Effect of the Electric Field Profile on the Accuracy of E-FISH Measurements in Ionization Waves

Electric field induced second harmonic (E-FISH) generation has emerged as a versatile tool for measuring absolute electric field strengths in time-varying, non-equilibrium plasmas and gas discharges. Yet recent work has demonstrated that the E-FISH signal, when produced with tightly focused laser beams, exhibits a strong dependence on both the length and shape of the applied electric field profile (along the axis of laser beam propagation). In this paper, we examine the effect of this dependence more meaningfully, by predicting what an E-FISH experiment would measure in a plasma, using 2D axisymmetric numerical fluid simulations as the true value. A pin-plane nanosecond discharge at atmospheric pressure is adopted as the test configuration, and the electric field evolution during the propagation of the ionization wave (IW) is specifically analyzed. We find that the various phases of this evolution (before and up to the front arrival, immediately behind the front and after the connection to the grounded plane) are quite accurately described by three unique electric field profile shapes, each of which produces a different response in the E-FISH signal. As a result, the accuracy of an E-FISH measurement is generally predicted to be comparable in the first and third phases of the IW evolution, and significantly poorer in the second (intermediate) phase. Fortunately, even though the absolute error in the field strength at certain time instants could be large, the overall shape of the field evolution curve is relatively well captured by E-FISH. Guided by the simulation results, we propose a procedure for estimating the error in the initial phase of the IW development, based on the presumption that the starting field profile mirrors that of its corresponding Laplacian conditions before evolving further. We expect that this approach may be readily generalized and applicable to other IW problems or phenomena, thus extending the utility of the E-FISH diagnostic.

Characterization of an ETV6-NTRK3 rearrangement with unusual, but highly significant FISH signal pattern in a secretory carcinoma of the salivary gland: a case report

Background Fusions of neurotrophic tropomyosin receptor kinase genes NTRK1, NTRK2 and NTRK3 with various partner genes occur in both common and rare tumours and are of paramount predictive value due to the availability of very effective pan-Trk inhibitors like Larotrectinib and Entrectinib. Detection of NTRK fusions is mainly performed by fluorescence in situ hybridization (FISH) and next generation sequencing (NGS). The case described here showed a very unusual, but highly significant FISH signal pattern with an NTRK3 break apart probe, indicative of a functional NTRK3 rearrangement. Case presentation We describe here the case of a male patient who was originally diagnosed with an adenocarcinoma of the parotid gland without evidence of metastases. After the development of multiple lung metastases, an extensive immunohistochemical and molecular examination of archived tumour tissue including analysis of NTRK was performed. NTRK expression was detected by immunohistochemistry (IHC) and then comprehensively analysed further by FISH, quantitative reverse transcription PCR (RT-qPCR), and NGS. NTRK3 break apart FISH showed multiple and very faint single 3′ signals in addition to fusion signals. Quantitative reverse transcription PCR and NGS confirmed an ETV6:exon5-NTRK3:exon15 fusion. Diagnosis was therefore revised to metastatic secretory carcinoma of the salivary gland, and the patient subsequently treated with Larotrectinib, resulting in persisting partial remission. Conclusions Our findings underline the importance to be aware of non-canonical signal patterns during FISH analysis for detection of NTRK rearrangements. Very faint single 3′ signals can indicate a functional NTRK rearrangement and therefore be of high predictive value.

Detection of Del(17p) in Hematological Malignancies By Imaging Flow Cytometry

Del(17p) in chronic lymphocytic leukemia (CLL) and plasma cell myeloma has a unique genomic profile leading to refractoriness to conventional therapies and poor overall survival. Detection is generally by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei, not necessarily all of being neoplastic cells. The small cell sample analyzed, and high threshold for a positive result (>5% positive cells) makes FISH a low precision test. This means false negative results are inevitable, especially if del(17p) is only in a minor clone, potentially leading to suboptimal treatment. To address this, we developed an automated FISH method with analysis on an imaging flow cytometer, an instrument with the functionality of a standard flow cytometer which generates high-resolution digital images of each cell. By combining immunophenotyping with FISH, on whole cells in a single test, we can detect chromosomal defects in cells with a specific phenotype. Aims: Our aim was to determine the capability of this automated integrated immunophenotyping-FISH imaging flow cytometry method to detect del(17p) in CLL and myeloma, the two commonest hematological malignancies, and in which this genetic defect encodes poor prognosis. We hypothesized that it would be more sensitive and specific than standard FISH. Methods: Bone marrow and/or blood samples in EDTA anticoagulant from 19 cases of CLL or myeloma, at diagnosis or on therapy, were studied. After red cell lysis, cells were incubated with fluorochrome-conjugated antibodies to CD3, CD5, CD19, CD38, and CD138 antigens (fluorochromes: BV480, BV605, AF647). Following fixation, cell membranes were permeabilized and double-stranded DNA denatured (78oC for 5 mins). FISH probes to the centromere of chromosome 17 (CEP17, Spectrum Green) and 17p12 locus (Orange-Red) were added and hybridized for 24 hours at 37oC. Nuclei were stained with SYTOX AADvanced. Data for up to 200,000 cells was collected on the Amnis® ImageStream®XMk II imaging flow cytometer. Digital images (x60) and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying the nuclei of CD5/CD19-positive CLL cells or CD38/CD138-positive plasma cells for each probe. Digital images and quantitative data were assessed for FISH signals within immunophenotyped cells. Results: Between 10,000 and 200,000 (mean 60,000) cells were analyzed per sample. The FISH signals were seen on the digital images and confirmed by quantitative mean channel fluorescence intensity of the probes. There were 12 CLL cases with one 17p FISH signal in the CD5/CD19-positive population, with the number of del(17p) CLL cells ranging from 2 - 35% (or 0.4 - 23% of all cells analyzed) (Fig 1). This amounted to between 270 and 35,441 cells in the analysed sample with del(17p). The lowest del(17p) burden was in a patient on cytoreductive therapy. All CLL cells had normal diploid spots for the control CEP17 probe, and the CD3/CD5-positive T cells had dual signals for both CEP17 and 17p12 probes. There were 5 myeloma cases with 1 FISH signal for 17p overlying the nucleus of the CD38/CD138-positive plasma cells (Fig 2). In these cases, 52-90% of cells (15,600-18,000 cells) had a plasma cell phenotype and 13-19% of these (or 2,340-2,964 CD38/CD138-positive cells) showed del(17p). This represented 1-5% of all cells in the sample. All gated plasma cells had 2 FISH spots for CEP17. Conclusion: This imaging flow cytometry method that integrates FISH with immunophenotyping could detect del(17p) in CLL and myeloma with a lowest limit of detection of 0.4% and 1% respectively. The high sensitivity was achieved as many thousands of cells were analyzed, and 17p was only assessed in gated cells with the phenotype of interest (i.e. CD5/CD19 or CD38/CD138). This method, that includes positive cell identification by phenotype, precludes the need for prior cell sorting or purification. Imaging flow cytometry for del(17p) by "immuno-flowFISH" brings a new dimension to FISH analysis at diagnosis and for disease monitoring. Its high precision and specificity will enable detection of del(17p), even when only present in minor sub-clones, for therapeutic decision making and prognostic stratification. This technique has a real place in clinical assessment of del(17p) in CLL and myeloma and could be applied for other significant chromosomal defects of therapeutic and prognostic significance. Figure 1 Disclosures Augustson: Roche: Other: Support of parent study and funding of editorial support. Cheah:Celgene, F. Hoffmann-La Roche, Abbvie, MSD: Research Funding; Celgene, F. Hoffmann-La Roche, MSD, Janssen, Gilead, Ascentage Pharma, Acerta, Loxo Oncology, TG therapeutics: Honoraria.

A universal chromosome identification system for maize and wild Zea species

Maize was one of the first eukaryotic species in which individual chromosomes can be identified cytologically, which made maize one of the oldest models for genetics and cytogenetics research. Nevertheless, consistent identification of all 10 chromosomes from different maize lines as well as from wild Zea species remains a challenge. We developed a new technique for maize chromosome identification based on fluorescence in situ hybridization (FISH). We developed two oligonucleotide-based probes that hybridize to 24 chromosomal regions. Individual maize chromosomes show distinct FISH signal patterns, which allow universal identification of all chromosomes from different Zea species. We developed karyotypes from three Zea mays subspecies and two additional wild Zea species based on individually identified chromosomes. A paracentric inversion was discovered on the long arm of chromosome 4 in Z. nicaraguensis and Z. luxurians based on modifications of the FISH signal patterns. Chromosomes from these two species also showed distinct distribution patterns of terminal knobs compared to other Zea species. These results support that Z. nicaraguensis and Z. luxurians are closely related species.

The Polymorphisms of Oligonucleotide Probes in Wheat Cultivars Determined by ND-FISH

Non-denaturing fluorescence in situ hybridization (ND-FISH) has been used to distinguish wheat chromosomes and to detect alien chromosomes in the wheat genome. In this study, five different oligonucleotide probes were used with ND-FISH to examine 21 wheat cultivars and lines. These oligonucleotide probes distinguished 42 wheat chromosomes and also detected rye chromatin in the wheat genome. Moreover, the signal patterns of the oligonucleotide probes Oligo-pTa535-1 and Oligo-pSc119.2-1 showed high polymorphism in the wheat chromosomes. A total of 17.6% of the A group chromosomes, 25.9% of the B group chromosomes and 8.9% of the D group chromosomes showed obvious mutations when they were compared to the standard ND-FISH signal patterns, and most of them were Oligo-pSc119.2-1 mutants. The results suggested that these polymorphisms could be induced by the crossing of wheat cultivars. The results provided more information for the further application of oligonucleotide probes and ND-FISH.

Automated nuclear cartography reveals conserved sperm chromosome territory localization across 2 million years of mouse evolution

Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the nucleus into zones, and manually scoring which zone a FISH signal lies in. This is time consuming, limiting the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and two autosomes in three mouse lineages (Mus musculus domesticus, Mus musculus musculus and Mus spretus). We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse evolution.

The genome structure of Arachis hypogaea (Linnaeus, 1753) and an induced Arachis allotetraploid revealed by molecular cytogenetics

Peanut,Arachishypogaea(Linnaeus, 1753) is an allotetraploid cultivated plant with two subgenomes derived from the hybridization between two diploid wild species,A.duranensis(Krapovickas & W. C. Gregory, 1994) andA.ipaensis(Krapovickas & W. C. Gregory, 1994), followed by spontaneous chromosomal duplication. To understand genome changes following polyploidy, the chromosomes ofA.hypogaea, IpaDur1, an induced allotetraploid (A.ipaensis×A.duranensis)4xand the diploid progenitor species were cytogenetically compared. The karyotypes of the allotetraploids share the number and general morphology of chromosomes; DAPI+bands pattern and number of 5S rDNA loci. However, one 5S rDNA locus presents a heteromorphic FISH signal in both allotetraploids, relative to corresponding progenitor. Whilst forA.hypogaeathe number of 45S rDNA loci was equivalent to the sum of those present in the diploid species, in IpaDur1, two loci have not been detected. Overall distribution of repetitive DNA sequences was similar in both allotetraploids, althoughA.hypogaeahad additional CMA3+bands and few slight differences in the LTR-retrotransposons distribution compared to IpaDur1. GISH showed that the chromosomes of both allotetraploids had preferential hybridization to their corresponding diploid genomes. Nevertheless, at least one pair of IpaDur1 chromosomes had a clear mosaic hybridization pattern indicating recombination between the subgenomes, clear evidence that the genome of IpaDur1 shows some instability comparing to the genome ofA.hypogaeathat shows no mosaic of subgenomes, although both allotetraploids derive from the same progenitor species. For some reasons, the chromosome structure ofA.hypogaeais inherently more stable, or, it has been at least, partially stabilized through genetic changes and selection.