1. Second Half
 Break!

2. 2. Basic IHC techniques,
Detections and Standardization
Attendees will gain basic knowledge of:
 Detection Methods
fluorescent and enzymatic Labels
 Standardization Methods
Validation techniques

3. Immunohistochemistry-
Detection Methods
Tissue on
a slide
+Primary antibody
(anti-antigen X)
+ Means of
Visualization
Also referred to as
“label” or “detection”
 Combines the fields of Immunology,
Histology and Chemistry

4. History of Immunohistochemistry-
Detection Systems
 1942 Coons, Creech, Jones, Berliner,
 Developed indirect immunofluorescence method in order to
demonstrate an antigen (pneumococcal antigen in tissue)
 1959 Singer
 Developed a method for conjugating antibodies to a label
(ferritin)
 1966 Graham & Karnovsky
 Developed a method for tagging an enzyme to an antibody
(horseradish peroxidase)
 1967 Nakane & Pierce
 Developed a direct enzyme-labeled antibody technique
[immunoperoxidase]

5. History of Immunohistochemistry-
Detection Systems
 1970 Sternberger
 Developed a multi-step method using unlabelled primary
antibody [peroxidase-antiperoxidase (PAP) technique]
 1971 Hokfelt
 Applied immunohistochemistry as a method to visualize
neurotransmitters
 1974 Heitzman & Richards
 Developed a different multi-step method for detection using
an unlabeled primary [avidin-antibiotin complex (ABC)
technique]

6. Detection Systems
What Is a Detection System?
 A way to visualize the staining (to see the antibody
reaction) Label
Primary
antibody
Tissue
antigen

7. Detection Systems
Labels
 The antibody molecule cannot be visualized by itself
 Therefore the site of antibody binding cannot be
visualized by itself
 A “label” is required to visualize this reaction
 “Label” is a molecule that can produce a visible
signal (a label)
 Two Common Types of Detection
 Direct
 Indirect
 Two most common types of labels are:
 Fluorescent labels
 Enzymatic labels

8. Detection Systems
Direct Labels
 Fluorescent Labels – Direct Label
 Fluorescent labels use dye molecules which fluoresce (emit
light) in different colors
 Color emitted is specific to each dye
 Most commonly used fluorescent dyes are:
 FITC (Fluorescein Isothyocyanate) - GREEN
 Texas Red - RED
 Rhodamine - RED
 Alexa dyes
 More recently available fluorescent dyes which emit a
stronger signal (higher quantum yield) than the traditional
fluorescent dyes and are more stable
 Fluorescent labels can be visualized by themselves using a
fluorescent scope

9. Patient
tissue
Primary
antibody
FITC
Immunofluorescence
Direct Labels

10. Patient
tissue
Primary
antibody
FITC
UV light
source
Fluorescence

11. Detection Systems
Direct Labels
Human Kidney:
1. Basement membrane of
glomerulus green with FITC
anti-IgG
Fluorescent Direct Labels
Human Skin:
1. Nerves red with Cy3
2. Basement membrane green
with Cy2
3. Epidermis and vasculature
blue with Cy5
Human Artery:
1. Nerves orange-red with a
Cy3
2. Collagen in basement
membrane green with Cy2
3. Endothelial cells blue with
Cy5

12. Sources of False Flourescent
Positive Staining
 Some tissue components have autofluorescent properties.
 They include:
 elastic fibers
 collagen fibers
 formalin fixed paraffin-embedded tissues
 Primary or secondary antibody non-specific binding
530 Long Pass 530 Long Pass
15 Seconds 4 Minutes

13. Detection Systems
Direct label Pitfalls
 Advantages:
 Rapid turn-around time
 Limits non-specific reactions due to the use of a single antibody
 Pitfalls:
 Less sensitive due to the use of only one layer of labeled
antibody (little signal amplification)
 To gain sensitivity requires:
 High expression of antigen
 Very sensitive label
 Not all antibodies can be direct-labeled
 Labeling of the primary antibody compromises the binding
activity
 Limited in primary antibody selection (limited variety)

14. Detection Systems
Indirect Labels
 Enzymatic Labels
 Use enzymes that react with other reagents to
produce a chemical reaction and generate a colored
end-product
 Two most commonly used enzymes are:
 Horseradish Peroxidase (HRP)
 Alkaline Phosphatase (AP)
 A substrate and a chromogen are required to generate
a colored reaction end-product
 The substrate and chromogen are a matched pair
 There may be more than one substrate/chromogen
set for each enzyme
 Colored end-product is visualized with a regular light
microscope

15. Detection Systems- Indirect Labels
Enzymes and Chromogens
 Horseradish Peroxidase
 Diaminobenzidine (DAB)
 produces chestnut brown color
 Aminoethylcarbazole (AEC)
 produces brick red color
 Alkaline Phosphatase
 Fast Red
 produces bright fuchsia color
 NBT
 produces dark blue color

16. Detection Systems
Indirect
 Avidin-Biotin
 Most common
 Uses a secondary antibody to link the primary antibody to the
rest of the detection molecules
 Employs the high affinity properties of biotin for avidin or
streptavidin
 Biotin is a naturally occurring vitamin found throughout the
body (especially in tissues like liver, spleen, heart and brain)
 Avidin is a protein found in egg white
 Streptavidin is a protein produced by the fungus streptomyces
avidinii
 It has basically the same properties as avidin but is
"cleaner"
 Uses the multi-valent properties of (strept)avidin to attach
multiple molecules of enzyme

17. Biotin-based Detection Kit
B
B
Biotinylated
Secondary Antibody
Primary Antibody
Antigen of Interest
Streptavidin HRP
** *
Chromogen

18. Detection Systems
Indirect label Pitfalls
 Avidin-Biotin
 Advantages:
 More sensitive due to multi-layer amplification
 Fast, stable complex due to high affinity between (strept)avidin conjugate and
biotin-labeled protein
 Less background (especially with streptavidin)
 Greater selection of primary antibodies available
 Pitfalls:
 Possibility of
 Non-specific reaction due to cross-reactivity of the secondary antibody (link)
 Non-specific reaction due to binding of endogenous biotin and (strept)avidin
 Background staining due to biotin binding (typically enhanced with antigen
retrieval)
 Protein carriers required in the diluent to eliminate background (or a separate
protein block step has to be employed)
 Cross-reactivity may occur with cell adhesion molecules (streptavidin has some
sequences that are homologous to fibronectin. Fibronectin is a general cell
adhesion molecule found throughout the body used to anchor cells to collagen
["molecular glue"])

19. How Does a Biotin-free (Polymer,
multimer) Detection Kit Work?
 The reagents in a biotin-free detection systems are applied in the
following order:
 1 - Endogenous peroxidase inhibitor is applied
 Quenches endogenous peroxidase
 2 - Primary antibody is applied
 Targets the antigen of interest
 3 – Polymer, multimer complex is applied
 Targets the primary antibody
 Contains both the secondary antibodies and enzyme
 4 - Chromogen/Substrate is applied
 Produces colored end product at sites where the polymer
complex is bound to the primary antibody

20. The End Result

21. Principal factors affecting the outcome of
immunohistochemical studies

22. What are the Principle Parameters
affecting the outcome of IHC staining?
All Parameters Affect IHC staining!!!
Analytic:
 Quality control in immunohistochemistry
 Primary antibody, clone, dilution
 Buffer, Time, Temperature
 Detection system
 Amplification
Preanalytic
 Pre-treatment
 Interpretation
 Localization
 Positive/Negative-cut-off level
Postanalytic
 Quality Control
 Internal/external
 Quantification
 Reporting
 Tissue, type, dimension
 Section Thickness
 Storage
 Drying
 Fixation, Time, Type, Volume, Decalcification
 Preparation
 Blocking
ETC…………

23. How do Histotechs avoid IHC
Pitfalls?
 Acquire a strong foundation of Basic
Immunohistochemistry, to avoid pitfalls (even
if you are running IHC on an autostainer!!)
 Standardization
Validation procedures, to ensure reproducible
results

24. Standardization
Attendees will gain a basic knowledge of
standardization through:
 Validation Methods
 Reproducibility

25. Validation
Laboratory validation requirements
come from 3 different sources:
 Clinical Laboratory Improvement Amendments of 1988
(CLIA ’88) section 493.1253 (b)(2)
 College of American Pathologists (CAP)
 Joint Commission for Accreditation of Healthcare
Organizations (JCAHO)

26. Purpose of Validation
 To establish and implement performance and
testing criteria for a particular assay in a
particular laboratory
 Verification of an established procedure
 Each laboratory should develop its own
validation protocols.

27. Verification of Established
Procedure
 Laboratories using commercially available devices
according to manufacturers instructions should perform
appropriate studies to verify that performance properties
established by the manufacturer are obtained in the
laboratory setting.
 Laboratories that modify commercially available devices
should fully validate the modified device’s performance.
 Laboratories using in-house developed procedures may
establish the performance parameters by verifying
against another laboratories performance, provided the
procedure has undergone full validation by the other
laboratory

28. Types of Validation
 New Test
-New Assay to the Laboratory
- New Methodology from Validated
Protocol
 Lot to Lot Validation

29. New Assay Validation
 Identify the critical aspects of a procedure that should be carefully
controlled and monitored to provide consistent and reliable results.
 Substantiate the application of the methodology to detect the target
in question including appropriate peer reviewed literature
references.
 Establish the minimum amount/quantity of tissue/specimen needed
to obtain reliable results (ref. Eric His paper, CLSI-standards
(formerly NCCLS), ASCO-CAP, CLIA)
 Validate each step of the analytic procedure, QC, equipment and
instrument.
 Assure characterization of all critical reagents (e.g. cell conditioner,
retrieval buffer, enzymes, etc.) utilized by the procedure

30. New Assay Validaiton
 Once staining protocol conditions have been optimized, the antibody
should be validated on a set of clinical cases designed to test the
limits of diagnostic utility (sensitivity and specificity) of the antibody.
- The package insert contains the starting point for
protocol optimization
 The test should be validated for all appropriate “specimen” types.
This includes:
- Appropriate tissue types
- Fixation ( if other than that validated by the
manufacturer)
 The use of multi-tissue blocks is a very idea! Include tumors
with high/low sensitivity and specificity with internal negative
controls.

31. New Assay Validation
 Verification of performance specifications
 Demonstrate that it can obtain performances
as established by manufacturer.
- Specificity
- Sensitivity
- Precision (Reproducibility) the use of multi-
tissue blocks is very helpful here!

32. New Method Validation
 Once the test system has been validated with a
particular method (i.e. a particular antibody and/or
detection), if you change antibody and/or detection,
the new method must be re-validated.
 The test should be validated for all appropriate
“specimen” types. This includes:
- Appropriate tissue types
- Fixation (if other than that validated by the
manufacturer).

33. New Method Validation
 New Assay to the Laboratory
 New Methodology from Validated Protocol
- Examples:
- Different Clone
- Same Clone, Different Vendor
- Different Detection
- Different Pretreatment
- Different Protocol (Deviation from PMA approved protocol)
- Different Fixation

34. Validation
Each one of the Validated processes
also varies depending upon its FDA
Antibody class. Get familiar with the classes! Know the difference
between RUO, ASR, IVD and PMA status.
http://www.fda.gov/cdrh/oivd/index.html
Use search engine by key word, i.e., antibody classes
Examples:
Class l
Class ll
Class lll

35. Lot to Lot Validation
 New lots of primary antibody or commercial detection
reagent should be compared to the previous lot.
 Use an appropriate panel of known positive and negative
control tissue.
- Example: 1 slide positive control, 1 slide negative
control or 1 slide that contains both run against previous
lot. Make sure to use same slide controls for
the comparison. Again, multi-tissue blocks can be used
here.
 Results of testing should be documented.

36. Reproducibility
 Once a new antibody or assay has been
validated. Be sure each step has been
documented for reproducible results
 Next, it is important to establish reliability
through reproducibility from day to day and
from slide to slide with in a staining chamber
or autostainer.
 Staining instruments have contributed greatly
in the past 10 years to reproducible results
therefore establishing “standardization”.

37. Artifacts in
Immunohistochemistry
 Desquamartifact - squamous cells that flake off from our skin and interact with a HMW
IHC stain
 Bubble-Microscopic bubbles that find their way on the tissue and “starve” the underlying
tissue of reagents
 Drying- slides may be completely negative, stain muddy brown in color. May have edge
chromogen deposition.
 Trappings- tissue flapping or becomes detached form the slide at some point of the
staining and reagents get trapped under resulting in foci chromogen deposition.
 Edge- The edges of the tissue stain, may be a variation in trapping artifact or poor
fixation or inappropriate antibody titers-
 Poor fixation- gradient staining to dark staining outside and no staining in the inside
and vice versa.
 Bacterial Contamination- bacteria may begin to grow in reagents, antibody or
instrumentation
 Graphite pencil- the use of graphite pencils can cause miniscule particles to
make their way onto the tissue or blood smear, showing up as an artifact
 Endogenous Biotin- Binding of avidin to endogenous biotin present in tissues
or cells can cause false position staining. Especially in liver and kidney.
 Precipitated DAB- When DAB chromogen is not filtered, small bits of DAB precipitate
may collect on the surface of the section.
 Mold- can grow on control slides that have stored for a long period of time.
Instrumentation that utilizes hydrophilic reagents and warm chambers may grow mold
over time if not decontaminated on a regular basis.

38. Conclusion-Rules to go by in
IHC
 Treat patient tissue as if it were your own
 Cut tissue thinly, don’t cram large tissue into cassettes and don’t
let it sit in formalin for extended periods of time
 IHC is not a silver bullet and all tumors will not necessarily react
like they are “expected” to. If IHC results do not make sense,
retrace steps and seek further opinions.
 Make multi-tissue (tumor) blocks and use them.
 If using concentrated antibodies, always titer a broad range rather
than what the manufacturer suggest, they could be way off line.
 Know the characteristics of a true positive and a false positive
stain. Always look at the stain (QC) prior to Pathology evaluation.
 Do not try to interpret poorly fixed tissue, edge artifact, necrotic
tissue or those containing strange staining in general as “true”
staining, as those usually exhibit random staining.
 Always Validate and Standardize new or changed reagents.