scholarly journals Histochemistry today: Detection and location of single molecules

2017 ◽  
Author(s):  
Carlo Pellicciari
Keyword(s):  
In Situ Hybridization ◽  
Quantitative Assessment ◽  
Recent Literature ◽  
Single Molecules ◽  
High Specificity ◽  
Mechanistic Explanation ◽  
Lectin Histochemistry ◽  
Cell Functions ◽  
Molecular Maps

Especially in the latest years, histochemical investigations have progressively been oriented toward the visualization and quantitative assessment of single molecules, thanks to the availability of stains, reactions and procedures allowing to detect in situ proteins, or carboydrates or nucleic acid sequences with high specificity. This is evident from the recent literature, where in the large majority of the published articles immunohistochemistry, lectin histochemistry or fluorescence in situ hybridization were used as experimental methodologies. Since in biomedical research it is crucial to specifically label and localize molecules there, where they exert their structural roles and activities, histochemistry will continue to provide scientists the most appropriate tools for tracing molecular maps suitable for reaching a mechanistic explanation of cell functions in tissues.

Iso-chromosome 12p Analysis by Fluorescent In-Situ Hybridization: An Academic Institutional Experience

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S72-S72
Author(s):  
P P Patwardhan ◽  
S Satturwar ◽  
R Dhir ◽  
G M Quiroga-Garza
Keyword(s):  
In Situ Hybridization ◽  
Germ Cell ◽  
Fluorescent In Situ Hybridization ◽  
Clinical Presentation ◽  
Germ Cell Tumors ◽  
High Specificity ◽  
Testicular Germ Cell ◽  
Chromosome 12P ◽  
Institutional Experience

Abstract Introduction/Objective Chromosome 12 abnormalities like iso-chromosome 12p (i12p) and amplification of 12p are seen in majority (89%) of the primary and metastatic testicular germ cell tumors (TGCTs). i12p can be detected by karyotyping, fluorescent in-situ hybridization (FISH) or reverse transcriptase polymerase chain reaction. The aim of this study was to review i12p FISH data at our institution and assess the clinical utility. Methods/Case Report Laboratory information system was queried over a period of 15 years to search for cases where i12p FISH test was requested. FISH test was performed using TelVysion 12p telomeric probe and CEP 12 centromere probe on paraffin-embedded tissue or cell blocks. A ratio of 12ptel/CEP12 signal of 1.4 or greater was considered as positive. Patient demographics, clinical presentation, pathologic findings, and follow-up data were documented and correlated. Results (if a Case Study enter NA) Total 58 cases were identified with an age range of 14 to 76 years. Majority were male (M=52, F=6). Of these cases, 15 were testicular and 43 extra-testicular cases that included resection (n=35), biopsy (n=20) and cell-blocks (n=3). i12p was detected in 8 out of 15 testicular cases while i12p was detected in 16 out of the 43 extra-testicular cases. The extra- testicular cases included 17 retroperitoneal lesions, 8 lesions from the mediastinum, 6 lymph nodes from other sites and 12 miscellaneous lesions. Using pathology diagnosis with immunohistochemistry as gold standard, overall sensitivity was 60% and specificity was 86%. There were 3 false positive cases [Benign testicular parenchyma (n=1), suspicious for germ cell neoplasia in-situ (n=1) and undifferentiated epithelioid neoplasm (n=1)]. Conclusion Our results show that although the sensitivity was limited, FISH test for i12p demonstrated high specificity(86%) for diagnosis of primary or metastatic TGCTs. As an adjunct test, i12p FISH can help identify and further characterize a significant number of GCTs with unusual morphology or clinical presentation.

scholarly journals Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1922-1928 ◽  
Author(s):  
M Bentz ◽  
G Cabot ◽  
M Moos ◽  
MR Speicher ◽  
A Ganser ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Acute Lymphoblastic Leukemia ◽  
Yeast Artificial Chromosome ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
High Specificity ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Blood Smears

Abstract The presence of BCR-ABL fusion genes has important diagnostic and prognostic implications in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The CML-specific chimeric BCR-ABL gene with a break involving the major breakpoint cluster region (M-bcr) of the BCR-gene has been detected by means of fluorescence in situ hybridization (FISH). In this study, we present a FISH protocol that allows the detection of breaks in both the major and the minor breakpoint cluster region (m-bcr). Three hybridization signals of D107F9, a yeast artificial chromosome (YAC)-derived probe spanning the breakpoint regions of the BCR gene, were indicative of the translocation events. To increase the specificity further, this probe was combined with cos-abl 8, a cosmid probe flanking the breakpoint within the ABL gene for dual-color hybridization. Samples of 21 patients with CML, the ALL-derived cell line SUP-B15, and of seven patients with Philadelphia chromosome (Ph1)-positive ALL (three of them with breakpoints within m-bcr) were examined. BCR-ABL fusion was detected in all cases with high specificity (false-positive nuclei: mean, 0.1%). On cytogenetic preparations, the percentages of BCR-ABL- positive interphase cells ranged from 53% to 91%. Comparable efficiencies were achieved on blood smears. In conclusion, hybridization with D107F9 and cos-abl 8 allows unambiguous diagnosis of BCR-ABL genes and is likely to become an important tool for the monitoring of therapies in patients with CML and ALL.

Horse Radish Peroxidase Labelled Oligonucleotides and Tyramide Signal Amplification for Fluorescence in Situ Hybridization to Low Abundant Cytokine Mrnas

1997 ◽  
Vol 3 (S2) ◽  
pp. 203-204
Author(s):  
Mariette van de Corput ◽  
Rob van Gijlswijk ◽  
Mark Bobrow ◽  
Tom Erickson ◽  
Roel Dirks ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Signal To Noise Ratio ◽  
High Specificity ◽  
Tyramide Signal Amplification ◽  
Horse Radish Peroxidase ◽  
Metaphase Chromosomes ◽  
Generation Capacity

In recent years, Tyramide Signal Amplification (TSA) has gained acclaim as a very sensitive detection method for immunocytochemsitry and fluorescence in situ hybridization (FISH). To maximally exploit the great signal generation capacity of TSA in mRNA-FISH, minimizing signals emanating from non-specifically bound nucleic acid probe becomes of prime importance, because a specificity check of the signals observed in the cytoplasm is virtually impossible. We reasoned that utilization of synthetic oligonucleotides (ONTs) in stead of commonly used cDNAs or cRNAs would diminish non-specific probe binding and that direct Horse Radish Peroxidase (HRP) labelling of ONTs and TSA would enable their in situ detection.This approach was first tested in metaphase DNA-FISH using chromosome-specific repeats as targets. Using bifunctional crosslinking chemistry and HPLC, 5’-hexylamino oligonucleotides for chromosome specific simple satellite and alphoid sequences were conjugated to HRP and purified. Following 15 - 20 min of situ hybridization of a single HRP-ONT probe to metaphase chromosomes and a direct flurochrome-tyramide detection step, such repeat targets could be visualized with high specificity and excellent signal-to noise ratio.

Epstein-Barr Virus (EBV)-Encoded RNA: Automated In-Situ Hybridization (ISH) Compared with Manual ISH and Immunohistochemistry for Detection of EBV in Pediatric Lymphoproliferative Disorders

2009 ◽  
Vol 12 (3) ◽  
pp. 195-199 ◽  
Author(s):  
Naim K. Fanaian ◽  
Cynthia Cohen ◽  
Sandra Waldrop ◽  
Jennifer Wang ◽  
Bahig M. Shehata
Keyword(s):  
In Situ Hybridization ◽  
Predictive Value ◽  
Epstein Barr Virus ◽  
High Sensitivity ◽  
High Specificity ◽  
Lymphoproliferative Disorders ◽  
Barr Virus ◽  
Epstein Barr ◽  
Low Sensitivity

Detection of Epstein-Barr virus (EBV) may be achieved by various methods, including EBV-encoded RNA (EBER) in-situ hybridization (ISH) and immunohistochemistry (IHC) for latent membrane protein (LMP-1). We compared novel automated ISH and IHC techniques in pediatric lymphoproliferative disorders with results obtained by manual ISH. Thirty-seven pediatric cases previously studied by manual EBER ISH (including 18 EBER-positive, 15 EBER-negative, and 4 EBER-equivocal cases) were used for the study. Automated EBER ISH and automated LMP-1 IHC were performed using the BondMax autostainer and prediluted EBER probe and EBV cell surface 1 to 4 at 1:50 dilution, respectively. Results of each of the automated techniques for EBV detection were compared with results by manual EBER ISH. Compared with manual EBER ISH as the gold standard, automated ISH had a sensitivity and specificity of 94% and 69%, respectively, accuracy of 83%, positive predictive value (PPV) of 79%, and negative predictive value (NPV) of 90%. Automated IHC had a sensitivity of 44%, specificity of 93%, accuracy of 67%, PPV of 88%, and NPV of 59%. Automated ISH and IHC correlated significantly ( P < 0.045). Automated ISH is useful for diagnosis of EBV-related pediatric neoplasms, being easy to perform and interpret and requiring only the technologist's time to set up and having a high sensitivity and NPV. The automated IHC protocol is of too low sensitivity for routine use, although results show high specificity and PPV.

The Utility of UroVysion Fluorescence In Situ Hybridization in Pancreatic Fine-Needle Aspiration Samples Directed and Obtained by Endoscopic Ultrasonography

2013 ◽  
Vol 137 (1) ◽  
pp. 64-71 ◽  
Author(s):  
David N. Henkes ◽  
Sandeep N. Patel ◽  
Laura A. Rosenkranz ◽  
Jose L. Escobedo
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Pancreatic Adenocarcinoma ◽  
Fine Needle Aspiration ◽  
False Negative ◽  
High Specificity ◽  
Needle Aspiration ◽  
Fine Needle

Context.—The diagnosis of pancreatic adenocarcinoma can be challenging for the pathologist. Endoscopic ultrasound-guided, fine-needle aspiration (EUS-FNA) can be used to obtain samples of pancreatic masses. UroVysion fluorescence in situ hybridization (UFISH) has been reported to increase the sensitivity and to be very specific for the diagnosis of adenocarcinoma when combined with cytology in the diagnosis of biliary brushings and washings. Objectives.—To determine the sensitivity and specificity of UFISH on tissues obtained from pancreatic lesions suggestive of adenocarcinoma obtained by EUS-FNA, compared against fine-needle aspiration (FNA) results. Additionally, to use patient follow-up data to evaluate UFISH results in FNA samples that showed significant atypia but did not meet the criteria for malignancy. Design.—Sixty consecutive cases of pancreatic EUS-FNA from our institution submitted for UFISH testing. Results.—Polysomic UFISH has a sensitivity of 93% and a specificity of 100% when compared against FNA results. Follow-up studies showed that adding UFISH to FNA increased the sensitivity for patients with true-positive results from 83% to 94% and increased specificity from 85% to 100%. For 7 patients with suspicious FNA results who had sufficient follow-up, UFISH was 100% sensitive and 100% specific. Conclusions.—UFISH can be used to confirm the diagnosis of malignancy in pancreatic adenocarcinoma. Because of the high specificity, polysomic UFISH may help establish a diagnosis of malignancy when the FNA features are suggestive of, but not conclusive for, malignancy. The most common cause for a false-negative UFISH result was insufficient numbers of malignant cells.

scholarly journals Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1922-1928 ◽  
Author(s):  
M Bentz ◽  
G Cabot ◽  
M Moos ◽  
MR Speicher ◽  
A Ganser ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Acute Lymphoblastic Leukemia ◽  
Yeast Artificial Chromosome ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
High Specificity ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Blood Smears

The presence of BCR-ABL fusion genes has important diagnostic and prognostic implications in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The CML-specific chimeric BCR-ABL gene with a break involving the major breakpoint cluster region (M-bcr) of the BCR-gene has been detected by means of fluorescence in situ hybridization (FISH). In this study, we present a FISH protocol that allows the detection of breaks in both the major and the minor breakpoint cluster region (m-bcr). Three hybridization signals of D107F9, a yeast artificial chromosome (YAC)-derived probe spanning the breakpoint regions of the BCR gene, were indicative of the translocation events. To increase the specificity further, this probe was combined with cos-abl 8, a cosmid probe flanking the breakpoint within the ABL gene for dual-color hybridization. Samples of 21 patients with CML, the ALL-derived cell line SUP-B15, and of seven patients with Philadelphia chromosome (Ph1)-positive ALL (three of them with breakpoints within m-bcr) were examined. BCR-ABL fusion was detected in all cases with high specificity (false-positive nuclei: mean, 0.1%). On cytogenetic preparations, the percentages of BCR-ABL- positive interphase cells ranged from 53% to 91%. Comparable efficiencies were achieved on blood smears. In conclusion, hybridization with D107F9 and cos-abl 8 allows unambiguous diagnosis of BCR-ABL genes and is likely to become an important tool for the monitoring of therapies in patients with CML and ALL.

scholarly journals Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.

1992 ◽  
Vol 40 (10) ◽  
pp. 1457-1463 ◽  
Author(s):  
J C Adams
Keyword(s):  
In Situ Hybridization ◽  
Horseradish Peroxidase ◽  
Lectin Histochemistry ◽  
Fixed Tissue ◽  
Enzyme Systems ◽  
Enzymatic Action ◽  
Hybridization Probes ◽  
Background Staining ◽  
Amplification Procedure

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.

Evaluation of serum HER2 extracellular domain in in metastatic gastric or gastroesophageal junction cancer: Correlation with HER2 status by immunohistochemistry and fluorescence in situ hybridization and clinicopathologic parameters.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 4107-4107
Author(s):  
Xin An ◽  
Shuqin Dai ◽  
Fang Wang ◽  
Qiong Shao ◽  
Cui Chen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Gastroesophageal Junction ◽  
Locally Advanced ◽  
Surrogate Marker ◽  
High Specificity ◽  
Extracellular Domain ◽  
High Serum ◽  
Clinicopathological Parameters ◽  
Her2 Status

4107 Background: To explore the association between serum human epidermal growth factor receptor-2 (HER2) extracellular domain (ECD) and tissue HER2 status, their relationship with clinicopathological parameters and impact on overall survival (OS) in metastatic gastric or gastro-oesophageal junction (GEJ) adenocarcinoma. Methods: A total of 219 histologically confirmed inoperable locally advanced, recurrent, or metastatic gastric or GEJ adenocarcinoma were included. Serum HER2 ECD was measured by chemiluminescent assay. Tissue HER2 status was accessed by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) assay. Results: The median serum ECD level was 9.3 ng/ml (range 3.0 to >350). Tissue HER2 status was positive (IHC3+ or 2+ with FISH amplification) in 37 patients (16.9%) and negative in 182 patients (83.1%). Statistically significant associations were found between serum HER2 ECD level and HER2 status assessed by IHC and FISH. The ROC-analysis suggested the cutoff of 16.35 ng/ml (24 of 219 patients had HER2 ECD >16.35 ng/ml ) could produce a sensitivity of 51.4% and a specificity of 97.3% to predict tissue HER2 status. If the cutoff value was increaseed to 22 ng/ml, then all 12 patients with serum HER2 ECD >22 ng/ml were HER 2 positive in primary tumor, corresponding to a specificity of 100% and a sensitivity of 32.4%. High serum HER2 ECD levels were strongly associated with liver metastasis (P < 0.001), the intestinal histologic type (Lauren's classification) (P =0.003), large number of metastais (>2) (P=0.012) and increased LDH level (P < 0.001) . High serum HER2 ECD levels were more common in GEJ adenocarcinoma although the difference had no stastical significance. HER2 ECD levels did not show a significant impact on OS. Conclusions: A significant association was observed between serum HER2 ECD levels and tissue HER2 status in metastasis gastric or GEJ adenocarcinoma. The high specificity of serum HER2 ECD assay to predict tissue HER2 status suggests its potential use as a surrogate marker of the HER2 status in gastric cancer.

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