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PubMed1999

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A by interphase fluorescence in situ hybridization.

Charcot-Marie-Tooth Disease (CMT) is the most common cause of peripheral neuropathy, with an incidence of 1:2500 persons affected. Previously, we reported the use of fluorescence in situ hybridization (FISH) to detect the common submicroscopic duplication of 17p12 found in more than 98 per cent of individuals with CMT1A. We found that FISH is a reliable means for the diagnosis of the duplication of 17p12 in peripheral blood and reported the validation of the FISH assay for amniotic fluid specimens. Herein, we report the validation of the FISH assay for use on chorionic villus samples (CVS) to prenatally diagnose CMT1A duplications and the testing of 17 prenatal specimens. Seven fetuses were found to carry the duplication and are predicted to be affected. FISH is a rapid assay in prenatal specimens, with a 9.3 day average turn-around time. Limited follow-up on pregnancies indicates that the duplication found in CMT1A is reliably diagnosed in the fetus, using FISH on either amniotic fluid specimens or CVS.

Charcot-Marie-Tooth Disease↗
PubMed1996

Automation of spot counting in interphase cytogenetics using brightfield microscopy.

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5-10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction.

Algorithms↗
PubMed1996

Detection of numerical chromosomal abnormalities by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes on archival cytologic samples.

Fluorescence in situ hybridization (FISH) is rapidly emerging as a tool for analyzing numerical and structural chromosomal abnormalities in both liquid and solid tumors. Most studies make use of fresh samples. To determine the feasibility of detecting numerical chromosomal abnormalities (NCA) by FISH using chromosome-specific probes 8, 12, 17, and X (Vysis, Inc., Downers Grove, IL) on archival cytologic preparations, we studied 23 patient samples, one Papanicolaou-and one Diff-Quik-stained slide per case (46 slides), and two additional unstained slides (fresh ascitic fluids) as controls. Included in this study were nine ascitic fluids (four benign and five malignant), four malignant pleural fluids, three benign bladder washes, and seven malignant fine-needle aspirates (FNA) from various sites. The slides ranged from 1-94 days old. After removal of coverslips using xylene, all slides were destained in a series of alcohol and water washes. Pretreatment of slides with pepsin was followed by the in situ hybridization procedure. Two hundred cells per slide were evaluated for distinct separate signals. Results showed the following: 1) all slides were evaluable except for eight (8/46) which had either too few cells or enough cells but with faint signals, 2) the oldest sample showed distinct signals, 3) previously Diff-Quik-stained slides showed relatively better signals than Papanicolaou-stained slides, 4) samples less than a month old showed relatively better signals, and 5) malignant samples showed various NCA, but not the benign samples. We conclude that FISH on archival cytologic preparation 1) is feasible, although age of the slide is a factor since better signals were seen in those less than a month old, 2) shows better results in previously Diff-Quik-stained slides, and 3) is a tool that can be used in the retrospective study of various liquid and solid neoplasms.

Cell Nucleus↗
PubMed1997

Numerical aberrations of chromosomes 1, 2, and 7 in astrocytomas studied by interphase cytogenetics.

For both juvenile astrocytomas and astrocytomas of adults, numerical and structural aberrations of chromosomes 1 and 7 have been described. To study the frequency of those aberrations in more detail and to exclude in vitro artifacts, we investigated directly prepared material from 18 juvenile and 26 astrocytomas of adults by fluorescence in situ hybridization with DNA probes specific for chromosome regions 1p36, 1q12, 2cen, and 7cen. Chromosome 2 was used as control in the hybridization with chromosome 7. To exclude tissue-specific alterations, we tested cerebral and cerebellar paraffin-embedded material from persons who had died from other diseases. In 13 of the juvenile astrocytomas, we found a loss of 1p36 in relation to 1q12 in 16 astrocytomas of adults, a gain of signals from 1p36 or both probes for chromosome 1 was seen. Gain of chromosome 7 was found in 25 cases. Unexpectedly, gain of chromosome 2 occurred in 32 cases. For both probes, there was no difference between astrocytomas of children and those of adults. Our data suggest that loss of 1p is an early event in the development of juvenile astrocytomas and that trisomy 7 is frequent in malignant tumors and tumors containing a potential of growing malignantly.

Adolescent↗
PubMed1999

Arylamine N-acetyltransferase type 2 (NAT2), chromosome 8 aneuploidy, and identification of a novel NAT1 cosmid clone: an investigation in bladder cancer by interphase FISH.

Two genes (arylamine N-acetyltransferase types 1 and 2, NAT1 and NAT2), which are known to metabolize bladder carcinogens, are located on chromosome band 8p22. Alterations in chromosome 8, including deletions of 8p, occur frequently in many epithelium-derived tumors. In this study, fluorescence in situ hybridization (FISH) was used for study of the relationship between chromosome 8 deletions in the region of NAT1 and NAT2 and grade and stage of tumor in bladder cancer. Cells from 52 bladder tumors were examined by dual-labeling FISH with a centromere 8-specific probe and a cosmid probe for NAT2. A more limited number were examined for loss with both the NAT2 probe and a newly constructed NAT1-specific cosmid. Loss of NAT2 was found in 6/52 patients in more than 30% of cells, and in 10/52 in 10%-30% of cells examined. Six samples also showed loss of NAT1, indicating that the region of deletion spans at least the distance of the two genes. No obvious correlation between loss of NAT genes with grade and stage of tumor was evident. Interestingly, 17/52 (32%) tumors showed an increased copy number of chromosome 8, with tumors of low stage showing relatively smaller increases of chromosome 8. Loss of 8p22 and genetic instability involving chromosome 8 indicate that this chromosome is important in bladder cancer and that NAT genes will act as important genetic landmarks in defining deletions in this disease. Genes Chromosomes Cancer 25:376-383, 1999.

Acetyltransferases↗
PubMed2001

Assessment of fetal status in multiple gestation pregnancies using interphase FISH.

The use of fluorescence in situ hybridization (FISH) for women with multiple gestation pregnancies has been evaluated. Women were referred for chromosome analysis because of advanced maternal age, abnormal ultrasound findings or a positive family history and/or prior to fetal reduction. FISH was successfully applied to all specimens obtained by amniocentesis or chorionic villus sampling (CVS). Based on FISH results, fetal-fetal contamination of specimens following CVS was 11.5% in twin pregnancies and 16% in triplet or higher multiples. FISH detected trisomy 21 in three cases with no false negatives or positives. Whereas FISH may provide rapid and useful assessment of fetal status in decision-making regarding fetal reduction, the present study also highlighted the obstetrical difficulty of ensuring a sample representative of each fetus following CVS in addition to the possibility of not identifying clinically significant chromosome aberrations using currently available FISH probes.

Amniotic Fluid↗
PubMed2005

Down syndrome with pure partial trisomy 21q22 due to a paternal insertion (4;21) uncovered by uncultured amniotic fluid interphase FISH.

OBJECTIVE: To emphasize the usefulness and reliability of fluorescence in situ hybridization (FISH) on uncultured amniotic fluid cells in the prenatal diagnosis of common chromosomal aneuploidies. METHODS: FISH analyses utilizing centromeric, locus-specific or whole chromosome paint DNA probes specific for chromosomes X, Y, 13, 18, 21, and 4 were performed on uncultured amniotic fluid cells or the peripheral blood specimen from the father. Routine chromosome analysis was carried out as well. RESULTS: A prenatal case with partial trisomy 21 due to a paternal cryptic insertion (4;21) was ascertained by a rapid overnight FISH on uncultured amniotic fluid cells. The fetus was delivered at term and had classical features of Down syndrome. CONCLUSION: Our results stress the importance of FISH on uncultured amniotic fluid cells to supplement routine cytogenetics, especially in cases with abnormal ultrasound findings.

Adult↗
PubMed2005

The utility of interphase fluorescence in situ hybridization for the detection of the translocation t(11;14)(q13;q32) in the diagnosis of mantle cell lymphoma on fine-needle aspiration specimens.

BACKGROUND: Mantle cell lymphoma can be difficult to differentiate cytologically from other small cell non-Hodgkin lymphomas. Nevertheless, the distinction is important, because mantle cell lymphoma is more aggressive than other small cell non-Hodgkin lymphomas. The purpose of this study was to determine whether fluorescence in situ hybridization (FISH) is helpful in diagnosing mantle cell lymphoma on fine-needle aspiration (FNA) specimens by detecting the t(11;14)(q13;q32) translocation that is characteristic of this tumor. METHODS: Fifty-five lymph node FNA specimens from 53 patients were analyzed using FISH. A 2-color FISH assay that employed probes at the 14q32 (immunoglobulin H) and 11q13 (dual-colored, directly labeled cyclin D1) loci was used. The number of single-fusion and double-fusion signals in 200 cells was counted. If > or = 14% single-fusion signals or > or = 1.5% double-fusion signals or both were present, then the sample was considered FISH positive. The findings were correlated with the cytologic, histologic, and immunophenotypic findings in each specimen. RESULTS: Of the 55 cytology specimens, 17 were mantle cell lymphomas, and 38 were nonmantle cell lymphomas, including 16 small lymphocytic lymphomas (9 of 16 in an accelerated phase), 5 large cell lymphomas, 5 follicular lymphomas, 7 transformed large cell lymphomas (Richter syndrome), 3 atypical lymphoid proliferations, and 2 low-grade B-cell lymphomas. All 17 mantle cell lymphomas were positive by FISH. In addition, there were six small lymphocytic lymphomas (two in accelerated phase), one transformed large cell lymphoma, and one large cell lymphoma of follicular origin positive by FISH. The mean number of single-fusion and double-fusion signals, respectively, was 36 and 33 in mantle cell lymphoma specimens and 19 and 3 in positive nonmantle cell lymphoma specimens. CONCLUSIONS: The detection of the t(11;14)(q13;q32) translocation by FISH analysis was helpful in diagnosing mantle cell lymphoma on FNA specimens. Double-fusion signals were more specific for mantle cell lymphoma than single-fusion signals. In rare instances, other non-Hodgkin lymphomas also showed increased numbers of single-fusion signals that were not necessarily indicative of the t(11;14)(q13;q32) translocation. Therefore, in an initial diagnosis of mantle cell lymphoma, significant numbers of double-fusion FISH signals should be identified and interpreted in conjunction with the cytologic and immunologic studies.

Biopsy, Fine-Needle↗
PubMed1987

Quantitative analysis of a nuclear antigen in interphase and mitotic cells.

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.

Antigen-Antibody Complex↗
PubMed1994

Bladder irrigation specimens assayed by fluorescence in situ hybridization to interphase nuclei.

Bladder irrigation specimens provide a sampling of the entire bladder urothelium and are the most practical sample for longitudinally monitoring patients. This study presents cross-sectional fluorescence in situ hybridization (FISH) analyses with correlated DNA cytometry data on 76 patients monitored for recurrent bladder tumors. FISH probes complementary to centromeric satellite sequences for chromosomes 1, 7, 9, 11, 15, and 17 were used. Aberrations in copy number were observed for chromosomes 1, 7, 11, and 17 principally in patients with aneuploid tumors. Monosomy of chromosome 9 was observed in 39% of the diploid and 31% of the specimens with high hyperdiploid fraction. Significantly, 24% of patients with a history of bladder cancer but with no clinical evidence of disease exhibited monosomy of chromosome 9. This suggests a persistent and significantly large population of abnormal cells in the absence of clinical evidence of disease. Loss of chromosome 9 relative to DNA ploidy was observed in 24% of patients with no evidence of disease, in 59% of patients with tumor, and in 79% of patients with histologically confirmed transitional cell carcinoma, grades 1-3. Loss of chromosome 15 was also observed in a large percentage of patients. Loss of chromosome 15 was observed in 41% of specimens from patients in whom no tumor was seen, in 38% of specimens from patients with tumor, and in 67% of specimens from patients with histologically confirmed transitional cell carcinoma. Results of this study document the use of bladder irrigation specimens as a specimen source for FISH analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2003

Detection of a subset of CD30+ anaplastic large cell lymphoma by interphase fluorescence in situ hybridization.

T/null-cell anaplastic large cell lymphoma (ALCL) is a morphologically and clinically heterogeneous group of non-Hodgkin's lymphoma; to date several morphologic variants have been described on histologic specimens. However, the cytologic features of these variants in the fine-needle aspiration (FNA) specimens have not been well evaluated. The t(2;5)(p23;q35) has been identified in a subset of T/null-ALCL and is known to be associated with a favorable prognosis. We reviewed the cytomorphologic characteristics in 24 FNA specimens of ALCL. In all cases, the diagnosis was confirmed on histologic specimens, and immunohistochemical studies for anaplastic lymphoma kinase (ALK) protein expression were performed on the aspirates. The presence of ALK breakpoints were evaluated in nine cases, using a DNA break-apart probe on chromosome 2 covering the ALK gene by fluorescence in situ hybridization (FISH) techniques. Two hundred cells per case were examined. The results were expressed as the percentage of cells containing more than two signals of chromosome 2 to the total number of cells counted. FNA sites included lymph nodes (20), lung (2), breast (1), and soft tissue (1). The median age of the patients was 56 yr (range, 17-75 yr). Twenty cases had systemic involvement; in four cases, skin was the primary site with secondary involvement of the lymph nodes. All cases were CD30(+) by immunohistochemistry; 20 were of T-cell phenotype and 4 were null cell type. The cytologic evaluation revealed typical anaplastic morphology (common type) with many "hallmark cells" in 16 (67%) cases. Other morphologic variants identified were small cell pattern in five cases, monomorphic pattern in two cases, and lymphohistiocytic pattern in one case. FISH studies showed that six (66.7%) of nine cases had at least two signals of chromosome 2, consistent with ALK breakpoints. With careful cytomorphologic evaluation in conjunction with appropriate immunohistochemical studies, a diagnosis of ALCL can be confidently made in the FNA specimens in the cellular aspirates and its morphologic variants also can be recognized. Furthermore, the FNA specimen is suitable in detecting ALK breakpoints by FISH study, permitting rapid identification of a subset of patients with ALCL, who may have a favorable prognosis. Using a commercially available probe, detection of ALK breakpoints in the FNA specimens is simple and can be a useful diagnostic adjunct in cases where distinction from other lymphomas or lymphoid lesions is morphologically difficult.

Activin Receptors, Type I↗
PubMed1990

Silver staining of interphase nucleolar organizer regions in cytologic smears previously stained by the Papanicolaou and May-Grünwald-Giemsa techniques.

Malignant cells are known to display a distinctive argyrophilic nucleolar organizer region (Ag-NOR) protein distribution compared with distribution in their benign counterparts. In this study, Ag-NOR staining was applied to smears from reactive and malignant serous effusions. The smears were either unstained or had been previously stained with the Papanicolaou or May-Grünwald-Giemsa (MGG) technique. The purpose of this study was to assess the feasibility and usefulness of Ag-NOR staining in diagnostic cytopathology and to set up a procedure that could be used on prestained smears in retrospective studies. The one-step silver staining method was applied to prestained smears after a weak destaining step in trichloracetic acid and to unstained smears after a postfixation step in Carnoy's fluid. Positive results were observed on both destained and unstained preparations, MGG-stained smears showing a better visualization of the silver deposits than Papanicolaou-stained smears. Major differences in size, shape, and distribution patterns of the Ag-NOR-positive granules were observed between neoplastic and reactive cells. Furthermore, the mean number of silver-stained dots per cell was significantly higher in malignant cells (33.32 +/- 2.23) than in reactive mesothelial cells (9.71 +/- 0.66). These data indicate that this Ag-NOR technique can be profitably applied to prestained cytologic smears to assist in the diagnosis of malignancy and that the technique has the advantage that cellular morphology and silver staining can be evaluated on the same slide.

Ascitic Fluid↗
PubMed2003

A study on the incidence of ABL gene deletion on derivative chromosome 9 in chronic myelogenous leukemia by interphase fluorescence in situ hybridization and its association with disease progression.

Fluorescence in situ hybridization for the BCR/ABL rearrangement in 138 bone marrow specimens from 59 Philadelphia(+) (Ph(+)) chronic myelogenous leukemia (CML) patients, 35 Ph(+) acute lymphoblastic leukemia (ALL) patients, and 57 Ph(-) ALL patients was used. Sixteen (27.1%) of the 59 CML patients had deletions of the residual ABL gene on the derivative chromosome 9. During the study period, 32 of the 59 CML patients progressed to blast crisis or accelerated phase. Of these, nine patients had residual ABL gene deletions on the derivative chromosomes 9 and 23 patients had no deletions. The mean duration from first diagnosis to blast crisis or accelerated phase for the nine patients with ABL deletions was 32.8 months, and for the 23 patients without ABL deletions, it was 62.4 months (P = 0.017). The overall survival time for the 16 patients with deletions was 32.8 months, and for the 43 patients without deletions, it was 60.1 months (P = 0.164). ABL deletions were not detected among the 35 ALL patients (17 with major BCR/ABL, 18 with minor BCR/ABL), and it appears that this deletion occurs rarely or not at all in Ph(+) ALL patients, which is in contrast to the CML patients (27.1%). However, we detected two ALL cases with ABL deletion but without BCR/ABL rearrangement among 49 Ph(-) ALL and 66 Ph(-) AML patients. In conclusion, patients with ABL deletions progress to blast crisis or accelerated phase in a significantly shorter time than do those without such deletions. It is therefore suggested that the ABL deletion is an indicator of a poor prognosis in CML.

Acute Disease↗
PubMed1992

Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization.

Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line J1 was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.

Burkitt Lymphoma↗
PubMed1993

In situ hybridization to interphase nuclei in acute leukemia.

Numerical chromosome abnormalities were studied in 17 acute lymphoblastic leukemias and one hyperdiploid acute myeloblastic leukemia by fluorescence in situ hybridization (FISH) using YAC clones specific to chromosomes 21 and 6. The results agreed well with cytogenetic findings. Hyperdiploid leukemias with more than 50 chromosomes usually had 4 copies of chromosome 21 and three of chromosome 6, while diploid and pseudodiploid cases were confirmed to have two copies of the two chromosomes. Interesting discrepancies were also observed. In one patient, trisomy 6 was detected by FISH but not by cytogenetics because of the probable inclusion of a chromosome 6 segment within a marker chromosome. The percentages of nuclei with 3 or 4 spots (chromosome 21) and three spots (chromosome 6) in hyperdiploid cells were significantly different in some patients, whereas they might be identical from cytogenetic data.

Adolescent↗