Joe Myers, M.S., CT(ASCP)

JMyers1@aol.com

Loosely defined, heat-induced epitope retrieval (HIER) is a pretreatment procedure often used prior to immunohistochemistry (IHC) or in situ hybridaization (ISH) procedures to improve staining by modifying the molecular conformation of ‘target’ proteins through an exposure of slide-mounted specimen material (sectioned tissue and other cellular preparations) to a heated buffer solution.  This modification process is necessary because, although aldehyde-based fixatives are excellent for preserving cellular morphology, they also cause protein cross-linking, resulting in the inability of some protein epitopes to bind complementary antibodies. HIER is commonly used in conjunction with enzyme digestion as a means of improving the reactivity of various antigens within IHC/ISH staining reactions.

Since a variety of techniques and devices have been used to perform HIER, this review may be useful to individuals interested in identifying the most ideal method or instrument for their laboratory, and assist them in making better informed decisions.  While a number of papers describing the advantages and disadvantages of various HIER methods for specific applications have been published (1,2), few have compared the technical differences between methods (3), or objectively presented the capabilities and shortcomings of existing HIER devices.

HIER, also known simply as antigen retrieval (AR), was pioneered in the early 1990’s (4,5) after it had become apparent that use of enzyme digestion alone to improve IHC staining was inadequate.  Although the expression “antigen retrieval” has gained widespread acceptance within the scientific community, alternative terms such as “protein unmasking”, “decloaking”, and “epitope recovery” have also been used to describe these procedures.  As a general rule, all HIER procedures involve heating slide-mounted specimen material in a buffer solution, followed by a cooling-off period.  Heat causes cross-linked protein epitopes to ‘unfold’ (in manner similar to DNA denaturation), while buffer solutions aid in maintaining the conformation of the unfolded protein.  The primary differences between various HIER methods are the means by which such solutions are heated and exposed or applied to slides.

During the early development of HIER, the most commonly employed methods for heating HIER solutions involved use of water baths, microwave ovens, and autoclaves.  By the mid-1990’s, however, scientists began to experiment with ‘kitchen-grade’ vegetable steamers and pressure cookers as heat sources, finding that equally good results could be obtained with these devices.  Recognizing that household appliances are limited in their ability to allow for protocol ‘customization’, development efforts in recent years have resulted in several devices designed specifically for performing HIER procedures in a laboratory environment.

In their simplest form, most HIER devices consist of a primary metal or plastic chamber (into which secondary reagent containers are placed), surrounded by a reliable mechanism for heating the liquid in the secondary containers.  The only limit to the number of slides (secondary reagent containers) that such a device can hold is the size of the primary chamber.  Considering the importance of producing consistent results, the ideal HIER device: A) incorporates a precision-controlled heat source, capable of maintaining temperatures at or above 100^oC; B) holds a reasonable volume of retrieval buffer and slides; and C) minimizes the potential for evaporation and boiling of the HIER solution.  These latter requirements are important because excessive evaporation: A)  causes the salt concentration of the buffer to fluctuate; B) boiling can cause specimen material to be released from the slide; and C) boil-over can lead to exposure of ‘raw’ specimen material to the atmosphere, resulting in inadequate retrieval and less than ideal morphology, due to drying artifact.  As a function of the inverse relationship between temperature and exposure time, devices that operate above 100^oC and prevent boiling (e.g. pressure cookers) have become very popular because they produce good results in the shortest possible timeframe.

Other important aspects of HIER devices include the ease with which time and temperature settings can be adjusted, the amount of time needed to complete a ‘run’, the amount of operator intervention that is required, and the potential for the operator to be scalded while handling the device or the retrieval solutions.  Ideally, HIER devices (or the protocols in which they are employed) should also provide a means of documenting performance, which ensures consistency and helps in meeting various regulatory agency-imposed ‘QC’ standards.  Table 1 provides a summary of the important characteristics of several common HIER methods.

Table 1. HIER Method Comparison

Comparative Characteristic	Water Bath	Vegetable Steamer	Microwave Oven	Pressure Cooker*
Temperature Range	25 to 100oC	85 to 95oC	50 to 100oC+	25 to 125oC
Heat-source Regulation	Good	Poor	Good	Excellent
Potential for Reagent Evaporation	Significant	Significant	Significant	Minimal
Potential for Boil-over	None	Minimal	Significant	Minimal
Potential to Scald Operator	Minimal	Moderate	Moderate	Significant
Maximum Slide Capacity per ‘Run’	(Variable)	72**	96**	96**
Maximum Reagent Capacity	(Variable)	750 mL***	1 Liter***	1 Liter***

Note:  All devices specified above are capable of holding both Coplin jars and TissueTek™-style slide racks/reagent containers

 *Refers to programmable devices, not ‘standard’ household appliances; see Device Comparison table for more information

 **Based on use of TissueTek™-style slide racks and reagent containers to hold retrieval solutions; although some models may hold more slides/containers than specified above, treatment of additional more slides may compromise performance characteristics (i.e. time, staining consistency)

 ***Based on use of TissueTek™-style slide racks/containers; use of Coplin jars results in a reduction in slide capacity and reagent ‘consumption’

Note: Tissue-Tek™ products are manufactured by Sakura Finetek

Recognizing that laboratorians needed to use reliable, inexpensive heat sources, scientists have employed a number of readily available devices, such as laboratory-grade water baths, autoclaves, and certain household appliances.  Although simple in design and operation, the primary drawback of water baths is the inability to achieve retrieval temperatures above 100^oC (resulting in longer protocols), the requirement for manual filling and draining of buffers (requiring significant ‘hands-on’ time’), and the potential for retrieval solutions to evaporate during extended, high-temperature protocols.

While the household-grade microwave oven also holds a prominent place in the field of HIER, it too has several shortcomings, most notably the difficulty in regulating temperature, the likelihood of boiling, and the fact that these units ‘loose power’ over time.  Vegetable steamers, like water baths, are often incapable of heating solutions above 95^oC, resulting in exceptionally long protocols.  In addition, vegetable steamers do not hold very many slides, relative to other devices.  Pressure cookers have become very popular, primarily because of their higher operating temperature and ‘closed system’ design, which allows for shorter protocols and eliminates the potential for boiling.  This is especially true of units that are retrofitted with a digital control panel and pressure gauge, since these modifications provide for better temperature regulation and permit the operator to monitor performance during the procedure.  The important technical aspects of these devices can be found in Table 2.

*Based on use of TissueTek™-style slide racks and reagent containers (or similar, proprietary products) to hold retrieval solutions

**Based on use of Autostainer™ slide racks

***Refers to amount of reagent consumed during an individual ‘run’, within applicable ‘reaction chambers’

Note: Autostainer™ systems are manufactured by Lab vision Corporation

Despite the fact that household appliances are commonly used within the laboratory environment, scientists agree that more standardization and specially-designed instruments are needed.  It is interesting to note that one prominent IHC vendor seems to have had some difficulty deciding which ‘technology’ provides the best HIER results.  A few years ago, they advocated use of a vegetable steamer, then began recommending use of a laboratory-grade water bath (possibly because of its application in the validation studies used to secure FDA approval for one of their proprietary IHC test kits); at present, they advocate use of and market a digitally-controlled pressure cooker.

After more than ten years without the availability of a true lab instrument to perform these procedures, there are now several commercially available devices designed specifically for HIER (See Table 2).  For example, PickCell Laboratories’ has introduced the 2100 Retriever™, which is a pressure cooker-like device (without the pressure gauge and time/temperature display found on similar units).  The EZ Retriever™, from BioGenex Laboratories, is essentially an industrial-grade microwave oven containing four Teflon® reaction chambers and a temperature-monitoring probe.  Like the devices upon which their operations are based (i.e. pressure cookers and microwave ovens), these new instruments possess many of the same shortcomings.  At present, the only device that can easily be classified as laboratory instrument is the PT (PreTreatment) Module™ from Lab Vision Corporation.  This semi-automated instrument was designed primarily for use with Lab Vision’s own Autostainer™ slide racks, which allows slides to be transferred directly from the PT Module™ to an Autostainer™ at the completion of the HIER procedure.  The primary shortcomings of this device are that it requires the operator to fill and drain reagents from two large, removable stainless steel ‘tanks’, and consumes a disproportionately large volume of retrieval solution.

No discussion of HIER methods and devices would be complete without mentioning the ability of some automated IHC slide staining systems to perform HIER.  At present, there are more than eight different IHC stainers on the market, of which only three offer ‘on-board’ HIER capability.  Unlike the devices described above, in which many slides are submerged in a heated buffer solution at the same time, HIER performed within IHC stainers usually involves processing slides individually.  For example, Ventana Medical Systems’s Benchmark® stainer essentially ‘sprays’ HIER solution unto individual, horizontally-oriented slides within a rotary ‘carousel’, then heat each slide to a preset temperature, and rotate the carousel in order to repeat this process on other slides.  In most protocols, the HIER solution is applied as many as nine times before HIER is considered completed.  In a similar fashion, Vision-Biosystems’ Bond-maX™ stainer dispenses HIER reagents onto rows of horizontally-arranged slides via a pipettor mechanism, and then whole rows are heated together.

As interesting as these features are, the addition of HIER capability to an IHC slide stainer does nothing but increase the likelihood of specimen loss and drive up the cost of performing this otherwise ‘inexpensive’ procedure.  The former problem is especially significant because the operator is usually not aware of specimen loss until an entire (expensive) IHC run has been completed, which usually requires repeating the IHC stain; the latter concern is discussed at greater length in the following section.

Another important aspect of HIER that deserves discussion is the cost-effectiveness of various methods and devices.  This is especially true when one considers that addition of HIER, or deparaffinization and HIER, to an automated protocol can substantially increase the overall cost of IHC (7).  One important characteristic of HIER devices is the amount of reagent that is consumed during a ‘run’ or processing batch (shown in Table 2), and there are significant differences between devices.  For example, previously-published data (8) have demonstrated that the cost of performing HIER on an automated IHC stainer can be as much as six times greater than performing HIER in a modified pressure cooker.  Another example of a major difference in cost between two vendor’s methods and devices is shown in Table 3.

Heat-induced epitope retrieval is a rather simple laboratory technique that has become an essential part of many IHC staining procedures.  Although there a only a few basic methods, there are variety of devices used for HIER, ranging from modified household appliances to semi-automated laboratory instruments.  Unfortunately, these variations also mean that HIER lacks standardization and cost controls.  The purpose of this review, then, is to educate the buying public on the technical and financial aspects of HIER, with the hope that this information can be used to make better method selection and device acquisition decisions.

References

1.   Taylor CR, Shi SR, et al: Comparative study of antigen retrieval heating methods: Microwave, microwave and pressure cooker, autoclave, and steamer.  Biotech Histochem 71(5): 263-70, 1996.

2.   Pileri SA, Roncador G, et al: Antigen retrieval techniques in immunohistochemistry: A comparison of different methods. J Pathol 183(1): 116-23, 1997.

3.   Tacha D and Teixeira M: History and overview of antigen retrieval: Methodologies and critical aspects. J Histotechnol 25(4): 237-42, 2002.

4.   Shin RW, Iwaki T, et al: Hydrated autoclave pretreatment enhances Tau immunoreactivity in formalin-fixed normal and Alzheimer’s disease brain tissues. Lab Invest 64(5):693-702, 1991.

5.   Shi S-R, Key ME and Kalra KL: Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39(6):741-48, 1991.

6.   Myers J: Automated slide stainers for special stains, immunohistochemistry and in situ hybridization: A review. Med Lab Observer 36(1): 28-30, 2004.

7.   Myers J: Reducing Immunohistochemistry Expense – Part 1. Advance for Administrators of the Laboratory 13 (9):16-18, 2004.

8.   Myers J: Reducing Immunohistochemistry Expense – Part 2. Advance for Administrators of the Laboratory 13 (10):18-22, 2004.