Theory-Based Digital Interventions to Improve Asthma Self-Management Outcomes: Systematic Review.

BACKGROUND: Asthma is a chronic disease requiring effective self-management to control it and prevent mortality. The use of theory-informed digital interventions promoting asthma self-management is increasing. However, there is limited knowledge concerning how and to what extent psychological theory has been applied to the development of digital interventions, or how using theory impacts outcomes. OBJECTIVE: The study aimed to examine the use and application of theory in the development of digital interventions to enhance asthma self-management and to evaluate the effectiveness of theory-based interventions in improving adherence, self-management, and clinical outcomes. METHODS: Electronic databases (CENTRAL, MEDLINE, EMBASE, and PsycINFO) were searched systematically using predetermined terms. Additional studies were identified by scanning references within relevant studies. Two researchers screened titles and abstracts against predefined inclusion criteria; a third resolved discrepancies. Full-text review was undertaken for relevant studies. Those meeting inclusion criteria were assessed for risk of bias using the Cochrane Collaboration tool. The review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. Study outcomes were classified as medication adherence, self-management, asthma control, clinical markers of health, quality of life, other quality of life outcomes, and health care utilization. Effectiveness was calculated as an average outcome score based on the study's reported significance. The Theory Coding Scheme (TCS) was used to establish the extent to which each intervention had applied theory and which theoretical constructs or behavioral determinants were addressed. Associations between TCS scores and asthma outcomes were described within a narrative synthesis. RESULTS: Fourteen studies evaluating 14 different digital interventions were included in this review. The most commonly cited theories were Social Cognitive Theory, Health Belief Model, and Self-Efficacy Theory. A greater use of theory in the development of interventions was correlated with effective outcomes (r=.657; P=.01): only the 3 studies that met >60% of the different uses of theory assessed by the TCS were effective on all behavioral and clinical outcomes measured. None of the 11 studies that met ≤60% of the TCS criteria were fully effective; however, 3 interventions were partially effective (ie, the intervention had a significant impact on some, but not all, of the outcomes measured). Most studies lacked detail on the theoretical constructs and how they were applied to the development and application of the intervention. CONCLUSIONS: These findings suggest that greater use of theory in the development and application of digital self-management interventions for asthma may increase their effectiveness. The application of theory alone may not be enough to yield a successful intervention, and other factors (eg, the context in which the intervention is used) should be considered. A systematic approach to the use of theory to guide the design, selection, and application of intervention techniques is needed.

Mutation burden and other molecular markers of prognosis in colorectal cancer treated with curative intent: results from the QUASAR 2 clinical trial and an Australian community-based series.

BACKGROUND: Molecular indicators of colorectal cancer prognosis have been assessed in several studies, but most analyses have been restricted to a handful of markers. We aimed to identify prognostic biomarkers for colorectal cancer by sequencing panels of multiple driver genes. METHODS: In stage II or III colorectal cancers from the QUASAR 2 open-label randomised phase 3 clinical trial and an Australian community-based series, we used targeted next-generation sequencing of 82 and 113 genes, respectively, including the main colorectal cancer drivers. We investigated molecular pathways of tumorigenesis, and analysed individual driver gene mutations, combinations of mutations, or global measures such as microsatellite instability (MSI) and mutation burden (total number of non-synonymous mutations and coding indels) for associations with relapse-free survival in univariable and multivariable models, principally Cox proportional hazards models. FINDINGS: In QUASAR 2 (511 tumours), TP53, KRAS, BRAF, and GNAS mutations were independently associated with shorter relapse-free survival (p<0·035 in all cases), and total somatic mutation burden with longer survival (hazard ratio [HR] 0·81 [95% CI 0·68-0·96]; p=0·014). MSI was not independently associated with survival (HR 1·12 [95% CI 0·57-2·19]; p=0·75). We successfully validated these associations in the Australian sample set (296 tumours). In a combined analysis of both the QUASAR 2 and the Australian sample sets, mutation burden was also associated with longer survival (HR 0·84 [95% CI 0·74-0·94]; p=0·004) after exclusion of MSI-positive and POLE mutant tumours. In an extended analysis of 1732 QUASAR 2 and Australian colorectal cancers for which KRAS, BRAF, and MSI status were available, KRAS and BRAF mutations were specifically associated with poor prognosis in MSI-negative cancers. MSI-positive cancers with KRAS or BRAF mutations had better prognosis than MSI-negative cancers that were wild-type for KRAS or BRAF. Mutations in the genes NF1 and NRAS from the MAPK pathway co-occurred, and mutations in the DNA damage-response genes TP53 and ATM were mutually exclusive. We compared a prognostic model based on the gold standard of clinicopathological variables and MSI with our new model incorporating clinicopathological variables, mutation burden, and driver mutations in KRAS, BRAF, and TP53. In both QUASAR 2 and the Australian cohort, our new model was significantly better (p=0·00004 and p=0·0057, respectively, based on a likelihood ratio test). INTERPRETATION: Multigene panels identified two previously unreported prognostic associations in colorectal cancer involving TP53 mutation and total mutation burden, and confirmed associations with KRAS and BRAF. Even a modest-sized gene panel can provide important information for use in clinical practice and outperform MSI-based prognostic models. FUNDING: UK Technology Strategy Board, National Institute for Health Research Oxford Biomedical Research Centre, Cancer Australia Project, Cancer Council Victoria, Ludwig Institute for Cancer Research, Victorian Government.

Treatment perceptions in patients with asthma: Synthesis of factors influencing adherence.

BACKGROUND: Non-adherence to asthma treatment is a contributing factor for poorly controlled asthma. AIM: The aim of this systematic review is to explore patients' perceptions of their inhaled asthma treatment, and how these relate to adherence, using both qualitative and quantitative data. METHODS: Pre-determined search terms and inclusion criteria were used to search electronic databases (The Cochrane Library, MEDLINE, EMBASE and PsycINFO). Two researchers screened titles and abstracts using the Rayyan web app and data were extracted in relation to psychological components (beliefs about, and attitudes towards, medicines) and adherence. RESULTS: Of 1638 papers, 36 met the inclusion criteria. Key themes were: Perceived need for treatment - all 12 studies using the BMQ to measure patients' perceived need for treatment found that patients' beliefs about their necessity for treatment were associated with adherence-; Concerns about treatment - immediate and long-term side effects (58%), worries about safety (19%), and potential addiction to asthma medication (31%)-; and Perceived social stigma - 22% of studies reported that embarrassment contributed to poor adherence. CONCLUSIONS: Acknowledging and addressing patient treatment beliefs and perceptual barriers to adherence is integral to designing adherence interventions for asthma patients. Further research is needed to better our understanding of the relationship between treatment perceptions and adherence.

The antibiotic resistome and microbiota landscape of refugees from Syria, Iraq and Afghanistan in Germany.

BACKGROUND: Multidrug-resistant bacteria represent a substantial global burden for human health, potentially fuelled by migration waves: in 2015, 476,649 refugees applied for asylum in Germany mostly as a result of the Syrian crisis. In Arabic countries, multiresistant bacteria cause significant problems for healthcare systems. Currently, no data exist describing antibiotic resistances in healthy refugees. Here, we assess the microbial landscape and presence of antibiotic resistance genes (ARGs) in refugees and German controls. To achieve this, a systematic study was conducted in 500 consecutive refugees, mainly from Syria, Iraq, and Afghanistan and 100 German controls. Stool samples were subjected to PCR-based quantification of 42 most relevant ARGs, 16S ribosomal RNA gene sequencing-based microbiota analysis, and culture-based validation of multidrug-resistant microorganisms. RESULTS: The fecal microbiota of refugees is substantially different from that of resident Germans. Three categories of resistance profiles were found: (i) ARGs independent of geographic origin of individuals comprising BIL/LAT/CMA, ErmB, and mefE; (ii) vanB with a high prevalence in Germany; and (iii) ARGs showing substantially increased prevalences in refugees comprising CTX-M group 1, SHV, vanC1, OXA-1, and QnrB. The majority of refugees carried five or more ARGs while the majority of German controls carried three or less ARGs, although the observed ARGs occurred independent of signatures of potential pathogens. CONCLUSIONS: Our results, for the first time, assess antibiotic resistance genes in refugees and demonstrate a substantially increased prevalence for most resistances compared to German controls. The antibiotic resistome in refugees may thus require particular attention in the healthcare system of host countries.

Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.

INTRODUCTION: The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. METHODS: A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). RESULTS: NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. CONCLUSION: NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.

How development and manufacturing will need to be structured--heads of development/manufacturing. May 20-21, 2014 Continuous Manufacturing Symposium.

Continuous manufacturing (CM) is a process technology that has been used in the chemical industry for large-scale mass production of chemicals in single-purpose plants with benefit for many years. Recent interest has been raised to expand CM into the low-volume, high-value pharmaceutical business with its unique requirements regarding readiness for human use and the required quality, supply chain, and liability constraints in this business context. Using a fairly abstract set of definitions, this paper derives technical consequences of CM in different scenarios along the development-launch-supply axis in different business models and how they compare to batch processes. Impact of CM on functions in development is discussed and several operational models suitable for originators and other business models are discussed and specific aspects of CM are deduced from CM's technical characteristics. Organizational structures of current operations typically can support CM implementations with just minor refinements if the CM technology is limited to single steps or small sequences (bin-to-bin approach) and if the appropriate technical skill set is available. In such cases, a small, dedicated group focused on CM is recommended. The manufacturing strategy, as centralized versus decentralized in light of CM processes, is discussed and the potential impact of significantly shortened supply lead times on the organization that runs these processes. The ultimate CM implementation may be seen by some as a totally integrated monolithic plant, one that unifies chemistry and pharmaceutical operations into one plant. The organization supporting this approach will have to reflect this change in scope and responsibility. The other extreme, admittedly futuristic at this point, would be a highly decentralized approach with multiple smaller hubs; this would require a new and different organizational structure. This processing approach would open up new opportunities for products that, because of stability constraints or individualization to patients, do not allow centralized manufacturing approaches at all. Again, the entire enterprise needs to be restructured accordingly. The situation of CM in an outsourced operation business model is discussed. Next steps for the industry are recommended. In summary, opportunistic implementation of isolated steps in existing portfolios can be implemented with minimal organizational changes; the availability of the appropriate skills is the determining factor. The implementation of more substantial sequences requires business processes that consider the portfolio, not just single products. Exploration and implementation of complete process chains with consequences for quality decisions do require appropriate organizational support.

Achieving Continuous Manufacturing: Technologies and Approaches for Synthesis, Workup, and Isolation of Drug Substance May 20-21, 2014 Continuous Manufacturing Symposium.

This whitepaper highlights current challenges and opportunities associated with continuous synthesis, workup, and crystallization of active pharmaceutical ingredients (drug substances). We describe the technologies and requirements at each stage and emphasize the different considerations for developing continuous processes compared with batch. In addition to the specific sequence of operations required to deliver the necessary chemical and physical transformations for continuous drug substance manufacture, consideration is also given to how adoption of continuous technologies may impact different manufacturing stages in development from discovery, process development, through scale-up and into full scale production. The impact of continuous manufacture on drug substance quality and the associated challenges for control and for process safety are also emphasized. In addition to the technology and operational considerations necessary for the adoption of continuous manufacturing (CM), this whitepaper also addresses the cultural, as well as skills and training, challenges that will need to be met by support from organizations in order to accommodate the new work flows. Specific action items for industry leaders are.

How Development and Manufacturing Will Need to Be Structured-Heads of Development/Manufacturing May 20-21, 2014 Continuous Manufacturing Symposium.

Continuous manufacturing (CM) is a process technology that has been used in the chemical industry for large-scale mass production of chemicals in single-purpose plants with benefit for many years. Recent interest has been raised to expand CM into the low-volume, high-value pharmaceutical business with its unique requirements regarding readiness for human use and the required quality, supply chain, and liability constraints in this business context. Using a fairly abstract set of definitions, this paper derives technical consequences of CM in different scenarios along the development-launch-supply axis in different business models and how they compare to batch processes. Impact of CM on functions in development is discussed and several operational models suitable for originators and other business models are discussed and specific aspects of CM are deduced from CM's technical characteristics. Organizational structures of current operations typically can support CM implementations with just minor refinements if the CM technology is limited to single steps or small sequences (bin-to-bin approach) and if the appropriate technical skill set is available. In such cases, a small, dedicated group focused on CM is recommended. The manufacturing strategy, as centralized versus decentralized in light of CM processes, is discussed and the potential impact of significantly shortened supply lead times on the organization that runs these processes. The ultimate CM implementation may be seen by some as a totally integrated monolithic plant, one that unifies chemistry and pharmaceutical operations into one plant. The organization supporting this approach will have to reflect this change in scope and responsibility. The other extreme, admittedly futuristic at this point, would be a highly decentralized approach with multiple smaller hubs; this would require a new and different organizational structure. This processing approach would open up new opportunities for products that, because of stability constraints or individualization to patients, do not allow centralized manufacturing approaches at all. Again, the entire enterprise needs to be restructured accordingly. The situation of CM in an outsourced operation business model is discussed. Next steps for the industry are recommended. In summary, opportunistic implementation of isolated steps in existing portfolios can be implemented with minimal organizational changes; the availability of the appropriate skills is the determining factor. The implementation of more substantial sequences requires business processes that consider the portfolio, not just single products. Exploration and implementation of complete process chains with consequences for quality decisions do require appropriate organizational support. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Achieving continuous manufacturing: technologies and approaches for synthesis, workup, and isolation of drug substance. May 20-21, 2014 Continuous Manufacturing Symposium.

This whitepaper highlights current challenges and opportunities associated with continuous synthesis, workup, and crystallization of active pharmaceutical ingredients (drug substances). We describe the technologies and requirements at each stage and emphasize the different considerations for developing continuous processes compared with batch. In addition to the specific sequence of operations required to deliver the necessary chemical and physical transformations for continuous drug substance manufacture, consideration is also given to how adoption of continuous technologies may impact different manufacturing stages in development from discovery, process development, through scale-up and into full scale production. The impact of continuous manufacture on drug substance quality and the associated challenges for control and for process safety are also emphasized. In addition to the technology and operational considerations necessary for the adoption of continuous manufacturing (CM), this whitepaper also addresses the cultural, as well as skills and training, challenges that will need to be met by support from organizations in order to accommodate the new work flows. Specific action items for industry leaders are: Develop flow chemistry toolboxes, exploiting the advantages of flow processing and including highly selective chemistries that allow use of simple and effective continuous workup technologies. Availability of modular or plug and play type equipment especially for workup to assist in straightforward deployment in the laboratory. As with learning from other industries, standardization is highly desirable and will require cooperation across industry and academia to develop and implement. Implement and exploit process analytical technologies (PAT) for real-time dynamic control of continuous processes. Develop modeling and simulation techniques to support continuous process development and control. Progress is required in multiphase systems such as crystallization. Involve all parts of the organization from discovery, research and development, and manufacturing in the implementation of CM. Engage with academia to develop the training provision to support the skills base for CM, particularly in flow chemistry, physical chemistry, and chemical engineering skills at the chemistry-process interface. Promote and encourage publication and dissemination of examples of CM across the sector to demonstrate capability, engage with regulatory comment, and establish benchmarks for performance and highlight challenges. Develop the economic case for CM of drug substance. This will involve various stakeholders at project and business level, however establishing the critical economic drivers is critical to driving the transformation in manufacturing.

Development and evaluation of a genomic signature for the prediction and mechanistic assessment of nongenotoxic hepatocarcinogens in the rat.

Evaluating the risk of chemical carcinogenesis has long been a challenge owing to the protracted nature of the pathology and the limited translatability of animal models. Although numerous short-term in vitro and in vivo assays have been developed, they have failed to reliably predict the carcinogenicity of nongenotoxic compounds. Extending upon previous microarray work (Fielden, M. R., Nie, A., McMillian, M., Elangbam, C. S., Trela, B. A., Yang, Y., Dunn, R. T., II, Dragan, Y., Fransson-Stehen, R., Bogdanffy, M., et al. (2008). Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat. Toxicol. Sci. 103, 28-34), we have developed and extensively evaluated a quantitative PCR-based signature to predict the potential for nongenotoxic compounds to induce liver tumors in the rat as a first step in the safety assessment of potential nongenotoxic carcinogens. The training set was derived from liver RNA from rats treated with 72 compounds and used to develop a 22-gene signature on the TaqMan array platform, providing an economical and standardized assay protocol. Independent testing on over 900 diverse samples (66 compounds) confirmed the interlaboratory precision of the assay and its ability to predict known nongenotoxic hepatocarcinogens (NGHCs). When tested under different experimental designs, strains, time points, dose setting criteria, and other preanalytical processes, the signature sensitivity and specificity was estimated to be 67% (95% confidence interval [CI] = 38-88%) and 59% (95% CI = 44-72%), respectively, with an area under the receiver operating characteristic curve of 0.65 (95% CI = 0.46-0.83%). Compounds were best classified using expression data from short-term repeat dose studies; however, the prognostic expression changes appeared to be preserved after longer term treatment. Exploratory evaluations also revealed that different modes of action for nongenotoxic and genotoxic compounds can be discriminated based on the expression of specific genes. These results support a potential early preclinical testing paradigm to catalyze broader understanding of putative NGHCs.

Further characterization of the first seminoma cell line TCam-2.

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

JKT-1 is not a human seminoma cell line.

The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

Global mRNA analysis to determine a transcriptome profile of cancer stemness in a mouse model.

BACKGROUND: Striking similarities between stem cells and cancer cells have led to the concept of the existence of a cancer stem cell, a concept that has since been documented in many tumours including breast, brain and prostate tumours. Teratocarcinomas are malignant tumours occurring predominantly in the testes composed of undifferentiated stem cells and mature tissues. Cancer stemness was studied using the teratocarcinoma model of tumourigenesis. MATERIALS AND METHODS: The gene expression profile of murine embryonic stem cell lines was compared to its malignant counterpart, murine teratocarcinoma cell lines. Validation was performed using real-time quantitative PCR. RESULTS: A list of 1170 differentially expressed genes was obtained. Significant pathways involved in cancer stemness included oxidative stress and angiogenesis. Transcription factors and extracellular matrix molecules appeared prominently. CONCLUSION: Novel molecules have been highlighted including decorin, an extracellular matrix protein, which may provide opportunities for the investigation of innovative strategies in the future treatment of cancer.

Single-cell sequencing and mini-sequencing for preimplantation genetic diagnosis.

There is increasing interest in the use of preimplantation genetic diagnosis (PGD) as an alternative to routine prenatal diagnosis. However, the costs associated with development and testing of new PGD protocols have forced some PGD centres to limit the number of diseases for which PGD is offered. One of the main factors in the design of new protocols, which affects cost and accuracy, is the choice of the mutation-detection technique. We have assessed the reliability of DNA sequencing and mini-sequencing for clinical diagnosis at the single-cell level and have found them to be rapid and accurate. Extensive optimisation for individual mutations is not usually necessary when employing these versatile techniques and consequently a smaller investment of time and resources should be required during development of new protocols. Additionally, we report single-cell protocols for the diagnoses of cystic fibrosis, sickle cell anaemia and beta-thalassaemia, which utilise mini-sequencing. Unlike most mutation-detection techniques, mini-sequencing permits analysis of very small DNA fragments. Small amplicons experience low allele dropout (ADO) rates, and consequently this approach could potentially improve the reliability of PGD.

Comparison between fluorescence in situ hybridization (FISH) and quantitative-fluorescent polymerase chain reaction (QF-PCR) for the detection of aneuploidies in single blastomeres.

OBJECTIVES: The aim of our investigation was to compare the efficiencies of the fluorescence in situ hybridization (FISH) and the quantitative-fluorescent PCR (QF-PCR) methods for the detection of sexing and numerical chromosome disorders in single blastomeres collected from the same preimplantation human embryos. METHODS: FISH analysis was carried out on 145 blastomeres from the 79 surplus embryos with probes specific for chromosomes 13, 18, 21, X, and Y. QF-PCR was performed with each one or two of the primers specific for the same chromosomes on 151 blastomeres from the same embryos obtained from patients undergoing IVF treatment. RESULTS: Analyses were possible on 135 blastomeres (93%) by FISH and on 117 blastomeres (77%) by QF-PCR. Of 65 embryos, which could be analyzed by both methods, 20 embryos (31%) were diagnosed as abnormal. CONCLUSION: The present study shows that FISH tests are more accurate than QF-PCR assays for the detection of numerical chromosome disorders when performed on single blastomeres.