This contribution was inspired by a comprehensive monograph on tissue processing for histology by M.D. McGavin, Professor Emeritus, Department of Pathology, College of Veterinary Medicine, University of Tennessee. It has been updated and partially rewritten by Roger Kelly and Paco Uzal.
GENERAL PHILOSOPHY
There is a tendency by inexperienced pathologists merely to whittle up an animal (especially one without any gross lesions), place the pieces in fixative and then trim in large numbers of tissues without thought to orientation, artifacts, optimal fixation, etc., in the hope of finding significant lesions.
In previous times, all trainees would have spent weeks or months in a histopathology laboratory, in order to acquire experience of the problems that occur on a routine basis. If a pathologist is to avoid making work difficult for the histotechnologist, she/he must understand the basic histological preparative processes:
* The relative risk of autolytic and pressure artifact in different tissues
* The chemical basis and principles of the various methods of tissue fixation
* The principles and practice of tissue embedment and microtomy
* The indications and limitations of the various histological stains.
This paper will deal with the first two of these operations: tissue sampling and fixation.
GENERAL PRINCIPLES OF SAMPLING, FIXATION AND TRIMMING
Fixation and the postmortem interval.
Most tissues contain some contractile component (muscle or collagen) and if very fresh will contract in contact with 10% buffered neutral formalin (BNF). The result of this contraction will cause distortion, e.g. small intestine rolls up like a roller blind, and muscle will develop contraction band artifacts.
But these are minor problems when set against the beauty of rapidly-fixed histological material. Enterocytes, for example, will begin to loosen and slide from the small intestinal villous basement membrane within 2 minutes after circulation ceases, so if small bowel disease is suspected, samples must be collected very rapidly after euthanasia, even if this means disruption of the usual dissection routine.
Dissection trauma.
Tissues such as skin and bone are tough enough to withstand quite rough handling without sustaining much pressure artifact. But it is almost impossible to handle parenchymatous organs and hollow viscera with fingers or forceps without physically damaging them to some degree. So pathologists must acquire the ability to handle tissues much more gently that, say, surgeons, about whom pathologists have been known to murmur: ‘Their mistakes have a chance to heal.’ It is desirable to handle tissues with fingers as little as possible. Manipulate soft tissues by using toothed forceps on the connective tissue adjacent to the tissue to be sampled. Why toothed forceps? Well, if a piece of tissue that has been crushed by toothed forceps should by mistake be included in a histological section, there will be no doubt as to the cause of the artifact so produced.
The sharper the knife, the less the pressure needed to transect tissue. This is obvious, but it is alarming how many residents and pathologists are entirely incapable of sharpening a knife. During cutting, the knife must be drawn towards the prosector so the actual cutting is a mixture of downward and tangential pressures. When slicing brain, the prosector should use little downward pressure but a tangential cut from a long brain-slicing knife.
Scissors – even sharp ones – crush rather than cut, so one should avoid including scissor-cut surfaces in histological sections, although this may not always be possible, especially when dealing with lung or blood vessels.
Tissue sampling and fixation.
The thinner the tissue sample, the quicker the fixation, obviously, but there are limits to the thinness one can shave off an organ, even with a very sharp knife. Five to 7mm from a solid organ is a reasonable compromise. It is obvious that fixation will be delayed in the center of bulky blocks of solid tissue, so if such samples are received from the field or inexperienced students, tissue must be trimmed from the surface of the block to obtain the best histology possible.
Brain fixation needs special consideration, as sometimes does lung. These will follow in later instalments.
The recommended ratio between volume of fixative needed, and volume of the sampled tissue is traditionally quoted at 10:1. But it is better to err generously; formalin is cheap.
Fixation time varies to some extent with the nature of the tissue and its thickness: the process can be accelerated by heat, agitation and microwaving. The mild heat in the tissue processor for a short while is excellent. These general remarks will apply to tissue fixation by formaldehyde in the form of 10% BNF. A saturated solution of formaldehyde gas in water (37% w/v) is known as “100% formalin”, so the 10% BNF fixative is 100% formalin diluted 10x in phosphate buffer. Without the buffer, the pH of the solution will often be less than 6, especially if formic acid has formed, as it does in industrial-grade formalin. At pH below 6, free haemoglobin polymerizes to brown artifactual ‘formalin pigment’ that resembles haemosiderin. Tissue samples received from field veterinarians will often be fixed in unbuffered formalin, so formalin pigment will often be a feature of these specimens, particularly after longer postmortem intervals. It can be removed by soaking in picric acid before staining.
Fixation of tissues with high mucin content.
Intestinal mucosa is easily damaged, both by delayed fixation (as mentioned above), and by difficulty in handling the slippery tissue after fixation. But fixatives that contain acetic acid as well as formalin – such as Bouins – fix the mucin and the cells, and these can be useful for slimy tissues. As a bonus, they tend to make certain intranuclear viral inclusion bodies more obvious. However, these acidic fixatives interfere with haematoxylin staining if the tissue stays in them too long, and they hemolyse erythrocytes. The fixation of surface mucin by Bouins takes only an hour, so after that the sample should be transferred to 10% NBF to complete fixation. This will improve subsequent H&E staining of the sections.
Bouin’s fixation has been discontinued by some laboratories because of its disadvantages, but it does have its proponents.
Trimming samples for paraffin embedment.
Since embedment involves the use of tissue cassettes, it is easy to ensure that the histotechnologist will always know from which surface to start cutting sections: you just make sure trainees know that the surface from which the sections are to be cut is always placed facing the bottom of the cassette.
Ideally, if surgeons want to be sure about the relationship between malignant tissue and surgical excision lines (surgical margins), they will paint the incised surfaces of a biopsy with a dye that will identify the excised surfaces in histological sections. If the sample is bulky, they may incise it one or more times after dyeing it; these incisions will then be recognizable as post-surgical. Unfortunately, as we all know, surgeons tend to be a law unto themselves and have been known to ignore pathologists’ advice. If they do paint cut surfaces, they are likely to do it as an afterthought, after having made a few incisions into the mass.
