Fluorescence in situ hybridization patterns of BCR/ABL1 fusion in chronic myelogenous leukemia at diagnosis.

Background : Chronic myelogenous leukemia (CML) is characterised by the t(9;22)(q34;q11.2) which results in the formation of the BCR/ABL1 fusion gene. Occasionally, the t(9;22) may be associated with submicroscopic deletions of chromosomes 9 and/or 22 which appear to be associated with a worse prognosis. Three or four-way variant t(9;22) may also occur. All these changes as well as gain of the Philadelphia chromosome which represents disease progression can be detected by fluorescence in situ hybridization (FISH) analysis. FISH analysis at presentation is used to determine the number of cells with BCR/ABL1 fusion and establish whether the patterns are typical or atypical. Response to therapy can then be monitored by serial testing. Patients and Methods : The study group consisted of all patients diagnosed or suspected to have CML who had interphase FISH analysis at presentation on peripheral blood/bone marrow using a commercially available BCR/ABL1 dual colour, dual fusion probe. The study was performed at a tertiary hospital in India between 2004 and 2010. Results: There were 1076 diagnostic samples which were positive for BCR/ABL1 fusion. Typical dual fusion signals (two fusions, one red and one green, 2F1R1G) were seen in 801 cases (74 %). Atypical signal patterns were seen in 275 cases (26%). These were: 1F1R2G (4%), 1F2R1G (2.5%) and 1F1R1G (11%) representing deletions of the derivative 9 involving chromosome 9 sequences, chromosome 22 sequences, or both respectively; 3F1R1G (6.5%) usually representing gain of an additional Philadelphia chromosome and 1F2R2G (1%) representing a three- or four-way variant translocation. More than one signal pattern was seen in 1%. Conclusions: Our findings were similar to the literature with respect to the distribution of signal patterns except that we had a lower number of patients with variant translocations. While each signal pattern is typically associated with a particular abnormality, there can be more than one explanation for each pattern. Hence, metaphase FISH analysis is the "gold standard" for the interpretation of signal patterns.

Electromagnetic field change the expression of osteogenesis genes in murine bone marrow mesenchymal stem cells / 华中科技大学学报（医学）（英德文版）

In order to identify the differentially expressing gene of bone marrow mesenchymal stem cells (MSCs) stimulated by electromagnetic field (EMF) with osteogenesis microarray analysis, the bone marrow MSCs of SD rats were isolated and cultured in vitro. The third-passage cells were stimulated by EMFs and total RNA was extracted, purified and then used for the synthesis of cDNA and cRNA. The cRNA of stimulated group and the control group was hybridized with the rat oligo osteogenesis microarray respectively. The hybridization signals were acquired by using X-ray film after chemiluminescent detection and the data obtained were analyzed by employing the web-based completely integrated GEArray Expression Analysis Suite. RT-PCR was used to identify the target genes: Bmp1, Bmp7, Egf and Egfr. The results showed that 19 differentially expressing genes were found between the stimulated group and the control group. There were 6 up-regulated genes and 13 down-regulated genes in the stimulated group. Semi-quantitative RT-PCR confirmed that the expressions of Bmp1, Bmp7 mRNA of the stimulated group were up-regulated (P<0.05) and those of Egf, Egfr were down-regulated (P<0.05). It was suggested that the gene expression profiles of osteogenesis of the bone marrow MSCs were changed after EMF treatment. It is concluded that the genes are involved in skeletal development, bone mineral metabolism, cell growth and differentiation, cell adhesion etc.

Application of nested polymerase chain reaction (nPCR) using MPB 64 gene primers to detect Mycobacterium tuberculosis DNA in clinical specimens from extrapulmonary tuberculosis patients.

BACKGROUND & OBJECTIVE: The conventional culture technique for diagnosis of extrapulmonary tuberculosis is time consuming. In order to find a sensitive and rapid technique nested polymerase chain reaction (nPCR) targeting the conserved MPB 64 gene of Mycobacterium tuberculosis was evaluated for detection of M. tuberculosis DNA directly from clinical specimens of extrapulmonary origin. METHODS: A total of 400 clinical specimens from clinically suspected cases of extrapulmonary tuberculosis and 30 control specimens of nontuberculous aetiology were processed by smear and culture and by nPCR technique for detection of M. tuberculosis. The specimens were divided into 3 groups, (group I--280 specimens [104 peritoneal fluid (PF), 120 cerebrospinal fluid (CSF), 44 lymph node biopsies 3 pericardial fluid and 9 other biopsy specimens], group II--120 aqueous humour (AH) from idiopathic granulomatous uveitis cases, and group III--30 control specimens (10 CSF and 20AH). RESULTS: The conventional culture was positive only in 16 of 400 specimens. The overall positivity of nPCR was 35.2 per cent (141/400). Among the 280 specimens from extrapulmonary lesions (group I), 15 were bacteriologically positive, while 115 of 265 bacteriologically negative specimens (43.4%) were positive by nPCR. All the 30 control specimens were negative by nPCR. INTERPRETATION & CONCLUSION: The nPCR using MPB64 gene primers might be a rapid and reliable diagnostic technique for detection of M. tuberculosis genome in clinically suspected extrapulmonary tuberculosis specimens, as compared to the conventional techniques.

Analysis of the factors affecting the accuracy of detection for single base alterations by oligonucleotide microarray

The oligonucleotide microarray, a high-throughput polymorphism detection technology, holds great promise for the characterization of complex genetic variance. To achieve greater sensitivity and specificity for it to be an effective platform technology we present results and discuss some of the factors influencing signal intensities and single-mismatch discrimination in array-based mutation/SNP detection. Probes with a series of concentrations were spotted onto the slide in order to find the optimal concentration with the identifiable satisfying signals and the stable ratios between matched and mismatched probes. It was found that under our experimental conditions, when the initial probe concentration is higher than the maximum immobilization capability of the slide (7.5 micrometer), the hybridization signal will be saturated and the ratio between matched and mismatched probes will be more stable than at a lower probe concentration. Considering the cost of probes and the systematic stability, a constant spotting concentration of 10 micrometer was selected. The stability of different types of mismatched oligo-DNA duplexes on the glass surface was also confirmed. The results show that the order of stability of mismatched oligo-DNA duplexes on a glass surface is in general agreement with previous reports conducted using liquid and polyacrylamide gel pads. This suggests that the influence of the mismatched base pair on the stability of the duplex in a solid hybridization system is similar to that in the solution hybridization environment.