Drying Chambers

Again, as described in Chapter 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

Fig. 2. 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 on-board programming, which eliminates the need for an external computer, reducing bench space requirements. (Courtesy of Genial Genetic Solutions.)

Fig. 2. 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 on-board programming, which eliminates the need for an external computer, reducing bench space requirements. (Courtesy of Genial Genetic Solutions.)

one or more drops of this suspension on a number of microscope slides, and it 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 the case with in situ harvesting. Most cytogenetics laboratories initiate four to six cultures from each sample, 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; in many cases, three 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 usable metaphases. The concept of discarding one and trying again, as is so often done 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.

Electric Smoker Home Depot
Fig. 3. Benchtop drying chamber. (Courtesy of Percival Scientific, Inc.)

As the 3:1 methanol:acetic acid fixative used in cytogenetics laboratories dries, it "pulls" the cell membrane across the slide or cover slip 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 airflow over the cells (and possibly, as suggested by some studies, the barometric pressure) all affect the rate of drying; therefore, when utilizing in situ processing, controlling these parameters is the only way to control chromosome spreading (1).

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.

There are probably as many solutions to this situation as there are cytogenetics laboratories. Some have constructed enclosed chambers in which airflow, humidity, and temperature can be varied, although these are typically prone to failure whenever the air conditioning breaks, because it is easy to warm the air inside the chamber but extremely difficult to cool it. Some labs have designed climate-controlled rooms; these frequently function well, but the drawbacks here are 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 conditions. Such rooms are also often costly to build.

Recently, several companies have developed self-contained chambers specifically for the purpose of drying in situ cultures; an example is shown in Fig. 3. 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

Thermotron Cytogenetic
Fig. 4. Floor model drying chamber. (Courtesy of Thermotron Industries.)

as heating), this chamber has been modified to control humidity as well, and fans have been installed to allow for control of airflow over the cover slips. 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.

A variation on this theme is shown in Fig. 4. Here, the entire drying process, including aspiration of fixative, can take place inside the chamber. The technologist sits at the unit and manipulates the processing with a glove-box approach. 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.

These condition-controlled chambers are gaining in popularity in cytogenetics laboratories, and some use them not only for in situ processing but also for routine slide making because of the consistency they provide.

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Responses

  • Hannu Ryyn
    What effect does humidity have on slidemaking in cytogenetics?
    25 days ago

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