Automated Paraffin Processing

Few histology laboratories today process tissues manually. It would be difficult to justify the extra delays that doing so would bring about in a diagnostic setting. Compared to the automated machines in other laboratory disciplines, automated tissue processors are relatively inexpensive, yet save considerable time. The major problem with manual processing is that no agitation or fluid changes can take place after the laboratory closes or on weekends, and this necessarily slows down the process.

Carousel Processors
The first automatic tissue processors were introduced during the first half of the 20th century. In the USA, they were produced under the name of "Auto-Technicon" and in the UK under the name of "Histokine", and later by other companies. They were referred to as carousel processors and consisted of a circular platform with beakers of processing fluids in sequence, and an electrical motor and circuitry which lifted a basket and moved it from beaker to beaker in sequence according to predetermined timing. They agitated the tissues in the fluids either with a small rotary motor attached to the basket holder, or by constantly raising and lowering the basket in the containers of fluid. Usually they had twelve stations, although one manufacturer (Technicon) made a model with two platforms and two sets of containers so that two loads could be processed simultaneously on one machine. There was provision for the final two containers to be heated so that infiltration could be done with molten paraffin wax.

Timing on these machines was determined by cutting a V shaped notch into a circular aluminum plate with a leading straight side and a trailing sloped side. The plate completed one revolution in 24 hours, and a notch was cut in the edge of the plate whenever the machine was to move the basket. As the plate revolved, a lever would fall into the notch and activate the motor which would move the basket one position clockwise, then disengage. The lever would reset as it moved out of the notch, which took about 15 minutes, which was the minimum time that could be set.

Some laboratories, in the interests of being frugal about purchasing machines for histology, put two baskets on a single machine in consecutive positions, effectively doubling capacity at the expense of processing quality and convenience. This was, I believe, the source of the practice of putting formalin in the first two containers instead of simply doubling the time in a single formalin container, an obviously more rational approach.

Fluid Transfer Processors
With the advent of modern electronics and the introduction of computers, there have been significant changes to automated processing machines. Processors now usually use fluid transfer, the fluids involved being processing reagents. The tissues are placed in cassettes then into a processing chamber. The processing fluids are pumped into the chamber and drained after the designated period. Agitation of some kind is used, perhaps magnetic stirring bars or by draining the chamber and refilling it. In addition, the chamber may be capable of being heated and the different processing fluids, including the fixative, may be able to be used warm.

The earliest of these more modern machines used individual timers for each station and the electronics switched from timer to timer sequentially and controlled the filling and draining of the processing chamber. As computer technology has advanced it is common now to have computer controlled timing. It is easy, therefore, to store more than one processing schedule in a computer database and simply call up the one that is wanted. Similarly, changes to a schedule are far more easily made than recutting a notched aluminum disk.

Maintenance of both types of processors is essential. Fluids must be both topped up daily and replaced on a regular schedule, whether that is based on the number of cassettes processed or on the number of processing runs between changes. It is usual to use a similar procedure as with manual processing, that is, fluids are moved down the sequence as appropriate and a fresh fluid inserted in the last place of a fluid type (dehydrant, clearant, wax). If large numbers of cassettes are processed, this may need to be done daily or on alternate days. It should be done frequently enough, based on practical experience, that poor processing effects are not seen. It is poor economy to attempt an extra run with a series of fluids, as at the least it may result in greater difficulty sectioning and poorer morphology, and at the worst may require reprocessing, with the attendant delays and costs.

One particular problem with fluid transfer processors is often a source of problems. That is precipitation of salts from neutral buffered formalin when the fixative is replaced with ethanol of a greater concentration than about 60%. There is a gradual buildup of salts in the ethanol, which eventually precipitate within the tissue and can cause problems during sectioning. Most processors stipulate a water flush of the processing chamber at periodic intervals so as to control this problem. The wash procedure should be done at least according to the minimum specified, and preferably more frequently.

Practicalities
It is preferable if the tissue is fixed completely before they are placed onto the processor. This was reasonably easy to accomplish when a 48 hour turn around time was used, but as 24 hour turn around times began to be used it caused a problem. Formalin fixation takes at least 24 hours for a thin slice of tissue, but surgical specimens taken during the day and required to be processed that same night cannot possibly be properly fixed during the available time. It is partially solved by instituting specimen reception cut off times, i.e. large specimens must be received by noon and small specimens by 2:00 p.m. if they are to be processed that same day.

This is not a complete resolution of the problem as it requires large specimens to be examined unfixed and samples taken fairly soon after reception so that a maximum period can be used for fixation. It is less of a problem for small biopsies. The problem can be minimised by placing poorly fixed and unfixed tissue into formalin in a wax oven at about 60°C-65°C for a few hours, until processing begins by placing the tissue basket on the processor. It is also a common practice to have fixative in the first container, and in fluid transfer processors the temperature of the fixative may be increased.

It is essential that tissues are trimmed to an appropriate thickness (3 mm maximum), and with an area that allows the slice to fit loosely in the cassette so that the processing fluids are not inhibited in their access. This is often not done properly, because the person selecting the tissue for examination forces too much tissue into a cassette, often so much that processing fluids can't penetrate. Add to that the fatty nature of some specimens and their poorly fixed or unfixed state, and it is easy to see how incomplete infiltration comes about. When not done properly, some specimens, such as large fatty breast lumps, are essentially raw. Tissues like that cannot be sectioned, and the diagnoses are consequently delayed, defeating the whole purpose.

In most laboratories the working day was from 8:00 a.m. to 5:00 p.m., or something similar, giving a period of 15 hours during which processing could take place. If processing could start at 4:00 p.m., we would have the traditional 16 hours equating to overnight treatment. Today, of course, starting times are much earlier in many laboratories and may extend later with staggered shifts. The schedules should be altered to reflect such differences.

 


 

Translate in
Google Translate
Instructions