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Histopath LEC > IMMUNOHISTOCHEMISTRY Prt 1 > Flashcards

IMMUNOHISTOCHEMISTRY Prt 1 Flashcards

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1
Q

Pre-analytical steps

A

A. Biopsy
B. Accessioning
C. Grossing
D. Tissue Processing and Embedding
E. Sectioning

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2
Q

Analytical Steps

A

A. Antigen Retrieval
B. Blocking
C. Primary Antibody
D. Enzyme-HRP
E. Counterstaining
F. Dehydration, Mounting, and Coverslipped

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3
Q

• Used in histology to detect the presence of specific protein marker that can assist with accurate tumor classification and diagnosis.

A

IMMUNOHISTOCHEMISTRY (IHC)

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4
Q

• It is used in the pathology laboratory as an aid in the differential diagnosis and classification of cancer, and for certain other diseases, including infections.

A

IMMUNOHISTOCHEMISTRY (IHC)

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5
Q

The factors that influence the_____ result start in the surgery operating room and end at the interpretation of the stain by the pathologist, which ultimately leads to treatment decision by the oncologist.

A

immunohistochemical staining

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6
Q

In the Accessioning room, the sample details are entered into the…

A

Laboratory Information System (LIS)

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7
Q

• A _____can ensure track and trace capabilities.

A

barcoded label

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8
Q

• The specimen is visually examined for suspicious areas that require further examination.

A

GROSSING / GROSS EXAMINATION

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9
Q

Samples from the specimen that require further microscopic testing are excised as tissue blocks and placed in..

A

barcoded cassettes

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10
Q

processed into a form and condition suitable for making ultrathin microscopic sections.
• Typically, the tissue is fixed in formalin and then dehydrated before it is embedded in paraffin.

A

TISSUE PROCESSING AND EMBEDDING
• The steps where the tissue block is

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11
Q

The fine art of cutting the paraffin-embedded tissue blocks into ultrathin (_____) sections and placing them onto glass slides.

A

-4 um

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12
Q

A_____ on the slide can ensure traceability and may also contain protocol information for the requested test for that particular section.

A

barcode

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13
Q

• After sectioning, the sample will be fished-out from the flotation bath and will undergo ______to know if the sample is positive or negative.

If positive =_______

A

H&E staining

request for IHC

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14
Q

END PRODUCTS OF PRE-ANALYTICAL STEPS:

If we already have these four, then we can request an IHC.

A
  1. Histopath result/report
  2. Blocks
  3. Slides -› read by the pathologist for confirmation.
  4. Histopath Worksheet (sometimes only)
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15
Q

• The analytical part of the IHC process.
• It encompasses antigen retrieval, application of the primary antibody and visualization system, and ending with counterstaining.

A

STAINING

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16
Q

STAINING

It encompasses…

A

antigen retrieval
application of the primary antibody and visualization system
ending with counterstaining.

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17
Q

ANALYTICAL STEPS

A
  1. Antigen Retrieval
  2. Blocking
  3. Primary Antibody
  4. Enzyme-HRP
  5. Counterstaining
  6. Dehydration, Mounting, and Coverslipped
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18
Q

• It is performed to recover the antigens that may have been altered by fixation.

A

ANTIGEN RETRIEVAL

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19
Q

• Endogenous enzymes are blocked (this step can also be performed after primary antibody incubation)

A

BLOCKING

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20
Q

Can be done before or after primary antibody.
Block nonspecific antibodies.

A

BLOCKING

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21
Q

• It is applied to specifically binds to the antigen of interest.

A

PRIMARY ANTIBODY

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22
Q

(HRP)

A

ENZYME-HORSERADISH PEROXIDASE

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23
Q

• The secondary antibody carries the label (enzyme)

A

ENZYME-HORSERADISH PEROXIDASE (HRP)

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24
Q

• Upon application, it binds to the primary antibody.

A

ENZYME-HORSERADISH PEROXIDASE (HRP)

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25
• It is performed to visualize nuclei and overall tissue architecture.
COUNTERSTAINING
26
But before counterstaining.____ is performed:
DAB+ Diaminobenzidine (DAB)
27
• A chromogen -> gives color to the dyes. Will bind to the secondarv antibody to give color. • If there will be a color, everything is identified as positive.
Diaminobenzidine (DAB)
28
There is also an H&E staining that does not utilize secondary antibody.
Antigen + Antibody + Color (DAB)
29
• Counterstain used for IHC.
HEMATOXYLIN
30
In H&E staining, it is a primary stain while in IHC it is a counterstain.
Hematoxylin
31
In H&E staining procedure, dehydration is…
95, 100, 100, Xylene, Xylene, then mount.
32
Since we used xylene, we have a high refractive index of the sample. The mounting media to be used should be a… _____mounting media is not allowed because of xylene. The sample will have a white/milky appearance.
resinous mounting media/mountant. Aqueous
33
IHC slides should be coverslipped because:
• It is expensive to perform • It uses expensive reagents • It should be long-lasting
34
POST-ANALYTICAL PROCESS • The_____ interprets the stains in context with positive and negative tissue controls, using bright field microscopy. • The results are reported to the____ for treatment decision.
pathologist oncologist
35
POSITIVE AND NEGATIVE TISSUE CONTROLS • When fishing out, the____ is placed only on half of the slide (bottom part). While the_____ is placed on the other half (upper part). • Every slide has a_____.
sample control
36
• IV. SECTIONING Use high-quality slides to provide High-Quality sections. Take particular care to use thin, flat sections that have been thoroughly dried onto the slide. Preferably use_______ or ______ for IHC
charged slides APES-coated slides
37
o Much more expensive and durable than frosted-end slides.
Charged slides
38
During IHC, the temperature used in the water bath is around… Withstand high temperatures…
95C Charged slides
39
• These controls must be processed identically to the specimen but contain the target protein.
INTERNAL TISSUE CONTROLS
40
In some cases, it will be advantageous to have this control tissue stain only _______as to monitor not only for the presence of the antigen but also for any possible loss of sensitivity.
marginally positive
41
IHC STAINING PROCEDURE MATERIALS
• Drying Oven • Pipettes and Tips • Control Tissue (+) • Tissue Paper • Humid Chamber • DAKO FLEX • Staining Jar • Dako Concentrated • Timers • Coverslips • Distilled Water • Dako Antibody Diluent • Charged Slides • Envision FLEX Target Retrieval Solution (TRS) (high pH) • Reagents For Deparaffinization • Envision FLEX Target Retrieval Solution (TRS) (low pH) • Water Bath › 95-99 Degrees Celcius • Temperature-controlled water bath should • Wash buffer • Hematoxylin • Dako pen
42
• the sensitivity and specificity are the core elements
PRIMARY ANTIBODY
43
• must provide both a both high sensitivity and a high specificity to produce accurate and robust IHC assay.
PRIMARY ANTIBODY
44
- they are able to detect or react immediately to the presence of antigen - the antibody must react to a specific antigen only and nothing else
Sensitive Specific
45
2 categories of primary antibody
(1) Monoclonal (2) Polyclonal
46
• they have become widely used because of their high specificity, consistency, purity and commercial availability.
MONOCLONAL ANTIBODIES
47
MONOCLONAL ANTIBODIES • produced in____ according to the in______ method developed by_____
mice vitro hybridoma Köhler
48
one: reacts to one antigen (Ag) thus it has high specificity since it only reacts to a specific type of antigen
Monoclonal
49
POLYCLONAL ANTIBODIES • produced in____ by ______techniques, with booster immunizations to maximize the reactivity against the target antigen
rabbits traditional immunization
50
• frequently give a higher sensitivity (avidity) compared to monoclonal antibodies, as the many antibody 'species' present react with more antigen sites
Polyclonal antibody
51
many: reacts to many antigen (Ag) thus it has high sensitivity since it is sensitive to many types of antigen
Polyclonal antibody
52
• the solution used in antigen retrieval, where it unmasks the antigens, for it to be visible and be binded by the primary antibodies
TARGET RETRIEVAL SOLUTION (TRS)
53
• basically, used to reveal antigenic sites/epitopes
TARGET RETRIEVAL SOLUTION (TRS)
54
the major drawback of Formalin-Fixed Paraffin-Embedded (FFPE) tissue
MASKING
55
when formalin-induced molecular modification of proteins (antigens) result in loss of the ability of the antibody to react with the antigen
MASKING
56
a loss that can only be corrected by the restoration (retrieval) of the 'formalin-modified' antigen molecular structure
Masking
57
TARGET RETRIEVAL SOLUTION (TRS) These improve antigen expression of samples by:
(1) breaking down formalin induced antigen cross-linking (2) re-exposing epitopes on the antigen to antibody binding
58
TRS Heat and enzyme retrieval are both employed, with_____, which is now being the most commonly used.
Heat-Induced Epitope Retrieval (HIER)
59
Heat-Induced Epitope Retrieval (HIER) Temperature: Buffer:
>95 pH6 or pH9
60
TRS • 2 categories:
• HIGH pH (pH 9) o blue color • LOW pH (pH 6) o pink color
61
• ER and PR can be processed together in TRS since both of them utilizes _____ pH • HER2/neu will be processed separately since it utilizes____ pH
high low
62
Machine used in trs
• Microwave • Pressure cooker • Water bath
63
HYDROPHOBIC PEN • Ex.
Dako Pen, Pap Pen
64
its purpose is to prevent the water from escaping within the encircled area
HYDROPHOBIC PEN
65
- read by MTs It refers to the percentage of the positive results in the sample
Proportion Score
66
- read by Pathologists
Intensity Score
67
Allred score = positive =
PS + IS 23

Histopath LEC (37 decks)

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