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Histopath LEC > IMMUNOHISTOCHEMISTRY Prt 1 > Flashcards

IMMUNOHISTOCHEMISTRY Prt 1 Flashcards

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
Q

• It is performed to visualize nuclei and overall tissue architecture.

A

COUNTERSTAINING

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26
Q

But before counterstaining.____ is performed:

A

DAB+

Diaminobenzidine (DAB)

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27
Q

• 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.

A

Diaminobenzidine (DAB)

28
Q

There is also an H&E staining that does not utilize secondary
antibody.

A

Antigen + Antibody + Color (DAB)

29
Q

• Counterstain used for IHC.

A

HEMATOXYLIN

30
Q

In H&E staining, it is a primary stain while in IHC it is a counterstain.

A

Hematoxylin

31
Q

In H&E staining procedure, dehydration is…

A

95, 100, 100, Xylene, Xylene, then mount.

32
Q

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.

A

resinous mounting media/mountant.

Aqueous

33
Q

IHC slides should be coverslipped because:

A

• It is expensive to perform
• It uses expensive reagents
• It should be long-lasting

34
Q

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.

A

pathologist

oncologist

35
Q

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_____.

A

sample

control

36
Q

•
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

A

charged slides
APES-coated slides

37
Q

o Much more expensive and durable than frosted-end slides.

A

Charged slides

38
Q

During IHC, the temperature used in the water bath is around…

Withstand high temperatures…

A

95C

Charged slides

39
Q

• These controls must be processed identically to the specimen but contain the target protein.

A

INTERNAL TISSUE CONTROLS

40
Q

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.

A

marginally positive

41
Q

IHC STAINING PROCEDURE MATERIALS

A

• 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
Q

• the sensitivity and specificity are the core elements

A

PRIMARY ANTIBODY

43
Q

• must provide both a both high sensitivity and a high specificity to produce accurate and robust
IHC assay.

A

PRIMARY ANTIBODY

44
Q
  • 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
A

Sensitive

Specific

45
Q

2 categories of primary antibody

A

(1) Monoclonal (2) Polyclonal

46
Q

• they have become widely used because of their high specificity, consistency, purity and commercial availability.

A

MONOCLONAL ANTIBODIES

47
Q

MONOCLONAL ANTIBODIES

• produced in____ according to the in______ method developed by_____

A

mice

vitro hybridoma

Köhler

48
Q

one: reacts to one antigen (Ag)

thus it has high specificity since it only reacts to a specific type of antigen

A

Monoclonal

49
Q

POLYCLONAL ANTIBODIES
• produced in____ by ______techniques, with booster immunizations to maximize the reactivity against the target antigen

A

rabbits

traditional immunization

50
Q

• frequently give a higher sensitivity (avidity) compared to monoclonal antibodies, as the many antibody ‘species’ present react with more antigen sites

A

Polyclonal antibody

51
Q

many: reacts to many antigen (Ag)
thus it has high sensitivity since it is sensitive to many types of antigen

A

Polyclonal antibody

52
Q

• the solution used in antigen retrieval, where it unmasks the antigens, for it to be visible and be binded by the primary antibodies

A

TARGET RETRIEVAL SOLUTION (TRS)

53
Q

• basically, used to reveal antigenic sites/epitopes

A

TARGET RETRIEVAL SOLUTION (TRS)

54
Q

the major drawback of
Formalin-Fixed
Paraffin-Embedded (FFPE) tissue

A

MASKING

55
Q

when formalin-induced molecular modification of proteins (antigens) result in loss of the ability of the antibody to react with the antigen

A

MASKING

56
Q

a loss that can only be corrected by the restoration (retrieval) of the ‘formalin-modified’ antigen molecular structure

A

Masking

57
Q

TARGET RETRIEVAL SOLUTION (TRS)

These improve antigen expression of samples by:

A

(1) breaking down formalin induced antigen cross-linking

(2) re-exposing epitopes on the antigen to antibody binding

58
Q

TRS

Heat and enzyme retrieval are both employed, with_____, which is now being the most commonly used.

A

Heat-Induced Epitope Retrieval (HIER)

59
Q

Heat-Induced Epitope Retrieval (HIER)

Temperature:
Buffer:

A

> 95

pH6 or pH9

60
Q

TRS

• 2 categories:

A

• HIGH pH (pH 9)
o blue color
• LOW pH (pH 6)
o pink color

61
Q

• 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

A

high

low

62
Q

Machine used in trs

A

• Microwave
• Pressure cooker
• Water bath

63
Q

HYDROPHOBIC PEN
• Ex.

A

Dako Pen, Pap Pen

64
Q

its purpose is to prevent the water from escaping within the encircled area

A

HYDROPHOBIC PEN

65
Q
  • read by MTs
    It refers to the percentage of the positive results in the sample
A

Proportion Score

66
Q
  • read by Pathologists
A

Intensity Score

67
Q

Allred score =

positive =

A

PS + IS

23

Histopath LEC (37 decks)

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