Overview Of Formalin Use In Pathology And Possible Alternatives

Overview Of Formalin Use In Pathology And Possible Alternatives

Introduction:

Formalin has been used as a tissue preservative in operating rooms and pathology laboratories for over one hundred years. Formalin is a solution of about 4% formaldehyde and water (known as 10% NBF) and is ubiquitous in clinical laboratories, pathology laboratories, and in operating rooms. It is an inexpensive reagent and effective at what it does. Unfortunately, it is hazardous. According to the U.S. Department of Labor’s Occupational Safety & Health Administration (“OSHA”), formalin/formaldehyde is a moderate fire and explosion hazard when exposed to heat or flame and when mixed with certain chemicals. More problematic are its health hazards. Formalin is highly irritating to the upper respiratory tract, eyes, and skin, and is a known carcinogen. “In humans, formaldehyde exposure has been associated with cancers of the lung, nasopharynx and oropharynx, and nasal passages.¹ It is also considered to be mutagenic. In addition to those problems, modern healthcare institutions often have multiple clinics, hospitals, operating rooms, and laboratories at separate geographic locations. This requires that surgical tissue samples, large or small, be transported over significant distances in formalin-filled containers, greatly increasing the risk of potentially dangerous formalin spills.

Here we have discussed the risks of formalin use, trends in formalin use and disposal, and how laboratory and operating room workflow is affected by it. Alternate methods for storing and preserving pathology specimens will be introduced along with descriptions of changes to workflow, benefits in terms of healthcare worker safety, and the safety and economic benefits of these changes.

The problem, as noted, is that if a high-volume laboratory or hospital processes thousands of anatomical samples yearly, they are utilizing literally thousands of gallons of formalin, with its inherent hazards. In addition, hospitals, health systems and laboratories are increasingly spread out over regional distances (sometimes nationally), which require tissue samples to be transported, increasing exposure and the risk of spillage. In addition, any changes to standard procedures, i.e., eliminating formalin altogether or using a different type of fixative, requires that the tests be validated for clinical utility by the laboratories.

Formalin - A Key Component Of The Histopathology And Pathology Laboratory.

Formalin, despite its known health hazards, remains a key component of the histopathology and pathology laboratory. Over the years alternative fixatives have been studied, such as Bouin and Hollande, and newer products such as GreenFix, UPM and CyMol. A study published in The European Journal of Histochemistry compared these fixatives “to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods.²

Each had their advantages and disadvantages, but ultimately none provided right combination of acceptable qualities to replace formalin. The point of that study, however, was to evaluate whether any of those reagents could replace formalin in terms of test validation. The focus was not safe use or ease of disposal. Due to issues of cost, re-validation and the need to evaluate long-term effects of alternate fixatives on tissue preservation, it is unlikely that total elimination of formalin is likely or even possible. In most cases, the pathologist or histologist has been trained to interpret characteristic tissue morphology that is created by formalin fixation. Laboratory scientists and pathologists looking to replace formalin fixation have several factors to consider:

• Toxicity of the fixative (both short-term and cumulative)

• Component volatility

• Flammability

• The effects of over-fixation on tissues

• Storage requirements if specimens cannot be left in fixative

• Compatibility of the fixative with instrumentation

• The practical, legal and financial requirements of disposal after use³

A more practical and realistic goal is to evaluate methods and approaches to minimize the use of formalin while still maintaining a high level of fixative effectiveness and flexibility, while decreasing healthcare workers’ exposure to formalin.

Gross Examination

Tissues biopsied from the patient are transported from the operating room to the pathology department. Traditionally the specimen is placed into a container filled with formalin then transported to the pathology laboratory. The pathologist or pathology assistant will describe the physical appearance of the tissue sample. Small representative samples of the tissue are placed into small plastic cassettes that hold tissue while it is processed. The processed tissue is then embedded into a paraffin block.

Fixation

If the tissue was not transported in a fixative (such as formalin), it typically will be fixed in formalin as a step during processing. According to the University of Utah Medical Library, “The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis.”⁴ There are five groups of fixatives—aldehydes, mercurial, alcohols, oxidizing agents, and picrates—but formalin is considered to be the most effective for all uses.

For the purposes of current discussion, the important steps occur between surgery and gross examination and processing, because those steps involve formalin. However, to give context to validation issues, it should be noted that, increasingly, tissue specimens can undergo a variety of molecular-based and immunohistochemical (antigen)-based tests that can be performed on samples taken from paraffin blocks.

Pathology Tissue Specimens Movement

Almost immediately after removal from the body, tissue specimens begin to degrade. The primary factors involved in tissue degradation are warm and cold ischemia, i.e., time and temperature, or the time between the chilling of the tissue, organ or body part after excision. Even within a single institution, there is transport time from removing the specimen from the patient and moving it to the laboratory. In facilities with greater geographic distances, such as hospitals and surgery centers at separate facilities from the laboratory, the time it takes to transport the specimen becomes a factor. This is why tissues are traditionally placed into a fixative, to halt the process of degradation. But, as described above, fixatives such as formalin pose a health and safety risk to healthcare providers.

Time

Even use of formalin does not cause “instant” fixation of tissues. The rule of thumb is, under ambient and passive exposure, it takes one hour for formalin to penetrate 1 millimeter into tissues. Although finding data regarding the amount of time a tissue specimen can remain viable at room temperature unfixed is hard to come by, “not very long” is the most common response. Numerous studies have been conducted on different types of tissues examining preanalytic ischemic times for specific antigen and protein expression data. A study by Neumeister et al. (2012), for example, focused on breast cancer tissue, concluding that “Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions.⁵ They also conclude that size and heterogeneity of the tissue plays a significant role. Creating even more time pressure on histologists and pathologists are DNA and RNA degradation times. A 2004 study by Spruessel et al. reported that, “Initial changes of gene and protein expression profiles were already observed 5-8 min after colon resection. Fifteen minutes after surgery, 10%-15% of molecules, and after 30 min, 20% of all detectable genes and proteins, respectively, differed significantly from the baseline values.⁶

Temperature

As suggested above, room temperature specimens have a very short period of viability for the majority of laboratory uses. If fresh specimens are not examined immediately, they are typically fixed as soon as possible. Even in various fixatives, time is a factor. Geoffrey Rolls points out in his article, “Fixation and Fixatives”, “For light microscopy initial fixation is usually carried out at room temperature and this may be followed by further fixation at temperatures up to 45°C during tissue processing.⁷

A study published by Yildiz-Aktas et al. (2012) evaluating cold ischemic time on IHC results for ER/PR and HER2 expression in breast tumors said, “Non-refrigerated samples are affected more by prolonged cold ischemic time than refrigerated samples. Cold ischemic time period of as short as one-half hour may occasionally impact the immunohistochemical (IHC) staining for progesterone receptor. Significant reduction in IHC staining for hormone receptors, and HER2, however, generally does not result until 4 hr for refrigerated samples and 2 h for non-refrigerated samples. The ASCO/CAP guideline of cold ischemic time period of <1 h is a prudent guideline to follow.⁸

Safety

Transporting specimens in formalin, obviously, expands the risk of exposure and spills. Containers for transport need to be well sealed, resistant to vibration and shock that may cause leakage or breakage. In the amounts typically found in the laboratory, OSHA has designated formalin spills under the category of Manageable Chemical Spill, which is short of Immediately Dangerous to Life and Health (IDLH). What this means, from a practical point of view, is that most formalin spills in a laboratory or transport environment (i.e., courier vehicle), can be cleaned up by someone who is not hazmat trained. However, it is flammable, potentially explosive, as well as carcinogenic and mutagenic. Standard procedure in most hospital or lab environments would include cordoning off the spill area during clean-up, and potentially evacuating personnel to avoid exposure to fumes. Any system that would minimize or eliminate the amount of fixative/formalin would be desirable.

New Pathology Tissue Management Technologies and Methods

As mentioned, formalin is a reliable, well-documented fixative. It is also hazardous, and any method that can decrease the amount of formalin used while maintaining current clinical viability would be welcome, both for healthcare provider safety and for decreased disposal efforts and costs. Two new approaches, both utilizing under-vacuum sealing, include:

• Delaying the introduction into formalin/fixative for controlled short-term transport and storage

• Standardizing formalin/fixative amounts based on weight and specimen type

Delaying (or Eliminating) Formalin Use During Specimen Transport—Milestone’s TissueSAFE

It is well known that tissues, once removed from the body and kept at room temperatures, begin to degrade quickly. Quick freezing or refrigeration is one option, although not suitable for all tissues or applications. A new alternative, however, for short-term storage of pathology tissues is a combination of vacuum storage and refrigeration. Milestone Medical (Sorisole, Italy) has developed a system for sealing tissue specimens in a medical grade vacuum bag, which is then stored refrigerated for a limited time. This system is called TissueSAFE. Immediately after excision the specimen is moved from the operating room to an adjoining room where the TissueSAFE system is located. The tissue is placed in a specimen bag and sealed under vacuum. Specimen bags can be labeled with critical case information. The tissue sample is then placed into a carryable “coolbag” for short distances or into a refrigerated transfer box. In this controlled environment, most tissue types are preserved “as fresh” for up to 72 hours. Once the specimen is delivered in this state to the laboratory, it is removed from the sealed bag, examined, and prepared for diagnostic testing, either on the fresh specimen or following fixation of the tissue. TissueSAFE benefits include:

• No transport of formalin
• Minimizes formalin in the operating room
• Reduce nursing staff exposure to formalin fumes
• Reduce or eliminate costly formalin spills
• Specimens are held “as fresh,” enabling improved visualization during grossing
• Tissues do not dry out
• Autolytic (degradation) process is slowed
• Tissue cools quicker in vacuum than in air
• Pathology department selects fixative type, fixation start time and duration, while improving documentation of this workflow step

Researchers at the Città della Salute e della Scienza of Torino (Italy) evaluated the TissueSAFE system. The authors state, “From a pathological standpoint no morphological or immunohistochemical drawbacks were encountered in a local series (more than 2000) of UV (under vacuum) processed cases.”9 Based on that study and others, the Italian Group of Mammary Pathology (GIPaM) of the Society of Pathology (SIAPEC) recognized the UV methods of tissue preservation and transport.¹⁰

Standardizing Formalin – Milestone’s SealSAFE

The SealSAFE system is similar to the TissueSAFE system except that it utilizes a specimen bag to which formalin can be added, then the specimen bag and the tissue it holds is vacuum-sealed. The generally accepted ratio of formalin used to tissue weight is 10:1. Unfortunately, this really is more dogma than scientific fact. A 2012 study by Buesa and Peshkov11 found that a 2:1 ratio at 45°C was adequate. In working with the Milestone SealSAFE system and experimenting with the formalin to tissue weight ratio, Dr. Richard J. Zarbo, MD, Senior Vice President and KD Ward Chair of Henry Ford Health System’s Pathology and Laboratory Medicine, has presented data indicating that a 1:1 ratio is adequate.^12,13

Using specially designed vacuum bags, the tissue is placed in the bag, then placed into the SealSAFE cavity. The device automatically weighs the tissue specimen. The laboratory assigns a preset ratio of formalin to tissue weight (1:1, 1:2, 1:2.5, 1:3, etc.), then fixative is dispensed into the bag while under a closed case, keeping splashing or fumes from the technologist or nurse performing the procedure. The final step seals the specimen bag, under vacuum, within the closed and ventilated cavity. The same bag can be opened and resealed up to three times from the operating room to final storage, if samples need to be taken for different types of testing. SealSAFE benefits include:

• Standardization and quality – the same amount of formalin to tissue is always used

• Less formalin – vacuum bags reduce formalin volume

• Safety – closed and ventilated system – reduces formalin exposure

• Improved archiving – prints labels with pertinent patient information

• Cost savings – decreased formalin volumes and use of bags versus rigid containers reduces costs of biohazard material disposal

• Enables flexibility in fixative choice – formalin, formalin substitutes, alcohols, and molecular fixatives.

Studies have validated both systems for histology, immunohistochemistry, and DNA and RNA-based molecular tests.^12,13

Conclusion

Concerns over formalin use in clinical laboratories, as well as its use in various manufacturing processes, are not new. It is unlikely that use of formalin will ever be completely eliminated in the anatomic and surgical pathology laboratory. As a fixative, formalin is highly functional and inexpensive. Because of its potential health hazards and disposal costs, it is important that healthcare institutions and laboratories make every effort to eliminate use of formalin when possible while still maintaining high quality healthcare services, or maximize methods that will minimize the amount of formalin used as well as providing technologies that guarantee healthcare worker safety. While using formalin, appropriate working practices include minimizing formaldehyde evaporation as the safety rationale. Formalin can remain for a long time as the ubiquitous fixative reagent in anatomical pathology.¹⁵

References:

1. United State Department of Labor. Occupational Safety & Health Administration (OSHA). Substance technical guidelines for formalin.

2. Gatta LB et al. Application of alternative fixatives to formalin in diagnostic pathology. Eur J Histochem. 2012 May 4:56(2):e12. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428961/

3. Rolls G. Fixation and Fixatives, Part 4 – Popular Fixative Solutions. Leica Biosystems, Wetzlar, Germany. March 6, 2012.

http://www.leicabiosystems.com/pathologyleaders/fixation-andfixatives-4-popular-fixative-solutions/

4. Histotechnology. University of Utah Medical Library.

http://library.med.utah.edu/WebPath/HISTHTML/HISTOTCH/HISTOTCH.html

5. Neumeister VM et al. Quantitative Assessment of Effect of Prenalytic Cold Ischemic Time on Protein Expression in Breast Cancer Tissues. J Natl Cancer Inst. 2012;104(23): 1815-24.

6. Spruessel A et al. Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision. BioTechniques. June 2004;36:1030-1037.

7. Rolls G. Fixation and Fixatives, Part 2 – Factors influencing chemical fixation, formaldehyde and glutaraldehyde. Leica Biosystems, Wetzlar, Germany. March 6, 2012.http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-

2-factors-influencing-chemical-fixation-formaldehyde-andglutaraldehyde/

8. Yildiz-Aktas IZ et al. The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Mod Pathol. 2012 Aug;25(8):1098-105.

9. Bussolati G et al. Tissue transfer to pathology labs: under vacuum is the safe alternative to foromalin. Virchows Arch. 2008 Feb;452(2):229-31.

10.http://www.siapec.it/content/file/3325/2013_DocumentoDEFINITIVO_GIP_3_.pdf

11. Buesa RJ, Peshkov MV. How much formalin is enough to fix tissues? Ann Diagn Pathol. 2012 Jun;16(3):202-9.

12. Zarbo RJ et al. Validation of Tissue Preservation Using a Vacuum Sealed Formalin-Free Transport System. United States and Canadian Academy of Pathology abstract #2096. Baltimore, MD, March 6, 2013. Mod Pathol. 2013;26:S1.

13. Chitale DA et al. Molecular Validation Study of Nucleic Acids Extraction from Vacuum Sealed Surgical Pathology Specimens. United States and Canadian Academy of Pathology abstract #2057. San Diego, CA, March 5, 2014. Mod Pathol. 2014, Suppl2;27:502A. S2.

14. Formalin. International Agency for Research on Cancer. 2006. http://monographs.iarc.fr/ENG/Monographs/vol100F/mono100F-29.pdf

15. Dimenstein IB. A Pragmatic Approach to Formalin Safety in Anatomical Pathology. Lab Medicine. December 2009;40(12):740-746,

16. Mark Terry (2014): Advances in Pathology Tissue Management Reduce Formalin Use, Improve Quality and Cut Costs. (Edit): Dave Sanford & Joel Servais Dark Intelligence Group, Inc.