Second Half Break!

2. Basic IHC techniques, Detections and Standardization

Attendees will gain basic knowledge of:

Detection Methodsfluorescent and enzymatic Labels

Standardization MethodsValidation techniques

Immunohistochemistry-Detection Methods

Tissue on a slide

+Primary antibody (anti-antigen X)

+ Means of VisualizationAlso referred to as“label” or “detection”

Combines the fields of Immunology, Histology and Chemistry

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]

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]

Detection SystemsWhat Is a Detection System? A way to visualize the staining (to see the antibody

reaction) Label

Primary antibody

Tissue antigen

Detection SystemsLabels 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

Detection SystemsDirect 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

Patient tissue

Primary antibody

FITC

Immunofluorescence Direct Labels

Patient tissue

Primary antibody

FITC

UV light source

Fluorescence

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 Cy32. Basement membrane green

with Cy23. Epidermis and vasculature

blue with Cy5

Human Artery:1. Nerves orange-red with a Cy32. Collagen in basement membrane green with Cy23. Endothelial cells blue with Cy5

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

Detection SystemsDirect 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)

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

Detection Systems- Indirect LabelsEnzymes 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

Detection SystemsIndirect 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

Biotin-based Detection Kit

B

B

Biotinylated Secondary Antibody

Primary Antibody

Antigen of Interest

Streptavidin HRP

** *Chromogen

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"])

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

The End Result

Principal factors affecting the outcome of immunohistochemical studies

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 AmplificationPreanalytic Pre-treatment Interpretation Localization Positive/Negative-cut-off levelPostanalytic Quality Control Internal/external Quantification Reporting Tissue, type, dimension Section Thickness Storage Drying Fixation, Time, Type, Volume, Decalcification Preparation Blocking

ETC…………

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

Standardization

Attendees will gain a basic knowledge of standardization through:

Validation Methods Reproducibility

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)

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.

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

Types of Validation New Test

-New Assay to the Laboratory - New Methodology from Validated Protocol

Lot to Lot Validation

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

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.

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!

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).

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

ValidationEach one of the Validated processes also varies depending upon its FDAAntibody class. Get familiar with the classes! Know the difference

between RUO, ASR, IVD and PMA status.

http://www.fda.gov/cdrh/oivd/index.htmlUse search engine by key word, i.e., antibody classes

Examples:Class lClass llClass lll

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.

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”.

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.

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.