A review of automated slide stainers for IHC and ISH

Medical Laboratory Observer, Jan, 2008 by Joe Myers

This review of automated slide stainers for immunohistochemistry (IHC) and in situ hybridization (ISH) is intended to serve as an update to a previous paper, (1) published in this journal approximately four years ago. While the focus of the earlier version was simply to describe the basic mechanical differences between the automated IHC/ISH systems available at that time, this review is designed to provide a more detailed analysis of the capabilities and, where practical, dispel some of the myths associated with certain instruments and functions in the hope that future acquisition decisions will be based more on "good science" and cost effectiveness than on marketing "hype." An effort has been throughout this paper to provide interested parties with the information necessary to consider available systems with an open mind, being aware that the perceived advantages of a particular system may overshadow its otherwise unknown shortcomings.

The bottom line is that there is no perfect system--each has strengths and weaknesses that should be considered during the objective evaluation that usually precedes acquisition of capital equipment like IHC/ISH systems. (2) One of the most difficult aspects of preparing such a review, as well as conducting actual system evaluations, is that there are so many ways in which IHC/ISH instruments can be classified. For example, one can look at systems on the basis of physical attributes such as slide capacity and layout (i.e., carousel or array), (1) the degree of reagent flexibility with which a system operates (i.e. closed or open), or the mechanisms employed to measure and apply reagents to slides. A summary of the most important features available on today's commercially available instruments is provided in Tables 1 and 2.

Applications and approaches

Although it may seem unnecessary to explain, IHC/ISH systems are, unlike instruments for hematological applications (e.g., Wright staining), specifically designed to perform more complex and time-consuming procedures. At this starting point, then, it is important to point out that there is actually very little difference between an instrument that is capable of performing IHC and one that is (also) capable of performing ISH, since both procedures involve, with one exception, the same fundamental mechanics. All IHC/ISH systems perform similar functions--that is: 1) provide an environment for reagents to react with specimen material during timed incubation periods, 2) apply unique reagents onto slides in a predetermined manner, and 3) apply rinse solutions onto slides at specified intervals (i.e., between incubation periods). The only additional function of instruments marketed specifically for ISH is the ability to apply controlled heat to slides for the purpose of denaturing DNA targets. Therefore, if the targets of a laboratory's ISH procedures are exclusively RNA, then nearly any system could be used, since such protocols do not require this slide-heating capability.

Another way of looking at IHC/ISH systems has to do with the approach taken by the manufacturer/distributor--that is, is a instrument designed to permit operators to use reagents from any source and allow them to create their own staining protocols, or does the system require them to use reagents obtained (with the exception of primary antibodies and probe solutions) exclusively from the system vendor. The issue here is freedom (i.e., can you use the instrument as you see fit, or are you forced to do things as the vendor sees fit?). Unfortunately, it is not feasible to classify and describe IHC/ISH systems by the degree of openness with which they can be operated, which is why the following information will be presented in another fashion: by examining some of the aspects that make certain instruments seem similar, and yet, in other ways, completely unique. The reader, is, therefore, encouraged to refer to Tables 1 and 2 for comparing and contrasting these systems.

Capacities and capabilities

For most people involved in the evaluation of IHC/ISH instruments, the primary criteria that are most often used for evaluation and/or acquisition decisions is the quantity of slides that a system can process in a single run and its functional capabilities, since these parameters seem easy to identify and understand. If only it were that simple; the reality is that even the processing capacity of an instrument is not static since some units can be continually fed additional slides once the initial run has been initiated. Objectively comparing the functionality of these systems is also complicated by the fact that every instrument has some mechanical or operational feature that makes it unique. The only way to effectively compare IHC/ISH systems is to classify them, at least partially, into categories on the basis of these unique features. For example, a great deal of emphasis has been placed on the ability (or inability) of an instrument to perform heat-induced epitope retrieval (HIER). (3) Admittedly, this capability is important because: 1) such pre-treatments are necessary in the vast majority of protocols involving formalin-fixed tissue, and 2) when not performed online, these procedures must be performed manually prior to loading slides onto an instrument. There are, however, other aspects of on-line HIER that are often overlooked, in particular the misconception that it greatly improves productivity and the fact that it can cost as much as six times more to perform HEIR within a stainer than it does to perform 'manual' HIER (i.e., within devices like pressure cookers, vegetable steamers, and microwave ovens). (4) Another example of an often overlooked capability of some IHC/ISH instruments is that of performing multiantigen immunostaining (MAIS) procedures, (5) which have become very popular among both clinical pathologists and research scientists. The important issue in this case is the ability of such systems to apply two or more substrate-chromogen solutions, particularly since some of these reagents must be prepared just prior to application (unlike other reagents, which are stable at room temperature for hours, days, and even weeks at a time).