Publications (15)19.62 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Systemic mastocytosis (SM) associated with t(8;21) acute myeloid leukemia (AML) is very rare, and the D816 mutation of the KIT gene has previously been detected only in adult patients. We herein report the case of a 5-year-old female presenting with AML harboring t(8;21)(q22;q22). Her AML was refractory to chemotherapy, and bone marrow mastocytosis developed simultaneously at the initial diagnosis and during chemotherapy. The D816A mutation of KIT was detected. SM associated with t(8;21) AML, accompanied by a KIT mutation in children may result in a poor prognosis, despite the fact that t(8;21) AML are generally considered to have a favorable risk. Pediatr Blood Cancer 2012; 59: 1313-1316. © 2012 Wiley Periodicals, Inc.
- [Show abstract] [Hide abstract] ABSTRACT: The advent of tyrosine kinase inhibitors as molecular target therapy has resulted in a marked change in the laboratory process for the diagnosis and therapeutic monitoring of chronic myelogenous leukemia. This includes defining the molecular typing of BCR-ABL1 to establish the diagnosis, a quantitative and/or high quality assay for minimal residual disease to evaluate the molecular response, and mutation analysis and chromosomal examination to assess its resistance to inhibitors. These processes should be used where appropriate for each patient. In the ongoing development and clinical use of novel agents for treatment of the leukemia, the quality assurance of each process of molecular-genetic testing, such as specimen handling, measurement, and reporting, has become increasingly important in the quality care of patients.
- [Show abstract] [Hide abstract] ABSTRACT: We herein report the findings of a 47-year-old Japanese female with chronic myeloid leukemia (CML) with a cryptic BCR-ABL1 transcript on chromosome 9 and a derivative chromosome 22 unrelated to BCR-ABL1. Although she achieved and continued to demonstrate a major molecular response to imatinib treatment following interferon-alpha, there was persistence of a derivative chromosome 22. A detailed chromosome/molecular studies, including serial karyotyping analysis, finally resulted in the karyotyping at the disease onset to be 47,XX,+del(22)(q11.2), with two genetic evens, namely a cryptic BCR-ABL1 transcript on chromosome 9 and derivative chromosome 22 unrelated to BCR-ABL1. This CML case with these two rare genetic events thus raises diagnostic issues such as the difficulty in making a concise evaluation of the chromosomal/molecular events and an accurate disease prognosis, as well as the difficulty in determining the disease remission status after treatment.
- [Show abstract] [Hide abstract] ABSTRACT: Genetic factors may be involved in the susceptibility to mycobacterium infection. We studied the association of polymorphisms in the candidate gene, SLC11A1 (solute carrier family 11 member 1; formerly NRAMP1), which controls immune reactions to intracellular pathogens, with susceptibility to active Mycobacterium tuberculosis and nontuberculous mycobacterium infections in a Japanese population, who was admitted to a local hospital in the Kanagawa prefecture. A case-control study was performed by comparing the frequency of polymorphisms in the SLC11A1 gene in patients with pulmonary tuberculosis (57 cases) or with Mycobacterium avium-intracellulare infection (17 cases) and ethnically matched healthy controls (51 cases). Three SLC11A1 genetic polymorphisms, G/C at intron 4 (496+14G/C), transformation of aspartic acid to asparagine at 543 codon (D543N), and TGTG deletion in 3′ untranslated region (1729+55del4) were examined, using restriction fragment length polymorphism analysis of polymerase chain reaction products. The polymorphism of D543N wasfound closely associated with both pulmonary tuberculosis (P = 0.009) and M. avium-intracellulare infection (P = 0.01). The polymorphism of intron 4 was closely associated only with pulmonary tuberculosis (P = 0.05). The polymorphisms in SLC11A1 may partly explain the susceptibility to active M. tuberculosis and M. avium-intracellulare infections without predisposing factors in a Japanese population.
- [Show abstract] [Hide abstract] ABSTRACT: Designing stem cell transplantation (SCT) conditioning regimens for Fanconi anemia (FA) has proved difficult because of hypersensitivity to the DNA cross-linking agents. We performed chromosome fragility tests with 56 FA patients and with 50 non-FA patients with severe aplastic anemia or myelodysplastic syndrome. We evaluated peripheral blood lymphocyte specimens cultured for 72 hours and treated with mitomycin C, diepoxybutane (DEB), cyclophosphamide (CY) metabolites, cytosine arabinoside (Ara-C), and fludarabine (Flu) metabolite (9-beta-D-arabinofuranosyl-2-fluoroadenine [2-F-Ara-A]). The DEB and CY metabolite tests were highly sensitive and specific for FA (P<10(-4)) for both tests), and the number of aberrations per cell for DEB correlated with that for the CY metabolite test (P < 10(-4)) but did not correlate with the number of aberrations per cell for the Ara-C and 2-F-Ara-A tests. The difference in breakage frequencies between FA and non-FA patients for cultures treated with 2-F-Ara-A was not statistically significant. Most of the breakages observed in cells treated with 2-F-Ara-A-and Ara-C were chromatid breaks. It may be possible to determine the appropriate CY dose in the preconditioning regimen for SCT in FA patients on the basis of the in vitro effects on fragility, and Flu or Ara-C may be a safer drug than high-dose CY for conditioning in FA patients.
- [Show abstract] [Hide abstract] ABSTRACT: A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log(10) (IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R(2) = 0.972, n = 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.
- [Show abstract] [Hide abstract] ABSTRACT: We developed and evaluated a prototype automated specimen preparation instrument for the specific capture of hepatitis C virus (HCV) RNA with probes and magnetic bead-fluid separation. HCV RNA was isolated from serum by lysis of virus particles with a chaotropic agent, followed by hybridization of the RNA with biotinylated probes and capture of the hybridized RNA with streptavidin-coated paramagnetic particles. After washing of the hybrid-particle complexes to remove nonspecifically bound materials, the particles were resuspended in a specimen diluent and were then ready for amplification and detection with a fully automated PCR system (COBAS AMPLICOR; Roche Diagnostic Systems). The analytical sensitivity in the dilution series was 33 copies per ml or greater. Comparison of the test results with those obtained by a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R; Sanko Junyaku Co., Ltd.) showed 93% (49 of 53 samples) sensitivity and 100% (12 of 12 samples) specificity. There was 94% overall agreement between results. When RNA was extracted by the manual method from serum containing 10(3) or 10(5) copies of HCV per ml in the presence of heparin, there was an inhibitory effect on detection of both HCV RNA and the internal control. In contrast, when RNA was extracted from the serum by the automated method, there was no inhibitory effect. This inhibitory effect of heparin on the manual method was also observed for a series of serum specimens from a hemodialysis patient, but the inhibitory effect was eliminated by the automated specimen preparation method. In summary, a fully automated RNA extraction system for PCR detection of HCV RNA by use of specific capture with probes and magnetic bead-fluid separation was shown to have performance similar to that of the conventional manual method. In addition, it successfully eliminated the inhibitory effect of the heparin in the serum and permitted the detection of HCV RNA in serum samples from a hemodialysis patient. The prototype automated RNA extraction system is suitable as a totally automated system, starting with RNA extraction to detection of HCV, if it was combined with the fully automated COBAS AMPLICOR PCR system.
- [Show abstract] [Hide abstract] ABSTRACT: Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate inhibitory effects of the therapeutic reagents on the PCR detection of HCV and to evaluate the efficacy of their elimination by RNA extraction methods. RNA was extracted using a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R, Sanko Junyaku) or an automated system based on specific capture of HCV-RNA with probes and magnetic bead/fluid (B/F) separation (Roche Molecular Systems). When RNA was extracted by SepaGene RV-R from serum containing 10(5) copies per ml of HCV and amplified for HCV-RNA in the presence of hemoglobin, bilirubin, or heparin by Amplicor HCV (Roche Molecular Systems), results tended to be negative. In addition to these, two therapeutic reagents, ATP and dextran sulfate sodium were also found to have inhibitory effects. When ATP at concentrations up to 5 mM was added to the sera and RNA was extracted with SepaGene RV-R, there were no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, when dextran sulfate sodium up to 1 mM was added to the sera, there was a dose dependent inhibitory effect on detection of both HCV-RNA and the internal control. When HCV-RNA was extracted by the automated system, the inhibitory effect of dextran sulfate sodium was successfully eliminated. In conclusion, dextran sulfate sodium and ATP were newly identified as inhibitors that may be present in serum, and the efficacy of eliminating these substances varied among RNA extraction methods.
- [Show abstract] [Hide abstract] ABSTRACT: Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, co-amplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.
- [Show abstract] [Hide abstract] ABSTRACT: In the direct detection of pathogens by polymerase chain reaction (PCR) from clinical samples, false negative results due to the presence of inhibitor are problematic. In order to monitor such an inhibitor, we evaluated the detection of the positive internal control in PCR assay for HCV-RNA in serum samples positive for anti-HCV antibodies, of which 41 samples were positive for HCV-RNA by competitive RT-PCR assay. The positive internal control was coamplified with HCV-RNA and hybridized to the specific probe on magnetic beads and then hybrids were detected by colorimetric measurement using automatic PCR machine (COBAS AMPLICOR). Detection of the positive internal control was negatively correlated with viral copy number in sera assayed by competitive RT-PCR. Five of 52 samples (9.6%) with high HCV-RNA copy number (10(7) or 10(8) copies/ml) showed negative results for the internal control. The negative results for the internal control turned out to be positive when the sera were diluted and re-assayed, suggesting competitively inhibitory effects of high viral copy number on amplification of the internal control. Addition of heparin in the serum sample showed an inhibitory effect with a dose dependent manner on the detection of both HCV-RNA and the internal control, with a more effect on the lower copy number of HCV. On the other hand, addition of hemoglobin in the sample with concentration of up to 450mg/dl had no inhibitory effect on the detection of either HCV-RNA or the internal control. Coamplification and detection of the positive internal control was demonstrated to be useful to estimate effects of inhibitors, which may be present in clinical samples, in the detection of HCV-RNA by PCR.
- [Show abstract] [Hide abstract] ABSTRACT: In definite diagnosis of mycobacterial infection, prompt and adequate differential diagnosis leads to an appropriate treatment. We developed and evaluated a PCR assay based on co-amplification of the insertion sequence IS6110 and groEL gene that are species-specific for Mycobacterium tuberculosis complex and genus-specific, respectively. The detection limit of the assay system for cultured M. tuberculosis was 2 cells/ml, as compared with 200 cells/ml by culture onto Ogawa's medium. To assess the value of the assay in routine laboratory works, the results obtained by PCR were compared with those by standard microbiological methods for 758 specimens collected for the examinations of mycobacterial infection. The PCR system for detection of mycobacteria gave overall positive rate of 27.6% (209/758), as compared to 6.1% (46/758) by smear and 7.7% (58/758) by culture onto Ogawa's medium. Sensitivity and specificity were 97.8% and 97.3%, respectively, for the IS6110 and groEL gene for detection of M. tuberculosis complex; 91.7% and 80.3%, respectively, for only the groEL gene for detection of atypical mycobacteria. The PCR assay based on co-amplification of the IS6110 and groEL gene would be useful for diagnosis of mycobacterial infections, allowing not only more sensitive detection of mycobacteria but also rapid discrimination between M. tuberculosis complex and atypical mycobacteria. This assay would help to eliminate time-consuming confirmation, and to avoid both unnecessary treatment and hospitalization of the patient.
- [Show abstract] [Hide abstract] ABSTRACT: We compared two primer sets (A: 167bp, B: 269bp) derived from highly conserved domains within the 5' noncoding region (5'NC) of the hepatitis C virus (HCV) genome for their ability to detect HCV-RNA in a nested cDNA polymerase chain reaction assay (nested-PCR) in sera from 31 patients suspected of having HCV infection. Seventeen (54%) of 31 patients were positive for HCV-RNA with both primer sets. Using primer set A, 14(93%) of 15 samples with positive and 3(19%) of 16 samples with negative anti-HCV antibody test gave positive results for HCV-RNA. With primer set B, 15(100%) of 15 antibody positive samples and 2(13%) of 16 negative samples were positive for HCV-RNA. One antibody negative sample from a patient with alcoholic liver cirrhosis was positive for HCV-RNA only with primer set A. Another sample with positive antibody test, from a patient with chronic renal failure, was positive for HCV-RNA only with primer set B. A combination of more than one set of primers directed to the highly conserved 5'NC region, as well as proper selection of the exact nucleotide sequences, are important in improving the detection rate of HCV-RNA by PCR in serum of infected patients.
- [Show abstract] [Hide abstract] ABSTRACT: In the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid detection of MRSA is extremely important. The mecA gene codes the new drug resistant polypeptides called penicillin-binding protein 2a (PBP2a) or 2'(PBP2'), which mediates the clinically relevant resistance to all beta-lactam antibiotics. This gene could be beneficial in the detection of MRSA using the polymerase chain reaction (PCR). However, the identical mecA gene has been found in both coagulase-positive and coagulase-negative Staphylococcus with the appropriate methicillin-resistant phenotype. The second gene related to the expression of methicillin-resistance has been called femA. In this study, we amplified both mecA and femA genes by PCR in 97 strains and 32 clinical specimens. The mecA gene was positive in all 63(100%) MRSA strains and 2(6%) of the 34 methicillin-sensitive Staphylococcus aureus (MSSA) strains. Two strains with the methicillin-sensitive phenotype and the mecA gene resulted in methicillin-resistance when cultured on an agar plate containing 4.5% NaCl. The mecA gene was also present in all 10(100%) coagulase-negative Staphylococcus strains with the methicillin-resistant phenotype. The femA gene was positive in all 97(100%) MRSA and MSSA strains. On the other hand, the femA gene was absent from coagulase -negative Staphylococcus strains with the methicillin-resistant phenotype. Although the mechanism by which the product of femA gene influences the expression of methicillin-resistance is unknown, the gene appears to be restricted only in coagulase-positive Staphylococcus, regardless of methicillin-resistance. In conclusion, in vitro enzymatic amplification of both mecA and femA genes would lead to rapid and definite diagnosis of the MRSA infection.
Hiratuka, Kanagawa, Japan
- Central Clinical Laboratory
Tokai Central HospitalTokitsu-chō, Gifu, Japan