In situ hybridization with human papillomavirus using biotinylated DNA probes on archival cervical smears.

We report a method of in situ hybridization (ISH) of 10-year-old archival cervical smears with a cocktail of nick-translated human papillomavirus (HPV) DNA types 6, 11, 16, 18, and 31. The method, which does not require destaining, results in excellent preservation of morphological detail with only 2% cell loss. Methods of smear treatment and detection of the biotinylated probe with a multistep avidin-biotin-immunoperoxidase method are described. Biotinylated PBR 322 plasmid and biotinylated human DNA were used as negative and positive controls in each run. Twenty-nine of 50 smears (58%) showing changes consistent with CIN I-II were positive for HPV. Fourteen corresponding cervical biopsies were also studied by ISH, seven corresponding to HPV-positive smears and seven to HPV-negative smears. HPV DNA was demonstrated in six of seven biopsies (87%) from the positive group but none could be demonstrated in the negative group. We conclude that retrospective study can be performed on routine alcohol-fixed, Papanicolaou-stained cervical smears with biotinylated HPV probes with excellent cell preservation, minimal cell loss, and high degrees of specificity.     

Human papillomavirus DNA detection in Papanicolaou-stained cervical smears with a nonradioactive, in situ hybridization assay.

Archived Papanicolaou-stained cervical smears from women with different cervical pathologies were processed for human papillomavirus (HPV) DNA detection and typing with an in situ hybridization (ISH) assay that employed commercial biotinylated HPV DNA probes. Two HPV DNA probes were utilized: one included HPV genotypes 6/11 and the other, 16/18. The method yielded positive results for HPV DNA 6/11 in 5 cases with condylomata acuminata (100%) and in 2 of 47 with flat warty lesions (4.2%), whereas HPV DNA 16/18 was detected in 29/47 of the latter group (61.7%). In cases with cervical intraepithelial III or invasive squamous cell carcinoma the yield was lower: positive results for HPV DNA 16/18 were obtained in only one of the five cases with one or the other cervical pathology (20%). An analysis of the results showed that the sensitivity of the assay correlated with evidence in the Papanicolaou specimens of pathognomonic cell injury from HPV infection. In the presence of such cytologic features, HPV DNA typing was possible in 37/52 cases (65.4%). In view of the modest difficulty and relatively quick execution of the nonradioactive ISH assay, the authors believe that Papanicolaou cervical smears with cytologic changes of HPV infection could be processed by this method in order to acquire information on the HPV type or types involved in the cervical infection.     

Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization, immunocytochemistry and radioimmunoassay.

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt2cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNAs (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt2cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt2cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt2cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.     

HOPE fixation of cytospin preparations of human cells for in situ hybridization and immunocytochemistry.

In primary or cultured cells, in situ hybridization (ISH) or immunocytochemistry (ICC) is often performed on tissue that has been fixed by paraformaldehyde or Carnoy's. Recently we reported an optimized HOPE (HEPES-glutamic acid buffer-mediated organic solvent protection effect) fixation protocol for ISH targeting mRNA in lung tissues. We have now examined whether HOPE fixation could also be used on in vitro cultured cells for targeting mRNA by ISH or proteins by ICC on cytospin preparations. Using the myeloid stem cell line KG-1a as a model system, we showed that HOPE fixation can be applied for ISH and ICC on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immunohistocytochemical and molecular techniques.     

Staining reaction for beta-galactosidase in immunocytochemistry and in situ hybridization.

beta-galactosidase, revealed by an indigo blue reaction product, represents a valid tracer in immunohistochemistry. Observations on the instability of the indigo precipitate led us to investigate this phenomenon. We conclude that avoiding of xylene, and mounting of the preparates in Histoclear (a xylene substitute) and Canada balsam (instead of synthetic resin mountants) yields a sharp and stable indigo precipitate. In addition, we propose a sensitive alternative staining procedure for beta-galactosidase, based on TNBT reduction and precipitation. These reactions can be used both in immunohistochemistry and in in situ hybridization.     

Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization,immunocytochemistry and radioimmunoassay

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt₂cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNA_s (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt₂cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt₂cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt²cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.     

HPV DNA detection by in situ hybridization with catalyzed signal amplification on thin-layer cervical smears.

Thin layer-based technology in cervical cancer screening now allows both Papanicolaou staining and HPV testing on the same sample. Here, we show that in situ hybridization with catalyzed reporter deposition is a powerful HPV detection method when applied on thin-layer cervical smears, allowing distinction between two staining patterns suggestive of two different physical states of HPV DNA, where diffuse signals are suggestive of episomes and punctate signals are suggestive of viral DNA integration.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry.

Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Glass slide models for immunocytochemistry and in situ hybridization

We have developed a model system based on the immobilization of microdroplets (0.1 l volumes) of antigens or of target (sense) oligonucleotides onto aminoalkylsilane-coated glass slides. Oligopeptide antigens need to be vapour-fixed in order to achieve efficient immobilization, while oligonucleotides do not require fixation. Protein antigens, exemplified by rabbit immunoglobulin, may be subjected to liquid fixation. The glass slide models are optically translucent and useful both for in situ hybridization and immunocytochemistry. The slides are compatible with detection systems of both light and fluorescence microscopy and permit measurement of staining intensities by microfluorimetry or computerized microdensitometry. The model systems can be used for comparisons of method sensitivities, for characterizing antibody and probe sensitivities and cross-reactivities, and as internal standards or quality controls for immunocytochemistry and in situ hybridization.     

Comparison of the detection of cervical human papillomavirus infection by filter DNA hybridization of cytologic specimens and by in situ DNA hybridization of tissues

Cellular samples and subsequent cone biopsy samples from the same site in 18 patients were screened for infection with human papillomavirus (HPV) types 16 and 18 (HPV 16/18) by DNA hybridization. Filter hybridization of cells collected using cervical swabs was significantly less sensitive (with only 4 positive results) in detecting HPV 16/18 DNA sequences than was in situ hybridization of tissue sections (with 16 positive results). The in situ hybridization results correlated well with the cytologic and histologic findings of cervical intraepithelial neoplasia of grades II (mild dysplasia) and III (severe dysplasia and carcinoma in situ).     

Clinical relevance of human papillomavirus testing in cytopathology

Cancer of the uterine cervix is the second most common cancer in women worldwide. Currently, cervical screening is based on cytology alone. Because infection with high-risk human papillomavirus types (hrHPVs) is a necessary cause of cervical cancer, it has been postulated that screening might become more efficient when it is based on combined cytology and hrHPV testing. In this review we will discuss the advantages of added HPV tests in cervical cancer screening, as a quality control for false-negative smears, in triage of women with equivocal smears, in follow-up of women treated for CIN3 or cervical cancer and for the detection of cervical adenocarcinoma.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry

Summary Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Applications of fluorescence in situ hybridization in cytopathology: a review

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that is a powerful and versatile research tool and an important adjunct to tumor diagnosis and prognosis. Akin to the recent role of immunohistochemistry, research and clinical applications of FISH in cytopathology have been growing enormously over the last few years. Furthermore, due to its feasibility for virtually all types of cytologic specimens, a new and exciting era in both investigative and clinical cytopathology is expected.     

Detection of human papillomavirus (HPV) in genital warts and carcinomas by DNA in situ hybridization in Chinese patients

A series of 51 genital biopsies from normal epithelium, condylomata acuminata, leucoplakia and squamous cell carcinoma from Chinese male and female patients were analysed for the presence of human papillomavirus (HPV) types 6, 11, 16 and 18 by DNA in situ hybridization. All of the nine genital condyloma acuminata were positive for HPV DNA, in which HPV 6 was found in six cases, HPV 11 in two cases and HPV 18 in one case. Twelve out of the 21 cases (57.1% of the total) of cervical squamous cell carcinoma were shown to contain HPV DNA; HPV 16 was found in nine cases, HPV 18 in two cases and HPV 16/18 in one case. Present results support the earlier concept that HPV 6/11 are closely associated with benign genital lesions, and HPV 16/18 are mostly confined to higher grade of intra-epithelial neoplasias and carcinoma.     

Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.     

Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ polymerase chain reaction

Classical in situ hybridization (ISH) with biotinylated probes makes it possible to detect and localize human papillomavirus (HPV) nucleic acid sequences in cytological and histological materials. This method is however of limited value in the detection of a few copies of the virus. Moreover the specificity of such a technique is not always convincing when ISH signals are small and/or of low intensity. Recently, much attention has been focused on the utility of the in vitro polymerase chain reaction (PCR) and especially on PCR-single strand conformation polymorphism (SSCP) to amplify small amounts of viral DNA with accurate hybrid specificity. But the latter method requires nucleic acid extraction and tissue destruction. Thus, correlation between the PCR results and histological findings is not possible. Hence, the aim of our current study was to apply to HeLa cells and cervical formalin-fixed and paraffin-embedded biopsies, a novel procedure of ISH signal amplification, the catalyzed signal amplification (CSA). Such a procedure is based on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probed target. The biotin accumulation is then detected with streptavidin peroxidase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification successfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a method was found simpler and faster than in situ PCR and tissue morphology was better preserved.