Clinical metagenomics.

Clinical metagenomic next-generation sequencing (mNGS), the comprehensive analysis of microbial and host genetic material (DNA and RNA) in samples from patients, is rapidly moving from research to clinical laboratories. This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology. Here, we focus on the challenges of implementing mNGS in the clinical laboratory and address potential solutions for maximizing its impact on patient care and public health.

Laboratory investigations.

This chapter deals with chemical and hematologic investigations which are often considered in the diagnostic workup of subacute to chronic cerebellar ataxias. Relevant investigations in blood (serum, plasma), urine, and cerebrospinal fluid are discussed. Particular attention is paid to early diagnosis of treatable metabolic ataxias (such as abetalipoproteinemia, coenzyme Q10 deficiency, cerebrotendinous xanthomatosis, glucose transporter type 1 deficiency, Refsum disease, and vitamin E deficiency), but autoimmune ataxias, other vitamin deficiencies, and endocrine disorders should also be kept in mind. Adequate interpretation of test results has to consider age-specific reference values. The selection of investigations should mainly be driven by the overall clinical context, considering gender, history, age, and mode of presentation, cerebellar and other neurologic as well as extraneurologic findings.

Discrepancies in central review re-testing of patients with ER-positive and HER2-negative breast cancer in the OPTIMA prelim randomised clinical trial.

BACKGROUND: There is limited data on results of central re-testing of samples from patients with invasive breast cancer categorised in their local hospital laboratories as oestrogen receptor (ER) positive and human epidermal growth factor receptor homologue 2 (HER2) negative. METHODS: The Optimal Personalised Treatment of early breast cancer usIng Multiparameter Analysis preliminary study (OPTIMA prelim) was the feasibility phase of a randomised controlled trial to validate the use of multiparameter assay-directed chemotherapy decisions in the UK National Health Service (NHS). Eligibility criteria included ER positivity and HER2 negativity. Central re-testing of receptor status was mandatory. RESULTS: Of the 431 patients tested centrally, discrepant results between central and local laboratory results were identified in only 19 (4.4%; 95% confidence interval 2.5-6.3%) patients (with 21 tumours). On central review, seven patients had cancers that were ER-negative (1.6%) and 13 (3.0%) patients with 15 tumours had HER2-positive disease, including one tumour discrepant for both biomarkers. CONCLUSIONS: Central re-testing of receptor status of invasive breast cancers in the UK NHS setting shows a high level of reproducibility in categorising tumours as ER-positive and HER2-negative, and raises questions regarding the cost effectiveness and clinical value of central re-testing in this sub-group of breast cancers in this setting.

Plasma-Treated Microplates with Enhanced Protein Recoveries and Minimized Extractables.

SiO2 Medical Products, Inc. (SiO) has developed a proprietary technology that greatly enhances protein recoveries and reduces extractables from commercial microplates used for bioanalytical assays and storage of biologics. SiO technology is based on plasma treatment that chemically modifies the surface of polypropylene with predominantly hydrogen-bond-acceptor uncharged polar groups. The resultant surface resists nonspecific protein adsorption over a wide range of protein concentrations, thereby eliminating the need to passivate (and hence potentially contaminate) the microplates with blocking proteins. High shelf-life stability and cleanliness of the plasma-treated microplates have been demonstrated using five different proteins for two common microplate formats. The protein recovery performance of plasma-treated microplates is found to be higher compared with commercial low-protein-binding microplates.

Implementing Non-Invasive Prenatal Diagnosis (NIPD) in a National Health Service Laboratory; From Dominant to Recessive Disorders.

Our UK National Health Service regional genetics laboratory offers NIPD for autosomal dominant and de novo conditions (achondroplasia, thanataphoric dysplasia, Apert syndrome), paternal mutation exclusion for cystic fibrosis and a range of bespoke tests. NIPD avoids the risks associated with invasive testing, making prenatal diagnosis more accessible to families at high genetic risk. However, the challenge remains in offering definitive diagnosis for autosomal recessive diseases, which is complicated by the predominance of the maternal mutant allele in the cell-free DNA sample and thus requires a variety of different approaches. Validation and diagnostic implementation for NIPD of congenital adrenal hyperplasia (CAH) is further complicated by presence of a pseudogene that requires a different approach. We have used an assay targeting approximately 6700 heterozygous SNPs around the CAH gene (CYP21A2) to construct the high-risk parental haplotypes and tested this approach in five cases, showing that inheritance of the parental alleles can be correctly identified using NIPD. We are evaluating various measures of the fetal fraction to help determine inheritance of parental mutations. We are currently exploring the utility of an NIPD multi-disorder panel for autosomal recessive disease, to make testing more widely applicable to families with a variety of serious genetic conditions.

Clinical applications of MS-based protein quantification.

Mass spectrometry-based assays are increasingly important in clinical laboratory medicine and nowadays are already commonly used in several areas of routine diagnostics. These include therapeutic drug monitoring, toxicology, endocrinology, pediatrics, and microbiology. Accordingly, some of the most common analyses are therapeutic drug monitoring of immunosuppressants, vitamin D, steroids, newborn screening, and bacterial identification. However, MS-based quantification of peptides and proteins for routine diagnostic use is rather rare up to now despite excellent analytical specificity and good sensitivity. Here, we want to give an overview over current fit-for-purpose assays for MS-based protein quantification. Advantages as well as challenges of this approach will be discussed with focus on feasibility for routine diagnostic use.

[Liquid Biopsy: Challenges and Possibilities Regarding Its Clinical Application.]

Cell-free DNA is "Fragmented DNA found in circulation in the Cell-free component of whole blood". Cell-free DNA derived from tumors is expressed as circulating tumor DNA. Examination of circulating tu- mor DNA for genetic alterations present in the tumor tissue is defined as liquid biopsy. Currently in the cancer field, Cell-free DNA or CTC (Circulating tumor cells) is the main target of "Liquid Biopsy". Acquir- ing Cell-free DNA or CTC presents little challenge because of the recent technological developments. However, we need to improve the efficiency of CTC retrieval, and we also need to establish how to culture the retrieved CTCs. For clinical applications, PGx (Pharmacogenomics) and PGt (Pharmacogenetics) fol- lowing NGS (Next Generation Sequencing) are attractive areas for new and future applications. The intro- duction of "Liquid Biopsy" to the area of clinical trials is already in progress. As an expert group, members of the JSLM (Japanese Society of Laboratory Medicine) need to indicate the presence of quality control or quality management in the area of "Liquid Biopsy". Besides these quality issues, we, as clinical pathologists, need to think about harmonizing our expertise with surgical pathologists, who have historically handled clas- sical biopsies of solid samples. The field of "Liquid Biopsy" has marked potential; however, we need to overcome various obstacles to realize this.

Erros pré-analíticos em medicina laboratorial: uma avaliação preliminar em diferentes laboratórios de análises clínicas / Pre-analytical erros in laboratory medicine: a preliminary evaluation in different clinical laboratories

Objetivo: Este trabalho teve como objetivo avaliar os conhecimentos sobre erros pré-analíticos em medicina laboratorial de profissionais que atuam nesta área. Métodos: O presente estudo foi realizado por meio de um questionário para avaliar a ocorrência de erros laboratoriais pré-analíticos em dez laboratórios de análises clínicas privados e de pequeno porte nas zonas norte, sul e oeste da cidade do Rio de Janeiro. Resultados: A média de idade dos entrevistados foi de 35-55 anos, que trabalham como flebotomistas, técnicos de laboratório e analistas com formação superior. Duas das nove perguntas doquestionário de erros pré-analíticos apresentaram um elevado índice de respostas incorretas, a primeira relacionada ao tempo máximo de aplicação do torniquete/garrote ea segunda sobre as fases da etapa pré-analítica, ambas com 74% de respostas incorretas. A pergunta que apresentou um percentual de 100% de acertos estava relacionada à hemólise como causa de erros pré-analíticos. Conclusão: A melhoria da fase pré-analítica permanece um desafio para muitos laboratórios clínicos, visto quetodos os laboratórios avaliados apresentaram elevada frequência de procedimentos incorretos. É provável que este fato ocorra pela dificuldade dos laboratórios em relatar e registrar os erros provenientes da fase pré-analítica, bem como pelo despreparo dos profissionais envolvidos. Portanto, é de extrema importância a implantação de um programa para capacitação dos profissionais, a fim de minimizar a ocorrência de tais erros.

[The laboratory medicine as a foundation for personalized medicine. The application of biochips in medicine].

The development and application of biochips provide possibility to drastically transform laboratory medicine and to implement studies of arrays of biomarkers realizing approaches and concepts of personalized medicine. The main areas of application of microbiochips in laboratory medicine such as laboratory diagnostic, classification and prognosis of course of diseases, analysis of mechanisms of biologic processes are considered. The identification of inherent mutations in human genome is considered as perspective direction. These mutations cause various pathology and first of all oncologic diseases responsible for biotransformation of pharmaceuticals applied in chemotherapy of tumors in particular and also simultaneous identification of various infection agents (viruses, microorganisms, fungi, etc.) and their antibiotic-resistant forms. The role of biochips as a tool is demonstrated in genetic studies, technologies of genetic typing, large-scale international projects of studies of genome-wide screening of associations (GWAS).

[Circulating tumor cells: a new challenge for laboratory medicine]. / Les cellules tumorales circulantes : un nouveau challenge pour la biologie clinique oncologique.

Circulating tumor cells (CTCs) can be detected in the blood of patients with nearly all types of locally and metastatic adenocarcinomas. CTCs are epithelial cells whose release from a primitive tumor or a metastatic localization may be mediated by an epithelial-mesenchymal transition. Their pro-metastatic potential is still under debate because their phenotypes may be very heterogeneous, even within the same patient (expression of stem-cells markers, apoptotic status...). They often exhibit discrepancies with the primitive tumor, especially concerning the molecular basis of sensitivity/resistance to targeted therapies (expression of HER2 and hormone receptors, mutations responsible for resistance to tyrosine-kinase inhibitors). Many methods for CTCs analysis are commercially available but very few are evaluated and standardized enough for clinical applications. The CellSearch device is the only one which is validated by the FDA for managing metastatic breast, prostate and colo-rectal cancer. It was used in most of the studies having demonstrated the prognostic and predictive value of CTCs in many tumoral localizations. Other studies are wanted to assess the ability of CTCs to optimize the therapeutic choices, to monitor drug efficiency in real-time as well as to become a surrogate end-point for evaluation of new therapies. Beyond CTCs enumeration, their biological features will need to be investigated.

The role of metabolomics in neonatal and pediatric laboratory medicine.

Metabolomics consists of the quantitative analysis of a large number of low molecular mass metabolites involving substrates or products in metabolic pathways existing in all living systems. The analysis of the metabolic profile detectable in a human biological fluid allows to instantly identify changes in the composition of endogenous and exogenous metabolites caused by the interaction between specific physiopathological states, gene expression, and environment. In pediatrics and neonatology, metabolomics offers new encouraging perspectives for the improvement of critically ill patient outcome, for the early recognition of metabolic profiles associated with the development of diseases in the adult life, and for delivery of individualized medicine. In this view, nutrimetabolomics, based on the recognition of specific cluster of metabolites associated with nutrition and pharmacometabolomics, based on the capacity to personalize drug therapy by analyzing metabolic modifications due to therapeutic treatment may open new frontiers in the prevention and in the treatment of pediatric and neonatal diseases. This review summarizes the most relevant results published in the literature on the application of metabolomics in pediatric and neonatal clinical settings. However, there is the urgent need to standardize physiological and preanalytical variables, analytical methods, data processing, and result presentation, before establishing the definitive clinical value of results.

Utility and applications of orthotopic models of human non-small cell lung cancer (NSCLC) for the evaluation of novel and emerging cancer therapeutics.

Lung cancer is a leading cause of cancer deaths worldwide. Despite advances in chemotherapy, radiation therapy, and surgery, lung cancer continues to have a low 5-year survival rate, highlighting a dire need for more effective means of prevention, diagnosis, prognosis, and treatment. Mouse models that recapitulate the clinical features of advanced human lung cancer are critical for testing novel therapeutic approaches. This unit describes a highly reproducible, easy-to-establish orthotopic murine model of lung cancer, provides methods for in vivo imaging and monitoring of tumor growth, and discusses the usefulness of this model for translational lung cancer research and the development of therapeutic strategies.

Dermatofibrosarcoma protuberans: dealing with slow Mohs procedures employing formalin-fixed, paraffin wax-embedded tissue in a busy diagnostic laboratory.

Dermatofibrosarcoma protuberans (DFSP) is a relatively uncommon tumour that arises in the dermis and underlying soft tissue. Surgical removal is the preferred treatment, with relatively wide clearance margins of 3 cm or more. Slow Mohs procedures are often employed successfully to treat patients with such tumours. Slow Mohs procedures offer the benefit of improved cure rates and maximal tissue conservation. However, dealing with such tissue successfully presents the laboratory with a host of technical problems. This report advocates a set protocol to follow for slow Mohs, based on the experience acquired from dealing with 37 cases of DFSP over a 12-year period. The report establishes the benefits of slow Mohs paraffin wax-embedded tissue over frozen sections in terms of improved morphology, tissue preservation and immunocytochemical labelling with anti-CD34.

Quality standards and samples in genetic testing.

The most critical performance indicator for medical laboratories is the delivery of accurate test results. In any laboratory, there is always the possibility that random or systematic errors may occur and place human health and welfare at risk. Laboratory quality assurance programmes continue to drive improvements in analytical accuracy. The most rigorously scrutinised data on laboratory errors, which come from transfusion medicine, reveal that the incidence of analytical errors has fallen to levels where most of the residual risk is now found in preanalytical links in the chain from patient to result, particularly activities associated with ordering of tests and sample collection. This insight is important for genetic testing because, like pretransfusion testing of patients with unknown blood groups, a substantial proportion of genotyping results cannot be immediately verified. An increasing number of clinical decisions, associated personal and social choices, and legal outcomes are now influenced by genetic test results in the absence of other confirmatory data. An incorrect test result may lead to unnecessary and irreversible interventions, which may in themselves have associated risks for the patient, inaccurate risk assessment regarding the disease, missed opportunities for disease prevention or even wrongful conviction in a court of law. Unfortunately, there is limited information available about the risk of preanalytical errors associated with, and few published guidelines regarding, sample collection for genetic testing. The growing number and range of important decisions made on the basis of genetic findings warrant a reappraisal of current standards to minimise risks in genetic testing.

Innovations in therapies for heart valve disease.

The burden of heart valve disease among adults is enormous in the developed world. Increased life expectancy and age-related valvular degeneration remain the predominant contributors to heart valve dysfunction, which if uncorrected lead to congestive heart failure and increased morbidity and mortality. Clinical evidence on the detrimental impact of valve disease on both pediatric and adult populations has fueled growing interest in diagnosis and therapy for heart valve disease, and also significant financial investment from hospitals and medical device manufacturers in hybrid operating rooms and novel medical device technologies. A wide array of surgical, minimally invasive and percutaneous heart valve technologies are available today, which have significantly enlarged the surgeon's armamentarium, and revolutionized the traditional role of a surgeon in correcting such lesions. Amid this revolution in heart valve technologies, we present recent advances in heart valve therapies, critically appraise their clinical need, and finally discuss the clinical experience and outcomes of some of these technologies. The expected outcome of this review is to provide the clinical reader with a reasonable scientific basis to enable appropriate adoption of these technologies into their clinical practice.

Convergence technology in cancer medicine.

The field of convergence technology may be defined as an area of technologic innovation in which multiple devices or functionalities are combined within a single platform in a way that adds functional, operational or economic synergies. Within the field of medical devices, this concept embodies many different types of novel combinations representing syntheses of therapeutic, diagnostic and digital information technologies. In the current era of healthcare reform, such combinatorial technologies will be pressed to demonstrate improvements in comparative effectiveness compared with the use of separate independent components. Moreover, the new more stringent regulatory environment will require much greater levels of pre- and post-market safety reviews conducted under the auspices and authority of the US FDA Office of Combination Products. This branch of the FDA scrutinizes submissions and divides them into drugs, devices and biological products and includes many submissions previously regulated by disparate centers, such as the Center for Devices and Radiologic Health and the Center for Drug Evaluation and Research. The field of convergence technologies already amounts to a worldwide market extending to tens of billions of dollars and this article will attempt to summarize some of the key elements of this continued push for added value and more personalized medicine.

Desarrollo de la Inmunohematología en el Instituto de Hematología e Inmunología / Development of the immunohematology in the Institute of Hematology and Immunology

El desarrollo de la Inmunohematología en el Instituto de Hematología e Inmunología en los últimos años se ha caracterizado por la introducción de los anticuerpos monoclonales hemoclasificadores y de nuevos ensayos para profundizar y optimizar el diagnóstico inmunohematológico, así como elevar el nivel científico de profesionales y técnicos vinculados con la especialidad. Los resultados más relevantes se obtuvieron en la generación de anticuerpos monoclonales y en el estudio de los auto y aloanticuerpos eritrocitarios y plaquetarios. Se ha destacado también la labor como Laboratorio de Referencia en Inmunohematología, así como los aportes a la docencia en medicina transfusional. Las proyecciones futuras de trabajo deberán estar encaminadas a la introducción de las técnicas moleculares en Inmunohematología y a profundizar en el estudio de los anticuerpos contra leucocitos y plaquetas

The development of the Immunohematology in the Institute of Hematology and Immunology in past years has been characterized by the introduction of hemoclassifying monoclonal antibodies and of new trials to deepen and to optimize the immunohematologic diagnosis, as well as to raise the scientific level of the professionals and technicians linked to specialty. The more relevant results were achieved in monoclonal antibodies generation and in the study of the erythrocyte and platetelet alloantibodies and autoantibodies. The work as Immunohematology Reference Laboratory has been emphasized as well as the contributions to transfusion medicine teaching. The future work projections must to be directed to introduction of molecular techniques in Immunology and to deepen in the study of antibodies to leukocytes and platelets

Advances in the field of nanooncology.

Nanooncology, the application of nanobiotechnology to the management of cancer, is currently the most important chapter of nanomedicine. Nanobiotechnology has refined and extended the limits of molecular diagnosis of cancer, for example, through the use of gold nanoparticles and quantum dots. Nanobiotechnology has also improved the discovery of cancer biomarkers, one such example being the sensitive detection of multiple protein biomarkers by nanobiosensors. Magnetic nanoparticles can capture circulating tumor cells in the bloodstream followed by rapid photoacoustic detection. Nanoparticles enable targeted drug delivery in cancer that increases efficacy and decreases adverse effects through reducing the dosage of anticancer drugs administered. Nanoparticulate anticancer drugs can cross some of the biological barriers and achieve therapeutic concentrations in tumor and spare the surrounding normal tissues from toxic effects. Nanoparticle constructs facilitate the delivery of various forms of energy for noninvasive thermal destruction of surgically inaccessible malignant tumors. Nanoparticle-based optical imaging of tumors as well as contrast agents to enhance detection of tumors by magnetic resonance imaging can be combined with delivery of therapeutic agents for cancer. Monoclonal antibody nanoparticle complexes are under investigation for diagnosis as well as targeted delivery of cancer therapy. Nanoparticle-based chemotherapeutic agents are already on the market, and several are in clinical trials. Personalization of cancer therapies is based on a better understanding of the disease at the molecular level, which is facilitated by nanobiotechnology. Nanobiotechnology will facilitate the combination of diagnostics with therapeutics, which is an important feature of a personalized medicine approach to cancer.

Association between physician specialty and uptake of new medical technologies: HPV tests in Florida Medicaid.

BACKGROUND: It is well established that specialists often adopt new medical technologies earlier than generalists, and that racial and ethnic minority patients are less likely than White patients to receive many procedures and prescription drugs. However, little is known about the role that specialists or generalists may play in reducing racial and ethnic disparities in uptake of new medical technologies. Human papillomavirus (HPV) DNA tests, introduced as a cervical cancer screening tool in 2000, present a rich context for exploring patterns of use across patient and provider subgroups. OBJECTIVE: To identify patient characteristics and the provider specialty associated with overall and appropriate use of HPV DNA tests over time, and to examine the associations between clinical guidelines and adoption of the test in an underserved population. DESIGN: Retrospective longitudinal study using Florida Medicaid administrative claims data. PARTICIPANTS: Cervical cancer screening test claims for 415,239 female beneficiaries ages 21 to 64 from July 2001 through June 2006. MAIN MEASURES: Overall and appropriate use of HPV DNA tests. KEY RESULTS: Although minority women were initially less likely than White women to receive HPV DNA tests, test use grew more rapidly among Black and Hispanic women compared to White women. Obstetricians/gynecologists were significantly more likely than primary care providers to administer HPV DNA tests. Release of the first set of clinical guidelines was associated with a large increase in the use of HPV DNA tests (adjusted odds ratio: 2.46, p<0.0001); subsequent guidelines were associated with more modest increases. CONCLUSIONS: Uptake of new cervical cancer screening protocols can occur quickly among traditionally underserved groups and may be aided by early adoption by specialists.