Whole-genome array-CGH screening in undiagnosed syndromic patients: old syndromes revisited and new alterations.

We report array-CGH screening of 95 syndromic patients with normal G-banded karyotypes and at least one of the following features: mental retardation, heart defects, deafness, obesity, craniofacial dysmorphisms or urogenital tract malformations. Chromosome imbalances not previously detected in normal controls were found in 30 patients (31%) and at least 16 of them (17%) seem to be causally related to the abnormal phenotypes. Eight of the causative imbalances had not been described previously and pointed to new chromosome regions and candidate genes for specific phenotypes, including a connective tissue disease locus on 2p16.3, another for obesity on 7q22.1-->q22.3, and a candidate gene for the 3q29 deletion syndrome manifestations. The other causative alterations had already been associated with well-defined phenotypes including Sotos syndrome, and the 1p36 and 22q11.21 microdeletion syndromes. However, the clinical features of these latter patients were either not typical or specific enough to allow diagnosis before detection of chromosome imbalances. For instance, three patients with overlapping deletions in 22q11.21 were ascertained through entirely different clinical features, i.e., heart defect, utero-vaginal aplasia, and mental retardation associated with psychotic disease. Our results demonstrate that ascertainment through whole-genome screening of syndromic patients by array-CGH leads not only to the description of new syndromes, but also to the recognition of a broader spectrum of features for already described syndromes. Furthermore, on the technical side, we have significantly reduced the amount of reagents used and costs involved in the array-CGH protocol, without evident reduction in efficiency, bringing the method more within reach of centers with limited budgets.

Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents.

BACKGROUND: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. OBJECTIVE AND METHODS: Array-CGH with approximately 3500 large insert clones spaced at approximately 1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. RESULTS: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. CONCLUSIONS: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

Supervised automated microscopy increases sensitivity and efficiency of detection of sentinel node micrometastases in patients with breast cancer.

AIMS: To investigate the practicality and sensitivity of supervised automated microscopy (AM) for the detection of micrometastasis in sentinel lymph nodes (SLNs) from patients with breast carcinoma. METHODS: In total, 440 SLN slides (immunohistochemically stained for cytokeratin) from 86 patients were obtained from two hospitals. Samples were selected on the basis of: (1) a pathology report mentioning micrometastases or isolated tumour cells (ITCs) and (2) reported as negative nodes (N0). RESULTS: From a test set of 29 slides (12 SLN positive patients, including positive and negative nodes), 18 slides were scored positive by supervised AM and 11 were negative. Routine examination revealed 17 positive slides and 12 negative. Subsequently, automated reanalysis of 187 slides (34 patients; institute I) and 216 slides (40 patients; institute II) from reported node negative (N0) patients showed that two and seven slides (from two and five patients, respectively) contained ITCs, respectively, all confirmed by the pathologists, corresponding to 5.9% and 12.5% missed patients. In four of the seven missed cases from institute II, AM also detected clusters of four to 30 cells, but all with a size < or = 0.2 mm. CONCLUSIONS: Supervised AM is a more sensitive method for detecting immunohistochemically stained micrometastasis and ITCs in SLNs than routine pathology. However, the clinical relevance of detecting cytokeratin positive cells in SLNs of patients with breast cancer is still an unresolved issue and is at the moment being validated in larger clinical trials.

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.

FISH and chips: automation of fluorescent dot counting in interphase cell nuclei.

Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analyzed, especially when the frequency of aberrant cells is low. Automation of dot counting is required because manual counting is tedious, fatiguing, and time-consuming. We developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes (seen as dots) in each cell nucleus. This system works with two fluorescent dyes, one for the DNA hybridization dots and one for the cell nucleus. After the stage has moved to a new field, the image is automatically focused, acquired by a Photometrics KAF 1400 camera (Photometrics Ltd., Tuscon, AZ, USA), and then analyzed on a Macintosh Quadra 840AV (Apple Computer, Inc., Cupertino, CA, USA) computer. After the required number of cells has been analyzed, the user may interact to correct the computer by working with a gallery of the cell images. The automated dot counter has been tested on a number of normal specimens where 4,'6-diamidino-2-phenylindole (DAPI) was used for the nucleus counterstain and a centromeric 8 probe was used to mark the desired chromosome. The slides contained lymphocytes from cultured blood. We compared the results of the dot counter with manual counting. Manually obtained results, published in the literature, were used as the "ground truth." For a normal specimen, 97.5% of cells will have two dots. Fully automated scanning of 13 slides showed that an average of 89% of all nuclei were counted correctly. In other words, an average of 11% has to be interactively corrected, using a monitor display. The machine accuracies, after interactive correction, are comparable to panels of human experts (manual). The fully automatically obtained results are biased with respect to manual counting. An error analysis is given, and different causes are discussed.

Automated assessment of numerical chromosomal aberrations in paraffin embedded prostate tumor cells stained by in situ hybridization.

We investigated the feasibility of automated counting of in situ hybridization signals (ISH) in interphase cells isolated from paraffin embedded prostate tissue. In total, 34 specimens from 7 patients with prostate cancer were stained with probes specific for the centromeric regions of chromosomes Y, 1, 7, 8, 10, and 15, using an immunoperoxidase based technique suitable for bright-field microscopy. Enumeration of the number of ISH spots of 500 nuclei per specimen was performed (1) using an automatic system developed without any human intervention and (2) using the same system, but including verification of the counts based on visual inspection of the stored images. As reference from each specimen, 200 cell nuclei were evaluated manually, using conventional microscopy. A typical analysis procedure (including user verification) took 35 min. The difference (root mean error) between the automated counting and the counting after visual interaction was relatively small (15%). The percentage of cells with incorrect counts by automated analysis was 20.2%, a number that could easily be improved by user interaction. Detection of cells with aneusomy proved to be more sensitive compared to the routine manual counting, in cases where aberrant frequencies were low. Automated counting of samples with low frequencies (< 10%) resulted in a higher frequency of aberrant cells in 9 of 11 cases, probably due to the fact that an unbiased cell selection is guaranteed. Automated assessment of ISH signals is considered useful for the evaluation of chromosomal aberrations in prostate tumor cells, provided that the counts are visually confirmed.

Sample preparation and in situ hybridization techniques for automated molecular cytogenetic analysis of white blood cells.

With the advent of in situ hybridization techniques for the analysis of chromosome copy number or structure in interphase cells, the diagnostic and prognostic potential of cytogenetics has been augmented considerably. In theory, the strategies for detection of cytogenetically aberrant cells by in situ hybridization are simple and straightforward. In practice, however, they are fallible, because false classification of hybridization spot number or patterns occurs. When a decision has to be made on molecular cytogenetic normalcy or abnormalcy of a cell sample, the problem of false classification becomes particularly prominent if the fraction of aberrant cells is relatively small. In such mosaic situations, often > 200 cells have to be evaluated to reach a statistical sound figure. The manual enumeration of in situ hybridization spots in many cells in many patient samples is tedious. Assistance in the evaluation process by automation of microscope functions and image analysis techniques is, therefore, strongly indicated. Next to research and development of microscope hardware, camera technology, and image analysis, the optimization of the specimen for the (semi)automated microscopic analysis is essential, since factors such as cell density, thickness, and overlap have dramatic influences on the speed and complexity of the analysis process. Here we describe experiments that have led to a protocol for blood cell specimen that results in microscope preparations that are well suited for automated molecular cytogenetic analysis.

Automation of spot counting in interphase cytogenetics using brightfield microscopy.

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5-10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction.

A system for fluorescence metaphase finding and scoring of chromosomal translocations visualized by in situ hybridization.

Full automation on the scoring of radiation-induced chromosomal aberrations in conventionally stained metaphase spreads cannot be achieved reliably due to the complex image analysis problems involved. More success may be obtained by using in situ hybridization staining of the chromosomes. We describe the development of a system to detect metaphases on the basis of a fluorescent counterstain and subsequently analyze the number of translocations with the aid of whole chromosome paints fluorescing in a different colour. The system consists of a Macintosh IIfx computer, an automated Ergolux microscope equipped for fluorescence, and a Sony CCD camera. The performance of the metaphase finder was measured on a small set of slides counterstained with DAPI, whereas the suitability of the system for scoring aberrations was tested in a small feasibility study for the detection or radiation-induced translocations involving chromosome 4. The potentialities of the system for the use of multiple colours are discussed.

Automated image cytometry for detection of rare, viral antigen-positive cells in peripheral blood.

A cell detection method based upon automated screening is described for recognition of low frequencies (1 in 100,000) of immuno-enzymatically labelled white blood cells in human peripheral blood. The used image cytometry instrumentation (LEYTAS) includes a wide-field, fully automated microscope (Autoplan) and a modular image analysis computer (MIAC), both from Leica, Wetzlar, Germany. The MIAC contains image boards for optimum use of mathematical morphology algorithms. Communication with the MIAC is via a personal computer. Programs for automated cell analysis have been written in C language. Main features of the system are fast analysis of large microscope fields including a count of all cells, selection of objects of interest (alarms), and display of digitally stored images of these alarms. We tested this system for the detection of white blood cells expressing antigen of cytomegalovirus (pp65) in 50 human blood smears from kidney transplant recipients. Immuno-enzymatic (peroxidase) staining was performed with DAB and counterstaining with hematoxylin. For determination of the sensitivity, a series of dilutions of a positive sample with a negative sample was performed. The lowest frequency detected was 1 antigen-positive cell/3 x 10(5) antigen-negative cells. Screening time was about 60 min for one million cells.

Applicability of a noncooled video-rated CCD camera for detection of fluorescence in situ hybridization signals.

Cooled CCD cameras provide good sensitivity and linearity with a high dynamic range and are therefore well suited for quantification of fluorescence in situ hybridization signals. However, for a fraction of the cost, conventional noncooled, video-rated CCD cameras can also be applied for most applications in the field of fluorescence in situ hybridization, provided that they allow for longer integration times. This paper describes the use of the Sony camera, model XC-77RR-CE, for this purpose. Tests were carried out to compare the dark current, linearity, and signal-to-noise ratio of this camera with a Photometrics cooled CCD camera model KAF 1400, and the suitability for quantitative measurements was investigated on a model system of fluorescent beads. It is shown that if the dark current of the video-rated camera is internally corrected, integration times of up to 30 s can be used; under such conditions good linearity is maintained. The camera was found suitable for the detection of in situ hybridization spots in interphase nuclei using centromere-specific probes. The fast readout rate of the camera offers interesting facilities for quickly locating objects with relatively strong fluorescence, such as counterstained metaphases. The less intense probe signals may then be analyzed at higher magnification.