We report a case of a female karyotype that was normal except for double minutes (dmin) in acute myeloid leukemia. Using fluorescence in situ hybridization, the amplification of C-MYC was detected in both interphase and metaphase cells. The patient of the present case had received only limited therapy with cytosine arabinoside, but lived for more than one year. It supports the recent notion that dmin may not necessarily be associated with a poor outcome.
Cytarabine ; Female ; Fluorescence ; Humans ; In Situ Hybridization ; Interphase ; Karyotype* ; Leukemia, Myeloid, Acute* ; Metaphase ; Oncogenes*

Fluorescence in situ hybridization (FISH) techniques allow the enumeration of chromosome abnormalities and from a great potential for many clinical applications. In order to produce quantitative and reproducible results, expensive tools such as a cooled CCD camera and a computer software are required. We have developed a Chromosome Image Processing System (Chips) using FISH that allows the detection and mapping of the genetic aberrations. The aim of our study, therefore, is to evaluate the capabilities of our original system using a black-and-white video camera. As a model system, three repetitive DNA probes (D18Zl, DXZI, and DYZ3) were hybridized to variety different clinical samples such as human metaphase spreads and interphase nuclei obtained from uncultured peripheral blood lymphocytes, uncultured amniocytes, and germ cells. The visualization of the FISH signals was performed using our system for image acquisition and pseudocoloring. FISH images were obtained by combining images from each of probes and DAPI counterstain captured separately. Using our original system, the aberrations of single or multiple chromosomes in a single hybridization experiment using chromosomes and interphase nuclei from a variety of cell types, including lymphocytes, amniocytes, sperm, and biopsied blastomeres, were enabled to evaluate. There were no differences in the image quality in accordance with FISH method, fluorochrome types, or different clinical samples. Always bright signals were detected using our system. Our system also yielded constant results. Our Chips would permit a level of performance of FISH analysis on metaphase chromosomes and interphase nuclei with unparalleled capabilities. Thus, it would be useful for clinical purposes.
Blastomeres ; Chromosome Aberrations ; DNA Probes ; Fluorescence* ; Germ Cells ; Humans ; In Situ Hybridization* ; Interphase ; Lymphocytes ; Metaphase ; Spermatozoa

Determmation of the chomosomal constitution of human spermatozoa has been camed out though the human-hamster interspecific in vitro fertilization(IVF) system. In recent years, the introduction of fluorescence in-situ hybridization(FISH) technique has provided an alternative approach to evaluate the cbmmosomal constitution of human spermatozoa. The nuclei of mature spermatozoa are highly condensed with interpmtamine disulphide bridges, therefore the success of FISH on interphase human spermatozoa relies on partial decondensation of the sperm chromatin. In early studies, dithioothreitol(DTT) has been known as an efficient decondensation agent. Since then, several different decondensation methods using D1T have been establisdhed, and in terms of decondensation, we were tried to fix the optimal decondensation protocol using DlT. In our study, the optimal concentration and treatment time were 1-mM and 30 min, respectively. We examined chromosome complements of human sperm to investigate the effect of paternal age on the hequency of nondisjunction in human sperm. We investgated sperm karyotypes ftom two diffaent age groups)28+/-0.5, 46+/-6), A minimum of 1000 spermatozoa for one patient were analyzed. The mean frequencies of YY, XX, XY, 21-disamy spermatozoa ware 0.04%, 0.45%, 0.40%, 0.45% respectively in young age group and 1.06%, 0.62%, 1.06%, 0.76% in old ages. The mean frequency of disomy spermatozoa was higher in old age poup compare with those of young age group.
Chromatin ; Chromosome Aberrations* ; Complement System Proteins ; Constitution and Bylaws ; Fluorescence ; Humans ; Interphase ; Karyotype ; Paternal Age ; Spermatozoa*

BACKGROUND: One of major challenge of cytogenetics is to obtain qualitative metaphases to achieve a meaningful analysis. Methotrexate (MTX) synchronization and the Giant Cell Tumor-Conditioned Medium (GCT-CM) have been used to improve the metaphase preparation from hematopoietic malignancies. The purpose of this study is to determine Mitotic Index (MI) of bone marrow samples under several culture conditions that may improve the quality of chromosome preparation. METHODS: Sixty nine bone marrow samples were cultured into 3 groups by traditional methods. The first group was tested for the effect of cell concentration on MI. The second, for the effect of MTX concentration on MI. The third group was classified into 4 subgroups as follows: 1) MTX only in 24 hour culture 2) MTX and GCT-CM in 24 hour culture 3) 48 hour culture without MTX 4) 48 hour culture and GCT-CM. MI was calculated as the ratio of metaphase to interphase cells in 1000 cells. Quality of metaphase was evaluated by classified the metaphase cell into 3 types. RESULTS: The first and second groups revealed no relationship between cell concentration, amount of MTX and MI, respectively. The third group showed significant differences among four subgroups. The MI increased in subgroups using GCT-CM and in the 48 hour culture, with greatest increase in group using 48 hour culture and GCT-CM simultaneously. CONCLUSIONS: The use of GCT-CM medium and prolonged culture improved the quality of metaphase cells and MI. It is therefore beneficial to use these conditions in cytogenetic studies on bone marrow in hematologic disease.
Bone Marrow* ; Cytogenetics ; Giant Cells* ; Hematologic Diseases ; Hematologic Neoplasms ; Interphase ; Metaphase ; Methotrexate ; Mitotic Index*

OBJECTIVETo explore the clonal evolution of monosomy 7 in patients with aplastic anemia (AA).

METHODSMonosomy 7 (-7) in 81 AA patients with normal karyotype at diagnosis and 46 AA treated with immunosuppressive therapy (IST) and more than 6 months of recombinant human granulocyte colony-stimulating factor (rhuG-CSF) were detected by interphase- fluorescence in situ hybridization (FISH) retrospectively.

RESULTSThere were 5.4% - 7.6% of -7 cells in 11 (13.6%) of 81 patients at diagnosis, the survival and response rate to IST in -7 positive patients did not differ significantly from that in -7 negative patients (P = 0.481, 0.865); -7 cells disappeared after IST in all of the 11 patients including 5 received long-term rhuG-CSF therapy, and none of them evolved to myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) at a median follow-up of 44 months. Serial assessments of -7 clones were performed in 46 patients, none of whom detected -7 clones 3-6 months after IST, but -7 recurrence in 5 patients 12 - 15 months after IST. At a median follow-up of 48 months, FISH identified 6 patients with -7 clones while the conventional cytogenetic analysis (CCA) recognized in 5. Moreover, the first demonstration of -7 by FISH was 3 - 18 months earlier than that by CCA. All of the 6 patients with FISH detected -7 evolved to MDS/AML with -7 and four of them were retrospectively analysed for in samples at -7 diagnosis of AA, but none of them was positive.

CONCLUSIONSMonosomy 7 exists in a part of AA patients, but the preexisting -7 cells seems neither associated with fatality nor evolvation to MDS/AML. rhuG-CSF might facilitate the expansion of -7 clones; It is necessary to monitor -7 in AA, especially when received long-term rhuG-CSF therapy.

In multiple myeloma (MM), hyperdiploidy (HD) is known to impart longer overall survival. However, it is unclear whether coexistent HD ameliorates the adverse effects of known high-risk cytogenetics in MM patients. To address this issue, we investigated the clinicopathological characteristics of HD with high-risk cytogenetics in MM. Ninety-seven patients with MM were included in the study. For metaphase cytogenetics (MC), unstimulated cells from bone marrow aspirates were cultured for either 24 or 48 hours. To detect HD by interphase fluorescence in situ hybridization (iFISH), we assessed trisomies of chromosomes 5, 7, 9, 11, 15, and 17. Of the 97 MM patients, 40 showed HD. The frequency of co-occurrence of HD and high-risk cytogenetics was 14% (14/97). When the clinicopathological characteristics were compared between the two groups of HD with high-risk cytogenetics vs. non-HD (NHD) with high-risk cytogenetics, the level of beta 2 microglobulin and stage distribution significantly differed (P=0.020, P=0.032, respectively). This study shows that some of the clinicopathological characteristics of MM patients with high-risk cytogenetics differ according to HD or NHD status.
beta 2-Microglobulin ; Bone Marrow ; Cytogenetics* ; Fluorescence ; Humans ; In Situ Hybridization ; Interphase ; Metaphase ; Multiple Myeloma* ; Trisomy

PURPOSE: FISH is suggested as a useful tool for rapid detection of specific aneuploidy in uncultured amniocytes abnormality in interphase nucleus. In this study, we are going to share our experience using FISH in prenatal diagnosis and suggest the criteria for the diagnosis of aneuploidy by analyzing the results of FISH test. METHODS: From January, 1999 to May, 2006, 8,613 tests in amniotic fluids obtained from 7,893 pregnant women were performed by using FISH for prenatal diagnosis of trisomy 21, trisomy 18 and trisomy 13. The indications of chromosome study were a screen positive for Down syndrome or Edwards syndrome in maternal serum marker screening test and an advanced maternal age (> or =35 years old). RESULTS: We have the 8,502 informative results from 8,613 tests (98.7%) which is submitted our criteria and the sensitivity is 98.2%. CONCLUSION: FISH on uncultured amniocytes is a rapid, clinically useful tool for prenatal diagnosis, with informative specimens being highly accurate. But the limitation of FISH is both expensive and labor-intensive.
Amniotic Fluid ; Aneuploidy* ; Biomarkers ; Diagnosis ; Down Syndrome ; Female ; Humans ; Interphase ; Mass Screening ; Maternal Age ; Pregnant Women ; Prenatal Diagnosis* ; Trisomy

Untill now, classical karyotyping remains the most reliable and conclusive method, but sometimes very rapid and sensitive method for the detection of chromosomal abnormality is needed. The primed in situ labelling(PRINS) is very rapid and sensitive method for the detection of chromosomal aneuploidy and X-linked disorder instead of fluorescen in situ hybridization. The purpose of this study was to prepent the application of PRINS protocol to the rapid detection of chromosomes X and Y in both metaphases and interphase nuclei. The PRINS reaction was done in 10 amniocytes. We compared the PRINS result with conventional cytogenetic karyotyping. The result was the same with cytogenetic karyotyping. In the future, PRINS will become very useful and sensitive method for the chromosomal aneuploidy detection and X-linked disorder diagnosis.
Aneuploidy ; Chromosome Aberrations ; Cytogenetics ; Diagnosis ; In Situ Hybridization ; Interphase ; Karyotyping ; Metaphase ; Polymerase Chain Reaction* ; Prenatal Diagnosis ; Primed In Situ Labeling

The nucleolus of human cell is a morphologically well recognizable nuclear organelle and the argyrophilic NORs (AgNORs) are nucleic acid-argyrophilic nonhistone protein complex in the nucleoli and the silver staining allows their identification and enumeration at the light microscopic level. The AgNOR counts are in parallel with mitotic activity and vary in different phase of cell cycle. It has been reported that human cells have one AgNOR during interphase and S-phase. However, the correlation between the number of AgNORs and S-phase markers is still controversial and they have never been studied simultaneously. In this study, AgNOR and PCNA were stained simultaneously to find out the relationship of AgNOR counts with cell cycle (S-phase) in human palatine tonsil, gastric carcinoma, liver and brain tissues. S-phase cells (PCNA-positive) were found predominantly in lymphoid follicles in palatine tonsil but gastric carcinoma showed diffuse immunoreactivity for PCNA. The AgNOR counts varied according to the type and locus of tissue. More than one AgNOR were identified in S-phase cells and some of hepatocytes and neurons in the brain which were not in S-phase contained two or more AgNORs. The above results suggest that the number of AgNOR is a characteristic feature of each type of cells and can be more than one even in S-phase.
Brain ; Cell Cycle ; Hepatocytes ; Humans* ; Interphase ; Liver ; Neurons ; Organelles ; Palatine Tonsil ; Proliferating Cell Nuclear Antigen ; Silver Staining

A 24-year-old female with primary amenorrhea was referred for a chromosome study. The karyotype of the patient was 46,X,der(X) under initial GTG-banding analysis. Fluorescence in situ hybridization (FISH) analysis with an LSI Kallmann (KAL) region probe [probes for Xp22.3(KAL) and CEP(X) for control] was carried out. The abnormal chromosome was KAL- and CEP(X)x2. In addition, interphase FISH analysis revealed the patient to be mosaic for two different cell lines: 90% of cells had three signals and 10% of the cells had only one signal for CEP(X). Based on these results, the karyotype of the patient was 45,X/46,X,psu idic(X)(p22.1), which is partial trisomy for Xqter-->Xp22.1 and partial monosomy for Xpter-->Xp22.1. This karyotype was considered a variant of Turner syndrome. In summary, Idic(X) and low-level mosaicism was successfully characterized by FISH analysis with a CEP(X) probe.
Amenorrhea ; Chromosome Deletion ; Female ; Fluorescence ; Humans ; In Situ Hybridization ; Interphase ; Karyotype ; Mosaicism ; Trisomy ; Turner Syndrome ; X Chromosome ; Young Adult