Instrumentation For Fish

Although FISH (see Chapter 17) represents one of the most exciting and clinically significant developments of the last decade, most of the steps involved in preparing samples for analysis are unremarkable and often repetitive and, therefore, lend themselves to automation. When one considers the enormous increase in FISH sample volume most cytogenetics laboratories are experiencing, any device that can reduce the labor component of the process becomes indispensable.

Fig. 5. VP 2000 processor. The device automates various laboratory protocols, such as a pretreatment or deparaffinization step prior to performing a FISH assay. (Courtesy of Vysis, Inc.)

Pretreatment

For many applications of FISH, the only thing one must do to prepare a sample for analysis is make one or more additional slides or, in some cases, destain a slide that has already been examined. However, newer applications of the technology (e.g., HER2 analysis for breast cancer or ploidy analysis for bladder cancer recurrence; see Chapter 17) utilize specimen types not routinely handled in the cytogenetics laboratory, such as slides cut from paraffin blocks or made from bladder wash/urine samples. Such sample types require deparaffinization or other pretreatment before any FISH procedure can be performed. Although not difficult or complicated, these procedures are repetitive and time-consuming. Fortunately for the laboratory, devices that automate such steps have been developed (see Fig. 5). These devices also offer the laboratory the flexibility of performing other FISH pretreatment procedures, and they can even be programmed to perform certain routine cytogenetic or cytological procedures, making them more cost efficient for certain institutions. This can be significant, as these instruments are not inexpensive.

Hybridization

As with any DNA hybridization procedure, FISH requires a series of heating and cooling steps to facilitate denaturation and renaturation/hybridization of probe and target DNA. Analogous to the thermocyclers utilized for the polymerase chain reaction (PCR) in the molecular genetics laboratory, devices are available that permit a technologist to add FISH probes to a sample slide, close the cover, initiate a preprogrammed series of temperature changes, and walk away. These instruments can handle

Fig. 6. HYBrite denaturation/hybridization system. Up to 12 slides can be placed in the device, which can be programmed to heat and cool as required for various FISH protocols. (Courtesy of Vysis, Inc.)

a modest number of slides at one time to facilitate volume testing, and they can store several user-defined programs for analytical flexibility. (See Fig. 6.)

The drawback to these devices is the large volume or frequent use of probes that require different programming, necessitating the purchase of more than one unit. They have, however, come down in price in recent years.

AUTOMATED IMAGING SYSTEMS Introduction

The traditional method of imaging chromosomes has always been photomicrography. A photograph of metaphase chromosomes is taken, the film is developed and photographs are printed in a darkroom, and the chromosomes are cut out and arranged to form a karyotype. Although a standard technique for a long time, this process increases the already time-consuming nature of clinical cyto-genetics. Because of increasing workload in cytogenetics laboratories around the world, automated imaging is increasing in popularity.

Automated imaging systems dramatically reduce the time it takes to produce a karyotype, and therefore can be seen as one of the most important developments in automation of the cytogenetics laboratory. Furthermore, the growth in fluorescent techniques such as multicolor FISH, interphase FISH, and comparative genomic hybridization (CGH) can also be attributed to automated imaging. (See Chapter 17.)

Currently, the primary application of an imaging system in a cytogenetics laboratory is still the production of karyotypes, either from brightfield (G-band) or fluorescence (Q-band or R-band) images, although the use of automated imaging systems in FISH (painting probes, single-locus probes, multicolor FISH, CGH, etc.) is rapidly gaining popularity. Rare event detection (e.g., automated metaphase finding or fluorescent spot counting) also represents a growing application for imaging systems in cytogenetics.

Benefits

Reduction in the time it takes to complete an analysis is unquestionably the major benefit of an automated imaging system. Laboratories can save operator time by automating metaphase scanning, karyotyping, and FISH applications, resulting in a faster turnaround time and higher throughput of cases. Reduction in labor also translates to reduced costs.

Another big advantage of digital images is easy and compact storage. Some states require storage of patient cases for up to 100 years! With current compression technologies and digital storage devices, this is easier and less space-consuming than with photographs. In addition, photographs can deteriorate over time, making them harder to re-examine if necessary.

Automated imaging systems also provide consistency, especially when performing interphase FISH assays. Whereas manual spot counting can be highly subjective and error-prone, an automated system will use predefined parameters for spot counting and, using those parameters, will produce consistent results.

Sharing of data is important in a clinical lab setting and is clearly facilitated by the use of digital images versus traditional photography. With the growing use of the Internet and electronic mail, digital images are more easily shared for consultation and discussion (2). However, with data sharing via the Internet comes the need for compression, and a more pressing need for patient record security. Partly to address this need for patient record security, the US Congress recently passed the Health Insurance Portability and Accountability Act (HIPAA) (J). See also Chapter 6.

Although traditional photographic techniques offer some degree of contrast and other image adjustment, automated imaging systems further offer easy image enhancements, visualization techniques, and quantification, providing additional information. As stated earlier, recent advances in cytoge-netic applications, especially in FISH applications such as M-FISH, CGH, and interphase FISH can be attributed to imaging systems.

Limitations

Of course, there are limitations to automation in the cytogenetics laboratory. Probably the greatest limitation is the interpretation of the karyotype or FISH results and the diagnosis based on the analysis of the image. This will still be a task for the Director. Another limitation is that despite image enhancement features, the quality of the final image is still dependent on the quality of the original microscope image (2). An image might be improved through background elimination, contrast, and color enhancement or even longer exposure times, but all of these will not make up for a poor image resulting from poor microscope configuration or slide preparation.

Imaging System Components

In general, an imaging system for cytogenetics contains the following components: a microscope with camera adapter, a camera, computer and software, a printer, and an archival device. (See Fig. 7.)

Microscope with Camera Adapter

A detailed discussion of microscopes and microscopy can be found in Chapter 5. As the name already implies, the camera adapter is the device designed to attach a camera to a microscope. This adapter also permits the microscope image to be projected onto the photosensitive area of the camera.

Camera

Although a wide range of camera options are available (analog, digital, cooled, uncooled, monochrome, color), the most commonly used camera on automated imaging systems for the cytogenetic laboratory is a black-and-white, uncooled CCD (charge-coupled device) camera (4).

Fig. 7. CytoVision automated chromosome imaging system. (Courtesy of Applied Imaging.)

Computer

Although both PC- and Macintosh-based systems have been available, the recent trend has been a move toward PC-based imaging systems. The computer(s) can be networked, allowing the actual analysis of the images to be performed off-line and to facilitate data sharing.

Software

The software for automated imaging systems for cytogenetics consists of at least two parts: acquisition or capture, and the actual analysis. These can be two distinct steps or can be seamlessly integrated into one application. The acquisition step drives the camera in order to take a digital picture (capture an image). It also includes image enhancement features such as contrast adjustment, background subtraction, and shading correction. After image capture and enhancement, the user can analyze the image using the analysis applications of the software.

Although there are many commercial packages available for image analysis, cytogenetics software, especially developed to address the specific requirements of a cytogenetics laboratory, includes several important features that are not available in conventional image analysis packages. Some of these features include the automatic generation of karyotypes and the automated scoring of interphase FISH slides.

Printer

A high-quality print of the image is still important in the cytogenetics laboratory. Although the trend might be moving to a so-called paperless laboratory, a hard-copy print is often needed to for diagnostic and/or archival purposes. In addition to the high-resolution black and white images of karyotypes prepared by all cytogenetics laboratories, a printer used for FISH applications must be capable of reproducing the range of colors generated by modern FISH software.

Archival Device

As mentioned earlier, there is a need (often legally imposed) for long-term archiving of patient data. With the use of automated imaging systems, the data are in digital form and are easier to store. There are three basic categories of archival devices for digital data: tape, optical disk, and magnetic disk. Which type of archival device is best depends on several factors, including the expected volume of data to store, the duration of storage, and how often the data need to be accessed in the future. Currently, DVDs are used more and more as the storage device of choice.

Cytogenetics Applications of Automated Imaging

Karyotyping

The predominant application of automated imaging systems is karyotyping. Karyotyping involves separating and classifying the chromosomes based on the length of the chromosome, location of the centromere, and the banding pattern (see Chapter 3). Automated systems for karyotyping need to provide at least the following benefits to the user: ease of use, speed, image quality, and accuracy (4).

Less automated systems require the user to "cut out" the chromosomes using the mouse and then to place them into a karyotype. In semiautomated systems, the system will "cut out" the chromosomes, and the user classifies them into a karyotype. On the other hand, a fully automated imaging system will capture the metaphase chromosomes (either brightfield for G-banding or fluorescent for Q-banding), separate or "cut out" the chromosomes, classify them, and arrange them in a karyotype (see Fig. 8). However, some metaphases contain very complex clusters of overlapping chromosomes, and the user might still need to intervene and manually separate the chromosomes using the mouse.

A fully automated karyotyping system can also be used in conjunction with a so-called metaphase finding capability. This means that the system will automatically scan the slide in search of good metaphase spread that can be used for karyotyping. This application will be discussed later in this chapter.

From a software perspective, automated karyotyping systems need to include the following capabilities:

• Separation of chromosomes. Chromosomes in a metaphase might be touching or overlapping, and the software will not be able to classify the individual chromosomes until they are separated. Cytogenetics software will include features such as "split," "overlap" and "draw axis" to allow for the segmentation of such chromosomes.

• Automatic classification of the chromosomes into a karyotype. Using pattern recognition, the system will assign the classification of each chromosome based on length of the chromosome, location of the centromere, and the banding pattern. However, in cases where there are chromosomal abnormalities, the system might not recognize a chromosome, so then the user can assign or change the classification.

• Image enhancement to facilitate the interpretation of the banding pattern. Image enhancement features include the ability to change the contrast and brightness to bring out the banding pattern.

Scanning and Metaphase Finding

Finding metaphases acceptable for analysis is an integral part of cytogenetics. In normal samples, good quality metaphases are abundant. However, in some samples, such as in cancer cytogenetics, cells are often of poor quality, and metaphase spreads acceptable for analysis are few and hard to find. A system that will automatically scan a slide for metaphase spreads can greatly reduce the time spent by a technologist on these samples looking for those metaphases.

The microscope in a metaphase finding system is outfitted with a motorized stage and focus drive for automated focusing. Although automatically scanning one slide saves the user time, it does not make much sense to continuously have to change slides for scanning. To increase the throughput of the system, many suppliers add a stage or even slide loader to the system that holds multiple slides

Metaphase Spreads
Fig. 8. An example of karyotyping software. The original metaphase is in the upper right. (Courtesy of Applied Imaging.)

(see Fig. 9). Based on several parameters, the system images metaphase spreads (fluorescent or brightfield) and presents them to the user for review and analysis (see Fig. 10).

Key factors for a metaphase finding system are the ability to recognize appropriate metaphases or cells, accuracy of relocation to a metaphase of interest, speed of scanning, and sensitivity (the percentage of metaphase cells found by the system).

Software features important for metaphase finding include the following:

• Definition of rare event classification parameters to ensure optimum scan results. The user can define the parameters that are utilized by the system to identify the rare event.

• Ability to quickly relocate to a metaphase or rare cell for review.

• Sort function to organize metaphases or cells after scanning based on specific parameters.

Because of the general nature of the scanning system, it can also be used in other applications that require scanning for particular cells (rare events), such as FISH spot counting (see below) for detection of tumor cells in body fluids.

Laboratories are using scanning systems more and more for streamlining their workflows. The systems are set up to continuously scan slides for metaphases or rare events while technologists are analyzing the detected metaphases or cells on remote review stations. This increases throughput while using the technologists' time where it is most valuable: analyzing cases.

Fig. 9. Automated slide scanning and metaphase finding system. (Courtesy of MetsSystems Group, Inc.)
Fig. 10. Software interface of a metaphase finding system, showing thumbnails of the metaphase spreads located by the system. (Courtesy of Applied Imaging.)

FISH and Fluorescent Spot Counting

Fluorescence in situ hybridization is based on fluorescently labeled probes that hybridize to unique DNA sequences along the chromosomes. There are many different applications of FISH; see Chapter 17 for more detail on this technology.

Fluorescence in situ hybridization can be performed on either metaphase preparations or interphase cells. One of the applications is fluorescent spot counting used for translocation and copy number analysis performed on interphase cells (see Fig. 11). An example of an interphase FISH kit is the Vysis UroVysion® kit for the detection of chromosomal abnormalities associated with the recurrence and progression of bladder cancer (see Chapter 17, Fig. 14).

Generally, an imaging system for FISH needs to be able to capture low-light-level images, quantify the number of each fluorescent signal, and estimate the intensity ratio of the different signals.

Because interphase cells are three-dimensional (3-D) structures, the fluorescent signals in interphase FISH and spot counting can be present in different focal planes. This means that to be able to see all signals, the user will need to focus on the different planes, making the presence of a motorized focus drive on an automated system imperative. The automated focusing allows for resolution of the multiple signals across a large focal depth. Images from different focal planes are captured, processed, and compiled into one pseudo-3-D image that shows all signals in focus. This 3-D image capture is often referred to as Z-stack.

To visualize the different fluorochromes, the system uses different bandpass filters and a single, epi-illuminating light source (see Chapter 5, Fig. 3). An image is acquired for each fluorescent label used in the protocol, and the computer combines those into a color image. If the system is not equipped with an automated microscope with motorized filter block changing, a motorized filter wheel that will hold the different filters is highly desirable (5,6). (See Fig. 12.)

The microscope focus, camera, and filter wheel are automatically controlled and synchronized by Z-stack software for multiplane, multicolor fluorescence image capture. Images in different focal planes are acquired and combined in a focused color image to ensure that faint signals that would otherwise be omitted are incorporated in subsequent analyses.

To ensure consistent scoring and analysis of interphase FISH, the software should include the following:

• Trainable classifiers to determine which cells to score, so users can "teach" the system to work with their own results and standards.

• User-definable parameters to determine the scoring rules. Such parameters include spot size and spot separation distances (measured three dimensional) and the number of cells to score.

• The ability to reprocess the images under different scoring rules without having to rescan the slide.

• A reporting function that presents the results for review by the clinician. Reports should be customizable to reflect the user's preferred data layout, and should include images of scored cells and different representations of the results, such as bar charts and scatter plots.

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