Specificity Of Fluorescence In Situ Hybridization Research Articles

Fish: A Promising Screening Tool for Malignancy After Augmentation Cystoplasty?

IntroductionMalignancy after augmentation cystoplasty (AC) is reported up to 5.5 %. We assessed the use of urine fluorescence in situ hybridization (FISH) screening for bladder malignancy after AC. Patients and methodsIn this study, 36/98 patients under follow-up who have completed tenth year after ileal AC were included prospectively. Twenty-four (66.7 %) patients were tested with FISH initially and overall 28 (77.8 %) patients with conventional cytology (CC). Twenty-four (66.7 %) patients with FISH analysis also had cytology analysis. Blinded from the cytology results, 32 (88.9 %) patients who were consented underwent cystoscopy with random biopsy (native bladder, ileal segment, ileovesical junction). Two patients those were tested with FISH did not consented cystoscopy. This study was registred to the government registry (No: 71146310). ResultsMean follow-up time after AC was 15.4 ± 4.8 years. 2/32 (5.6 %) patients were diagnosed with adenocarcinoma in cyctoscopic biopsy. FISH analysis of 3/24 (12.5 %) patients demonstrated abnormal findings consistent with malignancy. Two FISH malignant patients were patients who had adenocarcinoma. The third patient's biopsy was benign and the third year control cystoscopy was normal. 2/4 patients with malignant CC had adenocarcinoma and 2/4 patients had benign biopsy. The sensitivity and specificity of FISH in our series were 100 % and 95 % respectively. Whereas the sensitivity and specificity of CC was 100 % and 91.6 % respectively. ConclusionDespite limited number of patients in this study, FISH showed higher specificity than CC in this series. FISH is a promising tool for malignancy screening after AC. Type of StudyDiagnostic Studies. Level of EvidenceII.

The Role of Fluorescence In Situ Hybridization in the Surveillance of Non-Muscle Invasive Bladder Cancer: An Updated Systematic Review and Meta-Analysis.

Background: Fluorescence in situ hybridization (FISH) has become a popular biomarker for subsequent monitoring the recurrence of non-muscle invasive bladder cancer (NMIBC), several studies have investigated the ability of FISH to detect recurrence in the surveillance of NMIBC. However, the results were inconsistent. Methods: We conducted a systematic literature search extensively on authenticated databases including PubMed/Medline, Embase, Web of Science, Ovid, and Cochrane Library. Meta-analysis was performed to find out the sensitivity and specificity of FISH in predicting recurrence of NMIBC. Results: 15 studies were ultimately included in this meta-analysis, a total of 2941 FISH evaluations from 2385 NMIBC patients were available. The pooled sensitivity of FISH was 68% (95% CI: 0.58–0.76), and the pooled specificity was 64% (95% CI: 0.53–0.74). Subgroup analyses were performed in 7 studies without Bacillus Calmette–Guerin (BCG) treatment, the pooled sensitivity was 82% (95% CI: 0.68–0.90), and the pooled specificity was 63% (95% CI: 0.37–0.82). And in 9 studies using “UroVysion standard” to define positive FISH results showed a pooled sensitivity of 60% (95% CI: 0.50–0.70) and specificity of 70% (95% CI: 0.61–0.78). Conclusions: The findings of this study indicate that FISH has a satisfactory sensitivity (68%) and specificity (64%) and could be a potential biomarker in the surveillance of NMIBC. Moreover, BCG treatment and different FISH methods may have an impact on the sensitivity and specificity, these factors should be taken into account when making clinical strategy.

Identification of Stenotrophomonas maltophilia in Blood Cultures by Fluorescence in Situ Hybridization

Background Stenotrophomonas maltophilia is a serious causative agent for bacteremia. Rapid identification of S. maltophilia in blood cultures is important to enable a satisfactory pathogen-based antibiotic therapy at an early stage. The aim of this study was to evaluate fluorescence in situ hybridization (FISH) for the identification of S. maltophilia in blood culture specimens. Methods Three hundred positive blood culture specimens were examined by both FISH and conventional laboratory methods for the identification of S. maltophilia. The results of FISH were compared with the results of the conventional methods. Results By conventional cultural and biochemical methods, S. maltophilia was identified in 47 blood culture specimens. Fluorescence in situ hybridization identified S. maltophilia in 46 of these 47 S. maltophilia–positive blood cultures. Thus, the sensitivity and specificity of FISH were 97.9% and 100%, respectively. Conclusions Our findings suggest that FISH is a suitable method for the identification of S. maltophilia in blood cultures.

Evaluation of fluorescent in-situ hybridization technique for diagnosis of malaria in Ahero Sub-County hospital, Kenya

BackgroundMalaria is a major cause of morbidity and mortality. Treatment of malaria in a timely manner could avert deaths. Treatment ultimately relies on the rapid and accurate diagnosis. Fluorescence in situ hybridization (FISH), a cytogenetic technique based on detection of specific nucleic acid, has the potential to address the limitations of the current diagnostic approaches. This study investigates further the performance of FISH for the diagnosis of malaria in a rural setting in Western Kenya.MethodsBlood samples from 302 patients presenting with fever (temperature ≥ 37.5 °C) were examined for malaria using the Giemsa microscopy (GM), rapid diagnostic test (RDT), polymerase chain reaction (PCR) and FISH.ResultsThe sensitivity and specificity of FISH was 85.6% and 96.2% respectively, while the corresponding values for GM were 82.2% and 100% respectively. RDT and PCR had sensitivities of 91.1% and 98.9%, respectively with their specificities being 89.6 and 100%, respectively. The positive predictive values for RDT, GM, FISH and PCR were 78.8%, 100%, 90.6% and 100%, respectively. The negative predictive values for RDT, GM, FISH and PCR were 96.0%, 93.0%, 94.0% and 99.5%, respectively. Their respective diagnostic accuracies were 90.1%, 94.7% 93.0% and 99.7%.ConclusionThe present study demonstrates that the specificity and reproducibility of FISH assays are high, thus adding to the growing evidence on the potential of the technique as an effective tool for the detection of malaria parasites in remote settings.

“FISHing” for an Accurate Diagnostic Test for Cholangiocarcinoma

Navaneethan U, Njei B, Venkatesh PG, et al. Fluorescence in situ hybridization for diagnosis of cholangiocarcinoma in primary sclerosing cholangitis: a systematic review and meta-analysis. Gastrointest Endosc 2014;79:943–950. Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory condition epitomized by progressive obliteration of the biliary tree. Although the natural history is variable, secondary biliary cirrhosis and end-stage liver failure occur in ≤50% of patients. In addition to managing the symptoms of PSC (predominantly pruritus and recurrent acute cholangitis), clinicians and patients remain vigilant for the interval development of cholangiocarcinoma (CCA), the lifetime incidence of which is 6%–20% (Am J Gastroenterol 2007;102:107–114). With improvements in cross-sectional imaging, PSC diagnosis and surveillance can be accomplished without the routine use of endoscopic retrograde cholangiopancreatography (ERCP). However, individuals with symptoms or signs of disease progression are referred for ERCP to palliate dominant strictures via dilation with or without short-term stent placement, and tissue sampling to evaluate for CCA. In this context, traditional intraductal tissue sampling techniques include brushings for cytology and forceps biopsies for histology. These modalities, alone or in combination, have limited (20%–60%) sensitivity for confirming CCA owing to the desmoplastic nature of these lesions, so clinicians cannot definitively “rule out” CCA with a negative result (Gastrointest Endosc 1995;42:565–572). Because cytopathology and histopathology are essentially qualitative tests that rely on a subjective assessment of cellular morphology, fluorescence in situ hybridization (FISH) has been studied as a supplementary assay. FISH is performed on intraductal brushing samples and uses fluorescently labeled probes that target the centromeres of chromosome 3, 7, and 17, as well as the 9p21 band (P16), evaluating for aneuploidy (abnormal number of chromosomes) by counting the number of affected cells; therefore, it is a quantitative test that requires theoretically fewer cells than cytopathology and removes the subjectivity associated with cytopathologic interpretation. Navaneethan et al (Gastrointest Endosc 2014;79: 943–950. e3) performed a structured metaanalysis of studies assessing the diagnostic utility of FISH in diagnosing CCA among patients with PSC. The authors included studies that allowed construction of a 2 × 2 contingency table with true-positive, false-negative, false-positive, and true-negative results. The authors used standard metaanalytic techniques to calculate composite sensitivity, specificity, likelihood ratios, and diagnostic odds ratio. There was moderate statistical heterogeneity among included studies, so the authors used a random effects model. A positive FISH for malignancy was defined as either polysomy (≥5 cells with gains of ≥2 of 4 probes) or trisomy/tetrasomy (trisomy, ≥10 cells with 3 copies of chromosome 7 or 3; tetrasomy, ≥10 cells with 4 copies of all probes). Eight studies of moderate to high methodologic quality (n = 828 individuals with PSC) met eligibility criteria. The pooled analysis from these studies demonstrated that FISH had an overall sensitivity of 68% (95% CI, 61%–74%), and specificity of 70% (95% CI, 66%–73%) for diagnosing CCA. The pooled positive likelihood ratio was 2.69 (95% CI, 1.84–3.97), and the negative likelihood ratio was 0.47 (95% CI, 0.39–0.58). The pooled odds ratio to detect CCA was 7.24 (95% CI, 3.93–13.36). The positive predictive value was 0.42 (95% CI, 0.36–0.47), and the negative predictive value was 0.87 (95% CI, 0.84–0.90). Stated alternatively, an individual with PSC and positive FISH had a 42% posttest probability of CCA and an 87% probability of being cancer free after negative FISH. Two of the included studies described the accuracy of FISH in the setting of negative cytology, reporting sensitivities of 57% (Gastroenterology 2006;131:1064–1072) and 60% (Am J Gastroenterol 2008;103:1263–1273) and specificities of 87% and 71%. This is the context in which FISH would be considered most valuable: when CCA is present, but routine cytology is negative for malignancy, FISH will be positive in ≤60% of cases. However, there remains a possibility that FISH is falsely positive, as reflected its lower specificity. The specificity of FISH seemed to be optimized (93%) by defining a positive test using polysomy alone (6 of 8 included studies), but this compromised the technique’s sensitivity (51%). The authors systematically removed 1 dataset at a time and recalculated their outcomes; no single dataset carried sufficient weight to influence significantly the pooled performance of FISH for diagnosing CCA in the setting of PSC.

Endoscopic ultrasound guided fine needle aspiration with fluorescence in situ hybridization analysis in 104 patients with pancreatic mass

Diagnosis of pancreatic malignancy is often based on cytological specimens collected by endoscopic ultrasound guided fine needle aspiration (EUS FNA). Several factors can decrease sensitivity of EUS FNA for pancreatic cancer: well-differentiated tumors, pancreatitis, blood, necrosis and slides with low cellularity. The objective of this study is to report on the use of fluorescence in situ hybridization (FISH) analysis combined with cytology in pancreatic masses. EUS database and medical records of patients referred for EUS between January 2009 through august 2013 were reviewed. Data on cytology, FISH and surgical pathology were reviewed. Surgical pathology, death or extended clinical follow-up were used to verify correct diagnosis of malignancy. FISH performed using a four-set DNA probe for chromosomes 3, 7, 17, and band 9p21 in patients with inconclusive immediate cytology reading. Sensitivity of cytology and FISH were compared. Study cohort comprised of 104 patients with FISH analysis on EUS FNA specimens of pancreatic masses (74 adenocarcinoma, 7 neuroendocrine tumor and 23 benign. Sensitivity of cytology and FISH for carcinoma was respectively: 62% and 81%. Sensitivity of FISH + cytology was 89%. The specificity of FISH and cytology was 100%. The most common abnormality on FISH was a 9p21 deletion seen in 43 patients (58%) followed by polysomy of 7 (46%). FISH detected malignancy in 23 patients with negative cytology. In patients with inconclusive immediate cytology reading, FISH is superior to cytology and improves overall sensitivity. The 9p21 deletion is the most common abnormality seen in this cohort of patients with pancreatic cancer.

Identification of Streptococcus agalactiae by fluorescent in situ hybridization compared to culturing and the determination of prevalence of Streptococcus agalactiae colonization among pregnant women in Bushehr, Iran

BackgroundPregnant women colonized by Streptococcus agalactiae (group B streptococci [GBS]) may transfer this microorganism to their newborns. S. agalactiae is an important cause of pneumonia, sepsis, and meningitis in newborns. Fluorescent in situ hybridization (FISH) is considered as a method of identification in the field of diagnostic microbiology. In this paper, we have designed a study to compare the DNA FISH after 7 h Lim broth enrichment and culturing for the identification of S. agalactiae and to determine the prevalence of vaginal colonization by S. agalactiae among pregnant women in Bushehr, Iran.MethodsVaginal swab specimens were obtained from 285 pregnant women at 35 weeks or more than 35 weeks of gestation. The specimens were inoculated into Lim broth. In order to evaluate the sensitivity and specificity of GBS DNA FISH after 7 h Lim broth enrichment, the specimens were tested using both FISH and conventional culture methods. In addition, the prevalence of GBS colonization was determined.ResultsBased on the results of this study, both the sensitivity and specificity of FISH were 100%. S. agalactiae was detected by both culture and FISH in 27 of the 285 pregnant women. Thus, the prevalence of GBS colonization was 9.5%.ConclusionsSince short-term (7 h) Lim broth enrichment followed by FISH using oligonucleotide probes showed a high sensitivity and specificity, this protocol is therefore a highly accurate and relatively rapid method for the detection of S. agalactiae. Our analysis suggests that the use of DNA FISH to screen for S. agalactiae colonization in pregnant women may be considered in the absence of GBS culture availability.

Use of a modified fluorescentin situhybridization procedure to improve the identification ofStreptococcus pneumoniaein blood cultures

Streptococcus pneumoniae is an important causative agent for bacteremia. Fluorescent in situ hybridization (FISH) is a helpful molecular technique for the rapid identification of S. pneumoniae in positive blood cultures. There are many reports concerning the application of an enzymatic treatment with lysozyme in the FISH procedure for partial cell wall digestion of S. pneumoniae. However, this study was aimed to test the FISH procedure without enzymatic treatment for the identification of S. pneumoniae in blood culture specimens. Seventy-seven positive blood culture specimens containing Gram-positive cocci were examined by both the conventional laboratory methods and FISH. Detection of S. pneumoniae was performed by two FISH procedures: one procedure was performed with an enzymatic treatment step and the other one was done without enzymatic treatment. In addition, the specimens were tested by the FISH procedure with enzymatic treatment to detect Streptococcus pyogenes and Enterococcus. The specificity of FISH in comparison with conventional culture methods was 100%. The sensitivity of the FISH procedure with enzymatic treatment for the detection of S. pneumoniae was 90%, whereas, the sensitivity of the FISH procedure without enzymatic treatment was 100%. In fact, by omission of enzymatic treatment, detection of S. pneumoniae was improved in 6 specimens. The results of the FISH and culture methods for the detection of S. pyogenes and Enterococcus were compatible. Altogether, FISH procedure without enzymatic treatment step seems to improve the detection of S. pneumoniae in some cases. Thus, for successful detection of S. pneumoniae, we suggest the application of both FISH procedures (the procedure with enzymatic treatment and the procedure without enzymatic treatment) for each blood culture specimen.

Canadian ALK (CALK): A pan-Canadian multicenter study to optimize and standardize ALK immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for ALK gene rearrangements.

8096 Background: ALK gene rearrangement (ALK+) has been found in 3-5% of advanced non-small cell lung cancer patients. FISH is considered the “gold standard” for identification of ALK+ tumors, but its cost-effectiveness and adoption as a screening assay has been debated. Recent reports suggested that ALK IHC may serve as an alternative screening or possibly a diagnostic method. In this context, CALK was initiated to assess the feasibility of implementing ALK IHC and/or FISH assays across Canadian hospitals. Methods: FISH-confirmed 22 ALK+ and 6 ALK- tumors were used as study samples. Unstained sections and scanned images of HE-stained slides from each tumor block were distributed to participating centres. IHC protocols with best signal to noise ratio using the 5A4 (Novocastra) or ALK-1 (Dako) antibodies were developed for various auto-stainers and implemented to suit the existing conditions of the participating centres. A common FISH protocol using the ALK break-apart probe (Abbott Molecular, Chicago, IL) was developed based on published reports. H-score was used to assess IHC. FISH signals were scored in 100 tumor cells/case by 2-3 pre-trained persons. A second round IHC study using newly distributed slides was completed by 8 centres. Results: Independent IHC scores from 12 centres and FISH scores from 11 centres were collected and analysed. The intraclass correlation coefficients (ICC) between centres for IHC and FISH were 0.84 and 0.68, respectively. Following the analysis of initial IHC results, a second round study resulted in improved ICC of 0.94. One of 23 tumors revealed IHC-/FISH+ discrepancy, with the FISH revealing unusual signal configurations that suggested an atypical rearrangement. However, the sensitivity and specificity of FISH results across centres using the 15 aberrant signals cut-off ranged from 86.7-100% and 100%, respectively. Conclusions: Standardization across multiple centres for ALK testing by IHC and FISH can be achieved. IHC detected all FISH+ ALK tumors, except for one discrepant case with atypical FISH finding of unknown clinical implication. The study was supported by a Pfizer Canada grant.

Utility of fluorescence in situ hybridization for the diagnosis of bladder urothelial carcinoma

Objective To evaluate the clinical value of fluorescence in situ hybridization (FISH) method for diagnosis of bladder urothelial carcinoma.Methods Urine samples from 81 patients suspected of having bladder urothelial carcinoma were collected for immediate urine cytology and FISH analysis.All patients underwent cystoscopy for identification of bladder lesions.Urine samples from 8 patients with benign disease of urinary system werealso analyzed by means of FISH and cytology.The sensitivity and specificity of FISH were compared with cytology.Results 81 subjects of bladder urothelial carcinoma were verified with pathology,which included 34 non-muscle invasive carcinoma,14 cases with muscle invasive carcinoma,42 cases with high-grade carcinoma,and 24 cases with low-grade carcinoma.Residual cases included 12 verified benign disease of urinary system,and to serve as controls with 8 patients with benign disease of urinary system.The sensitivities of FISH,cytology and cystoscopy were 72.8％,27.2％ and 97.5％.The sensitivity of FISH was superior to cytology,but inferior to cystoscopy.The specificity and diagnostic concordance rate of FISH and cytology were 85.0％,100.0％ and 75.2％,41.6％,respectively.Conclusion FISH analysis as a non-invasive method has good sensitivity and specificity for diagnosing bladder urothelial carcinoma. Key words: Bladder neoplasms; Carcinoma; In situ hybridization, fluorescence; Urine cytology; Cystoscopy

Application of fluorescence in situ hybridization in the diagnosis of urothelial carcinoma

Objective To evaluate the results of fluorescence in situ hybridization (FISH) for detecting urothelial carcinoma.Methods Voided urine samples from 100 patients with haematuria were analyzed by FISH and cytology; labeled probes for chromosomes 3,7,17 and 9 p16 were used to assess chromosomal abnormalities.The gold standard was pathology diagnosis.The overall sensitivity and specificity of FISH were evaluated and compared with cytology.Results The sensitivity of FISH and cytology in detection urothelial cancer were 82.5％ and 49.2％ respectively,and the specificity was 86.7％ and 96.6％ respectively.There was a significant difference between the sensitivity( P ＜ 0.05 ),and the different between the specificity was not significant ( P ＞ 0.05 ).Conclusion Compared with urine cytology,FISH diagnosis of urothelial carcinoma with high sensitivity and a similar specificity. Key words: Fluorescence in situ hybridization; Urothelial carcinoma; Cytology

Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Detection of specific genetic abnormalities by fluorescence in situ hybridization in soft tissue tumors

For the detection of chromosome translocations/chimeric genes and specific genetic abnormalities in soft tissue tumors, we conducted fluorescence in situ hybridization (FISH) analysis on 280 cases of soft tissue and other tumors using formalin-fixed paraffin-embedded tissue sections. The detection rate of the FISH split-signal was 84% (129/154 cases) for the translocation-associated soft tissue tumors, such as Ewing's sarcoma/primitive neuroectodermal tumor, synovial sarcoma, alveolar rhabdomyosarcoma, myxoid liposarcoma, clear cell sarcoma and so forth. Positive split-signals from EWSR1, SS18 and FOXO1A probes were detected in 3% (2/64) of various histological types of carcinoma, lymphoma, melanoma, meningioma and soft tissue tumors. In FISH using the INI1/CEP22 probe, the INI1 deletion signal was detected in 100% (9/9) of epithelioid sarcoma. In well-differentiated and dedifferentiated liposarcomas, detection of MDM2 amplification signals in FISH using the MDM2/CEP12 probe were both as high as 85% (11/13) and 100% (13/13), respectively. In other adipocytic and non-adipocytic tumors requiring differentiation from these types, detection was only 13% (5/39), and CEP12 polysomy was frequently detected. As these results demonstrate the high sensitivity and specificity of FISH, we concluded FISH to be a useful pathological diagnostic adjunct for definite and differential diagnosis of soft tissue tumors.

Polysomy and p16 deletion by fluorescence in situ hybridization in the diagnosis of indeterminate biliary strictures

The diagnosis of indeterminate biliary strictures is limited because of the low sensitivity of cytology. However, an accurate diagnosis of malignancy is critical in the management of patients with suspected biliary malignancy. Testing for chromosomal aneuploidy by fluorescence in situ hybridization (FISH) may increase the yield. To evaluate the diagnostic accuracy of FISH in indeterminate biliary strictures and the additional value of including deletion of 9p21 (p16) in the diagnostic criteria of malignant biliary strictures. Retrospective review. Academic medical center. This study involved 76 consecutive patients who were seen for the evaluation of indeterminate strictures at our institution. These patients were screened, and 50 patients with either a final pathologic diagnosis or ≥ 12 months' conclusive follow-up were included in the analysis. Sensitivity, specificity, and area under the curve (AUC) analysis of cytology alone compared with the presence of FISH polysomy versus FISH polysomy and 9p21 deletion. The presence of increased copy numbers (polysomy) of chromosome 3, 7, or 17 by FISH increased the sensitivity of brush cytology from 21% to 58%, and when the presence of 9p21 deletion was included, the sensitivity increased to 89%. The specificity of FISH was 97% (vs 100% for cytology). The accuracy of cytology combined with FISH polysomy (AUC = 0.93) or p16 deletion was significantly greater than the accuracy of cytology alone (AUC 0.6; P < .001) or even cytology combined with FISH polysomy (AUC = 0.77; P ≤ .05). Sample size. There is a relatively high incidence of malignant biliary strictures in the entire cohort but low incidence among primary sclerosing cholangitis patients, and the majority of cancers are cholangiocarcinomas (as opposed to pancreatic). FISH significantly improves the diagnostic accuracy of brush cytology in indeterminate biliary strictures. In our series, the addition of 9p21 deletion to FISH polysomy and cytology further improved sensitivity. This suggests that 9p21 deletion may be added to the diagnostic criteria in indeterminate strictures.

Fluorescence in situ hybridization, a diagnostic aid in ambiguous melanocytic tumors: European study of 113 cases

Some melanocytic tumors are ambiguous, so the reproducible histopathological diagnosis of benign or malignant lesion is difficult. This study evaluated the contribution of fluorescence in situ hybridization (FISH) first in 43 non-equivocal melanomas and nevi, and then in 113 ambiguous melanocytic tumors selected by expert pathologists from six different European institutions. We included two groups of ambiguous tumors: patients without recurrence (5-year minimal follow-up) and with metastases. An independent triple-blind histopathological review was performed to classify tumors as ‘favor benign’ (A−) or ‘favor malignant’ (A+). A four-color probe set targeting 6p25, 6q23, 11q13 and CEP6 was used for FISH. In the 43 non-equivocal melanomas and nevi, sensitivity was 85% and specificity 90%. Ninety out of 95 ambiguous melanocytic tumors included were FISH interpretable (67 FISH negative and 23 FISH positive). Of the 90 patients, 69 presented no recurrence and 21/90 exhibited metastases. These ambiguous tumors were mostly spitzoid tumors (45/90). Histopathological reviewers classified these tumors as favor malignant (49/90) and favor benign (32/90), whereas nine cases had a discordant diagnosis. By comparison with outcome, the sensitivity and specificity of histopathological review were 95 and 52%, and the sensitivity and specificity of FISH were 43 and 80%. Compared with histopathological review, the sensitivity and specificity of FISH were 34.5 and 91%. Interestingly, by combining the histopathological diagnosis with FISH results, the diagnosis was optimized, especially by increasing specificity (76% instead of 52% for expert diagnosis alone) and by improving sensitivity compared with FISH alone (90 vs 43% for FISH result alone). The value of this FISH test is to add a reproducible demonstration of malignancy to the histopathological diagnosis, especially in doubtful/ambiguous melanocytic tumors. A positive FISH test reinforces the diagnosis of melanoma, allowing such tumors (particularly thick tumors) to be managed as melanomas.

Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance

Fluorescence in situ hybridization (FISH) is a powerful technique used in the detection of chromosomal abnormalities. The high sensi- tivity and specificity of FISH and the speed with which the assays can be performed have made FISH a pivotal cytogenetic technique that has provided significant advances in both the research and diagnosis of haematological malignancies and solid tumours. From a medical perspective, FISH can be applied to detect genetic abnormalities such as characteristic gene fusions, aneuploidy, loss of a chromosomal region or a whole chromosome or to monitor the progression of an aberration serving as a technique that can help in both the diagnosis of a genetic disease or suggesting prognostic outcomes. FISH can also be applied to such research applications as gene mapping or the identification of novel oncogenes or genetic aberrations that contribute towards various cancers. FISH is based on DNA probes annealing to specific target sequence of sample DNA. Attached to the probes are fluorescent reporter molecules which under fluorescence microscopy confirm the presence or absence of a particular genetic aberration when viewed under fluorescence microscopy. The tech- nique has recently evolved to allow screening of the whole genome simultaneously through multicolour whole-chromosome probe techniques such as multiplex FISH or spectral karyotyping, or through an array-based method using comparative genomic hybridization. This simple, yet effective, technique has revolutionized cytogenetics and has become well established in its potential as a diagnostic and discovery tool in the fight against cancer.

A Comparison of Extension Cytology and Fluorescence in Situ Hybridization (FISH) for the Detection of Malignant Bile Duct Strictures

Summary: The cytology through the brushing of the bile duct during endoscopic retrograde cholangiopancreatography, (ERCP) is the most popular technique used to diagnosis of cancer bile duct; their sensitivity varies from 15 to 63% with specificity from 97 to 100%. Recent studies show that FISH identify malignant cells in bile duct. Objective: To evaluate sensitivity and specificity of FISH and the cytology to detect malignant bile duct strictures. Material and Methods: A comparative transversal design of consecutive cases with obstructive jaundice that were subject to the ERCP between January and June 2007 in the Endoscopy Center of the National Institute of Cancerology of Mexico, with clinical evidence of malignancy that were confirmed through biopsy and/or constructo, with imaging (Computarized Axial Tomography, Magnetic Resonance, Cholangiopancreatography and/or ERCP) showing the presence of an alteration in the bile duct (tumor or stricture) with obstructive jaundice (biochemical changes that include al least 2 of the following: total bilirrubine = >1.5 mg/dl; alkaline phosphatase = >150 U/I; GOT = >100 U/I; GPT = >100 U/I) and/or CA19-9>100 ng/mL and follow-up with surveillance of every patient.

Detection of Pseudomonas aeruginosa in sputum samples by modified fluorescent in situ hybridization

Pseudomonas aeruginosa is the most common and dominant infectious agent that causes chronic pneumonia in patients with cystic fibrosis (CF). Fluorescent in situ hybridization (FISH) is a powerful molecular method for the specific and rapid diagnosis of bacteria, including the detection of P. aeruginosa in sputum samples from CF patients. High background fluorescence of viscous sputum samples obtained from CF patients may impede detection of microorganisms by FISH. The aim of this study was to test the application of biotin during FISH technique to reduce unspecific background fluorescence in sputum samples to facilitate and improve detection of P. aeruginosa. Sixty-three sputum samples from CF patients were tested by FISH to detect P. aeruginosa. All the 63 samples were also examined by a modified FISH procedure including biotin treatment. The FISH results were compared with those of conventional culture method. The specificity of FISH was 100%. The sensitivity of FISH for detection of P. aeruginosa from samples without biotin treatment was 83.3%, whereas in biotin-treated samples was 88.1%. Biotin reduced background fluorescence of 12 sputum samples of CF patients and it did not show any adverse effect on FISH results of the remaining sputum samples. Therefore, using of biotin in FISH procedure seems to facilitate and improve the detection of respiratory tract infections by P. aeruginosa in this population.

Clinical Utility of Fluorescence in Situ Hybridization for Voided Urine for the Diagnosis and Surveillance of Bladder Cancer

Purpose: To evaluate the clinical utility of performing fluorescence in situ hybridization (FISH) in voided urine specimens for the diagnosis and surveillance of bladder cancer, we compared the results of FISH with the results of urine cytology. Materials and Methods: Voided urine samples from 196 patients were obtained for performing FISH and urine cytology. The bladder cancer group consisted of 76 patients who had biopsy-proven bladder cancer, and the control group was 120 patients without bladder cancer. FISH was performed using the UroVysion kit. The sensitivity and specificity of FISH were determined and compared with that of urine cytology. Results: The overall sensitivity for FISH was significantly higher than the corresponding value for urine cytology (50% vs. 23.7%, respectively, p= 0.001). The specificities for FISH and cytology were 97.5% and 99.2%, respectively. When analyzing the results concerning the T-category, the sensitivity of FISH and cytology was 22.9% and 8.6% for pTa, 79.2% and 45.8% for pT1, and 54.5% and 9.1% for pT2-4 tumors, respectively. Concerning the tumor grade, the sensitivity was 22.2% and 5.6% for G1, 48.5% and 24.2% for G2, 70.8% and 33.3% for G3 for FISH and cytology, respectively. Conclusions: These findings show that the sensitivity of FISH for voided urine samples is higher than that of cytology, but the specificity was not significantly different. We recommend FISH as a useful diagnostic tool for patients who are suspected of having new bladder cancer and recurrent cancer, in combination with performing urine cytology. (Korean J Urol 2008;49:307-312) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏