Hans Vrolijk

Automated Acquisition of Stained Tissue Microarrays for High-Throughput Evaluation of Molecular Targets

At present, limiting factors in the use of tissue microarrays (TMAs) for high-throughput analysis relate to the visual evaluation of the staining patterns of each of the individual cores in the array and to the subsequent input of the results into a database. Such a database is essential to correlate the data with tumor type and outcome, and to evaluate the performance against other markers achieved in separate experiments. So far, these steps are mostly performed by hand, and consequently are time-consuming and potentially prone to bias and errors, respectively. This paper describes the use of a high-resolution flat-bed scanner for digitization of TMAs with a resolution of about 5 x 5 micro m(2). The arrays are acquired, the positions of the tissue cores are automatically determined, and measurement data including the images of the individual cores are archived. The program provides digital zooming of arrays for interactive verification of the results and rapid linkage of individual core images to data sets of other markers derived from the same array. Performance of the system was compared to manual classification for a representative set of arrays containing colorectal tumors stained with different markers.

Comprehensive analysis of human subtelomeres with combined binary ratio labelling fluorescence in situ hybridisation

Cryptic subtelomeric chromosome rearrangements play an important role in the aetiology of mental retardation, congenital anomalies, miscarriages and neoplasia. To facilitate a comprehensive molecular–cytogenetic analysis of these extremely gene-rich and mutation-prone chromosome regions, novel multicolour fluorescence in situ hybridisation (FISH) techniques are being developed. As yet, subtelomeric FISH methods have either had limited multiplicities, making it necessary to perform many hybridisations per patient, or a limited scope of analysable chromosome mutation types, thus not detecting some aberration types such as pericentric inversions or very small aberrations. COBRA (COmbined Binary RAtio) labelling is a generic multicolour FISH technique that combines ratio and combinatorial labelling to attain especially high multiplicities with few fluorochromes. The Subtelomere COBRA FISH method (‘S-COBRA FISH’) described here detects efficiently all 41 BAC and PAC FISH probes necessary for a complete subtelomere screening in only two hybridisations. It was applied to the analysis of 10 cases with known and partially known aberrations and successfully detected balanced and unbalanced translocations, deletions and an unbalanced pericentric inversion in a mosaic situation. The ability of S-COBRA FISH to efficiently detect all types of balanced and unbalanced subtelomeric chromosome aberrations makes it the most comprehensive diagnostic procedure for human subtelomeric chromosome regions described to date.

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.

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 fluorescent dot counting in cell nuclei

We have developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 20 minutes 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 (e.g. FITC) and one for the cell nucleus (e.g. DAPI). After the stage has moved to a new field the image is automatically focused, acquired by a Photometrics KAF 1400 camera, and then analyzed on a Macintosh Quadra 840AV 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 machine accuracies are equal to panels of human experts (manual) and limited by the overlapping of dots in the 3D cell as seen through the 2D projection.

Order of human hematopoietic growth factor and receptor genes on the long arm of chromosome 5, as determined by fluorescence in situ hybridization

SummaryA large number of human hematopoietic growth factor and growth factor receptor genes are localized at the long arm of chromosome 5. In this study we have determined the order of the human interleukin-3 (IL3), IL4, IL5, IL9, granulocyte macrophage-colony stimulating factor (GMCSF), and the MCSF receptor (MCSFR) genes by fluorescence in situ hybridization. Genomic λ-clones were isolated using polymerase chain reaction (PCR)-generated probes and labeled with biotin and/or digoxigenin. These clones were first individually mapped: IL3, IL4, IL5, IL9, and GMCSF to 5q31 and MCSFR to 5q33. For ordering purposes multiple probe combinations were hybridized to metaphase chromosomes and interphase nuclei. The interphase hybridizations were evaluated by image analysis, which also allowed the measurement of the physical distance between the hybridization spots. These mapping results suggest the gene order 5cen-IL3/GMCSF-IL5-IL4-IL9-MCSFR-qter. The known genomic distance between the IL4 and IL5 genes allowed the estimation of the physical distances between the 5q31-specific genes, demonstrating that they are all within about 1.5 Mb of DNA.

Effect of chromatic errors in microscopy on the visualization of multi-color fluorescence in situ hybridization.

The relevant microscopical conditions for the optimal visualization of ratio-color FISH stained cells were investigated. Special attention was given to the influence of the type of illumination, (semi)-critical vs. Köhler type illumination, in combination with the use of multi-band excitation and emission filters, on the registration of the colors of ratio labelled probes. Due to chromatic errors, many collecting lenses were found to cause a wavelength dependent excitation pattern with critical illumination. This resulted in a change of the observed color of microscopic objects when stained with a mixture of two dyes and excited with a dual band pass filter. A quantitative study of this effect for semi-critical illumination of FISH ratio-labelled chromosomes revealed a difference of 20% between highest and lowest ratio values depending on the position of the object in the microscopic field vs. only 2.5% for Köhler type of illumination. The impact of these errors on the identification of ratio-labelled probes and on the sensitivity of comparative genomic hybridization (CGH) to detect gene amplifications or losses is discussed. Standard preparations consisting of solutions of defined mixtures of fluorescent dyes or objects stained with defined ratios of fluorophores, are proposed to correct for the errors observed.

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.

Microscopy and image analysis of fibre‐FISH

In this paper the aspects of image acquisition, processing and analysis for DNA-fibre mapping are described. As the nature and the quality of the fibre-FISH signals (given its resolution range of 1–500 kb) may vary to a great extent, an interactive approach was chosen for the selection and analysis of the fibres. The accuracy of this fibre-FISH mapping approach was compared with restriction mapping on the basis of a map of seven cosmid contigs from the thyroglobulin gene, which spans about 300 kb. The results were in full agreement with restriction mapping. Standard errors for sizes of the cosmids, gaps, and overlaps were obtained between 2.0 and 6.2 kb. By alternately labelling the clones of the DNA map a colour barcode can be composed which eases the identification of gene rearrangements, as is illustrated on two patients with a deletion in the Duchenne muscular dystrophy (DMD) gene. The time needed for straightening a fibre and defining the distances between the different cosmids is dominated by the amount of human interaction and typically takes 1–2 min. From this study it is clear that fibre-FISH analysis is well suited for mapping cosmid contigs and defining breakpoints in patient material with the same or better accuracy as restriction mapping and PCR analysis.

Detection of tumor cells in bone marrow, peripheral blood and lymph nodes by automated imaging devices

The presence of tumor cells in bone marrow, peripheral blood and lymph nodes has proven its clinical and prognostic value. Since the frequency of these cells in bone marrow and blood is sometimes as low as 1 per million and due to the fact that for the analysis of lymph nodes many sectioning levels have to be analyzed, automated imaging devices have been suggested as an useful alternative to conventional manual screening of specimens. The aim of this paper is to review the performance of current equipment that is commercially available, based on literature published so far. Requirements for introducing this equipment for routine clinical practice are discussed.