Lecture 4: Importance of IHC Flashcards
Carcinoma
Develops from epithelial cells that line inner and outer surfaces
These are common including breast, colon, lung, etc.
Small-cell carcinoma is most common and highly malignant in the lung
Sarcoma
Cells of mesenchymal origin that transform
Such as bone, cartilage, fat, vasculature, and hematopoiteic (blood forming) cells
Melanoma
Cancer from pigment producing melanocytes,
Typically arises in the skin, but can also occur in the mouth, intestine, or eye
Anaplastic malignancies
Undifferentiated or poorly differentiated, and therefore difficult to diagnose because the cells bear minimal resemblance to the cell from which they arose
May be classified as a tumor of unknown origin
IHC is useful in diagnostics because?
You can better classify cells and tumors based on markers identified through IHC, such as lymphoma and small-cell carcinoma which receive very different therapies and prognoses for the patient
Why is H&E needed in addition to IHC?
Because IHC cannot determine the difference between normal and neoplastic or between benign and malignant cell types, only their external cell type marker
H&E slides are used for morphological studies so the pathologist can identify morphological abnormalities in cell populations
Etymological meaning of Immunohistochemistry (IHC)
Immuno: antigen/antibody
Histo: tissue based
Chemistry: reactions
What is IHC?
A method used to identify/stain for the presence of biomarkers in prepared sections of tissue
This information is then applied to determine the origin, prognosis, and treatment for a tumor
What are the types of IHC staining patterns?
Nuclear
Cytoplasmic
Membranous
Organisms (ex H. pylori)
What is critical when troubleshooting an IHC procedure?
Proper Control tissue
Proper staining pattern
Proper Antibody (and concentration/dilution)
Also consistency run to run
Pre-Analytics (How we handle tissue prior to performing IHC)
Biopsy or surgical removal of tissue Accessioning Gross examination Tissue processing and embedding Sectioning
Basic analytical steps of IHC staining
Antigen retrieval
Primary antibody
Visualization system
Counterstaining
Specific Steps in (indirect) IHC
Fix, embed, section tissue Antigen retrieval Blocking of endogenous enzymes in the tissue Blocking background/son-specific staining Apply primary antibody Apply secondary antibody Apply chromogen (ex DAB or AEC) Apply counterstain Dehydrate, mount, coverslip
Post-Analytic steps
Pathologist interprets slides against positive/negative controls (some tissues contain internal + and - ctrls)
Looks at staining patterns and quality using a microscope
Results are reported to the oncologist or physician for treatment
Paratope
the variable regions of the antibody that bind to the epitope on an antigen
Fixation
preserves cellular structures for examination
Antigen retrieval
Formalin cross-links proteins as a method of fixation, forming methylene bridges that mask antigen sites, and sometimes destroy epitopes entirely
Thus we must “un-mask” antigens affected by fixation so that the antibody can bind to its antigen
HIER antigen retrieval (heat)
Heat Induced Epitope Retrieval, uses a microwave and or pressure cooker
EIER antigen retrieval (enzyme)
Enzyme Induced Epitope Retrieval, utilizes enzyme digestion such as proteinase K, trypsin, or pepsin
This method has the potential to damage tissue
Combination heat and enzyme retrieval
Heat first, then use enzyme fro a short time, which helps to preserve tissue morphology
What are the advantages of antigen retrieval?
Antibodies can be applied at a more dilute concentration
More epitope sites are available to antibodies for binding/staining
More reactions necessitating a shorter incubation time
More uniform staining
Decreased background staining
Consistent staining (run to run)
Easier to standardize staining methodology
Blocking is done because?
it prevents non-specific binding of your antibody to related but off target sites in the tissue (noise/brown hazy stain)
Direct immunofluorescence (DIF)
Fast, easy
fewer steps
less sensitive detection method
ABC
Avidin-Biotin complex
A method of IHC detection
Indirect (IHC)
More steps
Primary + Secondary
More sensitive
Biotin
A component of the Vitamin B2 complex that binds strongly to both streptavidin and avidin
Biotinylated antibody
An antibody that has an attached biotin group
(Strept)avidin
A protein secreted by the bacterium Streptomyces avidinii
Avidin is a glycoprotein found in non-denatured egg whites
Both bind strongly to biotin
ABC method amplification
Attraction between biotin and (Strept)avidin makes it easy to form complexes between biotinylated antibodies and indicator molecules that contain streptavidin
One antibody can possess multiple biotin’s which allows large (strept)avidin/biotin complexes to form around the antibody, thus amplifying the signal for visual detection/staining and making the system very sensitive
Albumin (egg whites)
Act an an effective blocking agent
