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The Silence of the Pathologists: Fixation in Histology and Histopathology
4 December 2020
By Hannah Thomson

 

‘Welcome to Fight Club. The first rule of Fight Club is: you do not talk about Fight Club.’ (Chuck Palauniak 1996).

Fight Club and Histopathology may not overlap widely, but their intersection is not empty. Dr Wilfred Weinstein of Los Angeles spoke of ‘The Silence of the Pathologists’ regarding difficulties with the diagnosis and grading of dysplasia in the gastrointestinal tract, but he could have been talking about the subject of this blog.

Several things all have to be done right for good histology: tissue sampling, fixation, block preparation, processing, embedding, microtomy and staining. Some are more controllable than others. One of the most critical is fixation.

Tissue starts to deteriorate the moment it is taken out of its native environment, deprived of its blood supply, or the organism dies. Without oxygen and energy molecules the energy balance of living cells fails. Lysosomal enzymes leaking out begin the work of destruction, and things fall apart. Cooling slows but does not stop this autolysis. Hard freezing arrests it, but it will begin again on thawing, and ice crystals can cause troublesome artefacts.

Fixation & Formalin

Fixation arrests autolysis permanently, by cross-linking and stabilising macromolecules and inhibiting enzymes. The classic histological fixative is formaldehyde (formalin) in buffered aqueous solution. Formaldehyde is a reactive chemical with many undesirable qualities, including toxicity and possible carcinogenicity, but no-one has found a completely satisfactory substitute for general use in histology and histopathology.

It is invaluable to be able to put a biopsy into a jar of 4% neutral buffered formalin, secure the lid and send it to the laboratory knowing that even if it travels for a week, it will usually arrive well enough preserved for processing to histology and other tests including immunohistochemistry or molecular analysis.

Good though it is, 4% neutral buffered formalin has limitations. Tissue cannot fix unless the fixative is within it. How does it get there? By diffusion through the tissue from the outside. Which takes time. For small mucosal biopsies, needle biopsies and small excisional biopsies (up to ~5mm) the time taken for diffusion is not significant. But for larger excisional biopsies and surgical resections it becomes increasingly important.

How long does it take for fixatives to diffuse into tissue?

Eighty years ago Peter Medawar (1941) showed that the depth d to which fixatives penetrate a plasma coagulum acting as tissue-surrogate is K√t, where t is the time elapsed since specimen immersion in fixative. K is a proportionality constant specific to each particular fixative.

What does this relationship mean for the histologist? Just this: that the time taken for any fixative to diffuse into the centre of a portion of tissue is proportional to the square of the linear dimensions of that portion of tissue.

Every histologist needs to know this.

As fixation cannot even begin in the centre of a portion of tissue before the fixative gets there, this means that compared to a 4mm biopsy, the start of fixation in the centre of a 20mm tumour excision immersed in formalin will be delayed 25-fold, and 100-fold in the centre of a 50mm excision specimen.

This is no mere theoretical problem. Every histopathologist is familiar with excision of specimens which look fixed on the outside but which when incised are raw and completely unfixed in the centre. Bad histology follows.

Accurate breast cancer grading requires good fixation. A 5cm breast cancer excision or a mastectomy left for 48 hours in formalin will be unfixed in the centre, with significant deterioration of the histology due to autolysis. One sees this in actual diagnostic practice [Image A].

Histology Tissue Fixation

Image A. These two images are of the same breast carcinoma. The upper panel shows the primary lesion in the breast, and fixation is extremely poor. The lower panel shows the same carcinoma in a well-fixed axillary lymph node. Mitotic cells are obvious in the nodal metastasis but are unrecognisable in the primary. Accurate grading of the primary lesion is impossible.

The pathologist who does not appreciate the importance of the innocuous-looking relationship d = K√t is at a disadvantage which may even compromise the quality of care offered to the patient.

That this relationship is not widely comprehended is shown by a form of specimen abuse commonly practiced. It is reasonable and practical to leave a prepared (partly dissected) specimen in fixative overnight (~18-24 hours) prior to further dissection and block selection the following day.

Sometimes, though, pathologist or technical staff decide to leave the specimen in fixative for two days (up to 48 hours of fixation), as they know from experience it will not be adequately fixed after only one day. They must think this will improve matters.

They forget that if the specimen will not be fixed after 24 hours, another 24 hours is not only useless (in that d = K√t means that by the third day fixative will only have penetrated less than half as far again as after 24 hours), but probably worse than useless, all that is achieved being another 24 hours of unimpeded autolysis.

Anyone with eyes to see knows that 48 hours in fixative does not solve the diffusion problem, but if one does not understand what is going wrong, one cannot think what else to do, and the same ineffective strategy is applied over and over again.

Deciding that a specimen ought to be left in fixative for 48 hours is merely to admit that the prosector has failed to prepare the specimen properly!

The solution is to acquire the proficiency needed to prepare unfixed specimens so that fixative has free access to the tissue with no more than a few millimetres of diffusion being needed for it to reach any part of the tissue.

James Going

About the Author: Dr James Going

Senior Clinical Pathologist

James joined OracleBio in 2019 as Senior Clinical Pathologist. With over 39 years of pathology experience as a Clinical Pathologist in the NHS, he has a wide and deep knowledge of diagnostic histopathology with specialist expertise in breast and gastrointestinal tract.

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