Human chromosome classification using competitive support vector machine teams

Classification of chromosome is a challenging task and requires very precise autonomous classifier. This paper proposes to employ competing support vector machines (SVMs) placed in a grid. Each agent in cells of the grid is responsible to distinguish two classes. Overall output is determined by simple majority voting of SVMs. Relying same principle as the work by Palalic and Can [17], we compared the results obtained where the algorithms delivers better accuracy

Microfluidic Integrated Technology: A Potential Tool for Portable Radiation Biodosimetry

In the event of a mass radiological or nuclear incident, a large number of individuals would require rapid biodosimetric screening for proper medical care, mitigation and follow-up procedures. Mass radiological triage is critical after any such large-scale event because of the need for Dose assessment of suffered individuals at an early stage. With the increasing probability of such unprecedented incidents around the world, the need for modelling and development of new medical countermeasures for potential future chemical, biological, radiological and nuclear has been well established. Unfortunately, the capacity of most of these methods are still restricted to laboratory establishments due to resource limitations, need of high end expertise or general immobility of bulky instruments required for the same. So far there exists no rapid diagnostic technique that may reliably discriminate levels of ionising radiation exposure based on samples collected at a single time point. In classical clinical settings, complete blood count, particularly the lymphocyte count is based on temporal assessment. The diagnostic ‘gold standard’ in the field of radiation biodosimetry is the dicentric chromosome assay which happens to be highly labour-intensive and extensively time consuming, rendering it inefficient in case of mass casualty situations. Advanced technologies such as microfluidic platform, BioMEMS, μTAS carry the potential to make system highly portable, cost efficient and independent of high skilled expertise. In this review of the latest advances in portable biodosimetry we evaluate our progress and identify areas that still need to be addressed to achieve true field-deployment readiness

A microscopy scanning system for clinical chromosome diagnostics.

During the course of this research, the following papers were authored or co-authored: (1) X. Wang, B. Zheng, M. Wood, S. Li, W. R. Chen, and H. Liu, "Development and Evaluation of Automated Systems for Detection and Classification of Banded Chromosome: Current Status and Future Perspectives", Journal of Physics D: Applied Physics, vol 38, 2005, pp 2536-2542. (2) X. Wang, S. Li, H. Liu, M. Wood, W. R. Chen, and B. Zheng, "Automated Identification of Analyzable Metaphase Chromosomes Depicted on Microscopic Digital Images", Journal of Biomedical Informatics, 2007. (Accepted for publication).An important part of the diagnosis and treatment of leukemia is the visual examination of the patient's chromosomes. Chomosomal changes serve as indicators of the nature and severity of the disease. Clinical genetics laboratories acquire images of metaphase chromosomes using a microscope and camera system, usually by manual search of the tissue slides. Manual techniques are labor intensive, slow and costly. A computer controlled scanning system can be an important tool for automating and expediting the chromosome analysis process. Commercial systems have been developed, but fall short of providing an automated (or even semi-automated) computer aided diagnosis technique. This dissertation describes the design and development of a prototype scanning system, and studies the impact of the scanning speed on the image quality, with an eye towards the development of a Computer Aided Diagnostic (CAD) system. The system consists of a laboratory grade microscope, a high-precision motorized stage, a video imaging system, and controlling software. An entire slide can be imaged and captured into a digital file for later review. Fast scan rates make a system more productive, which is essential for clinical practice, but motion blur can render the images unusable for post image processing and computer assisted diagnosis. Experimentally in this research, clinical chromosome images and resolution patterns were scanned under different objective lens magnifications ranges from 10X to 100X, at different scanning speed from 0mm/sec to 4mm/sec. These images were reviewed by observers. Significant motion blurs were observed at high magnification and scanning speed. The impact of scanning speed was also quantified by objective parameters such as modulation transfer functions (MTF). For example, with an objective lens power of 10X, the essential structure of a metaphase spread can still be visually detected with a scan speed of 4 mm/sec, whereas at that speed, the image under 60X and higher objective power is not recognized. Accordingly, an optimal design strategy for an efficient clinical system should balance optical magnification, scanning speed, as well as the frame rate of the camera

Detection of deleterious on-target effects after CRISPR-mediated genome editing in human induced pluripotent stem cells

The CRISPR/Cas9 system is an exceedingly powerful technology for precise genome editing. Its ease of use, high editing efficiency and an ever-growing CRISPR-based toolbox has provided researchers with novel possibilities to unravel the molecular and systemic consequences of changes in the genetic code. For this reason, CRISPR is now applied for editing in a wide range of different cell lines and organisms for basic and translational research. Here, accurate and precise editing is an indispensable prerequisite to generate reliable research models. However, a lot remains to be understood about the molecular mechanism of double-strand-breaks (DSBs) in the DNA as introduced by the Cas9 nuclease during editing. In fact, CRISPR editing can be accompanied by inadvertent genomic changes at the targeted locus (on-target) as well as other genomic sites (off-target). These can have drastic consequences on gene activity or expression and therefore need to be carefully investigated. Characterizing and avoiding unwanted off-target effects (OffTE) has been the focus of several studies and reliable tools for their detection have been developed. This is, however, not the case for on-target effects (OnTE) that have only been reported very recently. These can be large deletions, large insertions, complex rearrangements, or regions of copy-neutral loss of heterozygosity (LOH) around the target site. Several studies have described frequent occurrence of OnTEs in mice, but it has not been investigated if clinically relevant human cells, such as induced pluripotent stem cells (iPSCs) are also affected. The main problem with OnTEs is that they often remain unnoticed in standard quality controls like Sanger genotyping of the target locus, and additional checks are lacking in most CRISPR-based studies. This is also because there are no simple detection tools available. Therefore, in this study, we developed simple and reliable tools for OnTE detection after CRISPR genome editing: Structural alterations like large deletions, large insertions or complex rearrangements can be identified by quantifying the number of intact alleles at the edited locus using our new method called quantitative genotyping PCR (qgPCR). In addition, we validated genotyping of neighboring single nucleotide polymorphisms (SNPs) either by Sanger sequencing or SNP microarrays to reveal editing-induced regions of LOH. The entire workflow is broadly applicable to different cell lines and organisms after editing by the NHEJ or HDR pathway. We have applied our newly established detection technology to human iPSCs after HDR-mediated editing and demonstrate universal occurrence of OnTEs at multiple loci in up to 40% of edited single-cell clones. Furthermore, using an in vitro model of Alzheimer’s disease, we illustrate deleterious consequences of OnTEs on expression of the edited gene that may reduce pathogenic effects and therefore interfere with experimental findings. Overall, the threat of undetected OnTEs undermining the reliability of CRISPR-based studies has not received sufficient attention in the field so far. With this thesis, we hope to raise further awareness and propose that our simple and reliable on-target quality control workflow should be an essential part of all relevant genome editing experiments

Digital image analysis of mitotic chromosomes

Změny v počtu a ve struktuře chromozomů jsou příčinou řady vážných onemocnění. K odhalení chromozomálních změn slouží cytogenetická vyšetření, která nejčastěji vedou k sestavení karyotypu. Pro účely cytogenetických analýz se chromozomy vizualizují pomocí vhodných metod a nejčastěji se následně sestavují do karyotypu. Protože ruční stanovení karyotypu je časově i finančně náročné, vyvíjí se přístupy k automatickému karyotypování pomocí počítačového softwaru. Automatické karyotypovací systémy klasifikují chromozomy do tříd na základě identifikačních znaků, specifických pro každý chromozom. Automatickou klasifikaci však nejvíce limituje přítomnost překrývající se a silně ohnutých chromozomů, přítomných v téměř každé mitóze. Přesnost a spolehlivost karyotypovacích systémů stále závisí na zásahu uživatele. Cílem vývoje nových přístupů k automatickému karyotypování je tedy zejména překonání výše zmíněných problémů a dále vývoj takových klasifikačních metod, které umožňí klasifikaci chromozomů do párů bez lidské kontroly.Changes in chromosome number and structure may cause serious diseases. Cytogenetic tests leadin to set of karyotype are done for detecting these abnormalities. Chromosomes are visualised with proper methods and karyotype is made up most often. Manual karyotyping is time-consuming and expensive task. Because of this, researchers have been developing automated karyotyping systems. Karyotyping systems classify chromosomes into classes based on their characteristic features. Overlapping and bent chromosomes are limitations for automatic classification since they ocur at almost every mitosis. Accuracy and reliability of karyotyping systems still depend on the human intervention. Overcoming of these problems and development of fully automated system is the aim of modern approaches.

COMPREHENSIVE PERFORMANCE EVALUATION AND OPTIMIZATION OF HIGH THROUGHPUT SCANNING MICROSCOPY FOR METAPHASE CHROMOSOME IMAGING

Specimen scanning is a critically important tool for diagnosing the genetic diseases in today’s hospital. In order to reduce the clinician’s work load, many investigations have been conducted on developing automatic sample screening techniques in the last twenty years. However, the currently commercialized scanners can only accomplish the low magnification sample screening (i.e. under 10× objective lens), and still require clinicians’ manual operation for the high magnification image acquisition and confirmation (i.e. under 100× objective lens). Therefore, a new high throughput scanning method is recently proposed to continuously scan the specimen and select the clinically analyzable cells. In the medical imaging lab, University of Oklahoma, a prototype of high throughput scanning microscopy is built based on the time delay integration (TDI) line scanning detector. This new scanning method, however, raises several technical challenges for evaluating and optimizing the performance. First, we need to use the clinical samples to compare this new prototype with the conventional two-step scanners. Second, the system DOF should be investigated to assess the impact on clinically analyzable metaphase chromosomes. Further, in order to achieve the optimal results, we should carefully assess and select the auto-focusing methods for the high throughput scanning system. Third, we need to optimize the scanning scheme by finding the optimal trade-off between the image quality and efficiency. Finally, analyzing the performance of the various image features is meaningful for improving the performance of the computer aided detection (CAD) scheme under the high throughput scanning condition. The purpose of this dissertation is to comprehensively evaluate the performance of the high throughput scanning prototype. The first technical challenge was solved by the first investigation, which utilized a number of 9 slides from five patients to compare the detecting performance of the high throughput scanning prototype. The second and third studies were performed for the second technical challenge. In the second study, we first theoretically computed the DOF of our prototype and then experimentally measured the system DOF. After that, the DOF impact was analyzed using cytogenetic images from different pathological specimens, under the condition of two objective lenses of 60× (dry, N.A. = 0.95) and 100× (oil, N.A. = 1.25). In the third study, five auto-focusing functions were investigated using metaphase chromosome images. The performance of these different functions was compared using four widely accepted criteria. The fourth and fifth investigations were designed for the third technical challenge. The fourth study objectively assessed chromosome band sharpness by a gradient sharpness function. The sharpness of the images captured from standard resolution target and several pathological chromosomes was objectively evaluated by the gradient sharpness function. The fifth study presented a new slide scanning scheme, which only applies the auto-focusing operations on limited locations. The focusing position was adjusted very quickly by linear interpolation for the other locations. The sixth study was aimed for the fourth technical challenge. The study investigated 9 different feature extraction methods for the CAD modules applied on our high throughput scanning prototype. A certain amount of images were first acquired from 200 bone marrow cells. Then the tested features were performed on these images and the images containing clinically meaningful chromosomes were selected using each feature individually. The identifying accuracy of each feature was evaluated using the receiver operating characteristic (ROC) method. In this dissertation, we have the following results. First, in most cases, we demonstrated that the high throughput scanning can select more diagnostic images depicting clinically analyzable metaphase chromosomes. These selected images were acquired with adequate spatial resolution for the following clinical interpretation. Second, our results showed that, for the commonly used pathological specimens, the metaphase chromosome band patterns are clinically recognizable when these chromosomes were obtained within 1.5 or 1.0 μm away from the focal plane, under the condition of applying the two 60× or 100× objective lenses, respectively. In addition, when scanning bone marrow and blood samples, the Brenner gradient and threshold pixel counting methods can achieve the optimal performance, respectively. Third, we illustrated that the optimal scanning speed of clinical samples is 0.8 mm/s, for which the captured image sharpness is optimized. When scanning the blood sample slide with an auto-focusing distance of 6.9 mm, the prototype obtained an adequate number of analyzable metaphase cells. More useful cells can be captured by increasing the auto-focusing operations, which may be needed for the high accuracy diagnosis. Finally, we found that the optimal feature for the online CAD scheme is the number of the labeled regions. When applying the offline CAD scheme, the satisfactory results can be achieved by combining four different features including the number of the labeled regions, average region area, average region pixel value, and the standard deviation of the either region circularity or distance. Although these investigations are encouraging, there exist several limitations. First, the number of the specimens is limited in most of the assessments. Second, some important impacts, such as the DOF of human eye and the sample thickness, are not considered. Third, more recently proposed algorithms and image features are not used for the evaluation. Therefore, several further studies are planned, which may provide more meaningful information for improving the scanning efficiency and image quality. In summary, we believe that the high throughput scanning may be extensively applied for diagnosing genetic diseases in the future

Application of Nested PCR, Whole Genome Amplification and Comparative Genomic Hybridisation for Single Cell Genetic Analysis

Single cell genetic analysis is a necessity in the field of preimplantation genetic diagnosis (PGD), non-invasive prenatal diagnosis and can be applied to isolated tumour cells in the blood. Current techniques used in PGD and non-invasive prenatal diagnosis are mainly based on either the polymerase chain reaction (PCR) or fluorescence in situ hybridisation (FISH). However, the genetic analysis at a specific locus by PCR has the drawback of sacrificing further detection of common chromosomal aberrations if there is only one cell available. The limitation of FISH using specific probes is that it provides information on only one or a few loci at a time. Comparative genomic hybridisation (CGH), a molecular cytogenetic technique, has the advantage over FISH in permitting a comprehensive analysis of chromosomal imbalances across the whole genome. Combining degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and CGH techniques, it is possible to study minute quantities of DNA prepared from a very few cells. The aim of this project is to verify the feasibility of applying these techniques to a single cell and investigate the prospect of a novel strategy using whole genome amplification (WGA), nested PCR and CGH to increase the scope and capacity of single cell genetic analysis. The work started first to prove the amplification power of DOP-PCR from a single cell. Various protocols aiming at whole genome amplification (WGA) have different efficiencies in terms of yield and genomic coverage. DOP- PCR is superior to primer extension preamplification (PEP) in producing more quantity of DNA from a single cell. The amplification power of DOP- PCR from a single cell (5 pg) resulted in 10 mug yield with the bulk of DOP- PCR products between 200-2000 bp. DOP-PCR is designed to faithfully amplify the genome, and provide sufficient DNA template. Thus, numerous specific loci and the imbalance of every chromosome can be assessed in a single cell. To investigate the feasibility of locus detection from WGA products, two sets of nested PCR aiming at sex determination or CF ?F508 detection were optimised and tested on a range of DNA sources. The results proved that a strategy of nested PCR could be used to determine the sex and CF status on DOP-PCR-amplified DNA derived from single cells. Nested PCR was also used in the genetic analysis directly on a single cell. In the study of single blastomeres, the sex could be determined in 55.6% of cases (10/18) when nested PCR was used directly on single blastomeres. The feasibility of single cell CGH in the diagnosis of major chromosome abnormalities such as sex chromosome anomaly, trisomy 18 and 21 was established in this study. To produce successful single cell CGH experiments, the normal reference DNA could be made from either amplified DNA or non-amplified DNA. This study demonstrated that a reliable non-amplified reference DNA could increase the success rate of single cell CGH and help to identify the underlying causes of failed CGH experiments. For quantitative analysis of CGH experiments, the fluorescence ratio cutoff value was usually set at 1.2 and 0.8 to represent chromosomal gains and chromosomal losses, respectively. However, the fluorescent ratio profiles using stringent cut-off values would reduce the false positives with the risk of failing to detect the true abnormalities. In this study, the average number of false positives was 5 when threshold of 1.2/0.8 was used. Apart from the objective quantitative analysis, single cell CGH diagnosis of trisomy 21, 18 and sex could also be qualitatively verified from CGH images. After the DOP-PCR/CGH techniques had been proved feasible from a single cell, the sensitivity and reliability of CGH was further tested on 10-20 stained cells scraped from slides in order to simulate the current strategy used in the non-invasive prenatal diagnosis. This part also involved firstly, the correct diagnosis of trisomy 18 and 21 and secondly, several coded samples of known sex were tested for the performance of XXI single cell CGH. The results showed that chromosomal aberrations such as isochromosome X and segmental aneuploidy could be reliably detected. However, a small deletion at single band level could not be detected by single cell CGH. Thirdly, a manufactured mosaicism designed by mixing trisomy 18 male cells with normal female cells was tested to see if at 50-70% mosaic level the trisomy could be detected by single cell CGH. The results illustrated that trisomy 18 could not be detected at the 70% mosaic level using the current protocol. This may illustrate that accurate identification of cells of fetal origin is mandatory if current DOP- PCR/CGH techniques are to be applied in the field of non-invasive prenatal diagnosis. The final part involved using CGH on blastomeres and tested the possibility of employing a shortened protocol for single cell CGH. The preliminary results confirm that chromosome abnormalities may be a common phenomenon in the early embryonic cells. The success of overnight CGH illustrates that further reduction of time is possible. This may allow an expansion of its future application in PGD. Overall, this study demonstrates that DOP-PCR/nested PCR/CGH has the potential to serve as a powerful supplement to the present genetic analysis from a single cell. Concomitant detailed chromosome analysis and specific locus detection may become feasible in the field of PGD and non-invasive prenatal diagnosis in the near future

Targeting the adaptability of heterogeneous aneuploidy population

Aneuploid genomes, characterized by unbalanced and diverse chromosome stoichiometry (karyotype), are associated with cancer malignancy and drug-resistance of human pathogenic fungi. My PhD projects studied three aspects of aneuploidy that are logically linked to each other: the production of aneuploidy by environmental stress; the impact of the heterogeneous aneuploidy population generated by stress on adaptability; the potential therapeutic strategy towards these heterogeneous aneuploidy populations with high adaptability, a root for the clinical challenge in treating aneuploidy diseases such as cancer. We investigated whether pleiotropic stress could induce the production of aneuploidy in budding yeast. We showed that while diverse stresses can induce an increase in chromosome instability (CIN), proteotoxic stress, caused by transient Hsp90 inhibition or heat-shock, drastically elevated CIN to produce karyotypically mosaic cell population. The latter effect is linked to an evolutionarily conserved role for Hsp90 chaperon complexes in kinetochore assembly. We found the induction of aneuploidy population potentiates adaptability. Continued growth in the presence of Hsp90 inhibitor resulted in emergence of drug-resistant colonies with chromosome XV gain. This drug-resistance phenotype is a quantitative trait involving copy number increases of at least two genes located on chromosome XV. Short-term exposure to Hsp90 stress, which produced an aneuploidy population with heterogeneous karyotypes, potentiated fast adaptation to unrelated cyto-toxic compounds through different aneuploid chromosome stoichiometries. We designed an evolutionary trap to harness the adaptability of heterogeneous aneuploidy populations with high adaptability. Using a combination of experimental data and a general statistical model, we showed that the degree of phenotypic variation, thus evolvability, escalates with the degree of overall growth suppression irrespective of stress mechanisms. Such scaling explains the challenge of treating aneuploidy diseases with diverse different karyotypes by imposing a single mode of inhibition, yet specific karyotype features can be highly targetable. Motivated by this finding, we proposed an "evolutionary trap" targeting both karyotypic diversity and fitness of the population. This strategy entails a selective condition "channeling" a karyotypically divergent population into one with a predominant and drugable karyotypic feature. We provided a proof-of-principle test with mechanistic explanation in budding yeast and demonstrated the potential efficacy of this strategy toward aneuploidy-based azole resistance in the human pathogen Candida albicans. Karyotype channeling also happens naturally in tumors, which is resulted from adaptation to the tissue micro-environment and/or the need for oncogenic transformation. This natural karyotypic selection may be leveraged by drug treatment targeting the selected karyotype feature. Thus, the strategy proposed here may be utilized for designing a class of treatment regime distinct from current therapies