Standardization of Diagnostic Immunohistochemistry

Abstract

Standardization of every step in the pre-analytic, analytic, and post-analytic phases is crucial to achieving reproducible and reliable immunohistochemistry results. This chapter delineates some critical points in the pre-analytic, analytic, and post-analytic phases; reiterates some important questions which may or may not have a consensus at this time; and updates the proposed guidelines on antibody validation from the College of American Pathologists (CAP) Pathology and Laboratory Quality Center. Additionally, the chapter shares Geisinger’s experience with (1) testing/optimizing a new antibody and troubleshooting; (2) using cell blocks containing a mixture of cultured cell lines as external positive controls; (3) interpreting and reporting immunohistochemistry assay results; (4) improving and implementing a total immunohistochemistry quality management program; and (5) developing the concept of best practices in immunohistochemistry.

Frequently Asked Questions

• 1. Summary of common variables impacting immunohistochemistry (IHC) standardization (Table 2.1).

• 2. What are the most important factors in the pre-analytic phase (Table 2.2)?

• 3. What are the utilities of multi-tissue tissue microarray (TMA) blocks in a clinical IHC laboratory?

• 4. Can cultured cell lines be used as IHC-positive controls?

• 5. What are the advantages of using cultured cell lines instead of multi-tissue TMA blocks as external positive controls?

• 6. What cell lines are recommended for use as IHC-positive controls (Table 2.3)?

• 7. How are cell blocks prepared from cultured cell lines (Table 2.4)?

• 8. What specific cell lines are recommended for constructing a set of TMA control blocks for selected biomarkers (Table 2.5)?

• 9. What are the recommended immunohistochemistry critical assay performance controls (iCAPC) for the commonly used IHC markers (Table 2.6)?

• 10. How do you select antibodies?

• 11. What are the results and common problems encountered when testing a new antibody (Table 2.7)?

• 12. What are the general approaches before getting into a demanding technical issue?

• 13. How do you optimize a new antibody?

• 14. What are the possible solutions for each specific technical problem in Table 2.7?

• 15. How do you determine whether or not a primary antibody works?

• 16. What are the commonly used antigen retrieval methods?

• 17. What are the commonly used antigen retrieval protocols?

• 18. What are the recommended guidelines for antibody validation?

• 19. How do you select an automated staining platform?

• 20. How do you interpret IHC assay results?

• 21. How do you report IHC assay results (Table 2.8)?

• 22. How do you improve a total IHC quality management program (Table 2.9)?

• 23. What is the role of digital pathology in an IHC laboratory?

• 24. What are the available proficiency testing programs (Table 2.10)?

• 25. What are the required qualifications for IHC personnel?

• 26. What is the CAP checklist for clinical IHC laboratories (Table 2.11)?

• 27. How do you implement best practices in immunohistochemistry (Table 2.12)?

1. Summary of common variables impacting immunohistochemistry (IHC) standardization.

Numerous variables have been identified in the process of IHC standardization, and some will significantly influence the quality of staining results. These factors may occur in pre-analytic, analytic, and post-analytic phases as summarized in Table 2.1 [1,2,3,4,5,6,7,8,9,10].

Table 2.1 Summary of common variables impacting IHC staining results

2. What are the most important factors in the pre-analytic phase?

It has been recommended that tissue be fixed in 10% neutral-pH, phosphate-buffered formalin for a minimum of 8 hours [5]. If formalin or a formalin–alcohol mixture is a component solution on the tissue processor instrument, tissue should be fixed in formalin for 6–12 hours before being loaded onto the tissue processor. Non-formalin fixatives and/or alternative fixation methodologies are strongly discouraged [5].

The study by Engel and Moore identified 27 variables that have been examined and reported in published literature [7]. Some of these pre-analytic factors which may or may not impact an IHC assay result are summarized in Table 2.2.

Table 2.2 Summary of factors with or without influence on IHC test results

Drying slides can be easily overlooked in an IHC laboratory, and IHC technologists must be educated to pay particular attention to drying time and temperature. It has been recommended that slides be dried at 50–60 °C for a minimum of 1 hour or at room temperature for 24 hours [7].

Decalcification may have a negative impact on an IHC assay for certain antigens [11, 12]. As such, CAP recommends that a disclaimer be included in the surgical pathology or FNA 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 [13].”

3. What are the utilities of multi-tissue tissue microarray (TMA) blocks in a clinical IHC laboratory?

TMA blocks containing various numbers of tumors and/or normal tissues have demonstrated great utilities in clinical IHC laboratories. TMA blocks may be potentially useful 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, four 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 50–100 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 5–10 cases of a specific type of tumor, which is useful for antibody testing and optimization;

• 4. A TMA block containing 5–10 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.

4. Can cultured cell lines be used as IHC positive controls?

Yes. In fact, cultured cell lines can be a better source for positive control blocks for selected biomarkers. At Geisinger IHC Laboratory, we have used cell blocks containing a mixture of cultured cancer cell lines for a significant number of antibodies. These cell blocks can be used for (1) external positive and negative control tissues; (2) new antibody testing and optimization; (3) antibody validation; and (4) continuous quality monitoring of commonly used antibodies.

5. What are the advantages of using cultured cell lines instead of multi-tissue TMA blocks as external positive controls?

Depending on the antibodies being ordered, IHC labs in the United States use either tumor tissue blocks or normal tissue blocks as external positive control slides. The positive control blocks can be constructed by each IHC lab or ordered from a commercial company. The cost for each positive control slide varies for a given antibody. Many IHC labs choose to build the majority of their positive control blocks, only purchasing positive control slides for rare antibodies. Multi-tissue TMA blocks are most commonly used as external positive and negative control blocks.

In contrast to TMA blocks, cell blocks containing cultured cancer cell lines provide several advantages. They (1) provide more consistent and reliable quality; (2) save expensive IHC tech time to build TMA blocks; and (3) avoid consuming valuable tumor blocks, which are important for future molecular testing, clinical trials, research, and biospecimen banking, from pathology archives.

6. What cell lines are recommended for use as IHC positive controls?

Geisinger IHC laboratory has tested many cell lines. The ordering information, growth condition, and growth properties of these cultured cell lines are summarized in Table 2.3.

Table 2.3 Ordering information, growth condition, and growth properties of cultured cell lines

7. How are cell blocks prepared from cultured cell lines?

Cultured cells are harvested, and cell pellets are prepared using standard techniques. The following is a brief example:

When the cell growth is near confluent (adherent growth, about 1 × 10⁷ cells per dish) or near 0.5 × 10⁸ cells per dish (suspended growth), harvest cells by EDTA digestion and centrifuge (adherent growth); or directly move the suspended growth cultures to 50 mL Falcon tubes. Eight large culture dishes (150 × 25 mm each; each dish containing 5–10 × 10⁷ cells) are collected for one cell block preparation.

For the preparation of a cell block with mixed cell lines, we cultured select cell lines simultaneously and mixed these cells at proper ratios, depending on the purpose of the cell block. Table 2.4 shows an example of a melanoma control block with three different cell lines in the proper ratios and cell counts.

Table 2.4 Cell lines for construction of a melanoma control block

Examples of the steps to prepare a cell pellet are as follows:

• (a) Centrifuge the cells to make a cell pellet.

• (b) Move the bottom cell pellet to a small glass vial (Cat. #72631-10, Electron Microscopy Sciences, Hatfield, PA), and then place the small glass vial into a 25 mL polyethylene vial (Cat. #72621-62, Electron Microscopy Sciences, Hatfield, PA).

• (c) Spin the cells at 1600 rpm for 7 minutes in a Beckman centrifuge with swinging-bucket rotors.

• (d) Remove the small glass vial from centrifuge for cell block preparation.

• (e) Pour off supernatant completely, and preserve the cell pellet at bottom of the small vial.

• (f) Add approximately 5–6 drops of plasma (obtained from the Blood Bank of Geisinger Medical Laboratories) to the cell pellet and re-suspend by gently vortexing; then, add approximately 5–6 drops of bovine thrombin (Cat. #23-306291, Fisher Scientific Pittsburgh, PA) into the cells and mix gently, and then let it stand for 10 minutes.

• (g) The cell pellet should become a semi-solid clot at room temperature. Under a fume hood, insert a 23-gauge needle with a syringe containing approximately 2–3 mL of 10% neutral-buffered formalin along the side at the bottom of the vial. While the formalin is slowly pushed through the syringe, the clotted cell pellet is slowly dislodged from the flat-bottom glass vial and floats to the surface.

• (h) Place the clotted pellet into a labeled cassette, fix it with 10% neutral-buffered formalin for 6–8 hours (no more than 24 hours), and then send it to the histology lab for tissue processing and paraffin embedding.

• (i) The cassette with the clotted cell pellet is processed in the tissue processor using long-run program as routine surgical specimens. After processing, embed the clotted cell pellet in 57–59 °C paraffin on the embedding workstation (Cat. # A81000002, HistoStar™, Thermo Scientific). At this step, the cell block is made and the diameter of the cell block is about 0.5 cm.

• (j) Cut the cell block into 4~5μm sections and check the quality of the cell block with hematoxylin and eosin stain (H&E).

8. What specific cell lines are recommended for constructing a set of TMA control blocks for selected biomarkers?

After testing numerous cell lines, those with high-level expression (strongly positive), low-level expression (weakly to moderately positive), and no expression (no staining) for a targeted antigen were selected for six specific cell blocks. These cell blocks include (1) breast cancer; (2) melanoma; (3) lymphoma; (4) germ cell tumor; (5) malignant small round cell tumor; (6) sarcoma; and (7) tumor of unknown primary. These seven cell blocks can potentially cover over 70% of the commonly used diagnostic and predictive markers at Geisinger’s IHC laboratory. We have found that identifying a cell line with low-level expression of a targeted antigen can be challenging. Therefore, for some targeted antigens, only cell lines with high-level expression and no expression of these antigens were included.

The specific cell lines, the ratio of each cell line, types of cancer to be covered, and targeted biomarkers with high-level expression are summarized in Table 2.5. Figure 2.1a–h show a mixture of three breast cancer cell lines expressing targeted biomarkers. Figure 2.2a–h show a mixture of three melanoma cell lines expressing targeted biomarkers.

Fig. 2.1

A mixture of cultured breast cancer cell lines on H&E stained section (a), and expression of ER (b), PR (c), HER 2 (d), GATA3 (e), p53 (f), GCDFP15 (g), and TFF3 (h)

Fig. 2.2

A mixture of melanoma cell lines on H&E stained section (a), and expression of S100 (b), HMB-45 (c), MART-1 (d), MiTF (e), SOX10 (f), SOX2 (g), and S100A6 (h)

Table 2.5 Summary of specific cell lines used to construct a set of TMA control blocks for selected biomarkers

9. What are the recommended immunohistochemistry critical assay performance controls (iCAPCs) for the commonly used IHC markers?

An ideal external positive control for a targeted IHC marker should consist of a tissue/or tissues with high-level expression, low-level expression, or no expression of that antigen. A set of well-characterized primary positive controls (immunohistochemistry critical assay performance controls [iCAPCs]) recommended by the International Ad Hoc Expert Committee to cover 20 commonly used antibodies in clinical IHC labs is summarized in Table 2.6. A tissue with high-level expression of the targeted antigen tends to show a moderate to strong immunohistochemical reaction; in contrast, a tissue with low-level expression of the targeted antigen tends to demonstrate a low to moderate immunohistochemical reaction, or the low limit of detection (LLOD), of this specific antigen. The LLOD in an “optimized” IHC stain is defined by an observed positive reaction (staining) in a tissue/cellular element that is known to express low levels of the evaluated marker [14].

Table 2.6 Summary of iCAPCs for commonly used IHC markers

10. How do you select antibodies?

How do you select the “right” antibodies for your IHC lab and your patients? Before you decide to bring a new antibody to your IHC lab, 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 lab?

• 4. Where can I get this antibody?

• 5. Is more than one antibody (clone) available?

• 6. Do I have the positive control tissues to test and validate this antibody?

• 7. How am I going to implement it?

The following combined approach may be helpful in adding a new antibody to your IHC lab:

• 1. Studying and tracking mature publications in popular peer-reviewed pathology journals, especially those publishing articles on diagnostic surgical pathology and cytopathology;

• 2. Attending major pathology society conferences, particularly the United States & Canadian Academy of Pathology (USCAP) annual meeting, or reading the abstract book;

• 3. Using IHC vendor recommendations and online catalogs;

• 4. Using free websites with a published antibody library such as Geisinger’s IHC website (http://www.ihcfaq.com) [15], NordiQC (Nordic Immunohistochemical Quality Control, Aalborg, Denmark, http:/www.nordiqc.org) [16], and IHC menus on the pathology department websites of major medical institutions and hospitals.

11. What are the results and common problems encountered when testing a new antibody?

Troubleshooting problems encountered in an immunohistochemical staining procedure can be straightforward or a very complicated task. Many articles and book chapters have addressed these potential issues in great detail; therefore, this chapter is not intended to be comprehensive or to substitute for published literature. Instead, it attempts to re-emphasize the key points to remember when working on these problems. The nine most likely immunostaining results and potential problems encountered when testing and optimizing a new antibody in a positive control tissue block are summarized in Table 2.7. The possible causes and solutions for each specific problem will be addressed in Question #14.

Table 2.7 Summary of possible staining results when testing a new antibody

12. What are the general approaches before getting into a demanding technical issue?

As a general rule, the best approach to avoid the technical issues listed in Table 2.7 is to follow this simple checklist:

• 1. Follow all steps and instructions in the manufacturer’s protocol.

• 2. Consult the data sheet for general recommendations, such as positive control tissue, antigen retrieval technique, antibody dilution, blocking reagent, etc.

• 3. Confirm the compatibility of a secondary antibody to the species and subclass immunoglobulin of a primary antibody (such as rabbit monoclonal antibody or mouse monoclonal antibody).

• 4. Be sure to use the “right” tissue or tumor as a positive control; it should contain abundant and well-preserved antigen to be tested.

• 5. Use a positive control block (such as tissue microarray block) containing multiple tissue sections if it is available.

• 6. Ensure the oven temperatures do not exceed 60 °C.

• 7. Perform all relevant blocking steps to eliminate background staining, including endogenous peroxidases and phosphatases.

• 8. Check all reagents for appropriate preparation, expiration date, and storage condition.

• 9. Be aware that inadequate fixation (under-fixation), inappropriate fixative (other than 10% neutral-buffered formalin), and high acidity or prolonged decalcification may result in a false-negative result for many antibodies.

13. How do you optimize a new antibody?

The ultimate goal is to achieve a strong staining signal with little or no background staining using the highest primary antibody dilution. A TMA block containing a small number of tumors and/or normal tissues with known positivity and negativity for the target antigen is a good choice to test a new antibody. There are many ways to test a new antibody. Our experience demonstrates that a false-negative result is more likely due to the wrong antigen retrieval method rather than a suboptimal antibody dilution and/or incubation time. We tend to determine the best antigen retrieval method first and then test the proper primary antibody dilution and incubation time. To achieve this, we start with five different antigen retrieval methods/solutions (heat-induced epitope retrieval with citrate buffer/pH 6.0, with ethylenediaminetetraacetic acid (EDTA)/pH 8, with Target Retrieval Solution (TRS)/pH 6.1, with High pH Target Retrieval Solution (HiTRS)/pH 9, or enzyme digestion such as proteinase K/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 1–5 ug/mL of primary antibody concentration is usually recommended for an initial titration [9]. After determining the antigen retrieval method, we will test different antibody dilutions (usually three different dilutions) and adjust the incubation time based on 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, however, is that it can be used at a much higher dilution (>1:1000 for many antibodies), which will reduce the cost.

A standard online tracking table is created for the testing and optimization of each new antibody. The initial testing is labeled as protocol #1. Detailed documentation of changes to the antibody testing parameters, such as retrieval condition, dilution, and incubation time, is kept in an online tracking form and labeled as protocols #2, #3, etc. When the testing condition is optimized, the IHC lab director or the pathologist who oversees the IHC testing process will verify the protocol and proceed to the antibody validation process.

14. What are the possible solutions for each specific technical problem in Table 2.7?

• A. If strong staining signal and no background staining is obtained

  • The next step is to test the primary antibody in multiple dilutions, obtain the highest dilution with the optimal result, to save the primary antibody and cut down the cost.

• B. If strong staining signal and weak background staining is obtained

  • Reduce the primary antibody concentration.

  • Shorten the primary antibody incubation time.

  • Shorten the secondary antibody incubation time.

  • Further block the background staining.

• C. If strong staining signal and strong background staining is obtained

  • Reduce the primary antibody concentration.

  • Shorten the primary antibody incubation time.

  • Shorten the secondary antibody incubation time.

  • Further block the background staining.

  • Try different antigen retrieval methods.

• D. If weak staining signal and no background staining is obtained

  • Increase the primary antibody concentration.

  • Increase the incubation time for the primary antibody.

  • Increase the incubation time for the secondary antibody.

  • Switch to a more sensitive secondary detecting system.

  • Try different antigen retrieval methods.

• E. If weak staining signal and weak background staining is obtained

  • Increase the primary antibody concentration and reduce the incubation time.

  • Further block background staining.

  • Increase the incubation time for the secondary antibody.

  • Switch to a more sensitive secondary detecting system.

  • Try different antigen retrieval methods.

  • Use a different primary antibody.

• F. If weak staining signal and strong background staining is obtained

  • Further block background staining.

  • Switch to a more sensitive secondary detecting system.

  • Try different antigen retrieval methods.

  • Use a different primary antibody.

• G. If no staining signal and no background staining is obtained

  • Follow the general approaches in Question 12 step by step.

  • Increase the primary antibody concentration and incubation time.

  • Try different antigen retrieval methods.

  • Switch to a more sensitive secondary detecting system.

  • Contact the technical department of the primary antibody supplier for assistance.

  • Use a different primary antibody.

• H. If no staining signal and weak background staining is obtained

  • Follow the general approaches in Question 12 step by step.

  • Increase the primary antibody concentration and incubation time.

  • Try different antigen retrieval methods.

  • Switch to a more sensitive secondary detecting system.

  • Contact the technical department of the primary antibody supplier for assistance.

  • Use a different primary antibody.

• I. If no staining signal and strong background staining is obtained

  • Follow the general approaches in Question 12 step by step.

  • Try different antigen retrieval methods.

  • Switch to a more sensitive secondary detecting system.

  • Contact the technical department of the primary antibody supplier for assistance.

  • Use a different primary antibody.

15. How do you determine whether or not a primary antibody works?

To get a quick idea of whether a primary antibody works on positive control tissue, refer to Fig. 2.3. If the testing result appears in zone I, II, or III, the primary antibody will work well after fine-tuning. If the testing result falls into zone IV, V, or VI, after additional testing and adjustments of the staining condition, the primary antibody is most likely working. If the testing result ends up in zone VII, VIII, or IX, the primary antibody is unlikely to work; therefore, to save time, a new antibody from a different vendor should be considered.

Fig. 2.3

Summary of possible staining results, which gives one a quick idea of whether a primary antibody works on positive control tissue

16. What are the commonly used antigen retrieval methods?

Antigen retrieval technique is a process to unmask an antibody-binding site for a specific antibody on formalin-fixed, paraffin-embedded tissue sections. It can significantly enhance the immunohistochemical staining signal. There are two main antigen retrieval techniques. One is called heat-induced epitope retrieval (HIER). Another method uses enzymatic digestion and is called proteolytic-induced epitope retrieval (PIER) [2, 17, 18].

Many enzymes can be used in PIER, such as trypsin, proteinase K, pepsin A, and pronase. The key factors to obtain an optimal result include enzyme concentration, time of digestion, temperature, and pH. Proteinase K will provide an effective enzymatic digestion for membrane antigens such as pVHL, CD31, and VWF. Over-digestion may result in poor tissue morphology and even a false-positive staining; in contrast, under-digestion may cause a false-negative result.

Microwave oven and water bath are the most commonly used heating devices for HIER in our lab. Other devices may include microwave pressure cooker, vegetable steamer, and decloaker device. A heating and cooling time of 20 minutes each appears to be adequate for many antibodies. EDTA at pH 8.0 is the most frequently used retrieval solution in our practice; citrate buffer at pH 6.0, target retrieval solution at pH 6.1, and target retrieval solution at pH 9.0 (high pH) are also suitable for some antibodies [2, 17, 18].

A combination of HIER and PIER is an alternative approach to unmask difficult antigens when other methods fail. It is especially useful when performing double or triple labeling for two or more antigens simultaneously. However, special attention should be paid because two retrieval methods may cause a false-negative staining result for one of the two antibodies, and sometimes a tissue section may fall off the slide due to the prolonged retrieval time.

17. What are the commonly used antigen retrieval protocols?

• Protocol #1: Citrate Buffer Antigen Retrieval Method [2, 17, 18]

• Solutions and Reagents:

Solution A: Citric acid monohydrate (Fisher Scientific, Catalog# A104-500)

10.505 g, dilute in 500 mL of distilled water, mix to dissolve (0.1 M)

Solution B: Sodium citrate (Fisher Scientific, Catalog # S93364)

14.704 g, dilute in 500 mL of distilled water, mix to dissolve (0.1 M)

Store the solutions at 4 ° C for longer storage. Fresh preparation of citrate buffer before use:

$\left.\begin{array}{l}9\ \mathrm{mL}\ \mathrm{of}\kern0.5em \mathrm{Solution}\ \mathrm{A}\\ {}41\ \mathrm{mL}\ \mathrm{of}\kern0.5em \mathrm{Solution}\ \mathrm{B}\end{array}\right\}\mathrm{Dilute}\ \mathrm{in}\ 500\ \mathrm{mL}\ \mathrm{of}\ \mathrm{distilled}\ \mathrm{water},\mathrm{mix}\ \mathrm{to}\ \mathrm{dissolve}$

Adjust pH to 6.0 with 1 N NaOH and 1 N HCl and mix well.

Formalin, or other aldehyde fixation, forms protein cross-linking that masks the antigenic sites in tissue specimens, which in turn gives weak or false-negative staining for immunohistochemical detection of certain proteins. The citrate-based solution is designed to break the protein cross-linking, thereby unmasking the antigens and epitopes in formalin-fixed and paraffin-embedded tissue sections and enhancing staining intensity of antibodies.

• Procedure:

  • 1. Dewax paraffin-embedded tissue sections with two changes of Histoclear^® solution (National Diagnostics, Atlanta, GA, product #HS-200) or xylene, 5 minutes each.

  • 2. Rehydrate the sections in two changes of 100% and 95% ethanol for 30 seconds each and 70% ethanol for 30 seconds; then rinse in distilled water.

  • 3. Insert the slides in a slide holder and immerse them into a microwave dish containing 500 mL of citrate buffer. Set the lid loosely on top of the microwave dish.

  • 4. Heat the dish for 5 minutes at high power (level 10) and 10 minutes at medium power (level 5).

  • 5. Allow the slides to cool for 20 minutes at room temperature.

  • 6. Rinse sections in cool running tap water; then put the slides into Tris-Buffered Saline with 0.05% Tween (TBST) solution.

  • 7. Continue with an appropriate antibody staining protocol.

• Protocol #2: EDTA Buffer Antigen Retrieval Protocol [2, 17, 18]

• Solutions and Reagents:

EDTA buffer (1 mM EDTA, pH 8.0)

${\displaystyle \begin{array}{cc}\mathrm{EDTA}\ \left(\mathrm{Fisher}\ \mathrm{Scientific},\mathrm{Cat}\# BP120\hbox{-} 500\right)& 0.372\ \mathrm{g}\\ {}\mathrm{Distilled}\ \mathrm{water}\ & \kern0.5em 1000\ \mathrm{mL}\end{array}}$

Mix to dissolve. Adjust pH to 8.0 using 1 N NaOH. Store the solution at room temperature for up to 3 months or at 4 °C for longer storage.

This buffer works well for many antibodies, but sometimes it gives high background staining; therefore, a primary antibody can often be diluted in a lower concentration. It is very useful for low-affinity antibodies or when tissue antigens are not abundant.

The EDTA solution is also designed to break the protein cross-links, thereby unmasking the antigens and epitopes in formalin-fixed and paraffin-embedded tissue sections, thus enhancing the staining intensity of the antibodies.

• Procedure:

  • 1. Dewax the sections in two changes of xylene, 5 minutes each.

  • 2. Rehydrate the slides in two changes of 100% and 95% ethanol for 30 seconds each and 70% ethanol for 30 seconds; then rinse in distilled water.

  • 3. Place the slides in a slider holder and immerse them in a microwave dish containing 500 mL of EDTA buffer. Set the lid loosely on top of the microwave dish.

  • 4. Heat the dish for 5 minutes at high power (level 10) and 10 minutes at medium power (level 5).

  • 5. Allow the slides to cool for 20 minutes at room temperature.

  • 6. Rinse sections in cool running tap water; then put the slides into Tris-Buffered Saline with 0.05% Tween (TBST) solution.

  • 7. Continue with an appropriate antibody staining protocol.

• Protocol #3: Target Retrieval Solution Buffer Antigen Retrieval Protocol [2, 17, 18]

• Solutions and Reagents:

Target retrieval solution, pH 6.1 (Dako, Carpenteria, CA, Catalog# S1700)

Target retrieval solution, pH 9.0 (Dako, Catalog# S2368)

These products are to be used on formalin-fixed paraffin-embedded tissue sections mounted on glass slides for target retrieval prior to IHC procedures.

The retrieval procedure involves incubating the sections in preheated target retrieval solution in a water bath for 20 minutes prior to IHC procedures. This results in an increase in staining intensity for many primary antibodies.

• Procedure:

  • 1. Fill a Coplin staining jar or other suitable containers with a sufficient quantity of target retrieval solution. Place the container in a water bath. Heat the water bath to 95–99 °C (do not boil).

  • 2. Dewax the sections in two changes of xylene, 5 minutes each.

  • 3. Rehydrate the slides in two changes of 100% and 95% ethanol for 30 seconds each and 70% ethanol for 30 seconds; then rinse in distilled water.

  • 4. Incubate the sections in preheated target retrieval solution in a water bath for 20 minutes.

  • 5. Remove the entire jar or container with slides from the water bath and allow the slides to cool for 20 minutes at room temperature.

  • 6. Rinse the sections in cool running tap water; then put the slides into Tris-Buffered Saline with 0.05% Tween (TBST) solution.

  • 7. Continue with an appropriate antibody staining protocol.

• Protocol #4: Retrieve-All Antigen Unmasking System Retrieval Protocol [2, 17, 18]

• Solutions and Reagents:

Retrieve-All Antigen Unmasking System 1: Universal, 1X (Covance, Princeton, NJ, Catalog# SIG-31912)

Retrieve-All is an antigen unmasking solution available in the following three pH formulas for use in heat-induced unmasking. Retrieve-All 1 (Universal pH 8) is the most frequently used in our immunohistochemical laboratory. Other Retrieve-All solutions include Retrieve-All 2 (basic pH 10) and Retrieve-All 3 (acidic pH 4.8).

• Procedure:

  • 1. Fill a Coplin staining jar or other suitable containers with a sufficient quantity of Retrieve-All 1 solution. Place the container in a water bath. Heat the water bath to 95–99 °C (do not boil).

  • 2. Dewax the sections in two changes of xylene solution, 5 minutes each.

  • 3. Rehydrate the sections in two changes of 100% and 95% ethanol for 30 seconds each and 70% ethanol for 30 seconds; then rinse in distilled water.

  • 4. Incubate the sections in preheated Retrieve-All 1 solution in a water bath for 10 minutes.

  • 5. Remove the entire jar or container with slides from the water bath and allow the slides to cool for 10 minutes at room temperature.

  • 6. Rinse sections in cool running tap water; then put the slides into Tris-Buffered Saline with 0.05% Tween (TBST) solution.

• Protocol #5: Proteinase K Antigen Retrieval Protocol [2, 17, 18]

• Solutions and Reagents:

Proteinase K Solution (Dako, Catalog# S3020)

Proteolytic enzyme solution diluted in 0.05 mol/L Tris-HCl, 0.015 mol/L sodium azide, pH 7.5.

Proteinase K is used for the proteolytic digestion of formalin-fixed, paraffin-embedded tissue sections prior to an immunohistochemical staining protocol.

Enzymatic digestion unmasks certain epitopes/sites which have been masked during the formalin-fixation process; therefore, unmasking the antigens and epitopes in formalin-fixed and paraffin-embedded tissue sections will enhance staining intensity of antibodies. This method may cause tissue damage if the tissue sections are under-fixed. It is crucial to select the appropriate incubation time (5–20 minutes) and temperature (20–60 °C) for a specific application and try to avoid over-digestion of the tissue sections. Proteinase K is a very useful antigen unmasking solution for some cell membrane antigens.

• Procedure:

  • 1. Dewax the sections in two changes of Histoclear^® or xylene solution, 5 minutes each.

  • 2. Rehydrate in two changes of 100% and 95% ethanol for 30 seconds each and 70% ethanol for 30 seconds; then rinse in distilled water.

  • 3. Transfer the sections into proteinase K working solution and incubate for 5–15 minutes at room temperature in a humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation).

  • 4. If you use a Dako Autostainer, after performing peroxidase block (Dako, Catalog# K4007) or avidin/biotin-blocking processing, you can set up a step for proteinase K incubation before primary antibody incubation. Certainly, a polymer detecting system is a more favorable choice.

  • 5. Continue with an appropriate antibody staining protocol.

18. What are the recommended guidelines for 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 [19]. 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 [19].

• 1. Laboratories must validate all IHC tests before placing them into clinical service—Recommendation.

• 2. For initial validation of every assay used clinically, with the exception of HER2, 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.

• 3. For initial analytic validation of non-predictive 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 (e.g., rare antigen), the rationale for that decision needs to be documented—Expert Consensus Opinion.

• 4. 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.

• 5. For a marker with both predictive and non-predictive applications, laboratories should validate it as a predictive marker if it is used as such—Recommendation.

• 6. When possible, laboratories should use validation tissues that have been processed using the same fixative and processing methods as cases that will be tested clinically—Recommendation.

• 7. If IHC is regularly done on cytologic specimens that are not processed in the same manner as the tissues used for assay validation (e.g., alcohol-fixed cell blocks, air-dried smears, formalin post-fixed 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 non-predictive markers to test—Expert Consensus Opinion.

• 8. If IHC is regularly done 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 non-predictive markers to test—Expert Consensus Opinion.

• 9. Laboratories may use whole sections, TMAs, and/or multi-tissue 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 are similar to clinical specimens—Recommendation.

• 10. 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 one known positive case and one known negative case— Expert Consensus Opinion.

• 11. Laboratories should confirm assay performance with at least two known positive and two 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.

• 12. 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, antigen retrieval method (e.g., change in pH, different buffers, different heat platforms), antigen detection system, tissue processing or testing equipment, environmental conditions of testing (e.g., laboratory relocation), and laboratory water supply. The laboratory medical director is responsible for determining how many predictive and non-predictive markers and how many positive and negative tissues to test—Expert Consensus Opinion.

• 13. 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.

• 14. 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 50–100 tumors or normal tissues which were fixed and processed under similar or identical conditions as other routine patient samples. Two punched cores of 1.5 mm or 2.0 mm each were usually taken from each case. After antibody testing and optimizing on a small TMA block containing 5–10 cases of tumor/normal tissues, the antibody validation process was followed. For instance, for validation of napsin A monoclonal antibody, three TMA blocks were selected, including lung adenocarcinomas, papillary renal cell carcinomas (RCCs), and lung squamous cell carcinomas. The positive reference range for napsin A was expected to be 75–80% in lung adenocarcinomas, 50–60% in papillary RCCs, and close to zero in lung squamous cell carcinomas. If the validation data were within the reference range, napsin A was included in the antibody library and implemented in our IHC lab. If the validation data showed that 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 returned 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.

19. How do you select an automated staining platform?

Before you select an automated IHC staining platform, there are several questions you should ask. What are the advantages and disadvantages of automated versus manual IHC? What are the strengths and weaknesses of each automated platform, such as the Ventana Benchmark Ultra (Ventana Medical Systems, Tucson, Arizona), Leica Bond III (Leica Biosystems, Buffalo Grove, Illinois), Dako Omnis (Dako), 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 (LISs). The details are addressed in Chap. 3.

20. How do you interpret IHC assay results?

There is no universal scoring system for an IHC assay result or general agreement on 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 (CMV), 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 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 (MLH1, PMS2, MSH2, 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 (less than 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 border between a strong and a weak staining signal.

21. How do you report IHC assay results?

We report IHC assay results in a tabulated format as in Table 2.8. For example, the following table 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 and can be potentially used for future research projects and enable one to further understand the clinical significance of the extent and intensity of each IHC stain.

Table 2.8 Summary of IHC assay results on right lung biopsy

22. How do you improve a total IHC quality management program (quality assurance, quality control, and quality improvement)?

There are many excellent, comprehensive review articles and book chapters discussing the quality assurance, quality control, and quality improvement in the field of immunohistochemistry [2, 4, 5, 9]. 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 maintain 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 containing a set of quality parameters (see Table 2.9) goes with every stain. After the review, the acceptable slides or deficient slides are determined based on these criteria. 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 QC result and pathologist’s feedback for each stain are entered into the IHC database, and the results are collected and reviewed at our weekly laboratory technical specialist meeting. Each issue is analyzed and corrective action is 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 brings the issues back to IHC technologists to resolve. 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 take corrective action before releasing the slide(s) to an ordering pathologist.

Table 2.9 Quality parameters for the IHC quality control worksheet

• Continuous Quality Monitoring

Each new antibody goes through a vigorous optimizing and validating process before being implemented in our IHC lab. 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 lab begins to receive complaints from ordering pathologists about weak staining, background staining, or incorrect component staining of the antibody. How can we be more proactive than reactive to potential issues? With the growing list of antibodies in our IHC lab, 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 based on the highest test volumes, the clinical importance of these antibodies, and the target antigen absence from normal tissues, such as TTF1, p40, p63, ER, PR, HER2, GATA3, VHL, arginase-1, glypican-3, PAX8, WT1, CDX2, NKX3.1, NKX2.2, CD31, ERG, CD34, STAT6, desmin, SATB2, S100, HMB-45, MART-1, INSM1, chromogranin, synaptophysin, SALL4, OCT4, calretinin, inhibin-alpha, etc. A special TMA block containing tissues to test all the antibodies mentioned above was constructed. Multiple identical blocks were built. IHC tests were performed on the TMA slides for each antibody, and the slides were scanned and stored online. Monthly, 10–15 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 carried out.

23. What is the role of digital pathology in an IHC laboratory?

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 [20,21,22,23,24,25,26]. With continued improvement of workflow and integration with the LIS, 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 Ki67). 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 HER-2 IHC assay results on gastrointestinal and breast cancer, a fluorescence in situ hybridization (FISH) 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 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 the extent and intensity of the stain.

24. What are the available proficiency testing programs?

A CAP-certified IHC lab is required to participate in the CAP External Quality Assessment (EQA) and Proficiency Testing (PT) program. CAP offers a number of PT programs specifically designed for IHC laboratories. The newly developed program, CAP/National Society for Histotechnology (NSH) HistoQIP—IHC (program code HQIHC), is designed to improve the preparation of IHC slides in all laboratories handling GI, skin, and GU 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 CAP for evaluation. As feedback, participants receive a performance evaluation packet that includes detailed statistical data about the entire survey. The participant should be able to compare its performance with all participating labs. These programs include MK (General Immunohistochemistry), PM2 (ER/PR), PM3 (CD20), PM1 (CD117), mismatch repair proteins (MMR), 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 PT testing requirement of the ASCO/CAP guideline for ER, PR, and HER2 assessment in breast carcinoma. Geisinger also participates in CD30, PD-L1, p16, and BRAF V600E CAP PT testing program. The PM5 program is uniquely designed to evaluate a number of commonly used IHC assays, including chromogranin, cyclin D1, CDX2, CD30, D2-40, CK20, Ki67, PAX2, PAX8, and p63. Nevertheless, for the vast majority of over 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.org) [16] and United Kingdom National External Quality Assessment Service (UK NEQAS, Sheffield, England, http://www.ukneqas.org.uk) [27] are two of the most reputable ones.

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 website contains useful information about IHC, including free recommended staining protocols for different antibodies on different platforms. Vyberg and Nielsen published the results of a large study of proficiency testing in immunohistochemistry [28]. In their study, more than 30,000 IHC slides evaluated at 700 laboratories from 80 countries during 2003–2015 were collected. Overall, about 20% of the IHC staining results in the breast cancer IHC module and about 30% in the general module have been assessed as insufficient for diagnostic use. About 90% of the insufficient stains were characterized by a too weak or even false-negative staining reaction in one or more cores, while the remaining were insufficient due to poor signal-to-noise ratio, false-positive, or combined false-negative and false-positive results. The most common causes for insufficient IHC staining results are summarized in Table 2.10, which is obtained from this article.

Table 2.10 Major causes of insufficient staining reactions

25. What are the required qualifications for IHC personnel?

Minimum standards for the qualifications for 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 the ability to determine what slides are acceptable and what slides should be rejected before releasing them to the ordering pathologist; this 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.

26. What is the CAP checklist for clinical IHC laboratories?

Table 2.11 summarizes the 2019 (09.17.2019) CAP checklist for immunohistochemical laboratories [13]. This checklist is subject to changes in the next updated CAP checklist for anatomic pathology.

Table 2.11 Summary of the revised (9/17/2019) College of American Pathologists (CAP) checklist for immunohistochemistry

27. How do you implement best practices in immunohistochemistry?

The majority of large IHC laboratories have an extensive test menu with over 200 antibodies. 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. Due to the large number of antibodies available in a given IHC lab, both underutilization and overutilization of IHC markers have gradually become issues. Here are a few tips which may potentially help an IHC lab implement the concept of best practices in immunohistochemistry. However, most importantly, before ordering any IHC stain, a pathologist should ask what value the stain(s) will add to the final diagnosis and patient care. If the answer is “none at all” or “I don’t know,” the stain should not be performed. Additionally, if either three stains or five stains will provide the same information, only order three stains. With this in mind, the concept of best practices in immunohistochemistry can be effectively implemented.

• 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 overutilize or underutilize it. 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 Chap. 12.

• Begin with a Limited Panel of IHC Markers (First IHC Panel)

A single IHC marker approach (other than for pathogens such as CMV 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 statistically nearly impossible. A small IHC panel is recommended. For example, TTF1 and p40 can be applied for distinction of lung adenocarcinomas from squamous cell carcinomas.

Table 2.12 Lung adenocarcinoma versus lung squamous cell carcinoma (first IHC panel)

• Continue with Second IHC Panels

If the first IHC panel for the specific differential diagnosis is inconclusive, the follow-up second IHC panel can be considered. For the above example, if both TTF1 and p40 fail to yield the expected result, additional markers such as napsin A, CK7, p63, and CK5/6 can be considered. If the second IHC panel fails to lead to a conclusion, reconsider broadening the differential diagnosis if that clinically makes sense.

• 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 with more sensitive and specific markers. The less sensitive and specific markers should be deleted from the diagnostic panels. For example, both SALL4 and OCT4 have been proven to be excellent markers to identify germ cell tumor; therefore, the less specific germ cell markers such as PLAP should be removed from the IHC library to reduce an unnecessary cost.

• Track and Compare the IHC Utilization Data

The utilization of immunohistochemical stains should be audited periodically for each subspecialty group and each pathologist. Comparing and contrasting IHC utilization among pathologists within the same subspecialty group should be performed, using the group average or median as the benchmark. For instance, within a group of six GU 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/tissue core) for each pathologist? The pathologists found to excessively use IHC tests should correct the overutilization issue using the group average as a reference. If national benchmarking data are available, they can be used as a reference for the specialty group. The another reason for overutilization is IHC markers being ordered on both FNA and surgical specimens from the same biopsy procedure.