IMMUNOHISTOCHEMISTRY_Bruce Flashcards
are now routinely used for the identification of specific or highly selective cellular epitopes or antigens in frozen or paraffin-embedded tissues.
Immunohistochemical techniques
can also be used to detect organisms in cytologic preparations such as fluids, sputum samples, and fine needle aspirates.
Immunocytochemistry
Antibodies belong to the class of serum proteins known as…
immunoglobulins
is the most commonly used antibody for immunocytochemistry
IgG
is the structural part of the antigen that reacts with an antibody.
epitope
combines anatomical, immunological and biochemical techniques to identify discrete tissue components by the interaction of target antigens with specific antibodies tagged with a visible label.
Immunohistochemistry (IHC)
makes it possible to visualize the distribution and localization of specific cellular components within cells and in the proper tissue context.
IHC
While there are multiple approaches and permutations in IHC methodology, all of the steps involved are separated into two groups:
sample preparation and labeling.
is used for disease diagnosis, drug development and biological research.
IHC
Using___________, physicians use IHC to diagnose a cancer as_____, determine the______ of a tumor, and identify the cell type and origin of a______ to find the site of the primary tumor.
specific tumor markers
benign or malignant
stage and grade
metastasis
IHC is also used in _______to test drug efficacy by detecting either the activity or the up- or down-regulation of disease targets.
drug development
Samples can be viewed by either____ or _____ microscopy, and advances in the last 15 years have improved the ability to capture images, quantitate mult-Oparametric IHC data and increase the collection of that data through high content screening.
light or fluorescence
are produced by immunizing an animal with a purified specific molecule (immunogen) that contains the antigen of interest, and collecting immunoglobulin-rich serum after the animal has produced humoral antibody against the antigen.
Polyclonal antibodies
The most frequently used animal for the production of polyclonal antibodies is the
rabbit, followed by goat, pig, sheep, horse, guinea pig and others.
Some of the_______ antibodies may cross-react with other molecules and cause nonspecific staining, requiring their purification by absorption with the appropriate antigen, or antibody dilution to eliminate the unwanted reaction.
polyclonal
are the products of an individual clone of plasma cells.
Monoclonal antibodies
techniques have been developed to produce monoclonal antibodies that do not cross-react with other molecules.
Hybridoma and cloning
Antibodies from a given clone are immunochemically identical and react with a specific epitope on the antigen against which they are raised.
Monoclonal
are currently used almost exclusively for the production of monoclonal antibodies.
Mice
In certain instances, the tissue must be prepared as a______ section and fixed for a few seconds in______ or _____, to preserve immunological activity and prevent destruction of some of the labile antigenic sites.
However,_______ and______ techniques may also be done on formaldehyde-fixed and paraffin embedded sections.
cryostat
absolute methanol or acetone
immunofluorescence
immuno-peroxidase
Many masked antigens can now be retrieved in routinely processed tissue by
(1) proteolytic enzyme digestion
(2) microwave antigen retrieval
(3) microwave and trypsin antigen retrieval
(4) pressure cooker antigen retrieval
Proteolytic Enzyme Digestion
Formalin fixed paraffin sections are usually pre-treated with_____ to break down formalin cross-linking, unmask and allow certain antigenic sites to be exposed.
proteolytic enzymes
_________ 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____ and _____
Proteolytic enzyme digestion
trypsin and protease
Proteolytic Enzyme Digestion
Before pretreatments are employed, the sections are______, 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.
deparaffinized
Proteolytic Enzyme Digestion
The trypsin method uses _____in _____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.
0.1% trypsin
0.1 % calcium chloride
Proteolytic Enzyme Digestion
The protease method uses______ 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.
0.05 to 0.1% protease
Proteolytic Enzyme Digestion
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.
Proteolytic Enzyme Digestion
Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded.
Unmasking
Proteolytic Enzyme Digestion
: 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.
Serum Blocking
Proteolytic Enzyme Digestion
Primary Antibody
: Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for__________ or ________
1 hour at room temperature
overnight at 4 °C
Proteolytic Enzyme Digestion
Peroxidase Blocking
: Incubate sections in peroxidase blocking solution for ________
10 minutes at room temperature.
Proteolytic Enzyme Digestion
Secondary Antibody: Incubate sections with biotinylated secondary antibody at appropriate dilution in PBS for_____
30 minutes at room temperature.
a simple laboratory technique that has become an essential part of many immunohistochemistry and in situ hybridization procedures, is a pretreatment method used to improve staining results.
Heat-induced epitope retrieval (HIER)
….., 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
Heat
Heat reverses the formaldehyde mediated chemical modifications of the antigen through either of the following processes.
First and foremost, thermal energy breaks the____ that bind surrounding proteins or peptides to the antigen which lead to the “opening” or “unmasking” the epitope.
And, second, thermal energy removes______ from the sites of cross-links since several HIER buffers, such as EDTA and citrate, act as calcium chelators.
crosslinks
bound calcium ions
HIER heating sources include the
microwave
vegetable steamer
pressure cooker
water bath
T or F
the higher the temperature of the HIER solutions, the more effective the recovery of the epitope is.
True
_______ distribution of heat is uneven or inconsistent which results in a lack of reproducibility as to staining intensities.
In contrast, the(3) produce uniform and consistent heat distribution.
microwave
pressure cooker, steamer and water bath
higher temperatures produced by the _______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.
pressure cooker
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).
Microwave antigen retrieval
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
Heat pre treatment
The optimal length of exposure to heat may vary from______ and depends to some extent on the length of formalin fixation.
The most satisfactory time period appears to be____ minutes for most antigens and fixation protocols
10 to 60 minutes
20mins
Care should be taken not to allow the sections to_____ after heating, as this destroys antigenicity.
_____of poorly fixed material often damages nuclear details.
_________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 10% formol saline
dry
Boiling
Fibrous and fatty
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_____, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr. of fixation time.
On amplifying____, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr. in the formalin-fixed tissue.
DNA
RNA
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.
Bouin’s and B-5 tissues
is another alternative that appears to be less time consuming and allows for more consistent recovery of many antigens, compared to large batch microwave oven technique.
Pressure cooking antigen retrieval
In the large batch_______, heating temperature is not uniformly distributed and slides are subjected to “hot spots” and “cold spots” resulting in inconsistent antigen recovery.
microwave oven technique
_______ 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 prolife-ration potential, identification of infectious agents, prognostic and therapeutic implications, and many other aspects of diagnostic pathology.
Primary antibodies
Epithelial tumor markers:
- Keratin
- EMA (Epithelial membrane antigen)
- CEA (Carcinoembryonic antigen)
- TTF-1 (Thyroid transcription factor-1)
- PSA (Prostate specific antigen)
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 _____, and may be distinguished from carcinoma by applying an additional
panel of antibodies.
Keratin
is more frequently found in carcinomas of the lung, breast, uterus and ovaries (serous tumors). These tumors
are typically negative for CK20.
CK7 (Cytokeratin 7)
is more common in carcinomas of the colon and stomach.
These tumors are usually negative for CK7.
CK20 (Cytokeratin 20)
Transitional cell carcinomas of the bladder and mucinous
ovarian tumors are usually_____ for both CK7 and CK20
positive
Renal cell carcinomas, hepatocellular carcinomas, prostatic
adenocarcinomas, thyroid carcinomas and squamous cell carcinomas (skin, lung and esophagus) are usually_____ for either CK7 or CK20.
negative
is a high molecular weight protein that is helpful in determining the site of tumor.
EMA (Epithelial membrane antigen)
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).
EMA
is an oncofetal antigen that is present in carcinomas of the gastrointestinal tract, pancreas, lung, breast, ovary,
uterus and cervix.
CEA (Carcinoembryonic antigen)
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.
CEA
is useful in distinguishing lung
adenocarcinomas from mesotheliomas.
TTF-1 (Thyroid transcription factor-1)
It is positive in thyroid, lung and neuroendocrine tumors (medullary thyroid carcinomas, carcinoid tumors and small cell tumors of the lung).
TTF-1
is extremely useful in the diagnosis of prostatic adenocarcinoma.
It is also positive in certain pancreatic and salivary gland tumors.
PSA (Prostate specific antigen)
Intermediate Filament Markers
- Actin
- Vimentin
- Desmin
- Glial fibrillary acidic protein (GFAP)
- Neurofilament (NF)
- S-100 protein
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
Actin
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).
Vimentin
Melanomas and schwannomas always stain positive for______, 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.
vimentin
is a 53 kD intermediate filament expressed by smooth and striated (skeletal and cardiac) muscle.
Desmin
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).
Desmin
is a 51 kD intermediate filament
protein expressed by central nervous system glial cells, particularly astrocytes.
Glial fibrillary acidic protein (GFAP)
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 _____.
Glial fibrillary acidic protein (GFAP)
is expressed in cells of neural origin, particularly neurons, neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla and neuroendocrine cells.
Neurofilament (NF)
Tumors that show neuronal or neuroendocrine differentiation (e.g., neuroblastomas, ganglioneuromas, neuromas, chemodectomas, and pheochromocytomas) will stain positive for _______.
Neurofilament
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.
S-100 protein
Neuroendocrine markers:
- Neuron-specific enolase (NSE)
- Chromogranin
- Synaptophysin
is an isoenzyme marker whose presence in tissue provides strong evidence of neural or neuroendocrine differentiation.
Neuron-specific enolase (NSE)
is found in the neural secretory granules of endocrine tissues, and is recognized as a marker for neuroendocrine differentiation.
Chromogranin
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
is a 38 kD transmembrane protein associated with presynaptic vesicles of neurons.
It has been identified in normal neurons and neuroendocrine cells.
Synaptophysin
Germ cell tumor markers
- HCG (Human chorionic gonadotropin)
- APP (Alpha-fetoprotein)
- PLAP (Placenta-like alkaline phosphatase)
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).
Germ cell tumor markers
is synthesized by placental syncytiotrophoblasts, and is a marker for choriocarcinoma.
HCG (Human chorionic gonadotropin)
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 ____.
APP (Alpha-fetoprotein)
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 (Placenta-like alkaline phosphatase)
is positive in majority of seminomas.
PLAP
Mesenchymal tumor markers
- Myogenic tumors
- Fibrohistiocytic tumors
- Vascular tumors
- Melanomas
- Lymphomas
- 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.
Myogenic 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.
Fibrohistiocytic tumors
- Endothelial markers for vascular tumors (such as angiosarcomas) include Factor VII-related antigen, CD31 and Ulex Europaeus 1 (UEA).
Vascular tumors
- 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.
Melanomas
is a widely used, highly sensitive and highly specific marker for the diagnosis of melanoma.
Melanosome (HMB-45)
also encodes a melanoma-specific antigen that is present in normal pigmented cells of skin and retina as well as in certain adrenocortical tumors.
Melan-A (MART-1)
- 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.
Lymphomas
