Unmasking antigens for immunohistochemistry
Unmasking antigens for immunohistochemistry*
Abstract: Preservation of tissue by fixation in a formalin solution, followed by dehydration and paraffin-wax embedding remains the predominant method of preparation for microscopic analysis of morphology. Whilst this may be optimal for morphological assessment, this technique has major disadvantages for subsequent immunohistochemical study as a result of the structural alteration of antigens that occurs during the processing procedure. However, the introduction of a range of antigen unmasking procedures has revolutionised immunohistochemistry, establishing the technique as a powerful tool in diagnostic pathology. Here, the development of enzymatic, non-enzymatic and heat-based antigen retrieval techniques is reviewed, and the methods in use currently are appraised.
Key words: Antigens. Immunohistochemistry. Heating. Microwaves. Peptide hydrolases.
The first use of fluorescent-labelled antibodies applied to histological preparations was described in 1950 by Coons and Kaplin.1 Over the next three decades, this technology became an important research tool, with over 45% of all investigations reported in general medical journals in 1978 involving immunohistological techniques.2
However, until the introduction of antigen– retrieval techniques, it was accepted that formalin fixation and routine paraffin-wax processing protocols created insurmountable problems in the immunohistochemical detection of all but a handful of antigens and intracellular immunoglobulins.3,4 This concept was accepted widely, with the majority of researchers and clinicians making use of cryostat sections for several decades, despite the many inherent disadvantages associated with the use of fresh frozen tissue.
In an effort to address the problems of antigen and morphological preservation, numerous alternatives were developed using a range of fixatives,2,5 cold fixatives6 and/or novel processing protocols;7-9 however, despite substantial research on the subject, results left researchers without a universally acceptable alternative fixative to formalin for immunohistochemistry, and it remained the fixative of choice as changing would have meant reassessing the recognised histopathological criteria employed for diagnosis.
Fixation remained a major obstacle until 1975, when Huang10 used pronase digestion and described the successful indirect immunostaining of hepatitis B core and surface antigens in paraffin sections. This was followed in 1976 by the first use of trypsin11 on formalin-fixed tissue for retrospective immunofluorescence staining of a number of antibodies.
These studies proved that many antigens are not destroyed by formalin fixation – as had been believed previously – and can be unveiled by careful manipulation. Finally, the unmasking of tissue antigens in formalin-fixed, paraffin-embedded tissue, and their demonstration by immunohistochemistry, had become a reality; and the door to the world’s archives of fixed material began to open.
Since the breakthrough in 1975, researchers have been provided with highly sensitive and permanent visualisation systems,12-15 and this has been coupled with advances in hybridoma technology.16
Consequently, unmasking techniques have developed in many directions, with some leading to a radical reassessment of our understanding of antigen preservation.
Mechanisms involved in formalin fixation
To aid understanding of the various antigen-retrieval techniques, it is pertinent to summarise scientific opinion regarding the major chemical changes that occur in tissues during formalin fixation.
Formalin is an aldehyde-based fixative produced when formaldehyde gas is dissolved in aqueous solution. The gas hydrates rapidly to form methylene glycol and a small amount of formaldehyde.17 The state of equilibrium between formaldehyde and methylene glycol lies firmly in favour of the latter; thus, the amount of formaldehyde available always remains low. Methylene glycol penetrates tissue rapidly, together with a fraction of the formaldehyde (as carbony] formaldehyde). It is this formaldehyde fraction that reacts with the tissue; and, as this is used up, a further small amount of formaldehyde is formed as methylene glycol dissociates.17,18
Time, therefore, is of fundamental importance in formalin fixation. Exposure for 24 hours at room temperature or 16 hours at 37 deg C is the minimum time necessary for small amounts of formaldehyde to form progressively and react with the tissue, and for the reaction to reach equilibrium and provide adequate protection.17,19 This explains the paradox of why tissue immersed in formalin is penetrated rapidly but fixes slowly.
Once formed, formaldehyde reacts with hydroxymethyl groups and previously unreacted amino-acid sidechains, creating the methylene bridges that are the cross-links of formaldehyde fixation.17,20,21 Reactions occur with, and between, a wide range of functional groups, including proteins,22-24 glycoproteins,22,25 nucleic acids,26,27 and proteins closely associated with polysaccharides.25 This cross-linking affects the binding sites of the globular proteins, changing the tertiary and quaternary structure but not the important primary and secondary structure.24
The practical effect of formalin fixation is such that many, although not all, protein structures (i.e. antigen sites) are protected from the denaturing effect of subsequent processing protocols, and, although altered (masked), remain viable and available for subsequent unmasking.24,28 Thus, masking of antigens is a progressive effect whereby immunoreactivity is lost progressively during the fixation process. However, much of the chemistry of fixation remains unknown, and this has led to divergence of opinion about why certain unmasking techniques actually work.
Enzymatic unmasking techniques
For the past 25 years, proteolytic enzymes (peptide hydrolases) have proved to be valuable unmasking agents, the most extensively used being pepsin, pronase, proteinase K and trypsin. The latter is used most commonly and is certainly the most written about. All proteolytic enzymes have similar enhancing effects, although individual authors vary in their preference. Proteolytic enzymes unmask antigens by ‘digesting’ the cross-links formed during formalin fixation, and trypsin is known to catalyse the hydrolysis of arginyl and lysyl peptide bonds.29
It is conjectured that exposure to proteolysis leads to increased tissue permeability.30 However, this enables the relatively large molecules used in immunohistochemical procedures to gain greater access by removal of large aggregates of cross-linked protein,24 and this view is supported by Bell et al.31 in their study of glial fibrillary acidic protein (GFAP)epitope sensitivity to formalin. They concluded that the sensitivity of some epitopes is due to the binding of other molecular structures to the epitope and not to the direct effect of formaldehyde fixation.
Proteolysis does have its limitations, however, and some antigens are susceptible to enzyme digestion. For those that are resistant to enzyme digestion, insufficient unmasking can result in poor or false-negative results, whilst over-digestion may lead to loss of tissue morphology and detachment of the tissue section.
As indicated previously, fixation is time-dependent and the amount of unmasking required must take this into account, with longer fixation times resulting in longer digestion times.4,32
The quality of enzymes varies and it is important that they be obtained from a reliable source, and individual batches tested for optimum activity. Solutions must be fresh – as enzyme reactivity decreases with age – and solutions should be prewarmed to the required temperature to ensure consistent results. Reported procedures for the use of proteolytic pretreatments vary widely, depending on individual laboratory procedures; however, the information in Table 1 may be taken as a useful general guide.
In summary, unmasking antigens with proteolytic enzymes requires a considerable amount of technical expertise, with critical factors such as time, temperature, pH, and length of fixation requiring careful control. However, the role of these enzymes in the rapidly advancing field of immunohistochemistry appears to be limited, and Leong et al.33 conclude that, apart from the cytokeratins and desmin, trypsin did not improve the staining of the other antigens studied substantially. This notwithstanding, there is a current trend towards the use of trypsin after heat treatment,34 although extreme care must be taken as tissue sections appear to be extremely sensitive to even very short periods of digestion (unpublished observation).
Non-enzymatic unmasking techniques
Such methods of antigen unmasking date back to the 1940s, with the use of strong alkali or acid22,23 to reverse formaldehyde-induced cross-links. The most simple method is to place formalin-fixed tissues in water for a long period of time.35 Elias36 made use of a similar technique by placing deparaffinised sections in 10% sucrose in phosphate-buffered saline (PBS) at 4 deg C for 16 hours prior to immunostaining.
In 1986, Costa et al.37 used the denaturing effect of guanidine (6-8 mol/L) or urea (8 mol/L) to analyse amyloid types in tissue sections, and, a year later, Kimato et al.38 used formic acid to etch amyloid in brain tissue to render it immunoreactive.
Shi et al.35,39 achieved dramatic results by using saturated sodium hydroxide in methanol on formalinfixed, acid-decalcified, celloidin-embedded sections as a sequential step after the use of heat; and, in 1997, Ganbo40 confirmed the usefulness of NaOH/methanol solutions to unmask a wide range of lymphocyte and macrophage antigens in formalin-fixed, acid- or EDTA-decalcified, paraffin-embedded tissues.
Cold chemical methods, although useful in narrow specific studies, have not been accepted widely and remain on the fringes of antigen unmasking.
In 1991, Shi et al.41 challenged the old dogma regarding the need for cold fixation and processing protocols to preserve antigens when they unmask antigens successfully by boiling formalin-fixed, routinely processed paraffin sections; however, the basis for this method had its origins back in the 1940s. During their biochemical studies, Fraenkel-Conrat et al.22 demonstrated that formalin cross-links could be reversed not only by the use of chemical methods but also by hightemperature heating (120 deg C).
Shi et al. were the first to use the term `antigen retrieval, and made use of a microwave oven as the heat source to boil sections in solutions of lead thiocyanate or zinc sulphate. Of the 52 antibodies they tested, a significant increase in immunostaining was seen in 39, no change in nine, and reduced staining in four.
Initial scientific reaction was one of scepticism, particularly when Momose et al.42 failed to reproduce the results, and concluded that no advantage was gained over the conventional use of trypsin, and advocated the use of Carnoy’s fixative to obviate the need for antigen retrieval procedures altogether. The safety aspects associated with boiling heavy metal salt solutions quickly dispelled any thoughts that the scientific community would embrace this particular concept; and scepticism remained high when Shin et al.43 published their work using distilled water in a hydrated autoclave technique to unmask tau antigenicity in brain sections and immunoblots.
In 1993, Gerdes et al.44 demonstrated that citrate buffer (0.01 mol/L, pH 6.0) enabled the demonstration of many antigens. Further reports of success with heatbased methods appeared, with a number of studies testing them against known enzyme methods.28,45.46 These made use of a range of solutions and finally convinced the sceptics that high temperature could make antigens, previously only visualised in frozen sections, available for demonstration in paraffin sections.
Microwave antigen retrieval became firmly established with reports of the technique’s many advantages, and these included increased sensitivity, use of higher dilutions of antibodies, lower background staining compared with enzyme techniques,45 and destruction of some endogenous enzyme activity.47 In addition, reduction in differences in staining between tissues fixed in formalin for varying times and in fixation zoning effects within individual tissues was noted.46,47 However, the major factor in the popularity of heat-based antigenretrieval techniques came with the ability to unmask a far greater range of antigens (over 200 by 1997),34 compared with that achieved with enzyme techniques.
Any thought that microwaves were responsible for the unmasking effect were dispelled quickly, as success with other heat-based methods were reported.28,48,49 Conventional heating methods, such as the use of an oven, a Bunsen burner, or preheated buffers,41,47 although able to reproduce the effect of microwaves, are reported to be less consistent.28,41 In addition, microwave ovens became important because they are able to produce heating to 100 deg C (superheating to temperatures above 100 deg C being achieved when used in conjunction with plastic pressure cookers50) in a fast, efficient and reproducible manner, and any domestic microwave with an output between 700W and 900W is suitable.
Heat is the most important factor for antigen unmasking,41,51,52 and, in general, higher temperatures for shorter periods yield better results (for example, 100 deg C for 20 min is equivalent to 70 deg C for 10 h^sup 21^ ).
Initial work with microwave ovens was hampered by the need to use Coplin jars. These required constant topping up, could hold only small numbers of slides, and hot or cold spots produced inconsistencies. The current practice of using larger plastic containers, coupled with rotating turntables, has solved most of these problems.
A variety of alternative heating methods have been used and these include the use of steam heaters,53,54 water baths,55 autoclaves,43,48 and the more widely accepted pressure cooker.49,56 Pressure cookers permit the staining of large batches of slides; they are cheap, easily obtainable, occupy a small amount of bench space, produce even heating of the solution, and allow temperatures of between 100 deg C and 120 deg C to be reached at full pressure.57 This last point is important as it permits further reduction in time, with unmasking possible after two minutes.46
Standard procedures for using microwave ovens are relatively simple, and require unmasking solutions to be brought to the boil before inserting the slides, then boiling for 10-20 minutes. Pressure cooker protocols are similar, with slides placed into a boiling solution, and timing commenced (normally 2-4 min) only once the correct pressure is reached.
Plastic pressure cookers have been introduced and can be placed directly into a microwave oven and operated in exactly the same way as a standard pressure cooker. However, they work at a reduced pressure compared with that obtainable in steel versions, and may not reach such high temperatures.
Cooling down after unmasking is one aspect that is not well documented and requires further investigation. Some researchers have reported that no enhancement resulted unless the slides are left to cool down to room temperature,45 whilst other reports from the USA (via the ‘Histonet’ histology discussion group on the Internet) have suggested that the longer slides are left to cool down the more detrimental the effect. One reporter commented that no staining was obtainable after cooling for more than 45 minutes (M. Rentsch, personal communication).
Although achieving high temperature undoubtedly is the most important aspect of antigen retrieval, the use of different unmasking solutions to `fine tune’ the result may be necessary; however, the choice of unmasking solution is vast. All the solutions suggested are claimed to have some benefit in heat-based antigen retrieval,47 with lead thiocyanate, zinc sulphate, aluminium chloride, sodium chloride, sodium fluoride, iron chloride, calcium chloride, nickel chloride, ammonium chloride, citrate, (hydroxymethyl) aminomethane (Tris), urea, ethylenediaminetetraacetic acid, (EDTA, tetrasodium salt dihydrate) or ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), glycine-HCl buffer, and periodic acid” the most widely described and used.
These solutions fall into three main groups: inorganic and organic salts; metals; and denaturing agents. Composition and molarity are additional factors that must be considered when selecting a solution. For example, 0.3 mol/L aluminium chloride can unmask some epitopes not unmasked by citrate buffer,28 and 4-6 mol/L urea can unmask antigens not detectable at concentrations of 0.01-0.8 mol/L.28,52 Although most salt solutions are effective if used below 0.1 mol/L, metal salts (e.g. lead) are more effective at higher concentrations.41 The variables become even more daunting when the additional complexity of pH is introduced.
A wide range of commercial target retrieval fluids also is available today; however, their compositions are unknown and therefore difficult to comment on. Cattoretti and Suurmeijer28 were one of the first groups to attempt to bring some order to the use of unmasking solutions. In a comprehensive and systematic review, they appraised and evaluated 20 unmasking solutions (nine carboxylic and organic compounds [pH 6.0], 10 metal and salt solutions, and distilled water), and compared the results with those obtained by enzyme treatments. The authors concluded that 0.01 mol/L citrate or bicarbonate buffer (pH 6.0) and 6 mol/L urea were superior to all other solutions tested, using a small panel of selected antibodies.
The study was extended, using citrate buffer, 6 mol/L urea, and enzyme digestion, to include antibodies against a further 256 antigens; and the authors claimed to have identified three groups of antigens: those that survive fixation and embedding but are destroyed by subsequent treatments; those that can be demonstrated through trypsin digestion or boiling; and those that depend on stronger denaturation by heat or high molarity. Disappointingly, they concluded that `there is not a simple rationale to predict the accessibility of an antibody to a given antigen in formalin-fixed, paraffin-embedded tissues’ – findings that remain true today, despite the vast amount of work reported in the literature.
Much of the literature on this subject appears contradictory. For example, Preston and Shousha58 state clearly that pH has an effect on antigen retrieval, regardless of the type of solution; however, Norton et al.,49 using an aluminium pressure cooker, noted dramatic swings from pH 6.0 to 9.1, but stated that this had no impact on the final result. Preston and Shousha provided further food for thought when they unmasked antigens successfully by reducing the temperature and increasing the time in the unmasking fluid, suggesting that superheating is not a critical factor.
Undoubtedly, heat-based antigen retrieval is still in its infancy and not without some drawbacks, with unmasking of endogenous biotin producing high background staining, non-specific nuclear staining, and destruction of some antigens being well-documented disadvantages.35,45 In addition, reactivities normally considered insignificant can become troublesome,49 and this raises the question of validity.
Many questions remain regarding standardisation of procedures and the establishment of optimal staining protocols. What are the optimal heating conditions? What gold standard can be applied to demonstrate the optimal score for prognostic markers?
Although highly desirable, true standardisation of unmasking protocols is unlikely to be achievable in the near future. The current generation of domestic microwave ovens used in the majority of laboratories all have the inherent disadvantage that the power generated decreases with use; thus, no two ovens – even those of identical make – will have exactly the same heating characteristics.
In the future, it is possible that the work of Man et al.59 and Preston and Shousha58 will prevail, and that immunohistochemical progress will involve the development of low-temperature unmasking solutions, thus obviating the need for heat sources.
Heat-based antigen retrieval undoubtedly works. Whether or not it works by heat denaturation and hydrolysis, self assembly of unfolded protein chains and the subsequent restoration of antigenic sites, chelation of calcium complexes,20,60 unfolding of protein structure by metallic salt or urea solutions, through dissociation of hydrogen bonds, or simply through the loss of diffusable blocking proteins and the last traces of wax remains the focus of much debate.
Reasoned arguments have been put forward in support of each theory and it would appear that the explanation for success will vary depending on which unmasking fluid is used and which antigen is being demonstrated. However, it would seem likely that protein denaturation and dismantling of formaldehyde-induced cross-links is the mechanism of action behind heat-based methods, and this is supported by the susceptibility of sections to enzymes following heat treatment.
For those investigating antigen unmasking for the first time, Cattoretti and Suurmeijer28 provide the most logical way forward and recommend enzymatic digestion (using trypsin, pronase or pepsin), heatbased unmasking using a solution with an acceptable average for most antigens (0.01 mol/L citrate buffer [pH 6.01 or 0.1 mol/L EDTA [pH 8.01) or 4% aluminium chloride or 6 mol/L urea. The possibility of an antigen escaping detection after the use of the above is low, provided that it is not destroyed by formalin fixation.
Unmasking antigens for immunohistochemistry is here to stay and, despite the many unanswered questions and a feeling that often it is more an art than a science, will continue to have a profound effect on improving diagnostic services and a considerable impact on patient care.
*Commissioned by the Editor, and based on a structured reading essay submitted as part of the IBMS Continuing Professional Development programme
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Edinburgh University Department of Veterinary Pathology, Easter Buch Veterinary Centre, Roslin, Midlothian EH25 9RG, UK
(Acceoted 22 March 2001)
Correspondence to: N. MacIntyre, Whincroft, 8 Rosetta Road, Peebles, Border Region EH45 8JU, UK.
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