Fluorescence In Situ Hybridization Technique Research Articles

Challenges and solutions in FISH for formalin-fixed paraffin-embedded tissue: A scoping review.

Fluorescence in situ hybridization (FISH) has revolutionized molecular cytogenetic analysis since the 1980s, enabling precise localization of DNA sequences in cells and tissues. Despite its relevance, applying FISH to formalin-fixed paraffin-embedded (FFPE) tissue samples encounters significant technical challenges. This review addresses the main issues encountered in this context, such as inadequate fixation, contamination, block and slide age, inadequate pretreatment, and FISH technique. Proposed solutions include optimized pretreatment protocols, monitoring of blockage, careful selection of probes, and thorough analysis of results. Implementing good laboratory practices and quality control strategies are essential to ensure reliable results. Additionally, the use of emerging technologies such as artificial intelligence and digital pathology offers new perspectives for improving the efficiency and accuracy of FISH in FFPE samples. This review highlights the importance of a careful and personalized approach to overcome the technical challenges associated with FISH in FFPE samples, strengthening its role in research and clinical diagnosis. RESEARCH HIGHLIGHTS: Few FISH studies on FFPE: The scarcity of studies specifically addressing FISH applications in FFPE tissues highlights a critical gap in the literature. Troubleshooting FISH in FFPE tissues: Identifying and addressing common challenges in FISH techniques when applied to FFPE samples, such as signal quality and hybridization efficiency. Critical aspects of FISH technique: Discuss the main technical considerations crucial for successful FISH in FFPE tissues, including sample preparation, probe selection, and protocol optimization.

Polymicrobial consortia in the pathogenesis of biofilm vaginosis visualized by FISH. Historic review outlining the basic principles of the polymicrobial infection theory

The manuscript disputes the exclusive mono-infectious way of thinking, which presumes that for every infection only one pathogen is responsible and sufficient, when infectious vectors, close contact and reduced immunity meet. In situations involving heavily colonized anatomical sites such an approach often ends in insoluble contradictions. Upon critical reflection and evaluation of 20 years research on spatial organization of vaginal microbiota it is apparent, that in some situations, pathogens may act and operate in permanent, structurally organized consortia, whereas its individual components may be innocuous and innocent, failing to express any pathogenic effect. In these cases, consortia are the true pathogens responsible for many infectious conditions, which usually remain unrecognized as long as improperly diagnosed.The structure of such consortia can be unraveled using ribosomal fluorescence in situ hybridization (FISH). FISH methodology, that not only offers an ex vivo opportunity to recognize bacterial species, but provides unique physical insight into their specific role in the pathogenesis of polymicrobial infections. Ribosomal FISH technique applied to both, women with bacterial vaginosis (BV) and their male partners, has added significantly to our understanding of the pathogenesis of this condition and contributed to appreciating the mechanisms of polymicrobial, community-based infection, potentially leading to therapeutic advances.

Molecular cytogenetic screening of sex chromosome abnormalities in young horse populations.

Chromosomal abnormalities occur in the equine population at a rate of approximately 2%. The use of molecular cytogenetic techniques allows a more accurate identification of chromosomal abnormalities, especially those with a low rate of abnormal metaphases, demonstrating that the actual incidence in equine populations is higher. Estimation of the number of carriers of karyotypic abnormalities in a sample from a population of young horses of various breeds, using molecular cytogenetic techniques. Cross-sectional. Venous blood samples were collected from 500 young horses representing 5 breeds (Purebred Arabian, Hucul, Polish primitive horse [Konik], Małopolska, Coldblood, Silesian). Chromosomes and DNA were obtained from blood lymphocytes and evaluated by fluorescence in situ hybridisation (FISH) and PCR, using probes and markers for the sex chromosomes and select autosomes. Nineteen horses, 18 mares and 1 stallion, were diagnosed with different chromosomal abnormalities: 17 cases of mosaic forms of sex chromosome aneuploidies with a very low incidence (0.6%-4.7%), one case of a SRY-negative 64,XY sex reversal mare, and one mare with X-autosome translocation. The percentage of sex chromosomal aberrations was established as 3.8% in the whole population, 6.08% in females and 0.49% in males. Limited sample size, confined to horses from Poland. The rate of sex chromosomal abnormalities we identified was almost double that reported in previous population studies that used classical chromosome staining techniques. FISH allowed the detection of aneuploid cell lines which had a very low incidence. The FISH technique is a faster and more precise method for karyotype examination; however, it is usually focused on only one or two chromosomes while banding karyotyping includes the entire chromosome set.

Clinical Significance of Genetic and Molecular Changes in Primary Myeloid Sarcoma

To investigate the clinical significance of genetic and molecular changes in primary myeloid sarcoma (MS). Fourteen patients with primary MS were selected in Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, The First People's Hospital of Lianyungang from September 2010 to December 2021. AML1-ETO fusion, PML-RARα fusion and CBFβ breakage were detected by fluorescence in situ hybridization (FISH), and the mutations of NPM1, CEBPA, FLT3, RUNX1, ASXL1, KIT and TP53 genes were detected by new generation sequencing (NGS). Among 14 patients, the MS occurred in bone, breast, epididymis, lung, chest wall, cervix, small intestine, ovary, lymph nodes and central nervous system. The tumor cells expressed MPO (13 cases), CD34 (7 cases), CD43 (8 cases), CD68 (7 cases), CD99 (8 cases) and CD117 (6 cases). Cytogenetic abnormalities were observed in 4 cases, including 3 cases of AML1-ETO fusion and 1 case of CBFβ breakage, while no PML-RARα fusion was detected. There were no significant differences in overall survival (OS) and leukemia-free survival (LFS) between patients with and without AML1-ETO fusion/CBFβ breakage (both P >0.05). Among the 14 patients, the number of NPM1, CEBPA, FLT3-ITD, RUNX1, ASXL1, KIT and TP53 gene mutations was 5, 3, 5, 3, 2, 2, 1, respectively, of which 7 cases had at least one mutation in FLT3-ITD, RUNX1, ASXL1 and TP53 gene. The OS and LFS of patients with FLT3-ITD, RUNX1, ASXL1 or TP53 mutation were shorter than those without mutations (both P <0.01). The genetic and molecular abnormalities of primary MS can be detected by FISH and NGS techniques. FLT3-ITD, RUNX1, ASXL1 or TP53 mutation indicates a worse prognosis, but further clinical studies are needed to confirm it.

Identificação de Brachyspira pilosicoli, Brachyspira hyodysenteriae e Brachyspira intermedia em aves de postura e matrizes comerciais pela técnica de hibridização fluorescente in situ (FISH) em tecidos incluídos em parafina

ABSTRACT: Bacteria of the genus Brachyspira can cause enteric diseases in birds; thus, this study evaluated the efficacy of the fluorescence in situ hybridization (FISH) technique for the identification of B. pilosicoli, B. hyodysenteriae and B. intermedia using cecum samples fixed in formaldehyde from laying hens and commercial broiler breeders. Samples were collected from 112 birds aged between 35 and 82 weeks that originated from commercial laying and broilers farms. For the initial evaluation, spirochaetes were isolated from the cloacal swabs. Positive samples were analysed using qPCR to identify pathogenic species. Formalized cecum segments of these same birds were then analyzed using the FISH technique with labelled probes specific to B. pilosicoli, B. hyodysenteriae and B. intermedia. Forty isolates characteristic of Brachyspira were obtained, of which 14 were identified as B. hyodysenteriae and seven were identified as B. intermedia by qPCR; two samples were positive for both species, and 21 were not characterized. Using the FISH technique, 52 samples were positive for Brachyspira spp., 22 were positive for B. hyodysenteriae, 28 were positive for B. intermedia, seven were positive for B. pilosicoli, and eight were positive for two species. The FISH technique was able to identify significantly more positive birds compared with bacterial isolation followed by qPCR. Thus, it is concluded that the FISH technique was effective for identifying the three Brachyspira species evaluated and can thus be used as a rapid and effective diagnostic tool.

Multiplexed Immunofluorescence and Single-Molecule RNA Fluorescence In Situ Hybridization in Mouse Skeletal Myofibers.

RNA fluorescence in situ hybridization (FISH) is a powerful method to determine the abundance and localization of mRNA molecules in cells. While modern RNA FISH techniques allow quantification at single molecule resolution, most methods are optimized for mammalian cell culture and are not easily applied to in vivo tissue settings. Single-molecule RNA detection in skeletal muscle cells has been particularly challenging due to the thickness and high autofluorescence of adult muscle tissue and a lack of in vitro models for mature muscle cells (myofibers). Here, we present a method for isolation of adult myofibers from mouse skeletal muscle and detection of single mRNA molecules and proteins using multiplexed RNA FISH and immunofluorescence.

Fluorescence in situ hybridisation for interphase chromosomal aberration-based biological dosimetry.

Metaphase spreads stained with Giemsa or painted with chromosome-specific probes by fluorescence in situ hybridisation (FISH) have been in use since long for retrospective dose assessment (biological dosimetry). However, in cases of accidental exposure to ionising radiation, the culturing of lymphocytes to obtain metaphase chromosomes and analysis of chromosomal aberrations is time-consuming and problematic after high radiation doses. Similarly, analysing chromosomal damage in G0/G1 cells or nondividing cells by premature chromosome condensation is laborious. Following large-scale radiological emergencies, the time required for analysis is more important than precision of dose estimate. Painting of whole chromosomes using chromosome-specific probes in interphase nuclei by the FISH technique will eliminate the time required for cell culture and allow a fast dose estimate, provided that a meaningful dose-response can be obtained by scoring the number of chromosomal domains visible in interphase nuclei. In order to test the applicability of interphase FISH for quick biological dosimetry, whole blood from a healthy donor was irradiated with 8Gy of gamma radiation. Irradiated whole blood was kept for 2h at 37°C to allow DNA repair and thereafter processed for FISH with probes specific for Chromosomes-1 and 2. Damaged chromosomal fragments, distinguished by extra color domains, were observed in interphase nuclei of lymphocytes irradiated with 8Gy. These fragments were efficiently detected and quantified by the FISH technique utilising both confocal and single plane fluorescence microscopy. Furthermore, a clear dose-response curve for interphase fragments was achieved following exposure to 0, 1, 2, 4 and 8Gy of gamma radiation. These results demonstrate interphase FISH as a promising test for biodosimetry and for studying cytogenetic effects of radiation in nondividing cells.

Novel role of fluorescent in situ hybridization technique (FISH) in recommended surgical margins of dermatofibrosarcoma protuberans: A preliminary study

Dermatofibrosarcoma protuberans (DFSP) is a rare skin tumor. There is no standard recommendation for its surgical management. The currently used histological analysis are HES (hematoxylin eosin saffron) staining and immunohistochemistry for CD34 expression in particular cases. Fluorescent in situ hybridization (FISH) technique is only used to qualify the DFSP as translocated or non-translocated and is not used as a diagnostic method. The aim of our study was to determine by FISH (as a diagnostic method) whether cancerous cells that could not be identified through HES staining±immunohistochemistry were present at the two-centimeter margins that were found to be tumor-free. Samples from patients who underwent surgery between 2010 and 2018 were collected. Intralesional and peripheral (at 2cm margins) paraffin slides were included. An average of 7.4 slides per specimen was analyzed. Firstly, the preselected slides were reread by a senior pathologist to confirm the absence of microscopic findings of DFSP at 2cm margins. Secondly a FISH analysis was used as a quantitative diagnostic approach, in order to find the t(17;22) translocation. Among the seven specimens that included 2cm margins, two samples presented one or more translocations, which were not visible in standard morphology assessments at two centimeters tumor-free margins. FISH analysis can have a new role in defining tumor-free margins. This would reduce the incidence of disease recurrence after resection and improve the post-operative complementary care.

Childhood B-lymphoblastic leukemia: Integrating cytogenetics with other prognostic parameters and correlation with post-induction remission

BackgroundCytogenetics is irrefutably one of the most significant prognostic markers which have imparted ease and feasibility in the classification of acute leukemia. It is an essential prognostic tool in major treatment protocols for all hematological malignancies. This study aimed to note the frequency of different cytogenetic aberrations in children with B-ALL, their association with risk factors, and impact on post-induction remission status. MethodsA retrospective study was conducted in the Hematology department of The Indus Hospital, Karachi. All newly diagnosed patients of B-ALL (aged 1–17 years) from October 2, 2020 to October 13, 2021 were included. They were treated with modified Children's Oncology Group (COG) protocol. Cytogenetic studies including conventional karyotyping and fluorescence in situ hybridization (FISH) for BCR::ABL1, ETV6::RUNX1, and MLL gene rearrangement were performed on semi-automated cell imaging. ResultsAmong 238 patients, cytogenetic abnormalities were identified in 146 (61.3%) cases. A normal karyotype was observed in 92 (38.7%), high hyperdiploidy in 56 (24%), random structural abnormalities in 22 (9.2%), t (1:19) in 21 (8.8%), t (9; 22) in 12 (5.0%), complex karyotype in 10 (4.2%), 11q abnormalities in 5 (2.1%) and monosomy 7 in 1 (0.4%) case(s). FISH positivity for MLL, BCR::ABL1 and ETV6::RUNX1 was observed in 6 (2.5%), 18 (7.5%), and 20 (8.4%), cases, respectively. Post induction morphological remission was observed in 98.3% of cases. ConclusionThe overall frequency of cytogenetic abnormalities in the current cohort is comparable to previously reported studies. Our data also suggest the value of combining the karyotype and FISH techniques to increase the likelihood of detection of recurrent cytogenetic abnormalities. Even though there was a lack of correlation between genetic abnormalities and early outcome, a follow-up may be useful to ascertain the correlation with long-term survival.

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells.

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

Evaluation of protein expression of microchimeric Y-chromosome in ovarian endometriosis

Introduction: Endometriosis is a benign chronic gynaecological disorder with the presence of endometrium-like tissue outside the uterus. It affects 10% of the reproductive age group of women. The symptoms include infertility 30 – 50%, and dysmenorrhea 40 – 60%. Thus, endometriosis is a global burden with a wide range of symptomatology and hence causes difficulty for women who suffer from the disease. But the pathogenesis of the disease is yet to be understood. The presence of fetal Y-chromosome microchimerism in endometriosis is first reported using the FISH (Fluorescence In-Situ Hybridization) technique. Later, with a similar technique, conflicting results have been reported. The detection probes used were Y-specific chromosomes Yp11.1 – q11.1-Alpha Satellite DNA and Yq12 Satellite III DNA. The genomic evidence of Y chromosome microchimerism is evaluated using microarray expression followed by RNA-seq and quantitative RT-PCR experiments of target inserts for selected targets for further validation. This study reported the presence of 21 non-coding, and 33 protein-coding genes present in the Male Specific region of Y-chromosome (MSY) in ovarian endometriosis. Genomic studies have been reported in the literature but protein expression from Y-chromosome genes in ovarian endometriosis has not been reported. So, we aimed to evaluate protein expressions of EIF1aY, NLGN4Y, and ZFY in the eutopic endometrium of ovarian endometriosis. Method: The 2 study groups are allotted; Group A (Control) and Group B (Endometriosis). The tissue samples collected were annotated, processed, and performed western blot for identification of EIF1aY, ZFY, and NLGN4Y gene expression. The blots were scanned and analyzed using Bio-Rad Pharos FX TM Plus Molecular Imager and NIH Image J software ( http://imagej.nih.gov/ij/ ) respectively. Result: There was a significant increase in the gene expression of EIF1aY in the endometriosis group when compared to the control group (p &lt; 0.01). The Spearman correlation scores for NLGN4Y and ZFY are strongly associated (r = 0.617). Conclusion: The ectopic site promotes the niche and eutopic tissue has an error which enhances the histogenesis. It appears from the present study that proteins coded from Y-chromosome genes, namely EIF1aY, NLGN4Y and ZFY are involved in the pathogenesis of ovarian endometriosis, which could be a potential therapeutic target in future. To the best of our knowledge, this is the first report suggesting the involvement of Y-microchimerism at the protein level in endometriosis. Further studies with larger sample sizes and different stages and phenotypes of endometriosis may bring forth more conclusive knowledge in this regard that seems to promote the histogenesis of endometriosis at ectopic sites. Department of Physiology, AIIMS, New Delhi. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A rare disorder of sex development; de la chapelle syndrome

Sex reversal syndromes can be summarized as an incompatibility of chromosomal sex and gonadal characteristics. A very rare syndrome. 46 XX testicular disorder was first described by De La Chapelle in 1964 in 46 XX karyotype male individuals. Generally, patients whose phenotype is male apply to the health center with infertility, impotence, loss of libido, or gynecomastia. Translating the part of the Y chromosome, including the SRY (sex-determining region Y) gene, to the X chromosome during paternal meiosis is responsible for etiopathogenesis. &#x0D; In our case, a 38-year-old male patient applied to our outpatient clinic complaining of enlargement in both breasts. His beard-mustache and body hair distribution was expected, he had bilateral grade 2 gynecomastia, his penis length was 7 cm, and his testicles were small and palpable in the scrotum. Laboratory values were compatible with hypogonadotropic hypogonadism, and in the sperm analysis, azoospermia was detected. Karyotype analysis was 46 XX, SRY was also studied with the FISH (Fluorescence in Situ Hybridization) technique. The patient was diagnosed with 46 XX Testicular Disorder (de la Chapelle Syndrome), and testosterone replacement therapy was started. &#x0D; We aimed to present the diagnosis and management of De La Chapelle Syndrome in our case.

RENEB Inter-Laboratory Comparison 2021: The FISH-Based Translocation Assay.

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 ± 0.03 and 1.47 ± 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 ± 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 ± 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.

Abstract 4294: Bridging the gap between targeted NGS and FISH gene-level CNV detection capabilities in hematologic malignancies

Abstract Background: Copy Number Variations (CNVs) are prominent features of cancer cells. From a clinical standpoint, their accurate detection at a low cost is a priority. With regular increases in the number of markers to be tested, the cost effectiveness and practicality of gold standard techniques like Fluorescence In Situ Hybridization (FISH) are slowly decreasing. Cost-efficient Next Generation Sequencing (NGS) targeted gene panels can be scaled up but accurately detecting CNVs from the resulting data remains challenging. We demonstrate large amounts of data and machine learning can help bridge the gap between the two techniques. Methods: We collected the sequencing data of 6,277 patients tested using a custom amplicon based NGS assay designed to detect somatic alterations in 297 hematological cancer relevant genes such that at least one concurrent FISH test was also performed. FISH results were used to infer the gain, loss, or normality information for each corresponding gene. The annotated genes were then used to curate a training set by extracting 20 features per gene from the alignment results. A 3-class random forest classifier was trained using this dataset. The selected model was evaluated on a distinct set of 2,738 patients. Results: Evaluation results are provided in Table 1 for 8 genes for which the FISH probe used to infer the gain, loss, or normality information directly spanned the gene region. The predicted CNVs are almost a perfect match with FISH for 6 of these genes with a limit of detection at 20% abnormal cells. In most cases, the model reduces discordant calls by over 50% compared to using existing CNV detection software only. Conclusion: We show the CNV detection capabilities of a targeted NGS assay can closely match the gold-standard FISH technique by analytically correcting the biases introduced by the targeting procedures. The model presented here is used to detect CNVs in ALL patients after a successful formal validation in our laboratory. Table 1. Evaluation results. Gene FISH Positive Cases (Gain/Loss) FISH Negative Cases (Normal) All FISH Cases Total Concordant Sensitivity Total Concordant Specificity Total Concordant Accuracy ATM 72 72 100.00% 641 629 98.13% 713 701 98.32% CBFB 23 22 95.65% 517 506 97.87% 540 528 97.78% EGR1 171 169 98.83% 1,541 1,538 99.81% 1,712 1,707 99.71% KMT2A 27 25 92.59% 474 472 99.58% 501 497 99.20% MET 113 113 100.00% 1,593 1,582 99.31% 1,706 1,695 99.36% NF1 15 15 100.00% 908 908 100.00% 923 923 100.00% TERT 10 10 100.00% 1,723 1,709 99.19% 1,733 1,719 99.19% TP53 76 73 96.05% 1,567 1,556 99.30% 1,643 1,629 99.15% Citation Format: Christophe N. Magnan, Hyunjun Nam, Shashikant Kulkarni, Segun C. Jung. Bridging the gap between targeted NGS and FISH gene-level CNV detection capabilities in hematologic malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4294.

Alveolar rhabdomyosarcoma: novel surrogate markers associated with oncogenic translocation

Anomalies of the FOXO1 gene in alveolar rhabdomyosarcoma are associated with a worse clinical prognosis, which determines the high value of studying the status of this gene when choosing a therapy strategy. The «gold standard» for determining FOXO1 gene rearrangements is currently the fluorescent in situ hybridization (FISH) technique. Study of the relationship between canonical FOXO1 translocation and immunohistochemical expression of new surrogate markers in alveolar rhabdomyosarcoma to determine their predictive value. 139 cases of rhabdomyosarcoma were retrospectively studied. The study used tissue matrix technology (TMA). On sections obtained from TMA blocks, the FISH technique was implemented using the locus-specific probe MetaSystems XL FOXO1 Break Apart (Metasystems, Germany). Immunohistochemical studies were performed on similar sections from TMA blocks with OLIG2 (Cell Marque Antibodies, clone 211F1.1) and MUC4 (Cell Marque Antibodies, clone 8G7) antibodies. The final expression analysis and statistical processing using a 2x2 contingency table and Fisher's exact test passed 111 cases (76 without FOXO1 rearrangement and 35 with rearrangement). The specificity of OLIG2 and MUC4 expression for FOXO1-rearranged alveolar rhabdomyosarcoma was 85.53% and 80.26%, respectively (p<0.01). The present study confirms the high predictive value of the expression of surrogate markers OLIG2 and MUC4 in determining the genetic status of alveolar rhabdomyosarcoma, which makes it possible to predict with high specificity the detection of the FOXO1 gene rearrangement.

Visualization of Oligonucleotide-Based Probes Along Pseudochromosomes Using RIdeogram, KaryoploteR, and Circlize (Circos).

Fluorescence in situ hybridization (FISH) using oligonucleotide-based probes is an innovative modification of classic FISH techniques, enabling karyotypic identifications. Here, we exemplarily describe the design and in silico visualization of oligonucleotide-based probes derived from the Cucumis sativus genome. Additionally, the probes are also plotted comparatively to the closely related Cucumis melo genome. The visualization process is realized in R using various libraries for linear or circular plots including RIdeogram, KaryoploteR, and Circlize.

Cytogenetic Diagnosis of a British Shorthair Tomcat with a 37, X/38, XY/39, XY+der(Y) Karyotype

In this study, the determination of three different cell lines in a two-year-old British shorthair tomcat, using fluorescence in situ hybridisation (FISH), is described. The FISH technique was instrumental in the identification of this chromosomal aberration, which had not been previously described in cats. The mosaic karyotype with three cell lines (37, X; 38, XY; and 39, XY+der(Y)), detected using X-, Yand autosomal B1-whole chromosome painting probes, were all visible in the metaphase and interphase nuclei. The ratios of the three cell lines were 3.6%, 85.6% and 10.8%, respectively. In addition, at the time of this examination, the cat had a temperamental disposition that had persisted beyond castration.

Unexpectedly High Prevalence of iAMP21 in Children with Acute Lymphoblastic Leukemia in Mexico: A Report from the "Mexico in Alliance with St. Jude” (MAS) Collaborative Group

Keywords: acute lymphoblastic leukemia, intra-amplification of chromosome 21, genetic characterization. Mexico in Alliance with St. Jude (MAS) has carried out a multi-center collaboration for children with acute lymphoblastic leukemia (ALL) in Mexico since 2019. The "Bridge Project” has granted access to diagnostic tests that allow genetic characterization of children with ALL in participating centers. The Molecular Genetics Laboratory of Hospital Infantil Teleton de Oncologia (HITO) serves as the referral laboratory. 253 newly diagnosed ALL patients from 5 hospitals in Mexico have utilized a comprehensive diagnostic panel that includes cytogenetic and molecular tests to identify relevant abnormalities. iAMP21 is recognized by the 2017 World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues as a characteristic genetic alteration of B-lineage Leukemia/Lymphoma, with a reported incidence of up to 2-5%. It involves amplification of the RUNX1 gene and/or duplication of chromosome 21 dup(21q), and defines a relatively new cytogenetic subgroup in B-lineage ALL with poor prognosis, derived from the genetic instability of chromosome 21.The heterogeneity of iAMP21 contributes to the difficulty in its detection with the sole use of conventional cytogenetic techniques, rendering Fluorescent In Situ Hybridization (FISH) as a better strategy for the distinction between iAMP21 (amplification in tandem) and the sporadic gain of copies of the RUNX1 gene. METHODS: We performed a comprehensive diagnostic panel for 253 patients with ALL; the FISH B-cell lymphoid panel included (KMT2A, ETV6 (TEL)/RUNX1 (AML1), BCR/ ABL, E2A/(TCF3)/(PBX1) and the T-cell lymphoid panel included (MLL, BCR / ABL, E2A / (TCF3) / (PBX1), TLX1, TLX3, CDKN2A and TRA / D). Diagnosis of iAMP21 was made using the interphase FISH technique with the LSI ETV6/RUNX1 (Vysis®) double color double fusion probe (Abbott Molecular®, Ill), and Cytocell probe (OGT Company®, NY). A finding of ≥ 5 RUNX1 gene signals in interphase FISH and/or more than 3 signals within chromosome 21 was considered positive for the identification of iAMP21. Samples were processed according to the specifications to the AGT Cytogenetics Laboratory Manual. FISH analysis was performed by analyzing 200 interphase cells. When ETV6/ RUNX1 probes are used, samples of patients carrying iAMP21 usually show two normal copies of the ETV6 signal and multiple RUNX1 signals (three or more additional signals) and are negative for the ETV6-RUNX1 fusion gene. RESULTS: Among 253 patients with ALL, 233 cases were of lineage B (92%) and 20 (8%) were of lineage T. FISH was reported negative in 57 (23%) and positive in 185 (73%) cases, 11 results were not available (4.3%). We detected iAMP21 in 28 (11%); these cases were classified as high risk. We also report t(12;21) in 41 (16%), MLL gene disruption t(4;11) in 10 (4%), t(1;19) in 14 (6%), and t(9;22) in 7 (3%); 75 (30%) patients had nonspecific genetic gains. The T lineage showed CDKN2A del(9)(p21) in 6 (2.5%) and TRA/D(14)(q11.2) rearrangement in 4 (1.7%) cases. CONCLUSION: Our experience shows that FISH analysis with the ETV6/RUNX1 probe is useful for identifying iAMP21, a chromosomal abnormality with prognostic significance. Genetic risk factors in the Hispanic population that adversely impact survival in Latin America are not yet fully identified. The Molecular genetic analysis and the fluorescence in situ hybridization (FISH) technique are the basic methods used to detect the presence of the most common genetic abnormalities, the presence or absence of which has an impact on the patient's classification into the appropriate risk group. Our findings from this cohort point to an increased prevalence of iAMP21 in ALL patients in Mexico. We will soon incorporate gene sequencing to the diagnostic panel and improve the characterization of these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Spectral imaging and nucleic acid mimics fluorescence in situ hybridization (SI-NAM-FISH) for multiplex detection of clinical pathogens.

The application of nucleic acid mimics (NAMs), such as locked nucleic acid (LNA) and 2′-O-methyl-RNA (2’OMe), has improved the performance of fluorescence in situ hybridization (FISH) methods for the detection/location of clinical pathogens since they provide design versatility and thermodynamic control. However, an important limitation of FISH techniques is the low number of distinguishable targets. The use of filters in fluorescence image acquisition limits the number of fluorochromes that can be simultaneously differentiated. Recent advances in fluorescence spectral image acquisition have allowed the unambiguous identification of several microorganisms in a single sample. In this work, we aimed to combine NAM-FISH and spectral image analysis to develop and validate a new FISH variant, the spectral imaging-NAM-FISH (SI-NAM-FISH), that allows a multiplexed, robust and rapid detection of clinical pathogens. In the first stage, to implement/validate the method, we have selected seven fluorochromes with distinct spectral properties and seven bacterial species (Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter calcoaceticus). As a strong variation in fluorescence intensities is found between species and between fluorochromes, seven versions of a EUB LNA/2’OMe probe, each conjugated to one of seven fluorochromes, were used to rank species/fluorochromes by FISH and then optimize species/fluorochrome pairing. Then, final validation tests were performed using mixed populations to evaluate the potential of the technique for separating/quantifying the different targets. Overall, validation tests with different proportions of bacteria labeled with the respective fluorochrome have shown the ability of the method to correctly distinguish the species.

Classical chromosome features and microsatellites repeat in Gekko petricolus (Reptilia, Gekkonidae) from Thailand

The objectives of this study were to examine size, shape, diploid number (2n), fundamental number (NF), NORs region, and distribution of microsatellite by using Fluorescence in situ hybridization technique (FISH) and to establish the karyotype and standard idiogram of sandstone geckos (Gekko petricolus Taylor, 1962). Sandstone gecko distributed in the sandstone mountains in Laos, Cambodia, and Thailand. Five male and five female specimens were collected from Ubon Ratchathani and Mukdahan provinces, Thailand. The metaphase cells were directly prepared from the bone marrow cells. Chromosomes were stained by conventional staining, NORs-banding and FISH techniques. The results found that the diploid number was 38 chromosomes. The fundamental number was 54. The karyotype composed of 4 large metacentric, 4 large acrocentric, 2 large telocentric, 4 medium acrocentric, 2 medium telocentric, 2 small submetacentric, 2 small acrocentric and 18 small telocentric chromosomes. No morphological difference was identified between sex chromosomes of male and female specimens. The NORs appeared to telomere of the long arm of chromosome pair 17. The study displayed that the distribution of microsatellite using (CA)15 and (GAA)10 probes distributed throughout the genome. However, (CA)15 sequences concentrated in the telomere. The karyotype formula G. petricolus is as follow: 2n (38) = Lm4+La4+Lt2+Ma4+Mt2+Ssm2+Sa2+St18.