Evaluation of the basic assay performance of the GeneSoc® rapid PCR testing system for detection of severe acute respiratory syndrome coronavirus 2.

In the ongoing coronavirus disease 2019 (COVID-19) pandemic, PCR has been widely used for screening patients displaying relevant symptoms. The rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enables prompt diagnosis and the implementation of proper precautionary and isolation measures for the patient. In the present study, we aimed to evaluate the basic assay performance of an innovative PCR system, GeneSoC® (Kyorin Pharmaceutical Co. Ltd., Tokyo, Japan). A total of 1,445 clinical samples were submitted to the clinical laboratory, including confirmed or suspected cases of COVID-19, from February 13 to August 31. Specimen types included nasopharyngeal swabs. The sampling was performed several times for each patient every 2-7 days. Using this system, sequences specific for SARS-CoV-2 RNA could be detected in a sample within 10-15 min using the microfluidic thermal cycling technology. Analytical sensitivity studies showed that GeneSoC® could detect the target sequence of the viral envelope and RNA-dependent RNA-polymerase (RdRp) genes at 5 and 10 copies/µL, respectively. The precision of the GeneSoC® measurements using clinical isolates of the virus at a concentration of 103 copies/µL was favorable for both the genes; within-run repeatability and between-run reproducibility coefficient of variation values were less than 3% and 2%, respectively; and the reproducibility of inter-detection units was less than 5%. Method comparison by LightCycler® 480 showed the positive and negative agreement to be 100% [(174/174) and (1271/1271), respectively]. GeneSoC® proved to be a rapid and reliable detection system for the prompt diagnosis of symptomatic COVID-19 patients and could help reduce the spread of infections and facilitate more rapid treatment of infected patients.

Hypodysfibrinogenemia with a Heterozygous Mutation of γCys326Ser by the Novel Transversion of TGT to TCT in a Patient with Pulmonary Thromboembolism and Right Ventricular Thrombus.

We encountered a 45-year-old Japanese man who suffered from pulmonary thromboembolism and huge right ventricular thrombus after inferior vena cava (IVC) filter implantation without apparent thrombus in either the deep veins or inside the IVC filter. The biochemical data showed a discrepancy in the level of fibrinogen between the immunological and thrombin time methods, suggesting hypodysfibrinogenemia. The sequencing of the fibrinogen γ-chain gene (FGG) revealed a novel heterozygous missense mutation in exon 8 - a TGT to TCT transversion in codon 326 - resulting in an amino acid substitution of serine for cysteine (γCys326Ser). The characterization of the protein did not show known mechanisms for thrombosis in dysfibrinogenemia, such as dimer or albumin-binding complex formation. In summary, the current case with a life-threatening thrombotic event was found to have a novel heterozygous missense mutation resulting in γCys326Ser, which was suggested as a predisposing factor of the thrombosis. Known mechanisms responsible for thrombosis in the current case were not demonstrated, suggesting other mechanisms including superimposing inherited and/or acquired risk factors. When a patient presents with unusual thrombosis such as breakthrough pulmonary embolism and huge thrombus in the right ventricle, as in the current case, the laboratory process for heritable thrombophilia should be considered.

Systemic mastocytosis associated with t(8;21) acute myeloid leukemia in a child: detection of the D816A mutation of KIT.

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.

[The quality practice of molecular-genetic testing in the era of molecular target therapy in chronic myelogenous leukemia].

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.

Persistence of derivative chromosome 22 after achieving a major molecular response in chronic myeloid leukemia with a cryptic BCR-ABL1 fusion gene.

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.

In vitro effect of fludarabine, cyclophosphamide, and cytosine arabinoside on chromosome breakage in Fanconi anemia patients: relevance to stem cell transplantation.

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.

Quantitative assay of hepatitis C virus RNA using an automated extraction system for specific capture with probes and paramagnetic particle separation.

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.