Lecture 4: Importance of IHC

Question 1

Carcinoma

Answer 1

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

Question 2

Sarcoma

Answer 2

Cells of mesenchymal origin that transform

Such as bone, cartilage, fat, vasculature, and hematopoiteic (blood forming) cells

Question 3

Melanoma

Answer 3

Cancer from pigment producing melanocytes,

Typically arises in the skin, but can also occur in the mouth, intestine, or eye

Question 4

Anaplastic malignancies

Answer 4

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

Question 5

IHC is useful in diagnostics because?

Answer 5

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

Question 6

Why is H&E needed in addition to IHC?

Answer 6

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

Question 7

Etymological meaning of Immunohistochemistry (IHC)

Answer 7

Immuno: antigen/antibody
Histo: tissue based
Chemistry: reactions

Question 8

What is IHC?

Answer 8

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

Question 9

What are the types of IHC staining patterns?

Answer 9

Nuclear
Cytoplasmic
Membranous
Organisms (ex H. pylori)

Question 10

What is critical when troubleshooting an IHC procedure?

Answer 10

Proper Control tissue
Proper staining pattern
Proper Antibody (and concentration/dilution)

Also consistency run to run

Question 11

Pre-Analytics (How we handle tissue prior to performing IHC)

Answer 11

Biopsy or surgical removal of tissue
Accessioning
Gross examination
Tissue processing and embedding
Sectioning

Question 12

Basic analytical steps of IHC staining

Answer 12

Antigen retrieval
Primary antibody
Visualization system
Counterstaining

Question 13

Specific Steps in (indirect) IHC

Answer 13

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

Question 14

Post-Analytic steps

Answer 14

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

Question 15

Paratope

Answer 15

the variable regions of the antibody that bind to the epitope on an antigen

Question 16

Fixation

Answer 16

preserves cellular structures for examination

Question 17

Antigen retrieval

Answer 17

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

Question 18

HIER antigen retrieval (heat)

Answer 18

Heat Induced Epitope Retrieval, uses a microwave and or pressure cooker

Question 19

EIER antigen retrieval (enzyme)

Answer 19

Enzyme Induced Epitope Retrieval, utilizes enzyme digestion such as proteinase K, trypsin, or pepsin

This method has the potential to damage tissue

Question 20

Combination heat and enzyme retrieval

Answer 20

Heat first, then use enzyme fro a short time, which helps to preserve tissue morphology

Question 21

What are the advantages of antigen retrieval?

Answer 21

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

Question 22

Blocking is done because?

Answer 22

it prevents non-specific binding of your antibody to related but off target sites in the tissue (noise/brown hazy stain)

Question 23

Direct immunofluorescence (DIF)

Answer 23

Fast, easy
fewer steps
less sensitive detection method

Question 24

ABC

Answer 24

Avidin-Biotin complex

A method of IHC detection

Question 25

Indirect (IHC)

Answer 25

More steps
Primary + Secondary
More sensitive

Question 26

Biotin

Answer 26

A component of the Vitamin B2 complex that binds strongly to both streptavidin and avidin

Question 27

Biotinylated antibody

Answer 27

An antibody that has an attached biotin group

Question 28

(Strept)avidin

Answer 28

A protein secreted by the bacterium Streptomyces avidinii

Avidin is a glycoprotein found in non-denatured egg whites

Both bind strongly to biotin

Question 29

ABC method amplification

Answer 29

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

Question 30

Albumin (egg whites)

Answer 30

Act an an effective blocking agent