Immunohistochemistry in the Central Nervous System Protocol

Overview

Introduction of IHC in the Central Nervous System Protocol

Immunohistochemistry (IHC) is a critical ancillary test that aids in the accurate diagnosis of the central nervous system neoplastic and nonneoplastic conditions, this method localize and demonstrate specific antigens of interest in tissues or cells based on antigen antibody interactions visualizing it with the aid of a chromogen and light microscopy.

The use of IHC has been a routine and indispensable facet of modern diagnostic cancer pathology practice. Newer IHC biomarkers are being continuously found as indispensable aids in differential pathologic diagnosis, prognosis, or prediction of response to molecular-targeted therapies.

The workflow of IHC begins with the preanalytic phase (i.e., cold ischemia) followed by analytic phase (e.g., antigen retrieval) and postanalytic phase (microscopic interpretation), with a strict quality control to reach optimal results.

This chapter focuses on the materials, methods, and techniques used in immunohistochemistry and the principles of quality control and validation.

Materials of IHC in the Central Nervous System Protocol

Formalin, 10% neutral buffered.
Charged/PLUS slides.
Electrical stove.
Xylene.
100% ethanol.
96% ethanol.
Distilled water.
Pressure cooker.
Target retrieval solution (either citrate pH 6.2 or EDTA pH 8.5 or Tris pH 9.5).
Tap water.
Hydrogen peroxide reagent.
Tris-buffered saline (TBS) and/or phosphate-buffered saline (PBS).
Antibodies: primary and secondary.
30-diaminobenzidene tetrahydrochloride (DAB).
Harris hematoxylin.
Lithium carbonate or diluted ammonium hydroxide.
Automated immunohistochemistry stainer.

Methods of IHC in the Central Nervous System Protocol

Preanalytic Phase

Fixation and Cold Ischemia

The most widely used fixative is 10% neutral buffered formalin (NBF). This fixative is an aldehyde fixative that is categorized as a "cross-linking" fixative. This fixative has a good preservation of morphology, of antigens and for small molecules such as hormones.

The time of fixation (6–72 h), the volume (10 times the specimen) and the cold ischemic time (less than 1 h) are critical for an optimal staining and to avoid false-positive or false-negative results. See Notes 1 and 2.

Microtomy and Slide Preparation

Tissue sections should be cut at 3–4 μm using charged/PLUS slides. This contributes to good adhesion of the tissue to the slide and accurate postanalytic interpretation. Thicker sections, folds and tears can cause false-positive or false-negative staining results.

Dewaxing and Hydrating

Put the section slides in the electrical stove for 5–20 min at 66–70°C. This process allows adequate adhesion and dewaxing.
Give the sections three changes of xylene 5 min each.
Give the sections two changes of each of the solutions: xylene, 100% ethanol and xylene (50/50), ethanol 100% and ethanol 96%. At the end rinse slides with distilled water to remove alcohol completely.

For automated processes it is not strictly necessary any of the changes in xylene and ethanol for dewaxing. Only follow step 1 and insert the section slides into the automated stainer and run a specific protocol.

Analytic Phase

Epitope Retrieval

Epitope retrieval is referred to unmasking the epitope. Options for epitope unmasking include heat-induced epitope retrieval (HIER) and proteolytic-induced epitope retrieval (PIER)/enzymatic retrieval. PIER was the first method developed to address the antigen-masking effects of formalin fixation, the enzymes used in this technique are protease, proteinase K, trypsin. These methods sometimes produce false-positive or false negative results.

HIER methods have greatly improved the quality and reproducibility of IHC staining on the application of heat to formalinfixed paraffin-embedded (FFEP) section, using retrieval solution of varying pH levels in order to break cross-linkages and expose epitopes. The pH is critical and the heat sources include microwave oven, pressure cooker and water bath or automated IHC staining instrument. Regardless of the method used, do not let the slides dry out.

We use the HIER method with a pressure cooker for manual process with the following steps:

Plug in the pressure cooker and place the pan inside.
Fill the pan of the pressure cooker with distilled water. See Note 3.
Place the slides into the container filled with target retrieval solution (either citrate pH 6.2 or EDTA pH 8.5 or Tris pH 9.5). The slides should be covered completely by the solution.
Place the container with slides into the center pan then place the heat shield in the center pan. Put the lid on and secure.
Check the displayed parameter and for brain tissue some pressure cookers recommend setting the temperature at 60°C for two hours, and others recommend setting it at 90°C. See Note 4.
Set the heating time between 30 s to 5 min, depending on the antigen you are using.
Before opening the cooker the pressure should read 0.
Open the lid and let the slides cool for several minutes.
Slowly rinse the slides in running tap water.
Transfer slides to rinse buffer. See Note 5.

Blocking Nonspecific Binding

Background staining is a frequent problem in IHC and is a cause of nonspecific binding related to endogenous biotin, endogenous enzyme activity, and hydrophobic interactions. If the nonspecific binding is not blocked it could lead to false-positive results. Endogenous avidin binding has been observed in all biotin-based staining methods. Endogenous biotin is bound to enzymes and protein is frequently seen in brain tissue. Blocking is achieved by applying unlabeled avidin followed by an application of unlabeled biotin. If enzymes similar to that of the antibody label are present in the tissue sample being stained, they may react with the substrate and cause background staining.

Hydrophobic interactions result from cross-linking of amino acids and proteins during formalin fixation. Tissues that exhibit background staining due to hydrophobic interactions include collagen, other connective tissues, epithelium, and adipose tissue.

Blocking Method

Put the sections in the hydrogen peroxide reagent for 10 min so that the endogenous biotin is blocked. See Note 6.
Rinse in distilled water.
Rinse in a phosphate buffer (PBS) for 5 min.

Antibodies

Primary Antibodies

Polyclonal antibodies: A combination of several high-affinity antibody clones directed against many different epitopes of the target antigen, thereby increasing sensitivity, these immunoglobulin molecules are obtained from different B cells. This is why these types of antibodies react with different epitopes on the target antigen and can cause significant background signal or nonspecific staining. They are less expensive to produce and are susceptible to lot-to-lot variation.
Monoclonal antibodies (mouse/rabbit): Monoclonal antibodies have become widely used because of their high specificity, consistency, purity, commercial availability, and minimal lot-to-lot variation. They are obtained from a single cell line of an individual clone of plasma cells, so they have a single specificity and single affinity; therefore, nonspecific staining is not an issue during assay optimization. The chief disadvantage of using monoclonal antibodies is some loss in sensitivity and that they are expensive to produce.

Some antibodies need to be diluted, the optimal dilution is when the concentration of the antibody produces an adequate staining with minimal background staining and minimal nonspecific binding. The antibodies are diluted with proteinaceous buffered solution known as a diluent.

Other antibodies are "ready to use" (RTU). The benefits of RTU antibodies are their relative lot-to-lot consistency, reproducibility, and exclusion of a possible source of laboratory error during antibody titration.

On the following tables we exemplify some of the antibodies used for glial tumors and medulloblastoma as well as what immunoreaction we expect to find on each tumor.

Method

Incubate sections with the primary antibody for 30 min. The duration and temperature vary depending on the antibody (see Note 7).
Rinse with phosphate buffer (PBS) for 5 min.

Secondary Antibody/Detection System

The secondary antibody is used in the indirect method, which is a multistep method with specificity against the unlabeled primary antibody to amplify and visualize the antibody-antigen reaction. This amplification is possible because multiple secondary antibodies bind to a single primary antibody. The sensitivity of detection of target antigens increases with the addition of secondary antibodies.

The direct method is a one-step staining method, in which the primary antibody is conjugated directly to the label. This method lacks sensitivity and that is why it is not widely used.

Labeled streptavidin-biotin (LSAB) method: The LSAB method uses streptavidin, a protein purified from the bacterium Streptomyces avidinii, that binds to the biotin. Streptavidin has a high capability in binding biotin and does not bind to tissue lectins that cause nonspecific staining. Use of streptavidin results in increased sensitivity and thus allows for a higher dilution of primary antibody. However, avidin–biotin-based detection systems are rapidly becoming obsolete because of the issue with nonspecific, false-positive staining caused by interactions with endogenous tissue biotin.
Polymer-based immunohistochemistry: These methods employ a dextran polymer chain that provides additional binding sites and ultra-sensitivity. Since these methods are biotin-free, there is no reaction with endogenous biotin. In addition to increased sensitivity, polymer-based IHC staining methods are shorter in the number of steps when compared to LSAB staining methods.

Method

Incubate sections with the secondary antibody for 20 min. The time of incubation varies depending on the detection system used.
Rinse with phosphate buffer (PBS) for 5 min.

Chromogen and Substrates

The detection of antigens using an antibody requires a chromogen to visualize the target antigen. The incubation of slides with a chromogen substrate (color-producing system) results in a stable, colored end-product suitable for visualization with light microscopy. Staining in tissue sections results from the reaction of a soluble substrate with an enzyme to produce an insoluble, colored product. The intensity of the color produced when the substrate is added correlates with the concentration of the primary antibody and respective tissue antigen.

The most common enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). The associated chromogens for HRP are 3,30-diaminobenzidene tetrahydrochloride (DAB) and 3-amino-9-ethylcarbazole (AEC).

Method

Incubate sections in the DAB solution for 1–2 min, or until you identify by light microscopy cellular staining. See Note 8.
Rinse the excess with distilled water.

Counterstaining

The counterstaining is a process that stains the section that did not participate in the histochemical reaction. It is critical to use a counterstain that is compatible with the color produced by chromogen. The most frequent stain used is hematoxylin as a counterstain for a histochemical reaction producing a brown or red color.

Method

Counterstain with Harris hematoxylin for 1 min.
Rinse with tap water.
Turn with lithium carbonate or diluted ammonium hydroxide.
Rinse with tap water.
Dehydrate using 96% ethanol (two changes, 3–5 min), 100% ethanol (two changes, 3–5 min), and xylene (three changes, 3–5 min).
Mount in synthetic mounting medium.

Controls

There are two specific types of controls that must be accounted for in each IHC run: positive controls to confirm the analytic sensitivity of the assay (that the assay detected the antigen) and negative "tissue" controls to confirm the analytic specificity of the assay (that the assay is negative in the absence of antigen).

Positive control tissue must contain the target antigen and should be fixed, processed, and embedded in the same manner as patient samples being tested. Also should reflect the intended use of the antibody in diagnostic practice. It is preferable to use a tissue with low levels of antigen staining to confirm adequate analytic sensitivity of the assay, as loss of a significant degree analytic sensitivity may not be detected using tissue with intense immunoreactivity, thus leading to false-negative interpretations of patient tissue samples. It is optimal to have the control sections mounted on each slide of patients' tissue to confirm that the IHC assay worked on that particular slide. See Note 9.

Negative tissue controls are those tissues known to be negative for the antigen of interest and processed in the same manner as test samples are used. Negative tissue controls assess the specificity of the primary antibody by verifying the absence of cross-reactivity. One control block may contain positive and negative tissue for the assay and it is possible to use in the same slide.

Figure 1. Schematic representation of the steps in the different phases of immunohistochemistry for the Central Nervous System.

Postanalytic Phase, Quality Control, and Principles of Validation

Interpretation

The interpretation of the staining depends on the antibody used; it is very important to define the cell localization, the intensity, and sometimes the percentage of positive tumor cells.

The staining in normal tissue elements, if present within the specimen, should be considered as an additional check on the IHC assay performance.

Quality Management

Quality management involves quality control (QC) and quality assurance (QA) programs, as well as quality improvement (QI) initiatives.

QC consists in optimizing each aspect of the IHC, from specimen arrival to reporting the results, and includes the validation of the IHC assay.

QA programs establish the policies and procedures for daily QC and external proficiency testing, in addition to monitoring other quality metrics during preanalytic, analytic, and postanalytic phases.

QI is the practice of continuously assessing and adjusting performance using statistically and scientifically accepted procedures.

Principles of Analytic Validation

The goal of the analytic validation is to define the reproducibility, reliability, and accuracy of the antibodies to detect the molecule or antigen in the tissue before placing it into clinical service.

The basic principles or recommendations for analytic validation are:

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.
For 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, the rationale for that decision needs to be documented. It is important to define the sensibility and specificity of the assay and is recommendable use tissue with high and low levels of staining.
For 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.
Laboratories should use validation tissues that have been processed by using the same fixative and processing methods as cases that will be tested clinically.
If IHC is regularly done on cytologic or decalcified tissue, 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.
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.
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).
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; Antigen detection system; Tissue processing or testing equipment; Environmental conditions of testing; 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.
Laboratories should run a full revalidation (equivalent to initial analytic validation) when the antibody clone is changed for an existing validated assay.
The laboratory must document all validations and verifications in compliance with regulatory and accreditation requirements.

Notes of IHC in the Central Nervous System Protocol

Cold ischemic time, volume and the type of fixative used are very important variables for a good staining. For example, refrigeration delays, but will not prevent proteolysis that can result in false-negative stains due to antigen degradation or high background staining to nonspecific binding. The deleterious effects of delayed fixation depend on the marker, it is adversely in membranous antigens.
Consider not interpretable cases when you have unexpected results and if you do not know the preanalytic phase. Sometimes it is possible to use vimentin to evaluate the quality of the tissue.
Is important to read the specifications of your pressure cooker, some use 500 ml of distilled water while other only need 100 ml. The important part is that the water is touching the container on the bottom.
The temperature of the pressure cookers runs between 60°C and 110°C. This depends on the tissue sections that you are using. We are using central nervous system sections that is why we need the temperature at 60°C during 2 h.
Some antibodies do not need epitope retrieval (e.g., S-100 protein).
Prepare the solution of hydrogen peroxide using 100 ml of hydrogen peroxide with 90 ml of distilled water and 10 ml of 3% hydrogen peroxide.
The incubation must be carried out in a moist setting (humidity chamber). Incubation duration and temperature (e.g., 37°C, 4°C) can be modified as necessary. Depending on the antibody we are going to use. Antibody concentrations are inversely proportionate as the higher the antibody concentration, the shorter the incubation period. Ten to thirty minutes is the most widely used primary antibody incubation time. The temperature of incubation periods is critical and can affect incubation time. Higher incubation temperatures allow for shorter incubation periods. Incubation temperature of 4°C is often used with overnight incubations, while automated IHC stainers provide for consistent incubation temperature of 37°C.
To prepare the DAB solution add one drop of the DAB in 1 ml of buffer substrate.
Control sections cut previously and stored at ambient conditions for several days may lose antigenicity. Some protocols for specific antibodies have recommendations of storing the slides.

Reference