[F] Week 13: Enzyme Histochemistry and Diagnostic Application - Part 2 Flashcards
What are the marker commonly requested in the laboratory?
- Keratin
- Epithelial Membrane Antigen (EMA)
- Carcinoembryogenic Antigen (CEA)
- Thyroid Transcription Factor-1 (TTF -1)
- Prostatic Specific Antigen (PSA)
A highly sensitive marker for epithelial cells, and is present in all carcinomas including:
- Epithelial tumors
- Non-epithelial tumors (such as mesotheliomas and non-seminomatous germ cell tumors)
Keratin
What are the types of keratin?
- CK7 (Cytokeratin 7)
- CK20 (Cytokeratin 20)
Keratin
Specify what CK
* colon
* lung, breast, uterus, RCC
and ovaries
- CK20
- CK7
Keratin
CK that are usually positve for:
- Urothelial carcinomas of the bladder
- Gastric
- Mucinous ovarian tumors
CK7 and CK20
Keratin
CK that are usually negative for:
- Hepatocellular carcinomas
- prostatic adenocarcinomas,
- squamous cell carcinomas (skin, lung, and esophagus)
CK7 and CK20
Determine what Tumor
CK7 = +
CK20 = +
- Transitional cell carcinoma of bladder
- mucinous ovarian tumors, stomach
- pancreas
- gallbladder
Determine what Tumor
CK7 = -
CK20 = -
- Renal cell carcinoma
- hepatocellular carcinoma
- prostatic adenocarcinoma
- thyroid carcinoma
- squamous cell carcinoma (skin, lungs, and esophagus)
Epithelial Tumor Markers
- High molecular weight protein that is helpful in determining the site of tumor
Epithelial Membrane Antigen (EMA)
Epithelial Membrane Antigen (EMA) is positive for?
adenocarcinomas of the breast, lung, and
kidneys
Epithelial Membrane Antigen (EMA) is non-reactive for?
hepatocellular carcinomas, adrenal
carcinomas or embryonal carcinomas, and non-epithelial tumors
Epithelial Tumor Markers
Oncofetal antigen that is present in carcinomas of the:
- gastrointestinal tract
- pancreas
- lung
- breast
- ovary
- uterus
- cervix
positive in adnocarcinoma
Carcinoembryogenic Antigen (CEA)
Epithelial Tumor Markers
- Mostly for adenocarcinomas
- Lung adenocarcinomas vs mesotheliomas (for lung)
Thyroid Transcription Factor-1 (TTF-1)
Epithelial Tumor Markers
POSITIVE: thyroid, lung, and neuroendocrine tumors (medullary thyroid carcinomas, carcinoid tumors, and small cell tumors of the lung)
Thyroid Transcription Factor-1 (TTF-1)
Epithelial Tumor Markers
- Extremely useful in the diagnosis of prostatic adenocarcinoma
- POSITIVE: certain pancreatic and salivary gland tumor
Prostatic Specific Antigen (PSA)
what are the Intermediate Filament Markers
- Actin
- Vimentin
- Desmin
- Glial fibrillary acidic protein (GFAP)
- Neurofilament (NF)
- S-100 protein
Intermediate Filament Markers
It is a sensitive marker for muscle
differentiation and can be used to identify tumors derived from smooth, skeletal, and cardiac muscle
Actin
Intermediate Filament Markers
- 57kD intermediate filament that is present in normal mesenchymal cells and their neoplastic counterparts
- Non-specific for mesenchymal cells
- Counterpart of AMA
Vimentin
Intermediate Filament Markers
- 53 kD intermediate filament expressed by smooth and striated (skeletal and cardiac muscle)
- Leiomyoma and rhabdomyosarcoma
Desmin
Intermediate Filament Markers
- 51 kD intermediate filament protein expressed by central nervous system gl cells (Brain tissue), particularly astrocytes
- Positive: Astrocytoma, ependymomas, oligodendrogliomas, medulloblastomas
- Non-CNS tumors (meningiomas, metastatic carcinomas, and lymphomas) stain negative for GAP
Glial Fibrillary Acidic Protein
(GFAP)
Intermediate Filament Markers
Expressed in cells of neural origin, particularly neurons, neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla, and neuroendocrine
cells
Neuro-filament (NF)
Intermediate Filament Markers
- Mostly for CNS
- Low molecular weight calcium-binding
protein - 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
What are the neuroendocrine markers
- Neuron Specific Enolase (NSE)
- Chromogranin
- Synaptophysin
NEUROENDOCRINE MARKERS
- Isoenzyme marker
- Neural or neuroendocrine differentiation
Neuron- Specific Enolase (NSE)
NEUROENDOCRINE MARKERS
- Neural secretory granules of endocrine tissues, and is recognized as a marker for neuroendocrine differentiation
Chromogranin
NEUROENDOCRINE MARKERS
38 kD transmembrane protein associated with presynaptic vesicles of neurons
Synaptophysin
What are the Germ cell tumor markers
- HCG (Human chorionic gonadotropin)
- APP (Alpha-fetoprotein)
- PLAP (Placenta-like alkaline phosphatase)
Germ cell tumor markers
- Synthesized by placental syncytiotrophoblasts
- Choriocarcinoma
HCG (Human chorionic gonadotropin)
Germ cell tumor markers
- Normal hepatocyte
- Endodermal sinus tumors showing yolk sac differentiation, embryonal carcinomas
Alpha-Fetoprotein
(APP)
Germ cell tumor markers
- Placental syncytiotrophoblasts in late pregnancy
- Germ cell tumors, particularly germinomas
- Seminoma
PLAP (Placenta-like alkaline phosphatase)
what are the Mesenchymal tumor markers
- Myogenic Tumors
- Fibrohistiocytic Tumors
- Vascular Tumors
- Melanomas
- Lymphomas
Mesenchymal Tumor Markers
- Tumors of skeletal muscle origin
- Muscle specific actin and desmin
Myogenic Tumors
Mesenchymal Tumor Markers
- CD68 or FAM 56
Fibrohistiocytic Tumors
Mesenchymal Tumor Markers
VII-related antigen, CD31, and Ulex europaeus 1 (UEA)
Vascular Tumors
Mesenchymal Tumor Markers
S100 protein, Melanosome (HMB-45), Melan-A (MART-1)
Melanomas
Mesenchymal Tumor Markers
- LCA (leukocyte common antigen) or CD45
- T cells (CD3, CD4, CDS)
- B cells (CD19, CD20, CD23)
- Reed-Sternberg cells (CD15, CD30)
- Immunoglobulin light and heavy chains
Lymphomas
Requested for abnormal mitotic proliferation since counting is sometimes difficult for identification of abnormal mitosis tumors especially spindle-shaped tumors.
Cell proliferation markers
Cell Proliferation Markers
the most common immunohistochemical markers
used to assess proliferation of
tumor cells
- Ki-67 (MIB-1)
- PCNA (proliferating cell nuclear antigen)
OTHERS
- P53, c-erbB-2, c-myc
- 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
- Cancer-associated genes
- Infectious Agent Markers
Tissue Controls
- Contains the target protein
- does not contain the relevant antigen
- “built in”; contains the target marker in the tumor & adjacent normal elements
- Positive control
- Negative control
- Internal tissue control
TISSUE CONTROLS
- Located near the label in
the glass slide - from supplier or known positive prepared by histotechs
Positive control
TISSUE CONTROLS
- Seen in the tissue; should not stain
- E.g in breast tissue, we have ERPR so in normal breast ducts should not be stained
Negative control
TISSUE CONTROLS
- Built in contains the target marker in the tumor and adjacent normal elements
- From the patient (not tumor cell)
Internal tissue
control
TISSUE CONTROLS
TOF
- Tissue should have positive malignancy and abnormal structures like normal glands in the slide
- The positive and Internal tissue control are the ones given to the laboratory coming from the patient
- processed identically as the unknown
- +/- controls test for specificity of antibodies and avoid misinterpretations due to false +/- results
- F (normal)
- F (negative)
- T
- T
CHROMOGENIC METHOD
TOF
Chromogenic (brightfield) and fluorescence detection techniques are used in the determination of the presence and cellular location of an increasing number of proteins within a single biopsy
F (subcellular not cellular)
Chromogenic multi immunohistochemical staining:
immunoenzymatic reaction with chromogen and enzyme
- Can generate dense deposits that are easy to detect but difficult to quantitate
- More sensitive method than immunofluorescence
- less convenient because it includes more incubation and blocking steps
Chromogenic Method
CHROMOGENIC METHOD
Usually incubated with a chromogen using standard histochemical method to produce a stable-colored
reaction.
ENZYME LABELING
CHROMOGENIC METHOD
Enzyme labeling of antibodies with
horseradish peroxidase
Enzyme labeling of antibodies with horseradish peroxidase, followed by staining with appropriate substrate or chromogen mixture such as
diaminobenzidine (DAB) - dark brown
CHROMOGENIC METHOD
Optimal incubation time for enzyme labelling
30 to 60 minutes at room
temperature
what are the IMMUNOHISTOCHEMISTRY TECHNIQUES
- Direct Technique
- Indirect Technique
Direct Technique
Conjugate the primary antibody directly to the label, such as?
` fluorochrome or horseradish peroxidase
Direct Technique
TOF
The main advantage is that it is simple and quick. It requires two application of the reagent, followed by the appropriate chromogen substrate solution.
False (one only)
why direct technique are not routinely used?
less sensitive and unstable
what is the technique used for direct labelling?
Enhanced Polymer One-Step Stainng (EPOS) method
not commonly used
IMMUNOHISTOCHEMISTRY TECHNIQUES
- More commonly used
-
Two (2) or three (3) step procedure that involves application of the unconjugated primary antibody, followed by a
labeled antibody directed against the first antibody.
Indirect Technique
IMMUNOHISTOCHEMISTRY TECHNIQUES
- It is inexpensive and more sensitive.
- Horseradish peroxidase is the most commonly used enzyme for indirect antibody enzyme-complex techniques.
Indirect Technique
Two or Three indirect tenchnique
- Unconjugated primary antibodies first bind to the antigen.
- There are two enzymes bound.
- An enzyme-labeled secondary antibody directed against the primary antibody (now the antigen) is then applied, followed by
the substrate - chromogen solution.
Two-step Indirect
Technique
Two or Three indirect tenchnique
- This procedure is considered as a better procedure.
- Instead of two enzymes, there are three enzymes bounded. The second one acts as the bridge to the third antibody.
- Second enzyme-conjugated antibody is added to the previously described indirect technique.
- Further amplify the signal.
Three-step Indirect
Technique
OTHER TECHNIQUES
- Utilize preformed soluble enzyme-anti-enzyme immune complex.
- In this method, the staining sequence involves an unconjugated primary antibody and then a secondary antibody which serves as a glue for binding of soluble enzyme-anti-enzyme complexes.
Soluble Enzyme Immune Complex Techniques (Unlabeled Antibody Techniques)
OTHER TECHNIQUES
- Most commonlyused in the laboratory
- Standard method among most laboratories, especially in combination with frozen section processing for immunofluorescence of renal and skin biopsies
- Alcohol is not widely used as a fixative for routine histological techniques because of its poor penetrating ability.
Paraffin Wax Section Immunoperoxidase Technique
OTHER TECHNIQUES
- Soluble peroxidase-antiperoxidase complex is bound to unconjugated primary antibody by a second layer of “bridging” antibody that then binds to both the primary antibody and the rabbit PAP complex.
- Bridge is used.
Peroxidase-Antiperoxidase (PAP) Technique
Binding of the primary antibody to amino acids other than those within the desired epitope of the antigen.
- Antibody is already attached where it should not be. Normal serum, non-fat dry milk, BSA or gelatin is used as a reagent for it to block and remove those that are not bound.
BLOCKING OF UNWANTED NON-SPECIFIC STAINING
Blocking of Unwanted Non-specific
Staining
In the context of antibody-mediated antigen detection, nonspecific binding causes?
high background staining that can mask the detection of the target antigen
Blocking of Unwanted Non-specific
Staining
TOF
Endogenous enzymes or fluorochromes, endogenous biotin,
endogenous antibody binding activity and cross reactivity of
the secondary reagents with endogenous proteins
True
