Fixation, Fixation, Fixation

 

.                                                                   “Oh stay! You are so beautiful!” J.W. Goethe, Faust

INTRODUCTION

Is histotechnology art or artisan? This question sparks discussions among histotechnologists. Most lean to the assumption that histohechnology is clearly artisan that can be performed with artistic elegance and beauty.

In artistic terms histology is closer to “still- life” or in French “nature morte” (literally dead nature). The flowers are plucked, but the goal of the artist is to stir up imagination of their scent.

Histotechnology has a more modest goal as to help the viewer, in most instances the pathologist, to generate an informed conclusion, namely the diagnosis. The real beauty of histology is in maintaining the immediate pre-removal structures. We have to kill the cell and the tissue to keep them like alive. The key word is LIKE not As. The process is called fixation. It tries to stop the moment. Appropriate and adequate fixation in surgical pathology delivers the beauty of complexity and variety of nature for diagnostic purposes.

Beauty, however, is within the eyes of the beholder. Beauty per se in histotechnology is not valuable if it distracts attention from the cognitive process. That is why pathologists, as consumers, seem to be somewhat unappreciative of histotechnologists artistic work. “Histochemistry: a case of unappreciated beauty?” wrote René J. Buesa on the pages of Journal of Histotechnology.1 The pathologists do not have time and desire to watch even an extraordinary beautiful slide if it is in contradiction with his/her image at the occipital lobes of their brain that allow them to make the diagnosis, sometimes by intuition. Now intuition is more substituted by IHC markers but cannot be dismissed completely. From practical point of view the main value of a histological slide is in the standard consistency of a descriptive microscopic picture. Complete fixation is the key for standard specimen processing.

With the assumption that readers of this website are familiar with the theory of fixation presented in many fundamental handbooks 2,3,4,5,6 , as well as in some theoretical articles.7,8 these materials are intended to offer a pragmatic approach to fixation, predominately in surgical pathology. In a loftier sense, the article aims to express unconventional approaches to fixation process based on the author’s experience and observations, though some issues, and among them safety question, require separate more substantiate argumentation.

Fixation as preservation

Many processes start immediately and simultaneously after the tissue is taken out from the body. Some of them are presented on the diagram 1.

Processes after tissue removal                              Diagram 1

Drying out is the first concern. In mucinous tissues or film like biopsies, for instance laryngeal, it is of great importance.

Autolysis due to the continuation of the enzyme metabolic activity and release of lysosome’s digestive enzymes starts immediately after tissue removal. Some tissue with intensive enzyme activity, for instance, small intestine, pancreas, tubular area on the kidney, can be severely damaged.

Decomposition by infection (bacteria, fungi) is a reality of the environment, although putrefaction and decay do not occur in practice due to the short time between the specimen removal and processing.   In general, the contemporary requirement to tissue processing leans to maximal sterility, or better to say minimal contamination, especially keeping in mind that some ancillary studies (cytogenetics, flow cytometry) use tissue culture methodology.

Fixation stops everything. This was the main rationale for the immediate pouring fixative. However, the classic notion to start fixation as soon as possible after the tissue is removed can interfere with modern variety of ancillary studies which broadens the definition of preservation. In practical sense, more frequent use of refrigeration and sometimes even snap freezing (for enzyme profile) ought to be implemented. As the condition of specimen preservation, knowledgeable pre-processing triage is the requirement keeping in mind the main medical motto: “Don’t do harm.” In this instance, do not impede the possibility of the best diagnostic outcome for the patient.

Dual goals of fixation

As preservation procedure, fixation is aimed to achieve two goals: maintain as much as possible organ/tissue integrity and cellular/extracellular substances structure that existed before the removal procedure.Fixation provides preservation of the integrity and morphology tissue and cells keeping in mind that histology processing includes many “unpleasant” for them physical and chemical processes like dehydration and clearing, as well as embedding and staining to some degree. These goals are presented on the diagram 2.

Dual goals of fixation                                                         Diagram 2

Perhaps, the word protection requires quotation marks. It would be appropriate to define protection of antigenicity as not masking (messing up), as much as possible the tissue protein’s epitope as the immuno-determinant region on the antigen surface to be recognized by the antibody’s binding site. Immunohistochemistry changed the game. By the way, Zeus (Mishel’s solution) transport media does preservation for immunofluorescence, as RPMI for flow cytometry or DMEM-F-12 for cytogenetics.

Fixation for grossing processing

Preservation of the tissue/organ integrity is one of the goals during grossing, or sampling, or cut in, depending on the terminology that is used to define the initial stage of processing. It does not matter if the tissue/organ were cut or not, but, of course, if the tissue were cut, the integrity of the tissue/organ should be the primary concern. The tendency of specimens sampling in fresh state is wide spread due to the pressure of turnaround time. Insufficient fixation prior to sampling is underestimated in current surgical pathology practice.  This is the reason that this article discusses fixation for grossing processing in detail.

It would be an anatomical triviality to state that in opposite to more or less homogeneous cytology material tissues/organs are combination of heterogeneous types of histological components that are in a loose connection with each other. Every intrusion changes these original connections. The goal is to minimize them during grossing when the tissue/organ has to be adjusted to procedural means (optimal presentation of areas of diagnostic interest, size of the cassette, convenience for embedding, etc.). Grossing manipulations move these tissue conglomerates by pulling, pressing, twisting during the manual process.

Fixation provides immobilization of tissue structures for grossing manipulations. JA Kiernan puts it as spatial displacement of structural proteins and other constituents of tissue.6 This process has an old commonly accepted definition, of tissue hardening.  It stems from the dawn of histology before the formalin era when alcohol fixatives hardened tissue.  Alcohol really hardens the tissue frame by dissolving lipoid structures and coagulating protein fibers. Hardening in the early days of histology technique served to improve micro section. As Frieda Carson wrote: “…since much of sectioning was done freehand. Today, with an array of embedding media to choose from, this hardening action is less important.” 5 However, the old macroscopic tactile term “hardening” approximately explains the immobilization of tissue structures for grossing. It is widely used and mutually understandable.

Why do we need “hardening”? Let’s take a simple analogy. The sharpness of the knife/blade is useless to cut a soft bread loaf just from oven. Bread only looks homogeneous, in reality it consists of threads of baked dough. The tissue treads are following the knife pulling the dough under the weight of the knife. If the loaf is not fresh the cutting is easier. The surgical pathology tissues are definitely heterogeneous with loose connections between adjacent structures. The goal is to minimize their relative “movements” towards each others during cutting tissues which are not “fresh”, namely after fixation .

Perhaps, to some degree colloquial, the term “hardening” will stay until a different definition can be found. While using it we always should understand that it is an immobilization method for grossing manipulation to prevent literally tissue/organ mutilation. Hardening contributes to uniformity of section. Fixation in this instance is a “tool” that equalizes deficiencies of grossing tools (knives, blades, forceps, etc.). The more fragile are tissues the more careful and complete should be fixation for grossing immobilization, e.g. “hardening”.

Hardening during fixation comes with a price. In some rare instances, fixation makes grossing more difficult, if the tissues are completely different in consistency. For example, bone and adjacent soft tissue lose natural adhesiveness because the soft tissue becomes more brittle. It is difficult to maintain the integrity of the specimen if grossing were done on the specimen after fixation. Even the finest saw tears soft tissue from the bone due to the principle of sawing. In this situation the pathologist cannot answer the main clinical question about tumor and bone relationship. Working with complicated bone specimens, especially after facial-maxillary surgery, this author recommends sectioning bone and adjacent soft tissue before fixation.9

It seems that lobulated colon polyps fall apart easier after fixation. Eye ball is better to gross in fresh state due to extreme heterogeneity and fragility of tissues. Those are exceptions from the general rule that grossing is more efficient after fixation.

Change in color of blood can be accepted as an approximate end point of formalin fixation sufficient for grossing purposes. Formation of acid formalin hematin makes blood tan brown, although some other conditions (change in acidity can also make brown colors). This is important in biopsies which arrive at different stage of fixation.

Fixation as cells and extracellular substances stabilization

The main goal of fixation is cells and extracellular substances stabilization. Fixation also serves the aim of making the intimate structures more visible by opening channels for staining (5) through complicated physical and chemical processes such as electrical conductivity, viscosity and solubility gradients of reagents. These goals ought to be carried out without masking antigenicity as much as possible.

By essence, fixation uses chemical process although some physical methods are employed, especially with the development of microwave assisted processing. Actually, microwave itself is not more than heat coagulation if the fixative penetration were not completed. Coagulant (alcohols, mercury, zinc, etc.) as well as non-coagulant (formaldehyde, acetic acid, etc.) fixatives interact with proteins by stabilizing them. The coagulant alcohols do not establish a chemical bond with proteins that make them attractive for immunochemistry. The non-coagulant acetic acid with its fast penetration preserves nucleoproteins and prevents shrinking.

Every histology textbook contains numerous recipes of fixation solutions which actually are not used now in practice. Complicated fixative solutions have disappeared gradually leaving only a few of them, just not as a common practice. Among them Bouin’s solution (this author’s favorite for many years, despite many months of ulcers on a finger caused by picric acid), Carnoy’s, B-5. The latter almost everywhere is replaced by Zn formalin or even formalin. Disappearance of complicated recipes has many reasons. Among them are, in the author’s view, environment considerations (mercury-Zenker, B-5), the necessity to ex tempore mixing ingredients, improvement of technology processing (VIP, microwave), proprietary stock solutions, the necessity close monitoring, …and, yes, lowering the bar of requirement to quality of microscopic picture owning in part to the help from IHC markers.

Two main groups of fixatives are in competition at the practical laboratories market: formalin and alcohol based solutions, although there are, of course, mixtures of them.

Formalin

Fixative recipes come and go. Formalin has stayed for a long time, although during Virchow’s time, in the 19yh century, it was not in use. Formaldehyde is a gas methanal that dissolves in water at a maximum concentration between 37-40% vol./vol. The 10% solution formaldehyde contains between 3.7 and 4.0% of formaldehyde and is called “formalin” in many countries and “formol” in Europe.  Formalin’s formula changed also, but the main idea of NBF (normal buffered formalin), as 10% solution, is ubiquitous. Now a company Azer Formagel even offered 10% Gel Neutral Bur\ffered Formalin with a brand name Formagel. The 7-15 % methanol added as a stabilizer not only delays formaldehydes polymerization but brings some positive fixative features of alcohol. Formalin stored in a cold place usually contains less than the 37% of formaldehyde stated on the label. The missing formaldehyde is in the white precipitate (which is paraformaldehyde) in the bottom of the bottle. Good structural preservation for electron microscopy is possible in solution made from formalin with optimal pH and salt concentrations. The small amount of methanol in a neutral buffered fixative made from formalin could have no fixative (coagulant) action. 10% NBF is not the same thing as 3.7-4%formaldehyde. Whether they can be used interchangeably has to be validated.

Details of formalin fixation are presented in the Buesa’s article “Histology without Formalin?” 10 The article provides a comprehensive approach to three sides of formaldehyde action: penetration, covalent binding, and cross-linking (actual fixation). These work simultaneously but at very different rates: penetration twelve times faster than binding, which in turn is about four times faster than cross-linking. Due to fast penetration, the thickness of the gross section does not matter substantially in the range between till 4 mm (the maximal depth of the standard processing cassette), but temperature at the rate 37-45C (definitely below 60 C) can accelerate fixation. By using laboratory microwave oven accelerated processing with temperature to 50-55 C, the time of fixation can be substantially below the standard 24-48.

Zinc formalin fixative is accepted in surgical pathology practice for better nuclear details, especially in bone marrow biopsies. As with B-5, it requires monitoring. The time of fixation is 1.5-2 times longer. Zinc formalin is praised as immunochemistry friendly fixative because it often made antigen retrieval unnecessary. Anatech Ltd developed Z-Fix, a buffered zinc formalin that prevents formalin pigment while maintaining IHC fixative quality.

The formalin disadvantage is incomplete DNA and mRNA recovery. Formalin is also unfriendly towards molecular biology. Two disadvantages are the slow pace of fixation (24-48 hours at the room temperature) and necessity for heat induced antigen retrieval to overcome the deleterious effects of formalin fixation. All these disadvantages are reasons for the search of a different fixative.

Alcohol fixatives

Actually, fixation always is finished by alcohols while processing for paraffin embedding if cross-linking were not completed before. The process of dehydration is part of the final fixation.

The main alcohol fixative that survived since 19th century is Carnoy’s fixative. Originally it contained 100% or 95% alcohol (6 parts), chloroform (3 parts) and acetic acid (1 part). All old timers can remember it as a pain in the neck (problems with absolute alcohol, evaporating narcotic chloroform, ex tempore preparation). However, the nuclear result was excellent. Then chloroform was eliminated (Clark’s fluid), and ethanol was substituted by methanol (6 parts). Methacarn replaced Carnoy fixative in practice with good results for fast penetration and nucleic acids preservation though it is not recommended for most surface antigens.

Methacarn is only one of numerous alcohol fixatives to name some of them (OmniFix, NeoFix, BoonFix, EthMeth, F13, MicroFix, UMFix, Uni-Fix, Fine Fix, etc.). The content of many of them is not disclosed. One of them UMFix has an ambitious name as Universal Molecular Fixative that is claimed as a molecular-friendly alcohol based fixative that preserves morphology.13  All these fixatives as well as non-alcoholic formalin substitutes ( Anatech’s Prefer, Preserve, Safe Fix II, NOTOXhisto, Glyo-Fixx, DMS, HistoCHOICE, etc.) are discussed in detail in the above mentioned article “Histology without formalin?” 10  Glyoxal, the active ingredient of Prefer, a promising alternative to formaldehyde. forms crosslinkages longer than formaldehyde. Prefer contains also a small amount of ethanol that helps in penetration through lipids. However, Prefer  has been more than a decade on the market, but still is not accepted by general practice.13

A combination of alcohol and less than 10% formalin, for example IBF (methanol and 2-propanol with 3% formalin) is an option for some biopsies (prostate). There are other low percentage formalin fixatives as S.T. F, Pen Fix, Stat Fix, actually mixtures with alcohols and other undisclosed components.

A buffered alcoholic formalin, CBA Formalin, is manufactured by Anatech Ltd www.anatechltdusa.com. This fixative has the advantage of formalin’s fast penetration as a small molecule and fat tissue dissolving by alcohol, that increases penetration through fatty thick sections. Before the microwave assisted technology, the combination of 10% buffered formalin and 70% alcohol was my favorite fixative. it seams that Anatech’s Alcoholic Z-fix Concentrate ( mixture with Zink formalin) is right , but unnecessary in large specimen, but for bone marrow and similar small specimens (lymph nodes) it is enough  Zn formalin because fatty part is taken out during gross sampling.

The amount of brand names of formalin substitutes is impressive. The list of them is incomplete. Who can even remember them, never mind to have informed conclusion of their quality? Sporadic articles about proprietary (some of them do not have a disclosed formula) fixatives with contradicting always positive recommendations cannot be used as a credible guide for practice.

Where to swim?

The ship of practical surgical pathology histotechnology, with a crew empowered by IHC, navigates its course between Scylla and Charybdis. Histology practice is facing the monster of enormously increased amounts of slides and the pressure of turnaround time while trying to avoid sinking in the whirlpools of untested commercial fixatives offers. There is an urgent call to determine the course in regard to this crucial component of histotechnology quality as fixation.

The CAP regulations, or recommendations or guidelines, regading fixation for the Her2/ER/PR validation change the game because it is necessary to have comparible results between different laboratories for breast cancer treatment. The bread loaf gross sections and sections in the cassettes are not allowed to be processed with less than 6 hours in the cassette plus processor time for formalin.  It is a rule if the breast is not in cassettes before 3PM it cannot go on the processor.   In most cases the breast is fixed overnight, loafed.  Then the sections are left in the cassette for processing within the 48 hour. maximum for Her2Neu.  If they are late on Friday we process Saturday and take the tissue off on Sunday so we bent no rule for the formalin fixation.
The use of any other fixative require a complete validation of the alternative fixative to prove results are identical with the alternative fixative compared to formalin-fixed tissue. However, formalin is the only FDA approved fixative for  Her2/ER/PR.

There is a necessity to conduct a serious, perhaps international (we are not alone in the world), pilot projects under the auspices and direct involvement of a major group, like NSH or ASCP, or CAP, to determine the commonly accepted fixative in practical surgical pathology leaving experimentation to the research facilities. This project should take in consideration many often contradictory issues. Among them are: universality in practical laboratories, minimal masking for IHC, cost, speed of fixation, molecular biology friendly, and, of course, safety. Grossing needs (hardening) should be taken into account. Although there were attempts to conduct workshops, for instance by CAP, orientated predominately for IHC, the problem requires a comprehensive solution.

Keeping in mind commercial and research interests, it would not be a simple task to reach a pragmatic consensus. However, despite clashes of personal preferences, commercial interests, and politically correct tendencies, this task should be carried out. This is for the sake of standardization of histotechnology as a major player in the diagnostic process.

Here are some notions which might be taken into account. In a sketchy form, they reflect how the author understands the multilateral approach to the problem.

Universality as much as possible for the use in practical laboratories is an urgent necessity. In opposite to napkins or greeting cards when diversity is a virtue, fixatives in surgical laboratory ought to be ubiquitous. Each fixative makes artifacts, or aberrations, in the microscopic picture. There is common and individual experience used to evaluate them. Many decades of experience of using formalin fixed microscopic image cannot be thrown out the window.

Minimal masking of antigenicity for IHC is a serious advantage of every fixative due to the crucial importance of antibody markers in the surgical pathology diagnostic process. Noadditive noncoagulant fixative would be a dream fixative. The more realistic option would be a noadditive coagulant alcoholic fixative with its disadvantages of shrinking and hardening. Modified methcarn (8 parts of methanol and 1 par of acetic acid) looks promising. There is a long way to the more or less commonly accepted IHC friendly fixative.

The cost of the fixative is not an irrelevant issue. Laboratories are also participants in the sky rocketing cost of medical services. The low cost of formalin is determined not only by simplicity of production, but also by the amount of demand that drives the cost down.

The speed of fixation is an important factor in choice of a fixative. Turnaround time (TAT) is a reality in the surgical pathology laboratories. It is unclear why the prostate biopsy should be reported the next day (some laboratories try to report the same day) if the patient, as a rule, were scheduled to see the doctor in a couple weeks. Everyone who works in the practical laboratory knows how often supervisors try to please the pathologist accelerating the processing. However, the rush epidemic is a fact of medical life. At least, fixation ought to be complete.

Molecular- friendly is also more a wish than a reality of every day practice in surgical pathology. No any of molecular fixatives can in this point meet main requirements to the universal fixative as cost, simplicity of the recipe, stability in storage, previous experience, the gold standard in Juan Rosai’s words, of generations of pathologists, who will not trade it at this time the elusive gains for diagnosis of molecular biology, even an individually targeted cancer treatment.15

Safety considerations are the most persuasive in arguments of fixative choice for understandable reasons. Formaldehyde is the primary target of safety concerns. Without any doubt it is chemically active. It irritates eyes, mucosa, and skin. This author had had two formalin burns of the eyes, loss of voice after cleaning up a formalin spill, and many other chemical injuries. There are people with allergic reaction even to minimal concentration of formaldehyde in the air.16

Among objections to formalin use, the major is formaldehyde’s cancerogenicity. However, the data about formaldehyde’s cancerogenicity are limited.15 Analysis of National Cancer Institute cohort studies presented the excess of nasopharyngeal cancer among workers in formaldehyde industries although data about formaldehyde exposure as a cause of leukemia and lung cancer are inconsistent.17 The safety question requires separate sober discussion. The question of formalin safety is discussed in detail in the article “A Pragmatic Approach to Formalin Safety in Anatomical Pathology”, as well as at this website (see the article at the link Safety in Grossing Room).18

Meanwhile we have to work with formalin. Our stern, but true friend will serve practical surgical pathology in foreseeable time using all reasonable safety measures. Not optimal but merely a compromise tissue fixative.

CONCLUSION

The problem of a pragmatic consensus fixative for surgical pathology remains. Appropriate fixation is the main precondition for a representative, informative, and standard microscopic picture under the pathologist’s microscope. If fixation had been done properly, deficiencies of processing are repairable in most instances.  As in real estate, the motto is: Fixation, Fixation, Fixation.

Acknowledgment

Many thanks to René J. Buesa BS, HLT (ASCP) for valuable comments.

References

1. Buesa RJ., Letter to the Editor. Histochemistry: a case of unappreciated beauty? J Histotechenol  26 (4):283, 2003

2. Pearse AGE. Histochemistry, 4th ed. 1980 Vol 1.Baltimore, Williams&Wilkins Co

3. Lillie RD & Fulmer HM 1976 Histologic Technique and Practical Histochemistry 1976New   York: McGraw-Hill, pp.52-53

4. Bancroft JD, Gamble M: Theory and Practice of Histological Techniques. 5th EdNew York,NY, Churchill -Livingstone, 2002.

5. Carson FL, Hladik Ch: Histotechnology: A Self-Instructional Text 3nd Ed. Chicago, Ill ASCP Press; 2009 pp 1-23.

6. Kiernan JA Histological and Histochemical Methods, 2008, 4rd ed. Oxford: Butterworth-Heimann.

7. Dapson RW Fixation for 1900s: a review of needs and accomplishments, Bioth & Histochemistry 68 (2) 75-82, 1993

8. Wenk PA Fixatives: Coagulatives vs. Non-Coagulative or Is it Additive vs. Non-Additive? Histologic December 39 (2): 36-39, 2006

9. Dimenstein IB: Bone Grossing: Helpful Hints and Techniques Annals of Diagnostic Pathology, Vol12 (3):193-198, 2008

10. Buesa RJ: Histology without formalin? Annals of Diagnostic Pathology Vol 12 (6):387-396, 2008

11. Dapson RW Formaldehyde: Past, Present & Future 2001 HistoLogic May; 34 (1): 5-8, 2001

12. Dapson RW: Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval. Biotech & Histochem: 82 (3) 161-166, 2007

13. Dapson, RW  ‘Macromolecular changes caused by formalin fixation and antigen retrieval’, Biotechnic and Histochemistry, 2007; 82:3, 133 -140

14 Nadji M, Nassiri M, Vincek V et al: Immunochemistry of tissue prepared by a molecular-friendly fixation and processing system. Appl Immunohistochem Mol Morphol, 13: 277-282, 2005

15. Juan Rosai, Why microscopy will remain a cornerstone of surgical pathology Laboratory Investigation, Volume 87, May: 403-408, 2007

16. Formaldehyde (CASRN-50-00-0) Integrated Risk Information System US-EPA, IRIS Summaries.

17. Hauptmann M, Lubin JH, Stewart P.A, et al: Mortality from solid cancers among workers in formaldehyde industries. American Journal of Epidemiology, 2004; 159(12): 1117-1130.

18. Dimenstein IB.: A Pragmatic Approach to Formalin Safety in Anatomical Pathology”. LabMedicine, 2009; Vol 40, (12): 740-746.

 

 

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