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HPCT > Immunohistochemical Techniques and Rapid Tissue Processing > Flashcards

Immunohistochemical Techniques and Rapid Tissue Processing Flashcards

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1
Q
  • used for identification of specific or highly selective
    cellular epitopes or antigens in frozen or paraffin
    embedded tissues
A

Immunohistochemical techniques

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2
Q

– detects organism in cytologic preparation

A

Immunocytochemistry

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3
Q

Makes use of Ag-Ab interaction
* direct labeling Ab
* secondary labeling method

A

Immunologic techniques–

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4
Q

– most commonly used Ab in immunocytochemisry

A
  • IgG
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5
Q
  • Produced by immunizing an animal with a purified specific molecule (__) that
    contains the antigen of interest,
A

Polyclonal antibodies
- immunogen

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6
Q

collecting Ig-rich serum after the animal produces humoral antibodies

A

Polyclonal antibodies

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7
Q
  • Products of an individual
    clone of plasma cells
  • Produced through _ and _
A

Monoclonal Antibodies
- hybridoma and cloning techniques

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8
Q

Tissue preparation for Immunohistochemistry may be prepared as

A

-Cryostat section
- Formaldehyde-fixed and paraffin embedded tissues

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9
Q

to preserve immunological activity– prevent destruction of some labile antigenic site

A

Cryostat section
* absolute methanol, or acetone–

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10
Q

Formaldehyde-fixed and paraffin embedded tissues

A
  • immuno flluorescence
  • immunoperoxidase techniques
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11
Q

Retrieval of Masked Antigens

A
    1. Proteolytic enzyme digestion
    1. Microwave Ag Retrieval
    1. Microwave and Trypsin Ag retrieval
    1. Pressure Cooker Ag Retrieval
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12
Q
  • Formalin fixed paraffin sections are pre-treated
    with___
    ex _ and _ – to break down formalin cross-linking, unmask and
    allow certain antigenic sites to be exposed
A

Proteolytic Enzyme Digestion
- proteolytic enzymes (e.g. trypsin and
protease)

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13
Q

Proteolytic Enzyme Digestion are Useful for demonstrating ,,_

A

heavy chain Igs,
complement, and specific antigens (cytokeratin)

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14
Q

Involves the boiling of formalin-fixed deparaffinized sections in
certain solutions such as:–

A

Microwave Antigen Retrieval
0.01 M Citrate buffer (pH 6.0)– EDTA (pH 8.0)–
Tris EDTA (pH 9.9 or 10.0

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15
Q

Microwave Antigen Retrieval
Antibodies–

A

Proliferation markers– hormone receptors–
growth factors receptors

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16
Q

Microwave Antigen Retrieval Optimal length of heat exposure: __
* __ - strong adhesives

A
  • 10-60 minutes (20 min is the
    most satisfactory)
  • vectabond
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17
Q
  • Less time consuming
  • More consistent recovery of many antigens, compared to large batch microwave technique
A

Pressure Cooking Antigen Retrieval

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18
Q

Antigens
* Uses:–

A

Diagnosis of tumor–Determination of tumor type–
Evaluation for potential proliferation–
Identification of infectious agents –
Prognostic and therapeutic implications

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19
Q

Epithelial Tumor Markers

A

Keratin
Epithelial Membrane Antigen (EMA)
Carcinoembryonic Antigen (CEA)
Thyroid Transcription Factor-1 (TTF-1)
Prostate-Specific Antigen (PSA)

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20
Q

– highly sensitive, present in epithelial tumors
(carcinoma)

A

Keratin

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21
Q

carcinomas of lung, breast, uterus, and ovaries (serous tumors)

A

CK7 (Cytokeratin 7) –

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22
Q

– carcinomas of colon and stomach

A

CK20

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23
Q

– Transitional Cell carcinomas of the
bladder and mucinous ovarian tumor

A

CK7 and CK20

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24
Q

– high molecular weight protein that is helpful in determining the site
of tumor. – It is positive for adenocarcinomas of the ,,_

A

Epithelial Membrane Antigen (EMA)
- breast, lungs, and kidneys

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25
oncofetal antigen present in carcinomas of the GIT, pancreas, lung, breast, ovary, uterus, and cervix. – It is useful for differentiating_,_
Carcinoembryonic Antigen (CEA) - adenocarcinoma (+) and mesothelioma (-)
26
– useful in distinguishing lung adenocarcinomas from mesotheliomas
Thyroid Transcription Factor-1 (TTF-1)
27
– useful in the diagnosis of prostatic adenocarcinoma
Prostate-Specific Antigen (PSA)
28
Intermediate filament markers
Actin Vimentin Desmin Glial fibrillary acidic protein (GFAP) Neurofilament (NF) S100 Protein
29
– sensitive marker for muscle differentiation and can be used to identify tumors derived from muscles
actin
30
– a 57kD intermediate filament that is present in normal mesenchymal cells and their neoplastic counterparts– _, _ (+) for it
vimentin - melanomas and schwannomas stain
31
– a 53kD intermediate filament expressed by smooth and striated muscles. It is highly specific for myogenic tumors.
Desmin
32
– a 51kD intermediate filament protein expressed by CNS glial cells, particularly _
Glial fibrillary acidic protein (GFAP) - astrocytes
33
– expressed in cells of neural origin, particularly neurons, neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla, and neuroendocrine cell
Neurofilament (NF)
34
– 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 glands
S100 Protein
35
Neuroendocrine Markers
Neuron-specific enolase (NSE) Chromogranin Synaptophysin
36
– provides strong evidence of neural or neuroendocrine differentiation
Neuron-specific enolase (NSE)
37
– a marker for neuroendocrine differentiation
Chromogranin
38
– a 38kD transmembrane protein associated with presynaptic vesicles of neurons
Synaptophysin
39
Germ Cell Tumor Markers
Human Chorionic Gonadotropin (HCG) Alpha-fetoprotein (AFP) Placenta-like alkaline phosphatase (PLAP)
40
synthesized by placental syncytiotrophoblasts – a marker for choriocarcinoma
HUman Chorionic Gonadotropin (HCG)–
41
– marker for endodermal sinus tumors showing yolk sac differentiation
Alpha-fetoprotein (AFP)
42
marker for germ cell tumors, particularly germinomas
Placenta-like alkaline phosphatase (PLAP) –
43
Mesenchymal Tumor Markers
Myogenic Tumors Fibrohistiocytic tumors Vascular Tumors Melanomas Lymphomas
44
* Myogenic Tumors
positive for muscle-specific actin, desmin, myo D1, myoglobin, and myogenin
45
* Fibrohistiocytic tumors
CD 68, FAM 56, alpha-1 antitrypsin, alpha-1-antichymotrypsin
46
* Vascular Tumors
– Factor VII-related antigen, CD 31, Ulex europaeus 1 (UEA)
47
Melanomas–
S100 protein, melanosome (HMB-45), Melan-A (MART-1)
48
Lymphomas
Leukocyte Common Antigen (LCA) or also called CD 45. Immunophenotypic, subclass: - - T cell : CD3, CD4, CD8 - B cells: CD19, CD20, CD23 - Reed-Sternberg cells: CD15, CD30, - Ig light and heavy chains
49
* Most common are __,__ * Increased expression = greater aggressiveness and higher chance of recurrence of metastasis
Cell Proliferation Markers Ki-67 (MIB-1) and PCNA (Proliferating Cell Nuclear Antigen)
50
in order to test for specificity of the antibodies involved,- to avoid misinterpretation due to false positive or false negative results
Immunohistochemistry Controls-
51
Ø Known tissue section Ø Contains the antigen
POSITIVE
52
Ø Paralel section from the tissue Ø Omitting the primary antibody Ø Replacing the specific primary antibody
NEGATIVE
53
Ø “built-in” control Ø Also contains the target antigen in both normal and abnormal tissue element
INTERNAL TISSUE CONTROL
54
Ø Usualy incubated with a chromogen Ø e.g. __ Ø Optimal incubation time = __
Enzyme Labeling - Horseradish peroxidase + Diaminobenzidine = INSOLUBLE DARK BROWN REACTION - 30-60 minutes @ room temp
55
Ø Conjugate the 1o antibody directly to the label Ø Simple and quick Ø Less sensitive Ø May not detect smaLl amounts of antigen
DIRECT TECHNIQUE
56
Reduced number of incubation steps Rapid staining is completed in 10 mins. More sensitive For both _ AND _ Eliminates non-specific staining activity
Enzyme Polymer One step staining (EPOS) - polyclonal and monoclonal antibodies
57
- inexpensive - more sensitive - most commonly enzyme __ maybe 2/3 steps
indirect tecnique - horseradish peroxidase
58
ØRecently introduced Ø4-8x more sensitive ØColor reaction is developed with appropriate substrate/ chromogen:
Labeled Streptavidin Avidin Biotin (LSAB) Technique ü1o rabbit antibody üBiotinylated anti-rabbit immunoglobulin üStreptavidin-enzyme conjugate
59
* It is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section
In – Situ Hybridization
60
* Shares some similarities with immunohistochemistry * Based on the specificity of the interaction of a probe with the target nucleic acid, rather than the target protein or immunogen * It has high sensitivity
In – Situ Hybridization
61
__ is simply a known fragment of nucleic acid which forms a hybrid molecule with a sample that has nucleic acids complementary to its sequence
In – Situ Hybridization - Probe
62
The process of searching a sample for specific nucleic acid sequences is called__
"Hybridization reaction_
63
RAPID TISSUE PROCESSING FIXATION
UMFIX (METHANOL AND PEG SOLUTION
64
RAPID TISSUE PROCESSING STAINING
METALLIC STAINS (METHANAMINE SILVER)
65
RAPID TISSUE PROCESSING IMMUNOHISTOCHEMISTRY
37 DEGREES CELCIUS FOR 2 3 MINS

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