[F] Week 13: Enzyme Histochemistry and Diagnostic Application - Part 2 Flashcards
(66 cards)
1
Q
What are the marker commonly requested in the laboratory?
A
- Keratin
- Epithelial Membrane Antigen (EMA)
- Carcinoembryogenic Antigen (CEA)
- Thyroid Transcription Factor-1 (TTF -1)
- Prostatic Specific Antigen (PSA)
2
Q
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)
A
Keratin
3
Q
What are the types of keratin?
A
- CK7 (Cytokeratin 7)
- CK20 (Cytokeratin 20)
4
Q
Keratin
Specify what CK
* colon
* lung, breast, uterus, RCC
and ovaries
A
- CK20
- CK7
5
Q
Keratin
CK that are usually positve for:
- Urothelial carcinomas of the bladder
- Gastric
- Mucinous ovarian tumors
A
CK7 and CK20
6
Q
Keratin
CK that are usually negative for:
- Hepatocellular carcinomas
- prostatic adenocarcinomas,
- squamous cell carcinomas (skin, lung, and esophagus)
A
CK7 and CK20
7
Q
Determine what Tumor
CK7 = +
CK20 = +
A
- Transitional cell carcinoma of bladder
- mucinous ovarian tumors, stomach
- pancreas
- gallbladder
8
Q
Determine what Tumor
CK7 = -
CK20 = -
A
- Renal cell carcinoma
- hepatocellular carcinoma
- prostatic adenocarcinoma
- thyroid carcinoma
- squamous cell carcinoma (skin, lungs, and esophagus)
9
Q
Epithelial Tumor Markers
- High molecular weight protein that is helpful in determining the site of tumor
A
Epithelial Membrane Antigen (EMA)
10
Q
Epithelial Membrane Antigen (EMA) is positive for?
A
adenocarcinomas of the breast, lung, and
kidneys
11
Q
Epithelial Membrane Antigen (EMA) is non-reactive for?
A
hepatocellular carcinomas, adrenal
carcinomas or embryonal carcinomas, and non-epithelial tumors
12
Q
Epithelial Tumor Markers
Oncofetal antigen that is present in carcinomas of the:
- gastrointestinal tract
- pancreas
- lung
- breast
- ovary
- uterus
- cervix
positive in adnocarcinoma
A
Carcinoembryogenic Antigen (CEA)
13
Q
Epithelial Tumor Markers
- Mostly for adenocarcinomas
- Lung adenocarcinomas vs mesotheliomas (for lung)
A
Thyroid Transcription Factor-1 (TTF-1)
14
Q
Epithelial Tumor Markers
POSITIVE: thyroid, lung, and neuroendocrine tumors (medullary thyroid carcinomas, carcinoid tumors, and small cell tumors of the lung)
A
Thyroid Transcription Factor-1 (TTF-1)
15
Q
Epithelial Tumor Markers
- Extremely useful in the diagnosis of prostatic adenocarcinoma
- POSITIVE: certain pancreatic and salivary gland tumor
A
Prostatic Specific Antigen (PSA)
16
Q
what are the Intermediate Filament Markers
A
- Actin
- Vimentin
- Desmin
- Glial fibrillary acidic protein (GFAP)
- Neurofilament (NF)
- S-100 protein
17
Q
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
A
Actin
18
Q
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
A
Vimentin
19
Q
Intermediate Filament Markers
- 53 kD intermediate filament expressed by smooth and striated (skeletal and cardiac muscle)
- Leiomyoma and rhabdomyosarcoma
A
Desmin
20
Q
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
A
Glial Fibrillary Acidic Protein
(GFAP)
21
Q
Intermediate Filament Markers
Expressed in cells of neural origin, particularly neurons, neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla, and neuroendocrine
cells
A
Neuro-filament (NF)
22
Q
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
A
S-100 protein
23
Q
What are the neuroendocrine markers
A
- Neuron Specific Enolase (NSE)
- Chromogranin
- Synaptophysin
24
Q
NEUROENDOCRINE MARKERS
- Isoenzyme marker
- Neural or neuroendocrine differentiation
A
Neuron- Specific Enolase (NSE)
25
# NEUROENDOCRINE MARKERS
* Neural secretory granules of endocrine tissues, and is recognized as a marker for neuroendocrine differentiation
Chromogranin
26
# NEUROENDOCRINE MARKERS
38 kD transmembrane protein associated with presynaptic vesicles of neurons
Synaptophysin
27
What are the Germ cell tumor markers
1. HCG (Human chorionic gonadotropin)
2. APP (Alpha-fetoprotein)
3. PLAP (Placenta-like alkaline phosphatase)
28
# Germ cell tumor markers
* Synthesized by **placental syncytiotrophoblasts**
* Choriocarcinoma
HCG (Human chorionic gonadotropin)
29
# Germ cell tumor markers
* Normal hepatocyte
* Endodermal sinus tumors showing **yolk sac differentiation**, embryonal carcinomas
Alpha-Fetoprotein
(APP)
30
# Germ cell tumor markers
* Placental syncytiotrophoblasts in late pregnancy
* Germ cell tumors, particularly germinomas
* Seminoma
PLAP (Placenta-like alkaline phosphatase)
31
what are the Mesenchymal tumor markers
- Myogenic Tumors
- Fibrohistiocytic Tumors
- Vascular Tumors
- Melanomas
- Lymphomas
32
# Mesenchymal Tumor Markers
* Tumors of skeletal muscle origin
* Muscle specific actin and desmin
Myogenic Tumors
33
# Mesenchymal Tumor Markers
* CD68 or FAM 56
Fibrohistiocytic Tumors
34
# Mesenchymal Tumor Markers
VII-related antigen, CD31, and Ulex europaeus 1 (UEA)
Vascular Tumors
35
# Mesenchymal Tumor Markers
S100 protein, Melanosome (HMB-45), Melan-A (MART-1)
Melanomas
36
# 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
37
Requested for **abnormal mitotic proliferation** since counting is sometimes difficult for identification of abnormal mitosis tumors especially spindle-shaped tumors.
Cell proliferation markers
38
# Cell Proliferation Markers
the most common immunohistochemical markers
used to assess proliferation of
tumor cells
- Ki-67 (MIB-1)
- PCNA (proliferating cell nuclear antigen)
39
# 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
40
# Tissue Controls
1. Contains the target protein
2. does not contain the relevant antigen
3. “built in”; contains the target marker in the tumor & adjacent normal elements
1. Positive control
2. Negative control
3. Internal tissue control
41
# TISSUE CONTROLS
- Located near the label in
the glass slide
- from supplier or known positive prepared by histotechs
Positive control
42
# 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
43
# TISSUE CONTROLS
- Built in contains the target marker in the tumor and adjacent normal elements
- From the patient (not tumor cell)
Internal tissue
control
44
# 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
45
# 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)
46
Chromogenic multi immunohistochemical staining:
immunoenzymatic reaction with chromogen and enzyme
47
* 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
48
# CHROMOGENIC METHOD
Usually incubated with a chromogen using standard histochemical method to produce a stable-colored
reaction.
ENZYME LABELING
49
# CHROMOGENIC METHOD
Enzyme labeling of antibodies with
horseradish peroxidase
50
Enzyme labeling of antibodies with horseradish peroxidase, followed by staining with appropriate substrate or chromogen mixture such as
diaminobenzidine (DAB) - dark brown
51
# CHROMOGENIC METHOD
Optimal incubation time for enzyme labelling
30 to 60 minutes at room
temperature
52
what are the IMMUNOHISTOCHEMISTRY TECHNIQUES
1. Direct Technique
2. Indirect Technique
53
# Direct Technique
Conjugate the primary antibody directly to the label, such as?
` fluorochrome or horseradish peroxidase
54
# 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)
55
why direct technique are not routinely used?
less sensitive and unstable
56
what is the technique used for direct labelling?
Enhanced Polymer One-Step Stainng (EPOS) method
| not commonly used
57
# 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
58
# IMMUNOHISTOCHEMISTRY TECHNIQUES
* It is inexpensive and more sensitive.
* Horseradish peroxidase is the most commonly used enzyme for indirect antibody enzyme-complex techniques.
Indirect Technique
59
# 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
60
# 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
61
# 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)
62
# OTHER TECHNIQUES
* **Most commonly**used 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
63
# 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
64
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
65
# 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
66
# 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
