Early detection of SARS-CoV-2 in circulating immune cells in a mouse model.

SARS-CoV-2 infects the respiratory tract, lung and then other organs. However, its pathogenesis remains largely unknown. We used RareScope™ Fluorescence Light Sheet Microscopy (FLSM) and fluorescent in situ hybridization of RNA (RNA-FISH) to detect SARS-CoV-2 RNA and dissemination kinetics in mouse blood circulation. By RNA-FISH, we found that SARS-CoV-2 RNA-positive leukocytes, including CD11c cells, appeared as early as one day after infection and continued through day 10 post infection. Our data suggest that SARS-CoV-2-permissive leukocytes contribute to systemic viral dissemination, and RNA-FISH combined with FLSM can be utilized as a sensitive tool for SARS-CoV-2 detection in blood specimens.

Amplification of the 3q chromosomal region as a specific marker in cervical cancer.

OBJECTIVE: Chromosome 3q gain has been consistently observed in cervical intraepithelial neoplasia grades 2 and 3 (CIN 2,3) and squamous cell carcinomas of the cervix. There are a number of potential clinical uses of testing for 3q gain in liquid cytology specimens, including the identification of subsets of women with atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion cytology who are at greatest risk of having CIN 2,3 and would thus benefit most from immediate colposcopy. The objective of this study was to establish the sensitivity and specificity of 3q gain for discriminating between CIN 2,3 and normal. STUDY DESIGN: Residual cytology specimens were collected from 199 women. Liquid-based cytology (LBC) was used for the selection of subjects, with women with high-grade squamous intraepithelial lesion or high-grade squamous intraepithelial lesion who had colposcopy and adjudicated biopsy-confirmed CIN 2,3 forming the disease-positive group (n = 28) and women doubly negative for both cytology and high-risk human papillomavirus (hrHPV) testing forming the disease-negative group (n = 171). A single slide was prepared from each residual LBC specimen and analyzed for 3q gain by fluorescent in situ hybridization, using a probe specific for the 3q26 region and a control probe for the chromosome 7 centromere. Two approaches were compared for the determination of 3q gain. The first was based on the analysis of an entire cervical cytology slide for the presence of rare cells with a high copy number (>4 copies) for the 3q locus. The second approach was based on the analysis of 400 cells to determine the percentage with 3 or more copies of the 3q locus. RESULTS: Using the approach based on the detection of rare cells with a high copy number (>4 copies) for the 3q locus, 26 of the specimens from women with CIN 2,3 and none of the 171 specimens from women who were both hrHPV and cytology negative was positive for 3q gain. This translates to a sensitivity of 92.9% (95% confidence interval [CI], 76.5-98.9%), a specificity of 100% (95% CI, 97.8-100%), a positive predictive value of 100% (95% CI, 86.7-100%), and a negative predictive value of 98.8% (95% CI, 95.9-99.8), for distinguishing CIN 2,3 from normal. CONCLUSION: These data support the potential clinical use of 3q gain for the evaluation of women in a number of clinical situations, including women with atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesion, and those who are hrHPV positive.

Efficiency of manual scanning in recovering rare cellular events identified by fluorescence in situ hybridization: simulation of the detection of fetal cells in maternal blood.

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1-10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Determination of HER2 gene status by fully automated fluorescence microscopy.

OBJECTIVE: To examine fluorescence in situ hybridization (FISH) in HER2 amplification in response rates to trastuzumab therapy and both taxane and anthracycline-based chemotherapy regimens. STUDY DESIGN: A total of 400 tumor sections were analyzed over an 8-month period. The sections were hybridized with probes for the HER2 gene and chromosome 17 centromere using standard FISH methods and analyzed on an automated fluorescence microscopy system. RESULTS: Reliable and valid methods for identification of the patients that will respond to treatment with trastuzumab are needed in order to achieve maximum therapy efficacy and maintain cost efficiency. FISH-based analysis is potentially an objective and reproducible approach to determination of HER2 gene status; however, manual FISH counting is a laborious task and subject to inter and intraobserver variability. CONCLUSION: The system described in this paper is a valuable tool in providing a consistent approach to the interpretation of breast tumor tissue analyzed by FISH analysis. In addition to consistency, an automated system provides a record of the images produced that can be of immediate benefit in multiple review of a difficult or equivocal case and long-term benefit in terms of providing a permanent case record.

Digitized microscopy in the diagnosis of bladder cancer: analysis of >3000 cases during a 7-month period.

BACKGROUND: Fluorescent in situ hybridization (FISH) analysis of urine samples has proven to be a valuable adjunctive test to urine cytology for both diagnosis and monitoring recurrence of urothelial carcinoma. Automated FISH analysis has the potential to improve laboratory efficiency and to reduce interobserver and intraobserver variability, resulting in more accurate, reproducible, assay performance. METHODS: A total of 3200 slides containing urine specimens, hybridized with the UroVysion Bladder Cancer Kit (Abbott Molecular, Des Plaines, Illinois), a 4-probe set for chromosomes 3, 7, 17, and 9p21, was evaluated at Acupath Laboratories. The slides were analyzed over a 7-month period, using the Ikoniscope - oncoFISH bladder Test System (Ikonisys, New Haven, Connecticut). RESULTS: Analysis included the incorporation of a "flagging" system developed by Acupath Laboratories to identify cases, based on specific criteria, likely to benefit from further manual review. By using US Food and Drug Administration (FDA)-cleared scoring criteria, 96.3% of the slides could be reported directly from the automated scan, requiring no manual review of the slide. For the remaining 3.7% of the samples (all of which were very hypocellular), a manual review of each slide subsequently allowed diagnoses to be successfully reported. The average scan time was 31.7 minutes, and the average slide scan review time was 8.3 minutes. CONCLUSIONS: This study demonstrated the value of an automated approach to the analysis of FISH slides, affording the benefit of high-throughput while providing the user with the necessary images and tools to quickly and accurately report a case.

Gain of 3q26: a genetic marker in low-grade squamous intraepithelial lesions (LSIL) of the uterine cervix.

OBJECTIVE: Physicians have few resources for determining which LSIL will progress to HSIL or regress. Recently the chromosome 3q26 region was found to be amplified in patients with cervical cancer. The frequency of this 3q gain increased with severity of dysplasia. The primary objective of this study was to evaluate an automated FISH assay for detection of 3q gain in liquid cytology samples as a potential tool for risk stratification and triaging. METHODS: Slides prepared from 257 liquid cytology specimens (97 Negative, 135 LSIL 25 HSIL) were hybridized with a single-copy probe for the chromosome 3q26 region and a probe for the centromeric alpha-repeat sequence of chromosome 7, using standard FISH methods. Using automated analysis, the total number of nuclei and the number of nuclei with >2 signals for 3q26 were determined, using a 20x objective. The nuclei were rank ordered based on number of 3q26 FISH signals. The 800 nuclei with the highest number of signals were scored using both FISH probes and nuclei with increased numbers of 3q signals were enumerated. RESULTS AND CONCLUSIONS: Analysis of 257 specimens demonstrated that a fully automated FISH scoring system can detect 3q gain in liquid cytology samples. A fully automated method for determination of 3q gain in liquid cytology may be the assay necessary to implement routine testing. Additional studies to validate the utility of this technology are needed.

Detection of circulating fetal cells utilizing automated microscopy: potential for noninvasive prenatal diagnosis of chromosomal aneuploidies.

OBJECTIVE: As fetal cells can be indisputably identified through detection of Y FISH signals, we utilized an automated microscopy system developed to identify and enumerate cells bearing X and Y FISH signals. We further investigated the potential of fetal hemoglobin expression as a gender independent marker for automated identification of fetal cells. METHOD: For FISH-based scanning, verified fetal cells were identified based on the presence of a single X-signal and individual signals for each of the two Y FISH probes. For cell identification based on fetal hemoglobin expression, putative fetal cells were verified based on the presence of signals for anti-gamma or anti-epsilon globin antibody, and FISH signals for the X- and Y- chromosomes. RESULTS: Fetal cells were identified, by FISH-based scanning, in 28 of the 29 maternal samples from pregnancies with male fetuses. Simple density gradient centrifugation achieved a 3- to 5-fold increase in the number of fetal cells detected. CONCLUSION: Automated microscopy identified fetal cells in both first and second trimester maternal blood samples. Although we were unable to detect fetal erythroblasts in numbers sufficient for clinical diagnosis, the ability to reliably detect fetal cells by FISH-based scanning opens the possibility for prenatal detection of chromosomal aberrations utilizing circulating fetal cells.

Fully automated FISH examination of amniotic fluid cells.

OBJECTIVE: Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. RESULTS: A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. CONCLUSION: These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes.

Automated microscopy of amniotic fluid cells: detection of FISH signals using the FastFISH imaging system.

OBJECTIVE: FISH (fluorescence in situ hybridization) analysis is a valuable adjunct to cytogenetics that provides a rapid screen for common abnormalities. However, FISH is expensive, labor-intensive, and requires a high skill level and subjective signal interpretation. A fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: In this study we blindly compared automated FISH signal acquisition and display against standard FISH analysis. A total of 62 amniocentesis samples were prepared using the AneuVysion multicolor DNA probe kit and probed for chromosomes 13, 18, 21, X, and Y. Two sets of slides were produced from each sample. Fifty cells were scored in each slide. One set was evaluated using standard manual microscopy and the other using the automated image acquisition and display capabilities of the Ikoniscope fastFISH amnio Test System. This system uses epifluorescence optics, along with optimized slide management to process slides automatically. RESULTS: A 100% concordance was observed between the results obtained using manual microscopy and the automated system. There was also 100% concordance between the FISH results and those obtained by conventional karyotyping. CONCLUSION: Our data suggest that the automated system is capable of providing accurate and rapid identification and display of cells and FISH signals.

Automated detection of rare fetal cells in maternal blood: eliminating the false-positive XY signals in XX pregnancies.

OBJECTIVE: The purpose of this study was to develop a new method to help differentiate XX from XY signals in maternal blood from women carrying XY fetuses. STUDY DESIGN: We have developed a system to scan automatically for cells that bear X and Y fluorescence in situ hybridization signals. These XY target cells are identified by scans at low (x20) magnification, and all identified targets are revisited and verified at high (x100) magnification. The viewer software component of the system displays x20 images of all cells and intracellular fluorescence in situ hybridization signals that are present in each of the 4000 optical fields per slide, along with x100 images of automatically detected target cells. RESULTS: We initially examined 36,000 fields from 18 slides in 12 pregnancies (6 male and 6 female) using our system that is based on fluorescence in situ hybridization with a single probe for the X-chromosome and a single probe for the Y-chromosome and found XY nuclei in all samples, regardless of fetal gender. In the second phase of the study, a refinement of the approach that incorporated 2 independent probes for the Y-chromosome resulted in a false-positive rate for detection of XY nuclei in XX cases <0.00005%. CONCLUSION: Our data suggest that this system may allow for excellent "signal to noise" separation, which is required absolutely for fetal cell methods to differentiate aneuploid from normal pregnancies. Quantitation of fetal cells in the maternal circulation and standardization of processes that have been developed for their enrichment are crucial to moving fetal cell assessment from esoteric basic science to applied new technology.