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Anatomic Pathology > Immunohistochemistry > Flashcards

Immunohistochemistry Flashcards

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
Q

Deparaffinization and rehydration (time)

A
  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)
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26
Q

Target retrieval

A

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

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27
Q

Hydrogen peroxide blocking (time)

A

5 minutes

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28
Q

Primary ab (time)

A

20 mins

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29
Q

Washing in buffer (time)

A
  1. Dip 10 times
  2. Dip 10 times
  3. Dip 1 min
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30
Q

Labelled secondary ab (time)

A

15 minutes

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31
Q

Chromogen (time)

A

10 mins

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32
Q

Types of chromogen

A 
Peroxidase
AEC
DAB (diaminobenzidine)
Vector S-G
Vector - VIP
Vector Nova Red
Alk Phos
New Fuchsin
Fast Red
BCIP/NBT
Vector red
Vector black
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33
Q

Wash in distilled water (time)

A

1 min

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34
Q

Counterstaining

A

Hematoxylin, Light green, methyl green for 30 sec to 1 min

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35
Q

False negative causes

A 
Antigen loss
Inadequate ag retrieval
Method not sensitive enough
Expired ab
Wrong reagents or wrong reagent sequence
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36
Q

A method for localizing specific ags in tissues or cells based on ag-ab recognition.

A

Immunohistochemistry

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37
Q

A pair of light chains and a heavy chain

A

Antibody

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38
Q

Any molecule that is sufficiently complex to induce ab formation

A

Antigen

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39
Q

Antigenic determinant.

The exact site on the molecule with which the ab combines.

A

Epitope

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40
Q

Heterogenous mix of abs that recognize several epitopes on an antigen

A

Polyclonal

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41
Q

Recognizes a single type of epitope

A

Monoclonal

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42
Q

Unconjugated (unlabelled) antibody, raised against the ag of interest

A

Primary ab

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43
Q

Ab raised against the primary abs, conjugated either to biotin or to an enzyme or to fluorescent agents

A

Secondary antibodies

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44
Q

Applications of IHC

A

Diagnostic
Theranostic
Genomic

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45
Q

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

A

Theranostic

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46
Q

IHC as theranostic

A

HER2/neu analysis
c-kit testing for GISTS
CD20 in B cell lymphomas

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47
Q

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

A

Genomic

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48
Q

Examples of IHC as genomic

A

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.

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49
Q

ER (+)
PR (+)
HER2 (-)

A

Luminal A

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50
Q

ER (+)
PR (+)
HER2 (+)

A

Luminal B

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51
Q

ER (-)
PR (-)
HER2 (+)

A

HER2

52
Q

ER (-)
PR (-)
HER2 (-)

A

Basal-like

Perform CK5/6 and EGFR to define more precisely tumors in basal-like group.

53
Q

Molecular category of Breast CA with high expression of hormone receptors and associated genes (luminal A > luminal B)

A

Luminal

54
Q

Molecular category of breast CA with high expression of HER2 and other genes in amplicon. Low expression of ER and associated genes.

A

HER2

55
Q

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.

A

Basal-like

56
Q

BRCA1-associated cancers

A

Basal-like

57
Q

Therapy for luminal type Breast CA

A 
Endocrine therapy (response may be different for luminal A and luminal B)
Chemotherapy variable (luminal B > luminal A)
58
Q

Therapy for HER2 type breast CA

A

Trastuzumab (herceptin)

Anthracycline-based chemotherapy

59
Q

Therapy for basal-like breast CA

A

Platinum-based chemotherapy

60
Q

Prognosis: luminal type breast ca

A

Favorable

61
Q

Prognosis: HER2 type breast ca

A

Poor

62
Q

Prognosis: Basal-like breast ca

A

Poor

63
Q

Cornerstone in tumor diagnosis

A

Morphology

64
Q

Uses of IHC

A

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
Q 
PanCK (+)
LCA (-)
S100/HMB45/MART-1 tyrosinase  (-)
Desmin (-)
Vimentin (-)
A

Carcinoma

66
Q 
PanCK (-)
LCA (+)
S100/HMB45/MART-1 tyrosinase  (-)
Desmin (-)
Vimentin (-)
A

Lymphoma

67
Q 
PanCK (-)
LCA (-)
S100/HMB45/MART-1 tyrosinase  (+)
Desmin (-)
Vimentin (+)
A

Melanoma

68
Q 
PanCK (-)
LCA (-)
S100/HMB45/MART-1 tyrosinase  (-)
Desmin (+)
Vimentin (+)
A

Sarcoma

69
Q

Mesenchymal

A

Vimentin

70
Q

CK, EMA

A

Epithelial

71
Q

Desmin, HHF35, SmActin

A

Smooth muscle

72
Q

Myoglobin

A

Skeletal muscle

73
Q

CD68, Factor XIIIa

A

Fibrohistiocyte

74
Q

Leu7, GFAP

A

Nerve sheath

75
Q

HMB45

A

Melanocyte

76
Q

Neurofilament

A

Neuronal

77
Q

Factor VIII, CD34, CD31

A

Endothelial, perivascular

78
Q

LCA, CD3, CD20

A

Hematopoietic

79
Q

NSE, Chromogranin

A

Neuroendocrine

80
Q

MIC-2(O-13), CD99

A

Ewing’s sarcoma/PNET

81
Q

Transbronchial FNA of a lung lesion showing mostly isolated small cells with pyknotic nuclei. Ddx?

A

Small cell CA: CK(+) LCA(-)

Lymphoma: CK(-) LCA (+)

82
Q

Patients having no identifiable primary site, despite a careful clinical hx, PE, radiologic imaging and biochemical and histologic investigations

A

Metastatic Carcinoma of Unknown Primary (MCUP)

83
Q

Diagnostic approach to MCUP

A

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
Q

CK7+/CK20+

A

Transitional cell carcinoma
Pancreatic carcinoma
Ovarian mucinous carcinoma
50% of gastric CA

85
Q

CL7-/CK20+

A

Colorectal adenoCA

Merkel cell carcinoma

86
Q

CK7+/CK20-

A 
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
Q

CK7-/CK20-

A 
Squamous cell CA of the lung
Prostate adenoCA
Renal cell CA
Hepatoma
Thymus
88
Q

Prostate (MCUP)

A

PSA
Se 100
Sp 99

89
Q

Lung (MCUP)

A

TTF-1
Se 91
Sp 98

90
Q

Colon

A

CDX-2
Se 83
Sp 96

CK20
Se 68
Sp 91

91
Q

Colon and stomach

A

CDX-2
Se 56
Sp 98

92
Q

Colon, stomach, pancreas

A

CK20
Se 36
Sp 97

93
Q

Breast

A

GCDFP-15
Se 54
Sp 96

94
Q

Breast and ovary

A

ER
Se 74
Sp 95

95
Q

Ovary and pancreas

A

CA 125
Se 88
Sp 88

Mesothelin
Se 85
Sp 85

96
Q

Stomach and pancreas

A

Lysozyme
Se 65
Sp 69

CK 7
Se 72
Sp 96

97
Q

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

A

Metastatic thyroid CA: TTF-1(+) nuclear
TGB(+) cytoplasmic, membranous

Lung adenoCA:
TTF-1(+) nuclear
TGB(-)

98
Q

Poorly differentiated non-small cell CA in bronchial brushings

A

Lung Squamous cell CA:
p63 (+)
TTF-1 (-)

Lung AdenoCA:
p63 (+)
TTF-1 (+)

99
Q

Pleural fluid in a 79yo male. The ddx includes reactive mesothelium, malignant mesothelioma, and adenoCA of lung

A

Malignant mesothelioma:
Calretinin (+)
TTF-1 (-)

Reactive mesothelium:
EMA- isolated reactive mesothelial cells are highlighted

100
Q

Pleural fluid cytology from a 64yo female with no prior hx of malignancy. The ddx includes adenoCA and mesothelioma.

A

Metastatic adenocarcinoma:
Calretinin (-)
TTF-1 (+)

101
Q

IHC in Hematopathology

A

Distinguishing reactive conditions from lymphomas.
Distinguishing lymphomas from other malignancies
Classification of lymphomas
Prognosis and staging of lymphoma
Assay of the therapeutic targets

102
Q

Work-up lineage for lymphoma

A

CD20+: B cell lymphoma
CD3+: T cell or NK cell lymphoma
CD20-/CD3-: nonhematolymphoid, plasmacytoma, anaplastic lymphoma, t cell lymphoma, lymphoblastic lymphoma

103
Q

Important markers for classifying lymphomas:

Precursor lymphoblastic lymphoma

A

TdT

104
Q

Important markers for classifying lymphomas:

Chronic lymphocytic lymphoma

A

CD5, CD23

105
Q

Important markers for classifying lymphomas:

Mantle cell lymphoma

A

Cyclin D1 (aka BCL-1 and PRAD): nuclear positivity

106
Q

Important markers for classifying lymphomas:

Follicular lymphoma

A

CD10 (or BCL-6)

107
Q

Important markers for classifying lymphomas:

Burkitt lymphoma

A

Ki67 (~100% proliferation index)

108
Q

Important markers for classifying lymphomas:

Angioimmunoblastic T cell lymphoma

A

CD10, follicular dendritic cell markers (extrafollicular meshworks)

109
Q

Important markers for classifying lymphomas:

Anaplastic large cell lymphoma

A

CD30, ALK

110
Q

Biomarkers in prostate CA

A

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
Q

GCDFP-15 meaning

A

Gross cystic disease fluid protein - 15

112
Q

IHC panel of small round cell tumors in children

A

CK - carcinoma
LCA - lymphoma
Desmin - rhabdomyosarcoma
CD99 - Ewings sarcoma/PNET

113
Q

IHC panel of small round cell tumors in adults

A

CK - carcinoma
LCA - lymphoma
Vimentin, S100 - sarcoma
Chromogranin/Synaptophysin - PNET

114
Q

IHC panel of endometrial and endocervical, primary

A

Both: CK7(-)/CK20(+)

Endocervical: p16, CEA
Endometrial: vimentin, ER

115
Q

IHC panel in subclassification of the most common lung cancers

A

SCCA: p63, CK5/6, CK7(-)
AdenoCA: TTF-1, CK7(+)
Small cell: chromogranin/synaptophysin, variable CK positivity

116
Q

IHC panel of atypical small acinar proliferation in prostate biopsy cases.

A

AMACR

Myoepithelial markers: p63, 34BA12 or HMWCK, SMA

117
Q

IHC panel of DCIS vs Invasive Ductal Carcinoma breast biopsy cases

A

Myoepithelial markers: p63, actin, SMA

CK5/6: (+)ADH with low grade DCIS

118
Q

IHC panel of mature B cell neoplasm

A 
Follicular lymphoma: Bcl-2
MALT: cytokeratin
3
4
5
119
Q

IHC panel in differentiating HL from NHL

A 
LCA
CD30: golgi and cytoplastmic membrane
CD15: golgi and cytoplasmic membrane
CD20
CD3
Fascin (cytoplasmic expression in HL)
120
Q

IHC panel in differentiating lung, thyroid, hepatocellular, primary.

A

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
Q

Pattern of bcl-2 expression in reactive lymphadenopathy

A

Interfollicular and paracortical

122
Q

Pattern of bcl-2 expression in follicular lymphoma

A

Within the follicles

123
Q

Olfactory neuroblastoma

A

CD56 (NCAM)

NSE

124
Q

Medium-sized lymphoma

A

Lymphoblastic lymphoma: TdT

Follicular lymphoma

125
Q

Positive for HMB-45

A

Epitheloid angiomyolipoma

Melanoma

126
Q

Renal cell CA

A

RCC, CD10

Anatomic Pathology (3 decks)

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