46,XX Testicular Disorder of Sex Development (DSD): A Case Report and Systematic Review.

Background and objectives: XX male syndrome is part of the disorders of sex development (DSD). The patients generally have normal external genitalia and discover their pathology in adulthood because of infertility. There are no guidelines regarding XX male syndrome, so the aim of our study was to evaluate the literature evidence in order to guide the physicians in the management of these type of patients. Materials and Methods: We performed a systematic review of the available literature in September 2018, using MEDLINE, Web of Science, Embase and Google Scholar database to search for all published studies regarding XX male syndrome according to PRISMA guidelines. The following search terms were used: "46 XX male", "DSD", "infertility", "hypogonadism". Results: After appropriate screening we selected 37 papers. Mean (SD) age was 33.14 (11.4) years. Hair distribution was normal in 29/39 patients (74.3%), gynecomastia was absent in 22/39 cases (56.4%), normal testes volume was reported in 0/14, penis size was normal in 26/32 cases (81.2%), pubic hair had a normal development in 6/7 patients (85.7%), normal erectile function was present in 27/30 cases (90%) and libido was preserved in 20/20 patients (100%). The data revealed the common presence of hypergonadotropic hypogonadism. All patients had a 46,XX karyotype. The sex-determining region Y (SRY) gene was detected in 51/57 cases. The position of the SRY was on the Xp in the 97% of the cases. Conclusions: An appropriate physical examination should include the evaluation of genitalia to detect cryptorchidism, hypospadias, penis size, and gynecomastia; it is important to use a validated questionnaire to evaluate erectile dysfunction, such as the International Index of Erectile Function (IIEF). Semen analysis is mandatory and so is the karyotype test. Abdominal ultrasound is useful in order to exclude residual Müllerian structures. Genetic and endocrine consultations are necessary to assess a possible hypergonadotropic hypogonadism. Testicular sperm extraction is not recommended, and adoption or in vitro fertilization with a sperm donor are fertility options.

Culture and Harvest of CpG-Stimulated Peripheral Blood or Bone Marrow in Chronic Lymphocytic Leukemia.

Chromosome analysis of chronic lymphocytic leukemia (CLL) is an important clinical tool for evaluating prognosis and disease progression. Visualizing chromosomes microscopically using traditional cytogenetic techniques requires dividing cells to be arrested during metaphase. The major challenge for performing this analysis on CLL samples is stimulating the cells to divide in culture. Stimulation of CLL cells with CpG oligodeoxynucleotides has improved our ability to perform chromosome analysis for this leukemia. This protocol should help the reader successfully culture CLL samples for clinical chromosome analysis.

[Application for prenatal diagnosis using both chromosomal karyotype analysis and BACs-on-Beads assay].

OBJECTIVE: To assess the application value in prenatal diagnosis using karyotype analysis combined with BACs-on-Beads (BoBs) assay. METHODS: Nine hundred sixty five pregnant women were subjected to amniocentesis, chromosomal karyotype analysis and detection of BoBs were employed simultaneously for abnormal number of chromosomes and 9 chromosome microdeletion syndrome in prenatal diagnosis. RESULTS: Fifty cases common chromosome aneupoidies were successfully detected by both karyotype analysis and BoBs which included 31 cases of trisomy 21,10 cases of trisomy 18 and 9 cases with sex chromosome abnormality. BoBs in addition detected 1 case of DiGeorge-1 microdeletion syndrome and 1 case of 7q11.23 microduplication syndrome. All 9 fetuses with chromosome abnormalities detected by karyotyping were missed by BoBs, including 2 cases of marker chromosomes,4 cases of chromosomal translocation,1 case of chromosomal inversion, 2 cases of Sex chromosome mosaicism; 2 cases of fetal inherited from the parents,7 cases for novel mutations. CONCLUSION: Karyotype analysis combined with BoBs dedtection is a rapid, effective and highly accurate prenatal diagnosis model that may should be widely used in clinical diagnosis.

[The value of a novel prenatal diagnosis model with combination of karyotyping and BACs-on-BeadsTM technique].

OBJECTIVE: To explore the value of a novel prenatal diagnosis model using combined chromosomal karyotyping and BACs-on-BeadsTM(BoBs), a newly-developed technique. METHODS: 1048 single pregnancy pregnant women with various diagnostic indications were performed amniocentesis for prenatal diagnosis with karyotyping and BoBs simultaneously. RESULTS: Among 1047 successfully cultured specimens, 50 chromosomal abnormalities were identified with BoBs, including 43 common chromosomal trisomies, 3 chimeric chromosomes and 4 structural abnormalities, of which 3 microdeletions/microduplications were not detected with karyotyping. Except for extra yield of 1 Robertsonian translocation, the other numerical chromosomal abnormalities were detected with both karyotyping and BoBs. Ten fetal chromosome abnormalities were confirmed with karyotyping, including 8 structural abnormalities and 2 chimeric chromosomes. CONCLUSION: Combination of karyotyping and BoBs turned out to be a rapid, complementary and effective diagnostic model for fetal chromosomal abnormalities and microdeletion syndromes, which could yield a higher detection rate of fetal chromosomal abnormalities and chromosomal microdeletions/microduplications.

Array-based molecular karyotyping in fetal brain malformations: Identification of novel candidate genes and chromosomal regions.

BACKGROUND: For the majority of congenital brain malformations, the underlying cause remains unknown. Recent studies have implicated rare copy number variations (CNVs) in their etiology. METHODS: Here, we used array-based molecular karyotyping to search for causative CNVs in 33 fetuses of terminated pregnancies with prenatally detected brain malformations and additional extracerebral anomalies. RESULTS: In 11 fetuses, we identified 15 CNVs (0.08 Mb to 29.59 Mb), comprising four duplications and eleven deletions. All larger CNVs (> 5 Mb) had also been detected by prenatal conventional karyotyping. None of these CNVs was present in our 1307 healthy in-house controls (frequency < 0.0008). Among these CNVs, we prioritized six chromosomal regions (1q25.1, 5q35.1, 6q25.3-qter, 11p14.3, 15q11.2-q13.1, 18q21.1) due to their previous association with human brain malformations or owing to the presence of a single gene expressed in human brain. Prioritized genes within these regions were UBTD2, SKA1, SVIP, and, most convincingly, GPR52. However, re-sequencing of GPR52 in 100 samples from fetuses with brain malformations or patients with intellectual disability and brain malformations revealed no disease-causing mutation. CONCLUSION: Our study suggests chromosomal regions 1q25.1, 5q35.1, 6q25.3-qter, 11p14.3, 15q11.2-q13.1, and 18q21.1 to be involved in human brain development. Within three of these regions, we suggest UBTD2, GPR52, and SKA1 as possible candidate genes. Because the overall detection rate of array-based molecular karyotyping was slightly higher (23%) than that of conventional prenatal karyotyping (20%), we suggest it's use for prenatal diagnostic testing in fetuses with nonisolated brain malformations.

[Classifier of domestic pig (Sus scrofa domesticus) automatic chromosome identification].

Automated chromosome classification is an essential task in cytogenetics of animals and plants. Until now the automatic karyotyping systems were obtained only for human chromosomes. The main aim of this study was to develop the automatic pig chromosome classifier using image processing software "VideoTest-Karyo 3.1". To solve this problem 1578 chromosomes from 47 metaphases were used. The constructed classifier was checked with the use of additional sample of metaphases classified in fully automatic regime: error rate was 8.2%, this corresponds to 3.12 ± 0.26 errors per metaphase plate (these values are within acceptable limits for such kind of studies). In further studies the extra sample of pig acrocentric chromosomes was added to classifier up to 1807 chromosomes. This addition reduced the error rate up to 6.1%, which correspondes to 2.78 ± 0.18 errors per metaphase plate. It should be underlined that the revealed errors can immediately be corrected by an operator on every stage of analysis. The classifier was also verified using the chromosomes of boar with rcp(1p-; 11p+) in fully automatic regime and routine stained metaphases of Siberian minipigs with rob(16;17) in semi automatic regime. In both cases the chromosomes were identified correctly. The area of application of obtained pig automatic chromosome classifier is discussed.

The impact of the condenser on cytogenetic image quality in digital microscope system.

BACKGROUND: Optimizing operational parameters of the digital microscope system is an important technique to acquire high quality cytogenetic images and facilitate the process of karyotyping so that the efficiency and accuracy of diagnosis can be improved. OBJECTIVE: This study investigated the impact of the condenser on cytogenetic image quality and system working performance using a prototype digital microscope image scanning system. METHODS: Both theoretical analysis and experimental validations through objectively evaluating a resolution test chart and subjectively observing large numbers of specimen were conducted. RESULTS: The results show that the optimal image quality and large depth of field (DOF) are simultaneously obtained when the numerical aperture of condenser is set as 60%-70% of the corresponding objective. Under this condition, more analyzable chromosomes and diagnostic information are obtained. As a result, the system shows higher working stability and less restriction for the implementation of algorithms such as autofocusing especially when the system is designed to achieve high throughput continuous image scanning. CONCLUSIONS: Although the above quantitative results were obtained using a specific prototype system under the experimental conditions reported in this paper, the presented evaluation methodologies can provide valuable guidelines for optimizing operational parameters in cytogenetic imaging using the high throughput continuous scanning microscopes in clinical practice.

Diagnóstico prenatal y array-CGH II: gestaciones de bajo riesgo / Array-CGH in prenatal diagnosis II: low-risk pregnancies

Recientemente, la tecnología conocida como array-CGH se ha establecido como una herramienta diagnóstica de primer orden para el estudio de pacientes con anomalías congénitas, retraso mental no filiado y otras enfermedades neurológicas. Sin embargo, su utilidad como técnica de primer uso en el campo prenatal está actualmente en fase de evaluación, especialmente en embarazos de bajo riesgo. En una población de 530 gestantes con embarazos de bajo riesgo se realizó, simultáneamente, cariotipo convencional y un estudio de array-CGH para el diagnóstico prenatal. Mientras que el cariotipo detectó 3 casos (0,5%) con alteraciones citogéneticas no equilibradas (una de ellas no definida), el array-CGH detectó 8 casos con este tipo de alteraciones (1,5%), identificando el cambio indefinido detectado por cariotipo. Este estudio demuestra positivamente que el array-CGH puede ser una herramienta útil en el diagnóstico prenatal en embarazos de bajo riesgo(AU)

The array-CGH technique has recently been established as a first-tier diagnostic test for studying patients with congenital anomalies, idiopathic mental retardation and other neurological disorders. However, its use in prenatal diagnosis is still being evaluated, especially in low-risk pregnancies. A study was conducted on a population of 530 low-risk pregnancy women using both conventional karyotype and array-CGH for prenatal diagnosis. Whereas conventional karyotype detected 3 foetuses (0.5%) with unbalanced cytogenetic aberrations (one of them was undefined), array-CGH detected 8 foetuses with copy number aberrations (1.5%), and positively identified the undefined cytogenetic aberration detected using karyotype. In conclusion, this study proposes array-CGH as a useful tool in prenatal diagnosis for low-risk pregnancies(AU)

Bone marrow cytogenetics workup: Application of lean management system to determine if additional cell workup is helpful and necessary to analysis.

INTRODUCTION: High workload volumes in a Cytogenetics laboratory can lead to long result turn-around times (TAT). This study aimed to improve laboratory efficiency by adopting Lean Management System initiatives to increase productivity through the elimination of wastes. This study examined if the prerequisite 20-cell analysis was sufficient for a conclusive result or if additional cell workup was necessary to ascertain the presence of a previous chromosome abnormality among cases on follow-up, or when a single abnormal cell was encountered during the analysis to determine the presence of a clone. MATERIALS AND METHODS: The karyotype results of cases that had additional workup were retrieved from among 8040 bone marrow cases of various haematological disorders performed between June 2003 and June 2008. RESULTS: Of 8040 cases analysed, 2915 cases (36.3%) had additional cell workup. Only 49 cases (1.7%) led to the establishment of a clone. The majority of these cases could have been resolved without the additional workup, especially if fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR)-based assays had been utilised. CONCLUSION: This study shows that the additional workup procedure is redundant. The time saved by discontinuing the workup procedure can be used to analyse other cases, leading to increased laboratory efficiency and a faster TAT without compromise to patient care. The practice of additional workup over and above the 20- cell analysis should be dispensed with as little benefit was derived for the amount of additional manpower expended. FISH or PCR-based assays should be utilised to elucidate a case further.

Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250K Nsp, and SNP6.0 arrays.

Specific chromosomal alterations are recognized as important prognostic factors in chronic lymphocytic leukemia (CLL). Array-based karyotyping is gaining acceptance as an alternative to the standard fluorescence in situ hybridization (FISH) panel for detecting these aberrations. This study explores the optimum single nucleotide polymorphism (SNP) array probe density for routine clinical use, presents clinical validation results for the 250K Nsp Affymetrix SNP array, and highlights clinically actionable genetic lesions missed by FISH and conventional cytogenetics. CLL samples were processed on low (10K2.0), medium (250K Nsp), and high (SNP6.0) probe density Affymetrix SNP arrays. Break point definition and detection rates for clinically relevant genetic lesions were compared. The 250K Nsp array was subsequently validated for routine clinical use and demonstrated 98.5% concordance with the standard CLL FISH panel. SNP array karyotyping detected genomic complexity and/or acquired uniparental disomy not detected by the FISH panel. In particular, a region of acquired uniparental disomy on 17p was shown to harbor two mutated copies of TP53 that would have gone undetected by FISH, conventional cytogenetics, or array comparative genomic hybridization. SNP array karyotyping allows genome-wide, high resolution detection of copy number and uniparental disomy at genomic regions with established prognostic significance in CLL, detects lesions missed by FISH, and provides insight into gene dosage at these loci.

Computer simulations improve university instructional laboratories.

Laboratory classes are commonplace and essential in biology departments but can sometimes be cumbersome, unreliable, and a drain on time and resources. As university intakes increase, pressure on budgets and staff time can often lead to reduction in practical class provision. Frequently, the ability to use laboratory equipment, mix solutions, and manipulate test animals are essential learning outcomes, and "wet" laboratory classes are thus appropriate. In others, however, interpretation and manipulation of the data are the primary learning outcomes, and here, computer-based simulations can provide a cheaper, easier, and less time- and labor-intensive alternative. We report the evaluation of two computer-based simulations of practical exercises: the first in chromosome analysis, the second in bioinformatics. Simulations can provide significant time savings to students (by a factor of four in our first case study) without affecting learning, as measured by performance in assessment. Moreover, under certain circumstances, performance can be improved by the use of simulations (by 7% in our second case study). We concluded that the introduction of these simulations can significantly enhance student learning where consideration of the learning outcomes indicates that it might be appropriate. In addition, they can offer significant benefits to teaching staff.

Description of an ultrathin multiwire proportional chamber-based detector and application to the characterization of the Spraguea lophii (Microspora) two-dimensional genome fingerprint.

Multiwire proportional chamber is a useful technology to build detectors that supersede the lack of interactivity of autoradiography in molecular biology experiments. Some drawbacks still limited the diffusion of existing instruments in biological laboratories. The major competitors are storage phosphor imaging systems. The simplified description of a radio-chromato-imager prototype (RCI) based on an original ultrathin multiwire proportional chamber is presented. It combines the advantage of the different existing technologies to present competitive properties in terms of efficiency, spatial resolution, robustness, manipulation easiness and production cost. Application of the RCI detector to molecular biology was performed by the analysis of karyotype and restriction display two-dimensional pulsed-field gel electrophoresis (KARD 2-D PFGE) data which are used to describe small eukaryotic genome structures. The comparative analysis with autoradiography was performed with the PDQuest software on Spraguea lophii (Microspora) genome fingerprints. The spot detection procedure applied to the different images leads to a similar conclusion considering the genome structure of S. lophii which appeared to be composed of 15 chromosomes for 13 karyotypic bands (200-880 kbp).

MicroMeasure: a new computer program for the collection and analysis of cytogenetic data.

The ability to identify individual chromosomes in cytological preparations is an essential component of many investigations. While several computer software applications have been used to facilitate such quantitative karyotype analysis, most of these programs are limited by design for specific types of analyses, or can be used only with specific hardware configurations. MicroMeasure is a new image analysis application that may be used to collect data for a wide variety of chromosomal parameters from electronically captured or scanned images. Unlike similar applications, MicroMeasure may be individually configured by the end user to suit a wide variety of research needs. This program can be used with most common personal computers, and requires no unusual or specific hardware. MicroMeasure is made available to the research community without cost by the Department of Biology at Colorado State University via the World Wide Web at http://www.biology.colostate.edu/MicroMeasure.

Sensitivity of multiple color spectral karyotyping in detecting small interchromosomal rearrangements.

Multiple color spectral karyotyping (SKY) has been proven to be a very useful tool for characterization of the complex rearrangements in cancer cells and the de novo constitutional structural abnormalities. The sensitivity of SKY in detecting interchromosomal alterations was assessed with 10 constitutional translocations involving subtelomeric regions. Among the 13 small segments tested, 9 were clearly visualized and 8 were unambiguously identified by SKY. Fluorescence in situ hybridizations (FISH) with subtelomeric probes confirmed the reciprocity in three of the four translocations in which a small segment was not detectable by SKY. On the basis of resolution level of G-banding and the information obtained from the FISH analysis, the minimum alteration that SKY can detect is estimated to be 1,000-2,000 kbp in size with the currently available probes. This study has demonstrated the power, but also the limitations, of SKY in detecting small interchromosomal alterations, particularly those in subtelomeric regions.

[Analysis program for quantitative detection of chromosome aberrations using comparative genomic hybridization]. / Analyseprogramm zur quantitativen Erfassung chromosomaler Aberrationen mittels komparativer genomischer Hybridisierung (CGH).

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for the detection of chromosomal imbalances between a tumor and a normal genome. In order to produce quantitative and reproducible results, we developed an image analysis program that allows the detection and mapping of the genetic alterations. The result is represented as a CGH sum karyogram in which the genetic changes are documented as color coded-chromosomes. The aim of our investigation is to correlate the genotype with the phenotype on the basis of CGH sum karyograms and thus to achieve a genetic characterization that will supplement the morphological tumor description.

Bivariate flow karyotyping with air-cooled lasers.

An experimental flow cytometer was constructed using a quartz flow cell optically coupled to a 1.22 NA lens. A pair of crossed cylindrical quartz lenses allowed multilaser excitation. Two helium-cadmium (HeCd) lasers, emitting 16 mW at 442 nm and 35 mW at 325 nm, were used to excite chromomycin A3 and Hoechst 33258 fluorescence, respectively. Bivariate flow karyotypes from normal human male chromosomes and from the Daudi cell line were obtained and were compared to those from a standard instrument using dual water-cooled lasers. The new experimental instrument exhibited comparable resolution to that from the standard instrument. In further experiments with Daudi chromosomes, the 35 mW HeCd laser was replaced with a 10 mW HeCd laser, and the system still gave good, though slightly decreased, resolution.