Fixation is a key step in the practice of diagnostic pathology, and even in modern times, is intimately linked to the use of formaldehyde (HCHO), a gas commercially available in concentrated solution (37%), stabilized with ethanol, and named Formalin. A 10% dilution of concentrated formalin (in tap water or buffers with final concentrations of formaldehyde of approximately 4%) is named 10% formalin
The main motives for the popularity of formalin among pathologists are: low cost, straightforward laboratory preparation procedure, long-standing tradition and international use.
There is a general consensus that formalin is the best fixative and there is therefore no need for improvement for any reason, generating what has been defined by some authors as “the formalin dogma”
, a somewhat fatalistic and “addictive” approach that has severely hampered the search for alternatives to formalin in fixation procedures.
Political changes in recent decades, along with the generalized shift towards a “market-regulated economy”, have introduced economy-driven research, and the proprietary use of innovative and scientific observations, along with a more open approach as well as the free exchange of scientific knowledge. However, the free marketing of alternative fixatives with registered and proprietary formulas have in fact acted as an obstacle to the diffusion and sustained use of registered products
. It has also discouraged the quest and testing of formalin substitutes for every-day use, and the widespread scientific research and investigation into new fixatives.
The toxicity of formaldehyde is emerging as the main issue for its abolition as a general fixative used in large quantities in pathology laboratories (a medium-large structure may annually use more than 3,500 liters of ready-to-use formalin).
In 1987, the U.S. Environmental Protection Agency (EPA) classified formaldehyde as a probable human carcinogen under conditions of unusually high or prolonged exposure
 and a few years later the permissible exposure level was lowered from 1 ppm to 0.75 ppm (average daily exposure)
 and the potential toxicity of formaldehyde exposure to technicians and pathologists was stressed, meanwhile promoting the search for alternative fixatives.
More recently, from the initial IARC report linking formaldehyde exposure to human nasopharyngeal carcinoma
, a successive link between formaldehyde exposure and leukemia was put forward in 2009
; these statements were reinforced in the 2012 report
 by the same agency. These data of the carcinogenicity for humans of formaldehyde should act as a potent stimulus to reconsider the “formalin dogma” and to evaluate with an open mind the use of alternative fixatives.
The U.S. Occupational Safety and Health Administration (OSHA) stated that employers must reduce worker exposure to formaldehyde at, or below, permissible exposure limits (PEL) and the TWA (time-weighted average) should be less than or equal to 0.75 ppm. The 15-min short term exposure limit (STEL) is 2 ppm.
International and National agencies set stringent limits of formaldehyde exposure ranging from 0.016 ppm TWA (National Institute for Occupational Safety and Health, USA) to 2 ppm (OSHA and Australia) for STEL
Moreover, the formation of DNA-Protein cross-links (DPC or DPX) possibly represents a permanent “signature” of exposition to formalin
. More recently chromosomal alterations have been detected in health workers and linked to formalin use in Pathology Wards
. This information was largely ignored by the pathologist community who assumed that the use of formaldehyde as a fixative is mandatory and thus completely safe when used with adequate procedures
Recent reports comparing a group of pathologists and industrial workers, and using individual devices to monitor exposure to formaldehyde demonstrated that environmental exposure in pathology departments is trivial in most areas while the sampling activity in the gross room may result in exposure to toxic levels that exceed the recommended values
[15, 16]. Individual exposure to formaldehyde was monitored by measuring Malondialdehyde-deoxyguanosine adducts on peripheral leukocytes and the alkylation of hemoglobin to form a terminal N-methylene valine residue. These data show that formaldehyde exposure in the gross room is comparable to that of workers in plastic factories and that perfectly efficient aspiration devices may not in fact avoid individual exposure.
Nevertheless, law restrictions on the use of human carcinogenic substances, mainly directed at industrial production and use, are also valid for pathology laboratories. In Italy, safety labels on formaldehyde preparations have not changed from irritant (X) to carcinogenic substance (skull pictogram). One of the aims of our study is to clarify the necessity of general awareness of the scientific and ethical issues related to the abolition of formaldehyde use in pathology laboratories, and to contribute to the quest for valid alternatives, either homemade or commercially-available.
On the other hand, this classification into a Class I substance makes surveillance of exposed personal mandatory, and may involve Directors of Surgical Pathology Departments as well as Hospital Administrators responsible for the health of workers. The delay in introducing this new information as a National Standard has led to the generation of the idea that the IARC warning was limited to industrial environments and not mandatory for pathology laboratories.
However, a recent European legislation (e.g. in Italy n° 81/2008) clearly states that carcinogens should only be used in closed work-cycles and must be substituted when technically possible (Art. 235). European Union legislation is moving in the same direction.
While industries (e.g. wood and furniture) are rapidly adhering to law prescriptions (and are certifying the absence of formaldehyde vapors), in the medical field an unusual delay is still present.
A rational, open-minded and innovative approach to fixation, with the scope of abolishing formalin use altogether,
[17, 18] or at least limiting its use to very rare situations in which there are no available alternatives (like Formaldehyde-induced fluorescence for Catelcholamines or formaldehyde-vapor fixation in certain histo-enzymatic determinations
The use of formalin in surgical pathology laboratories may be partially justified by the large size of the surgical specimen, putatively easier to handle after formalin fixation and supposedly better fixed in comparison to alternative fixatives. However, it must be stressed that manipulation of large resection materials, along with increasing demands of more detailed macroscopical descriptions and extended sampling (as required by modern staging of tumors) may increase the exposure of pathologists to formaldehyde vapors.
However, when studying experimental animals of small dimensions no rational explanation may be proposed for the continuation of the use of formalin-fixed tissue for microscopical evaluation.
Nevertheless, the National Toxicology Program of the U.S. – probably the largest program in this field - requires the use of buffered formalin
. Conversely, an European Institution (Cesare Maltoni Cancer Research Center, European Ramazzini Foundation of Oncology and Environmental Sciences, Bologna, Italy. firstname.lastname@example.org) suggests alcoholic fixation of all organs of rats apart from bones
. Curiously, for de-calcification the same protocol suggest a solution containing formaldehyde and formic acid, despite there being several formaldehyde-free methods of decalcification available, as reported in literature and on the market.
As these formaldehyde-free procedures are effective in human pathology for large bony samples, there is no logical reason for the continued use of formaldehyde - formic acid formulations for decalcification of bones of much smaller experimental animals.
A second more recent, but preeminent issue, is the search for a more effective way of fixing tissues in order to preserve nucleic acids and proteins for molecular biology techniques
[21–25], with the perspective of utilization of the vast surgical pathology archives of tissues, for scientific research on tumors or other relevant human diseases, and to integrate these determinations into clinical diagnoses.
The importance of good preservation of tissues, not only for microscopic evaluation, but also for Histochemistry, Immunohistochemistry and Molecular Biology is taken into consideration
[17, 21, 26, 27]. The fatalistic approach is that that once a specimen is dropped in formalin, no other manipulations or control procedures are necessary. This is the case even for very large specimens that will never be adequately fixed in their central parts unless proper slicing (or alternative maneuvers) is applied. It is also given a low profile which underestimates the potential of new tools (like nucleic acid technology or proteomics) in the diagnostic process.
As highlighted in a study on the influence of fixation and processing parameters
, the final fixation of many tissue of large specimens primarily fixed with formalin is frequently achieved during processing and is actually an alcoholic fixation. This note - contrary to efforts to standardize formaldehyde fixation and processing
 - is a further argument against the “formalin dogma”, but it also an important rational explanation of the fact that paraffin-embedded tissues are very stable and that tissue reactivity is influenced by primary fixative, but is stabilized by alcoholic dehydration and clearing before embedding. For instance, the deleterious action of Picric Acid (a constituent of Bouin's Fixative) mainly affects DNA bases and is not related to improper fixation.
Is worth noting that a practical application of the “formalin dogma”, the acronym FFPE (formalin- fixed paraffin embedded) enforced the dogma than no other fixative but formalin should be used
The use of microwaves may be a interesting way for obtaining a better fixation either as a step in fixation–processing procedures, or as an upgrade of automatic processing instruments.
Commercially-available alternatives to formalin are fairly numerous and in different supply formats either ready-for-use or in concentrated forms. While ready-to–use formulas are very convenient, concentrated formulas may allow some degree of manipulation and custom tailored modifications.
As noted by Kiernan
 the use of “secret mixtures”, that is- the registered proprietary formulas of poorly detailed components- is a severe limitation to a rigorous scientific approach to fixation, and despite being expensive, may be a straightforward solution, at least in the short term.
Less numerous are the non- proprietary formulas proposed in recent scientific literature while the wealth of information on time-honored fixatives reported in scientific journals and histochemistry text books may be worth revisiting as they give useful clues for new improvements. The use of Zinc salt-based fixatives as alternatives to formalin was recently reported by a Swedish group
 and subsequently upgraded by other scientists
The availability of commercial alternatives to formalin is mainly restricted to Western countries, being widespread in North America, and to a lesser degree in Europe. The possibility of preparing effective substitutes in the laboratory may be of particular interest, considering the high cost of an effective and durable aspiration chain of formalin vapors (from aspiration hoods, aspirated cabinets and final proper disposal), and the limited economical resources of developing countries that frequently lack proper servicing of instruments
The low cost of formalin, being less expensive than brand substitute products, and also being readily available in the market by many suppliers and in different convenient formats has hampered the diffusion and research of alternatives. Indirect costs of formalin fixation (for example: adequate disposal of used fixative and fixed specimens; potential toxicity of products from incineration of residual specimens; and the imperative necessity of perfect maintenance of aspirations devices, such as hoods and aspirated cabinets) should also be considered in economical evaluation of costs. Moreover the necessity of adequate health surveillance of workers exposed to a human carcinogen should be also considered as an additional and non-trivial economical cost.
Recently, new devices were proposed by industrial firms to avoid the exposure to formaldehyde in pathology laboratories during grossing activities
[33, 34] however a more radical approach for completely abolishing formaldehyde and for obligatory use of alternative fixatives not based on gas in solution seems more appropriate.
In the present study we started from the standard practice of our pathology laboratory at the Ospedale Infantile Regina Margherita (OIRM) disestablished in 2009, in which formalin use as a fixative was limited to autopsy practice, a small activity in comparison to histology and cytology.
The decision to substitute formalin was the only available possibility to protect lab personnel from gas exposure when faced with the refusal of the hospital administration board to upgrade the aspiration hoods in a reasonable time.
From 1992, formalin was substituted with a commercial formula distributed in Italy as “Biolina” but corresponding to “Histochoice” (Amresco, OH, USA). This glyoxale-based fixative with ethyl alcohol (20-40% in the ready-to-use fixative) gave an acceptable morphology in our hands, and was used for almost 3 years as a routine fixative. In the gradual process of formalin substitution, 0.5 ml of methylene blue was added to a liter of Biolina, obtaining a light blue color. This simple method permitted detection, at a glance, of the presence of the substitute fixative, and avoided the use of smelling when in doubt. The use of this “light blue fixative” was widely accepted, especially in surgical theaters. In the autopsy practice, Biolina was unsatisfactory due to a slower tissue penetration and the lack of tissue hardening that made further trimming difficult. Therefore, Formalin use was limited to post-mortem examinations with cautious procedures to limit vapor diffusion.
In order to protect the proprietary formula, the safety sheet did not fulfill the European standards on safety instructions for chemical products for laboratory or industrial use (16 points are required to be clearly stated)
. Subsequently Biolina was retracted from commercial use in Italy, and all known deposits of the reagent were sequestrated.
Abruptly, it was necessary to face a difficult situation in which no commercial alternative was available in the national market, and the temptation to return to formalin was therefore very high.
From a rapid consultation of the scientific literature we discovered the interesting investigations of Bostwick
 with a commercial substitute of formalin (Stat Fix). From the published components, we were able to prepare a mixture of PolyethylenGlycol (PEG), acetic Acid, Glycerol and ethyl alcohol (PAGA) in the lab, and this formulation was used with decent morphological results and no problems in Immunohistochemistry or Histochemistry. For almost one year PAGA was used in the substitution of formalin. Although PAGA was satisfactory as a routine fixative and not particularly expensive, an abrupt shortage of technical help led to a shift to a commercial alternative to formalin. No-Tox (EarthSafe Industries, Inc., Belle Mead, NJ 08502), a new commercial substitute of formalin was introduced in the market and was available in Italy. Unfortunately some difficulties were encountered with Immunohistochemistry (particularly Mib-1) and no suggestions were available from the producer in order to resolve this staining problem, and antigen retrieval experience was still limited
[28, 36]. We tested another new substitute of formalin, produced and marketed by Merck (Neo-Fix), which was an alcohol-based fixative (50% ethyl alcohol) and PEG. Since tissue shrinking was still acceptable and Mib-1 was easily detected, we shifted to the use of Neo-Fix and used it for more than 6 years, but due to commercial problems this fixative was no longer commercially available in Italy at the end of 2003. At that time we tested the commercially available alternatives to formalin present in the Italian market and selected FineFixx (Milestone, Italy) as the most suitable. FineFixx is an isopropyl and ethylic alcohol-based fixative (70% ethyl alcohol and tensioactive agents). The high alcohol percentage has evident shrinking artifacts but, despite this, morphology is acceptable. Immunohistochemistry and Histochemistry were also satisfactory.
In the quest for formaldehyde abolition, we also faced the problem of finding an effective substitute for the formaldehyde-formic acid mixture for a de-calcifying formula suitable for large orthopedic specimens. We obtained the recipe of de-calcifying fluid that was used routinely at the Surgical Pathology Dept. of Pini Orthopedic Hospital of Milan (Italy) (courtesy of A. Parafioriti M.D.) This method, based on Formic and Hydrochloric acids, was as effective as the former and easy to prepare in the laboratory.