Abstract

Context.—Immunohistochemistry has taken a central role in the field of pathology, and its role is destined to increase as companion diagnostics analogous to the HercepTest are required for new targeted therapeutics. However, the inherent subjectivity of the assessment of an objective value (the in situ protein concentration) suggests that new technologies to measure the protein concentration may be required to achieve the accuracy required for companion diagnostics.

Objective.—This article discusses the state of immunohistochemistry and reviews the currently available devices for quantitative in situ assessment of protein expression.

Data Sources.—Data for this work were collected from the published literature, the Internet, and from information provided by device vendors.

Conclusions.—Although there is a long history of efforts to quantify immunohistochemistry, there has been a lack of broad acceptance because the resultant objective accuracy has not significantly improved outcome measures compared with the traditional, conventional analysis by eye. As the demand grows for companion diagnostics with complex assessment requirements, we are likely to see increased usage of quantitative platforms, especially those with the capacity to do multiplexed analysis. This most likely will be driven by a requirement for outcomes that cannot be achieved by traditional methods.

During the past 30 years, the number of laboratory tests available to physicians has grown exponentially. The number and quality of tests used in anatomic pathology with the goal of improvement of patient care have also increased. These technologies range from special histochemical stains to molecular assays at the DNA, RNA, and protein levels. However, histopathology remains the gold standard for most diagnostic and therapeutic decisions in surgical and autopsy pathology. The interpretation of histologic sections, however, is an inherently subjective process based primarily on morphologic features. With a goal of making pathologic examinations less subjective, the widespread use of immunohistochemistry (IHC) initially assisted pathologists in making diagnoses, adding to or complementing morphologic information with molecular information. More recently, it has been used to predict response to targeted therapy. This new role for IHC, in predicting response for new targeted therapies, is likely to place new demands on the quality, reproducibility, and accuracy of this technology. This review examines the current state of the technology of in situ assessment of protein expression.

TECHNICAL ASPECTS OF IMMUNOHISTOCHEMISTRY

Although the discovery of IHC is credited to Coons and Jones,1 who established an immunofluorescent technique for the detection of bacteria, it was not until the late 1970s that IHC became a standard tool in diagnostic pathology. Immunohistochemistry involves a series of uniform steps, typically beginning with antigen retrieval. Methods of antigen retrieval vary in terms of reagents and methods. The process may involve pressure cooking, protease treatment, microwaving, or heating histologic sections in baths of appropriate buffers, with the standard goal of unmasking antigens hidden by formalin cross-links or other fixation. The molecular mechanisms of antigen retrieval are not fully understood and remain the subject of many articles2–4 and books.5,6 The first definitive step of IHC following antigen retrieval is the application of a specific primary antibody (typically produced by immunizing a mouse or rabbit with a peptide/antigen of interest), followed by extensive washing to remove excess amounts of primary antibody. A species-specific secondary antibody is then applied, which binds to the primary antibody. The secondary antibody is typically conjugated to biotin, horseradish peroxidase, or some other tag. Finally, a detection reagent is applied that includes a chromagen or a fluorescently tagged molecule to visualize the localization of the primary antibody.7 

TOWARD STANDARDIZATION OF IMMUNOHISTOCHEMISTRY

Immunohistochemistry has become a standard assay in surgical pathology, despite the fact that it often lacks reproducibility and standardization. Sources of variability include fixation conditions, specimen pretreatment, reagents, detection methods, and interpretation of results. With the increasing use of IHC assays and the increased importance of the results, there is a need for standardization of the assay. In the early 1980s, the Biologic Stain Commission attempted to improve the standardization of IHC by sponsoring workshops for pathologists, manufacturers, and representatives of the Food and Drug Administration.8 Although it may not be possible to standardize all the potential variables in IHC, the interpretation of IHC results may be standardized through the use of new quantitative methods. A simple system of labeling specimens as either positive or negative is not a biologically accurate method of analysis.8 A laboratory measurement of gene expression (measured at the mRNA or protein level) is most commonly a continuous rather than ordinal measurement. The use of the pathologist's 3- or 4-point subjective scoring method is less accurate and reproducible. Thus it is likely that we will see progressive introduction of more objective quantitative scoring methods using automated systems in the analysis of IHC.

With the introduction of tissue microarrays (TMAs),9,10 the opportunities for IHC standardization have become more tangible. Using specialized equipment (Beecher Instruments, Inc, Sun Prairie, Wis) with coring needles ranging from 0.6 to 2.0 mm in diameter, a TMA is produced by relocating tissue from conventional histologic paraffin blocks into one recipient paraffin block. The recipient block can be processed in the same fashion as any standard histologic paraffin block, providing the opportunity for slide-based analyses of tissue from multiple patients. The TMAs permit an investigator to complete studies that previously spanned months and comprised hundreds of whole tissue sections now in a matter of days on one microscope slide. This tool allows for rapid biomarker development and validation. It also provides a mechanism to limit some of the inherent variabilities of IHC as hundreds of tissues (located on the same slide) are treated in the exact fashion. A number of standardization studies have illustrated the value of this platform.11,12 The subtleties of pretreatment, antigen retrieval, antibody incubation times and temperatures, and detection techniques become less significant because all tissues on the arrays are exposed to identical conditions. There are variables that are still difficult to control, including prefixation treatment of the specimen in the operating room, inconsistent fixation modalities and times,13 and antigen oxidation.14 

Although it may suffice for hematoxylin-eosin (and other “special”) stains to have the histotechnologist stain the slides to the relative satisfaction of the pathologist or laboratory, the application of this subjective technique to IHC is a major weakness.8 To increase the accuracy of IHC evaluation, variability should be eliminated at all possible levels, from staining variability to processing and interpretation. This issue was addressed in a recent Specialized Programs of Research Excellence meeting and is summarized by Bast et al.15 A number of companies are introducing instruments to help automate each of the processes associated with IHC. There are numerous automated slide-staining devices on the market, as well as automated microscopes and automated analysis programs. The preparation steps of IHC, including antibody validation, tissue processing, and antigen retrieval, are not addressed in this review because of the size and scope of this topic. Rather, we focus strictly on the analysis or interpretation phase of the process.

MACHINES FOR AUTOMATED ANALYSIS OF IN SITU PROTEIN EXPRESSION

The diagnostic pathologist typically interprets IHC by using a binary positive-negative end point or a 3- to 4-point scale. The H SCORE16 and criteria of Allred et al17 represent further attempts to produce a more continuous scale. However, none of these subjective approaches provide a truly continuous measurement of protein expression. Additionally, these methods are rarely reproducible, with high levels of intraobserver and interobserver variability. With the assistance of a computer, automated analysis programs help eliminate the inherent variability of pathologist-based scoring and may also increase the sensitivity and dynamic range of in situ measurement of protein expression. Thus, automated objective analysis has been the goal of a number of companies that market products to pathology laboratories.

Assays for molecular quantification have been in existence for decades. In particular, popular techniques include reverse transcriptase–polymerase chain reaction for quantification of nucleic acids and colorimetric (eg, Bradford assay,18 Lowry assay19) or antibody-based (eg, enzyme-linked immunosorbent assay) methods for protein quantification. A major drawback to these tools is that they require the maceration of tissues and cells to quantitatively assess the amount of a particular biomolecule present, which leads to loss of critical spatial information.

Although chromogenic or fluorescence techniques are widely used in quantitative histochemistry, the latter may be preferable because it is more sensitive and permits multiparametric analysis.20 The basic tenet of immunofluorescence is that a fluorescent product is deposited at the site of the antigen. The antigen may be located in a specific cellular (eg, nuclear, organellular, cytoplasmic, membranous) or extracellular locale. After photographic capture, the reaction product may be quantified by image-analysis software.21–23 

There are a number of computer-based programs designed specifically for the quantitative analysis of IHC. Systems for which publications can be found include BLISS and IHCscore of Bacus Laboratories, Inc (Lombard, Ill); ACIS of Clarient, Inc (San Juan Capistrano, Calif); iVision and GenoMx of BioGenex (San Ramon, Calif); ScanScope of Aperio Technologies (Vista, Calif); Ariol SL-50 of Applied Imaging Corporation (San Jose, Calif); LSC Laser Scanning Cytometer of CompuCyte Corporation (Cambridge, Mass); and AQUA of HistoRx Inc (New Haven, Conn) (Table). In addition, a recent report demonstrates that tools within Adobe Photoshop (Adobe Systems Incorporated, San Jose, Calif) can be used for quantitative analysis of immunofluorescent signals.24 The following section provides an overview of each system, as assessed in the fall of 2005. Given the rapid changes that occur in this field, it is likely that some of this information may soon be outdated as new devices are developed and older devices are improved or abandoned.

Commercially Available Automated Analysis Systems

Commercially Available Automated Analysis Systems
Commercially Available Automated Analysis Systems

The BLISS Slide Scanner (Bacus Laboratories Inc) was one of the first automated machines used for image acquisition of TMAs. The system automates image acquisition of standard histologic slides and TMAs. This device is a grandchild of the CAS 200 system,25 which represents the earliest and most commercially (and scientifically) successful attempt at quantitative in situ analysis. The current system is optimized for virtual slide and Internet control of the microscope. There are 8 to 10 US patents owned by Bacus Laboratories related to this device and its methods, which appear to be optimized for virtual slides rather than quantitative analysis.

The device captures a prescan of an entire slide with a ×1.25 objective. This scan is used for navigational control of the slide. Next, the system digitizes the microscope slide at different objective levels ranging from ×1.25 to ×63 and producing a virtual slide for further assessment by the pathologist.26 This system requires the end-user to identify regions of interest. Individual high-power images can then be scored using Bacus Laboratories' IHCscore software. The IHCscore is an image-analysis system designed for quantitative tissue scoring. There are 4 different stain measurements, 21 Markovian texture measurements, and 8 other morphologic measurements. Measurements are overlaid directly on the region of interest on the virtual slide.

Clarient (formerly Chromavision) markets a device called the ACIS (automated cellular imaging system), which is a digital microscope system with the ability to detect, count, and classify cells based on color, shape, and size. This device was originally designed as a rare-event detector, but then reworked as a method for quantitative analysis of IHC directed toward analysis of HER-2.27 Subsequently, software was improved to also allow analysis of TMAs. The ACIS system detects levels of hue, saturation, and luminosity.28 Through the use of a digital camera, a voltage signal is produced proportional to transmitted light intensity. This signal is then converted into a numerical density measurement.29 The ACIS advanced color space transformation software provides identification of the color of interest and a pattern-recognition imaging technology allows identification of morphologic change.29 The system also has algorithms built into the software that allow it to analyze data and produce numeric scores for various parameters defined by the user. This device has been demonstrated in the scientific and pathology literature with many publications citing the utility of ACIS-assisted analysis in the analysis of IHC.30–35 This system has enjoyed the greatest commercial success, although market penetration has been estimated only around the 5% level.

BioGenex has developed the iVision system and GenoMx vision systems. The iVision system is used for standard IHC and in situ hybridization staining. Numerous slides can be loaded into cassette towers to be automatically scanned by the iVision system.36 After scanning, a color image of the assay is selected by the operator along with the score for inclusion in the iVision. GenoMx is designed for TMA core-image acquisition, archiving/retrieval, multiparameter detection, and quantification. This system can be used for both chromogenic and fluorescence-based staining. It has numerous analysis programs with user-defined parameters. In addition to the analysis of IHC assays, the GenoMx can also assess chromogenic and fluorescent in situ hybridization assays. This device appears to be more popular in biotechnology or use in confidential applications because MEDLINE searches for iVision and GenoMx revealed no published references.

Aperio designed a device called the ScanScope, which is now marketed by DakoCytomation (Carpinteria, Calif). It is capable of high-speed digital slide creation, management, and analysis. The ScanScope system combines a linear array detector with high-performance optomechanics to digitize an entire slide at high resolution within minutes. This system efficiently organizes data into manageable file folders to be analyzed manually or automatically using Aperio's image-analysis algorithms. Aperio has 4 basic algorithms that are used for image analysis. The first algorithm is the positive pixel algorithm. This algorithm looks for positive pixel areas and shades them orange; the negative pixels are shaded blue and neutral pixels are shaded white. Aperio also has algorithms incorporated to perform nuclear IHC analysis, membrane IHC analysis, and micrometastasis analysis.37 The results of algorithmic analyses are displayed as overlays on slide images. Aperio also has several end-user applications. MEDLINE searches for Aperio and ScanScope revealed no published references.

Applied Imaging produces the Ariol SL-50, a high-throughput, automated image-analysis system for the quantification of biomarkers. It is capable of analyzing brightfield and fluorescent imaging with special capabilities for automated analysis of fluorescent in situ hybridization. The system captures and quantifies images of IHC, fluorescent in situ hybridization, and immunofluorescence and has software for analysis of TMAs. It is also capable of rare cell detection and measures of angiogenesis and DNA ploidy.38 A demonstration of this system was performed by Jiang et al34 in which they examined the utility of α-methylacyl-CoA racemase as a biomarker of prostate cancer. Using the Ariol SL-50 and the Clarient ACIS, they examined more than 800 prostate cancer specimens by means of TMA technology. By measuring both immunostaining intensity and percentage, they demonstrated that both systems provide reproducible, objective measurements of immunoreactivity.

CompuCyte has produced an automated analysis system using LSC Laser Scanning Cytometer and iCyte software. This instrument, an Olympus BX50 fluorescent microscope coupled with argon, helium neon, and violet lasers coupled to a computer-controlled optics unit, is described as a cross between a flow cytometer and a static image cytometer. The lasers are used to simultaneously excite different fluorochromes in cellular specimens that emit discrete wavelengths detected by a set of photomultiplier tubes. Together these features permit the ability to generate high-content stoichiometric data on heterogeneous populations of large numbers of cells. Thus, the LSC is used much like a fluorescent-activated cell sorter to obtain 3-color immunofluorescence intensity information from TMAs.

The LSC allows light scatter and fluorescence measurements, but it also records the position of each measurement. The device can measure multicolor fluorescence and light scatter on a single cell basis. This is of particular use when cell quantities are low, as in a fine-needle aspirates. The iCyte software uses multiple algorithms to define subcellular compartments and eliminate extraneous information. The biggest difference between this technology and the other methods of automated analysis is that the philosophy is more similar to flow cytometry than that of other systems. That is, levels of expression are used to define a threshold that is then used to count the number of entities above the threshold. As with flow cytometry, the result is a count of events rather than an “intensity” proportional to a protein concentration. Numerous publications demonstrate the utility and popularity of the LSC system (more than 200 at the time of review), although most are in applications analogous to flow cytometry (not solid tissue-based). Of particular interest, CompuCyte validated its system by quantifying HER-2 protein expression on a breast cancer TMA, demonstrating excellent correlation with reference fluorescent in situ hybridization results.39 

HistoRx has commercialized the AQUA (automated quantitative analysis) technology developed by Camp et al40 in our laboratory at Yale. This system was designed specifically for quantification of IHC on TMAs. The system relies solely on molecular interactions (effectively colocalization) rather than feature extraction algorithms, which use contrast-generating edges. It defines subcellular compartments and makes tumor/stroma distinctions on the basis of the expression of specific proteins or other molecular markers. For example, the nucleus is not the round structure in the middle of the cell, but rather any region that is above a certain threshold for staining with a DNA-binding fluorescent stain (eg, DAPI); the tumor cells are defined from stroma by the expression of tumor-specific proteins (eg, cytokeratin in the case of carcinomas, S100 in the case of melanomas). Using fluorescence also provides a broader dynamic range of signal intensity than absorbance-based “brown stain” readers.41 The AQUA system utilizes an automated image-capture system that starts by taking low-resolution images with a ×4 objective (64 × 64 pixels) that are merged together to create a whole-slide image. Next, rows and columns are identified and spot placement is edited to form a grid. Monochromatic high-resolution images of each spot are then taken using a ×10 objective (1024 × 1024 pixels); images 8 μm below the spot are also acquired. The images are saved and analyzed as 8-bit TIFF (tagged image file format) images.40 

Once the images are captured, the AQUA analysis is run using a set of algorithms: rapid exponential subtraction algorithm and pixel-based locale assignment for compartmentalization of expression. Rapid exponential subtraction algorithm, based on the principles of deconvolution theory, attempts to reduce the “out-of-focus” noise from parts of the specimen that are above and below the plane of focus. By subtracting an out-of-focus image (from 8 μm below the focal plane) from an in-focus image, rapid exponential subtraction algorithm improves the resolution of the image, improving the signal-to-noise ratio. Pixel-based locale assignment for compartmentalization of expression utilizes fluorescent tags to distinguish tumor cells from stroma, as well as to define subcellular compartments. This algorithm also measures expression of a marker of interest within cellular and subcellular compartments. The output variable (AQUA score) is a “pixel intensity/pixel area” value ranging from 0 to 255, based on the average intensity for all pixels evaluated.40 

This system has been extensively used and tested in our laboratory, where we have shown that AQUA scores are directly proportional to in situ protein concentrations. Our laboratory, and collaborators within and outside our institution, have published a number of articles using this system,42–49 but the system has only recently become available to other users. One recent study that demonstrates the prognostic significance of subcellular localization of the activator protein 2-α in melanoma, employed the LSC and AQUA systems. The LSC device estimated the number of cells with stronger nuclear versus cytoplasmic expression of activator protein 2-α (based on a user-defined threshold) and, the AQUA system calculated overall melanoma-specific cytoplasmic expression relative to melanoma-specific nuclear expression of activator protein 2-α. The results of the study indicate that stronger relative expression of activator protein 2-α in the nucleus is a favorable indication in early malignant melanoma.50 

All of these systems and methods have a common goal: the standardization of IHC interpretation. Standardized, controlled assays, in combination with a device that provides quantitative and objective output, can dramatically improve the quality of the data obtained from IHC studies. They allow increased sensitivity in scoring and provide a more reliable and reproducible analysis of protein expression in situ. With the advent of these new systems and a controlled approach, the goal of standardizing IHC, from the fixation of tissues to the analysis of IHC results, is achievable.

Acknowledgments

This study was supported by the Gloria Whitlock Research Award from the Breast Cancer Alliance of Greenwich, Conn, and grants R33 CA 110511 and R33 CA106709 from the National Institutes of Health (Dr Rimm).

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Dr Rimm declares a duality of interest as a founder, consultant, and stockholder of a new biotechnology company called HistoRx, which is mentioned in this work. This company was founded by Yale University and is the exclusive Yale licensee of the AQUA technology developed in Dr Rimm's laboratory. The other authors have no relevant financial interest in the products or companies described in this article.

Author notes

Reprints: David L. Rimm, MD, PhD, Department of Pathology, Yale University School of Medicine, 310 Cedar St, New Haven, CT 06520-8023 (david.rimm@yale.edu)