Manual Paraffin Processing

Embedding tissues to make paraffin blocks is now almost a reference standard in diagnostic histology. It can be done relatively fast and enables comparatively thin sections to be cut with good definition. It is capable of that, anyway, although the mark is often missed in the desire for a quickly produced microscope slide so that a diagnosis may be issued. It is difficult to be critical of the desire to help patients, but it must be acknowledged that the wish to have a prepared H&E stained slide in a shorter and shorter time causes stress on the technical preparation of the material.

In particular, fixation is given short shrift, and in many cases dehydration, clearing and infiltration are barely adequate, simply compounding any problem. This in turn causes problems with sectioning and tissue morphology, both anatomical and cytological. The fact is that initial fixation must be adequate, even if not thorough. The tissues must be denatured enough to resist the fluids which follow. Unfortunately, half an hour in neutral buffered formalin does not do that, even if the tissue is discoloured. Fixation is a chemical process, and like any chemical process a period of time is required for it to go to completion, or even just a significant part of the way. The point is that for proper processing, complete fixation is required.

This section gives manual processing schedules. They are based on the author's experiences in different laboratories, predominantly as a student technologist. Some laboratories still processed manually for special purposes in those days (early 1960s), usually when the highest quality sections were required or when larger than usual tissues were to be processed (fetal lungs, slice through whole adult kidney etc). Always the tissue was thoroughly fixed first, sometimes for weeks or months in a 10% formalin variant.

The fixative used is not particularly important from the perspective of processing schedules, although it will affect the final appearance of the sections considerably. It is more important that fixation be complete, rather than which one is used. Many fixatives give complete fixation overnight, others may take much longer, but whichever one is used, do make sure that the tissue is properly fixed before processing begins. In the schedules the references to fixation, in most cases, assume 10% neutral buffered formalin. If a different fixative is used, substitute the appropriate treatment for that fixative.

As processing machines were not all that reliable in those days, most laboratories also had a rapid schedule to permit continuation of the work should a processing machine break down and capacity not be enough to meet the daily workload requirements. The most urgent cases were processed on the remaining processors, while less urgent cases would be processed manually. The crunch came, of course, when the processor had been repaired and a double workload required sectioning and staining.

Occasionally it was necessary to prepare a section from a surgical specimen that was removed from a patient early in the morning, because a frozen section was inadequate for diagnosis. There was a schedule for that, too. It may still be useful today, sometimes.

There were at least two approaches to setting up manual processing. One setup used 1 litre beakers with metal lids, each filled with one of the fluids. The tissues were placed into cassettes (round in those days) with an ID label and either placed directly into the beaker or placed into a basket and then placed in the beaker. Sometimes the basket was suspended in the beaker from a slowly turning rotary motor for gentle agitation, otherwise agitation was achieved by removing the fluid and immediately pouring it back in, or lifting the basket out and lowering it again a few times. This was done every hour or so. Fluids were usually filtered after the tissues were taken out and moved to the next fluid. This ensured there was no tissue carry over. Of course, the basket or cassettes were well drained on absorbent paper between fluids to minimise mixing of the fluids.

Those who have used the early type of automatic tissue processor, such as the Auto-Technicon or the Histokine, will recognise that the sytem just described is very similar to how they operated. Those early tissue processors simply automated an existing system of processing.

The second approach required a series of small jars, usually 50 mL or 100 mL glass specimen containers with screw cap lids. Trimmed tissues from a single case were placed into a container together with an ID label, then almost filled with fluid. Agitation was achieved by inverting or gently swirling the glass jar every hour or so. This system was felt to be more thorough since it avoided any restrictions to fluid penetration from the cassettes, as agitation was not continuous. It did require a significant number of jars, particularly if the processing schedule took more than one day and a few day's work was being processed at the same time. Often each day's specimens were placed into seperate trays to identify them, or the lids were marked for identification. Reservoirs of the processing fluids were usually stored in 1 litre glass bottles with stoppered lids. The fluids were filtered back into these as the fluids were changed to ensure no carry over. Contamination between fluids was minimised by placing absorbent paper over the emptied jar and inverting for a few seconds.

In both systems fluid replenishment was done similarly. The early fixatives and ethanol were discarded and replaced with fresh fluid. The first absolute ethanol was discarded, then each absolute ethanol was moved down one position, and fresh absolute ethanol put in the last place. The same was done with the clearing agent and paraffin wax. The frequency of replenishment of the fluids depended very much on the throughput. A laboratory which had a basket full of cassettes every day might replenish daily or on alternate days. The need to replenish more frequently became obvious very quickly as the tissues would not infiltrate properly, so most laboratories erred on the frequent side to avoid any problems.

 


 

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