Fig. 7.1
Multiprep robotic harvester. This device was designed specifically for cytogenetics laboratories, with enhancements such as automatic fixative mixing, integral fume extraction, multiple dispensing, and aspiration probes to reduce the risk that blockage will ruin a harvest, and onboard programming, which eliminates the need for an external computer, reducing bench space requirements (Photo courtesy of Genial Genetic Solutions)
Robotic processors are also available for the blood/bone marrow harvest procedure. Because these devices must be capable of both liquid handling and centrifugation, they require a substantial amount of space and are designed to be freestanding rather than benchtop units (Fig. 7.2).
Fig. 7.2
Hanabi-PII metaphase chromosome harvester (Photo courtesy of Transgenomic, Inc.)
Drying Chambers/Slide Makers
Again, as described in Chap.4, the typical end product of the cytogenetic harvest is a centrifuge tube with fixed cells, both mitotic and nonmitotic. Spreading of chromosomes is achieved by placing one or more drops of this suspension on a number of microscope slides and is controlled by the height from which the suspension is dropped, the temperature and condition of the slide, and any number of manipulations while the slide is drying (including the ambient conditions in the laboratory). Results are monitored with phase contrast microscopy, and any slide that is not satisfactory can be discarded and replaced; trained individuals can determine the adjustments necessary to improve drying and spreading. Provided that such adjustments are made properly and quickly, running out of cell suspension is generally not a problem.
This is not always true, however, particularly with small volume bone marrow aspirates, and it is never the case with in situ culture and harvesting, typically utilized for prenatal diagnosis. Most cytogenetics laboratories initiate four to six in situ cultures from each amniotic fluid or CVS sample (see Chap. 12), depending on the condition of the specimen upon receipt. Regulations and good clinical sense require that cells from at least two of these are examined, and in many cases, three or more cultures are required. When one considers that at least one culture or some other form of backup should be retained against an unexpected need for additional testing, it becomes evident that every culture dish must produce useable metaphases. The concept of discarding one and trying again, possible in many cases when making slides from cell suspensions, does not apply. Further complication is introduced by the fact that the physical force generated by dropping the cells onto a glass slide is not available when in situ processing is used, and so spreading of chromosomes is accomplished solely by the manner in which the cultures are dried.
As the 3:1 absolute methanol:glacial acetic acid fixative used in cytogenetics laboratories dries, it “pulls” the cell membrane across the slide or coverslip with it, allowing the chromosomes of mitotic cells to separate. If this process is viewed with a phase contrast microscope, the metaphases appear to open much like a flower blossom. Clearly, the ambient temperature and humidity, as well as air flow over the cells (and possibly, as suggested by some studies, the barometric pressure), all affect the rate of drying and the ultimate quality of the chromosome preparation [1, 2]. When utilizing in situ processing, controlling these parameters is the only way to control chromosome spreading.
In fact, of greatest importance is not merely controlling conditions, but maintaining them with a high degree of consistency. With each change in any one parameter, drying and spreading of chromosomes changes; once the correct combination is achieved, it is of paramount importance that it be maintained throughout the entire harvest. This is true regardless of the specimen type or harvest method.
There have probably been almost as many solutions to this situation as there are cytogenetics laboratories. Some constructed enclosed chambers in which air flow, humidity, and temperature could be varied, although these were typically prone to failure whenever the air-conditioning broke down since it is easy to warm the air inside the chamber but almost impossible to cool it. Some labs designed climate-controlled rooms; these frequently functioned well, but the drawbacks here were the need to maintain conditions while properly venting out fixative fumes (an engineering challenge, but certainly possible) and the potential to expose the technologist to uncomfortable if not unhealthy conditions. Such rooms were also costly to build.
The availability of specialized equipment has all but eliminated the need for such homegrown solutions.
Several companies have developed self-contained chambers specifically for the purpose of drying in situ cultures; an example is shown in Fig. 7.3. The advantages to this type of hardware are its ability to maintain conditions, quick recovery time after opening the chamber to insert or remove dishes, and potential for external venting if necessary. The disadvantage is the necessity to remove the fixative prior to placing the dishes in the chamber, creating the potential for drying to begin under noncontrolled conditions if there is any delay in getting the dishes into the chamber.
Fig. 7.3
Benchtop drying chamber. Initially developed for the culture of insect cells (which are grown at room temperature, and so the incubator must be capable of cooling as well as heating), this chamber has been modified to control humidity as well, and fans have been installed to allow for control of airflow over coverslips or slides (Photo courtesy of Percival Scientific, Inc.)
A variation on this theme is shown in Fig. 7.4. Here, the entire drying process, including aspiration of fixative, can take place inside a freestanding chamber. The technologist sits at the unit and manipulates the processing with a glove-box approach. The advantage here is that the drying process takes place under controlled conditions from the instant the fixative is removed; there is no need to rush to get dishes or slides into the chamber. The drawback to this concept is the large size of the unit, and a somewhat more cumbersome and limiting setup; removing one or more cultures for examination (an absolute requirement) can be more intrusive to the workflow.
Fig. 7.4
Floor model drying chamber (Photo courtesy of Thermotron Industries)
A benchtop device that combines advanced computer control capability for precise control and monitoring of conditions with the ability to perform aspiration inside the chamber is also available (Fig. 7.5).
Fig. 7.5
Monalisa® ambient conditions chamber. Exact specifications for multiple programs, to accommodate different tissue types, are software-controlled via a laptop computer for ease of operation (Photo courtesy of elja, Inc.)
These condition-controlled chambers are gaining in popularity in cytogenetics laboratories, and some use them not only for in situ processing but for routine slide making as well due to the consistency they provide. For this reason, devices designed specifically for slide making are now also available (Fig. 7.6), and one of these actually automates the entire slide-making process (Fig. 7.7).
Fig. 7.6
Hanabi metaphase spreader. Temperature, humidity, and airflow are set and rapidly stabilized so when chromosome preparations are placed on microscope slides, they are dried in a consistent and reproducible manner. Multiple slides can be created simultaneously (Photo courtesy of Transgenomic, Inc.)
Fig. 7.7
Hanabi-PIV automated slide maker (Photo courtesy of Transgenomic, Inc.)
Instrumentation for Fish
While fluorescence in situ hybridization (FISH, see Chap.17) represents one of the most exciting and clinically significant developments in cytogenetics, 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 FISH sample volume that many cytogenetics laboratories receive, any device that can reduce the labor component of the process becomes indispensable.
The entire FISH process can be performed automatically (Fig. 7.8), but laboratories also have the option of utilizing instruments that are specifically designed for different aspects of the procedure.
Fig. 7.8
Xmatrx™ automated FISH processing system (Photo courtesy of Abbott Molecular, 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 so as to be able to interpret the results of hybridization to already-analyzed metaphases. However, some applications of the technology (e.g., ERBB2 analysis for breast cancer or FISH for bladder cancer recurrence; see Chap.17) utilize specimen types that are not processed for chromosome analysis, 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. While not difficult or complicated, these procedures are repetitive and time consuming. Fortunately for the laboratory, devices that automate such steps are available (Fig. 7.9). 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.
Fig. 7.9
VP 2000 Processor. This device automates various laboratory protocols, such as a pretreatment or deparaffinization step prior to performing a FISH assay (Photo courtesy of Abbott Molecular, Inc.)
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 a modest number of slides at one time, and store several user-defined programs for analytical flexibility. Newer models include fluid handling capabilities so that various pretreatment steps can also be performed prior to hybridization (Fig. 7.10a, b).
Fig. 7.10
(a) Thermobrite StatSpin® programmable temperature controlled slide processing 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 (Photo courtesy of Abbott Molecular, Inc.). (b) Thermobrite® Elite Automated Laboratory Assistant. This device adds an automated fluidic system to facilitate pretreatment of different specimen types (Photo courtesy of Iris Sample Processing)
The drawback to these devices is that large volume or frequent use of probes that require different programming necessitate the purchase of more than one unit. Some have, however, come down in price in recent years.
Enrichment
Eliminating the need for actively dividing cells notwithstanding, FISH assays can still suffer from difficulties in detecting abnormal cell populations if these are present in small numbers. Techniques to enrich the population of target cells prior to performing FISH are gaining popularity and are expected soon to become standard of care for certain neoplasms (see also Chap.17). One approach to enrichment is to chemically couple magnetic beads with an antibody to a specific cell surface marker. Target cells can then be magnetically separated from a specimen prior to a FISH assay. Instruments to automate this process are now available; an example is shown in Fig. 7.11.
