Storing self-contained gel capillary cassettes for POC medical diagnostics

For effective clinical uptake of the lab on a chip/point of care technology (LOC-POC), in addition to cost advantages LOC-POC devices should offer multiple patient screening panels for related diseases as well as cold-chain transportation and storage abilities. We recently described a device that performs polymerase chain reaction (PCR) to simultaneously screen raw clinical samples from up to 16 patients for multiple infectious agents (Manage et al., Lab Chip, 2013, 13, 2576–2584). This cassette contains glass capillaries with desiccated semi-solid acrylamide gels that include all the reagents except for the sample, with integrated quality control. Here we report the development of protocols to store assembled PCR cassettes at room temperature, 4 uC or 220 uC as well as at +40 uC. We show that our cassettes are stable, with no loss of activity for at least 3 months at RT and at least 7 months at 4 uC and 220 uC. However, the activity of desiccated cassettes degrades when stored for more than 2 weeks at 40 uC, insufficient time for postmanufacture delivery and use of cassette PCR. To address this, we have evaluated two stage storage protocols. PCR cassettes can initially be stored at 4 uC and 220 uC for prolonged periods of time and removed for shorter term storage at RT, retaining activity for at least a month, which would facilitate transport to remote areas for testing. Effective use of cassette PCR in high temperature regions of the world, for experimental purposes defined here as 40 uC, appears to be feasible only after a first stage storage in the cold, followed by no more than 1 week at 40 uC. This should allow sufficient time for delivery by the manufacturer to a central area well served by power and refrigeration, for later ambient temperature transport and use in under-resourced areas that lack refrigeration

Phenotypic Detection of Clonotypic B Cells in Multiple Myeloma by Specific Immunoglobulin Ligands Reveals their Rarity in Multiple Myeloma

In multiple myeloma, circulating “clonotypic” B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential “feeder” cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10−3, this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology

Sanidade.

Introdução. Comportamento e sinais clínicos. Doenças virais. Varíola da carpa. Herpevírus da necrose hematopoiética do kinguio. Herpevírus da carpa. Viremia-primaveril da carpa. Linfocistose. Doenças bacterianas. Infecções por Aeromonas spp. Infecções por Flavobacterium columnare. Infecções por Edwardsiella spp. Infecções por Vibrio spp. Infecções por Streptococcus spp. Infecções por Mycobacterium spp. Infecções por Francisella spp. Doenças parasitárias causadas por protozoários: Doença do buraco na cabeça. Tricodiníase. Tetrahymenose ou doença dos guppies. Ictioftiríase ou doença dos pontos brancos. Piscinoodiníase ou doença do veludo. Doenças parasitárias causadas por helmintos: Monogenea. Digenea. Cestoda. Nematoda. Acantocephala. Doenças parasitárias causadas por crustáceos: Copépodes. Branquiúros. Isópodes. Doenças causadas por fungos: Saprolegniose. Doenças causadas por microsporídeos: Microsporídeos. Profilaxia. Aclimatação. Quarentena. Tratamento com quimioterápicos. Cuidados na aplicação de produtos e medicamentos. Seleção do método de aplicação. Considerações finais.bitstream/item/225453/1/pl-cap4.pd

An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics

This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format can simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis. To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma Urealyticum (UU) and Mycoplasma Homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50'. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for most clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will permit on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices

A Single Nucleotide Polymorphism in the RASGRF2 Gene Is Associated with Alcoholic Liver Cirrhosis in Men

Background Genetic polymorphisms in the RAS gene family are associated with different diseases, which may include alcohol-related disorders. Previous studies showed an association of the allelic variant rs26907 in RASGRF2 gene with higher alcohol intake. Additionally, the rs61764370 polymorphism in the KRAS gene is located in a binding site for the let-7 micro-RNA family, which is potentially involved in alcohol-induced inflammation. Therefore, this study was designed to explore the association between these two polymorphisms and susceptibility to alcoholism or alcoholic liver disease (ALD). Methods We enrolled 301 male alcoholic patients and 156 healthy male volunteers in this study. Polymorphisms were genotyped by using TaqMan® PCR assays for allelic discrimination. Allelic and genotypic frequencies were compared between the two groups. Logistic regression analysis was performed to analyze the inheritance model. Results The A allele of the RASGRF2 polymorphism (rs26907) was significantly more prevalent among alcoholic patients with cirrhosis (23.2%) compared to alcoholic patients without ALD (14.2%). This difference remained significant in the group of patients with alcohol dependence (28.8% vs. 14.3%) but not in those with alcohol abuse (15.1% vs. 14.4%). Multivariable logistic regression analysis showed that the A allele of this polymorphism (AA or GA genotype) was associated with alcoholic cirrhosis both in the total group of alcoholics (odds ratio [OR]: 2.33, 95% confidence interval [CI]: 1.32–4.11; P = 0.002) and in the group of patients with alcohol dependence (OR: 3.1, 95% CI: 1.50–6.20; P = 0.001). Allelic distributions of the KRAS polymorphism (rs61764370) did not differ between the groups. Conclusions To our knowledge, this genetic association study represents the first to show an association of the RASGRF2 G>A (rs26907) polymorphism with ALD in men, particularly in the subgroup of patients with AD. The findings suggest the potential relevance of the RAS gene family in alcoholism and ALD

Live lecture versus video podcast in undergraduate medical education: A randomised controlled trial

<p>Abstract</p> <p>Background</p> <p>Information technology is finding an increasing role in the training of medical students. We compared information recall and student experience and preference after live lectures and video podcasts in undergraduate medical education.</p> <p>Methods</p> <p>We performed a crossover randomised controlled trial. 100 students were randomised to live lecture or video podcast for one clinical topic. Live lectures were given by the same instructor as the narrator of the video podcasts. The video podcasts comprised Powerpoint™ slides narrated using the same script as the lecture. They were then switched to the other group for a second clinical topic. Knowledge was assessed using multiple choice questions and qualitative information was collected using a questionnaire.</p> <p>Results</p> <p>No significant difference was found on multiple choice questioning immediately after the session. The subjects enjoyed the convenience of the video podcast and the ability to stop, review and repeat it, but found it less engaging as a teaching method. They expressed a clear preference for the live lecture format.</p> <p>Conclusions</p> <p>We suggest that video podcasts are not ready to replace traditional teaching methods, but may have an important role in reinforcing learning and aiding revision.</p

Can energy self‑sufficiency be achieved? Case study of Warmińsko‑Mazurskie Voivodeship (Poland)

An analysis was carried out to show whether the Warmińsko-Mazurskie Voivodeship (Poland) could become energy selfsufficient. The technical potential of electricity and heat from renewable sources has been calculated. The calculated values are 6.93 TWh/year of electricity and 15.84 PJ/year of heat—these amounts would ensure the energy independence of the Voivodeship. The Warmińsko-Mazurskie Voivodeship is an example of transformation towards “green” energy, it shows that such transformation is also possible in Poland even in short term. This would reduce air pollution as well as limit the import of energy resources. It is very important, it allows us to think with optimism and implement Poland’s energy transformation towards renewable energy (RE). Additionally, a SWOT analysis of each type of RE in the Warmińsko-Mazurskie Voivodeship was presented. The SWOT analysis makes it possible to identify the strengths, weaknesses, prospects and threats for RE in the Voivodeship and the whole country. It has been found that there is a great interest of investors in RE in the Voivodeship, there is usually a great public support for new energy sources, and the biggest barriers are high investment costs and complicated law in Poland