Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis

<p>Abstract</p> <p>Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma.</p> <p>Introduction</p> <p>Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year <abbrgrp><abbr bid="B2">2</abbr></abbrgrp>. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation <abbrgrp><abbr bid="B3">3</abbr><abbr bid="B4">4</abbr></abbrgrp>. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis <abbrgrp><abbr bid="B5">5</abbr><abbr bid="B6">6</abbr></abbrgrp>.</p> <p>GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost <abbrgrp><abbr bid="B7">7</abbr></abbrgrp>. Therefore, rapid and more cost effective technique are being sought.</p> <p>LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye <abbrgrp><abbr bid="B8">8</abbr><abbr bid="B9">9</abbr></abbrgrp>. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product <abbrgrp><abbr bid="B10">10</abbr><abbr bid="B11">11</abbr></abbrgrp>. Therefore, current investigations using melting curve analysis are being developed <abbrgrp><abbr bid="B12">12</abbr><abbr bid="B13">13</abbr></abbrgrp>.</p> <p>In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.</p

Poor Thermal Care Practices among Home Births in Nepal: Further Analysis of Nepal Demographic and Health Survey 2011

Introduction - Hypothermia is a major factor associated with neonatal mortality in low and middle income countries. Thermal care protection of newborn through a series of measures taken at birth and during the initial days of life is recommended to reduce the hypothermia and associated neonatal mortality. This study aimed to identify the prevalence of and the factors associated with receiving ‘optimum thermal care’ among home born newborns of Nepal. Methods - Data from the Nepal Demographic and Health Surveys (NDHS) 2011 were used for this study. Women who reported a home birth for their most recent childbirth was included in the study. Factors associated with optimum thermal care were examined using Chi-square test followed by logistic regression. Results - A total of 2464 newborns were included in the study. A total of 57.6 % were dried before the placenta was delivered; 60.3% were wrapped; 24.5% had not bathing during the first 24 hours, and 63.9% were breastfed within one hour of birth. Overall, only 248 (10.7%; 95% CI (8.8 %, 12.9%)) newborns received optimum thermal care. Newborns whose mothers had achieved higher education (OR 2.810; 95% CI (1.132, 6.976)), attended four or more antenatal care visits (OR 2.563; 95% CI (1.309, 5.017)), and those whose birth were attended by skilled attendants (OR 2.178; 95% CI (1.428, 3.323)) were likely to receive optimum thermal care. Conclusion - The current study showed that only one in ten newborns in Nepal received optimum thermal care. Future newborn survival programs should focus on those mothers who are uneducated; who do not attend the recommended four or more attend antenatal care visits; and those who deliver without the assistance of skilled birth attendants to reduce the risk of neonatal hypothermia in Nepal

Stochastically Gating Ion Channels Enable Patterned Spike Firing through Activity-Dependent Modulation of Spike Probability

The transformation of synaptic input into patterns of spike output is a fundamental operation that is determined by the particular complement of ion channels that a neuron expresses. Although it is well established that individual ion channel proteins make stochastic transitions between conducting and non-conducting states, most models of synaptic integration are deterministic, and relatively little is known about the functional consequences of interactions between stochastically gating ion channels. Here, we show that a model of stellate neurons from layer II of the medial entorhinal cortex implemented with either stochastic or deterministically gating ion channels can reproduce the resting membrane properties of stellate neurons, but only the stochastic version of the model can fully account for perithreshold membrane potential fluctuations and clustered patterns of spike output that are recorded from stellate neurons during depolarized states. We demonstrate that the stochastic model implements an example of a general mechanism for patterning of neuronal output through activity-dependent changes in the probability of spike firing. Unlike deterministic mechanisms that generate spike patterns through slow changes in the state of model parameters, this general stochastic mechanism does not require retention of information beyond the duration of a single spike and its associated afterhyperpolarization. Instead, clustered patterns of spikes emerge in the stochastic model of stellate neurons as a result of a transient increase in firing probability driven by activation of HCN channels during recovery from the spike afterhyperpolarization. Using this model, we infer conditions in which stochastic ion channel gating may influence firing patterns in vivo and predict consequences of modifications of HCN channel function for in vivo firing patterns

Determinants of facility delivery after implementation of safer mother programme in Nepal: A prospective cohort study

Background: There are several barriers for pregnant women to deliver in a health care facility. This prospective cohort study investigated factors affecting facility delivery and reasons for unplanned place of delivery after implementation of the safer mother programme in Nepal. Methods: Baseline interviews using a validated questionnaire were conducted on a sample of 700 pregnant women representative of the Kaski district in central Nepal. Follow-up interviews of the cohort were then conducted within 45 days postpartum. Stepwise logistic regression analysis was performed to determine factors associated with the facility delivery outcome. Results: Of the 644 pregnant women whose delivery location had been identified, 547 (85%) gave birth in a health care facility. Women were more likely to deliver in a health facility if they were educated especially with higher secondary or above qualification (adjusted odds ratio (OR) 12.39, 95% confidence interval (CI) 5.09 to 30.17), attended 4 or more antenatal care visits (OR 2.15, 95% CI 1.25 to 3.69), and lived within 30 minutes to the facility (OR 11.61, 95% CI 5.77 to 24.04). For the 97 women who delivered at home, 72 (74.2%) were unplanned, mainly due to quick precipitation of labour making it impossible to reach a health facility. Conclusions: It appeared that facility delivery occurs more frequent among educated women and those who live nearby, even though maternity services are now freely available in Nepal. Because of the difficult terrain and transportation problem in rural areas, interventions that make maternity service physically accessible during antenatal period are needed to increase the utilisation of health facility for child birth

Accurate path integration in continuous attractor network models of grid cells

Grid cells in the rat entorhinal cortex display strikingly regular firing responses to the animal's position in 2-D space and have been hypothesized to form the neural substrate for dead-reckoning. However, errors accumulate rapidly when velocity inputs are integrated in existing models of grid cell activity. To produce grid-cell-like responses, these models would require frequent resets triggered by external sensory cues. Such inadequacies, shared by various models, cast doubt on the dead-reckoning potential of the grid cell system. Here we focus on the question of accurate path integration, specifically in continuous attractor models of grid cell activity. We show, in contrast to previous models, that continuous attractor models can generate regular triangular grid responses, based on inputs that encode only the rat's velocity and heading direction. We consider the role of the network boundary in the integration performance of the network and show that both periodic and aperiodic networks are capable of accurate path integration, despite important differences in their attractor manifolds. We quantify the rate at which errors in the velocity integration accumulate as a function of network size and intrinsic noise within the network. With a plausible range of parameters and the inclusion of spike variability, our model networks can accurately integrate velocity inputs over a maximum of ~10–100 meters and ~1–10 minutes. These findings form a proof-of-concept that continuous attractor dynamics may underlie velocity integration in the dorsolateral medial entorhinal cortex. The simulations also generate pertinent upper bounds on the accuracy of integration that may be achieved by continuous attractor dynamics in the grid cell network. We suggest experiments to test the continuous attractor model and differentiate it from models in which single cells establish their responses independently of each other

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Task shifting for emergency obstetric surgery in district hospitals in Senegal

Peer reviewedPostprin