Detection of 47, XYY Trophoblast Fetal Cells in Maternal Blood by Fluorescence in situ Hybridization after Using Immunomagnetic Lymphocyte Depletion and Flow Cytometry Sorting

1992 ◽  
Vol 7 (3-4) ◽  
pp. 190-194 ◽  
Author(s):  
V. Cacheux ◽  
C. Milesi-Fluet ◽  
G. Tachdjian ◽  
L. Druart ◽  
J.F. Bruch ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Maternal Blood ◽  
Lymphocyte Depletion ◽  
Fetal Cells ◽  
Flow Cytometry Sorting

268 IDENTIFICATION OF X- AND Y-BEARING SPERMATOZOA IN SORTED BUFFALO (BUBALUS BUBALIS) SEMEN BY FLUORESCENCE IN SITU HYBRIDIZATION

2009 ◽  
Vol 21 (1) ◽  
pp. 231
Author(s):  
M. Zhang ◽  
X. J. Zhuang ◽  
Y. Q. Lu ◽  
C. H. Hu ◽  
S. S. Lu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Y Chromosome ◽  
X Chromosome ◽  
Chromosome Painting ◽  
Fish Method ◽  
Painting Probes ◽  
Flow Cytometry Sorting

Flow cytometry sorting technology has been successfully used to sort the X- and Y-chromosome bearing sperm. Previous studies showed that fluorescence in situ hybridization (FISH) method was a simple and reliable procedure for assessing the effectiveness of separation of X- and Y-sperm in the swine (Kawarasaki T et al. 1998 Theriogenology 50, 625–635) and the bovine (Rens W et al. 2001 Reproduction 121, 541–546). Reports of sex-preselection by flow-cytometry sorting of the X- and Y-sperm were also seen in the buffalo (Presicce GA et al. 2005 Reprod. Dom. Anim. 40, 73–75; Lu YQ et al. 2006 Anim. Reprod. Sci. 100, 192–196). There was, however, no report to date for using the FISH method to assess the purity of the sorted buffalo sperm. The objective of the present study was to verify the purity of flow cytometrically-sorted buffalo X- and Y-sperm by FISH using bovine X- and Y- chromosome painting probes prepared by microdissection. The X- and Y- chromosomes of bovidea were microdissected respectively from the metaphase spreads of Holstein blood cells with a glass needle controlled by a micromanipulator and amplified by degenerate oligo-nucleotide primer-PCR (DOP-PCR) (Mariela N et al. 2005 Genet. Mol. Res. 4, 675–683). The DOP-PCR products of X- and Y- chromosome were labeled with CY3-dUTP and Biotin-11-dUTP, respectively. The buffalo X- or Y-sperm DNA from unsorted semen and sorted semen were hybridized to the labeled probes, respectively. The results showed that the hybridized signals were clearly visible in the metaphase karyotype of bovine and buffalo semen samples. About 47.7% (594/1246) and 48.9% (683/1396) of the unsorted buffalo sperm emitted strong fluorescent signals when assessed by Y- and X-chromosome painting probes, respectively, which was conformed to the sex ratio in normal buffalo sperm (50%:50%). About 86.1% (1529/1776) hybridization signals of the sperm in the sorted X-semen assessed by X-chromosome painting probes were detected, while 82.2% (2232/2716) of the Y-sorted buffalo sperm emitted strong fluorescent signals when assessed by Y-chromosome painting probe. The results of the flow cytometer re-analysis revealed that the proportions of X- and Y-bearing sperm in the sorted semen were 89.6% and 86.7%, respectively. There were no apparent differences between the two assessment methods of sperm separation by flow cytometry re-analysis and by FISH with the X-Y paint probe. In conclusion, bovine X- and Y-chromosome painting probes prepared using microisolation method could be used to verify the purity of the sorted sperm in the buffalo. This study was supported by the Guangxi Department of Science and Technology (0626001-3-1) and National Key Technology R&D Program, The People’s Republic of China (2006BAD04A18). The authors (M. Zhang, X.J. Zhuang, and Y.Q. Lu) contributed equally to this work.

scholarly journals FISH Analysis of All Fetal Nucleated Cells in Maternal Whole Blood: Improved Specificity by the Use of Two Y-chromosome Probes

2005 ◽  
Vol 53 (3) ◽  
pp. 319-322 ◽  
Author(s):  
Susanne Mergenthaler ◽  
Tatiana Babochkina ◽  
Vivian Kiefer ◽  
Olaf Lapaire ◽  
Wolfgang Holzgreve ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Y Chromosome ◽  
Blood Cells ◽  
Maternal Blood ◽  
Fish Analysis ◽  
Blood Samples ◽  
Fetal Cells ◽  
Nucleated Red Blood Cells ◽  
Noninvasive Prenatal Diagnosis

Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12–20 male fetal cells/ml of maternal blood were identified by XY- and YY-FISH, respectively.

scholarly journals Telomere analysis by fluorescence in situ hybridization and flow cytometry

1998 ◽  
Vol 26 (16) ◽  
pp. 3651-3656 ◽  
Author(s):  
M. Hultdin ◽  
E. Gronlund ◽  
K.- F. Norrback ◽  
E. Eriksson-Lindstrom ◽  
G. Roos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry

Genome ◽  
2003 ◽  
Vol 46 (5) ◽  
pp. 893-905 ◽  
Author(s):  
M Kubaláková ◽  
M Valárik ◽  
J Bartoš ◽  
J Vrána ◽  
J Cíhalíková ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Physical Mapping ◽  
Secale Cereale ◽  
B Chromosomes ◽  
Addition Lines ◽  
Large Numbers ◽  
Secale Cereale L

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R–7R) could be discriminated and sorted from individual wheat–rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.Key words: chromosome isolation, chromosome sorting, fluorescence in situ hybridization, repetitive DNA sequences, wheat-rye addition lines, B chromosomes, physical mapping.

A Novel t(9;22;11) Translocation Variant Involving 11q23 in a Patient with Chronic Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Lin Yuehui ◽  
Qinglong Zheng ◽  
Tong Wu ◽  
DAN LIU
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Chronic Myeloid Leukemia ◽  
Fluorescence In Situ Hybridization ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Philadelphia Chromosome ◽  
Flow Cytometry Analysis

Background:The progression of Philadelphia chromosome positive chronic myeloid leukemia (CML) is frequently accompanied by cytogenetic evolution, commonly unbalanced chromosomal changes. but balanced chromosomal translocations are very rare in CML, especially translocations involving the 11q23. The few reported cases with blast phase (BP) of CML carrying a 11q23 rearrangement results in insufficient responses to tyrosine kinase inhibitors (TKIs) and possess a poor prognosis. Methods:Cytogenetic analysis , fluorescence in situ hybridization (FISH), RNA sequencing (RNA-seq), targeted genomic sequencing and multi-parametric flow cytometry analysis were performed to identify the chromosome translocations and pathogenic gene alterations in a 36-year-old female with myeloid BP of CML. Results: In BP, the bone marrow (BM) aspiration showed 61% myeloid blasts; Multi-parametric flow cytometry analysis revealed the abnormal myeloid blasts expression of the following antigens: CD117, CD13, CD33, CD38, partially expressed CD15, CD64. Chromosome analysis revealed a t(11;22)(q23;q11) translocation in addition to the t(9;22)(q34;q11). Fluorescence in situ hybridization (FISH) test confirmed that the t(11;22)(q23;q11) involved the mixed lineage leukemia(MLL) gene on 11q23 and RNA sequence revealed MLL-SEPT5 and BCR/ABL1(p210) fusion transcripts positive. Mutations on 339 commonly mutated genes in hematologic malignancies were analyzed by targeted next-generation sequencing showed ASXL1 p.G949Vfs*2 mutation. The patient failed to respond to both imatinib and dasatinib despite the absence of resistance-associated mutations in the BCR/ABL1 gene and she had a myeloid blast crisis at 19 months after initiation of first- and second-generation TKI treatment. After BP, she received ponatinb, a third-generation TKI with chemotherapy. Regretly she didn't achieve a complete remission(CR) and was in the process of salvage transplantation at present. Conclusions:The presence of 11q23 rearrangements in BC of CML is rare and most likely accounts for the adverse clinical outcome. We first report a patient who diagnosed CML with t(11;22)(q23;q11) and MLL-SEPT5 fusion gene positive in BP of CML. The clinical course was aggressive, and therapy was poorly tolerated. Disclosures No relevant conflicts of interest to declare.

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