2-color Fluorescence Research Articles

Analytical validation of the LungLB test: a 4-color fluorescence in-situ hybridization assay for the evaluation of indeterminate pulmonary nodules

BackgroundEvaluation of indeterminate pulmonary nodules (IPNs) often creates a diagnostic conundrum which may delay the early detection of lung cancer. Rare circulating genetically abnormal cells (CGAC) have previously demonstrated utility as a biomarker for discriminating benign from malignant small IPNs in the LungLB assay. CGAC are identified using a unique 4-color fluorescence in-situ hybridization (FISH) assay and are thought to reflect early cell-based events in lung cancer pathogenesis and the anti-tumor immune response. LungLB is a prognostic tool that combines the CGAC biomarker and clinical features to aid in IPN evaluation by improving the stratification of patient risk of malignancy.MethodsHerein we describe the analytical performance of the LungLB blood test. Analytical validation was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with adaptations for rare cell-based assays. Multiple operators, reagent lots, and assay runs were tested to examine accuracy, precision, reproducibility, and interfering factors.ResultsThe FISH probes used in the LungLB assay demonstrate 100% sensitivity and specificity for their intended chromosomal loci (3q29, 3p22.1, 10q22.3 and 10cen). LungLB demonstrates analytical sensitivity of 10 CGAC per 10,000 lymphocytes analyzed, 100% analytical specificity, and high linearity (R2 = 0.9971). Within run measurements across 100 samples demonstrated 96% reproducibility. Interfering factors normally found in blood (lipemia, biotin) and exposure to adverse temperatures (-20ºC or 37ºC) did not interfere with results. Sample stability was validated to 96 hours.ConclusionThe analytical performance of LungLB in this validation study successfully demonstrates it is robust and suitable for everyday clinical use.

Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy.

Understanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30nm spatial resolution. We developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30nm lateral resolution. We used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM). We demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites. Our optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.

The presence of circulating genetically abnormal cells in blood predicts risk of lung cancer in individuals with indeterminate pulmonary nodules

PurposeComputed tomography is the standard method by which pulmonary nodules are detected. Greater than 40% of pulmonary biopsies are not lung cancer and therefore not necessary, suggesting that improved diagnostic tools are needed. The LungLB™ blood test was developed to aid the clinical assessment of indeterminate nodules suspicious for lung cancer. LungLB™ identifies circulating genetically abnormal cells (CGACs) that are present early in lung cancer pathogenesis.MethodsLungLB™ is a 4-color fluorescence in-situ hybridization assay for detecting CGACs from peripheral blood. A prospective correlational study was performed on 151 participants scheduled for a pulmonary nodule biopsy. Mann-Whitney, Fisher’s Exact and Chi-Square tests were used to assess participant demographics and correlation of LungLB™ with biopsy results, and sensitivity and specificity were also evaluated.ResultsParticipants from Mount Sinai Hospital (n = 83) and MD Anderson (n = 68), scheduled for a pulmonary biopsy were enrolled to have a LungLB™ test. Additional clinical variables including smoking history, previous cancer, lesion size, and nodule appearance were also collected. LungLB™ achieved 77% sensitivity and 72% specificity with an AUC of 0.78 for predicting lung cancer in the associated needle biopsy. Multivariate analysis found that clinical and radiological factors commonly used in malignancy prediction models did not impact the test performance. High test performance was observed across all participant characteristics, including clinical categories where other tests perform poorly (Mayo Clinic Model, AUC = 0.52).ConclusionEarly clinical performance of the LungLB™ test supports a role in the discrimination of benign from malignant pulmonary nodules. Extended studies are underway.

Primary Cutaneous Marginal Zone Lymphoproliferative Disorder of Donor Origin after Allogeneic Hematopoietic Stem Cell Transplantation.

Primary cutaneous posttransplant lymphoproliferative disorders (PTLDs) after allogeneic hematopoietic stem cell transplant (allo-HSCT) are exceedingly rare, with only 6 published cases, all of them consisting in T-cell neoplasms. In this report, we present for the first time a donor-derived B-cell PTLD consisting in a primary, cutaneous, B-cell, marginal zone, lymphoproliferative disorder (PCMZLPD). The patient, a 37-year-old woman with a history of Hodgkin lymphoma received an allo-HSCT from her healthy, matched, related father, achieving complete host chimerism in the bone marrow and peripheral blood. However, 8 years after the allo-HSCT, she presented asymptomatic skin lesions consisting in oval, well-defined, slightly raised erythematous plaques, located on the arms, trunk, and legs. Skin biopsies of 2 lesions demonstrated a class-switched IgG+, EBV-, PCMZLPD, showing kappa light chain restriction and monoclonal rearrangement of the IgH gene. Microsatellite genotyping and 2-color fluorescence in situ hybridization (X and Y chromosomes) confirmed that the origin of the neoplastic cells was the donor graft. The lesions showed an indolent behavior, good response to topical corticosteroids, and no need for systemic treatment. Our case broadens the spectrum of PTLD, a diverse group of lymphoid and/or plasmacytic proliferations with variable clinical presentations and histopathological features.

A Lightweight and Robust Framework for Circulating Genetically Abnormal Cells (CACs) Identification Using 4-Color Fluorescence In Situ Hybridization (FISH) Image and Deep Refined Learning.

Circulating genetically abnormal cells (CACs) constitute an important biomarker for cancer diagnosis and prognosis. This biomarker offers high safety, low cost, and high repeatability, which can serve as a key reference in clinical diagnosis. These cells are identified by counting fluorescence signals using 4-color fluorescence in situ hybridization (FISH) technology, which has a high level of stability, sensitivity, and specificity. However, there are some challenges in CACs identification, due to the difference in the morphology and intensity of staining signals. In this concern, we developed a deep learning network (FISH-Net) based on 4-color FISH image for CACs identification. Firstly, a lightweight object detection network based on the statistical information of signal size was designed to improve the clinical detection rate. Secondly, the rotated Gaussian heatmap with a covariance matrix was defined to standardize the staining signals with different morphologies. Then, the heatmap refinement model was proposed to solve the fluorescent noise interference of 4-color FISH image. Finally, an online repetitive training strategy was used to improve the model's feature extraction ability for hard samples (i.e., fracture signal, weak signal, and adjacent signals). The results showed that the precision was superior to 96%, and the sensitivity was higher than 98%, for fluorescent signal detection. Additionally, validation was performed using the clinical samples of 853 patients from 10 centers. The sensitivity was 97.18% (CI 96.72-97.64%) for CACs identification. The number of parameters of FISH-Net was 2.24M, compared to 36.9M for the popularly used lightweight network (YOLO-V7s). The detection speed was about 800 times greater than that of a pathologist. In summary, the proposed network was lightweight and robust for CACs identification. It could greatly increase the review accuracy, enhance the efficiency of reviewers, and reduce the review turnaround time during CACs identification.

Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T cells.

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor-mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins.

Attention Mask R-CNN with edge refinement algorithm for identifying circulating genetically abnormal cells.

Recent studies have suggested that circulating tumor cells with abnormalities in gene copy numbers in mononuclear cell-enriched peripheral blood samples, such as circulating genetically abnormal cells (CACs), can be used as a non-invasive tool to detect patients with benign pulmonary nodules. These cells are identified through fluorescence signals counting by using 4-color fluorescence in situ hybridization (FISH) technology that exhibits high stability, sensitivity, and specificity. When FISH data are analyzed, the overlapping cells and fluorescence noise is a great challenge for identifying of CACs, thereby seriously affecting the efficiency of clinical diagnosis. To address this problem, in this study, we proposed an end-to-end FISH-based method (CACNET) for CAC identification. CACNET achieved nuclear segmentation and counted 4-color staining signals through improved Mask region-based convolutional neural network (R-CNN), followed by cell category (normal cell, deletion cell, gain cell, or CAC) according to pathological criteria. Firstly, the segmentation accuracy of overlapping nuclei was improved by adding an edge constraint head during training. Then, the interference of fluorescence noise was reduced by fusing non-local module to reconstruct the feature extraction network of Mask R-CNN. We trained and tested the proposed model on a dataset comprising 700 frames with 58,083 nuclei. The Accuracy, Sensitivity, and Specificity (overall performance metric for the algorithm) of CAC identification with CACNET were 94.06%, 92.1%, and 99.8%, respectively. Moreover, the developed method exhibited approximately identification speed of approximately 0.22 s per frames. The results showed that the proposed method outperformed the existing CAC identification methods, making it a promising approach for early screening of lung cancer.

Detection of circulating genetically abnormal cells using 4-color fluorescence in situ hybridization for the early detection of lung cancer

PurposeAvailable biomarkers lack sensitivity for an early lung cancer. Circulating genetically abnormal cells (CACs) occur early in tumorigenesis. To determine the diagnostic value of CACs in blood detected by 4-color fluorescence in situ hybridization (FISH) for lung cancer.MethodsThis was a prospective study of patients with pulmonary nodules ≤ 30 mm detected between 10/2019 and 01/2020 at four tertiary hospitals in China. All patients underwent a pathological examination of lung nodules found by imaging and were grouped as malignant and benign. CACs were detected by 4-color FISH. Patients were divided into the training and validation cohorts. Receiver operating characteristics analysis was used to analyze the diagnosis value of CACs.ResultsA total of 205 participants were enrolled. Using a cut-off value of ≥ 3, blood CACs achieved areas under the curve (AUCs) of 0.887, 0.823, and 0.823 for lung cancer in the training and validation cohorts, and all patients, respectively. CACs had high diagnostic values across all tumor sizes and imaging lesion types. CACs were decreased after surgery (median, 4 vs. 1, P < 0.001) in the validation set. The CAC status between blood and tissues was highly consistent (kappa = 0.909, P < 0.001). The AUC of CAC (0.823) was higher than that of CEA (0.478), SCC (0.516), NSE (0.506), ProGRP (0.519), and CYFRA21-1 (0.535) (all P < 0.001).ConclusionCACs might have a high value for the early diagnosis of lung cancer. These findings might need to be validated in future studies. Evidence suggested homology in genetic aberrations between the CACs and the tumor cells.

Identification of circulating tumor cells using 4-color fluorescence in situ hybridization: Validation of a noninvasive aid for ruling out lung cancer in patients with low-dose computed tomography-detected lung nodules.

Approximately one third of needle biopsies that are performed to rule out malignancy of indeterminate pulmonary nodules detected radiologically during lung cancer screening are negative, thus exposing cancer-free patients to risks of pneumothorax, bleeding, and infection. A noninvasive confirmatory tool (eg, liquid biopsy) is urgently needed in the lung cancer diagnosis setting to stratify patients who should receive biopsy versus those who should be monitored. A novel antigen-independent, 4-color fluorescence in situ hybridization (FISH)-based method was developed to detect circulating tumor cells (CTCs) with abnormalities in gene copy numbers in mononuclear cell-enriched peripheral blood samples from patients with (n=107) and without (n=100) lung cancer. Identification of CTCs using FISH probes at 10q22.3/CEP10 and 3p22.1/3q29 detected lung cancer cases with 94.2% accuracy, 89% sensitivity, and 100% specificity compared with biopsy. The high accuracy of this liquid biopsy method suggests that it may be used as a noninvasive decision tool to reduce the frequency of unnecessary needle biopsy in patients with benign pulmonary lesions.

Optimized FRET Pairs and Quantification Approaches To Detect the Activation of Aurora Kinase A at Mitosis.

Genetically encoded Förster's Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signaling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must be optimized to monitor multiple biochemical activities simultaneously and in single cells. In addition, their sensitivity must be increased to follow their activation even when the abundance of the biosensor is low. We describe here the development of a second generation of Aurora kinase A/AURKA biosensors. First, we adapt the original AURKA biosensor-GFP-AURKA-mCherry-to multiplex FRET by using dark acceptors as ShadowG or ShadowY. Then, we use the novel superYFP acceptor protein to measure FRET by 2-color Fluorescence Cross-Correlation Spectroscopy, in cytosolic regions where the abundance of AURKA is extremely low and undetectable with the original AURKA biosensor. These results pave the way to the use of FRET biosensors to follow AURKA activation in conjunction with substrate-based activity biosensors. In addition, they open up the possibility of tracking the activation of small pools of AURKA and its interaction with novel substrates, which would otherwise remain undetectable with classical biochemical approaches.

Wavelength Separation Tunable 2-Color Soliton Generation and Its Application to 2-Color Fluorescence Multiphoton Microscopy

Optical solitons generated through soliton self-frequency shift in optical waveguides, has found the widespread applications in multiphoton microscopy (MPM). The multicolor fluorescence MPM, involving the simultaneous imaging of multiple structures labeled by different fluorophores, typically necessitates multiple-color pulses whose wavelengths match the peak excitation wavelengths of the fluorophores. Here we demonstrate experimentally an efficient method of generating wavelength separation tunable 2-color solitons, based on our recently proposed theoretical scheme of prechirping to manipulate the soliton order of the excitation pulse. Using this method, we reach a maximum soliton separation tuning range of 190 nm, with 1550-nm excitation. As an experimental demonstration of the applicability of this technique, we further show the MPM results using this 2-color soliton source for 2-color, 3-photon fluorescence imaging of fluorescent beads.

CELL‐MATRIX INTERACTIONS AND MECHANOSENSITIVE PATHWAYS IN UTERINE LEIOMYOMA (FIBROID) PROLIFERATION

BackgroundUterine leiomyomas (fibroids; UF) are benign myometrial neoplasms that produce clinically‐relevant symptoms in &gt;25% of all women including excessive menstrual bleeding and infertility, and represent the leading cause of hysterectomy (~600,000/year in the US). Despite their importance, the mechanisms that promote UF growth are still not well‐understood. UFs are comprised of smooth muscle cells and fibroblasts with substantial extracellular matrix (ECM). While cells within UFs produce ECM, the role of ECM per se in modulating cell properties towards overall tumor growth is not known. This study tests the hypothesis that UF cells are mechanically sensitive, and that the ECM provides a positive feedback mechanism which further promotes UF growth.MethodsCells were enzymatically isolated from UFs of patients undergoing myomectomy at Mayo Clinic Rochester (IRB approved). Fibroid cells were plated onto soft (0.2kPa) vs. stiff (64kPa) polydimethylsiloxane (PDMS) elastomer substrates or standard tissue culture plastic substrates (~100GPa) for 24 h in DMEM containing 10% FBS prior to RNA isolation and qPCR analysis. Transcript levels for known ECM and profibrotic genes were determined. Nuclear localization of YAP and TAZ was visualized using immunofluorescence and quantified using a Cytation5 imaging system. Separately, 2‐color fluorescence immunohistochemistry for YAP and TAZ was performed on formalin fixed; paraffin embedded normal myometrium and fibroid tissue sections. Transciption level and nuclear localization calculations were performed with Kruskall‐Wallis and Wilcoxon Sign‐Rank tests, respectively.ResultsSignificant increases in transcripts for ECM gene Collagen 1 (COL1A1) were noted for UF cells plated on stiff PDMS and standard plastic compared to soft PDMS. The pro‐fibrotic genes Connective Tissue Growth Factor (CTGF), alpha smooth muscle actin (ACTA2), Endothelin 1 (EDN1), and plasminogen activator inhibitor 1 (SERPINE1) were progressively increased in expression going from soft PDMS substrate to plastic. The anti‐fibrotic gene, cyclooxygenase 2 (PTGS2) was decreased on stiff PDMS and plastic compared to soft PDMS. In cells plated on plastic, there was a significant decrease in YAP/TAZ nuclear localization in cells treated with the Rho‐kinase inhibitor Y‐27632, consistent with a mechanobiologic feedback. Immunohistochemistry showed a two‐fold increase in YAP/TAZ nuclear localization for fibroid tissue compared to normal myometrium.ConclusionsBased on the growing appreciation that ECM mechanical properties contribute to UF pathobiology and fibrotic matrix deposition, we show enhanced ECM and profibrotic gene expression by fibroid cells as a function of substrate stiffness. These results demonstrate a feedback mechanism by which UFs progress through matrix stiffness‐mediated ECM deposition. Here, the mechanosensitive pathways of YAP and TAZ may be involved in transducing ECM signals to UF growth.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pathogenetic aspects in precursor lesions of gastrointestinal tumors

The pathogenesis of precursor lesions of gastrointestinal tumors is manifested in many ways. In the esophagus an aberrant genetic expression of intestinal transcription factors, such as CDX2 is initiated by local environment factors. During the subsequent dysplasia to carcinoma sequence, chromosomal gain and loss of genes occurs. A4-color fluorescence in situ hybridization (FISH) assay can be applied in dysplasia as well as in Barrett's adenocarcinoma to define prognostic marker combinations. In the gastric carcinogenesis sequence the gene expression of CDX1 is regulatively dependent on an interplay between inflammation and promotor methylation. In the colon sessile serrated adenomas show asequence with initial BRAF mutation and late onset of MLH1 promotor hypermethylation with consecutive potential cancer progression. This event is accompanied by an increase of intraepithelial lymphocytes, which is an easy to use tool for routine diagnostics using H&Esections. Next generation sequencing (NGS) investigations of germline mutations in colorectal cancer revealed aspectrum of mutations with low penetration in the field of mismatch repair proteins as well as the APC gene. An individual risk stratification for penetration of these germline mutations is necessary. In conclusion, genetics, phenotypes and terminology of gastrointestinal precursor lesions are unified to amutually influencing concept within medicine.

Differences in the Induction of Induced Human CD4+ CD25+ FoxP3+ T-Regulatory Cells and CD3+ CD8+ CD28− T-Suppressor Cells Subset Phenotypes In Vitro: Comparison of Phorbol 12-Myristate 13-Acetate/Ionomycin and Phytohemagglutinin Stimulation

CD4+ CD25+ FoxP3+ T-regulatory cells (Treg) and CD3+ CD8+ CD28− T-suppressor cells (Ts) were shown to have immunosuppressive function in vivo and in vitro. However, the in vitro inducibility of Ts subsets is rather unclear. We investigated the induction of Treg and Ts subsets in peripheral blood mononuclear cells of 5 healthy control individuals during stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin or phytohemagglutinin (PHA). Phenotypes were analyzed 0, 4, 8, 16, and 24 hours after initiation of cell culture using 4-color fluorescence flow-cytometry. Number of CD4+ CD25+ FoxP3+ CD127− Treg increased during PMA/ionomycin or PHA stimulation (P < .01). CD4+ CD25+ FoxP3+ Treg coexpressed the phenotypes interleukin (IL)-2−, IL-10+, and/or transforming growth factor (TGF)-β+ after stimulation (all P < .01). Interferon (IFN)-γ production was induced only by PMA/ionomycin (P < .01) but not by PHA (P = NS). IFN-γ-secreting Treg were detectable at 4 hours whereas IL-2−, IL-10+ and/or TGF-β+ Treg required 16 hours of stimulation. In contrast, CD3+ CD8+ CD28− Ts phenotypes were not inducible during 24-hour PMA/ionomycin or PHA stimulation (all P = NS). However, Ts coexpressed IL-10 and/or TGF-β during polyclonal stimulation (all P < .01), whereas the proportion of IL-2− Ts remained stable during the cell culture period (P = NS). Similar to Treg, IFN-γ-secreting Ts were detected only during PMA/ionomycin stimulation (P < .01), but not during PHA stimulation (P = NS). We conclude that the proportion of CD3+ CD8+ CD28− Ts remains stable during polyclonal stimulation. They modify only the cytokine pattern indicating activation of the Ts. In contrast, CD4+ CD25+ FoxP3+ CD127− Treg are inducible by PMA/ionomycin and PHA stimulation. IFN-γ- secreting Treg form the first line of immunoregulatory T cells during an initiated immune response followed by IL-2−, IL-10+, and/or TGF-β+ Treg.

Chromosomal Aberrations by 4-Color Fluorescence In Situ Hybridization Not Detected in Spitz Nevi of Older Individuals

Chromosomal Aberrations by 4-Color Fluorescence In Situ Hybridization Not Detected in Spitz Nevi of Older Individuals

Chromosomal Aberrations by 4-Color Fluorescence In Situ Hybridization Not Detected in Spitz Nevi of Older Individuals

To investigate whether Spitz nevi with typical histopathological features in older patients demonstrate chromosomal aberrations by 4-color fluorescence in situ hybridization (FISH). Retrospective medical record review, with prospective masked histopathological and cytogenetic analyses. University-affiliated dermatology and dermatopathology setting. Twenty-five patients 50 years or older with melanocytic nevi showing histopathological features typical of Spitz nevi. Three dermatopathologists masked to the patients' ages reviewed histopathological sections of melanocytic lesions for features typical of Spitz nevi. FISH was performed on samples with typical histopathological features by a 4-color FISH probe set used for the evaluation of malignant melanocytic neoplasms. None of the study cases showing histopathological features typical of Spitz nevi had detectable chromosomal abnormalities by FISH. Spitz nevi in older patients demonstrate molecular features similar to those of Spitz nevi in younger age groups. The findings of normal karyotypes in combination with typical histopathological features are reassuring of Spitz nevus diagnoses in older patients and suggest no correlation of increased malignant potential with advanced age per se.

Fluorescence In Situ Hybridization (FISH) Analysis of Melanocytic Nevi and Melanomas

A 4-color fluorescence in situ hybridization (FISH) assay, using probes to chromosomes 11q, 6p, 6q, and 6 cent, has recently been proposed as an ancillary tool for the diagnosis of melanoma. The authors report herein their experience with this assay. To determine the sensitivity and specificity of the assay for histopathologically unequivocal cases, they analyzed 50 melanocytic nevi, 50 primary melanomas, and 15 metastatic melanomas. Of 50 melanocytic nevi, 47 were FISH negative on initial readout (test sensitivity, 94%); 49 were FISH negative after correction for tetraploidy (test specificity, 98%). Of 50 primary melanomas, 41 were FISH positive (test sensitivity, 82%). Of 15 metastatic lesions, 13 were FISH positive (test sensitivity, 85%). Of the 9 FISH-negative melanomas, 6 metastasized. The tumors of the 5 patients who had survived thick primary melanoma for more than 5 years without recurrence were all FISH positive. Half of the patients whose primary melanoma was tested by FISH had undergone sentinel lymph node (SLN) biopsy. When the authors compared the FISH results of those 25 melanomas with the SLN status, no statistically significant correlation was found. These findings document limitations of the current FISH assay. A rare nevus may be FISH positive. Some primary metastasizing melanomas are FISH negative. Even metastatic melanomas can be FISH negative. Awareness of the limitations in test sensitivity and specificity of the FISH assay is important to avoid an erroneous diagnosis by overreliance on cytogenetic findings. Correlation with clinical and histopathological findings is paramount for accurate diagnosis.

Gain of the Human Telomerase RNA Gene TERC at 3q26 is Strongly Associated With Cervical Intraepithelial Neoplasia and Carcinoma

This study investigated the gain of the human telomerase RNA gene TERC at 3q26 in patients with uterine cervix disease in the southern part of China and assessed the relationship between TERC gain and cervical pathological findings. One hundred ten cervical specimens, which were collected from patients with various kinds of uterine cervix disease that was subsequently diagnosed as chronic cervicitis and with examination results negative for intraepithelial lesion or malignancy (NILM, n = 23), mild dysplasia (cervical intraepithelial neoplasia type 1 [CIN1], n = 37), moderate dysplasia (CIN2, n = 12), severe dysplasia (CIN3, n = 10), and squamous cell carcinoma (SCA, n = 28) confirmed by histologic diagnosis, were analyzed for the proportion of abnormal cells with TERC gain using a commercially available 2-color fluorescence in situ hybridization (FISH) probe. The cases with a higher proportion of abnormal cells than the threshold evaluated by NILM were recorded as positive TERC gain. The chi and Kruskal-Wallis tests were used to assess the associations between FISH findings and diagnoses. The incidence of positive TERC gain in the cases diagnosed as NILM or CIN1 was significantly lower than that in cases diagnosed as CIN2, CIN3, or SCA (P < 0.01). In addition, a significantly higher proportion of abnormal cells with TERC gain was found as a pathological change from CIN1 to SCA (P < 0.01). We conclude that the TERC gain seems to be an important associated genetic event in CIN and carcinoma; FISH is a potential tool for the diagnoses of uterine cervix disease.

Integrated Portable Polymerase Chain Reaction-Capillary Electrophoresis Microsystem for Rapid Forensic Short Tandem Repeat Typing

A portable forensic genetic analysis system consisting of a microfluidic device for amplification and separation of short tandem repeat (STR) fragments as well as an instrument for chip operation and four-color fluorescence detection has been developed. The microdevice performs polymerase chain reaction (PCR) in a 160-nL chamber and capillary electrophoresis (CE) in a 7-cm-long separation channel. The instrumental design integrates PCR thermal cycling, electrophoretic separation, pneumatic valve fluidic control, and four-color laser excited fluorescence detection. A quadruplex Y-chromosome STR typing system consisting of amelogenin and three Y STR loci (DYS390, DYS393, DYS439) was developed and used for validation studies. The multiplex amplification of these 4 loci with 35 PCR cycles followed by CE separation and 4-color fluorescence detection was completed in 1.5 h. All the amplicons can be detected with a limit of detection of 20 copies of male standard DNA in the reactor. Real-world forensic analyses of oral swab and human bone extracts from case evidence were also successfully performed. Mixture analysis demonstrated that a balanced profile can be obtained even at a male-to-female template ratio of 1:10. The successful development and operation of this portable PCR-CE system establishes the feasibility of rapid point-of-analysis DNA typing of forensic casework, of mass disaster samples or of individuals at a security checkpoint.

Bacterial Adherence to Tantalum Versus Commonly Used Orthopedic Metallic Implant Materials

Evaluation of bacterial adhesion to pure tantalum and tantalum-coated stainless steel versus commercially pure titanium, titanium alloy (Ti-6Al-4V), and grit-blasted and polished stainless steel. Experimental in vitro cell culture study using Staphylococcus aureus and Staphylococcus epidermidis to evaluate qualitatively and quantitatively bacterial adherence to metallic implants. A bacterial adhesion assay was performed by culturing S. aureus (ATCC 6538) and S. epidermidis (clinical isolate) for one hour with tantalum, tantalum-coated stainless steel, titanium, titanium alloy, grit-blasted and polished stainless steel metallic implant discs. Adhered living and dead bacteria were stained using a 2-color fluorescence assay. Adherence was then quantitatively evaluated by fluorescence microscopy and digital image processing. Qualitative adherence of the bacteria was analyzed with a scanning electron microscope. The quantitative data were related to the implant surface roughness (Pa-value) as measured by confocal laser scanning microscopy. Bacterial adherence of S. aureus varied significantly (p = 0.0035) with the type of metallic implant. Pure tantalum presented with significantly (p < 0.05) lower S. aureus adhesion compared to titanium alloy, polished stainless steel, and tantalum-coated stainless steel. Furthermore, pure tantalum had a lower, though not significantly, adhesion than commercially pure titanium and grit-blasted stainless steel. Additionally, there was a significantly higher S. aureus adherence to titanium alloy than to commercially pure titanium (p = 0.014). S. epidermidis adherence was not significantly different among the tested materials. There was no statistically significant correlation between bacterial adherence and surface roughness of the tested implants. Pure tantalum presents with a lower or similar S. aureus and S. epidermidis adhesion when compared with commonly used materials in orthopedic implants. Because bacterial adhesion is an important predisposing factor in the development of clinical implant infection, tantalum may offer benefits as an adjunct or alternative material compared with current materials commonly used for orthopedic implants.