Feature Representation and Signal Classification in Fluorescence In-Situ Hybridization Image Analysis

Fast and accurate analysis of fluorescence in-situ hybridization (FISH) images for signal counting will depend mainly upon two components: a classifier to discriminate between artifacts and valid signals of several fluorophores (colors), and well discriminating features to represent the signals. Our previous work has focused on the first component. To investigate the second component, we evaluate candidate feature sets by illustrating the probability density functions (pdfs) and scatter plots for the features. The analysis provides first insight into dependencies between features, indicates the relative importance of members of a feature set, and helps in identifying sources of potential classification errors. Class separability yielded by different feature subsets is evaluated using the accuracy of several neural network (NN)-based classification strategies, some of them hierarchical, as well as using a feature selection technique making use of a scatter criterion. The complete analysis recommends several intensity and hue features for representing FISH signals. Represented by these features, around 90% of valid signals and artifacts of two fluorophores are correctly classified using the NN. Although applied to cytogenetics, the paper presents a comprehensive, unifying methodology of qualitative and quantitative evaluation of pattern feature representation essential for accurate image classification. This methodology is applicable to many other real-world pattern recognition problems

Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel™. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD