Collection Of Biological Material Research Articles

An assessment of tape-lifts

Tape-lifting is a non-destructive alternative to swabbing for collection of biological materials deposited on surfaces, especially on porous substrates. While there have been a number of studies looking at the efficiency of tapes in terms of recovery and their effect on downstream processes, none has been able to visually monitor cellular material collection.We report on a comparative study of a range of tapes regarding their collection efficiency of cellular material visualised using fluorescence microscopy, their background fluorescence after staining with DD diluted with three different solvent types and inhibition of subsequent PCR using direct PCR. Three of the fourteen tapes were selected for further testing. These were brown packing tape (Packmate™), clear tape (Sellotape®), and DNA-free tape (Lovell Surgical Solutions). These did not inhibit direct STR amplification; the other eleven tapes exhibited either high background fluorescence and/or inhibition of subsequent PCR. The effectiveness of the three tapes for the collection of cellular material was tested after tape-lifting a fingermark once, twice and ten times. The amount of cellular material within fingermarks stained with Diamond™ dye (DD) was recorded using fluorescence microscopy before and after tape-liftings. The DNA-free tape (Lovell) used in many forensic laboratories gave poor recovery compared to the other two tapes. After a tape-lift, an average of 30 % of cellular material was recovered by using DNA-free tape (Lovell), contrasting with an average recovery of 59.5 % for the clear tape (Sellotape®) and 88.8 % for the brown packing tape (Packmate™). The results presented show that standard crime scene sampling tape does collect DNA but is less effective than shop-bought tapes. Full DNA profiles can be generated from all of touched fabric samples that were collected DNA using the three tapes, triaged by DD staining and amplified by direct PCR approach.

Instrument to streamline the process of sending biological samples to an epidemiological surveillance center, from a Reference Unit for infectious and parasitic diseases.

Introduction: The sending of biological materials for specific exams to the Public Health Laboratory of Amazonas (LACEN-AM) depends on documents containing essential information to guide the performance of the tests, which need to go through a quality process and in most cases the lack of data compromises the streamflow to LACEN-AM. Objective: Create a checklist for the process of sending samples to the Public Health Laboratory (LACEN-AM). Methods: The Health Surveillance Guide (GVS), Integrated Manual for Epidemiological Surveillance of botulism, technical notes, reference technicians for information on registration and tests performed at LACEN-AM were consulted and the sequence of activities was also observed. carried out in the notification epidemiology sector, the orientation of the collection of biological material until the sending of the samples. Results: Between 2017 and 2019, 6 lists were sent to send samples to LACEN-AM, one list for each disease (Paracoccidioidomycosis, Hydatidosis, Severe Acute Respiratory Syndrome, Acute Flaccid Paralysis, Human Rabies and Botulism). Conclusion: It is necessary to simplify the procedures related to the notification activities for sending biological material samples, improving operationalization, especially for paracocociodiomycosis, hydatidosis and SRAG, diseases that are not included in the GVS, making it difficult to understand the flow of sending biological materials when requested or, in the cases of (PFA), human rabies and botulism which, because they are in the process of monitoring, have their flows difficult to discern. Therefore, disseminating these procedures by means of an instrument can contribute to the improvement and understanding of the technicians who work in epidemiological surveillance, and will certainly speed up their actions, ensuring speed in sending the samples.

Abstract A075: SPECTA: The EORTC translational research platform for Europe

Abstract SPECTA is the translational research platform from the European Organisation for Research and Treatment of Cancer (EORTC), which facilitates pan-European research projects by providing a comprehensive infrastructure: a single protocol and Patient Informed Consent to enrol patients with cancer, a centralised database for clinical data collection (including long-term follow-up and quality of life), a centralised biobank and quality control sample workflow, expert molecular laboratories dedicated to the needs of the translational research projects. So far, 11 countries with 36 sites are open for recruitment with 43 authorized investigators. Two SPECTA projects focus on rare populations. The first, AYA (Adolescent and Young Adults) enrols patients aged between 12 and 29 years old, diagnosed with high grade glioma or sarcoma. AYA is a pilot study with a strong collaboration, between DKFZ and EORTC, aiming at diagnosis confirmation (central pathology review by pathologist experts and methylation diagnosis), and extensive molecular analysis (WES, RNAseq and methylation array). The aim is not only to have a direct impact on patients through the clinical advice provided to the treating physician by the molecular tumor board, but also to build knowledge for future clinical trials in this population with unmet needs. The second, Arcagen, is a collaborative project with the EUropean network for RAre CANcer (EURACAN), the European network for rare cancer. This project, with the support of Roche, aims at building a clinically annotated genomic database (NGS panels for FMI) of 1,000 rare cancer patients from the 10 EURACAN domains. So far, 81 patients have been recruited: sarcoma (n=43), thymic malignancies (n=16), germ cell tumor (n=10), and rare head and neck tumor (n=15). Druggable alterations were found in about 15% of patients. Molecular reports will be sent to local clinicians, the study results will be used for designing for the next generation of clinical trials in adult rare cancers. The last project currently enrolling in SPECTA is IMMUcan, an Innovative Medicine Initiative project. IMMUcan will generate broad molecular (WES and RNAseq) and cellular profiling data (multiplex immunofluorescence and imaging mass cytometry) of tumor and its microenvironment. Integration of this information with clinical data, will promote understanding on how the immune system and tumors interact, and the impact of current therapeutic interventions. For this purpose, we will enrol 3,000 patients with NSCLC, HNSCC, breast, colorectal and renal cell cancers. For all projects, we faced recurrent issues regarding tumor and peripheral sample quality. We overcomed those issues by performing optimization studies for pan European collection of biological material (such as PBMCs, ctDNA, vesicles or FFPE slides stability for imaging). SPECTA is an academic solution for next level precision medicine, with all the clinical, operational and scientific challenges that it implies. Citation Format: Marie Vinches, Teresa de Rojas, Aleksandra Stevovic, Martin Mccabe, Jean Yves Blay, Vassilis Golfinopoulos, Sabine Tejpar, Marie Morfouace. SPECTA: The EORTC translational research platform for Europe [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A075. doi:10.1158/1535-7163.TARG-19-A075

The main directions of use biological material to provide the efficiency of laboratory supply in agricultural products testing in Ukraine

Ukraine has significant resource potential for the agrarian sector development and improving the efficiency of agricultural production. An effective mechanism for controlling agro-industrial products is monitoring of indicators of quality and safety not only of finished products but also of all components involved in its manufacturing (soil and water as the main resource for agricultural production, plant protection products, mineral and organic fertilizers, seeds of agricultural cultures, etc. We analyzed normative documents on the accreditation of laboratories and international recommendations concerning the rational use of standardized biological material in the testing activity. We conducted the analysis of the testing activity results of the Ukrainian laboratory of quality and safety of agricultural products (ULQSAP). So, the expediency of optimizing the functioning of biological material collections in scientific institutions substantiated. The conducted analysis testifies to expediency of creation the national bioresource center, which will unite informationally all the functioning collections of biological material in order to increase the efficiency of its innovative potential.

Правове регулювання поняття біологічних матеріалів як складової біобанку та їх співвідношення з анатомічними матеріалами

Правове регулювання поняття біологічних матеріалів як складової біобанку та їх співвідношення з анатомічними матеріалами

PECULIARITIES OF THE NATURAL ORIGIN OF TRICHINELLOSIS IN THE CHUKOT PENINSULA TERRITORY

Trichinellosis is a natural focal nontransmissive zoonotic disease, the causative agent of which is the nematode Trichinella nativa (T2). The collection of biological material was carried out on the territory of the coastal settlements of the Chukot Peninsula. The objects of the presented work were the representatives of the order carnivore (Carnivora) of the following families: canids (Canidae), feline (Felidae), bears (Ursidae), walrus (Odobenidae), seal (Phocidae) and the whale (Сetacea). Larvae detection from the muscle tissue of animals was carried out by standard methods. Infection rates were determined by the extensiveness (EI) and intensity of invasion (II).During the evolution Trichinella have acquired a wide range of hosts with similar trophic relations, and therefore they ensure the long-term functioning of the natural focus. The organism of the animals, which are host species, plays the role of reservoir (the reservation phase). There were the following components of the guest component among the studied host species: 5 species of terrestrials, and 4 species of marine mammals. The animals’ contamination ranged from 60.0% to 100% in terrestrials, and it ranged from 0.9% to 4.3% in marine animals. Successful circulation of Trichinella nativa (dispersion phase, distribution) under the extreme conditions of Arctic region became possible due to several reasons. They are: getting tolerated to low temperatures in the process of evolution, the process of decay and a number of other adaptive properties that allow them to maintain viability and invasive properties in the corpses of terrestrial carnivores and marine mammals during the long winter. The natural focus of trichinellosis on the territory of the Chukot Peninsula is unique and multifaceted in nature, and it is of interest for researchers to learn environmental and general biological laws, taking into account the traditional economic activities of the indigenous population.

Affected for good or for evil: The formation of issue-publics that relate to the UK National DNA Database.

The United Kingdom has a long tradition of collecting and storing DNA data for criminal identification purposes. The development of the UK National Criminal Intelligence DNA Database has been accompanied by public controversies. Building on recent developments in Science and Technology Studies on public engagement, we elaborate on the concept of emergent and co-produced issue-publics. We explore which different types of issues affect and mobilize publics along the historical development of the National Criminal Intelligence DNA Database, and how publics take shape alongside the institutionalization of regulatory and governance solutions. We identify three related issue-publics: a ‘biological citizen issue-public’ concerned with human and civil rights regarding the collection of biological material; a ‘watchdog issue-public’ that emerges to identify the problems surrounding a lack of civic accountability; and a ‘co-decision making issue-public’, including the stakeholders who advise on decisions relating to the database.

The effect of biotin interference on the results of blood hormone assays.

Approximately 70% of medical decisions are made based on results of laboratory investigations. Immunochemical methods are used most commonly in routine laboratory diagnostics of endocrine disorders. Those methods are limited by susceptibility of the immunochemical reaction to various interferences. Interference may be caused by the presence of autologous antibodies, heterophilic antibodies, or paraproteins in the blood serum, by cross-reactions with similar reagents, haemolysis, significant lipidaemia, or hyperbilirubinaemia. Some recent reports have indicated a significant effect of biotin on the reliability of laboratory investigations. Biotin is a water-soluble vitamin belonging to the B group. It is present in popular dietary supplements - alone or as a component of multi-vitamin formulas - and it is advertised as a remedy to falling out and fragility of hair and nails. Due to its low molecular weight and a strong affinity to streptavidin, biotin is used in many immunochemical tests. Due to a strong and stable bond of streptavidin and biotin, analytical methods using the streptavidin (avidin)-biotin system are characterised by superior sensitivity, and they allow determination of very low levels of the tested substance in biological material. The presence of exogenous biotin in a sample may cause interference when using tests that utilise the streptavidin (avidin)-biotin system. Interference of biotin with immunochemical tests depends on several factors: the construction of the immunochemical test, the dose used by the patient, the biotin concentration in the sample, and most of all - the time from the last dose to the collection of biological material for laboratory testing. In this paper we present some practical recommendations and a procedure to be followed in the case of suspected interference of biotin in immunochemical assays, for clinicians and laboratory diagnosticians.

Glomerular Hypertrophy and Alterations in Renin-Angiotensin System Activation Are Associated with Diminished Systemic Blood Pressure in Aging Mice with Sickle Cell Anemia

Sickle cell nephropathy is a major and severe clinical complication of sickle cell anemia (SCA). The hypoxic microenvironment of the renal medulla leads to hyperosmolarity and acidosis, which favors deoxygenated hemoglobin S (HbS) polymerization and subsequent sickling of erythrocytes, promoting vaso-occlusive processes in the organ. Renal ischemia and glomerular blood flow can modulate angiotensin II (AngII) production, a vasoconstrictor hormone derivative of the Renin-Angiotensin System (RAS), which regulates blood pressure (BP), among other functions. Considering the elevated blood volume and cardiac output in individuals with SCA, an increase in BP might be expected; however SCA patients often present lower PB (Pikilidou et al., J Clin Hypertens., 2015), which modifies with aging. Reduced plasma AngII in male mice with SCA has been reported previously (Dos Santos et al., Life Sci., 2014), thus we aimed to investigate changes in PB and RAS, in association with aging and renal alterations in an animal model of SCA. Male chimeric mice without SCA (QCON) and male chimeric mice with SCA (QSCA) were generated by the irradiation of 8-week old C57BL6 mice followed by bone marrow cell transplantation from C57BL6 or transgenic Berkeley SCA mice, respectively. As circulating and tissue RAS are markedly affected by estrogens, we used only male mice for all protocols. Animals were subdivided into young adults (5 months of age) and adults (8 - 9 months of age). After confirming the phenotype of the transplanted SCA mice, BP was measured by a noninvasive method (NBP - AdInstruments) and animals were sacrificed at the age of 5/9 months for biological material collection. Plasma AngII and Angiotensin Convertor Enzyme (ACE) were quantified by ELISA. For histological analysis of the kidneys, organ specimens were fixed and embedded in historesin, sectioned and stained with hematoxylin and eosin. Renal AngI/II was quantitated by Western Blot from snap-frozen kidneys. The mean BPs of QSCA at 5 and 8 months old were significantly lower (87.9 ± 1.9 mmHg, n=12; and 94.31 ± 4.4 mmHg, n=2; respectively) than those of QCON of the same age (108.9 ± 5 mmHg, n=10; and 110.6 ± 2.2 mmHg, n=9; respectively), indicating that this model mimics the pathological changes in the blood flow that occur in SCA patients. Given the close relationship between AngII and PB regulation, we investigated whether the RAS is altered in QSCA mice at these ages. Both young adult and adult male QSCA mice presented lower plasma AngII concentrations (young adult, 24.18 ± 3 pg/ml, n= 11; adult, 16.44 ± 2 pg/ml, n=4), compared to QCON animals (young adult, 38.77 ±9, n= 10; adult, 35.52 ± 5.3 pg/ml, n=10, P<0.05, compared to QSCA); however no significant correlation between reduced BP and reduced plasma AngII was observed. AngII is produced by the cleavage of AngI by ACE; plasma ACE concentrations were also reduced in young adult QSCA mice (12.5 ± 1.8 pg/ml, n=11), compared to young adult QCON mice (21.9 ± 4.9 pg/ml, n=9). Moreover, reduced ACE concentrations in young adult/adult QSCA correlated with plasma levels of AngII (rs=0.785, P<0.001, n=15). Findings imply that AngII generation could be compromised in the organs of SCA mice; indeed, intra-renal AngI/II protein expression analysis showed that adult QSCA mice presented a significantly reduced expression of renal AngI/II (0.71 ± 0.2 AU, normalized to β-actin) compared to adult QCON mice (1.6 ± 0.2 AU), suggesting a low production of this hormone at advanced age in SCA. Glomerular hypertrophy increases in cardiovascular and renal disease models, including in hypertension and SCA. Quantitative morphometric analyses showed expansion of the glomerular-capillary tuff area of QSCA mice, which increased with age (young-adult: 372.2 ± 12.8 µm2; adult: 412.9 ±12.1 µm2, n=3), compared to young-adult and adult QCON mice (324.1 ± 10.8 µm2; 332.8 ± 12.4 µm2, n=3, p<0.001). As such, glomerular hypertrophy may reflect hyperfiltration and renal damage in the SCA mice, which apparently augments with age. Subsequent alterations in glomerular pressure may incur adaptive responses in the RAS, in turn diminishing ACE and AngII production, in association with alterations in systemic blood pressure. An understanding of the RAS in SCA may improve our understanding of renal damage and its systemic consequences in SCA and will be important for determining how ACE inhibitor and AngII receptor blockers drugs may be best utilized in these patients. DisclosuresNo relevant conflicts of interest to declare.

Biobank linked to SWEDEHEART quality registry—routine blood sample collection opens new opportunities for cardiovascular research

High-quality biobanking within routine health services, through the use of existing health-care practices and infrastructure, with respect to safety and integrity of patients in line with the Swedish Biobank Act, enables large-scale collection of biological material at reasonable costs. Complementing the extensive information on myocardial infarction patients from a national registry gives unique opportunities for research focusing on better understanding of cardiovascular disease occurrence and prognosis, developing of new diagnostic methods, and personalized treatments with greater efficacy and fewer side effects.

Biological material collection to advance translational research and treatment of children with CNS tumours: position paper from the SIOPE Brain Tumour Group

Paediatric CNS tumours are the most common cause of childhood cancer-related morbidity and mortality, and improvements in their diagnosis and treatment are needed. New genetic and epigenetic information about paediatric CNS tumours is transforming the field dramatically. For most paediatric CNS tumour entities, subgroups with distinct biological characteristics have been identified, and these characteristics are increasingly used to facilitate accurate diagnoses and therapeutic recommendations. Future treatments will be further tailored to specific molecular subtypes of disease, specific tumour predisposition syndromes, and other biological criteria. Successful biomaterial collection is a key requirement for the application of contemporary methodologies for the validation of candidate prognostic factors, the discovery of new biomarkers, the establishment of appropriate preclinical research models for targeted agents, a quicker clinical implementation of precision medicine, and for other therapeutic uses (eg, for immunotherapies). However, deficits in organisational structures and interdisciplinary cooperation are impeding the collection of high-quality biomaterial from CNS tumours in most centres. Practical, legal, and ethical guidelines for consent, storage, material transfer, biobanking, data sharing, and funding should be established by research consortia and local institutions to allow optimal collection of primary and subsequent tumour tissue, body fluids, and normal tissue. Procedures for the collection and storage of biomaterials and related data should be implemented according to the individual and organisational structures of the local institutions.

High frequency of M. leprae DNA detection in asymptomatic household contacts

BackgroundCharacterization of the Mycobacterium leprae genome has made possible the development of Polymerase Chain Reaction (PCR) systems that can amplify different genomic regions. Increased reliability and technical efficiency of quantitative PCR (qPCR) makes it a promising tool for early diagnosis of leprosy. Index cases that are multibacillary spread the bacillus silently, even before they are clinically diagnosed. Early detection and treatment could prevent transmission in endemic areas.MethodsIn this study, the qPCR technique is used to detect DNA of M. leprae in samples of slit skin smears (SSS) of the ear lobe and blood of leprosy patients and their asymptomatic household contacts residing in Governador Valadares, MG, Brazil, a hyperendemic area for leprosy. A total of 164 subjects participated in the study: 43 index cases, 113 household contacts, and, as negative controls, 8 individuals who reported no contact with patients nor history of leprosy in the family. The qPCR was performed to amplify 16S rRNA fragments and was specifically designed for M. leprae.ResultsOf asymptomatic household contacts, 23.89% showed bacillary DNA by qPCR in samples of SSS and blood. Also, 48.84% of patients diagnosed with leprosy were positive for qPCR while the bacillary load was positive in only 30.23% of patients. It is important to note that most patients were already receiving treatment when the collection of biological material for qPCR was performed. The level of bacillary DNA from household contacts was similar to the DNA levels detected in the group of paucibacillary patients.ConclusionConsidering that household contacts comprise a recognizable group of individuals with a high risk of disease, as they live in close proximity to a source of infection, qPCR can be used to estimate the risk of progress towards leprosy among household contacts and as a routine screening method for a chemoprophylactic protocol.

Frecuencia de errores preanalíticos ocurridos en la Medicina Veterinaria

The laboratory tests are extremely important for a definitive diagnosis, they assist in 70% in medical decision making. As in any circumstance, there are likelihood of errors, which are often occasioned by negligence or lack of knowledge of the subject. The present study aimed to demonstrate the main pre-analytical errors in veterinary medicine. The main errors reported were related to the non-use of procedure gloves, followed by failure to perform the tricotomy at the blood collection site. It was possible to verify that the veterinarians know about the best tube to be used in the clinical routine of harvesting and sending blood to the laboratory, however, there are still gaps in the club time. Pre-analytical errors are known as a methodology for a collection of biological material.

Quality-Assured Biobanking: The Leiden University Medical Center Model.

Prospective or "de novo" biobanking is becoming increasingly popular. Biobanks are installed to provide large collections of biological materials for future medical research. Quality assurance of biobank samples is an important aspect of biobanking. Therefore, it is vital that all samples are collected and processed in a similar manner according to standardized procedures to ensure high-quality samples and reduce variability in the analytical process. We describe the processes of the centralized biobanking facility at the Leiden University Medical Center (LUMC).

An approach to the floristics and faunistics of the intertidal zone in El Pulpo rocky shore, Cazones, Veracruz, Mexico.

The rocky intertidal of Veracruz, Mexico is a coastal ecosystem with high biodiversity. However,its relevance has not been recognized compared with other marine ecosystems. The objective of this study was todevelop the first floristic and faunistic lists of the rocky intertidal of El Pulpo beach, located in the municipalityof Cazones, Veracruz. Samples were taken from 2013 to 2016 in the rocky intertidal strip in periods of low tidein order to record the marine biota and the collection of biological material for their taxonomic identification. Inaddition, a bibliographic review was performed to complement the species inventory. Fifty-one species of benthicmacroalgae, one species of phanerogam, 186 species of invertebrates, and five species of vertebrates were recorded totaling 243 intertidal marine species. The Rhodophyta are the most representative of the phycoflora with 29species, while the phylum Mollusca are the most representative of the fauna with 139 species. Most of the marinespecies were recorded in the middle and lower intertidal. Consequently, the lowest richness was recorded in theupper intertidal which consists of filamentous and foliose algae, mollusks and crustaceans with both browsing andfilter habits. This distribution coincides with those described for other rocky intertidal environments. The numberof species recorded in the rocky intertidal of El Pulpo beach demonstrates the relevance of this ecosystem. It isthus necessary to encourage actions for its protection and conservation.

Bloody infrastructures!: Exploring challenges in cord blood collection maintenance

ABSTRACTThe collection of umbilical cord blood, a source of stem cells for cancer treatment, has become a highly strategised process. STS scholarship has explored the moral/economic tensions of this case but focuses less on questions of infrastructure. This paper aims to flesh out our understanding of how stem cell collections maintain usefulness whilst clinical requirements change. It borrows from literature on studying ‘infrastructure’ to analyse qualitative data on the UK context, exploring how it might help to think of these collections not simply as banks, but as infrastructures. It attends to how maintenance relies on alertness to the shifting standards of ‘users’, and demonstrates that infrastructural thinking offers the heuristic richness needed to explore these important aspects of maintaining collections of biological material and sustaining them into the future. It thus provides a contribution to the STS literature on tissue banking and the growing interdisciplinary corpus on issues of infrastructure.

Features of organization of medico-legal expert researches in the cases of the mass injuring of victims in the salon of bus

Features of organization of medico-legal expert researches in the cases of the mass injuring of victims in the salon of bus

Bayesian estimation of sensitivity and specificity of a milk pregnancy-associated glycoprotein-based ELISA and of transrectal ultrasonographic exam for diagnosis of pregnancy at 28–45 days following breeding in dairy cows

Using a milk sample for pregnancy diagnosis in dairy cattle is extremely convenient due to the low technical inputs required for collection of biological materials. Determining accuracy of a novel pregnancy diagnostic test that relies on a milk sample is, however, difficult since no gold standard test is available for comparison. The objective of the current study was to estimate diagnostic accuracy of the milk PAG-based ELISA and of transrectal ultrasonographic (TUS) exam for determining pregnancy status of individual dairy cows using a methodology suited for test validation in the absence of gold standard. Secondary objectives were to evaluate whether test accuracy varies with cow’s characteristics and to identify the optimal ELISA optical density threshold for PAG test interpretation. Cows (n=519) from 18 commercial dairies tested with both TUS and PAG between 28 and 45days following breeding were included in the study. Other covariates (number of days since breeding, parity, and daily milk production) hypothesized to affect TUS or PAG test accuracy were measured. A Bayesian hierarchical latent class model (LCM) methodology assuming conditional independence between tests was used to obtain estimates of tests’ sensitivities (Se) and specificities (Sp), to evaluate impact of covariates on these, and to compute misclassification costs across a range of ELISA thresholds. Very little disagreement was observed between tests with only 23 cows yielding discordant results. Using the LCM model with non-informative priors for tests accuracy parameters, median (95% credibility intervals [CI]) TUS Se and Sp estimates of 0.96 (0.91, 1.00) and 0.99 (0.97, 1.0) were obtained. For the PAG test, median (95% CI) Se of 0.99 (0.98, 1.00) and Sp of 0.95 (0.89, 1.0) were observed. The impact of adjusting for conditional dependence between tests was negligible. Test accuracy of the PAG test varied slightly by parity number. When assuming false negative to false positive costs ratio≥3:1, the optimal ELISA optical density threshold allowing minimization of misclassification costs was 0.25. In conclusion, both TUS and PAG showed excellent accuracy for pregnancy diagnosis in dairy cows. When using the PAG test, a threshold of 0.25 could be used for test interpretation.

Standardization and validation of Dot-ELISA assay for Paracoccidioides brasiliensis antibody detection

BackgroundParacoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI).MethodsIn order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI.ResultsThe DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization.ConclusionDot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life – membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories – and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.

Maintenance of a Drosophila melanogaster Population Cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.