of cervical scrapings gave good signal-to-noise ratios for Ki-67 and p53 in dysplastic groups (Fig 3BC).
In Situ Hybridization
ISH results for the centromere of chromosome 1 (cen 1) and HPV Type 16 (HPV 16) were evaluated on the basis of signal intensity, signal shape, nuclear morphology, and number of ISH signals as known from the literature (Table 1). To facilitate probe penetration, crosslinked proteins were removed by a proteolytic enzyme pretreatment. To standardize the experimental protocol, the differently fixed AgarCytos were all pretreated equally with sodium isothiocyanate and pepsin. Fixation for 2 hr in ethanol/carbowax or CytoRich resulted in moderate to strong ISH signals and good nuclear morphology. However, fixation for 16 or 64 hr resulted in fuzzy ISH signals that were difficult to discern. Ethanol/carbowax/formaldehyde fixation for 2 hr showed better results: discrete ISH signals and good nuclear morphology were observed, but the signal intensity decreased rapidly with prolonged fixation. Neutral-buffered formaldehyde resulted in strong and discrete ISH signals, and although overall cell morphology was poor (cell shrinkage), the nuclear morphology was fair. Best results were obtained with Unifix after 64 hr of fixation, because strong and discrete ISH signals were observed and overall cell morphology was very well maintained. Consistent ISH results for cen 1 and HPV 16 were obtained on AgarCyto sections of cervical scrapings that underwent the Unifix fixation protocol. In dysplastic cell groups, the ploidy status for cen 1 could be determined ( Fig 3D). HPV 16 ISH combined with CARD signal amplification specifically detects HPV 16 sequences in individual cells and is able to discriminate episomal from integrated infection ( Fig 4).
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LiPAPCR
DNA isolated from a single 4-µm-thick AgarCyto section has proved sufficient for reliable HPV screening with LiPAPCR, irrespective of the type of fixative (Table 1). A431 that does not contain any HPV sequences was always negative. Examples of HPV detection and typing by LiPAPCR in AgarCyto cervical scrapings are given in Fig 5.
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DNA Image Cytometry
To allow DNA ICM of whole nonsectioned cells, a small fraction of the primarily-fixed cell suspension was reserved for cytocentrifugation before further processing into AgarCytos (Fig 2). We compared two fixatives (70% ethanol and Unifix) with the standard fixative (50% ethanol/2% carbowax) concerning the DNA index (DI) on Molt-4 and A431 cell lines (Table 2). After ethanol/carbowax fixation, the DI of Molt-4 cells was 2.17 and of A431 cells was 2.02. The DI of Molt-4 was 2.16 after 70% ethanol fixation and 2.00 after Unifix fixation, which is lower than the reference value. For A431 cells, the DI values differed from their reference DI value more prominently, i.e., 1.92 for 70% ethanol and 1.86 for Unifix. Moreover, Unifix-fixed cells did not stick well to the glass slides. Ethanol/carbowax fixation gave the best results for DNA ICM. For further processing of the remaining cells into AgarCytos, a secondary fixation in Unifix is required within 16 hr to maintain good cytomorphology and successful application of ICC and ISH.
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Discussion |
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We have developed the AgarCyto cell processing method for multiple diagnostic analyses of a single brush sample from the uterine cervix. The AgarCyto processing method includes preparation of a routine Pap smear and the collection and processing of the remaining cells on the brush in paraffin. Multiple microscopic specimens can be produced from a single scraping for the application of standard H&E staining, ICC, and ISH. Furthermore, a single AgarCyto section can serve as DNA input for LiPAPCR and, before AgarCyto embedding, cytospin preparations can be produced for DNA image cytometry.
In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Up to now, diagnostic molecular markers for cervical cancer have been identified from retrospective studies on histological material or re-stained archival Pap smears. Prospective studies on cytological material are hampered by insufficient amounts of diagnostic specimens from the same patient. Cell block processing methods of cytological material may solve this problem, but none of the developed methods describes comprehensive application of molecular techniques in a routine setting. Our methodological research has focused on the application of a wide range of molecular diagnostic techniques, using both in situ techniques, while maintaining cell morphology, and biochemical techniques.
The protocol for AgarCyto processing of cervical scraping material is summarized in Fig 2. We recommend primary fixation of the cells in ethanol/carbowax. The cell suspension can be transported and, if so desired, cytospin preparations can be produced for DNA image cytometry. Cells must be secondarily fixed in Unifix within 16 hr, however, to ensure good cell morphology in ICC and ISH staining of the AgarCyto. Therefore, if DNA image cytometry is not performed, primary fixation in Unifix is preferred.
The fixative that yielded the best overall results was Unifix, a 4% formaldehyde-based fixative containing zinc sulfate, methanol, and other constituents. We used standard protocols for ICC and ISH with each fixation type. It is conceivable, however, that by adjusting the ICC and ISH protocols optimal results would have been obtained with each fixation. In particular, ethanol/carbowax and ethanol/carbowax/formaldehyde fixation for 2 hr yielded good overall results that may need slight modification of the ICC protocol. However, short fixation times are not preferred in routine laboratory practice. Cervical scrapings are usually obtained in the clinic and must be transported to the diagnostic laboratory. Therefore, 16-hr (i.e., overnight) ethanol/carbowax and subsequent Unifix fixation, which is allowed to last 64 hr (i.e., over the weekend), provides ideal logistics and yields the best overall results.
Sensitivity of the immunostaining procedure for p53 was dependent on the use of fixation and antigen retrieval methods (
HPV typing of the cervical cell lines by LiPAPCR was highly sensitive and specific, regardless of the type of fixation. This result underlines the robustness of the LiPAPCR technique. The application of LiPA-PCR, using input DNA from AgarCytos of a large group of women diagnosed with a cervical neoplasia, will be discussed elsewhere (
Our findings with Unifix fixation are in accordance with earlier reports on the use of Zn-containing formaldehyde fixatives. Good results have been obtained with ISH for HPV 16 DNA in CaSki and SiHa cells (
The AgarCytos could be valuable for routine cytomorphological diagnosis. Although the preparation procedure is more time-consuming than smears or monolayers and the cytomorphology of AgarCytos looks somewhat different from routine smear preparations due to sectioning, a favorable feature of AgarCyto morphology is the histology-like appearance (Fig 3A). Cell clumping, as often observed in smears and causing dubious diagnoses, are easier to interpret in AgarCyto sections because cell polarity and differentiation can be examined just as in histology. AgarCyto diagnosis may be of use if smear preparations are inadequate because of too many cell clumps. However, clinical studies need to be performed to validate cytopathological diagnosis based on AgarCytos. Once the diagnostic cytomorphological criteria of AgarCytos have been determined, AgarCytos will be the ideal vehicle for molecular inventory studies. Both retrospective and prospective studies can be employed to identify biomarkers of premalignant lesions of cervical cancer and other types of cancer that depend on screening of cytological material. In our experience, AgarCyto cell processing can be applied equally well to uterine cervix brushes, oral cavity brushes, urine, bladder washes, lung sputa, bronchial washes, and fine-needle aspiration samples. Therefore, AgarCytos cell processing should become a routine cytological laboratory practice.
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Footnotes |
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1 Present address: Division of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
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Acknowledgments |
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Supported by the Dutch Cancer Society KUN 97-1486.
We wish to thank A. Gemmink and S. Wienk for excellent technical assistance, I. Cornelissen for culturing cells, H. De Leeuw for DNA image cytometry measurements, and Prof Dr J. Merkus, Dr B. Keijzer, Dr L. Massuger, and C. Schijf for valuable scientific discussions.
Received for publication December 6, 1999; accepted January 19, 2000.
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