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Anatomic Pathology > Immunohistochemistry > Flashcards

Immunohistochemistry Flashcards

(126 cards)

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1
Q

4 steps of IHC staining

A
  1. Fixation and processing
  2. Blocking non-specific background staining
  3. Detection systems
  4. Antigen retrieval
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2
Q

Purpose of Fixation in IHC

A

To optimize preservation because this affects morphologic and immunohistologic results.

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3
Q

Qualities of a good fixative

A
  1. Should preserve antigenic integrity
  2. Should limit extraction, diffusion or displacement of antigen during processing
  3. Should give good preservation of morphology
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4
Q

Coagulant fixative

A

Ethanol

Produce fewer changes in intermediate filaments and immunoglobulins, than cross-linking fixative

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5
Q

Cross-linking fixative

A

Formaldehyde

Alter the IHC results for a significant number of antigens

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6
Q

Advantages of formalin as fixative

A

(Fixation time dependent)

  1. Preservation of morphology
  2. Economical
  3. Sterilize tissue specimen
  4. Carbohydrate antigens are better preserved
  5. Low molecular weight antigens are well-preserved
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7
Q

2 aspects to blocking of background staining of tissues

A
  1. Nonspecific antibody binding

2. Presence of endogenous enzymes

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8
Q

Methods of blocking nonspecific background staining

A
  1. Quenching endogenous peroxidase by an H202 methanol solution
  2. Blocking endogenous biotin by using the avidin-biotin blocking agent, or skim milk
  3. Normal serum will act as a secondary Ab to block nonspecific binding sites
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9
Q

2 types of detection system

A
  1. Direct

2. Indirect

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10
Q

Principle of detection system

A

Attach certain labels or flags to antibodies in order to visualize the target ab-ag localization in the tissue sections

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11
Q

Properties of direct conjugate-labeled antibody method

A

Uses one labelled ab, which binds directly to ag.
Purity or specificity.
Rapid, easy.
Monoclonal.
Sensitivity is low due to low signal amplification.

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12
Q

Properties of indirect or sandwich detection system

A

Versatility is increased.
The primary ab can often be used at a higher working dilution to achieve successful staining.
The secondary ab is prepared with high order of specificity and affinity.
More sensitive due to signal amplification through several secondary ab reactions with different ag sites in the primary ab.

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13
Q

Streptavidin-peroxidase complex as detection system

A

Peroxidase enzyme oxidizes DAB, turning it into a brown pigment.
This pigment precipitates out of solution as a brown solif, located at the site of the antigen.

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14
Q

2 types of antigen retrieval or epitope unmasking

A

Enzyme (trypsin, pepsin, pronase, ficin, prot K, protease 24, saponin.
HIER (WB, Pressure-cooker, microwave, autoclave, etc)

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15
Q

HIER retrieval solutions / buffers

A

Na Citrate pH 6
EDTA pH 8
citric acid buffers
Tris buff pH 9.5

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16
Q

Consistent results in terms of ag preservation

A

10% NBF
10% zinc formalin
10% formal saline

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17
Q

The first antigen retrieval method to enhance staining

A

Enzymatic digestion

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18
Q

Light to Med at 37C for AE1/AE3, B72.3, Factor XIIIa, HAM-56

A

Trypsin

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19
Q

Strong at 37C for CK7, CK20, Pan CK, Lu-5(7’)

A

Pepsin

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20
Q

Extra strong at room temp for EGFR (5’)

A

Protease 24

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21
Q

Very very strong.

Conc x time study necessary

A

Protinase K

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22
Q

3 broad categories with respect to the importance of pH in Ag Retrieval

A
  1. Most ag showed no sig variation using AR solutions with pH ranging 1-10.
  2. Nuclear ag (ER) showed dramatic decrease in intensity at mid-range pH but optimal at low pH.
  3. Some ag (HMB 45) are weak at low pH (1-2) but excellent in the high range.
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23
Q

IHC staining steps (shortcut)

A 
Deparaffinization
Ag retrieval
Block unoccupied sites
Add primary abs
Add secondary abs
Add substrates for color development
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24
Q

IHC staining Steps (Long steps)

A 
Deparaffinization and rehydration
Rinse with buffer
Target retrieval - cool
Rinse in tap water and distilled water
Block endogenous peroxidase
Rinse with buffer
Primary ab
Rinse with buffer 
Secondary ab
Rinse with buffer
DAB
Rinse with distilled water
Hematoxylin
Rinse with tap water
Coveralip with aqueous resin
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25
Deparaffinization and rehydration (time)
1. Xylene - 5mins 2. Xylene - 5mins 3. Absolute ethanol - 3mins 4. Absolute ethanol - 3mins 5. 95% ethanol - 3mins 6. 95% ethanol - 3mins 7. Distilled water (30 secs to 1 min)
26
Target retrieval
Total: 15-18mins plus 20 mins cooling time = 35-38 mins ``` High - 5mins Med High/Low - 5mins Med - 5mins Med - 3mins Cooling time - 20mins ``` In 700-800 watts microwave oven
27
Hydrogen peroxide blocking (time)
5 minutes
28
Primary ab (time)
20 mins
29
Washing in buffer (time)
1. Dip 10 times 2. Dip 10 times 3. Dip 1 min
30
Labelled secondary ab (time)
15 minutes
31
Chromogen (time)
10 mins
32
Types of chromogen
``` Peroxidase AEC DAB (diaminobenzidine) Vector S-G Vector - VIP Vector Nova Red Alk Phos New Fuchsin Fast Red BCIP/NBT Vector red Vector black ```
33
Wash in distilled water (time)
1 min
34
Counterstaining
Hematoxylin, Light green, methyl green for 30 sec to 1 min
35
False negative causes
``` Antigen loss Inadequate ag retrieval Method not sensitive enough Expired ab Wrong reagents or wrong reagent sequence ```
36
A method for localizing specific ags in tissues or cells based on ag-ab recognition.
Immunohistochemistry
37
A pair of light chains and a heavy chain
Antibody
38
Any molecule that is sufficiently complex to induce ab formation
Antigen
39
Antigenic determinant. | The exact site on the molecule with which the ab combines.
Epitope
40
Heterogenous mix of abs that recognize several epitopes on an antigen
Polyclonal
41
Recognizes a single type of epitope
Monoclonal
42
Unconjugated (unlabelled) antibody, raised against the ag of interest
Primary ab
43
Ab raised against the primary abs, conjugated either to biotin or to an enzyme or to fluorescent agents
Secondary antibodies
44
Applications of IHC
Diagnostic Theranostic Genomic
45
Used to describe the proposed process of diagnostic therapy for individual patients - to test them fir possible reactions to a new medication or to tailor a treatment based on the test resultr
Theranostic
46
IHC as theranostic
HER2/neu analysis c-kit testing for GISTS CD20 in B cell lymphomas
47
Facilitate recognition of specific genomic aberrations in the patient's tissues by identifying (or not identifying) the presence or absence of specific aberration or gene signature
Genomic
48
Examples of IHC as genomic
Testing for microsatellite stability in colorectal CA (MLH1, MSH2, MSH6 or PMS2). Identification of basal-like breast CA (high grade, ER/PR/HER2 (-), CK5/CK14/CK17 (+), variable EGFR expression.
49
ER (+) PR (+) HER2 (-)
Luminal A
50
ER (+) PR (+) HER2 (+)
Luminal B
51
ER (-) PR (-) HER2 (+)
HER2
52
ER (-) PR (-) HER2 (-)
Basal-like | Perform CK5/6 and EGFR to define more precisely tumors in basal-like group.
53
Molecular category of Breast CA with high expression of hormone receptors and associated genes (luminal A > luminal B)
Luminal
54
Molecular category of breast CA with high expression of HER2 and other genes in amplicon. Low expression of ER and associated genes.
HER2
55
Molecular category of breast CA with high expression of basal epithelial genes, basal cytokeratins; low expression of ER and associated genes; low expression of HER2.
Basal-like
56
BRCA1-associated cancers
Basal-like
57
Therapy for luminal type Breast CA
``` Endocrine therapy (response may be different for luminal A and luminal B) Chemotherapy variable (luminal B > luminal A) ```
58
Therapy for HER2 type breast CA
Trastuzumab (herceptin) | Anthracycline-based chemotherapy
59
Therapy for basal-like breast CA
Platinum-based chemotherapy
60
Prognosis: luminal type breast ca
Favorable
61
Prognosis: HER2 type breast ca
Poor
62
Prognosis: Basal-like breast ca
Poor
63
Cornerstone in tumor diagnosis
Morphology
64
Uses of IHC
Unknown tumor type (ie carcinoma vs lymphoma) Metastatic CA of unknown primary Further classification of CA and lymphomas Identification of in situ lesions vs invasive carcinomas for breast and prostate cancers Grading of gliomas ie Ki-68 Predictive factors to guide specific therapy ie c-Kit, ER/PR, Her2/neu Identification of extacellular material Identificatiin of infectious agents
65
``` PanCK (+) LCA (-) S100/HMB45/MART-1 tyrosinase (-) Desmin (-) Vimentin (-) ```
Carcinoma
66
``` PanCK (-) LCA (+) S100/HMB45/MART-1 tyrosinase (-) Desmin (-) Vimentin (-) ```
Lymphoma
67
``` PanCK (-) LCA (-) S100/HMB45/MART-1 tyrosinase (+) Desmin (-) Vimentin (+) ```
Melanoma
68
``` PanCK (-) LCA (-) S100/HMB45/MART-1 tyrosinase (-) Desmin (+) Vimentin (+) ```
Sarcoma
69
Mesenchymal
Vimentin
70
CK, EMA
Epithelial
71
Desmin, HHF35, SmActin
Smooth muscle
72
Myoglobin
Skeletal muscle
73
CD68, Factor XIIIa
Fibrohistiocyte
74
Leu7, GFAP
Nerve sheath
75
HMB45
Melanocyte
76
Neurofilament
Neuronal
77
Factor VIII, CD34, CD31
Endothelial, perivascular
78
LCA, CD3, CD20
Hematopoietic
79
NSE, Chromogranin
Neuroendocrine
80
MIC-2(O-13), CD99
Ewing's sarcoma/PNET
81
Transbronchial FNA of a lung lesion showing mostly isolated small cells with pyknotic nuclei. Ddx?
Small cell CA: CK(+) LCA(-) | Lymphoma: CK(-) LCA (+)
82
Patients having no identifiable primary site, despite a careful clinical hx, PE, radiologic imaging and biochemical and histologic investigations
Metastatic Carcinoma of Unknown Primary (MCUP)
83
Diagnostic approach to MCUP
Det the cell line of differentiation using major lineage markers Det the CK type or types of distribution in tumor cells Det if there is co-expression of vimentin Der if there is expression of supplemental ag of epithelial or germ cell derivation (CEA, EMA, PLAP) Det if there is expression of cell-specific products, cell-specific structures or receptors that are unique identifiers of cell types (GCDFP, PSA, TTF-1)
84
CK7+/CK20+
Transitional cell carcinoma Pancreatic carcinoma Ovarian mucinous carcinoma 50% of gastric CA
85
CL7-/CK20+
Colorectal adenoCA | Merkel cell carcinoma
86
CK7+/CK20-
``` Non-small cell CA of the lung Small cell CA of the lung Breast CA, ductal and lobular Nonmucinous ovarian CA Endometrial adenoCA Mesothelioma Squamous cell CA of the cervix ```
87
CK7-/CK20-
``` Squamous cell CA of the lung Prostate adenoCA Renal cell CA Hepatoma Thymus ```
88
Prostate (MCUP)
PSA Se 100 Sp 99
89
Lung (MCUP)
TTF-1 Se 91 Sp 98
90
Colon
CDX-2 Se 83 Sp 96 CK20 Se 68 Sp 91
91
Colon and stomach
CDX-2 Se 56 Sp 98
92
Colon, stomach, pancreas
CK20 Se 36 Sp 97
93
Breast
GCDFP-15 Se 54 Sp 96
94
Breast and ovary
ER Se 74 Sp 95
95
Ovary and pancreas
CA 125 Se 88 Sp 88 Mesothelin Se 85 Sp 85
96
Stomach and pancreas
Lysozyme Se 65 Sp 69 CK 7 Se 72 Sp 96
97
FNA of a cervical lymph node in a patient with no known primary cancer. Cytology is suggestive of a low grade adenoCA, lung (bronchoalveolar type) or thyroid origin
Metastatic thyroid CA: TTF-1(+) nuclear TGB(+) cytoplasmic, membranous Lung adenoCA: TTF-1(+) nuclear TGB(-)
98
Poorly differentiated non-small cell CA in bronchial brushings
Lung Squamous cell CA: p63 (+) TTF-1 (-) Lung AdenoCA: p63 (+) TTF-1 (+)
99
Pleural fluid in a 79yo male. The ddx includes reactive mesothelium, malignant mesothelioma, and adenoCA of lung
Malignant mesothelioma: Calretinin (+) TTF-1 (-) Reactive mesothelium: EMA- isolated reactive mesothelial cells are highlighted
100
Pleural fluid cytology from a 64yo female with no prior hx of malignancy. The ddx includes adenoCA and mesothelioma.
Metastatic adenocarcinoma: Calretinin (-) TTF-1 (+)
101
IHC in Hematopathology
Distinguishing reactive conditions from lymphomas. Distinguishing lymphomas from other malignancies Classification of lymphomas Prognosis and staging of lymphoma Assay of the therapeutic targets
102
Work-up lineage for lymphoma
CD20+: B cell lymphoma CD3+: T cell or NK cell lymphoma CD20-/CD3-: nonhematolymphoid, plasmacytoma, anaplastic lymphoma, t cell lymphoma, lymphoblastic lymphoma
103
Important markers for classifying lymphomas: | Precursor lymphoblastic lymphoma
TdT
104
Important markers for classifying lymphomas: | Chronic lymphocytic lymphoma
CD5, CD23
105
Important markers for classifying lymphomas: | Mantle cell lymphoma
Cyclin D1 (aka BCL-1 and PRAD): nuclear positivity
106
Important markers for classifying lymphomas: | Follicular lymphoma
CD10 (or BCL-6)
107
Important markers for classifying lymphomas: | Burkitt lymphoma
Ki67 (~100% proliferation index)
108
Important markers for classifying lymphomas: | Angioimmunoblastic T cell lymphoma
CD10, follicular dendritic cell markers (extrafollicular meshworks)
109
Important markers for classifying lymphomas: | Anaplastic large cell lymphoma
CD30, ALK
110
Biomarkers in prostate CA
PSA - cytoplasmic expression in glandular epithelium Basal cell markers - p63, HMW-CK Alpha-methyl-CoA racemase (AMACR) - recently identified as being overexpressed in prostatic adenocarcinoma (80%)
111
GCDFP-15 meaning
Gross cystic disease fluid protein - 15
112
IHC panel of small round cell tumors in children
CK - carcinoma LCA - lymphoma Desmin - rhabdomyosarcoma CD99 - Ewings sarcoma/PNET
113
IHC panel of small round cell tumors in adults
CK - carcinoma LCA - lymphoma Vimentin, S100 - sarcoma Chromogranin/Synaptophysin - PNET
114
IHC panel of endometrial and endocervical, primary
Both: CK7(-)/CK20(+) Endocervical: p16, CEA Endometrial: vimentin, ER
115
IHC panel in subclassification of the most common lung cancers
SCCA: p63, CK5/6, CK7(-) AdenoCA: TTF-1, CK7(+) Small cell: chromogranin/synaptophysin, variable CK positivity
116
IHC panel of atypical small acinar proliferation in prostate biopsy cases.
AMACR | Myoepithelial markers: p63, 34BA12 or HMWCK, SMA
117
IHC panel of DCIS vs Invasive Ductal Carcinoma breast biopsy cases
Myoepithelial markers: p63, actin, SMA | CK5/6: (+)ADH with low grade DCIS
118
IHC panel of mature B cell neoplasm
``` Follicular lymphoma: Bcl-2 MALT: cytokeratin 3 4 5 ```
119
IHC panel in differentiating HL from NHL
``` LCA CD30: golgi and cytoplastmic membrane CD15: golgi and cytoplasmic membrane CD20 CD3 Fascin (cytoplasmic expression in HL) ```
120
IHC panel in differentiating lung, thyroid, hepatocellular, primary.
Thyroid: CK7+/CK20- TTF-1(+): nuclear TGB(+): cytoplasmic Lung: CK7+/CK20- TTF-1(+): nuclear TGB(-): cytoplasmic Liver: CK7-/CK20- AFP: poorly diff hepatocellular CA Hepar-1: well to mod diff hepatocellular CA
121
Pattern of bcl-2 expression in reactive lymphadenopathy
Interfollicular and paracortical
122
Pattern of bcl-2 expression in follicular lymphoma
Within the follicles
123
Olfactory neuroblastoma
CD56 (NCAM) | NSE
124
Medium-sized lymphoma
Lymphoblastic lymphoma: TdT | Follicular lymphoma
125
Positive for HMB-45
Epitheloid angiomyolipoma | Melanoma
126
Renal cell CA
RCC, CD10

Anatomic Pathology (3 decks)

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