Rapid quantification of hepatitis B virus DNA by automated sample preparation and real-time PCR

Monitoring of hepatitis B virus (HBV) DNA in serum by molecular methods has become the standard for assessment of the replicative activity of HBV. Several molecular assays for the detection and quantification of HBV DNA have been described. However, they usually lack automated sample preparation. Moreover, those assays, which are based on PCR, are limited by a short dynamic range (2 to 3 log units). In the present study, the use of RealArt HBV LC PCR Reagents in conjunction with automated extraction on the COBAS AMPLIPREP analyzer was evaluated. Members of an HBV proficiency program panel were tested; linearity, interassay, and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was evaluated with a total of 117 clinical specimens. When members of the HBV proficiency program panel were tested by the new molecular assay, the results were found to be within +/-0.5 log unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve over more than 6 log units. The interassay variation of the RealArt HBV LC PCR Reagents by use of the automated sample preparation protocol ranged from 16 to 73%, and the intra-assay variation ranged from 9 to 40%. When clinical samples were tested by the new assay with the automated sample preparation protocol and the results were compared with those obtained by the COBAS AMPLICOR HBV MONITOR Test with manual sample preparation, the results for 76% of all samples with positive results by both tests were found to be within +/-0.5 log unit and the results for another 18% were found to be within between 0.5 and 1.0 log unit. In conclusion, the real-time PCR assay with automated sample preparation proved to be suitable for the routine molecular laboratory and required less hands-on time.     

Evaluation of automated sample preparation and quantitative PCR LCx assay for determination of human immunodeficiency virus type 1 RNA

Efforts have been made to achieve full automation of molecular assays for quantitative detection of human immunodeficiency virus type 1 (HIV-1). In the present study, the Abbott LCx HIV RNA Quantitative assay was evaluated in conjunction with automated HIV-1 RNA extraction on the MagNA Pure LC instrument and compared to the conventional LCx HIV RNA Quantitative assay, which uses a manual nucleic acid extraction protocol. Accuracy, linearity, and interassay and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was tested with a total of 105 clinical specimens. When the accuracy of the LCx HIV RNA Quantitative assay with the automated sample preparation protocol was tested, all results were found to be within +/- 0.5 log unit of the expected results. Determination of linearity resulted in a quasilinear curve over 3.5 log units. For determination of interassay variation, coefficients of variation were found to be between 21 and 66% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 10 and 69% for the LCx HIV RNA Quantitative assay with manual sample preparation. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 25% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 7 and 19% for the LCx HIV RNA Quantitative assay with manual sample preparation. When clinical samples were tested by the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and the results were compared with those of the LCx HIV RNA Quantitative assay with manual sample preparation, 95% of all positive results were found to be within +/- 0.5 log unit. In conclusion, the assay with automated sample preparation proved to be suitable for use in the routine diagnostic laboratory and required significantly less hands-on time.     

Gene expression profiling after radiation-induced DNA damage is strongly predictive of BRCA1 mutation carrier status.

PURPOSE: The impact of the presence of a germ-line BRCA1 mutation on gene expression in normal breast fibroblasts after radiation-induced DNA damage has been investigated. EXPERIMENTAL DESIGN: High-density cDNA microarray technology was used to identify differential responses to DNA damage in fibroblasts from nine heterozygous BRCA1 mutation carriers compared with five control samples without personal or family history of any cancer. Fibroblast cultures were irradiated, and their expression profile was compared using intensity ratios of the cDNA microarrays representing 5603 IMAGE clones. RESULTS: Class comparison and class prediction analysis has shown that BRCA1 mutation carriers can be distinguished from controls with high probability (approximately 85%). Significance analysis of microarrays and the support vector machine classifier identified gene sets that discriminate the samples according to their mutation status. These include genes already known to interact with BRCA1 such as CDKN1B, ATR, and RAD51. CONCLUSIONS: The results of this initial study suggest that normal cells from heterozygous BRCA1 mutation carriers display a different gene expression profile from controls in response to DNA damage. Adaptations of this pilot result to other cell types could result in the development of a functional assay for BRCA1 mutation status.     

Polyoma tumor development in neonatally polyoma-virus-infected CD4−/− and CD8−/− single knockout and CD4−/−8−/− double knockout mice

CD4−/− or CD8−/− single knockout as well as CD4−/−8−/− double knockout mice were infected with polyoma virus as newborns or 1 week after birth. The animals were followed for tumor development and virus persistence. Double knockout mice developed tumors at a higher incidence (29%) than either the CD8−/− or CD4−/− single knockout mice (11% and 2%, respectively). Persistence of polyoma virus was examined by PCR in one third of all animals included in the study. Seven of the 17 CD4−/−8−/− double knockout mice gave positive evidence of virus persistence up to 6 months p.i. where virus DNA was present in most organs. Corresponding tests in single knockout mice gave positive results of persistent viral DNA in 2 of the 19 CD8−/− and 2 of the 7 CD4−/− mice. In the single knockout mice polyoma DNA could only be detected in a more limited variety of organs compared to the double knockouts. © 1996 Wiley-Liss, Inc.     

Mosaicplasty: long-term follow-up

The successful treatment of chondral and osteochondral defects of the weightbearing surfaces is a challenge for orthopaedic surgeons. Autologous osteochondral transplantation is one method that can be used to create a hyaline or hyaline-like repair in the defect area. Ten years of clinical experience with autologous osteochondral mosaicplasty are described. Clinical scores, imaging techniques, arthroscopy, histological examination of biopsy samples, and cartilage stiffness measurements were used to evaluate the clinical outcomes and quality of the transplanted cartilage in a total of 831 patients who underwent mosaicplasty. According to our investigations, good-to-excellent results were achieved in 92% of the patients treated with femoral condylar implantations, in 87% of those treated with tibial resurfacing, in 79% of those treated with patellar and/or trochlear mosaicplasties, and in 94% of those treated with talarprocedures. Long-term donor-site disturbances, which were assessed using the Bandi score, showed that patients had 3% morbidity after mosaicplasty. Sixty-nine of 83 patients who were followed arthroscopically showed congruent gliding surfaces, histological evidence of the survival of the transplanted hyaline cartilage, and fibrocartilage filling of the donor sites. Four deep infections and 36 painful postoperative hemarthroses were experienced as complications arising from the surgical procedures. On the basis of both these promising results and also those of other similar studies, autologous osteochondral mosaicplasty would appears to be an alternative for the treatment of small and medium-sized focal chondral and osteochondral defects of the weightbearing surfaces of the knee and other weightbearing synovial joints.     

Mediastinal bulky tumour in Hodgkin's disease and prognostic value of positron emission tomography in the evaluation of post-treatment residual masses

Among the 193 patients (82 female, 111 male) treated primarily for Hodgkin's disease at our clinic between 1990 and 2001 and followed up until 2003, 42 (22%) had mediastinal bulky tumours (MBTs) by the Cotswolds criteria. The rate of MBT diagnosis was significantly greater in the early stage of the disease, these patients were younger and--in contrast to the other group--they all received combined therapy. No significant differences were found in the overall and relapse-free survival rate in the two groups, but relapse and death rates were lower in the patients with bulky tumours. Of the total number of patients, 27 underwent a total of 31 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) examinations, mainly for the evaluation of post-treatment residual mass viability. In the 12 positive cases, the majority of patients received further therapy. During the mean follow-up time of 58 months (range 5-98 months) after obtaining negative results, progression of the disease was found in 2 cases 14 and 23 months later, respectively. Based on our results, we conclude that FDG-PET examinations show a good correlation with clinical follow-up results.     

Incidence and clinical relevance of abnormal complete blood counts in long-term survivors of childhood cancer

BACKGROUND: The purpose of the study was to determine the incidence and clinical significance of abnormal complete blood counts (CBCs) obtained during follow-up of childhood cancer survivors. METHODS: A retrospective cohort study was conducted on 193 survivors, diagnosed between 1970-1986, who were followed in our center's After Cancer Experience Program and are participants in the Childhood Cancer Survivor Study. Of these patients, 49% were female and 25% were racial/ethnic minorities. The primary outcome was determination of the cumulative percentage of patients having an abnormal CBC by 2 or 3 standard deviations (SDs). Four components of the CBC were examined and employed to define an abnormal CBC: low white blood cell count (WBC), high mean corpuscular volume (MCV), low platelet count, and low hemoglobin concentration. Association of treatment exposures to abnormal values was assessed with a multilevel logistic model. RESULTS: There were 1297 patient visits during 1401 person-years of follow-up. The mean number of visits per survivor was 6.7 (SD 4.2). The cumulative percentage of subjects with at least one abnormal CBC was 70%. The cumulative percent of subjects with a value abnormal by 2 SD was WBC = 23%, MCV = 37%, platelets = 9%, hemoglobin = 49%. For values abnormal by 3 SD, the frequencies were WBC = 3%, MCV = 20%, platelets = 1%, hemoglobin = 27%. None of the patients developed myelodysplastic syndrome or a secondary leukemia during the follow-up period. Exposure to epipodophyllotoxins was associated with an increased risk of having abnormally high MCV values. CONCLUSIONS: Mildly abnormal CBC values are common in survivors of childhood cancer. Abnormal values are often of questionable significance but seem to persist over time. Epipodophyllotoxin therapy was found to be associated with increased frequency of high MCV levels.     

Laterality of deep white matter hyperintensities correlates with basilar artery bending and vertebral artery dominance

AimTo investigate whether vertebrobasilar geometry contributes to the presence, severity, and laterality of white matter hyperintensities (WMH).MethodsWe retrospectively reviewed 290 cerebral scans of patients who underwent time-of-flight and fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) between 2017 and 2018. WMH were counted, localized, and grouped according to laterality on the FLAIR sequence. A 3D mesh of the posterior circulation was reconstructed (with ITK SNAP software) and the morphology of the vertebrobasilar system analyzed with an in-house software written in Python.ResultsPatients were assigned into a group with WMH (n = 204) and a group without WMH (n = 86). The severity of WMH burden was mainly affected by age and hypertension, while the localization of the WMH (or laterality) was mainly affected by the vertebrobasilar system morphology. Basilar artery morphology only affected the parieto-occipital region significantly if both posterior communicating arteries were hypoplastic or absent. The dominant vertebral artery and basilar artery curve had an opposite directional relationship.ConclusionsAn unequal vertebral artery flow is an important hemodynamic contributor to basilar bending. Increased basilar artery curvature and increased infratentorial WMH burden may signal inadequate blood flow and predict cerebrovascular events.

Cerebrovascular diseases, predominantly stroke, remain the leading cause of death and functional disability worldwide (1). White matter hyperintensities (WMH) on cerebral T2-weighted fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) sequences have been linked to cerebrovascular disease outcomes and ischemic stroke (2,3) (Figure 1). WMH, subclinical cerebral alterations caused by changes in cerebral vascular morphology, may lead to cerebral histological changes long before the manifestation of neurological deficits or cognitive decline (4). Multiple studies suggested that ischemia plays a crucial role in the development of tortuous arterioles and reduced cerebral blood flow. Despite the two decades of advancements in the understanding of the etiology of these processes, much uncertainty remains (2). Chronic hypoperfusion and arteriolosclerosis are considered key features in the development of WMH. The presence of WMH triples the risk of stroke and doubles the risk of dementia (5). Previous publications showed a significant correlation between cerebral artery morphology and stroke localization (6). However, the connection between WMH and large cerebral artery morphology is not yet fully understood.Open in a separate windowFigure 1White matter hyperintensity on T2 fluid-attenuated inversion recovery magnetic resonance imaging sequence (scale bar, 5 cm; source: Medical Imaging Centre, Semmelweis University). Based on our white matter hyperintensities evaluation protocol, this patient was categorized as posterior cerebral artery region right dominant.Approximately 20% of strokes occur in the region supplied by the vertebrobasilar system. Unlike most systemic arteries, which are characterized by a tree-like branching pattern, the basilar artery is unique as it is created from the confluence of the two vertebral arteries. The vertebral arteries are mostly asymmetric and left-dominant. The varying vessel morphologies may cause asymmetrical blood flow in the basilar artery, which over time possibly leads to bending. This phenomenon, named basilar curve or basilar bending, may create microvascular damage to the surrounding brain areas.Therefore, our first aim was to investigate if basilar artery geometry affected the existence and laterality of WMH. Our second aim was to investigate the influence of the vertebral artery dominance on basilar artery geometric indices.