Table of Contents
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Many of today’s researchers prefer to use FFPE samples of tissues for their IHC, histological or in-situ genomic analyses. FFPE stands for “Formalin-Fixed Paraffin-Embedded” and describes the two key features of this method of tissue preservation. Formalin is a formaldehyde solution and has been in use since the discovery in the late 1800s of the preservative effects of formaldehyde by the German physician Ferdinand Blum (Fox, et al., 1985). Paraffin is infused into the tissue following fixation with formaldehyde, and the tissue is also surrounded with a paraffin casing to help support it and protect it from oxidation. Professionally fixed and embedded FFPE samples and the biomolecules they contain are reasonably stable at ambient temperatures. Structurally, the fixed and embedded tissues are resilient and can be used for microscopic anatomy studies almost indefinitely, but over time, the antigenicity of some proteins will degrade limiting IHC studies to samples no older than a few decades. Double stranded DNA is surprisingly stable in FFPE blocks, but other less stable biomolecules such as RNA may degrade within a decade or less, especially once sections have been cut.
Archives that accumulate large numbers of FFPE samples are an especially rich source of data when comparison of many cases (i.e. FFPE samples from multiple donors) is desirable in order to draw statistically robust conclusions about the characteristics of particular indications. If the FFPE samples are going to be used in “high stakes” biomedical research, then it is important that every stage of the preparation process is performed by fully trained and certified professionals, preferably in a CLIA certified (i.e government inspected) or CAP accredited (College of American Pathologists) laboratory.
It should be noted that the FFPE process is not universally standardized, and institutions across the globe may use slightly different protocols with different formalin solutions or otherwise tailor the process to their preferences and requirements. What follows is an overview of the process and descriptions of what each step entails, as well as some common variations.
Prior to tissue processing is the necessary step of tissue acquisition. This is actually the most difficult element to control because it is intertwined with patient care, 41CFR46 compliance, and in the US, internal review board (IRB) oversight. The specifics of the medical procedure and the conventions of standard care are important because they can affect variables such as time intervals between the administration of anesthesia and ligation of vessels, removal of the tissue and the elapsed time before fixation, any of which may impact the quality of the specimen. For example, changes in RNA and proteins are likely to occur during the time interval, known as warm ischemia time, between ligation of the blood supply and tissue removal (Dash, et al., 2002). This ischemia time can range from minutes to hours depending on the organ, the standard operating procedures of the institution, the surgeon and other healthcare personnel involved, and the surgical approach. For that reason, it has been recommended that this time be recorded for each specimen and that it be kept to a minimum (Hewitt, et al., 2008).
FFPE Tissue Processing
Once the tissue sample has been acquired, additional triage, dissection or micro-dissection (collectively referred to as grossing) may be needed to isolate and prepare the specific portion of tissue that will be passed through the remaining processing steps. Tissue processing today is often completely automated up to the final step, embedding, which may be manually accomplished. There are many tissue processors available, but all will follow the sequence of the first four steps shown above. The automation of the various steps helps to eliminate variations in the procedure as a source of variation of experimental results between different FFPE samples.
Fixation is the initial step in FFPE tissue processing. Critical factors for consideration include the solution to be used, the length of time allowed for fixation, and the thickness and histological properties of the tissue sample to be fixed.
The fixative that is used by most laboratories is neutral-buffered, 10% formalin. Formalin is a liquid that is 37-40% formaldehyde and 10% methanol (by weight) in water; thus a 10% formalin solution contains about 3.7% – 4% formaldehyde and 1% methanol (Hewitt, et al., 2008; Kiernan, 2000). In actuality, formaldehyde exists in solution primarily as monomers or small polymers of methylene glycol (methanediol, formaldehyde monohydrate) which is formed by the reversible reaction of formaldehyde with water. High weight methylene glycol polymers are termed paraformaldehyde and will precipitate out of solution, but methanol slows this process of polymerization which is the reason for its inclusion in formalin solutions. Formalin diluted with water (such as 10% formalin)—and especially if it is a buffer solution—will contain principally monomers as the vast number of water molecules break up any methylene glycol polymers that would normally have existed in a solution of higher formaldehyde concentration (Kiernan, 2000). Buffers, the remaining component of formalin, are also added because formaldehyde has a tendency to oxidize to formic acid (Fox, et al., 1985). Formic acid in tissue fixation can result in the precipitation of formalin pigment (acid hematin) granules which can resemble pigments produced by some parasites (Pizzolato, 1976). Buffering of formalin prevents this from happening. Common buffering agents include magnesium carbonate, calcium carbonate, citrate, Tris, and phosphate buffers (Hewitt, et. al., 2008). Commercial formalin often contains phosphate buffers. “Neutral-buffered,” which is typically how formalin is prepared, simply means that the pH is kept to about 7.
• Process and Mechanism
Because of the low molecular weights of formaldehyde and methylene glycol, formalin (primarily methylene glycol in solution) penetrates tissues relatively quickly, at a rate that depends on tissue type but with an average rate of 1mm/hour (Hewitt, et al., 2008). Protocols for fixation will thus vary depending on what type of tissue is to be fixed. Once inside the tissue, the fraction of formaldehyde molecules that are present in solution will begin to cross-link with proteins, but this happens at a much slower rate than the rate of diffusion. The reaction of formaldehyde with the tissue draws it out of solution and tugs that reversible formaldehyde-to-methylene glycol reaction back in the direction of formaldehyde so that more of it is produced even as it is consumed (Fox, et al., 1985). Lysine amino side chains are the most reactive with formaldehyde, but others that are somewhat reactive with formaldehyde include arginine, asparagine, histidine, glutamine, serine, and tyrosine (Howat and Wilson, 2014). Following reaction, the resulting hydroxymethyl group attached to the complex can then further react with the same protein or other proteins, thus forming stable methylene bridges (protein-CH2-protein) (Kiernan, 2000). It is this that produces the insolubility and rigidity of tissues that have been formalin-fixed and thus preserved. About 24-48 hours are required for this cross-linking to sufficiently occur throughout the tissue (Thavarajah, et al., 2012), but it has been recommended that no more than 36 hours be allowed for this process as fixation for a longer period of time will decrease the quality of biomolecules in the FFPE samples, such as by shortening the length of nucleic acids that can be extracted (Hewitt, et al., 2008).
The major benefit of using formaldehyde—especially as neutral-buffered, 10% formalin—is that it preserves a wide range of tissues and components. However, it does have limitations. For instance, a combination of formaldehyde with glutaraldehyde (C5H8O2), or a solution of glutaraldehyde alone, is typically used for electron microscopy (Kiernan, 2000) as those solutions are better for preserving tissue structures for that purpose. Note that tissues prepared with a glutaraldehyde or glutaraldehyde-aldehyde solution will not be considered FFPE samples. Another challenge is the retrieval of DNA and RNA molecules from FFPE tissue as formalin tends to modify nucleic acids—even to the point of introducing artificial mutations in DNA—and break them up into small fragments (Srinivasan, Sedmak, and Jewell, 2002). Yet it is possible to extract DNA and RNA fragments from FFPE tissue that are suitable for analysis, as described in a protocol written by Tang, et al. (2009), with a key factor in nucleic acid recovery being appropriate protease digestion. Commercial kits for nucleic acid extraction from FFPE samples are available as well.
• Specimen Thickness
One other major point to consider regarding formalin fixation is the thickness of the tissue sample. While formalin does penetrate tissue relatively quickly, as mentioned above, the thickness of the tissue will affect the quality of the fixed sample. More time will be required for formalin to reach the center of thicker tissue samples. While formalin is gradually diffusing into the innermost reaches of the sample, the outer regions will already have begun the process of fixation so that the biomolecules there are more likely to degrade in the protracted length of time required for total fixation. Additionally, if time limits are adhered to (such as a maximum of 36 hours as mentioned above), then there is the possibility that the tissue sample will not be sufficiently fixed (preserved) in its entirety. For this reason, tissues for fixation that are larger in width than several millimeters or perhaps one or two centimeters would be better dissected into thinner segments for optimum fixation (Hewitt, et al., 2008). Institutions may have different protocols for the length of time and volume of fixative required for tissue samples of different widths, but in general the ratio of the volume of fixative to the volume of tissue should be 10:1 (Klatt, 2016).
The second step in FFPE specimen processing is dehydration. Because paraffin is immiscible with water, all the water from the formalin solution must be removed from the tissue before paraffin can infiltrate it. A series of alcohol (often ethanol) solutions, frequently beginning with 70% and progressing to 100% alcohol, will be used to remove essentially all of the water from the tissue. Using fresh solutions or frequently replacing used solutions is required for optimum dehydration because the alcohol will become increasingly diluted with use. It is also imperative that this step be completed fully (with sufficient time set aside for this step) because insufficient dehydration can lead to tissue degradation (Klatt, 2016; Hewitt, et al., 2008).
Since paraffin is actually not miscible in alcohols, either, the next step is to remove the alcohol with a substance that is miscible with paraffin. This step is known as clearing, and xylene is the clearing agent most often used (Klatt, 2016; Hewitt, et al., 2008).
Following adequate fixation, dehydration, and clearing, the tissue can finally undergo paraffin infiltration (or impregnation). This step is also not standardized, and institutions from around the world may use different paraffins and wax mixtures of varied composition. Paraffins have different melting points and textures that impact the final tissue block and its characteristics. In the United States as well as in Western Europe, synthetic paraffins with low melting points (55°C-63°C) are normally used for the best outcomes. Latex, dimethyl sulfoxide, and proprietary “plasticizers” may be included in the formulation in order to modify the texture and malleability of the final tissue sample (Hewitt, et al., 2008).
Nations from other parts of the world will often incorporate beeswax into their formulations in order to improve the malleability and decrease the melting temperature of low quality paraffins used. Beeswax contains contaminants such as pollen, however, and it decreases the quality of the final sample. Higher melting temperatures are to be avoided because they later result in decreased and insufficient deparaffinization of the tissue specimen as well as a decrease in the amount of nucleic acids recovered from the tissue (Hewitt, et al., 2008).
The last step in processing FFPE samples is paraffin embedding. The tissue specimen infiltrated with paraffin is surrounded with a coat of paraffin. Care must be taken to correctly orient the tissue block in the mold (“cassette”) because this impacts the plane of sectioning when thin sections are sliced off the block with a microtome (Klatt, 2016). Once the paraffin casing has solidified, the FFPE block is ready for storage and will remain well preserved for years, even up to decades (Hewitt, et al., 2008).
Once the tissue is embedded, it is ready for sectioning on the microtome. Since the tissue is not always completely flat against the front of the paraffin, the histotechnologist typically must “face” into the block. The tissue block is put into the block holder of the microtome and, while adjusting the angle of the block holder so as to waste as little tissue as possible, the histotechnologist turns the handwheel, moving the block up and down against a sharp blade. The block is cut until a full representative section of the tissue is at the forefront of the block. Once the tissue is faced, the block is put onto ice to cool, which will help facilitate the cutting of thin sections; paraffin that is too warm will create compressed tissue with wrinkled sections. When the block is cooled, it is placed back into the block holder. The technician again turns the handwheel, this time at a slow, even pace to create consecutive thin sections that stick to each other, forming a “ribbon.” The ribbon is placed onto a warm water bath (the temperature is kept just below the melting point of the paraffin) to smooth out any wrinkles that have formed. The technician then places a microscope slide into the water bath and “scoops up” the chosen section(s) so that they stick to the slide.
The most common thickness for tissue sections is between 3-5 micrometers (or microns, for short), which is the thickness of a single cell. Slides with 3-5 micron sections are normally used for staining, whether it be the standard Hematoxylin & Eosin (H&E) stain, special stain, or immunohistochemical (IHC) stain. Researchers will often request “curls” in lieu of thin sections on slides. Curls are thicker sections (10 microns or greater) that are put into Eppendorf tubes and are generally used whenever RNA or DNA needs to be extracted from the FFPE samples. Thick sections can also be put onto slides instead of tubes; this is optimal when only a portion of the tissue will be used for extraction. In this instance, the tissue is scraped off the slide and put into a tube. FFPE samples that have been sectioned are structurally stable, and if treated and stored properly they can be used for microscopic analysis for some time after the sectioning has occurred, but if chemical extractions are to be performed, then generally the sections should be used as soon as possible after cutting to prevent oxidative and biological degradation of the exposed target molecules.
Looking for information about the histology and procurement of FFPE blocks representing specific indications? Check out our histology resource page. Lab-Ally is an industry leader in 45CFR46 and 21CFR11 compliance, ethically sourced human biospecimens, IHC, ISH and FISH lab services, bioinformatics, scientific data management and more.
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