Immunohistochemistry Notes

Immunohistochemistry

Immunohistochemistry (IHC) techniques are used to identify specific cellular epitopes or antigens in frozen or paraffin-embedded tissues and organisms in cytologic preparations.

Basic Principles

IHC makes use of antigen-antibody interactions.
The site of antigen binding is demonstrated by direct labeling of the antibody or by a secondary labeling method.
Antibodies belong to the class of serum proteins known as immunoglobulins.
IgG is the most commonly used antibody for immunocytochemistry.
An epitope is the structural part of the antigen that reacts with an antibody.
IHC combines anatomical, immunological, and biochemical techniques to identify discrete tissue components.
It allows the visualization of the distribution and localization of specific cellular components within cells and in the proper tissue context.
The steps involved are separated into two groups: sample preparation and labeling.

Applications of IHC

IHC is used for:

Disease diagnosis
Drug development
Biological research

Disease Diagnosis

Using specific tumor markers, physicians use IHC to:
- Diagnose a cancer as benign or malignant.
- Determine the stage and grade of a tumor.
- Identify the cell type and origin of a metastasis to find the site of the primary tumor.

Drug Development

IHC is used to test drug efficacy by detecting either the activity or the up- or down-regulation of disease targets.
Immunofluorescence is often performed on frozen tissue due to the high background auto-fluorescence seen in formalin-fixed paraffin-embedded tissue.
Samples are prepared on individual slides, or multiple samples can be arranged on a single slide for comparative analysis, such as with tissue microarrays.
IHC slides can be processed and stained manually, while technological advances now provide automation for high-throughput sample preparation and staining.
Samples can be viewed by either light or fluorescence microscopy.
Advances have improved the ability to capture images, quantitate multi-parametric IHC data, and increase the collection of that data through high content screening.

Polyclonal Antibodies

Produced by immunizing an animal with a purified specific molecule (immunogen) that contains the antigen of interest.
Immunoglobulin-rich serum is collected after the animal has produced humoral antibody against the antigen.
The most frequently used animal for the production of polyclonal antibodies is the rabbit, followed by goat, pig, sheep, horse, guinea pig, and others.
Because polyclonal antibodies are produced by different cells of the animal, they are immunochemically not identical to each other.
They react with various epitopes on the antigen against which they are raised.
Some of the polyclonal antibodies may cross-react with other molecules and cause non-specific staining, requiring their purification by absorption with the appropriate antigen or antibody dilution to eliminate the unwanted reaction.

Monoclonal Antibodies

Animals immunized with the specific immunogen will produce numerous clones of plasma cells (polyclonal) that in turn will produce the antibody.
Monoclonal antibodies are the products of an individual clone of plasma cells.
Hybridoma and cloning techniques have been developed to produce monoclonal antibodies that do not cross-react with other molecules.
Antibodies from a given clone are immunochemically identical and react with a specific epitope on the antigen against which they are raised.
Mice are currently used almost exclusively for the production of monoclonal antibodies.
Propagation can be carried out in culture medium or by transplantation of the hybridoma into the peritoneal cavity of syngeneic mice from where the antibodies are harvested.
This has dramatically increased the quantities and number of specific monoclonal antibodies available for immunohistochemistry.

Preparing Tissue for Immunohistochemistry

In certain instances, the tissue must be prepared as a cryostat section and fixed for a few seconds in absolute methanol or acetone to preserve immunological activity and prevent destruction of some of the labile antigenic sites.
However, immunofluorescence and immuno-peroxidase techniques may also be done on formaldehyde-fixed and paraffin-embedded sections.
Many masked antigens can now be retrieved in routinely processed tissue by:
- Proteolytic enzyme digestion
- Microwave antigen retrieval
- Microwave and trypsin antigen retrieval
- Pressure cooker antigen retrieval

Proteolytic Enzyme Digestion

Formalin-fixed paraffin sections are usually pre-treated with proteolytic enzymes to break down formalin cross-linking, unmask and allow certain antigenic sites to be exposed.
Proteolytic enzyme digestion is especially useful for demonstrating heavy chain immunoglobulins, complement, and specific antigens (such as cytokeratin) in formalin-fixed paraffin-embedded biopsies.
The most common enzymes used are trypsin and protease.
Before pretreatments are employed, the sections are deparaffinized, taken to alcohol and, in the case of peroxidase labeling, treated with 0.5% methanol in hydrogen peroxide for 10 to 15 minutes to destroy endogenous peroxidase activity.
The slides are then washed in running water and taken to distilled water.

Trypsin Method

Uses 0.1% trypsin in 0.1% calcium chloride in distilled water, adjusted to pH 7.8 with sodium hydroxide, and preheated at 37°C.
The slides are also preheated at 37°C in distilled water before placing in freshly prepared trypsin solution.
After a predetermined period of time, the slide is transferred to cold running water to terminate enzyme digestion.

Protease Method

Uses 0.05 to 0.1% protease in distilled water, adjusted to pH 7.8 with sodium hydroxide.
The section is preheated at 37°C in distilled water and placed in protease solution for a shorter period of time due to its faster rate of enzyme digestion.

Paraffin Sections Procedure

Deparaffinize sections in xylene 2 times for 5 minutes each time.
Hydrate with 100% ethanol 2 times for 3 minutes each time.
Hydrate with 95% ethanol for 1 minute.
Rinse in distilled water.
Follow procedure for pretreatment as required.

Pre-treatment of Tissue Sections

Antigenic determinants masked by formalin-fixation and paraffin-embedding often may be exposed by epitope unmasking, enzymatic digestion, or saponin, etc.
Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded.

Procedure:

Rinse sections in PBS-Tween 2 times for 2 minutes each time.
Serum Blocking: Incubate sections with normal serum block – species same as secondary antibody, for 30 minutes to block non-specific binding of immunoglobulin. Note: This protocol uses avidin-biotin detection system. Avidin- biotin block may be needed based on tissue type. Normal serum block should be used prior to avidin-biotin block.
Primary Antibody: Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
Rinse in PBS-Tween 20.
Peroxidase Blocking: Incubate sections in peroxidase blocking solution for 10 minutes at room temperature.
Rinse in PBS-Tween 20.
Secondary Antibody: Incubate sections with biotinylated secondary antibody at appropriate dilution in PBS for 30 minutes at room temperature.
Rinse in PBS-Tween 20 3 times for 2 minutes each time.
Detection: Incubate sections in streptavidin-HRP in PBS for 30 minutes at room temperature.
Rinse in TBS 3 times for 2 minutes each time.
Chromogen/Substrate: Incubate sections in DAB solution for 1-3 minutes.
Rinse in PBS-Tween 20 2 times for 2 minutes each time.
Counterstain if desired.
Rinse in distilled water.
Dehydrate through 95% ethanol for 2 minutes, then 100% ethanol for 2 times 3 minutes each time.
Clear in xylene.
Coverslip with mounting medium.

Heat-Induced Epitope Retrieval (HIER)

HIER is a pretreatment method used to improve staining results.
Heat, coupled with specific buffered solutions, is utilized to recover antigen reactivity in formalin-fixed paraffin-embedded tissue.
It reverses the formaldehyde-mediated chemical modifications of the antigen through either of the following processes:
- Thermal energy breaks the crosslinks that bind surrounding proteins or peptides to the antigen which lead to the “opening” or “unmasking” of the epitope.
- Thermal energy removes bound calcium ions from the sites of cross-links since several HIER buffers, such as EDTA and citrate, act as calcium chelators.
HIER heating sources include the microwave, vegetable steamer, pressure cooker, and water bath.
In general, the higher the temperature of the HIER solutions, the more effective the recovery of the epitope is.
While each of these heating sources is suitable for HIER, there are advantages as well as drawbacks associated with each source.
- For example, often, the microwave distribution of heat within a microwave is uneven or inconsistent which results in a lack of reproducibility as to staining intensities.
- In contrast, the pressure cooker, steamer, and water bath produce uniform and consistent heat distribution.
- However, while the higher temperatures produced by the pressure cooker are advantageous since, in a short period of time, an effective recovery of epitope reactivity can be readily achieved, damage or distortion to the morphology of connective tissues can also occur.

Reagents Required for Heat-induced Epitope Retrieval:

10 x Antigen Retrieval Solution:
- Antigen Retrieval Reagent-Basic
- Antigen Retrieval Reagent-Acidic
- Antigen Retrieval Reagent-Universal
Deionized H2O
1 x PBS: 0.137 M NaCl, 0.05 M $NaH<em>2PO</em>4$ , pH 7.4

Equipment:

Polypropylene Coplin staining jar (or equivalent)
Water bath at 92-95°C

Procedure:

Make working dilutions by mixing 1 part of 10X Antigen Retrieval concentrate with 9 parts of deionized water.
Preheat retrieval solution to 92-95°C. This may be achieved by placing a polypropylene Coplin staining jar filled with retrieval solution into a water bath. Note: Heating may cause cracking of glass staining dishes.
Immerse slides into preheated retrieval solution for 2-10 minutes. Note: Since the effect of antigen retrieval reagents depends on their temperature (90-100 °C) and incubation time (up to 30 minutes), optimal conditions should be determined by the individual investigator. Cryostat sections are more sensitive to damage by retrieval solution than paraffin-embedded tissues. To avoid tissue damage, it may be necessary to shorten the incubation time to 2-5 minutes.
After the incubation is finished, remove the Coplin jar with retrieval solution and slides from the water bath, and let it cool to room temperature.
Gently rinse the slides with deionized water and then with PBS. Note: Because tissues may be loosened after the retrieval procedure, avoid vigorous rinsing to prevent detachment from the slides.

Microwave Antigen Retrieval

Microwave antigen retrieval is a relatively new technique that involves the boiling of formalin-fixed deparaffinized sections in certain solutions, such as 0.01 M-citrate buffer (pH 6.0), EDTA at pH 8.0 or Tris EDTA (pH or 10.0).
Many antigens thought previously to be either lost or destroyed by routine histological processing techniques can be retrieved by microwave oven heating.
Antibodies such as the proliferation markers (Ki-67 and MIB-1), hormone receptors (ER and PR), growth factor receptors (HER-2/neu) and others which were previously thought to be applicable only to frozen sections, are demonstrated well on paraffin sections after heat pre-treatment.
Most antigen retrieval methods apply temperatures near the boiling point of water.
The optimal length of exposure to heat may vary from 10 to 60 minutes and depends to some extent on the length of formalin fixation.
The most satisfactory time period appears to be 20 minutes for most antigens and fixation protocols.
Care should be taken not to allow the sections to dry after heating, as this destroys antigenicity.
Boiling of poorly fixed material often damages nuclear details.
Fibrous and fatty tissues tend to detach from the slide.
This can be prevented by mounting the sections on slides with a strong adhesive (such as Vectabond), or dipping Vectabond-coated slides in I0% formol saline for 1 to 2 minutes and air drying before picking up sections.
Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations.
On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr. of fixation time.
On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr. in the formalin-fixed tissue.
Bouin's and B-5 tissues give consistent results only after 6 hr. of fixation.
The choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.

Pressure Cooking Antigen Retrieval

Pressure cooking antigen retrieval is another alternative that appears to be less time-consuming and allows for more consistent recovery of many antigens, compared to the large batch microwave oven technique.
In the large batch microwave oven technique, heating temperature is not uniformly distributed and slides are subjected to "hot spots" and "cold spots" resulting in inconsistent antigen recovery.

Antigens

Primary antibodies against numerous antigens are now available in the market and are widely used for diagnosis of tumors, determination of tumor type, the evaluation of proliferation potential, identification of infectious agents, prognostic and therapeutic implications, and many other aspects of diagnostic pathology.

Epithelial tumor markers:

Keratin is a highly sensitive marker for epithelial cells and is present in epithelial tumors (carcinoma). Certain non-epithelial tumors (such as mesotheliomas and non-seminomatous germ cell tumors) also stain positive for keratin and may be distinguished from carcinoma by applying an additional panel of antibodies.
a) CK7 (Cytokeratin 7) is more frequently found in carcinomas of the lung, breast, uterus, and ovaries (serous tumors). These tumors are typically negative for CK20.
b) CK20 (Cytokeratin 20) is more common in carcinomas of the colon and stomach. These tumors are usually negative for CK7.
c) Transitional cell carcinomas of the bladder and mucinous ovarian tumors are usually positive for both CK7 and CK20.
d) Renal cell carcinomas, hepatocellular carcinomas, prostatic adenocarcinomas, thyroid carcinomas, and squamous cell carcinomas (skin, lung and esophagus) are usually negative for either CK7 or CK20.
EMA (Epithelial membrane antigen) is a high molecular weight protein that is helpful in determining the site of tumor. It is positive for adenocarcinomas of the breast, lung, and kidneys but more often nonreactive for hepatocellular carcinomas, adrenal carcinomas, or embryonal carcinomas, and negative for non-epithelial tumors (sarcomas, lymphomas, melanomas) and other tumors (meningiomas, mesotheliomas, anaplastic large cell lymphomas, and plasma cell tumors).
CEA (Carcinoembryonic antigen) is an oncofetal antigen that is present in carcinomas of the gastrointestinal tract, pancreas, lung, breast, ovary, uterus, and cervix. It is especially useful for differentiating between adenocarcinoma (CEA-positive) and mesothelioma (CEA-negative). Prostate, thyroid, and renal carcinomas are usually non-reactive to CEA.
TTF-1 (Thyroid transcription factor-1) is useful in distinguishing lung adenocarcinomas from mesotheliomas. It is positive in thyroid, lung, and neuroendocrine tumors (medullary thyroid carcinomas, carcinoid tumors and small cell tumors of the lung).
PSA (Prostate specific antigen) is extremely useful in the diagnosis of prostatic adenocarcinoma. It is also positive in certain pancreatic and salivary gland tumors.

Intermediate Filament Markers

Actin is a contractile intermediate filament protein present in muscle and some non-muscle tissue. It is a sensitive marker for muscle differentiation and can be used to identify tumors derived from smooth, skeletal, and cardiac muscle.
Vimentin is a 57kD intermediate filament that is present in normal mesenchymal cells and their neoplastic counterparts (i.e., sarcoma, melanoma, lymphoma, leukemia, seminoma, and some neural tumors). Melanomas and schwannomas always stain positive for vimentin, so that a negative staining may be used to exclude the diagnosis. It is almost always present in tissue sections because of the background stromal elements, and has limited use as a stand-alone stain, but it can be very helpful when combined with other specific tumor markers.
Desmin is a 53 kD intermediate filament expressed by smooth and striated (skeletal and cardiac) muscle. It is considered to be highly specific for myogenic tumors, including leiomyoma (smooth muscle tumor) and rhabdomyosarcoma (skeletal muscle tumor). It is also used to demonstrate the myogenic component of mixed tumors (i.e., carcinosarcomas or malignant mixed mesodermal tumors).
Glial fibrillary acidic protein (GFAP) is a 51 kD intermediate filament protein expressed by central nervous system glial cells, particularly astrocytes. It is most widely used to confirm the diagnosis of astrocytoma (but may also be present in certain cases of ependymomas, oligodendrogliomas and medulloblastomas). Non-CNS tumors (meningiomas, metastatic carcinomas and lymphomas) stain negative for GFAP.
Neurofilament (NF) is expressed in cells of neural origin, particularly neurons, neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla and neuroendocrine cells. Tumors that show neuronal or neuroendocrine differentiation (e.g., neuroblastomas, ganglioneuromas, neuromas, chemodectomas, and pheochromocytomas) will stain positive for neurofilament.
S-100 protein is a low molecular weight calcium-binding protein that is expressed in CNS glial cells, Schwann cells, melanocytes, histiocytes, chondrocytes, skeletal and cardiac muscle, myoepithelial cells and some epithelial cells of breast, salivary and sweat gland epithelium.

Neuroendocrine markers:

Neuron-specific enolase (NSE) is an isoenzyme marker whose presence in tissue provides strong evidence of neural or neuroendocrine differentiation.
Chromogranin is found in the neural secretory granules of endocrine tissues and is recognized as a marker for neuroendocrine differentiation. Immunoreactivity is typically granular and its distribution is similar to that seen with silver staining methods such as Grimelius stain. A combination of keratin and chromogranin positivity is typical of neuroendocrine carcinoma. Chromogranin positivity with a negative keratin stain is typical of paraganglioma.
Synaptophysin is a 38 kD transmembrane protein associated with presynaptic vesicles of neurons. It has been identified in normal neurons and neuroendocrine cells.

Germ cell tumor markers

Non-seminomatous germ cell tumors (i.e. embryonal carcinomas, teratomas, choriocarcinomas, and endodermal sinus or yolk sac tumors) generally stain positive for epithelial markers (keratin). For more specific classification, the following germ cell tumor markers are used:

HCG (Human chorionic gonadotropin) is synthesized by placental syncytiotrophoblasts and is a marker for choriocarcinoma.
AFP (Alpha-fetoprotein) is synthesized by normal liver hepatocytes, and is used as a marker for endodermal sinus tumors showing yolk sac differentiation. Embryonal carcinomas and teratomas containing these elements, as well as hepatocellular carcinomas will also stain positive for AFP.
PLAP (Placenta-like alkaline phosphatase) is produced by the placental syncytiotrophoblasts in late pregnancy and is used as a marker for germ cell tumors, particularly germinomas. Most embryonal carcinomas, choriocarcinomas and endo-dermal sinus tumors will also stain positive for this antibody. PLAP is positive in the majority of seminomas.

Mesenchymal tumor markers

Myogenic tumors - Tumors of skeletal muscle origin are positive for muscle-specific actin and desmin and/or other muscle markers such as myo-D1, myoglobin and myogenin.
Fibrohistiocytic tumors - The use of histiocytic markers such as CD68, or FAM 56, combined with more nonspecific proteolytic enzymes such as alpha-1-antitrypsin and alpha-1-antichymotrypsin may be helpful in the diagnosis of malignant fibrohistiocytic sarcomas. An undifferentiated component of sarcoma may react only with vimentin.
Vascular tumors - Endothelial markers for vascular tumors (such as angiosarcomas) include Factor VII-related antigen, CD31 and Ulex Europaeus 1 (UEA).
Melanomas - Melanocytes are derived from neural crest and will be reactive for S100 protein. The intensity of staining for S100 is usually inversely proportional to the melanin content of the tumor. Melanosome (HMB-45) is a widely used, highly sensitive and highly specific marker for the diagnosis of melanoma. Melan-A (MART-1) also encodes a melanoma-specific antigen that is present in normal pigmented cells of skin and retina as well as in certain adrenocortical tumors.
Lymphomas - The best screening marker for lymphoma is LCA (leukocyte common antigen), also known as CD45. For immunophenotypic subclassification of lymphoma, the most common markers used include those for T cells (CD3, CD4, CDS), B cells (CD19, CD20, CD23), Reed-Sternberg cells (CD15, CD30), and immunoglobulin light and heavy chains.

Cell Proliferation Markers

Ki-67 (MIB-1) and PCNA (proliferating cell nuclear antigen) are the most common immunohistochemical markers used to assess the proliferation of tumor cells.
Increased expression of these antigens is usually associated with greater aggressiveness and a higher likelihood of recurrence of metastasis.

Cancer-associated genes

The development and progression of a malignant phenotype of human tumors is related to abnormalities of structure or activity of proto-oncogenes and/or mutation of tumor suppressor genes such as p53.
Many cellular oncogenes, including c-erbB-2, c-myc and ras have been found to be activated in cancer, particularly of the breast.

Infectious Agent Markers

Antigenic markers are now available for a number of infectious agents, including hepatitis A virus, hepatitis B surface and core antigens, hepatitis C virus, human papilloma virus, cytomegalovirus, Epstein-Barr virus, toxoplasma, pneumocystis carinii, helicobacter pylori, cryptosporidium, cryptococcus neoformans, histoplasma, entamoeba histolytica, and mycobacteria.
For mycobacteria, immunohistochemical techniques are more sensitive, the results are obtained faster than with tissue culture, and they are easier to read than acid-fast stains.

Controls

It is essential to use positive and negative controls when processing tissue sections for immunohistochemistry in order to test for the specificity of the antibodies involved and to avoid misinterpretations due to false positive or false negative results.
To be specific, the immunohistochemical technique must not cause staining in the absence of the primary antiserum.
The staining should be inhibited when the primary antibody is absorbed by the relevant antigen prior to its use, but it should not be inhibited when the primary antibody is absorbed by other related or unrelated antigen.

Positive Control: It is always advisable to use, as a positive control, a section that is known and proven to contain the antigen in question because the absence of staining in a test section does not necessarily mean that the antigen is absent in the tissue being studied.
Negative Control: This can be done using a parallel section from the tissue, and either omitting the primary antibody from the staining schedule or replacing the specific primary antibody by an immunoglobulin that is directed against an unrelated antigen.
Internal Tissue Control: Also named as "built in" control, this eliminates the variable of tissue fixation between specimens and controls but it contains the target antigen, not only in the tissue elements under investigation, e.g., tumors, but also in adjacent normal tissue element. One example is the presence of S-100 protein in both melanoma and normal tissue elements, such as peripheral nerves and melanocytes.

Chromogenic Method

Chromogenic (brightfield) and fluorescence detection techniques are used in the determination of the presence and subcellular location of an increasing number of proteins within a single biopsy.
Chromogenic multi-immunohistochemical staining is based on immunoenzymatic reaction with chromogen and enzyme.
Chromogenic IHC staining can generate dense deposits that are easy to detect but difficult to quantitate, because of nonlinear optical effects and low achievable dynamic ranges.
In facilitating chromogenic detection, the primary antibody, secondary antibody, or streptavidin is conjugated to an enzyme.
Horseradish peroxidase (HRP) and alkaline phosphatase, which convert 3,3' diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), into brown and red end products, respectively are commonly used enzymes.
When a soluble organic substrate is applied, the enzyme reacts with the substrate to generate an insoluble colored product that is localized to the sites of antigen expression.
Chromogenic detection is considered to be a more sensitive method than immunofluorescence.
It requires only a typical light microscope unlike fluorescence microscopy which requires a specialized light source and filter sets.
Chromogenic detection, however, is less convenient because it includes more incubation and blocking steps.
Like immunofluorescence, it allows for the visualization of multiple antigens, but only if the antigens are confined to different locations in the cell and tissue because overlapping colors may obscure results.
An advantage of DAB chromogenic staining is that the colored precipitate formed during the reaction between HRP and DAB is not sensitive to light and the slides can be stored for many years.

Enzyme Labeling

Enzymes are widely used in immunohistochemistry and are usually incubated with a chromogen using standard histochemical methods to produce a stable colored reaction.
Enzyme labeling of antibodies with horseradish peroxidase, followed by staining with an appropriate substrate or chromogen mixture such as diaminobenzidine (DAB), will produce an insoluble dark brown reaction end product and allow labeled cells to be counterstained with hematoxylin and other nuclear stains.
The optimal incubation time for linking antibodies with peroxidase conjugates is 30 to 60 minutes at room temperature.

Direct Technique

The traditional direct technique of doing immunohistochemistry is to conjugate the primary antibody directly to the label, such as a fluorochrome or horseradish peroxidase.
The main advantage of using directly labeled antibody is that it is simple and quick because it requires only one application of the reagent, followed by the appropriate chromogen substrate solution.
However, it is less sensitive compared to indirect techniques that involve 2 or 3 stages of conjugation and staining.
This carries the risk of not detecting small amounts of antigen that could be crucial in making the diagnosis.
The method is no longer sufficiently sensitive for today's demands.

Enhanced Polymer One-Step Staining (EPOS) method

The Enhanced Polymer One-Step Staining (EPOS) method, marketed by Dako A/S, is a new direct technique whereby a large number of primary antibody molecules and peroxidase enzymes are attached to a dextran polymer