Immunohistochemistry has become an indispensable ancillary technique in anatomic pathology laboratories. Standardization of every step in preanalytic, analytic, and postanalytic phases is crucial to achieve reproducible and reliable immunohistochemistry test results.
To standardize immunohistochemistry tests from preanalytic, analytic, to postanalytic phases.
Literature review and Geisinger (Geisinger Medical Center, Danville, Pennsylvania) experience.
This review article delineates some critical points in preanalytic, analytic, and postanalytic phases; reiterates some important questions, which may or may not have a consensus at this time; and updates the newly proposed guidelines on antibody validation from the College of American Pathologists Pathology and Laboratory Quality Center. Additionally, the article intends to share Geisinger's experience with (1) testing/optimizing a new antibody and troubleshooting; (2) interpreting and reporting immunohistochemistry assay results; (3) improving and implementing a total immunohistochemistry quality management program; and (4) developing best practices in immunohistochemistry.
Immunohistochemistry (IHC) has become an indispensable ancillary technique in anatomic pathology laboratories, especially in the last decade. There are 5 milestones in the development and advancement of the IHC field, including (1) the discovery of monoclonal antibodies that significantly increase diagnostic specificity1; (2) a heat-induced antigen retrieval (AR) method that makes it possible to most effectively run IHC assays on formalin-fixed and paraffin-embedded surgical and cytologic specimens2–10; (3) a highly sensitive secondary detection system, which enables one to detect trace amounts of proteins on formalin-fixed and paraffin-embedded tissue with little background staining11–25; (4) an automated staining system, which enables one to run hundreds of IHC slides on the same day, in the same laboratory, with reproducible and accurate results26–28 ; and (5) digital pathology with imaging analysis, which allows one to use digital slides (whole slide scan) for quality control purposes and to further improve turnaround time by delivering digital images (electronic slides) via Web sites.29–35 The IHC assay is a high-complexity test and encompasses many complicated working steps in the preanalytic, analytic, and postanalytic phases. Any potential errors occurring in any of these steps will result in unreproducible and unreliable results. The key steps are summarized in Table 1.
In general, IHC is a highly reproducible and reliable assay when an IHC test is run on adequately fixed and processed paraffin-embedded tissue under the identical staining procedure as that of the antibody validation process. However, owing to the presence of numerous variables that may influence an IHC test result and the lack of standardization among clinical IHC laboratories, and even within a laboratory, inconsistent IHC assay results have been frequently reported in the literature. The challenging questions are: Is it possible to standardize diagnostic IHC tests among clinical IHC laboratories? How does one improve and implement a total IHC quality management program? And how does one implement best practices in immunohistochemistry?
This article is not intended to be a comprehensive review of each step involving preanalytic, analytic, and postanalytic phases. This has been well addressed in many original publications,2–28,36–61 review articles and society recommendations/guidelines,62–84 and book chapters.85–89 Instead, it highlights some critical points, reiterates some important questions, which may or may not have a consensus at this time, and updates some newly proposed guidelines and recommendations for antibody validation from the College of American Pathologists (CAP) Pathology and Laboratory Quality Center.90 Additionally, we will share Geisinger's experience and express our opinions on (1) testing/optimizing a new antibody and troubleshooting; (2) interpreting and reporting IHC assay results; (3) a total IHC quality management program (quality assurance, quality control, and quality improvement); and (4) best practices in immunohistochemistry.
This discussion will focus on class I IHC assays, which encompass most IHC markers in a clinical IHC laboratory, with the exception of prognostic and predictive IHC markers, including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu), which are classified as class II IHC assays and required to be reported to an ordering clinician as stand-alone test results. Of note, one should be aware that the same IHC marker can be designated as class I or class II, depending upon how the test result is interpreted. For example, an IHC assay for ER, when working on a tumor of unknown primary, will be treated as a class I IHC assay; in contrast, if ER is used as a predictive marker, the IHC assay will be considered a class II test. The recommendations from the College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) for ER, PR, and HER2/neu, which have been widely accepted by both pathologists and clinicians and implemented in all CAP-certified anatomic pathology laboratories, will not be discussed in this article.91–94
PREANALYTIC PHASE
It has been recommended that tissue should be fixed in 10% neutral pH, phosphate-buffered formalin for a minimum of 8 hours.36 If formalin or a formalin-alcohol mixture was a component solution on the tissue processor instrument, tissue should be fixed in formalin for 6 to 12 hours before being loaded onto the tissue processor. Nonformalin fixatives and/or alternative fixation methodologies are strongly discouraged.36
Engel and Moore82 identified 62 preanalytic variables, 27 of which have been examined and reported in published literature. Among the 27 preanalytic variables, they concluded that 15 factors (including fixation delay, fixative type, fixative concentration, pH and buffer, time in fixative, reagents and conditions of dehydration, clearing reagent and temperature, paraffin-embedding temperature and duration, and condition of slide drying and storage) were capable of impacting an IHC test; in contrast, 12 factors (including tissue to fixative ratio, the type of processor used, type of paraffin, postfixation washing duration, the number and position of specimens during dehydration, and the duration of paraffin block storage) had no reported influence on the IHC assay.82
Drying slides can be easily overlooked in an IHC laboratory, and IHC technologists need to be educated to pay particular attention to drying time and temperature at all times. It has been recommended that slides be dried at 50°C to 60°C for a minimum of 1 hour or at room temperature for 24 hours.82 Drying slides at 60°C for 4 hours may reduce immunostaining intensity in 23% of target antigens, compared with 37°C overnight drying,82 and 69% of target antigens display reduced staining intensity or increased nonspecific background staining after incubation of the slides at 70°C for 8 hours.82
Decalcification may have a negative impact on an IHC assay for certain antigens.72,95,96 As such, CAP recommends that a disclaimer be included in the surgical pathology or fine-needle aspiration report, which may read as follows: “This IHC assay has not been validated on decalcified tissues. Results should be interpreted with caution given the likelihood of false negativity on decalcified specimens.” 97
ANALYTIC PHASE
Utility of Multitissue Tissue Microarray Blocks
Tissue microarray (TMA) blocks containing various numbers of tumors and/or normal tissues have proven to be extremely valuable in the field of biomarker research and also have demonstrated great utility in clinical IHC laboratories. At Geisinger, we have used TMA blocks for (1) antibody testing and optimization; (2) antibody validation or verification; (3) positive and negative control tissues; (4) quality control; and (5) new biomarker discovery. Depending upon the need, 4 different prototypes of TMA blocks can be constructed: (1) a TMA block containing a broad spectrum of tumors and/or normal tissues from various organs, which is useful for screening a new biomarker; (2) a TMA block containing 50 to 100 tumors with a specific diagnosis, such as lung adenocarcinoma, which is useful for antibody validation, revalidation, and research purposes in determining the diagnostic sensitivity and specificity of a newly discovered antibody; (3) a TMA block containing 5 to 10 cases of a specific type of tumor, which is useful for antibody testing and optimization; and (4) a TMA block containing 5 to 10 cases of selected, mixed tumors and/or normal tissues from various organs, which can be used as external positive and negative control tissues for each antibody. The detailed application of each type of TMA block will be discussed throughout this article.
Antibody Selection
More than 200 diagnostic antibodies are generally available in a large clinical IHC laboratory, and hundreds of antibodies are usually available in research laboratories. The list of new antibodies is growing rapidly with the discovery of new biomarkers by molecular methodologies. How do you select the “right” antibodies for your IHC laboratory and your patients? Before you decide to bring a new antibody to your IHC laboratory, here is a set of questions that you may want to raise: (1) Why do I need this antibody, and what is its clinical application? (2) What is the diagnostic sensitivity and specificity of this antibody? (3) What is the likely test volume in my IHC laboratory? (4) Where can I get this antibody? (5) Is more than 1 antibody (clone) available? (6) Do I have the positive control tissues to test and validate this antibody? and (7) How am I going to implement it?
At Geisinger, we have been taking a combined approach to adding new antibodies to our IHC laboratory: (1) studying and tracking mature publications in popular peer-reviewed pathology journals, especially those publishing articles on diagnostic surgical pathology and cytopathology, such as American Journal of Surgical Pathology, Archives of Pathology & Laboratory Medicine, American Journal of Clinical Pathology, Human Pathology, Modern Pathology, Histopathology, and Applied Immunohistochemistry & Molecular Morphology; (2) attending major pathology society conferences, particularly the United States & Canadian Academy of Pathology annual meeting, or reading the abstract book. It is also helpful to see the posters and discuss the antibody with the poster presenters to obtain additional information (such as clone, dilution, AR method, control tissue, and expected results) and inquire if there is any potential technical difficulty in working up this antibody, which is not usually detailed in a poster, a presentation, or even in a publication; (3) IHC vendor recommendations and online catalogs; and (4) free Web sites with a published antibody library, such as Geisinger's IHC Web site (http://www.ihcfaq.com),98 NordiQC (Nordic Immunohistochemical Quality Control, Aalborg, Denmark, http://www.nordiqc.org),77 and IHC menus on the pathology department Web sites of major medical institutions and hospitals.
Antibody Optimization and Troubleshooting
We routinely use a TMA block containing a small number of tumors and/or normal tissues with known positivity and negativity for the target antigen to test a new antibody. There are many different ways to test a new antibody. The ultimate goal is to achieve a strong staining signal and little or no background staining when using the highest primary antibody dilution (to save primary antibody and reduce cost). Before testing a new antibody, consult the product data sheet carefully for general recommendations, such as positive control tissue, AR method, antibody dilution range, and incubation time. Be sure to confirm the compatibility of a secondary antibody to the species and subclass immunoglobulin of a primary antibody (such as mouse monoclonal antibody or rabbit monoclonal antibody). Perform all relevant blocking steps to eliminate background staining, including endogenous peroxidase and phosphatase. We tend to determine the best AR method first and then test the proper primary antibody dilution and incubation time. Our experience demonstrates that a false-negative result is more likely due to the wrong AR method rather than a suboptimal antibody dilution and/or incubation time. To achieve this, we start with 5 different AR methods/solutions (heat-induced epitope retrieval with citrate buffer/pH 6.0, with ethylenediaminetetraacetic acid/pH 8, with Tris/pH 6, with high pH solution, or enzyme digestion such as proteinase K Tris/pH 7.5 solution) and a fixed, high concentration of primary antibody (if a recommended dilution range is 1:100 to 1:500, we will start with 1:100). A range of 1 to 5 μg/mL of primary antibody concentration is usually recommended for an initial titration.69 After determining the AR method, we will test the different antibody dilutions (usually 3 different dilutions) and adjust the incubation time on the basis of the initial test result. A polyclonal antibody is less specific and may cross-react with other antigens; therefore, using a polyclonal antibody is discouraged unless a monoclonal antibody is not commercially available. The advantage of using a polyclonal antibody is that it can be used at a much higher dilution (>1:1000 for many antibodies), which will reduce the cost.
Table 2 will give you a quick idea whether or not a primary antibody works on the positive control tissue. If the IHC assay result appears in rows 1, 2, or 3, the primary antibody will work well after fine-tuning. If the test result falls into rows 4, 5, or 6, after additional testing and adjustments of the staining condition, the primary antibody is most likely working. If the test result ends up in rows 7, 8, or 9, the primary antibody is unlikely to work; therefore, to save time, a new antibody from a different vendor should be considered.10,99
A standard online tracking table is created for the testing and optimization of each new antibody. The initial testing is labeled as protocol No. 1. Detailed documentation of changes to the antibody testing parameters, such as retrieval condition, dilution, and incubation time, are kept in an online tracking form and labeled as protocols No. 2, No. 3, etc. When the testing condition is optimized, the IHC laboratory director or the pathologist who oversees the IHC testing process will verify the protocol and proceed to the antibody validation process.
Antibody Validation
The CAP Pathology and Laboratory Quality Center gathered a team of pathologists and histotechnologists with expertise in immunohistochemistry to develop guidelines for validation of immunohistochemical assays.90 Following review of 126 related articles, open comments, panel discussion, and expert opinions, 14 guideline statements, including 4 recommendations and 10 expert consensus opinions, were proposed as listed below.90
Laboratories must validate all IHC tests before placing into clinical service.—Recommendation
For initial validation of every assay used clinically, with the exception of HER2/neu, ER, and PR (for which established validation guidelines already exist), laboratories should achieve at least 90% overall concordance between the new test and the comparator test or expected results. If concordance is less than 90%, laboratories need to investigate the cause of low concordance.—Recommendation
For initial analytic validation of nonpredictive factor assays, laboratories should test a minimum of 10 positive and 10 negative tissues. When the laboratory medical director determines that fewer than 20 validation cases are sufficient for a specific marker (eg, rare antigen), the rationale for that decision needs to be documented.—Expert Consensus Opinion
For initial analytic validation of all laboratory-developed predictive marker assays, laboratories should test a minimum of 20 positive and 20 negative tissues. When the laboratory medical director determines that fewer than 40 validation tissues are sufficient for a specific marker, the rationale for that decision needs to be documented.—Expert Consensus Opinion
For a marker with both predictive and nonpredictive applications, laboratories should validate it as a predictive marker if it is used as such.—Recommendation
When possible, laboratories should use validation tissues that have been processed with the same fixative and processing methods as cases that will be tested clinically.—Recommendation
If IHC is regularly done on cytologic specimens that are not processed in the same manner as the tissues used for assay validation (eg, alcohol-fixed cell blocks, air-dried smears, formalin postfixed specimens), laboratories should test a sufficient number of such cases to ensure that assays consistently achieve expected results. The laboratory medical director is responsible for determining the number of positive and negative cases and the number of predictive and nonpredictive markers to test.—Expert Consensus Opinion
If IHC is regularly performed on decalcified tissues, laboratories should test a sufficient number of such tissues to ensure that assays consistently achieve expected results. The laboratory medical director is responsible for determining the number of positive and negative tissues and the number of predictive and nonpredictive markers to test.—Expert Consensus Opinion
Laboratories may use whole sections, TMAs, and/or multitissue blocks (MTBs) in their validation sets as appropriate. Whole sections should be used if TMAs/MTBs are not appropriate for the targeted antigen or if the laboratory medical director cannot confirm that the fixation and processing of TMAs/MTBs is similar to clinical specimens.—Recommendation
When a new reagent lot is placed into clinical service for an existing validated assay, laboratories should confirm the assay's performance with at least 1 known positive case and 1 known negative case.—Expert Consensus Opinion
Laboratories should confirm assay performance with at least 2 known positive and 2 known negative cases when an existing validated assay has changed in any one of the following ways: antibody dilution, antibody vendor (same clone), incubation or retrieval times (same method).—Expert Consensus Opinion
Laboratories should confirm assay performance by testing a sufficient number of cases to ensure that assays consistently achieve expected results when any of the following have changed: fixative type, AR method (eg, change in pH, different buffer, different heat platform), antigen detection system, tissue processing or testing equipment, environmental conditions of testing (eg, laboratory relocation), laboratory water supply. The laboratory medical director is responsible for determining how many predictive and nonpredictive markers and how many positive and negative tissues to test.—Expert Consensus Opinion
Laboratories should run a full revalidation (equivalent to initial analytic validation) when the antibody clone is changed for an existing validated assay.—Expert Consensus Opinion
The laboratory must document all validations and verifications in compliance with regulatory and accreditation requirements.—Expert Consensus Opinion
At Geisinger, we have established a large TMA bank containing thousands of tumors and normal tissues from various organs. Each TMA block typically contains 50 to 100 tumors or normal tissues that are fixed and processed under similar or identical conditions as other routine patient samples. Two punched cores of 1.5 or 2.0 mm each have usually been taken from each case. After antibody testing and optimizing on a small TMA block containing 5 to 10 cases of tumor/normal tissues, the antibody validation process is followed. For instance, for validation of napsin A monoclonal antibody, 3 TMA blocks are selected, including lung adenocarcinomas, papillary renal cell carcinomas, and lung squamous cell carcinomas. The positive reference range for napsin A is expected to be 75% to 80% in lung adenocarcinomas, 50% to 60% in papillary renal cell carcinomas, and close to zero in lung squamous cell carcinomas.100 If the validation data are within the reference range, napsin A is included in the antibody library and implemented in our IHC laboratory. If the validation data showed the positive percentage was significantly below the reference range (below 70% in this case), we repeated the validation process in 10 cases of lung adenocarcinoma on routine sections to eliminate the possibility of focal staining on TMA sections, which consequently resulted in a lower diagnostic sensitivity. If the positive rate (sensitivity) continued to be low, we would return to the antibody testing and optimizing step to increase the positive staining signal and subsequently increase the diagnostic sensitivity to the reference range if possible.
Multitissue TMA Blocks for External Positive and Negative Tissue Controls
External positive control (EPC) is an important method of quality control for the IHC staining process and is required by CAP and many IHC vendors. External positive control is an invaluable tool to assess the appropriateness of antibody immunoreactivity, especially when an internal control is not available on testing tissue. There is no clear definition or general agreement on what specimens should be used for either positive or negative controls. How many positive and negative controls are needed? Should they be tumor and/or normal tissues?
In the past, the Geisinger IHC laboratory has relied on a single tissue block or multitissue sausage block for this purpose. To cover a large and expanding antibody inventory, which currently contains more than 200 antibodies, the IHC laboratory has had to keep a long list of tissue blocks for EPC. Sometimes, it became challenging for technologists to identify the correct EPC for an antibody. Another challenge was too much tissue consumption, particularly for tumor tissues. From a quality control point, the lack of staining consistency and reproducibility was also problematic because different tissue blocks were used for different antibody runs.
To overcome these shortcomings, the laboratory started to invest time and effort into building a set of TMA blocks for EPC use. These blocks each contain multiple 1.5- or 2-mm cores of normal and/or tumor tissue. They are specifically designed to either maximize tissue representation for broad antibody coverage or target unique pathologic conditions, such as tumor of unknown primary, melanoma, hematopoietic neoplasm, germ cell tumor, small round cell tumor, and sarcoma. Tissues are taken exclusively from selected blocks with demonstrable positive immunoreactivity for desired antibodies.
The new TMA EPCs appear on IHC-stained slides as neatly arranged rows of circular tissue, which only occupy a small space at one end of a slide and leave ample space for a tested specimen. Not only do they show a superior visual appearance, they are also extremely easy to read under the microscope. Their multitissue composition empowers users to simultaneously compare immunoreactivity not only in 1 but in many normal tissues or tumor types, which usually provides both external positive and negative controls on a same TMA section. The use of normal tissues with known antigen expression is highly recommended, instead of tumor tissues, which may demonstrate variable antigen expression from one area to another. Each TMA block can cover multiple markers. In fact, 6 TMA control blocks cover most antibodies (>200) in our IHC laboratory, which used to be covered by almost 50 external positive control blocks. For example, the tumor of unknown primary control block containing normal colon, pancreas, skin, stomach, liver, kidney, and testis will cover many frequently used IHC markers as listed in Table 3. The germ cell tumor control block containing seminoma, embryonal carcinoma, yolk sac tumor, and placental tissues covers the most frequently used germ cell tumor markers, including sal-like protein 4 (SALL4), octamer-binding transcription factor 4 (OCT4), placental alkaline phosphatase (PLAP), sex-determining region Y box 2 (SOX2), NANOG homeobox (NANOG), cluster of differentiation (CD) 30, α-fetoprotein (AFP), glypican-3, CD117, podoplanin (D2-40), and cytokeratin (CK). Ideally, a positive control block should contain high, intermediate, and low expression of the tested antigen (epitope). For convenience, a digital image library to categorize the staining pattern of a specific antibody with its corresponding TMA EPC is available online. It can be used as a reference when questions about staining quality are encountered. The images in the library can also serve as the gold standard for routine quality control purpose.
Currently, TMA EPCs are used for most antibodies in the laboratory, with exceptions for pathogen-specific antibodies, quantitative antibodies, and some esoteric antibodies. Since the introduction of TMA EPCs, IHC technologists have provided uniformly positive feedback because it simplifies the workflow and minimizes potential error. Tissue microarray EPCs have also demonstrated superior performance in staining consistency and reproducibility, compared to the old-fashioned EPCs. Since TMA EPCs use small tissue cores at construction, it is predicted that they will also save precious tumor tissues in the long run.
It should be mentioned here that the revised CAP Anatomic Pathology Checklist Item ANP.22570 eliminates the requirement for negative reagent controls in immunohistochemistry as long as the detection system does not use an avidin-biotin linkage. This would include detection products marketed as “polymer” as well as “multimer.” 97
Automated Staining Platform Selection
Before you select an automated IHC staining platform, there are several questions you should ask. What are the advantages and disadvantages of automating IHC versus manual IHC? What are the strengths and weaknesses of each automated IHC staining platform, such as the Ventana Benchmark Ultra (Ventana Medical Systems, Tucson, Arizona), Leica Bond III (Leica Biosystems, Buffalo Grove, Illinois), Dako Omnis (Dako North America, Carpinteria, California), and Biocare intelliPath FLX (Biocare Medical, Concord, Massachusetts)? The parameters that need to be considered are user-friendliness, capacity, turnaround time, amount of reagent/antibody used, waste disposal control, quality of stains, ability to run multiplex and in situ hybridization, and flexibility of integration with other laboratory information systems. The details are addressed in the accompanying review article entitled “Overview of Automated Immunohistochemistry.”
POSTANALYTIC PHASE
Interpreting IHC Assay Results
There is no universal scoring system for an IHC assay result and also no general agreement on what is the cutoff point to render a positive or a negative IHC test result. In fact, it is unlikely and impractical to have an absolute cutoff value for all diagnostic immunomarkers. In general, we use 5% as a cutoff point to determine a positive or a negative staining result, especially for cytoplasmic and membranous staining markers. Many factors may influence the interpretation and should be taken into consideration when interpreting an IHC test result: (1) small biopsy and cell block versus large resection specimen; (2) the amount of target antigen in the tested tissue; (3) the specificity of the particular antigen; (4) the sensitivity of a primary antibody; (5) localization of the target antigen such as nuclear staining versus cytoplasmic staining; (6) the staining intensity of the internal and external positive controls; and (7) how well the tissue has been fixed and processed.
A false-negative result is far more common than a false-positive result. Adequate internal positive staining is the best way to exclude false-negative staining, and a good internal negative staining is the best way to rule out a false-positive staining. In general, nuclear staining is more reliable than cytoplasmic staining. Any nuclear staining, especially in a small tissue biopsy or cell block preparation, should be regarded as a significant finding. For pathogen staining, such as BK virus and cytomegalovirus, any nuclear staining (even a single nucleus stained) in the right context should be regarded as a positive result. If the known internal and external positive tissues are only weakly positive, then weak staining in the target tissue should be read as positive, or the IHC assay should be repeated. If the target tissue is only weakly positive in the presence of background staining, caution should be taken to render the IHC test result as positive. If the known internal and/or external positive controls are negative, the target tissue with no immunoreactivity should be repeated. Some IHC test results, such as integrase interactor 1 (INI1) and mismatch repair (MMR) proteins (MutL homolog 1 [MLH1], postmeiotic segregation increased 2, MutS protein homolog [MSH] 2, and MSH6) are significant when loss of expression occurs. In this instance, the presence of positive internal controls, such as lymphoid cells, endothelial cells, and stromal cells, is imperative before concluding loss of expression.
At Geisinger, we use a scoring system based on the extent and intensity of the stain. The extent of the stain is recorded as 0 (<5% of the target cells stained), 1+ (5%–25% of the target cells stained), 2+ (26%–50% of the target cells stained), 3+ (51%–75% of the target cells stained), or 4+ (>75% of the target cells stained). The staining signal is recorded as weak, intermediate, or strong. A strong signal can be easily seen on low magnification; a weak signal is usually observed on high magnification; an intermediate signal borders between a strong and a weak staining signal.
Reporting IHC Assay Results
We report IHC assay results in a tabulated format as in Table 4. For example, Table 4 illustrates a panel of antibodies used to differentiate a metastatic breast carcinoma from a primary lung adenocarcinoma; the IHC assay result below supports the diagnosis of lung adenocarcinoma. The following elements (antibody, result, clone, localization, tissue type, and paraffin block number) are recommended to be included in the pathology report. The positive staining result can range from 1+ to 4+, with weak, intermediate, or strong staining intensity. The detailed staining results (such as 4+, strong) are recorded in our database within the CoPath system (Cerner Corporation, Kansas City, Missouri), which can be potentially used for future research projects and can enable one to further understand the clinical significance of the extent and intensity of each IHC stain.
TOTAL IHC QUALITY MANAGEMENT PROGRAM (QUALITY ASSURANCE, QUALITY CONTROL, AND QUALITY IMPROVEMENT)
There are many excellent, comprehensive review articles and book chapters discussing quality assurance, quality control, and quality improvement in the field of immunohistochemistry.36,69,81,87 The following are some additional steps we have taken in our IHC laboratory to ensure consistent, reproducible, and reliable IHC test results on every antibody, every time.
Quality Control of Each Stain
To stay on top of high-quality service, one needs to be proactive rather than reactive to potential quality issues. At Geisinger, every IHC stain (both patient tissues and external positive and negative control tissues) is reviewed for quality control purposes before releasing it to an ordering pathologist. A Quality Control Worksheet (Figure) containing a set of quality parameters goes with every stain. After the review, the box of acceptable slides or deficient slides will be checked. If a slide is determined to be deficient by an IHC technician, the stain may be repeated to save time. A comment section is left for the ordering pathologist. The quality control result and pathologist's feedback for each stain will be entered into the IHC database, and the results will be collected and reviewed at a weekly laboratory technical specialist meeting. Each issue will be analyzed, and corrective action will be taken. Is the issue caused by the instrument, reagent, staining protocol, personnel, or other factors? More importantly, is this an isolated incident or a trend of poor quality? The IHC technical specialist will bring the issues back to IHC technologists and resolve them. The Quality Control Worksheet has been well received by both IHC technologists and pathologists. It has proven to be a crucial step in identifying early problems and is an effective way to educate IHC technologists, which in turn will make them more vigilant to poor-quality slides, and to take corrective action before releasing the slide(s) to an ordering pathologist.
Geisinger Medical Center's Immunohistochemistry Laboratory Quality Control Worksheet. Abbreviation: QC, quality control.
Geisinger Medical Center's Immunohistochemistry Laboratory Quality Control Worksheet. Abbreviation: QC, quality control.
Continuous Quality Monitoring
Each new antibody has to go through a vigorous optimizing and validating process before being implemented in our IHC laboratory. The quality of the IHC test result for a newly introduced antibody is usually excellent at the beginning. However, weeks or months after introduction, the quality of the IHC assay for a particular antibody may be unstable or even deteriorate. The IHC laboratory begins to receive complaints from ordering pathologists about weak staining, background staining, or wrong localization of the antibody; perhaps this sounds like a familiar scenario to you. The question is, how can we be more proactive than reactive to potential issues? With the growing list of antibodies in our IHC laboratory, we started a new initiative in monitoring a select number of antibodies on a regular basis, using TMA sections. The top 50 antibodies were chosen by the highest test volumes, the clinical importance of these antibodies, and the target antigen absence from normal tissues, such as thyroid transcription factor 1 (TTF1), napsin A, ER, PR, HER2/neu, GATA-binding protein 3 (GATA3), renal cell carcinoma marker (RCCma), α-methylacyl-CoA racemase (P504S), carbonic anhydrase IX (CAIX), von Hippel–Lindau tumor suppressor (pVHL), arginase-1, glypican-3, paired box gene 8 (PAX8), Wilms tumor 1 (WT1), caudal type homeobox 2 (CDX2), special AT-rich sequence–binding protein 2 (SATB2), S100, human melanoma black 45 (HMB-45), melanoma-associated antigen recognized by T cells 1 (Mart-1), CD3, CD20, CD10, CD15, CD30, myogenin, desmin, smooth muscle actin (SMA), chromogranin, synaptophysin, NK3 homeobox 1 (NKX3.1), NK2 homeobox 2 (NKX2.2), CD117, CD34, p16, p53, p40, CK 5/6, p63, SALL4, OCT4, calretinin, and inhibin-α. A special TMA block was constructed that contained tissues to test all of the antibodies mentioned above. Multiple identical blocks were built. Immunohistochemistry tests were performed on the TMA slides for each antibody, and the slides were scanned and stored online. Monthly, 10 to 15 selected antibodies are tested on the same TMA block and the results (extent and intensity of the stain) are reviewed, recorded, and compared to the previously scanned images stained with the same antibody. Imaging analysis can be applied to quantitate the staining signal (extent and intensity) if needed. If a suboptimal stain, such as weak staining or background staining, is observed in the newly stained slide, the IHC test will be repeated. If the stain remains suboptimal, the antibody will be withdrawn from the test menu, and a full investigation will be performed.
Digital Pathology
Digital pathology (whole slide scan), in conjunction with imaging analysis algorithms, is a great innovation in the field of anatomic pathology. This allows pathologists to review the same slides anywhere and anytime with superb and identical quality. Digital pathology has been applied to remote frozen section diagnosis, fine-needle aspiration specimen adequacy assessment, slide consultation, slide archiving, and standardization of educational course material.29–35 With continued improvement of workflow and integration with the laboratory information system, digital pathology may eventually replace the traditional microscope and revolutionize the field of anatomic pathology. With regard to the field of IHC, digital pathology together with imaging analysis will provide consistent, reproducible, and quantitative IHC assay results, especially for multiplex staining (such as double staining) and counting nuclear staining signals (such as Ki-67). Furthermore, digital pathology will reduce turnaround time in a situation wherein the IHC slides need to be delivered to another hospital or pathology office. For equivocal HER2/neu IHC assay results on gastrointestinal and breast cancer, a fluorescence in situ hybridization assay can be ordered before the glass slide is delivered. For a difficult case of tumor of unknown primary, a second panel of antibodies can be ordered in a timely fashion before the glass slide is delivered. For quality control purposes, digital pathology enables multiple slides to be viewed simultaneously. A standard TMA slide (for validation, positive control, or continuous quality-monitoring purposes) can be aligned side by side with the test slide for easy comparison of staining results (extent and intensity of the stain).
Proficiency Testing
A CAP-certified IHC laboratory is required to participate in the CAP External Quality Assessment (EQA) and Proficiency Testing Program. CAP offers a number of proficiency testing programs specifically designed for IHC laboratories. The newly developed program, CAP/National Society for Histotechnology HistoQIP–IHC (program code HQIHC), is designed to improve the preparation of IHC slides in all laboratories handling gastrointestinal, skin, and genitourological biopsies. It requires participants to submit IHC-stained slides for review by a panel of experts. Most other programs are geared to evaluate IHC assay methodology and performance. These programs require participants to perform specific IHC assays (analytes) on centrally prepared slides provided by CAP, interpret the results, and then report to the CAP for evaluation. As feedback, participants receive a performance evaluation packet in which detailed statistical data of the entire survey are included. The participating laboratory should be able to compare its performance with all participating laboratories. These programs include MK (General Immunohistochemistry), PM2 (ER/PR), PM3 (CD20), PM1 (CD117), MMR (Mismatch Repair Proteins), HER2 (breast), GHER2 (gastric HER2), and PM5 (in which markers vary by year). Among these programs, PM2 and HER2 (breast) are specifically designed to fulfill proficiency testing requirements of the ASCO/CAP guideline for ER, PR, and HER2/neu assessment in breast carcinoma. The PM5 program is uniquely designed to evaluate a number of commonly used IHC assays, including chromogranin, cyclin D1, CDX2, CD30, D2-40, CK20, Ki-67, PAX2, PAX8, and p63. Nevertheless, currently, for the vast majority of more than 200 diagnostic antibodies (class I tests) in a clinical IHC laboratory, there are still no standard CAP proficiency testing programs available.
Other international EQA programs specifically designed for IHC are also available. NordiQC (http://www.nordiqc.org77) and United Kingdom National External Quality Assessment Service (UK NEQAS, Sheffield, England, http://www.ukneqas.org.uk76) are two of the most reputable programs. NordiQC is an independent scientific organization in Denmark that dedicates itself to promoting high-quality IHC by arranging EQA services to IHC laboratories all around the world (mainly European and Asian countries) and providing recommendations for improvement based on testing results. Its Web site contains some useful information about IHC, including free recommended staining protocols for different antibodies on different platforms.
Qualifications of IHC Personnel
Minimum standards for the qualifications of immunohistochemistry staff, including laboratory directors, are not yet established by CAP or other regulatory agencies. Certified histotechnologists who receive adequate in-house IHC training are qualified to perform an IHC test. The American Society for Clinical Pathology (ASCP) offers an additional certificate program to histotechnologists who demonstrate advanced knowledge in both theory and practical experience in IHC. Our IHC technologists are encouraged to obtain Qualification in Immunohistochemistry (QIHC) certification from the ASCP. Additionally, competency assessment of each IHC technologist should be performed and documented annually. However, the most critical requirement for the IHC staff is to be able to determine what slides are acceptable and what slides should be rejected before releasing to the ordering pathologist, which will prevent the release of poor-quality slides and delays in turnaround time. These parameters are listed on the Quality Control Worksheet. Specifically, the staff should be able to recognize appropriate or inappropriate control reactions, tissue quality, and staining artifacts. Equally importantly, the staff should be able to resolve these issues after identifying the problem.
CAP Checklist for Clinical IHC Laboratories
Table 5 summarizes the 2013 (07.29.2013) CAP checklist for immunohistochemical laboratories.97 This checklist is subject to changes in the next updated CAP Checklist for Anatomic Pathology.
BEST PRACTICES IN IHC
Numerous IHC markers have been discovered and introduced into clinical IHC laboratories, especially in the last decade. Most large IHC laboratories have an extensive test menu with more than 200 antibodies. Centers for Medicare and Medicaid Services data show that billing for CPT (current procedural terminology) code 88342 for IHC tests has significantly increased in the last 6 years, with an average annual growth rate of approximately 15%, and the growth trend continues.101 Diagnostic IHC is an indispensable ancillary technique to anatomic pathology laboratories and has provided ample scientific evidence to objectively support and confirm the histologic impression of some very challenging cases. It was much easier to digest and master the detailed application of each antibody when an IHC laboratory carried only 30 to 50 available antibodies a decade ago. It is obviously much more challenging to grasp knowledge of more than 200 antibodies now. Therefore, both underutilization and overutilization of IHC markers have gradually become issues in anatomic pathology laboratories. To find a middle ground or best practice in immunohistochemistry, in the past few years the CAP IHC Committee has published a small series of articles attempting to address this problematic area.102–106 We have faced the same issue in our laboratory and would like to share some initiatives we have taken to tackle this problem.
Know the Diagnostic Sensitivity and Specificity of Each IHC Marker
The more you know about the characteristics of each IHC marker, the less likely you will be to overuse or underuse it. Table 6 summarizes the frequently used IHC markers in identifying a tissue-specific origin or in differentiating a malignant tumor from its benign counterpart. Importantly, an entirely sensitive and specific IHC marker rarely exists. A small panel of IHC markers rather than a single marker is strongly recommended to avoid a potential diagnostic pitfall. For the step-by-step approach to an undifferentiated neoplasm/tumor of uncertain origin, please refer to the accompanying review article entitled “Immunohistochemistry in Undifferentiated Neoplasm/Tumor of Uncertain Origin.”
Begin With a Limited Panel of IHC Markers (First IHC Panel)
We have constructed more than 100 small IHC panels to address the frequently asked questions in diagnosis and differential diagnosis of specific entities. These diagnostic panels are based on literature, Geisinger IHC data, and personal experience. A single IHC marker approach (other than for pathogens such as cytomegalovirus or BK virus) is strongly discouraged since aberrant expression of a highly specific IHC marker can rarely occur. However, aberrant expression of the entire panel of highly specific IHC markers is nearly statistically impossible. For Geisinger IHC data, more than 5000 TMA slides containing tumors from various organs have been stained with selected IHC markers. These data have been collected, analyzed, and compared to the published data. A few examples of first IHC panels are listed in Tables 7 through 10.
Continue With Second IHC Panels
Update IHC Panels With New Data
With additional studies and publications, many IHC markers initially believed to be highly specific gradually lost their specificities. However, additional novel biomarkers are emerging continuously. Both first and second IHC panels for a specific differential diagnosis should be kept updated. The diagnostic sensitivity and specificity of an IHC panel for a specific entity will increase over a period of time. For example, CD99 was initially considered as a highly specific IHC marker for Ewing sarcoma and lymphoblastic lymphoma. However, after extensive tests, CD99 is now considered a very nonspecific marker for Ewing sarcoma. In fact, CD99 has been used as a marker to exclude a diagnosis of Ewing sarcoma when it shows negativity, rather than confirming the diagnosis. A new nuclear marker, NKX2.2, has been demonstrated to be a more specific marker for Ewing sarcoma.107
Track and Compare the IHC Utilization Data
As aforementioned, we have created more than 100 small IHC panels and built these panels into the CoPath system, which allows pathologists to order the entire IHC panel as a group stain if they wish. The utilization of immunohistochemical stains is audited periodically for each subspecialty group and each pathologist; IHC utilization among pathologists within the same subspecialty group is compared and contrasted, using the group average or median as the benchmark. For instance, within the genitourological group of 6 pathologists, what is the percentage of IHC stains (PIN4) ordered in prostatic core biopsy cases (percentage of cases with IHC stains) for each pathologist? What is the percentage of IHC stains (PIN4) ordered per prostatic tissue core (stain per tissue core) for each pathologist? What is the percentage of IHC stains ordered in bladder specimens (percentage of cases with stains and stains per case) for each pathologist? The same auditing process applies to other subspecialty areas as well. The pathologists found to excessively use IHC tests will be informed to correct the overutilization issue, using the group average as a reference. The other reason for overutilization is the ordering of IHC markers on both fine-needle aspiration and surgical specimens from the same biopsy procedure. By using the recommended small IHC panels and performing periodic audits, we have begun to appreciate some improvement in the overutilization issue with no negative impact on patient care. However, most importantly, before ordering any IHC stain, a pathologist should ask what value this stain(s) will add to the final diagnosis and patient care. If the answer is “none at all” or “I don't know,” the staining procedure should not be performed. Additionally, if either 3 stains or 5 stains will provide the same information, you should only order 3 stains. With this in mind, the concept of best practices in immunohistochemistry can be effectively implemented.
In summary, this review article delineates some critical points in preanalytic, analytic, and postanalytic phases; reiterates some important questions, which may or may not have a consensus at this time; and updates the newly proposed guidelines on antibody validation from the CAP Pathology and Laboratory Quality Center. Additionally, the article shares Geisinger's experience with (1) testing/optimizing a new antibody and troubleshooting; (2) interpreting and reporting IHC assay results; (3) improving and implementing a total IHC quality management program; and (4) developing best practices in immunohistochemistry.
The authors would like to thank Kathy Fenstermacher, BA, for editing this manuscript.