Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Immunohistochemistry (IHC) uses antibodies to recognize specific antigens found in preserved tissue. Traditionally, thin slices of formalin-fixed, paraffin-embedded (FFPE) tissue—from a tumor or biopsy, for example—are cut, mounted onto a slide and processed so that an antibody can find its target and generate a visible signal. This labor-intensive and time-consuming process requires the skills of a trained technologist [1].
During the 1980s, many of these processes began to be automated, and today clinical-pathology laboratories routinely take slides containing FFPE tissue slices "and have the instrument perform hands-free drying, dewaxing, cell conditioning or pretreatment and application of antibodies, detection and counterstain chemistry such that when the slide is removed from the instrument it is ready to be coverslipped and sent to the pathologist for diagnosis,” explains Dave Magner, senior international product manager for advanced staining platforms at Ventana Medical Systems.
There are about a dozen automated IHC staining systems on the market—both benchtop and floor models—from about half-dozen vendors. Although it may not be easy to tell apart the slides that ultimately emerge from them, the instruments themselves certainly differ vis-à-vis their inherent capacity, throughput and hands-on time required; their ability to accept third-party reagents; or their flexibility and ease-of-use, among other attributes.
Who needs it?
According to Amber Nelson, Thermo Fisher Scientific’s IHC and cryostats product manager for the Americas, the company’s “key market segments” include “hospitals, specialty laboratories, reference laboratories, private labs, university/research, and pharmaceutical/biotech companies.”
Bonnie Whitaker, anatomical pathology operations director at The Ohio State University Medical Center Department of Pathology and chair of the National Society for Histotechnology (NSH) IHC Resource Group, suggests technology has advanced such that “any lab that wanted to do these clinically probably needs to be doing so on an automated stainer.” Such instruments enable far better run-to-run consistency and are prone to far fewer mistakes—especially with instruments that are hooked into laboratory information systems that use barcoded slides and reagents—than is possible when performing manual procedures day-to-day.
And there are multiple levels that can trip up IHC processing. Because IHC reagents—particularly antibodies—are expensive, IHC stainers should consistently deliver only the amount of reagent required. Because each slide may be stained with a different antibody, it’s important to avoid cross-contamination between slides. And because antibody can be depleted quickly from a solution, fresh reagent should be applied to each slide. Thus, automated IHC stainers are endowed with robotics and process control to perform far more complex and intricate tasks than the more common bulk dip-and-dunk hematoxylin and eosin (H&E) stainers.
Open and shut
Automated IHC staining systems tend to be categorized as open or closed,. Open “has come to mean that you can use reagents from any vendor on an open machine—to mix and match as you see fit—much [to] the desire of the research lab,” says Joe Myers, a board-certified lab technologist who is the previous chair of NSH’s IHC Resource Group and senior technical sales specialist for Biocare Medical. A closed system typically restricts not only reagents but also the ability to alter or design protocols. But that has an advantage in terms of troubleshooting, says Myers: “If something goes awry you only have one person to call.”
Most instruments on the market allow the use of third-party antibodies, says Magner, “although detection chemistry tends to be closed.”
For Whitaker, it’s a mixed bag. Open systems are typically a little less expensive to run and are more flexible. On the other hand, “the closed system also takes less technologist time and is not as likely to have mistakes occur,” she says. “My personal preference would be that all labs have one of each.”
Online, offline
For the antibody to gain access to the tissue in IHC processing, paraffin must be removed (typically with xylene or a similar organic reagent). In the vast majority of instances an additional heat-induced antigen (epitope) retrieval (HIER) step is also required that“heats slides to a high temperature to break the formalin bonds,” says Myers. These steps can be performed either prior to introducing the slides into the stainer or as a part of the automated process. Instruments capable of the latter are far more expensive. He notes that the typical sale price of a machine with offline retrieval is $85,000 to $90,000, whereas a machine with online retrieval ranges from $125,000 to $150,000, and “the cost of producing the slide is 30[% to] 40% more for the convenience of online retrieval.”
On the other hand, “online HIER is so incredibly helpful and time-saving—I don’t think people take the technician’s time into consideration enough—and another place that you can remove a chance for a mistake to be made,” says Whitaker.
Throughput and continuity
Sometimes deparaffinization and HIER are performed off-line, even when the instrument is capable of doing it online, to better accommodate batch scheduling, in which all the slides are loaded and off-loaded at the same time.
At one time, all automated IHC stainers were operated in batch mode. This allowed for less hands-on time and more efficient use of the instrument, including less reagent lost to dead space compared with a sequential or continuous modality and the ability to use a single set of controls for the entire batch, Whitaker notes. “But the tradeoff for doing that is that it’s not as timely—if you are running each case [set of slides from an individual patient, typically stained with different antibodies] on a case-by-case basis as [it is] requested and cut, of course your turnaround time is much improved.”
“We’re starting to see a big trend—especially as more labs embrace LEAN philosophies—toward smaller batches and more of a continuous-flow-type workflow scenario,” says Bryan Sieber, U.S. product manager for Dako. In the latter, cases generally can be added at any time and often prioritized, and time to first result is reduced.
The instruments’ slide capacity (as well as the number of reagents and the capacity of the reagent containers) varies greatly—from 30 to 84—although this is obviously less of an issue for those that operate in continuous mode.
Whether your lab needs to process 20 slides per day or can keep a staff employed throughout the clock, automated IHC stainers will save a great deal of time and effort and afford a level of reproducibility not possible with manual IHC. Now it’s just a matter of finding the best one to meet your needs.
Reference
[1] Taylor, CR, Rudbeck, L (editors), “Immunohistochemical Staining Methods,” Sixth Edition, Dako Denmark A/S, An Agilent Technologies Company, 2013.
Image: iStockPhoto