Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry

Abstract

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical Smears. Q 1994 Wiley-Liss, Inc.Key terms: Fluorescent in situ hybridization, cervical smears, human papillomavirus, automated fluorescence image cytometry Cervical dysplasia and carcinoma are common among women throughout the world. Approximately 500,000 women worldwide are diagnosed annually as having cervical cancer (24) and the mortality rate associated with this malignancy is second to breast cancer (20). In the United States, it is estimated that 13,000 women develop invasive cervical cancer annually (25).Substantial evidence has accumulated associating specific human papillomaviruses (HPVs) with human anogenital disorders, most notably cervical cancer (3,11,13,35). To date, over 60 different types of HPV have been described, of which about 20 have been associated with anogenital lesions (8). These 20 HPVs have been divided into "high risk" and "low risk" groups based on their association with benign vs. ma-'Supported by grants from NIH (CA51323 and the Office of Research on Womens Health), the Whitaker Foundation, and a contract with American Innovision, Inc.

Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

“…Labeling of the full-length DNA insert with digoxigenin using nick-translation (Boehringer Mannheim) resulted in a lower signal in CaSki cells compared with the D-DNA probe (data not shown). Method 1 enabled us to detect as few as one to five copies of HPV-16 in 100% of SiHa cells ( Figure 2C) compared with 60% for Method 2 (data not shown) (22). Lack of signal in C33-A and HT-3 cells (data not shown) with the D-oligo ( Figure 2D) and the D-DNA probes (data not shown) demonstrates the specificity of the probe labeling.…”

“…This assessment demonstrated that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one to five HPV genomes per nucleus (22). Because of the linearity of AFIC, we were able to collect images with different integration times, normalize the data, and subsequently compare them (Table 2).…”

“…we did not apply them in this study because the oligonucleotide probes showed sufficient sensitivity without amplification. In this investigation, we used a fluorescent substrate for alkaline phosphatase (Vector red substrate) for the following reasons: the quantity of its reaction product in the cell nuclei could be controlled by the incubation time; it is visible under both brightfield and fluorescence illumination; it does not photobleach significantly; the signal remains stable for several months; and its fluorescent intensity is directly proportional to the HPV copy number per cell (22). Overall, our data indicate that Method 1, using oligonucleotide tail-labeled probes, had the highest sensitivity compared with the other methods.…”

Automatic detection of clustered, fluorescent‐stained nuclei by digital image‐based cytometry

“…The images are stored digitally in computer memory from where they are accessible for analysis by software algorithms. IC is now a primary technique for understanding normal and pathological cellular mechanisms, because it is possible to quantitatively and nondestructively measure wide ranges of biochemical, morphological, densitometric, and contextual parameters on the individual cells and nuclei in the specimens (2,10,21,23,24,25,29,38,40).The convenient measurement of many individual nuclei requires image analysis algorithms that automatically locate every nucleus. This i s usually accomplished by staining the nuclei in such a way that their corresponding image intensities are significantly different from the background intensities.…”

“…The images are stored digitally in computer memory from where they are accessible for analysis by software algorithms. IC is now a primary technique for understanding normal and pathological cellular mechanisms, because it is possible to quantitatively and nondestructively measure wide ranges of biochemical, morphological, densitometric, and contextual parameters on the individual cells and nuclei in the specimens (2,10,21,23,24,25,29,38,40).…”