PREPARING
FOR THE
IHC QUALIFICATION
EXAM
1
Presented by:
Ethel R Macrea, HT (ASCP)QIHC
Southwest Skin Pathology Services
Tucson, AZ
dawnbrok@earthlink.net
2
OBJECTIVES
1.
Provide basic information regarding the exam
2.
Review in depth the outline provided by ASCP
3.
Delve into concepts and principles of troubleshooting
4.
Time permitting, play an IHC version of the game
JEOPARDY!
3
PREPARING FOR THE QIHC EXAM

The information to be reviewed is intended to aid the
applicant in study and preparation for ASCP’s Qualification
in Immunohistochemistry exam.

In and of itself, this is not a study guide.

The ASCP has not provided any information, suggestions
or recommendations for this workshop.

Studying for the exam is the responsibility of the
applicant.
4
33 West Monroe, Suite 1600
Chicago, IL 60603
Phone: 312-541-4999
800-267-ASCP (2727)
Fax: 312-541-4998
General Information e-mail: info@ascp.org
5
WHERE TO START

The best place to begin is actually at the ASCP Board of Registry Website
http://www.ascp.org/FunctionalNavigation/certification/GetQualified.aspx

Review the information regarding the exam
1.
Fees
2.
Eligibility requirements and routes
3.
Required experience
4.
Application process and forms
5.
Scheduling the exam
6.
Studying for the exam
7.
Results
8.
Revalidation
6
Here’s is what the website
will look like if you use the
link that was provided.
STEP 1

Identify the Qualification you’re applying for and
determine your eligibility.

For each qualification category, you’ll find a choice of routes
that you can take to verify your eligibility for the qualification,
depending on the education and training you’ve completed.
Find out the eligibility requirements for your qualification—
 Qualification
in Cytometry, QCYM
 Qualification in Immunohistochemistry, QIHC
 Qualification in Laboratory Compliance, QLC
 Qualification in Laboratory Informatics, QLI

*Requirements and fees stated are subject to change without notice
8
ELIGIBILITY REQUIREMENTS

To be eligible for this Qualification, an applicant must satisfy the requirements
of at least one of the following routes:

Route 1: ASCP technologist or specialist certification and six months full time
acceptable experience in immunohistochemistry in the U.S., Canada or a
CAP/The Joint Commission (JCAHO) accredited laboratory within the last five
years

Route 2: ASCP technician certification (HT, MLT) and twelve months full time
acceptable experience in immunohistochemistry in the U.S., Canada or a
CAP/The Joint Commission (JCAHO) accredited laboratory within the last five
years

Route 3: Baccalaureate degree or higher from a regionally accredited college/
university, and eighteen months full time acceptable experience in
immunohistochemistry in the U.S., Canada or a CAP/The Joint Commission
(JCAHO) accredited laboratory within the last five years.
9

If you work in a research laboratory, you can take the exam if
you meet any one of Eligibility routes.

If you work in veterinary histology, you can take the exam if you
meet any one of Eligibility routes

If you have worked in any combination of research, clinical,
veterinary histology/immunohistochemistry, you can take the
exam if you meet any one of Eligibility routes

Excperience in IHC does not have to be consecutive time but
must meet the total months of experience within the past five
years from the date the application is submitted.
10
STEP 2

Gather your education and experience
documentation.
 You’ll
need to provide documentation of your education
and training and specified experience requirements that
you’ve fulfilled in your area of specialization.
 Before you apply for the qualification, you should assemble
all the documentation needed to—
Verify your academic education
 Verify your experience (if required)

11
EXPERIENCE

Full and Part-Time Work Experience Defined


Full-time experience is defined as a minimum of thirty-five (35) hours
per week.
Individuals who have part-time experience may be permitted to
utilize prorated part-time experience to meet the work experience
requirements.

The number of years of experience required must be completed
within the time frame, specified in the requirements, calculated
from the date of application

Simultaneous experience at two different places of employment
in excess of 35 hours per week is not counted toward the
experience requirement.
12

QIHC Experience

Applicants must have experience in the following areas:

Immunohistochemical and/or Immunofluorescence Preparations




Staining technique
Selection of proper control material
Titration of immunologic reagents
Quality Assurance





Method selection, validation, documentation
Quality control
Reagent selection, preparation, storage, disposal
Safety
Specimen fixation, processing, microtomy
13
STEP 3

Apply for Qualification.

Complete and submit the application form with the nonrefundable application fee, ($200.00) along with all the
documentation you gathered in Step 2

Apply by mail and pay by credit card, check or money
order.

QIHC Application (PDF)
14
15
STEP 4

Schedule your examination.

Once your application and eligibility documentation
has been approved by BOR, you’ll receive an
admission letter to the examination within a 60 day
time period.

When the date arrives, you will need to access the
testing site.
16
17
THE TEST

This qualification can be earned by meeting the eligibility
requirements and taking a 50-item examination on your own
computer.

(more about this in a moment)

There may be images of stained slides with relevant questions
regarding antibody labeling, pattern of staining, artifacts,
endogenous background, etc.

It is timed – 90 minutes from start to finish

It is all multiple choice
18
FAQ’S ABOUT THE TEST
Q.
Q.
What is a passing grade?
A. One must have 70% correct answers to pass.
Does the computer tell us if we have passed or not?
A. No, you have to wait to be notified by mail.
Q.
Is there any kind of simulation software available to give an applicant some
experience before actually taking the exam?
A. Not at this time.
Q.
Can you skip a question and come back to it or change an answer?
A. Yes, you can do both until you click “Finished” for the entire test.
Q.
If I fail the first time I take the test, can I re-take it?
A. Yes, you have a limited time to re-take the test, but I believe the fee must be paid
again.
19
FAQ’S ABOUT COMPUTERS
Q. What software do I need to take a test?
A. Software compatibility is primarily determined by the browser you are using. This
site supports Internet Explorer 5.01 or later, Netscape 7.2 or later, Mozilla 1.7 or
later, AOL 6.0 or later, Firefox 1.0 or later, and Safari 1.2 or later. Most Windows
operating systems are supported, including Windows 98, Windows NT, Windows
2000, and Windows XP. MAC is also supported.
Q. What hardware do I need to take a test?
A. In addition to having a computer that supports one of the required browsers, you
will need to have a color monitor capable of displaying 800x600 with 32-bit color.
For best results, use a 17” or larger monitor that supports 1024 x 768 resolution.
Q. Will the test work over a dial-up connection?
A. You can take the test using a dial-up modem, but the performance will be slower.
For an optimal experience tests must be taken using a broadband connection,
such as DSL or a cable modem.
20
Q. Will my answers be lost if my computer crashes?
A. No. All answers are saved each time you go to the next
question. When you restart the test will start on the question that
was displayed when you crashed.
Q. I’m having problems. Where can I get help?
A. First make sure you are using a supported browser and
computer. If you are, check the list of common problems and
solutions under the technical support link to see if your problem is
listed. If you can not find an answer, please contact technical
support at ASCPOnline@testsys.com and we will be happy to
assist you.
Q. Can I take this test using paper and pencil?
A. No. This test is only available using a computer.
21
STEP 5

Study for Qualification –BOR recommendations-

Bancroft, J.D. & Gamble, M. (2007). Theory and Practice of Histological Techniques (6th ed.). New
York: Churchill Livingstone. Link to Purchase

Carson, F. (1997). Histotechnology: A Self-Instructional Text (2nd ed.). Chicago: ASCP Press. Link to
Purchase

Dabbs, D.J. (2006). Diagnostic Immunohistochemistry (2nd ed.). New York: Churchill Livingstone.
Link to Purchase

Elias, J.M. (2003). Immunohistopathology: A Practical Approach to Diagnosis (2nd ed.). Chicago:
ASCP Press. Link to Purchase

Shi, S., Gu, J., and Taylor, C.R. (2000). Antigen Retrieval Techniques: Immunohistochemistry and
Molecular Morphology. Natick, MA: Eaton Publishing Company. Link to Purchase

Taylor, C.R. and Cote, R.J. (2006). Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologists
(3rd ed). Philadelphia: W.B. Saunders Company. Link to Purchase
22
MY RECOMMENDATIONS

NSH has written a series of fourteen (14) self
assessment examination books which provides
questions with descriptive answers for preparation
of the certification exams.
Books:
$15.00 -NSH member
$35.00 -Non member
CD’s
$100.00 -NSH member
$175.00 -Non member
http://www.nsh.org/organizations.php3?action=printContentItem&orgid=111&typeID=1157&itemID=18326

NSH also offers a CD Rom version of the self assessment series.

All fourteen books are contained on this interactive CD. Each topic
provides 2 interaction modes:


Study (which provides explanations for answers as you go)
Quiz (which allows you to take a practice test).
23
NSH’S - SELF ASSESSMENT #5

IMMUNOHISTOCHEMISTRY, ENZYME HISTOCHEMISTRY, FLOW CYTOMETRY,
IN-SITU HYBRIDIZATION AND ELECTRON MICROSCOPY (2nd Edition)

Developed for teaching as well as self-assessment purposes.

Basic through advanced levels of immunology theory and immunologic
staining are included along with an introduction to in-situ hybridization,
flow cytometry and references for polymerase chain reaction techniques.

Included as well, are basic, intermediate and advanced questions on
electron microscopy.
24
NSH’S - SELF ASSESSMENT #11:

LABORATORY OPERATIONS

Contains questions for experienced and inexperienced
histotechnicians and histotechnologists on instrumentation, lab
math, education, management and regulations.

The book is divided into three levels of knowledge - basic,
intermediate and advanced.
I

t is one of the best sources of material for not only those
preparing for the HT and HTL examinations, but also for
supervisors and managers in histopathology.
25
NSH’S - SELF ASSESSMENT #13:

CYTOPREPARATORY TECHNIQUES

Provides the theory and practical application of various facets
of cytopreparatory techniques.

Included are specimen collection and receipt, processing and
staining, and troubleshooting and maintenance.

The booklet is not divided into three areas of difficulty.

Instead, it is divided into the various preparatory areas.
26
NSH’S - SELF ASSESSMENT #14:

LABORATORY SAFETY
 Provides
a comprehensive review of safety topics
applicable to laboratory personnel.
 Included
are Agencies, Organizations, Regulations,
Precautions.
 Chemical
hazards and biohazards as well as general safety
precautions such as electrical, fire, first aid, shipping,
ergonomics and radiation are included.
27
CAP CHECKLISTS

Latest: Anatomic Pathology Checklist
 IHC
is found in this section

Latest: General Laboratory Checklist

These are not study guides but still lists resources,
information and commentaries that may prove useful
28
CLSI

Quality Assurance for Immunocytochemistry;
Approved Guideline (MM4-A) 1999

This document provides recommendations for the performance of
immunocytochemical assays on cytologic and surgical pathology specimens.

It is intended to promote a better understanding of the requirements,
capabilities, and limitations of these diagnostic methods; to improve their
intra- and inter-laboratory reproducibility; and to improve their positive and
negative predictive values in the diagnosis of disease.

Members $60
Nonmembers $120

http://www.clsi.org/source/orders/index.cfm?task=0&Section=Online_Store

29
DAKO USA HANDBOOKS
FREE
Immunochemical Staining Methods Handbook ; 3rd Edition
(if you have it)
This is out of print and is no longer available.
Immunohistochemical Staining Methods Educational
Guide, 4th Edition- # 08002
Educational Guide to Demasking of Antigens; 2nd Edition
- # 00092

http://www.dako.com/prod_productrelatedinformation?url=support_ihc_handbook.htm

http://pri.dako.com/00092_demasking_antigens_row.pdf
30
Lessons in Immunohistochemistry
Anatech Ltd
http://www.anatechltdusa.com/index.html
One of the best sources of information
regarding principles of fixation, various
fixatives and the effects on IHC
This text addresses neutral buffered
formalin, zinc formalin, glyoxal
fixatives, post fixation and more.
FREE
31
$154.00

2nd edition, by Leong, Cooper and Leong. 2003
Introduction to Immunocytochemistry,


$119.20
Manual of Diagnostic Antibodies for Immunohistology,


$70.50
3rd edition, by Polak and Van Noorden
Microscopy, Immunohistochemistry, and Antigen Retrieval Methods:
For Light and Electron Microscopy, M.A. Havat

http://www.amazon.com
32
Immunohistochemistry: Methods Express Series
(Methods Express) edited by Renshaw
Price:$75.00 (40 £)

http://www.scionpublishing.com/shop/product_display.asp?currencyid=1&productid
=9781904842033
33
QUALIFICATION IN
IMMUNOHISTOCHEMISTRY
(QIHC)
ASCP’s Topic Outline
34
I. GENERAL IMMUNOLOGY (10%)
A. Antigen
B. Antibody
1. Monoclonal antibodies
2. Polyclonal antibodies
3. Antibody classes
4. Antibody structure
35
II. DETECTION SYSTEMS (10%)
A. Immunofluorescence
B. Immunohistochemistry
1. Substrates
2. Enzymes
3. Chromogens
4. Blocking reactions
36
III. SPECIMEN HANDLING (10%)
A. Fixation
B. Processing
C. Microtomy/Slide Preparation
D. Cytology Specimens
E. Immunofluorescence Specimens
F. Frozen Sections
37
IV. EPITOPE ENHANCEMENT (ANTIGEN RETRIEVAL) (20%)
A. Methods, Principles and Techniques
1. Heat induced epitope retrieval
2. Enzyme induced epitope retrieval
3. Combined heat and enzyme methods
38
V. STAINING (30%)
A. Principles and Mechanisms
1. Direct
2. Indirect
3. Avidin-Biotin
B. Tissues
1. Morphology/anatomy
2. Cell/component
demonstration
a. Staining patterns
b. Microorganisms
C. Stain Components
/Characteristics
1. Concentrated antibody
2. Pre-diluted antibody
3. Titrations
4. Reagents
D. Troubleshooting
E. Mounting Procedures
3. Pathology
39
VI. LABORATORY OPERATIONS (20%)
A. Quality Control/Quality Assurance
1. Documentation
a. Procedures
b. Quality control records
c. Personnel
d. Reagents/antibody lots
2. Selection, utilization and evaluation of control tissue
3. Slide storage
4. Method selection and validation
5. Troubleshooting
40
B. Safety
1. Storage
2. Disposal
3. Hazards
4. Regulations
5. Procedures
D. Ancillary Equipment
Instruments (e.g., microwave,
computers, pH meter, solvent
recovery)
E. Regulations
1. Federal government
2. Accrediting agencies
C. Laboratory Mathematics
41
BEGINNING TO STUDY

Look at each section in the outline


Search texts for basic information



Note the ‘weight’ given to each section
Pick out key words and understand their meaning
Get different perspectives from more than one text
Entertain some ways in which you might troubleshoot an
issue or recognize staining artifacts, background etc.




What would a problem look like?
How would you figure it out?
What would you do to fix it?
How can it be prevented moving forward?
42
STEP 6

Watch for your results and certificate.
 You’ll
receive your examination scores along with your
certificate within ten business days of your examination
date.
 No
information is provided as to which questions were
answered incorrectly.
43
WHEN YOU ARE QUALIFIED…

Individuals who have been qualified may indicate this qualification
through the use of initials in the following manner:

Jane Doe, QIHC
 for individuals qualified in immunohistochemistry

Jane Doe, HT(ASCP)QIHC
 for individuals who are qualified and who are also ASCP certified

Naturally, if you have degrees; BS, MA, etc these can also be listed.
Just add QIHC after your name in the sequence. Order does not
matter.
44
STEP 7


Revalidate after five years.

In order to maintain your qualification you must revalidate every five years.

Thirty (30) hours of continuing education is required to renew the IHC
Qualification after 5 years.
 If this criteria is not met, one would have to take the exam again to become
Qualified.

Presently, renewal cost is $50.00

View instructions (PDF) for completing the Qualification Revalidation Application
Form for QIHC
Revalidation Application Form (QIHC)
45
CONTINUING EDUCATION;
1. Immunohistochemical Preparations
10. Immunoproliferative Disorders
2. Immunofluorescence Preparations
11. Transplantation
3. Immunophenotyping
12. Histology
4. Quality Control
13. Immunology
5. Safety
14. Pathology of Diseases for which IHC is a
6. Laboratory Information Systems
Dx Procedure
7. Supervision/Management
15. In-situ Hybridization
8. Quality Assurance
16. In-situ Polymerase Chain Reaction
9. Immunodeficiency
17. Electron Microscopy
46
UNSURE ABOUT WHAT IS GOING TO BE ACCEPTED?

It is your responsibility to submit certificates to the Board
of Registry that list the provider of the course, the subject
of the course and the contact hours awarded for each
course and dates.

If you are unsure about the acceptability of a topic, please
contact the Board of Registry.

There is a live chat room

Or you may call into ASCP/BOR
47
WHAT OTHERS HAVE SAID ABOUT THE TEST…

Detection kits

There have been questions regarding polymer technology detection kits
but it is not specifically been called out in the outline

Not all polymer kits use the same technology, be sure to explore this
with those who make the kits.

Background or cross-reactivity may be very different and based on
specific polymer technologies

Polymer kits are all proprietary, so gaining insights will be a bit of a
chore
48
CYTOLOGY AND IHC

Questions have cropped up about IHC on cytological samples
such as FNA’s

Cytology samples are not fixed nor processed like tissue

Fixatives such as alcohols exert influences over pretreatments,
titers and backgrounds with IHC

To understand more about cyto-prepping, spend some time with a
cytotech or in cytology
49

Fixatives
 Not
everything revolves around formalin fixatives
 Know
about glyoxal fixatives
 Alcoholic fixatives

Know your regulatory labeling and its implications




ASR vs. IVD vs. RUO
PMA (IVD) and what it means to the laboratory
ISO and CE Mark
Understand the impact that HIER techniques have on IHC,
background, false positives etc
50

Feedback from those who have taken the exam regarding
best sources to use as study guides

DAKO handbooks
 Theory

NSH Self Assessment booklets
 Q & A with explanations

CLSI (NCCLS) MM4-A document
 QA and reasons behind it

CAP checklists
 Lots of basic information especially regarding FDA labeling and
controls

Do not limit yourself to these alone!
51
GOING THROUGH THE OUTLINE

This next segment, we will go through the topic
outline provided by ASCP.

This is intended to help you figure out what to focus
on more specifically as you study and prepare for the
exam.

These are suggestions only, and in no way is
predictive of the questions that will be asked on the
exam.
52
1. IMMUNOLOGY (10%)

There are terrific websites with an overview of
immunology concepts and principles.
Go to:
 University of South Carolina’s On-line lecture.


http://pathmicro.med.sc.edu/mayer/antigens2000.htm
Dalhousie University; Faculty of Medicine,Immunology
Bookcase
 http://pim.medicine.dal.ca/atg.htm
53
GENERAL IMMUNOLOGY

What is immunology?
 a science that deals with the immune system and the cell-mediated and
humoral aspects of immunity and immune responses (Merriam Webster
Dictionary)

What is innate immunity?
 Innate immunity is present from birth and offers the first line of defense against
“insults”. This layer of defense includes the skin, gastric system, lysozymes etc

What is acquired immunity?
 Acquired immunity is that which is acquired against specific insults and unique
to that one agent. It is both specific and remembered.

Immune responses are caused by the activation of resting lymphocytes, followed by
proliferation and differentiation. (Antibody Techniques, p 6)
54
ANTIGENS

An antigen is a substance capable of inducing a specific
immune response.

The term is derived from the generation of antibodies to
such substances.

Specific immune responses require recognition
molecules like the T cell receptor or antibodies which
recognize the antigen, or parts of it, and stimulate a
response by the specific arm of the immune response (T
or B cells).
55

Each part of the antigen that is recognized by either an
antibody or a T cell receptor is known as an epitope.

Depending on the size of the protein or polysaccharide,
there may be hundreds of B cell epitopes (recognized by
different antibodies) or T cell epitopes (presented by
antigen presenting cells to different T cells) in the same
molecule.

This actually helps the body have a better response to the
antigen as many T and B cells can be activated to
respond to a single target.
56

An antigen is usually a protein that binds to the antibody.

Usually, antigens are proteins, but can be any molecule, large or small,
associated with a carrier protein.


E.g.; FITC, Biotin, digoxigenin etc
A peptide is a small piece of protein consisting of a linear molecule
made up of two or more amino acids linked by peptide bonds

A hapten is a small molecule that cannot induce an immune
response by themselves but which can when coupled to a carrier
molecule.
Free haptens can react with products of the immune response after such
products have been elicited.
 Haptens have the property of antigenicity but not immunogenic.


Immunogen - A substance that provokes the immune response when
introduced into the body.

An immunogen is always a macromolecule
57

Antigens are the subject of IHC in a two very key ways.

First is that the antibody is targeted against a specific antigen.

This antigen is within the cell or tissue.



It can be on the membrane, the cytoplasm, nucleus or transmembranes, etc
This is why antibodies have a prefix, anti (α) in front of them.
Secondly, an antigen is what causes the immune response in the host
such as a mouse or rabbit, which then responds by producing
antibodies.



The most important thing to remember is that the object of IHC is to get an
antibody to locate its complimentary antigen.
The specific area of the antigen that the antibody is to bind to is called the
epitope.
An epitope is the unique portion of the overall antigen.
58
TROUBLES WITH ANTIGENS

What could possibly go wrong with an antigen since it is localized right
in the tissue?

Antigen preservation requires fixation or stabilizing and they must be
available for staining.

Antigens may be compromised by;
 Drug therapies
 Prolonged fixation in formalin
 Improper handling of the specimen for that particular antigen
 Time and storage conditions

Only by the process of elimination can one presume this to be the case.
59
POINTS FOR DISCUSSION

How might one determine that an antibody was not
working due to loss or compromised antigenicity?
 Fixation
 Decalcification
 Specimen
handling
 Patient therapies
60
61
IMMUNOLOGY & ANTIGENS – KEY WORDS
Immunology
Humoral immunity
Innate immunity
Acquired immunity
Lymphocytes
T – cell
B – cell
Antigen
antigenic determinant
epitope
peptide
hapten
immunogen
target
Antigenicity
 Factors impacting antigenicity
62
ANTIBODIES

A good websites to use for learning and practicing questions
about antibodies is:

THE BIOLOGY PROJECT; Immunology, Tutorial.
http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/prob_set/01t.html

Other good websites are;


CELLS ALIVE - http://www.cellsalive.com/antibody.htm
ENZYNE @ARTICLES - http://ezinearticles.com/?What-are-Antibodies&id=334858
63

In mammals there are five main types of antibodies including: IgA, IgD, IgE, IgG, and
IgM.


There are 4 IgG and 2 IgA subtypes present in humans.
Antibodies are created by plasma cells which are derived from the B-cells in the
immune system.

Due to the fact that antibodies exist freely in the bloodstream or bound to cell membranes,
they are said to be part of the humoral immune system.

Every different antibody recognizes a specific foreign antigen.

This is because the two tips of its "Y" are different to each antibody are allow different
antibodies to bind to different foreign antigens.

When the antibody binds to a bacteria, it tags the microbe or virus for attack by the
immune system such as killer T-cells.


Sometimes, antibodies can directly neutralize the foreign body.
The production of antibodies by B-cells is the main function of the humoral immune
system.
64
STRUCTURE OF AN ANTIBODY
Each antibody molecule is made up
of two types of protein chains; one
light chain pair one heavy chain pair.
The pronged portion has both a
segment of heavy and light chains
on each arm of the prong.
These are referred to as the Fab
fragment.
The opposite end of the antibody,
looking like the arm from which the
antibody forks, is made up of heavy
chain only.
The Fc fragment is
the part of the
antibody that is one of
these; IgG, IgA, IgM,
IgD or IgE.
It is this
immunoglobulin
designation that
describes the
antibody’s subclass or
isotype.
This end is called the Fc
fragment.
http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/prob
65
ANTIBODIES

Antibodies are available as monoclonal antibodies or
polyclonal antibodies

Either of these may be supplied as a prediluted
antibody, a concentrated antibody, or a lyophilized
antibody.

Species vary but mouse and rabbit are the most
common, although goat, guinea pig, donkey, horse,
chicken etc have been used too.
66

A protein from a human or animal can be taken and injected to a host animal.

Immediately the animal’s immune system recognizes that the protein is neither
his own, nor normal for him.

The immunized animal will form an antibody to the substance with which it had
been injected.

In the case of a polyclonal antibody, the animal is bled and the antibodies are
extracted from the serum or ascities fluid.


These might then be purified or adsorbed for commercial use.
For monoclonal antibodies, the spleen is removed from the immunized mouse or
other host, and fused with a myeloma cell line thus forming a hybridoma.
67

Individual clones are identified, and then tested for affinity and avidity.

This helps to determine the niche for commercialization.

Monoclonal antibodies can be raised as ascities or tissue culture supernatant.

Antibodies supplied by tissue culture supernatant are often reported as cleaner
(less background or crossreactivity) because the host’s immunoglobulins are
directed solely against the antigen of interest.

Ascities comes from implanting the hybridoma into the peritoneal cavity of the
host, and the resulting body fluid contains the secreted antibodies.

If the hybridoma is grown in cell culture, the antibodies are secreted into the
tissue culture supernatant.

Different vendors may offer the same clone but supply it as either tissue culture
supernatant or ascities.
68

In Immunohistochemistry (IHC), the application of two antibodies is common
practice.

The first antibody to be applied is called the primary antibody (1° ab).


The second antibody to be applied is called the secondary or linking antibody (2°
ab).



The primary antibody is the essentially the antibody of interest.
This antibody recognizes the species and isotype of the primary antibody, instead of
an epitope within the cell.
For this reason it is dubbed anti-mouse or anti-rabbit and so on.
Many times the secondary antibody has a tag or label on it, such as biotin or FITC
etc.


This is to aid in the linking up of the kit to the bound primary/secondary antibody
complex.
That is why it is often referred to as the linking antibody.
69

Antibodies are usually categorized as monoclonal or
polyclonal.

There are some significant differences between the two in
the way they are made as well as some advantages and
limitations to each.

Monoclonal Ab’s are most often derived from mice and now
monoclonal rabbit antibodies are increasing steadily.
 almost any species can be used to produce a monoclonal
or a polyclonal antibody.
70


Monoclonal antibodies are further identified by their isotype, which is
either IgG or IgM for the most part.

IgM’s are from the initial immune response, usually within a week of
immunization.

IgG is more prevalent after the second week.
IgG can be further described by their hinge regions of the molecule.


This contributes to IgG having further designations such as IgG1, IgG2a etc.
The antibodies are sorted into groups that recognize a specific region
of the antigen, or epitope.

The targeted epitope is now identified by the clone name for the antibody.
71
The clone name

The group name or family name is what the general name of the
antibody is.

The individuals have their own unique name or identifier called a
clone.

Clones of the same family may exhibit differences in affinity, avidity
and pretreatment requirements.

Because of the characteristics of these individual clones, random
substitution of one for the other should not be considered without
due diligence.
72

Monoclonal antibodies are known for the specific staining properties and very low
incidence of a false positive staining event.

If a tissue stained with the monoclonal antibody is negative, it does not necessarily
mean that the antigen is not there, but only that the specific epitope targeted by
the monoclonal antibody was not available for binding.

This may happen as fixatives, decalcification, tissue processing and perhaps some
patient treatment regimens distort the antigens or as tumors and other pathologic
processes undergo a number of mutations.

In light of this, a monoclonal antibody may have a specific limitation since it
cannot identify other epitopes of the same antigen
73
POLYCLONAL ANTIBODIES

Polyclonal antibodies have traditionally been raised in rabbits
 Any other species can be used, and often have.
 Polyclonal antibodies are generally IgG.

Prior to immunization, rabbits in particular, are screened to see if they already have
a detectable amount of natural antibodies that cross-react with humans.
 If they do, they are not a good candidate for immunization.

Only the host with the least amount of natural antibodies is usually selected for
antibody production.

This screening of the host prior to immunization produces the negative control
serum, or preimmune serum.
74

A polyclonal antibody may be supplied from rabbit (or other species)
serum.

Often, polyclonal antibodies are pooled from a number of hosts who
have been immunized against the same antigen.

Frequently some purification will be done prior to sale.

Immunoglobulin fractions are common, using salts and ion exchange
chromatography to eliminate unwanted proteins or antibodies.

Affinity purification or isolation is done by immunoaffinity chromatography,
adsorbing and eluting these contaminating proteins.

Dako Cytomation Catalog, 2005/06, pp 298 – 99.
75

Unpurified polyclonal antibodies may have a few contaminating antibodies also,
and these may contribute to some unexpected cross reactivity.

Occasionally, the host(s) will begin to produce a new antibody in response to some
accidental or unintended immunogen.

If this newly developed antibody goes undetected in the manufacturing process,
an unexpected cross reactivity may appear.


This has been one of the criticisms of polyclonal antibodies.
When the original host(s) is no longer available to produce the antibody, new hosts
have to be identified and immunized.


There is no guarantee that the new batch will be identical to the first.
So variability may occur and under some extreme circumstances, the antibody
becomes unavailable.
76

Polyclonal antibodies are especially sensitive since the
can bind with a number of epitopes.

On occasion, this can lead to the impression of a false
positive result since it may also attach to similar antigens.

Purification and adsorption minimizes this risk.

Diluting the antibody out to the optimal titer will help
tremendously too.
77
OTHER KINDS OF ANTIBODIES

Besides antibodies the commonly used antibodies made in mice
and rabbits and animals. There are a couple of others to be aware
of.

These are:
 Lectins
 Chimeras
78
LECTINS:

A lectin is any of several plant proteins that bind to specific
carbohydrate groups on proteins or on cell membranes and are
used in the laboratory to isolate glycoproteins, to stimulate
proliferation of lymphocytes, and to agglutinate red blood cells.

Lectins: detect carbohydrates, sugars
 Needs diluent specifically formulated for them (not PBS) Tris
with Ca and Mg added
 Cannot be detected in the traditional manner unless labeled
(such as biotin)
 Requires a method of linking the antibody to a kit; rabbit antilectin to link it to a detection kit
79
CHIMERA: HUMANIZED ANTIBODIES

“Scientists developed "chimeric antibodies," which still contain mouse protein
sequences (approximately 33%) but also contain human protein sequences
(approximately 66%).

Although chimeric antibodies are "more human" and theoretically, less likely to
trigger an immune reaction, they nonetheless can trigger a human anti-chimera
antibody response by the human immune system.

Scientists then developed CDR-grafted or "humanized" antibodies which contain
approximately 5% to 10% mouse protein sequences.”
Medarex: http://www.medarex.com/Development/Evolution.htm
80
CHIMERA

The portion that binds to the cell is mouse etc
 The
Fc portion has been modified to be human
 Cannot be detected in the traditional manner

Needs a secondary component that is anti-human
 The
greatest interest is the use of these antibodies to treat patients
with an antibody that specifically targets a tumor cell
Medarex Antibody Evolution
81
LABELED ANTIBODIES

An primary antibody can be labeled in several ways so as to
dramatically reduce the opportunity for background staining,
crossreactivity, and to save time performing multiple steps.

These methods are not really direct, but require two or three steps to
achieve the desired signal

Commonly used labels for antibodies are:


Polymer, Biotin, HRP, Alk PHs, and fluorophores
There is a great synopsis of these in the Dako catalog

Dako Cytomation Catalog, 2005/06, pp 299 - 304
82
TROUBLES WITH ANTIBODIES

Antibodies have very specific needs in order to work appropriately.

A few antibodies will tolerate a particular diluent for a time but
begin to break down sooner than they would otherwise had been
made up in a different diluent.

If a new antibody does not appear to work well, perhaps substituting a
different diluent may be the remedy.

Sometimes purchasing a different formula of a PBS or Tris with higher or lower
salt concentrations or pH may also yield better results.

Monoclonal antibodies recognize and bind with a single epitope, or
binding site.
83

A monoclonal antibody has the advantage of being very specific, which
occasionally can lead to a negative result.
 It does not mean that the antigen is not there, but only that the specific
epitope was not available for binding with an antibody.

This can happen as fixatives and tissue processing distort the antigens, as tumors
and other pathologic processes undergo a number of mutations or as a result to
some of the newer drug therapies.
 ER or PR after Tamoxifen®

Polyclonal antibodies have the advantage of being more sensitive, but less
specific.


Sometimes it is a little sticky, attaching itself to similar antigens.
A disadvantage of a polyclonal antibody is that each pooling of serum may contain
a different mix of antibody clones because the mix of rabbits may be different.


This may be evident in new lots of the antibody.
Or when purchasing a polyclonal antibody from another source.
84
TROUBLES WITH ANTIBODIES

How would one identify a problem with an antibody vs
antigen?

What about affinity and avidity of an antibody?

What factors might favor an antibody or not?
Diluent
 Concentration
 Pretreatment


What is a prozone effect?
85
ANTIBODIES – KEY WORDS
Antibody
Monoclonal
Polyclonal
Clone
Conjugated antibodies
Structure
F(ab)
Fc
Variable region
Light chain
Heavy chain
Kappa
Lambda
Types
IgG, IgM
Ascities
Serum
Tissue culture supernatant
Lyophilized
Prediluted or ready to use
Lectins
Chimera
Affinity purification
Immunoglobulin fractions
Specificity
Affinity
Avidity
Sensitivity
Prozone
86
87
II. DETECTION SYSTEMS (10%)

Detection systems are reagent products designed to identify, and
then attach to a bound antibody, directly or indirectly.

This enables the minute antigen-antibody reaction to be visible for
microscopy when applied to IHC.

These methods are loosely grouped as

direct methods

indirect methods
88
DIRECT METHODS

Direct methods use an antibody that is labeled with something that
is visible with either bright field microscopy or fluorescence.

Two very common ways are with a fluorescent label such as;
 FITC (there are many more possibilities)


Requires a fluorescent microscope to visualize staining
Labeled polymer

Visualized with a bright field microscope.
89
FLUORESCENCE

Fluorescence is the original method of performing IHC

It is a single step way to detect an antigen, quickly and easily

Today, it is done primarily on frozen sections for autoimmune diseases such as
Lupus, pemphigus / pemphigoid lesions and some kidney biopsies

Dermatology labs use this method extensively, as do those doing ISH for
chromosomal studies, and multiple antibody or gene labeling.

Looking at tissue stained with fluorescence requires some skill and training.

Unlike bright field methods, one cannot see all the same details of nuclear
staining, and the architecture of the tissue.
90

Notes:

Fluorescent labels are subject to fading over time

Photo-bleaching is an artifact of microscopic evaluation

Require a specific mounting media

There is no amplification of the antibody-antigen binding site unless
using a labeled /antibody/polymer

Tissue morphology can be enhanced by using a fluorescent
counterstain. There are two commonly used counterstains for FITC
primary antibody staining.


Propidium iodide (red)

DAPI (blue)
Double, triple and quadruple labeling is easily done with fluorescence
since they do not tend to mask one another.
91
QDOTS

Fundamentally, Qdot® nanocrystals are fluorophores—
substances that absorb photons of light, then re-emit
photons at a different wavelength.

Qdot® nanocrystals are nanometer-scale (roughly
protein-sized) atom clusters, containing from a few
hundred to a few thousand atoms of a semiconductor
material (cadmium mixed with selenium or tellurium),
which has been coated with an additional
semiconductor shell (zinc sulfide) to improve the
optical properties of the material.

These particles fluoresce in a completely different way
than do traditional fluorophores, without the
involvement of electronic transitions.
92

Qdot® bioconjugate is a generic term to describe Qdot®
nanocrystals coupled to proteins, oligonucleotides, small
molecules, etc., which are used to direct binding of the
quantum dots to targets of interest.

Examples of Qdot® bioconjugates include streptavidin, protein A, and
biotin families of conjugates.

Standard fluorescence microscopes are an excellent and widely
available tool for the detection of Qdot® bioconjugates.

These microscopes are often fitted with bright white light lamps
and filter arrangements; Qdot® nanocrystals efficiently absorb
white light using broad excitation filters, and the outstanding
photostability of Qdot® bioconjugates allows the microscopist
more time for image optimization.
http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Qdot/TechnologyOverview.html#history
93
LABELED ANTIBODIES

A primary antibody can be labeled in several ways so as to
dramatically reduce the opportunity for background staining,
crossreactivity and to save time performing multiple steps.

These methods are not really direct, but require two or three steps
to achieve the desired signal

Commonly used labels for antibodies are:


Polymer, Biotin, HRP, Alk PHs, and fluorophores
There is a great synopsis of these in the Dako catalog

Dako Cytomation Catalog, 2005/06, pp 299 - 304
94
INDIRECT METHODS

Indirect methods imply that two or three steps may be required to visualize the
antibody.

In some cases, as with double labeling, many more steps will be required

This has been done in a number of ways such as PAP, APAAP, ABC, Avidin-Biotin,
Strept-avidin biotin, polymer and branching and technologies (non-strept
avidin).

These may use a secondary antibody, a tertiary reagent and ultimately a
chromogen substrate.


Many use enzymes such as horseradish peroxidase and alkaline phosphatase.
Polymers and branching technologies are usually proprietary and/or patented
such that the actual technology may be less obvious.
95
COMMONLY NEEDED PARTS TO A DETECTION

Most detection kits commonly used consist of a secondary and
tertiary reagent.


Enzymes


A protein molecule that speeds up a chemical reaction in a living
organism.
 Enzymes act as catalysts for specific chemical reactions, converting a
specific set of reactants (called substrates) into specific products.
Substrates


Of these, the following are employed in one way or another
The substance acted on by an enzyme
Chromogens

A substance capable of being converted into a pigment or dye
96
COMMON METHODS

The most commonly used detection methods have been;






PAP and/or APAAP
 Similar concepts
Biotin – Avidin complexed (ABC)
Biotin labeled secondary antibody followed by strept-avidin + enzyme
conjugate
Polymers
 Dextran and many more proprietary polymers
Branching technologies
 Not polymer, but do not attach to endogenous biotin
Double and triple staining methods
97

Dako has an excellent reference for getting to understand
so many ways to detect an antibody, along with visual
characterizations of how these detection systems work.

All methods have limitations but as time goes on, many
limitations are either eliminated or a work-around is
found.

Ref:


Handbook; Immunochemical Staining Methods, 3rd Ed. Pp 26 – 33
Education Guide, Immunohistochemical Staining Methods, 4th ed. Dako,
Chapter 9
98


For example –

PAP technique- has been cited as producing prozone effects

Avidin/strept avidin methods detect endogenous biotin

ABC methods do not have a stable conjugate

Strept avidin methods bind to endogenous biotin

Fluorescent methods photo bleach and are not achievable.
In many aspects polymer technology has solved many of these
issues by not being able to stain endogenous biotin, using an
enzyme conjugate with a stable chromogen, and in many instances
provides amplification of the antigen-antibody reaction.
99
BLOCKING REACTIONS

Because we do so much to the tissue in the process of IHC
staining, it is inevitable that unwanted staining can occur by some
other mechanisms than the primary antibody.

To overcome this, we use blocking agents.

The choices of blocking agents is done in conjunction with the
chemistry that we are using to detect our antibody and knowing
what is likely to stain in the tissue as a result of this.
100

These unwanted staining reactions can be dubbed “background”.

Background staining has many causes, and as such, need to be
recognized and handled accordingly.






Cross-reactivity
Non-specific staining
Endogenous background
Fc receptors
Antigen diffusion
Two excellent sources of information regarding this are found in


Dako’s Handbook: Immunochemical Staining Methods, 3rd ed, pp 34
– 38
JM Polak’s and S Van Noorden’s Introduction to
Immunocytochemistry, 2nd, pp 55 – 62.
101
PROBLEMS WITH DETECTION SYSTEMS

Visualization of the antibody is wholly dependent on the sequential
application of reagents in the detection kit

careful attention needs to be paid to the carrying out of these steps in the
precise manner described by the package insert.

In a number of instances, the same slides can be restained even after
coverslipping.

One has to have a sense of where the procedure went wrong.

Do not repeat any pretreatment steps since the effects of this process
are still in place.
102

Depending on the kits being used, some reagents may need to be
made up fresh for each event, such as the chromogen.

If this is the case, be mindful of the recommended time allowed
before the solution begins to break down.

If the slides will not likely be ready for this step within the allowed
time, postpone making it until closer to the time it is needed.

If one is using automation, set up errors may occur.

If it had, the chances are that it will impact a number of
slides/antibodies
103
DETECTION SYSTEMS - KEY WORDS
Fluorescence
direct staining
Photo bleaching
Propidium iodide
DAPI
Labeled antibodies
Indirect staining
ABC
Avidin-biotin
Strept-avidin
Polymer
Chromogen
Substrate
PAP APAAP
Branching technology
Conjugate
Secondary antibody
Horseradish peroxidase
Alkaline phosphatase
Prozone
Blocking reagents
Hydrogen peroxide
Levamisole
Biotin blocking
Background
Endogenous biotin
Endogenous alk-phos
Endogenous peroxidase
Cross-reactivity
Non-specific staining
Fc receptors
Antigen diffusion
104
105
III. SPECIMEN HANDLING (10%)
FIXATION

An excellent review of fixation and fixatives if provided in Anatech’s
“Lessons In Immunohistochemistry”. 2005. pp 2 – 9

Fixation is the most influential process that directly affects the
outcome of IHC on paraffin sections and cytology preps.

Each fixative lends a different advantage or disadvantage to
performing IHC.

There are a variety of fixatives to choose from and any deviation
from a formula may have an impact on IHC.
106


Formalin is still the most commonly used fixative.

There are a variety of formulations!

NBF is slow to denature

Incomplete fixation in formalin will result in further (but different)
denaturization with alcohols while processing tissue
Even traditional 10% NBF formalin has several formulations and
something as simple as changing the vendor may provide a slight
formulation change.

This may be attributed to the buffering salts used and the addition of methanol
during the manufacturing of the formalin.
107

Zinc formalins do not crosslink even over time

It is a strong and rapid fixative

May impact the pretreatment steps routinely prescribed for a
number of antibodies needing less, not more.

May be used unbuffered or buffered, contain zinc sulfate vs. zinc
chloride.

NBF and Zinc formalin will form a precipitate if combined, so
tissue sections or cassettes need to be rinsed off before
combining on processor.
108

Glyoxal fixatives
 These
are aldehyde fixatives that do not crosslink.
 Glyoxal
changes the shape of one amino acid, arginine.
 This
may inhibit an epitope that is rich in arginine in which
case a particular retrieval method might be needed.
 These
are more often found in combination with nuclear
histones.
Anatech’s Lessons In Immunohistochemistry, pp10 & 16-17.
109
 HIER
solutions will be needed from time to time, but
glyoxal fixed tissue respond best to pH ranges 8.5 – 9.
pH’s above and below that range can damage tissue
severely.
 Generally
compatible with antibodies that are used for
formalin fixed tissue.
 Does
not respond well to proteolytic enzyme digestion
methods such as trypsin etc
110

Adequate and consistent fixation safeguards IHC results, allows for
predictable pretreatment regimens, and provides adequate
morphology.

Fixation also stabilizes the antigens that are to be detected.

Uneven or incomplete fixation contributes substantially to mixed
results for IHC, and is exacerbated by pretreatments such as with
proteolytic enzymes and antigen recovery methods.
111

Incomplete fixation with the primary fixative, such as formalin,
places the tissue at risk of being fixed in yet another or secondary
fixative such as alcohol in the tissue processor.

This in turns makes it almost impossible to have a pretreatment
application that addresses the specimen uniformly.

Note: Variable and incomplete fixation is one of the most common
causes of problems in IHC.
112
TISSUE PROCESSING

In addition to adequate fixation, adequate processing of tissue is
essential.

The dehydrants, usually an alcohol, will not only dehydrate tissue but
continue to fix tissue as well.


This impact is most evident when fixation with the primary fixative
is incomplete.
A very common practice is to include a formalin alternative in the
tissue processor or hold specimens in a reagent other than formalin
when delayed schedules are in place.
113


Delay schedules on tissue processors may hold tissue in a reagent
other than the primary fixative over the weekend or holiday.

This variant may exert some influence over IHC performance and
should be evaluated before assuming that there is none.

It has sometimes been recommended to hold cassettes in 70%
alcohol after the fixative in order to minimize the impact of prolonged
fixation in a cross linking fixative.
Today, there are the traditional tissue processors as well as microwave
tissue processing.
114

One needs to assess the impact that this alternative provides for IHC.

Some use proprietary reagents whose chemistry is unknown or unevaluated
for IHC.

In most instances fixation time is markedly shortened, therefore the tissue
may have a negative response to a number of pretreatments such as
proteolytic enzyme digestion and HIER.

Additionally, incubation times may be impacted, backgrounds may be higher
and titers may need to be extended out.

Validation of new types of reagents is crucial for every application that the
tissue may be subjected to.
115
DECALCIFICATION

May sacrifice antigen of interest!

Adequate or complete fixation prior to decalcification is the only way
to minimize this risk.

Strong acids (HCl, nitric and trichloroacetic acids) have been blamed
for loss of more antigens than weaker acids (formic and acetic) in
addition to the chelating agent EDTA have been better suited for IHC.

These are kinder to morphology as well.
116
PROBLEMS WITH TISSUE HANDLING

The tissue needs to be fixed well, first and foremost.

Without this elementary and essential step, there is no limit to what
can go wrong.

If a formalin fixative had not completely penetrated the tissue, then
the alcohols in the processor will complete the task.

Gradient staining across a tissue sample due to the activity of two
fixatives is very common and problematic.

When the procurer of the specimen changes something, it may
impact our success with IHC.
117
Endoscopy test for H. pylori
118
Microtomy and Slide Preparation

Paraffin sections for IHC are best cut between 3-5 µm.

This allows for adequate antigen content and cellular detail.

Very thin sections may produce very pale staining. Sections
thinner than 3 µm will produce very pale staining.

Slides are either dried at 37° C overnight or ~60° C for 1-2 hours prior to
staining.

Care should be taken to avoid prolonged high temperatures since this may
compromise antigens.
119

The usual gelatins in the water bath often prove inadequate for IHC.

Slides used for IHC usually have a positive charge.


This can be achieved in several ways.





These help tissue to adhere in spite of pH ranges, temperature and
multiple applications of reagents
Commercially available slides with a positive charge
poly-L-lysine
aminopropyltriethoxysilane, (APES)
There are other unique ways of producing a charge on glass slides as
well, such as etching.
Tissue need to have adequate time to dry and adhere to the slides
prior to staining.

Times may vary according to season, and even between techs.
120

It is important to note that the intent of the charged glass slides is
to hold onto the tissue which may be subjected to the rigors of IHC
staining, such as HIER, proteolytic enzyme digestion and extremes
in pH.

These slides are highly charged, and will be altered easily enough
with improper handling.

Humidity and inadequate drying times can contribute to tissue
lifting and loss.
121
CYTOLOGY SPECIMENS

Cytology preps, smears and touch preps are often fixed but certainly not in the
same manner as tissue that is destined to be embedded in paraffin.

Body fluids, and fine needle aspirates (FNA’s) are usually handled very differently
from cell blocks and the traditional PAP smear or GYN’s.

It is important to keep this in mind when developing protocols to be used across
more than one application.

Additionally, cytology preps have whole, uncut cells, with intact membranes unlike
their paraffin counterparts.

Cytology may use several fixatives including alcohol, carbowax and other
proprietary reagents.
122

Each of these fixatives exerts their respective influences on the antigens of
interest and may or may not need any pretreatment prior to staining with an
antibody.

Some of the cytology instruments for making monolayer preps have proprietary
fixatives which are usually an alcohol base.

Different alcohols, such as ethanol, methanol and reagent alcohol have varying
impacts on the antigen and the antibody protocol, just as different formulae of
formalin do.

In many cases, but certainly not all, the need for pretreatments are either
eliminated or modified.
123

Certain kinds of endogenous entities, such as endogenous biotin are not
masked.

Therefore blocking for this may be necessary if a labeled strept-avidin detection
kit is to be used.

Cell blocks are often fixed or post fixed in formalin after centrifugation and
making the button.

It is often the case that these fluids could have been collected in containers
containing anticoagulants such as sodium heparin or EDTA.

These may on occasion influence the antigenicity of the cells
124
Immunofluorescence Specimens

These specimens are often kidney or skin for autoimmune diseases and pemphigus /
pemphigoid lesions.

Specimens for Immunofluorescence are often collected in transport media such as
Michel’s or Zeus reagents.


They specimens may stay viable in this media for several weeks

These are then washed in a wash buffer, and the tissue is then frozen for sectioning.
Sections are cut in a cryostat and placed into ice cold, -20˚ C(+/- 2˚) for ~ 10 min.

They can be air dried after this, or kept in acetone if staining is immanent
125

They are then placed into IHC buffer such as PBS or Tris and stained
with fluorescent antibodies.

Circulating autoimmune antibodies can also be detected using
Immunofluorescence

The patient’s serum is applied to frozen normal tissue and allowed to bind.

These auto antibodies are then detected using fluorescent labeled antihuman antibodies

This is called Goodpasture’s serum technique
126

Another application for IF is in organ transplant work.

Frozen section biopsies from transplanted organs can be
stained with specific FITC or other labeled markers that help
to discern organ rejection from infection due to the patient’s
immuno-compromised condition

These often stat because the patient may be suffering from
rejection or infection.

These are collected and cut in much the same manner as
any other frozen section specimen with the addition of
acetone fixation.
127
Frozen Sections

Frozen section can be either fixed and then frozen or frozen and then fixed.

The first is usually done in the research setting with the host having undergone
perfusion with a fixative prior to necropsy, while the latter is typical of the clinical
setting.

There are a number of reasons to rely on frozen sections but perhaps the most
notable is that the antigens are more like the native state.

There are some antigens that can only be detected in fresh, frozen, unfixed
sections.

The most prevalent uses are in hematopathology, Dermpath (autoimmune
diseases) and organ transplant.
128

Cryoprotection, or the avoidance of ice crystals, is necessary to
preserve morphology of fixed, frozen sections.

This can be accomplished by immersing slides in 30% sucrose 1224 hours prior to staining.

The subsequent ice crystals are not detectable with light
microscopy.

Slides are often air dried after this until ready to stain, or stored at 80° C until needed.

Fixation with acetone also helps to prevent the diffusion of the
antigens that are to be stained.
129

Background staining in frozen sections is often different than that which might
be seen with paraffin sections.

Endogenous peroxidase is an artifact associated with formalin fixation and is
less likely to be an issue with frozens that are fixed in acetone.

Endogenous biotin is usually masked with formalin but not with acetone fixation.

It is important to note that either of these can only be a problem if one is using
an HRP label (endogenous peroxidase) or strept-avidin conjugates (endogenous
biotin).

Staining on the Fc receptor is sometimes a problem in frozen sections as well.

This can be blocked by using an Fc receptor blocker.
130
SPECIMEN HANDLING - KEY WORDS
Fixatives
NBF
Alcohol
Zinc formalin
Glyoxal fixatives
Precipitant fixatives
Coagulative fixatives
Cross-linking fixatives
Combination fixatives
Microwave processing
Microwave processing
Cytology specimens
monolayer techniques
FNA’s
cytology fixatives
Touch preps
Immunofluorescence
Autoimmune
Frozen sections
Cryoprotection
131
132
IV. EPITOPE ENHANCEMENT (ANTIGEN RETRIEVAL) 20%
Methods, Principles and Techniques

Pretreatments are measures that are taken to make an antigen
more accessible to the antibody.

Pretreatment refers to a number of approaches which include, but
are not limited to
Wow,


proteolytic enzyme digestion
antigen unmasking techniques
 Antigen Retrieval
 HIER (heat induced epitope retrieval)
20%?
133

Pretreatment alter the tissue to make the antigen more
accessible.

For this reason, the process needs to be very carefully
controlled because too much pretreatment may induce a false
positive staining result, while too little may yield no staining at
all.

Approximately half of the problems encountered while
performing IHC can be traced directly to this process being out
of control
134

A proteolytic enzyme is one that breaks up proteins into
smaller segments.

This will allow for the antibody to make its way down to the
antigen it seeks.

There are a wide variety of these proteolytic enzymes,
including trypsin, pepsin, ficin, pronase and protease.

These are made in a buffer, and usually performed by
placing the slides in this solution at 37°C, though temps
vary re. to enzyme.

The duration of this step needs to be controlled so that
over-digestion does not occur.
135

The amount required is actually dependent on the extent of fixation
and the fixative itself.

Excessive digestion will literally eat away the connective tissue
leaving a number of isolated cells that are no longer cohesive.

Some of the enzyme solutions are not stable for more than a few
hours so the reagent needs to be made fresh just before use,
trypsin is such an enzyme.

Many others, including protease, ficin and pepsin are commercially
available as stable solutions, thus making it convenient for the user.
136

Antigen Retrieval® - Dr. Shi et al is credited with introducing this concept of
antigen retrieval.

The original approach employed high temperature heating of paraffin embedded
samples in a microwave.

The pH of the buffers range from as low as 3.5 to as high as 10.

In the last decade a vast number of modifications have been made to the original
approach.

Heat sources come from microwaves, microwave pressure cookers, bench top
electric pressure cookers, autoclaves, water baths, ovens and hot plates.

Antigen unmasking is usually performed as a counter measure to fixation,
especially formalin.

They are done prior to the antibody application or any blocking steps.
137

The exact mode under which antigens become unmasked is as yet not fully
understood, but many have found that salts, metals, pH and temperatures play a
significant role in their recovery and in varying degrees for different antigens.

Hypotheses include concepts such as:







A loosening or breaking of cross linkages
Protein denaturization: observations that some antigens or endogenous
enzymes activities may be lost during AR
A number of pathways, including breaking up cross linkages, dissolving
some blocking proteins, precipitation of proteins, and/or rehydration of
tissue
Removal of the residual traces of paraffin due to heated solution
Hydrolysis of Schiff bases
Chelation of calcium complexes bound to protein during fixation
Reversal of protein confirmation due to fixation
138

Antigen recovery technologies, including the now patented Antigen Retrieval®
method, and HIER are decidedly the discovery of the 1990’s, paving the way for
new areas of research and the continual expansion of clinically significant
antibodies that were previously prohibitive.

In fact, the majority of the newly developed antibodies recommend pretreatment
of the tissue with an antigen recovery method.

Although antigen unmasking techniques are absolutely indispensable in the
performance of IHC techniques today, excessive or inappropriate antigen
unmasking may induce the impression of positive staining even without a primary
antibody.

There is no step requiring more quality control than this one.

Too much can produce false positive staining.
139
Combination of Proteolytic enzyme with HIER

Many are now finding that combining a milder form of proteolytic enzyme with a
milder HIER protocol can produce some very good results.

There are antigens that respond favorably if the pretreatment with the enzyme
precedes the HIER while yet others seem to prefer the reverse order.

When combining the two applications, even more care needs to be taken to avoid
going too far.

Both Anatech and Dako present very useful information regarding antigen
retrieval.
 Anatech’s “Lesson In Immunohistochemistry”, pp 13 – 20
 Dako’s Handbook: Immunochemical Staining Methods, 3rd end. pp 23- 25
140
EPITOPE ENHANCEMENT - KEY WORDS
HIER
Proteolytic enzymes
Antigen Retrieval
pH
Enzyme activity
Epitope
Buffer
141
142
V. STAINING 30%

Immunohistochemistry is the application of an antibody to a
corresponding antigen in tissue.

The antibody-antigen interaction is done at the molecular level, so the
bound antibody needs to be “stained” in order to make it visible for
microscopy.

This can be done in a variety of ways using a detection system.

These are generally kits, having a series of reagents that when
applied, incubated and rinsed with buffer in the appropriate order,
produce a color end product and often amplifying the antibodyantigen complex.
143

A Detection System is one or more steps that label the bound antibody, producing a
visible signal either by fluorescent microscopy or bright field microscopy.

Direct staining implies that the antibody itself has an agent attached or labeled to it.




This agent will in and of itself produce a visible signal.
This method is usually a fluorescent label or fluorophore.
Direct methods are one step procedures which do not amplify the antibody-antigen
binding site.
This method is quick.

Indirect staining implies that the antibody-antigen binding site is somehow amplified
by using a series of two or more agents to accomplish this.

Most detection systems used in clinical settings today use indirect methods.

The most common detection systems today are;




Fluorescence
Enzyme
Polymer
Branching
144
Detection systems or kits

Fluorescent labels use dye molecules which fluoresce (emit light) in
different colors at different wave lengths.



The color emitted is specific to each fluorophore (fluorescent dye)
chosen.
Can be used in a direct or indirect application
This technique as the following advantages




rapid technique, double- or triple-staining (multi-color) is easy to
interpret
dyes do not mask each other
more sensitive than enzymatic techniques
fluorescent labels are more readily conjugated to antibodies because
they are typically smaller in size.
145

There are some significant disadvantages as well.

They are:

fluorescent signal fades with storage, cannot be permanently
coverslipped

requires aqueous, oxygen scavenging mounting medium

difficult to visualize cell morphology

counterstain is fluorescent and has same issues as label

requires fluorescent microscope

requires specific filter sets for each fluorophores

The bulb “life” can compromise visualization
146

Enzymatic Labels use enzymes that react with other reagents to produce a
chemical reaction and generate a colored end-product.

The two most commonly used enzymes are:
 Horseradish Peroxidase (HRP)
 Alkaline Phosphatase (AP)

A substrate (the substance acted on by an enzyme) and a chromogen (a
compound that can be converted to a pigment) are required to generate a
colored reaction end-product.

The substrate and chromogen is a matched pair and there may be more than one
substrate/chromogen set for each enzyme.

The colored end-product is visualized with a regular light microscope.
147

Enzymes, substrates and chromogens combinations are:
 Horseradish Peroxidase (enzyme) uses these
chromogens Diaminobenzidine
(DAB) - produces chestnut brown color
 Aminoethylcarbazole (AEC) - produces brick red color
 Alkaline
Phosphatase (enzyme) uses these
chromogens Fast
Red - produces bright fuchsia color
 Fuchsin – produces bright fuchsia color
 BCIP/NBT – produces dark blue color
148


Some of the advantages to this technique are

the label is permanent (relative to fluorescent labels)

it’s easy to see the morphology

tissue structures are simultaneously visualized with antigen labeled

requires a light microscope

is usually compatible with current image analysis systems.
Some of the disadvantages are

that the time required to perform this technique is longer than
fluorescence

multi-color staining interpretation is more difficult but impossible

some chromogens are hazardous or toxic.
149

Polymer Detection systems uses a polymer backbone to attach enzymes and/or
secondary antibodies.

A polymer is a string of repeating molecular units of a specific length, such as
protein (e.g. Strept-avidin, Protein A), a carbohydrate (e.g. Dextran), or amino
acids (e.g. Lysine).

Polymer detection system have the advantages

decreased background due to absence of biotin binding or
endogenous biotin

rapid turn-around time (two steps instead of three in avidin-biotin
method)

sensitivity typically comparable to avidin-biotin methods or better.
150

The disadvantages are relatively few depending on the polymer being
used

Some of these rarely experienced limitations might be




temperature sensitive - must be at room temperature
some are species-specific (mouse or rabbit) but "universal" polymers
are available
some claim that diffuse staining may be more likely if the polymer is too
large.
There are more than one polymer technology available and they are
not created equal.
151

This second antibody “links” the first or primary antibody to the steps that will
follow it.

This second antibody was made to identify the species (not the antigen in the
tissue) of the primary antibody.
 It does this by attaching to the opposite end of the primary antibody where
the mouse or rabbit IgG (or IgM) is located.

The secondary (second antibody that recognized mouse or rabbit etc) antibody is
labeled or tagged with something that the next reagent in the kit can attach to.

The labels are usually biotin or a polymer.

This label will allow for the next reagent in the kit to attach to it.
 In the case of a biotin labeled secondary antibody, the next reagent is usually
an enzyme conjugated strept-avidin product.
152

The strept-avidin has as much affinity for the biotin as the primary
antibody has for the antigen.

This makes for a very strong binding reaction.

The enzyme that is attached to the strept-avidin is usually
horseradish peroxidase (HRP) or alkaline phosphatase (AP), but
other enzymes can be used.

When a secondary antibody is polymer labeled, the enzyme, usually
horseradish peroxidase, is already attached to the polymer along
with the secondary antibody.

The final step is to take advantage of the enzyme, either
horseradish peroxidase or alkaline phosphatase and apply the
chromogen and substrate.
153

The enzyme itself determines what your chromogen (color
precipitate) choices are here.
 If horseradish peroxidase is the enzyme, then 3,3'
Diaminobenzidine (DAB) or 3-Amino-9-Ethylcarbazole (AEC) is
your choices.
 If alkaline phosphatase is the enzyme, then Fast Red, New
Fuchsin or 5-Bromo-4-Chloro-3-Indolyl Phosphate/nitro blue
Tetrazolium (BCIP/NBT).

The substrate is a solution that an enzyme requires in the presence
of the chromogen.
 for HRP it is hydrogen peroxide
154
Wash Buffers

When performing IHC procedures it is imperative to use the appropriate wash
buffer (a solution which resists changes in pH).

The performance of the detection kits and the actual antibody itself are absolutely
dependent on the appropriate pH.

The most commonly used wash buffers are phosphate buffered saline (PBS) or tris
saline buffer (TBS) and Tris hydrochloric buffer (TRIS-HCl).

The recommended pH is right around 7.6 +/- .2.

The pH of these buffers should be checked each time they are made up and over
extended periods of time.

Some buffers may begin to support microbial growth if an antimicrobial agent is not
used in the formula.
155

Commercially available wash buffers often compensate for this by the addition of such an
agent.

Most commercial wash buffers contain a fraction of detergent.

This improves the wettability of the wash buffer and improves it effectiveness in cleaning
the slides.

A specific recommendation for a wash buffer may be made in the package insert for the
detection system.

Deviation from this may cause problems with the staining outcomes.

Wash buffers are used at the conclusion of the incubation period for each of the reagents
when performing the IHC protocols.

This step needs to be carried out effectively to cleanse the slide of any residual reagent
before the application of the next one.

This will help to minimize unwanted background staining.
156

Buffers should be monitored for pH drift.

This is best accomplished by using a pH meter.

Tris is a bit of a challenge to ph and requires a specific kind of electrode to do it
reliably.


Calomel pH probes are recommended for tris containing buffers as well
as protein solutions since either of these can be problematic for
silver/silver chloride junctions on probes.
In literature it has been reported that probes using a “linen” string in the probe give
errors in the results as well as there being problems of premature failure of probes
using the standard silver/silver chloride reference electrode and only a single
ceramic junction.
157

Apparently the silver ions in the junction cause precipitates that block the
junction and hence cause an unwanted pH potential).

This latter would be manifested by slow response and/or drift in the electrode.

The pH of tris buffers is also affected by temperatures, (-0.028 pH per +1°C
temperature change).

Therefore a pH meter which compensates for the temperature of the buffer can
be very advantageous to have, even for other reagents.

Needless to say, this kind is a bit more expensive than the pocket variety or pH
strips
158

The quality of the water used to make buffers is also a critical
factor.

The usual recommendation is NCCLS Type II water, deionized, with
low conductivity (available ions) and low bioburden load.

Distilled water is not necessarily deionized or vice versa.

Neither distilled nor deionized water implies that the water is sterile
or microbial free.

If you are not sure about the quality of the water available to your
lab, consider purchasing water in order to avoid countless hours of
poor staining results.
159
TISSUES

Morphology and anatomy

Tissue used to optimize a antibody protocol should be as similar to
patient tissue as possible in


Fixation

Processing

and relative time schemes.
Autopsy tissue should be avoided whenever an alternate solution is
available.

Multiple tissues should be used to optimize protocols to be sure that
the antibody is targeting all that it is expected to stain.
160




Cell component demonstration
Staining patterns
 Package inserts for antibodies describe the anticipated pattern of
staining, the cells that should be positive for the antibody, and even
distribution for some disease states.
 Staining will be described as membrane, transmembrane,
cytoplasmic, nuclear or nuclear membrane.
Known cross reactivities are also mentioned.
 Microorganisms
 When using antibodies against micro organisms, be clear as to
what they actually label.
Pathology
 The reason we do IHC in the clinical setting to determine various
aspects of pathology.
 Procedure must be sensitive enough to do this routinely and reliably
time and time again.
161
STAIN COMPONENTS & CHARACTERISTICS
Concentrated antibody
 Pre diluted antibody
 Titrations
 Reagents
 Troubleshooting
 Mounting Procedures

162
CONCENTRATED ANTIBODIES

Concentrated antibodies are very high in titer (antibody concentration),
containing a large proportion of the specific antibody.

They may be supplied as lyophilized or in liquid form.

A lyophilized antibody is in powder form and needs to be reconstituted before use.

Generally, far too much antibody exists to use as is or neat, so it must be diluted
out to determine the dilution or titer for routine use.

The data sheets supplied by the manufacturer of the antibody usually suggests
the range where the dilution occurs most often.
163

The optimal dilution needs to be determined within the context of
the detection methods used in each lab and the fixation and
processing of the samples.

Antibodies should be diluted using an appropriate antibody diluent.


Some antibodies are very particular about salt and protein concentrations,
along with buffer.
It is important to prevent the growth of bacteria etc. by adding an
ingredient to inhibit microbial growth if one intends to keep diluted
antibodies for an extended period of time
164
TITRATIONS

The “right” titer for an antibody is one that provides
the highest dilution with the best specific staining
and the least amount of background staining.

The test or optimization conditions must be
controlled so that the titer remains useful once it is
established.

All of this is influenced by time, concentration and
temperature.
165
DILUTION OF ANTIBODIES:

Start with the package insert for the antibody. The recommended starting dilution
for an antibody is usually expressed with two numbers, such as 1:50 - 1:100, or
another may be 1:500 - 1:1000.

These paired numbers represent a series, or serial dilution of the antibody where
the titer works out most often.

The first number is where the first dilution will occur, and the second number
represents the first number, diluted in half.

Example 1:




A 1:10 dilution means one part out of ten parts is antibody, or
one part antibody added to nine parts antibody diluent.
A 1:20 dilution means one part out of twenty parts is antibody or
one part antibody added to nineteen parts of antibody diluent.
166

Another term for a concentrated antibody is neat.

When talking about an antibody in the concentrated form, it is referred to as a
1:1, or one part out of one part is antibody, or one part added to no additional
parts.
 In other words, it is all antibody.

This 1:1 relationship is important when doing the calculations for the dilution of
that antibody.

Antibody dilutions are always expressed as 1:xx, (one to...). It is representative of
one part of antibody and its relevance to an expressed amount of diluent, :20,
:50, :100 etc.
167
UNITS OF MEASURE:

Antibodies are measured in microliters or lambda. The symbol for
microliters or lambda is µL.

One microliter is equal to 1/1000 ml. One ml. is equal to 1,000 µL.



1 ml = 1,000 µL.
5 ml = 5,000 µL.
10 ml = 10,000 µL.
168
FORMULA FOR DILUTIONS:

Antibodies are diluted in much the same way as formalin is, except
the amounts measured are microscopic compared to that which is
used when diluting formalin.

Consider how we make a 10% solution of formalin from a starting
solution of 37% formalin.

We dilute the 37% formalin to be 1 in 10 to make a solution
containing 10% of the 37% solution.

The resulting solution is 10% of the starting solution but contains
only 3.7% formalin.
169
FORMULA FOR DILUTIONS:

The same principle applies to making antibody dilutions.

Concentrated antibodies will have various concentrations depending on
where they are purchased and how the antibody is raised.

Whatever the concentration of the antibody at the start, we consider it
as if it were absolute, or 100%, just like we address 37% formalin.

From that starting point we dilute the antibody in an antibody diluent to
a working dilution that is compatible with the technology that will be
used to stain for the antibody.
170
FORMULA FOR DILUTIONS:
Step 1:
CONCENTRATED ANTIBODY x VOL. NEEDED = AMT OF ANTIBODY
DIVIDE BY DILUTION NEEDED
Example:
1:1 x
1:50
1,000 µL = 20 µL of concentrated antibody
Step 2:
VOL. NEEDED - THE AMT. OF ANTIBODY = AMT. OF DILUENT
Example:
1,000 µL – 20 µL of concentrated antibody = 980 µL diluent
171
EXAMPLE:
WE HAVE AN ALIQUOT (1:5), AND WE NEED TO MAKE A 1:100. 1 ML (1,000 ΜL) VOLUME IS
NEEDED
N.B.
Aliquot = 1:5; or one part in five parts, but like formalin, we treat this as our
concentrate.
Step 1:
1:5 x 1,000 µl = 50 µl of 1:5 (of the1:5 aliquot)
1:100
Step 2:
1,000 µl - 50 µl = 950 µl diluent
172
EXAMPLE:
WE HAVE A PREDILUTED ANTIBODY, AND WE NEED TO MAKE DILUTE IT IN HALF. MAKE 2
ML. OF DILUTED PREDILUTE.
N.B.
Predilute = 1:1; use neat or as supplied, just as if it were a concentrated antibody
Step 1:
1:1 x 2,000 μl = 1,000 μl of predilute
1:2
Step 2:
2,000 μl - 1,000 μl = 1,000 μl diluent
173
LIMITATIONS DUE TO MICROPIPETTES


This next example demonstrates how the
limitations of the micropipette will cause us
to rethink our approach to diluting this
antibody.
Wait a
minute
!
One would have to choose between making
a larger volume of diluted antibody or
make an aliquot from which the desired
dilution will be made.
174
OPTION 1MAKE A LARGER VOLUME, SUCH AS 5000ΜL :
Step 1
1:1 x 5,000 μl = 2 μl (of 1:1)
1:2500
Step 2
5,000 μl - 2 μl = 4,998 μl of diluent
Note: that the math looks better but the quantities are still quite small and may still be
cumbersome for some pipettes.
175
OPTION 2:
MAKE AN ALIQUOT SOLUTION FIRST, SUCH AS A 1:100:
1:1 x 500 μl = 5 μl (of 1:1)
1:100
500 μl - 5 μl = 495 μl of diluent
Now use the 1:100 solution to make 2 ml. of the 1:2500)
1:100 x 2000 μl = 80 μl (of 1:100)
1:2500
2000 μl - 80 μl = 1,920 μl of diluent
176
SERIAL DILUTIONS

The optimal dilution of an antibody has to be determined in conjunction with the
tissue fixation/processing and the detection technology to be used.

The dilutions recommended on a package insert are for the conditions unique to the
manufacturer and may or may not be the same for each lab.

They should be viewed as starting points only. For this reason, a series of dilutions
is looked at to determine the optimal conditions for each antibody.

To make serial dilutions, one simply takes a specified volume of diluted antibody
and adds it to an equal amount of antibody diluent.

This essentially dilutes the previous dilution in half.
177

For example, if one takes .5 ml (500 µl) of a 1:25 and adds that to .5 ml
(500 µl) of antibody diluent, the resulting dilution is a 1:50.

We now have one part antibody out of fifty parts instead of one part
antibody out of twenty-five parts.

If one took .25 ml (250 µl) of a 1:50 and added that to .25 ml (250 µl) of
antibody diluent, the resulting dilution would be 1:100.

The progression goes on into infinity, adding equal amounts of previous
dilution of an antibody and antibody diluent.

By adding an existing antibody dilution to an equal amount of antibody
diluent, we have cut the antibody concentration in half, making it twice as
dilute.
178

This exercise can be repeated for each antibody dilution thus creating a series.

A typical progression of serial dilutions would be...
 1:25, 1:50, 1:100, 1:200, 1:400, 1:800 etc.
 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 etc.
 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 etc.

One appreciable advantage to making a series of dilutions, (serial dilutions), is that
the math needs to be done only for the first dilution, or starting dilution.

After that, one is always removing a fixed amount of diluted antibody and adding it to
a fixed, equal amount of antibody diluent.

From the first dilution, one can literally make a series of dilutions into infinity
179

When trying to determine the best dilution of an antibody for routine use, several
dilutions would be done.

For example, to determine the best dilution of LCA, the manufacturer suggests 1:50 1:100.

One might start at one serial dilution below the recommended 1:50, and exceed the
recommended limit of 1:100 by one more serial dilution.

This would mean running a serial dilution set of 1:25, 1:50, 1:100, and 1:200.

Four control slides that were cut serially or consecutively would be selected for the test
run.

Each slide would be labeled for LCA, and each one would have a different dilution of that
antibody to be applied to it.

All other conditions for the run would be exactly the same.

The dilution of the antibody that represents the best signal versus the least or negative
background noise would be the most appropriate dilution of the antibody in a given
laboratory.
180
PROCEDURE
1. Make 1 ml of LCA diluted to 1:25. Place this in the first tube that is
labeled in like manner.
1:1 x 1,000 μl = 40 μl of 1:1
1:25
1st
1,000 μl - 40 μl = 960 μl of diluent
1 ml of LCA @ 1:25 dilution
181
PROCEDURE
2.
Set up three more tubes, and label them for the next dilutions, 1:50,
1:100, and 1:200 respectively.
2nd
1:50
3rd
1:100
4th
1:200
182
PROCEDURE
3.
Into each of these subsequent tubes, add 500 μl of
antibody diluent.
Note: this amount is one half the volume of the amount
placed in the first tube.
183
PROCEDURE
From the first tube containing 1ml of LCA diluted to
1:25, remove 500 μl.
4.



Place this into the 500 μl of antibody diluent in the
second tube.
Mix well.
Note: since we have removed
one half of the diluted antibody from
the first tube, there is now
1 ml of LCA diluted to 1:50.
1st
2nd
1:25
1:50184
PROCEDURE
5.
From the second tube containing 1 ml of LCA
diluted to 1:50, pipette out 500 μl.


Place this into the 500 μl of antibody diluent in the
third tube.
2nd
Mix well.
3rd

Note: there is now 1 ml
of LCA diluted to 1:100.
1:50
1:100 185
PROCEDURE
6.
From the third tube containing 1 ml of LCA diluted to
1:100, pipette out 500 μl.


Place this into the 500 μl of antibody diluent
in the fourth tube.
Mix well.
3rd

4th
1:100
1:200
Note: there is now 1 ml
of LCA diluted to a final
dilution of 1:200.
186
PROCEDURE
7. Label four slides, one for each of
the dilutions
LCA
1:25
LCA
1:50
LCA
1:100
LCA
1:200
8. When it is time for the antibody to
be applied to the slide, remove a
predetermined amount of antibody
from a tube and dispense it over
the tissue on the corresponding
slide.
9. Repeat for each dilution of the
antibody, applying the same fixed
volume to each slide.
187
TECHNICAL NOTE:

It is critical to keep the volume of antibody applied to
each slide consistent as well as the incubation times.

Variability in this technique may lead to variable results
when implemented for routine use.

Mimic what will be put into practice once the final
dilution has been determined.
188
MAKING A COCKTAIL OF MONOCLONAL ANTIBODIES:

From time to time it has been desirable and/or advantageous to make a cocktail of
antibodies.

Some familiar one are Keratin; AE1/AE3, and LCA; PD7/26 & 2B11.

If one wanted to, one can make cocktails of antibodies in one's own lab.

First one must obtain the clones to be cocktailed to together.

Each one of these clones would have to be titered individually.

Let's arbitrarily call these antibodies X, Y and Z.
189

Let's suppose that the antibody data sheet for each of these recommends dilutions as
follows:
X = 1:25 - 1:50 (serial dilutions would be 1:25, 1:50, 1:100 etc.)
Y = 1:100 - 1:200 (serial dilutions would be 1:50, 1:100, 1:200, 1:400 etc.)
Z = 1:40 - 1:80
(serial dilutions would be 1:20, 1:40, 1:80, 1:160 etc.)

Let's suppose after diluting these antibodies, and running them, the following titers are
determined to be appropriate for this lab;
X = 1:100
Y = 1:200
Z = 1:40

The next step would be to make a solution of a specified volume which will contain all of
these antibodies at their determined titers.
 One may elect to make 10 ml (10,000 µl).

To make this cocktail, one must first determine how much of each antibody will be present
in a 10 ml (10,000 l) µl solution.

The formula stays the same for a cocktail as it does for a single antibody.
 The only difference is one must consider the total amount of antibody before following
through with this formula.
190
Do the following steps:
antibody X:
1:1 x 10,000 μl = 100 μl of antibody X
1:100
antibody Y
1:1 x 10,000 μl = 50 μl of antibody Y
1:200
antibody Z
1:1 x 10,000 μl = 250 μl of antibody Z
1:40
191

Now add the amounts of each primary antibody together to
determine the total volume of antibody.
100 μl (X) + 50 μl (Y) + 250 μl (Z) = 400 μl (X+Y+Z)

Then subtract the total amount of antibody from the volume
to be made to determine the amount of diluent to add to this
mixture.
10,000 μl - 400 μl = 9,600 μl of diluent
192
TECHNICAL NOTE:

The pretreatment conditions must be identical for each antibody in the cocktail.

Stain a separate control slide for each antibody to be placed into the cocktail.

Stain a control slide with the cocktail and compare all that is stained with the
cocktail to the separate control slides.


The cocktailed antibody should stain cumulatively for each of the
antibodies.
On occasion, antibody that are placed together in a cocktail aggregate.

When this happens there will be less sensitivity noted on the stained
slide instead of more, and in serious cases, no staining at all.
193
PREDILUTED ANTIBODIES

Prediluted antibodies are already at the right titer, or dilution.
 The right titer or dilution is determined by which detection method it was titered
with.

Predilutes have the advantage of already having been worked out for titer, and
requires no additional effort from the tech.

It has the disadvantage of costing a bit more, and if staining is too light or too dark,
adjusting the titer is not an option.

Not all primary antibodies work universally on paraffin sections, frozen sections,
fresh preps (cytological preps) etc.
 When selecting the antibody, know what it will be used to stain before ordering
it.
 If a lab does cytological preps other than paraffin embedded cell blocks,
consider an antibody that is compatible with both.
194
BASIC IHC TROUBLESHOOTING
IHC is a complex procedure, in fact, it is a complex concept.

By definition, “complex is a conceptual whole made of complicated and
related parts” (http://www.thefreedictionary.com).

Given this explanation, is it any wonder that there are opportunities for
things to go astray?

For this reason, and many others, skill at troubleshooting is invaluable.
195

It has often been presumed that certain individuals have an innate
aptitude for this while others simply cannot.

The fact is that while some can do this instinctively, any one can,
and must, troubleshoot from time to time.

A troubleshooter is a problem solver.

The definition of problem solving is “the area of cognitive
psychology that studies the processes involved in solving problems
or the thought process involved in solving the problem” (WordNet®
1.6, 1997 Princeton University).

In other words it is a deliberate, systematic approach to identify the
problem, its causes, and how to remedy it.
196

There is a phenomenal website that one can visit with a myriad of
information regarding the skills and processes regarding these
techniques


On this website, two very basic troubleshooting principles are noted:



It is www.Troubleshooters.Com.
1) analyze the problem
2) analyze the solution
On this page Steve Litt recommends the following:




Describe the problem's symptoms, not your guesses
Describe your problem's symptoms in chronological order
Be explicit about the question you have
Courtesy never hurts, and sometimes helps
197

These suggestions really are key especially when trying to convey the
problem to another and also guiding oneself to a solution.

Steve suggest ten steps toward the universal approach to
troubleshooting.

While these might be more extensive than many will venture, it still
makes sense to grasp what is really needed to solve and prevent a
recurrance of the problem
198
The 10 step
Universal Troubleshooting Process
1. Get the attitude
2. Get a complete and accurate symptom description
3. Make a damage control plan
4. Reproduce the symptom
5. Do the appropriate general maintenance
6. Narrow it down to the root cause
7. Repair or replace the defective component
8. Test
9. Take pride in your solution
10. Prevent future occurrence of the problem
Troubleshooters.Com(R) Copyright (C) 1996-2002 by Steve Litt
199

There are two areas that have the greatest potential for problems
with IHC and in practice possibly account for more than 50% of calls
made each year to technical support departments.

These are tissue fixation/processing, and the pretreatments.

Knowing this, it becomes evident where the greatest attention to
detail needs to be.

If it is well understood how the tissue is fixed and processed, and
the pretreatment steps are carefully designed to match the tissue
conditions, the rest is easy.
200

When everything has been working well, and there is suddenly a
problem, many techs immediately go in crisis overload mode.

Ask yourself a few questions, but before you do, take a break to step
back and collect your thoughts.

First rule; detach yourself emotionally from the issue.

Get a complete and accurate description of the problem, just the
facts.

In the interim, is there a way to get the patient samples stained
while the problem is being investigated?
201

Could anything or anyone have changed something anywhere in the
process from beginning to end?
 This
 Is
includes the OR, doctor’s office, couriers etc
anyone new and on a learning curve?

Even if something does not appear to have changed, the mere fact
that something suddenly does not work appropriately indicates that
something, knowingly or unknowingly, did change.

The question: what, why, when, how and where
202
THE QUESTIONS:

When did it actually start?

Is it on the control, or just the patient or both?

Once you think the problem has been identified, can it be reproduced or
confirmed?

Can the root cause be mitigated to avoid repeat incidences?

Does something need to be replaced, remade or outsourced
203
TROUBLESHOOTING GUIDES

Many vendors have troubleshooting guides on their websites
or in their manuals, package inserts etc.

Dako’s Handbook of Immunochemical Staining Methods has
a whole section dedicated to troubleshooting. In the 3rd end,
it can be found on pp 47 – 61.
204
205
VI. LABORATORY OPERATIONS (20%)

This section is less technical but absolutely essential.

Much of these areas should be addressed in any lab that is
engaged in patient testing or even research.

Several references should the be focal point.

Self Assessment #11, Laboratory Operations
 Available from NSH
 Has questions and answers

CAP checklist for both the General Lab and Anatomic Pathology
 IHC is in the AP section.

http://www.cap.org/apps/docs/laboratory_accreditation/checklists/checklistftp.html
206

NCCLS GUIDELINES FOR QUALITY ASSRUANCE FOR IMMUNOCYTOCHEMSITRY cat
# MM4-A
 NCCLS is now known as Clinical and Laboratory Standards Institute
 http://www.nccls.org/

JCAHO
 http://www.jcaho.org/

FDA
 http://www.fda.gov/

CE


http://www.techintl.com/cemarking.cfm?OVRAW=CE%20mark&OVKEY=ce%20mark&O
VMTC=standard
CLIA
 http://www.fda.gov/cdrh/clia/
207

Quality Control and Quality Assurance
 Documentation
 Procedures
 QC
records
 Personnel
 Reagents and antibody lots
 Selection,
utilization and evaluation of control tissue
 Slide storage
 Method selection and validation
 Troubleshooting
208
SAFETY

Self Assessment #14, Laboratory Safety
 Available
from NSH
 Has questions and answers

Anatech’s book; Hazardous Materials in the Histopathology
Laboratory; Regulations, Risks, Handling and Disposal. Dapson R,
and Crookham – Dapson J. 4th Ed.





Storage
Disposal
Hazards
Regulations
Procedures
209
LABORATORY MATHEMATICS

Freida Carson’s book, A Self Instructional Text

Laboratory Mathematics and Solution Preparation, 1st end,
Chapter 5, pp79 – 86

Think about antibody dilutions, making buffers, molar and
molal solutions, percent solutions.
210
ANCILLARY EQUIPMENT/INSTRUMENTS

Dako’s Handbook of Immunochemical Staining Methods, 3rd Ed, pp 39 -40.

Preventive maintenance is a big one!

Records of service performed on instrumentation must be kept

Calibrations for devices such as micropipettes, thermometers etc must be
performed in accordance with regulatory agencies and/or accrediting bodies

Don’t forget water purification systems

Look at the CAP checklist and see what is usually asked regarding equipment.
211
REGULATIONS

In the U. S., clinical laboratories are governed by both the FDA
and CLIA

Accrediting agencies are either CLIA or agencies given
deemed status, such as CAP, COLA, and JCAHO

Internationally we have agencies such as ISO
212
FDA’S FINAL RULE

FDA requirements for labeling antibodies:
ASR – analyte specific reagent
 IVD – in vitro diagnostic use
 510 k (simple filing for most antibodies)
 PMA (highest filing and approval for antibodies that
determine patient’s treatment, both eligibility and response)
 RUO – should be confined to research applications, and is
not reimbursable under Medicare

213
214
WHY TAKE THE EXAM?

You are without a doubt your most valuable asset.
1.
Passing this test will promote your personal and professional growth
1.
2.
It says a lot about your personally
May open new doors of opportunity
2.
It will certainly make you expand your knowledge base of IHC
3.
You will be the better for it, even if no one pays you more because you
did it
4.
Looks great on the resume, just in case you want to use that for a new
career opportunity
215
ON A FINAL NOTE

It is hoped that this will serve to merely point the applicant in the
right direction in order to prepare for the exam.

There are no Q & A books on this topic comprehensive enough to use
as a study guide

Be sure to look at texts and information that are relatively current in this
field since old information may no longer apply

The Internet can be very valuable in looking up information
 Try

key words in your searches
The greatest challenge lies in the fact that this field is ever changing
and full of proprietary technologies
216
217