Advanced Laboratory Methods Research Articles

Оценка некоторых закономерностей процессов перекисного окисления липидов при увеитах у детей

Original article Assessment of some patterns of lipid peroxidation processes in uveitis in children O.S. Komarov, M.R. Guseva, S.A. Obrubov, N.M. Galkina Pirogov Russian National Research Medical University, Moscow, Russian Federation Uveitis is characterized by a variety of clinical forms, chronic and recurrent course, severe outcomes, and not always effective therapy. In addition, etiological diagnosis and treatment of patients require more subtle and advanced laboratory methods and deciphering the incompletely studied mechanisms of the pathogenesis of the disease. The work, based on significant clinical material, shows that the activation of polymorphonuclear leukocytes (PMN) in the blood is one of the main reasons for the increased processes of lipid peroxidation (LPO) in uveitis. The activity levels of PMN and LPO clearly correlate and reflect the degree of depth and dynamics of the inflammatory process in uveitis. These indicators more subtly characterize the course of the disease and the dynamics of recovery compared to traditional generally accepted criteria for assessing inflammation. Simultaneous study of the intensity of PMN and LPO on the one hand and assessment of the antioxidant activity (AOA) of the system makes it possible to predict the course of the disease, prescribe adequate therapy and judge the severity and completeness of the process. It has been proven that one of the main blood antioxidant proteins, ceruloplasmin, changes parallel to the clinical picture of uveitis, which can be used as a basis for prescribing antioxidant therapy or for assessing the effectiveness of treatment. Key words: uveitis, lipid peroxidation, antioxidants, ceruloplasmin, polymorphonuclear leukocytes

Breakthrough Measles among Vaccinated Adults Born during the Post-Soviet Transition Period in Mongolia.

Mongolia experienced a nationwide measles outbreak during 1 March 2015-31 December 2016, with 49,077 cases reported to the WHO; many were among vaccinated young adults, suggesting a possible role of vaccine failure. Advanced laboratory methods, coupled with detailed epidemiological investigations, can help classify cases as vaccine failure, failure to vaccinate, or both. In this report, we conducted a study of cases to identify risk factors for breakthrough infection for a subset of laboratory-confirmed measles cases. Of the 193 cases analyzed, only 19 (9.8%) reported measles vaccination history, and 170 (88%) were uncertain. Measles-specific IgG avidity testing classified 120 (62%) cases as low IgG avidity, indicating no prior exposure to measles. Ten of these cases with low IgG avidity had a history of measles vaccination, indicating primary vaccine failure. Overall, sixty cases (31%) had high IgG avidity, indicating breakthrough infection after prior exposure to measles antigen through vaccination or natural infection, but the IgG avidity results were highly age-dependent. This study found that among young children aged 9 months-5 years, breakthrough infection was rare (4/82, 5%); however, among young adults aged 15-25 years, breakthrough infection due to secondary vaccine failure (SVF) occurred on a large scale during this outbreak, accounting for the majority of cases (42/69 cases, 61%). The study found that large-scale secondary vaccine failure occurred in Mongolia, which highlights the potential for sustained outbreaks in post-elimination settings due to "hidden" cohorts of young adults who may have experienced waning immunity. This phenomenon may have implications for the sustainability of measles elimination in countries that remain vulnerable to the importation of the virus from areas where it is still endemic. Until global measles elimination is achieved, enhanced surveillance and preparedness for future outbreaks in post- or peri-elimination countries may be required.

Shigella and childhood stunting: Evidence, gaps, and future research directions.

Early childhood growth deficits have been shown to have lifelong health and economic impacts, yet their connection to one of their underlying causes, diarrheal diseases, has remained difficult to characterize. Identifying the processes and mechanisms that underlie this link has remained a challenge due to the complexity of the relationship and limitations in access to more advanced laboratory methods. In recent years, however, several large-scale, multisite studies have extensively investigated and reported the prevalence, etiology, and impacts of diarrheal diseases in children under 5 years (CU5) in low- to middle-income countries (LMICs). These studies, in combination with several single-site studies, have applied more advanced laboratory methods to uncover the etiology, true prevalence, infection mechanisms, and inflammation biomarkers of diarrheal disease. Of the multiple pathogens that have been shown to be strongly associated with diarrheal disease in CU5, Shigella is one of the more prevalent and impactful of these pathogens. In this narrative review, we highlight key insights from these studies and identify knowledge gaps and directions for future research. According to these studies, Shigella is most commonly detected in toddlers and young children; however, it can cause more severe disease and has a greater impact on linear growth for infants. Shigella often has a stronger relationship to linear growth faltering (LGF) than other enteropathogens, with higher Shigella loads resulting in greater growth deficits. Future studies should employ more Shigella-specific molecular assays and identify diarrheal etiologies using standardized diagnostics to improve child anthropometric and Shigella surveillance. Also, they should focus on uncovering the mechanisms of the relationship underlying Shigella and growth faltering to better characterize the role of asymptomatic infections and intestinal inflammation in this relationship.

A Review on the Cytotoxicity and Antibacterial Effect of Marine Organisms of Persian Gulf.

Marine organisms contain several natural products and bioactive compounds, including hydrolyzed proteins, antioxidant peptides, gelatin, collagen, ω-3 unsaturated fatty acids, vitamin A, vitamin D, calcium phosphate, hydroxyapatite, chitosan, lectin, and various toxins. They can inhibit diverse diseases, be used in pharmaceutical compounds, or as antibiotics and pigments. In this regard, these microorganisms are of crucial medicinal and economical importance. Thanks to new technologies and advanced laboratory methods, bioactive compounds can be extracted from aquatic organisms. In this review study, the cytotoxicity (IC50) and antibacterial effect of various extracts from marine organisms of the Persian Gulf are explored, compiled, and compared. Due to their easy accessibility, most of the studies are green, red, and brown algae.

Zebrafish Embryos and Larvae as Alternative Animal Models for Toxicity Testing.

Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.

Advanced Laboratory Methods for Detecting Yellow Fever Pathogen

Yellow fever is an acute infectious disease of viral nature, the causative agent of which is vector-borne –is transmitted through the bites of infected mosquitoes. Massive epidemics caused by the yellow fever virus are observed in the countries of Africa, South and Central America annually. Imported cases are also registered in non-endemic territories. The review presents the currently available data on the distribution, structure and classification of the yellow fever virus, the identification of its genetic variants depending on the geographical distribution, as well as modern methods of detection and identification of the pathogen in samples taken from sick and dead people. It considers the possibility of using virological, immunoserological and molecular-genetic methods for the diagnosis of yellow fever in different periods from the onset of the disease and in retrospective studies. The lists of diagnostic drugs of domestic and foreign production for the detection of agent markers (antigen, RNA), as well as specific antibodies of IgM and IgG classes, approved for use on the territory of the Russian Federation, are provided. The relevance of further development, improvement and introduction into laboratory practice of reagent kits that allow to detect the yellow fever virus in samples from sick people in a short time, with high efficiency and specificity is demonstrated. This will help to establish a diagnosis promptly and conduct timely anti-epidemic measures, as well as to determine the level of the population stratum immune to the pathogen in endemic regions and evaluate the effectiveness of immunization for the vaccinated contingent.

Coagulopathy associated with Hepato-renal disorders and its significance in critically ill patients

Coagulopathy has a high prevalence among critically ill patients and is the result of the derangement of both procoagulant and anticoagulant components of the coagulation system. Liver and renal function tests are some of the most commonly performed blood tests which assess the liver and kidney injury and these are one of the commonest causes of deranged coagulation parameters in these patients. This prospective observational study was conducted for two years in Department of Pathology in a tertiary care hospital in university medical college in western India. 219 cases with underlying liver and renal disorders were included in this study. Complete blood counts and coagulation studies including Prothrombin time (PT), Activated Partial Thromboplastin time (APTT), Fibrinogen were done. Statistical analysis was done to evaluate the correlation between various parameters. In our study, hepatic encephalopathy was the commonest cause followed by alcoholic liver disease and liver cirrhosis. Chronic kidney disease was common cause in patients with renal disorders. Deranged Bilirubin levels had a significant statistical association with PT while APTT had a significant association with blood urea levels. Advanced diagnostic and laboratory methods, early recognition of the signs and symptoms of coagulopathy and the complicating factors in liver and renal disorders in these patients is possible today. Liver and renal disorders are one of the important underlying causes for development of coagulopathies in criticially ill patients. Prompt and correct identification of these disorders and associated coagulopathy is important for proper management and improving outcome in these patients.

Resolving Differential Diagnostic Problems in von Willebrand Disease, in Fibrinogen Disorders, in Prekallikrein Deficiency and in Hereditary Hemorrhagic Telangiectasia by Next-Generation Sequencing

Diagnosis of rare bleeding disorders is challenging and there are several differential diagnostics issues. Next-generation sequencing (NGS) is a useful tool to overcome these problems. The aim of this study was to demonstrate the usefulness of molecular genetic investigations by summarizing the diagnostic work on cases with certain bleeding disorders. Here we report only those, in whom NGS was indicated due to uncertainty of diagnosis or if genetic confirmation of initial diagnosis was required. Based on clinical and/or laboratory suspicion of von Willebrand disease (vWD, n = 63), hypo-or dysfibrinogenemia (n = 27), hereditary hemorrhagic telangiectasia (HHT, n = 10) and unexplained activated partial thromboplastin time (APTT) prolongation (n = 1), NGS using Illumina platform was performed. Gene panel covered 14 genes (ACVRL1, ENG, MADH4, GDF2, RASA1, F5, F8, FGA, FGB, FGG, KLKB1, ADAMTS13, GP1BA and VWF) selected on the basis of laboratory results. We identified forty-seven mutations, n = 29 (6 novel) in vWD, n = 4 mutations leading to hemophilia A, n = 10 (2 novel) in fibrinogen disorders, n = 2 novel mutations in HHT phenotype and two mutations (1 novel) leading to prekallikrein deficiency. By reporting well-characterized cases using standardized, advanced laboratory methods we add new pieces of data to the continuously developing “bleeding disorders databases”, which are excellent supports for clinical patient management.

Extrapolating sparse gold standard cause of death designations to characterize broader catchment areas

Abstract Objectives The Child Health and Mortality Prevention Surveillance (CHAMPS) Network is designed to elucidate and track causes of under-5 child mortality and stillbirth in multiple sites in sub-Saharan Africa and South Asia using advanced surveillance, laboratory and pathology methods. Expert panels provide an arguable gold standard determination of underlying cause of death (CoD) on a subset of child deaths, in part through examining tissue obtained via minimally invasive tissue sampling (MITS) procedures. We consider estimating a population-level distribution of CoDs based on this sparse but precise data, in conjunction with data on subgrouping characteristics that are measured on the broader population of cases and are potentially associated with selection for MITS and with cause-specific mortality. Methods We illustrate how estimation of each underlying CoD proportion using all available data can be addressed equivalently in terms of a Horvitz-Thompson adjustment or a direct standardization, uncovering insights relevant to the designation of appropriate subgroups to adjust for non-representative sampling. Taking advantage of the functional form of the result when expressed as a multinomial distribution-based maximum likelihood estimator, we propose small-sample adjustments to Bayesian credible intervals based on Jeffreys or related weakly informative Dirichlet prior distributions. Results Our analyses of early data from CHAMPS sites in Kenya and Mozambique and accompanying simulation studies demonstrate the validity of the adjustment approach under attendant assumptions, together with marked performance improvements associated with the proposed adjusted Bayesian credible intervals. Conclusions Adjustment for non-representative sampling of those validated via gold standard diagnostic methods is a critical endeavor for epidemiologic studies like CHAMPS that seek extrapolation of CoD proportion estimates.

Splanchnic vein closures

Closures in the splanchnic venous system (SVS) represent abroad medical problem. Anatomically, individual or even multiple sections of SVS may be affected at the same time. Main sections of SVS include the venous liver outflow system, the portal vein, and the upper mesenteric vein and its basin. Thrombosis is clearly the predominant cause of closure. The closures can present as acute, subacute, chronic occult or chronic manifest. The main pathological and anatomical units are the Budd-Chiari syndrome (BCS), extrahepatic portal vein obstruction (EHPVO) and mesenteric vein thrombosis (MVT). Advanced laboratory, imaging and intervention methods substantially modify the approach to prevention, diagnosis and treatment; surgical approach also plays arole. The problem of SVS closures is interdisciplinary.

Outbreak of Shiga toxin-producing Escherichia coli O157:H7 linked to raw drinking milk resolved by rapid application of advanced pathogen characterisation methods, England, August to October 2017.

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 occurred on the Isle of Wight between August and October 2017. Of the seven cases linked to the outbreak, five were identified through the statutory notification process and two were identified through national surveillance of whole genome sequencing data. Enhanced surveillance questionnaires established a common link to a farm, and link to the likely food vehicle, raw drinking milk (RDM). Microbiological investigations, including PCR, identified the presence of STEC O157:H7 in samples of RDM. Analysis of core genome single nucleotide polymorphism (SNP) data of STEC O157:H7 from human stool specimens, animal faecal samples and RDM demonstrated a one SNP difference between isolates, and therefore close genetic relatedness. Control measures that were put in place included suspension of sales and recall of RDM, as well as restrictions on public access to parts of the farm. Successful integration of traditional epidemiological surveillance and advanced laboratory methods for the detection and characterisation of STEC O157:H7 from human, animal and environmental samples enabled prompt identification of the outbreak vehicle and provided evidence to support the outbreak control team’s decision-making, leading to implementation of effective control measures in a timely manner.

Pharmacological sedation management in the paediatric intensive care unit.

This review addresses sedation management on paediatric intensive care units and possible gaps in the knowledge of optimal sedation strategies. We present an overview of the commonly used sedatives and their pharmacokinetic and pharmacodynamic considerations in children, as well as the ongoing studies in this field. Also, sedation guidelines and current sedation strategies and assessment methods are addressed. This review shows that evidence and pharmacokinetic data are scarce, but fortunately, there is an active research scene with promising new PK and PD data of sedatives in children using new study designs with application of advanced laboratory methods and modelling. The lack of evidence is increasingly being recognized by authorities and legislative offices such as the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). The population in question is very heterogeneous and this overview can aid clinicians and researchers in moving from practice-based sedation management towards more evidence- or model-based practice. Still, paediatric sedation management can be improved in other ways than pharmacology only, so future research should aim on sedation assessment and implementation strategies of protocolized sedation as well.

Heavy metal removal mechanisms of sorptive filter materials for road runoff treatment and remobilization under de-icing salt applications

The objective of this research study was to elucidate the removal and remobilization behaviors of five heavy metals (i.e., Cd, Cu, Ni, Pb, and Zn) that had been fixed onto sorptive filter materials used in decentralized stormwater treatment systems receiving traffic area runoff. Six filter materials (i.e., granular activated carbon, a mixture of granular activated alumina and porous concrete, granular activated lignite, half-burnt dolomite, and two granular ferric hydroxides) were evaluated in column experiments. First, a simultaneous preloading with the heavy metals was performed for each filter material. Subsequently, the remobilization effect was tested by three de-icing salt experiments in duplicate using pure NaCl, a mixture of NaCl and CaCl2, and a mixture of NaCl and MgCl2. Three layers of each column were separated to specify the attenuation of heavy metals as a function of depth. Cu and Pb were retained best by most of the selected filter materials, and Cu was often released the least of all metals by the three de-icing salts. The mixture of NaCl and CaCl2 resulted in a stronger effect upon remobilization than the other two de-icing salts. For the material with the highest retention, the effect of the preloading level upon remobilization was measured. The removal mechanisms of all filter materials were determined by advanced laboratory methods. For example, the different intrusions of heavy metals into the particles were determined. Findings of this study can result in improved filter materials used in decentralized stormwater treatment systems.

Revisiting Mantoux test in the era of immunization and Multi-drug resistant tuberculosis

This review reinvents the role of mantoux test/tuberculin skin test (TST) in the laboratory diagnosis of tuberculosis (TB). TB has been the most significant infectious disease responsible for severe morbidity and mortality worldwide. Asia and African continents constitute most of the burden of world TB. Although a treatable infectious disease, the exposed individuals do not always show clinical signs of infection. TB may be present latent in many infected population. Clinical, radiological, cultural and staining methods, molecular techniques and many other advanced laboratory methods are available for the diagnosis of TB. Among the various laboratory techniques available for the diagnosis of TB, Mantoux TST is a low cost and easily done test that if performed and interpreted appropriately can be used for diagnosis of TB.

Skeletal dysplasias from an endocrine point of view

Skeletal dysplasias are complex group of diseases with different clinical and radiologic findings involving bone, and/or cartilage. Although skeletal dysplasias are rarely seen when considered individually, general incidence in the population is 1/3000-5000. Skeletal dysplasias are classified in 40 main groups on the basis of clinical, laboratory, radiological and molecular evaluations, and congenital malformations, spinal abnormalities, short extremities, and disproportionate short stature are seen in sekondereletal dysplasias. In addition to bone abnormalities, endocrine problems including hyperglycemia, hypo/hypercalcemia, adrenal failure, pubertal alterations, and disorders of sexual development which are mostly due to genetic disorders may accompany skeletal dysplasias. Differential diagnosis of skeletal dysplasias requires detailed history and physical examination, skeletal X-rays, genetic analysis, some advanced laboratory methods, and multidisciplinary aproach. In this review, it is aimed to present a general approach to skeletal dysplasias, endocrine abnormalities that may accompany, and the most recent classification published in 2010.

Revisiting Mantoux test in the era of immunization and Multi-drug resistant tuberculosis (MDR-TB)

This review reinvents the role of mantoux test/tuberculin skin test (TST) in the laboratory diagnosis of tuberculosis (TB). Tuberculosis has been the most significant infectious disease responsible for severe morbidity and mortality worldwide. Asia and African continents constitute most of the burden of world tuberculosis. Although a treatable infectious disease, the exposed individuals do not always show clinical signs of infection. TB may be present latent in many infected population. Clinical, radiological, cultural and staining methods, molecular techniques and many other advanced laboratory methods are available for the diagnosis of tuberculosis. Among the various laboratory techniques available for the diagnosis of tuberculosis, Mantoux tuberculin skin test is a low cost and easily done test that if performed and interpreted appropriately can be used for diagnosis of TB.

Utility Of 9-Color, 11-Parameter Flow Cytometry For Detection Of Plasma Cell Neoplasms: A Comparison With Bone Marrow Morphologic Findings and Concurrent M-Protein Studies In Serum and Urine

BackgroundMultiparameter flow cytometry (MFC) is a widely available laboratory platform used for primary diagnosis of mature B-cell and plasma cell (PC) neoplasms. Ongoing advances in myeloma therapeutics have drawn attention to the need for advanced laboratory methods for monitoring disease response and defining disease eradication. Recent literature indicates that achievement of remission based on sensitive MFC criteria is prognostically superior to remissions defined by older criteria based on monitoring of paraproteins in blood and urine. As such, the International Myeloma Working Group (IMWG) recommends MFC for minimal residual disease (MRD) testing during clinical trials for multiple myeloma (MM). This study validates a sensitive 9-color, 11-parameter, two-tube MFC assay suitable for both the initial diagnosis of patients with paraproteinemias and MRD monitoring in MM. MethodWe established and validated a 9-color, 11-parameter MFC assay in our laboratory. The 9-color tube contained cytoplasmic κ/cytoplasmic λ/CD45/CD38/CD56/CD138/CD19/CD117/CD20. Results of the MFC were compared to bone marrow microscopic examinations, immunohistochemical (IHC) studies and serum/urine M-protein measurements ordered on 363 samples from patients with documented or suspected PC neoplasms and IgM paraproteinemias. ResultsTotal preparation time is approximately 90 minutes (“hands-on” time under 30 minutes) for up to 3 concurrent specimens. The assay is performed in two tubes (control and PC panel) with analysis of up to 1.8 x 106 total bone marrow cells/tube. The mean instrument analysis time was ∼5 minutes/tube (range 1-9 minutes). MFC detected clonal PCs in 19% and 23% of cases in which IHC or morphologic evaluation, respectively, failed to show a clonal plasma cell population. The MFC assay consistently detected clonal PC in bone marrow aspirates when serum M-protein levels were above 1g/dl. The frequency of clonal PC detection by MFC fell in concert with M-protein levels. However, in 11% of patients, MFC detected clonal PC after serum and urine immunofixation studies turned negative. ConclusionWe conclude that the assay described herein is suitable when immunologic studies are indicated for either primary diagnosis or MRD detection in PC neoplasms. It equals or exceeds sensitivities reported in the literature and it is readily integrated into a high volume, multipurpose clinical flow cytometry laboratory. Disclosures:Keren:Sebia, Inc (Norcross, GA). : Consultancy.

Books and booklets in brief

In the history of plant pathology, few universities have been as influential as the University of Wisconsin –Madison. Published to mark the Centennial in 2010 of the great Department of Plant Pathology there, the present book is a relatively informal depiction of the first century of the institution’s life, captured through photographs and anecdotes, together with the personal reminiscences of faculty, staff and alumni. It should be read in conjunction with the companion volume, With One Foot in the Furrow (eds. P. H. Williams and M. Marosy, Kendall Hunt, Dubuque, Iowa, 1986), a formal, exhaustive analysis of the history of the Plant Pathology Department from its foundation in 1910 until its 75th anniversary in 1985. And One Hand on the Bench will, of course, be a source of endless fascination for all those who have worked at or been associated with the Plant Pathology Department at Madison. In addition, however, it is of very great significance to historians of science, since many of the great names of 20th century plant pathology worked or studied there including, from the 1960s onwards (the period most strongly represented in the book), many of the pioneers of physiological and molecular plant pathology. Finally, the titles of this book and of its earlier companion volume, taken together, convey an important message: that in research in plant pathology, although it is important to “keep one foot in the furrow”, as J. C. Walker emphasised to his student, Paul Williams, it is of equal importance to deploy the most advanced laboratory methods available, as John Andrews’ title now reminds us.

Counterfeit drugs: analytical techniques for their identification

In recent years, the number of counterfeit drugs has increased dramatically, including not only "lifestyle" products but also vital medicines. Besides the threat to public health, the financial and reputational damage to pharmaceutical companies is substantial. The lack of robust information on the prevalence of fake drugs is an obstacle in the fight against drug counterfeiting. It is generally accepted that approximately 10% of drugs worldwide could be counterfeit, but it is also well known that this number covers very different situations depending on the country, the places where the drugs are purchased, and the definition of what constitutes a counterfeit drug. The chemical analysis of drugs suspected to be fake is a crucial step as counterfeiters are becoming increasingly sophisticated, rendering visual inspection insufficient to distinguish the genuine products from the counterfeit ones. This article critically reviews the recent analytical methods employed to control the quality of drug formulations, using as an example artemisinin derivatives, medicines particularly targeted by counterfeiters. Indeed, a broad panel of techniques have been reported for their analysis, ranging from simple and cheap in-field ones (colorimetry and thin-layer chromatography) to more advanced laboratory methods (mass spectrometry, nuclear magnetic resonance, and vibrational spectroscopies) through chromatographic methods, which remain the most widely used. The conclusion section of the article highlights the questions to be posed before selecting the most appropriate analytical approach.

Reducing remediation costs, schedule, and environmental liability at TVA

AbstractTwo RCRA solid waste management unit/underground storage tank remediations were scheduled at the Tennessee Valley Authority (TVA) Environmental Research Center, Muscle Shoals, Alabama. By negotiating health risk‐based alternative cleanup levels (ACLs), using advanced laboratory methods, and employing bioremediation technology, ENSR provided a solution that satisfied the state agency and saved TVA time and money. Eliminating the need for transportation and off‐site disposal, TVA's environmental liabilities were also mitigated. This article presents a “how‐to” case study for effectively managing remediation projects and costs.