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Part I: The Staining Process
Introduction to Immunohistochemistry Clive R. Taylor, MD, D.Phil
Im • mu • no • his • to • chem • is • try (n.) Microscopic localization of specific antigens in tissues by staining with antibodies labeled with fluorescent or pigmented material. The American Heritage® Medical Dictionary
Click here for all chapters
Introduction to Immunohistochemistry | Chapter 1
Chapter 1.1 Introduction
tochemical staining result start in the surgery operating room and end at the interpretation of the stain by the pathologist,
Immunohistochemistry (IHC) is a method used to determine
which ultimately leads to treatment decision by the oncologist.
the expression of biomarkers in tissue. This educational guidebook will describe immunohistochemistry as it is used in the
For those new to the world of immunohistochemistry here
pathology laboratory as an aid in the differential diagnosis
is a brief outline of the steps needed to localize antigens in
and classification of cancer, and for certain other diseases,
tissues using antibodies for cancer diagnosis:
including infections. The factors that influence the immunohis-
Control Internal/external, critical stain quality indicators
complex; f. Counterstaining is performed to visualize nuclei and overall tissue architecture;
Reporting Diagnostic context
g. Sections are dehydrated, mounted and coverslipped. Figure 1.2 Many factors may influence the IHC staining result. With just 3 choices at each of 14 steps there are 4.8 million different procedures!
Introduction to Immunohistochemistry | Chapter 1
Chapter 1.2 History of immunohistochemistry
cein and visualized by ultraviolet light (fluorescence microscopy) (1). During the next 25 years, the Coons method was used with
This IHC Educational Guidebook will describe the potentials
different modifications, including labeling with heavy metals, but
and pitfalls in the immunohistochemical staining process from
it was not until the introduction of enzyme-labeled antibodies (2)
biopsy to interpretation, with special attention to the analytical
that the method overcame many of the inherent issues with fluo-
processes and how to improve certainty in the staining result by
rescein and heavy metal labeling of antibodies. In the early 1970s,
employing standardization to the processes.
application of the ‘immunoperoxidase’ method to formalin paraffin embedded tissues by Taylor, Mason and colleagues in Oxford,
Before immunohistochemistry reached its now widespread use
was a critical step in extending use of the method into ‘routine’
as an important method in routine cancer diagnosis, the tech-
diagnosis in anatomic pathology. The direct labeling method had
nology had a long history of technological developments out-
the drawback that each individual primary antibody, or the secon-
lined in the table below.
dary antibody, had to be labeled with enzyme. That problem was circumvented by the development of an unlabeled antibody en-
Table 1.1 The major milestones in the history of immunohistochemistry.
zyme method, the peroxidase anti-peroxidase (PAP) method, which had the further advantage of increased sensitivity, facili-
Fluorescence-labeled primary antibodies
Coons et al (1)
Enzyme-labeled primary antibodies
Nakane & Pierce (2)
Secondary un-labeled antibodies (PAP)
Sternberger et al (3)
and improved detection systems for visualization of antigens in
Detection of antigens on ultrathin sections
Kawarai & Nakane (4)
tissue, IHC suffered from lack of reproducibility, due in part to poor
Application to routine formalin paraffin sections
Taylor et al (5-7)
verse effects of fixation.
Invention of monoclonal antibodies
Köhler & Milstein (8)
Increased demand led to better quality reagents from the com-
Double staining using un-labeled antibodies (APAAP)
Mason & Sammons (9)
mercial sector, with improved quality control of production
Monoclonal antibodies to human antigens
McMichael et al (10)
Capillary gap semi-automated staining
Brigati et al (11)
Heat-induced antigen retrieval
Shi et al (12)
Dr. Niels Harboe, founder of Dako, realized in the early 1970s
Standardization efforts as ‘Total Tests’
the need for standardized antibody preparations for safe and
Dextran-polymer-based detection system
reproducible diagnoses and began producing purified poly-
Immunohistochemistry as companion diagnostics
Recommendations for improved standardization of IHC
Goldstein et al (14) Wolff et al (15)
Even with the purified and highly specific polyclonal antibodies
Molecular HER2 CISH Tests in the IHC lab
there was a need for improved specificity of antibodies and a
tating use in routine tissues. A related parallel development was the introduction of the alkaline phosphatase anti-alkaline phosphatase (APAAP) in 1978 (9). Even with the development of new
quality antibody reagents, and in part to the inconsistent and ad-
methods. Polyclonal antibody preparations differ between serum samples in affinity and specificity, as the immuneresponse changes with time and immunization preparations, and as one animal is replaced by another as the source.
clonal antibodies that had the same strength (as measured by titer) from batch to batch.
greater variety in terms of target proteins. The invention, in 1975, of hybridomas that could produce monoclonal antibodies (8) resulted in the production of the first monoclonal antibody that was
Professor Albert H. Coons and co-workers demonstrated in 1941
highly specific for human thymocytes using hybridoma technolo-
that it was possible to localize antigens in tissue slices using an-
gy (10). Monoclonal antibodies paved the way for a rapid growth
tibodies against Streptococcus pneumoniae labeled with fluores-
in the use of IHC in research and diagnosis of cancer.
Chapter 1 | Introduction to Immunohistochemistry
One other consequence of the lack of reproducibility was the development of automated instruments (11). Automation was
invented with the fundamental thought that a properly functioning and maintained instrument will consistently perform its
pre-programmed instructions in the same way – slide after slide-
Direct method (one step) The primary antibody (green) is labeled with an enzyme or fluorescence.
which is the principal reason why an instrument potentially can give superior reproducibility, compared with manual methods. However, progress was slow until 1991, when Shi et al (12) in-
troduced ‘antigen retrieval’ (or heat-induced epitope retrieval), thereby facilitating extension of IHC to a much broader range
of applications in formalin paraffin sections, but at the cost of adding yet another variable to the process. This important publication on antigen retrieval thus gave new insights and impetus to efforts in standardization of IHC, leading to the intro-
Indirect method (two steps) An enzyme-labeled secondary antibody reacts with unconjugated primary antibody bound to tissue antigen.
Primary antibody Tissue antigen
duction of the ‘Total Test’ concept (13) as a result of a series of meetings sponsored by the Biological Stain Commission and the FDA in the early 1990s. Peroxidase anti-peroxidase complex
The standardization efforts, coupled with attempts to use IHC in a semi-quantitative setting raised demands to a new level, exemplified by the introduction, in 1998, of the HercepTest™ (Dako), which was the first cancer companion diagnostic, in this instance designed for selection of breast cancer patients
for treatment with the new drug Herceptin® (Genentech/Roche). Clinical trials had shown that patients whose tumors over-
expressed HER2 would benefit the most from Herceptin® treat-
ment. The HercepTest™ assay uses IHC on patient samples,
Unlabeled method Pre-formed enzyme immune complex reacts with secondary antibody.
in combination with control cell lines having known HER2 expression to determine if a breast cancer overexpresses HER2. Some 15 years later, this assay together with similar HER2 assays from other vendors, still serves as a rare example of a
Dextran backbone HRP enzyme
semi-quantitative IHC assay used in routine clinical pathology. The polymer-based visualization system, introduced shortly before HercepTest™, is the most widely used detection method in IHC today, with advantages of stability and high sensitivity. The technical advances in IHC in the last decade have been
incremental, with little impact on the basics of the method.
Automation has become more advanced, including laboratory information system integration, with track and trace of samples, while whole slide digital imaging is slowly being integrated into the analysis of stain result. These advances can best be regarded as improvements in standardization; a process that started back
Secondary antibody, mouse/rabbit
Labeled polymer A long dextran polymer is labeled with both the secondary antibody and multiple enzyme molecules.
Figure 1.3 The development of detection systems used for IHC. Please see Chapter 6 for a full description of the many different detection methods.
Introduction to Immunohistochemistry | Chapter 1
in the early 1990s and was re-emphasized in the 2007 publications by Goldstein et al (14) and Wolff et al (15), but also by the work being done e.g. estrogen receptor assessment (16, 17). The
Chapter 1.3 Standardization in Clinical Immunochemistry vs. Immunohistochemistry
critical importance of IHC standardization became evident with the revelation of disturbingly high numbers of false negative or
For more than 30 years, clinical immunochemistry has em-
false positive results in IHC determinations of ER (estrogen recep-
ployed blood or urine samples to determine the concentra-
tor) and PR (progesterone receptor) expression, and also HER2.
tion of certain biomarkers, e.g. creatinine and cystatin C for
In one example, a re-testing in 2007 of 1,023 breast cancer sam-
evaluation of kidney function, and C-reactive protein as a
ples from Newfoundland revealed that approximately 1 out of 3
marker of inflammation. Although clinical immunochemis-
samples was scored falsely ER negative (17). As a consequence
try covers a multitude of assay types, most of these tests
of the false negative ER test results, these women were not ac-
are based on the ELISA (enzyme-linked immunosorbent as-
corded the potential benefit of anti-hormonal therapy.
say) method, a method that closely parallels IHC in principle. One major difference is that International Reference Materials and Calibrators are used in clinical immunochemistry
(ELISA) to achieve quantitative results from these assays.
140.000 122198 115006
Immunohistochemistry is based on principles similar to the ELISA method, yet it is at best a semi-quantitative method for
determination of the expression of biomarkers in tissue sam-
ples. However, IHC should not be regarded as simply another ‘special stain’, like a PAS stain or a silver stain. IHC is es-
sentially an ELISA method applied to a tissue section. In this
respect, when correctly performed, IHC has the potential
ELISA assay; much more than a simple stain. That the IHC
to perform as a reproducible and quantitative tissue-based
9044 3009 7423
method does not perform to this level, reflects deficiencies in the application of the method, specifically inconsistent sample preparation, lack of reference or calibration standards,
Figure 1.4 The number of IHC publications in the last 50 years. The data are from Pubmed using the search term “immunohistochemistry”.
and inadequate validation of reagents (18, 19). If ELISA can use a standard curve to convert the measured immunoreactivity into a quantitative amount of tested protein, then IHC – in theory – can also convert the IHC intensity observed in
The latest development in cancer diagnosis is the inclusion
FFPE tissue sections into the amount of tested protein by an
of molecular tests (FISH/CISH) in anatomic pathology labs,
equivalent standard ruler. Comparative studies of IHC inten-
driven by HER2 assessment requirements. Other techno-
sity on frozen tissue vs. FFPE tissue have shown identical
logies also are entering into the pathology lab and into rou-
intensity by using an optimized AR protocol (20, 21), and
tine diagnosis, and technologies such as array comparative
similar protein quality is evident when examined by mass
genomic hybridization or next generation sequencing will likely
spectrometry (22), leaving no theoretical reason for lack of
be a fundamental part of cancer diagnosis in the future. One
ongoing goal is to interface these newer methods of molecu-
says are at best no more than semi-quantitative, for reasons
lar analysis with existing and improved morphologic criteria, a
that are more of a practical nature.
field termed ‘Molecular Morphology’.
Chapter 1 | Introduction to Immunohistochemistry
Chapter 1.4 Growing Consensus for Standardization
Table 1.2 The Total Test: An IHC stain should be managed in the same rigorous manner as a clinical laboratory analysis. Modified from Taylor (14, 24).
From the beginning there has been concern relating to the reproducibility of immunohistochemical methods as applied
to formalin-fixed, paraffin-embedded (FFPE) tissue sections.
A consequence of not controlling all parameters (in fixation,
Acquisition, pre-fixation/transport time
processing and staining) is poor day to day reproducibility within a single laboratory, and poor reproducibility among
Fixation, type and total time Processing, temperature
different laboratories. In recent years these concerns have increased and lack of standardization, well shown in inter-
laboratory quality assurance surveys performed by NordiQC
Antigen retrieval procedure
and UK NEQAS, is now recognized as a major impediment to basic research, clinical trials, and direct patient care. Over the past three decades a number of conferences have been
Selection of primary antibodies Protocol; labeling reagents
held to address this topic and to seek constructive resolutions.
Among the most productive were a series of meetings spon-
sored by the Biological Stain Commission and the FDA in the
early 1990s, that led to recommendations for manufacturers concerning the precise description and validation of IHC rea-
Laboratory certification / QA programs
gents (23), and also highlighted the necessity to pay attention
to all aspects of the IHC test procedure. The latter recommen-
Assessment of control performance
dation, borrowed from the much more rigorous protocols ap-
Description of results
plied to immunologic assays in clinical laboratories, became known as the ‘Total Test’ approach (Table 1) (23, 24). A decade later a meeting of the FDA and NIST (National Institute of
Interpretation/reporting Pathologist, experience and CME specific to IHC
Standards and technology) focused upon standardization of HER2 IHC assays, and the need for universal control materials
In the analytical steps, antigen retrieval is the first challenge.
(reference standards) (25).
Different antigens require different antigen retrieval for optimal staining results, and the different variations of the AR process
Chapter 1.5 Standardization Starts in the Surgery Room
add another variable that must be controlled. Antigen retrieval is described in detail in Chapter 3. Selecting the right antibody for the right marker is one of the
While Table 1.2 only mentions a few of the major steps in a To-
key steps in the analytical process. Some monoclonal anti-
tal Test, the pre-analytical process alone contains at least 62
body clones are more specific than others against the same
identifiable steps of which 27 have been examined in published
biomarker. In other cases a polyclonal antibody may be the
research. Out of these 27 steps, 15 pre-analytical variables are
best choice. Selection of the primary antibody is described in
capable of impacting the immunohistochemistry staining result
detail in Chapter 4.
including fixation delay, fixative type, time in fixative, reagents
and conditions of dehydration, clearing, paraffin impregnation
Using a protocol that is optimized for the detection of the bio-
and conditions of slide drying and storage (26). Pre-analytical
marker is vital. The optimal protocol must be able to identify
variables are described in detail in Chapter 2.
the antigen of interest in cells and structures with both low and
Introduction to Immunohistochemistry | Chapter 1
high expression. Optimization of the staining protocol is de-
Post-analytical standardization is essential for prognostic or pre-
scribed in detail in Chapter 5.
dictive biomarkers, e.g. HER2 and ER/PR, adhering to specified stain interpretation guidelines to give the sample a scaled score
The final step of the analytical process is the visualization of the
(e.g. from 0-3+). However, most biomarkers are used for cell line-
antigen/antibody reaction. Here the selection of the detection
age and tissue identification, where expression levels are usually
system must consider the complexity of the visualization and the
not as critical and interpretation is not linked to a semi-quantita-
required amplification needed to visualize the biomarker. The var-
tive scoring system, but is reported as a binary ‘Yes’ or ‘No’ sys-
ious detection systems are described in detail in Chapter 6.
tem (positive or negative) for the tested biomarker. Digital analysis of IHC stains is described in Chapter 7.
Chapter 1.6 Future Aspects for Standardization of Immunohistochemistry
Table 1.3 Major steps affecting the immunohistochemistry staining result. Step
Effect on IHC
Depending on the suspected cancer type, tissue samples can be obtained in different ways such as punch/core biopsy, excisional/incisional biopsy, etc. Tissue degradation begins at the time of sample removal.
Sectioning and Mounting
The sample should be fixed as soon as possible after surgery, ideally within less than an hour. The chemical fixation crosslink proteins in the sample thereby stopping the degradation process. Too short or too long fixation can affect the staining result.
The consensus arising from the standardization efforts is that the reliability and reproducibility of IHC methods in routine surgical pathology have been greatly hindered by two key factors. 1. While reagents available for IHC have increased in quality, there has been an even greater increase in number of
sources and variety of staining methods. This plentitude of
After fixation, the sample is embedded in paraffin for long-term storage and to enable sectioning for subsequent staining. Once embedded in paraffin, samples can be stored (almost) indefinitely.
reagents contributes to lack of standardization in signifi-
Formalin-fixed, paraffin-embedded tissues are sectioned into thin slices (4-5 μm) with a microtome. The sections are then mounted onto adhesive-coated glass slides.
to requirements for such high standards of excellence in
Due to the fixation process, an antigen retrieval treatment is applied to unmask the epitopes, either by heat (heat-induced epitope retrieval; HIER) or enzymatic degradation (proteolytic-induced epitope retrieval; PIER). Incorrect antigen retrieval for the biomarker of interest will adversely affect the staining result.
cant ways, that in theory are manageable by good tech nique and use of proper controls, but in practice have led the technical process, that many laboratories cannot find
sufficient, or sufficiently skilled, staff to comply.
2. The usual method of sample preparation for tissue remains as formalin fixation and paraffin embedment (FFPE). This
venerable approach may be satisfactory for the preservation
of morphologic detail, but does adversely affect the antigenicity
of many target molecules in the tissue, to degrees that are
unknown. The enormous variation in protocols (including
An antibody with specificity for the biomarker of interest is applied. The specificity and sensitivity of the antibody affect the staining result.
fixation times) employed for FFPE among different laborato-
ries, or within the same laboratory from specimen to speci-
The antigen/antibody complex signal is amplified and visualized using a detection system. The strength of amplification of the reaction affects the staining result (intensity).
The staining pattern is assessed by a pathologist in context with other biomarkers, controls and other tests (e.g. H&E, special stains. Inter- and intra-observer variability is common, especially for semi-quantitative assays. This variability highlights the importance of training and inter-calibration.
men, compounds the problem and contributes to the current poor reproducibility.
While several decades have passed, these issues have not been satisfactorily addressed. Legions of investigators, and many manufacturers, have addressed different aspects of the problem, focusing upon better sample preparation (fixation), more effective methods of antigen retrieval, improved
Chapter 1 | Introduction to Immunohistochemistry
reagents, more sophisticated automated platforms, more sen-
high-quality reagents are available, with highly sensitive
sitive detection methods, and the development of reference
detection methods, but they must be employed pro-
standards or controls (13, 23-25).
perly in controlled fashion, and currently often are not.
In order to improve the quality and reproducibility from sample to
sample, and lab to lab, the accreditation process for many pa-
there is a pressing need for tissue-based IHC controls (or
thology laboratories now includes participation in external quality
‘reference standards’) (19, 25) that can be made available
assurance (EQA) schemes. EQA organizations, like NordiQC, UK
to all laboratories performing IHC assays, somewhat ana-
NEQAS and CAP, are independent organizations not associated
logous to the international reference standards and cali-
with commercial suppliers. Their role is to promote the quality of
brators that are available to clinical laboratories performing
immunohistochemistry (and in situ hybridization) by arranging
Participation in EQA schemes can help laboratories improve the reproducibility;
external QA schemes for pathology laboratories. Similar EQA schemes are now available in many countries and regions around
From this brief discussion it follows that to improve standardi-
the world. The purpose of EQA schemes is to improve the quality
zation to the point that all laboratories would carry out the IHC
of staining results in the participating laboratories; thus it is the
in identical fashion for every phase of the ‘Total Test’; it would
individual labs that are being assessed. It is their choice of an-
require them to use the same fixative and fixation time (adjusted
tibody, visualization system, instrumentation and protocol that is
to tissue type), the same antigen retrieval process, the same pri-
the basis for the EQA organization's evaluation and feedback. A
mary antibodies and detection systems, with the same automat-
lab volunteers to participate in the assessment runs. Laboratories
ed stainer and common controls. Clearly this perfect option will
typically enroll for a year, during which they receive approximate-
never happen, and we therefore must do what we can to reduce
ly 16 unstained tissue slides (NordiQC), or 7-8 different modules,
the consequences of the variables in the process.
where each module usually has two tissue slides (UK NEQAS), to stain using their own internal standard protocols for those markers
Ultimately the overriding factor in effecting significant change
designated by the QA organization. The labs return the stained
must be to transform the mindset of pathologists, at least of
slides to the QA organization for assessment, which is conducted
the next generation, to the view that the end result of an IHC
by experts engaged by the organization. The labs receive either
protocol is not just a ‘stain’, with intensity to be adjusted at the
a “Passed” rating or “Not Passed” rating. Both NordiQC and UK
whim of the pathologist. Rather IHC is a precise immunoassay
NEQAS inform all participants of their individual scores and pro-
that is strictly quantifiable, and must be performed only with a
vide suggestions for protocol optimization when required. Both or-
degree of technical rigor and control that matches any other
ganizations present the anonymous results on their web sites, with
immunologically-based assay of like principle (namely ELISA).
statistics and best method for the particular marker.
ELISA is a ‘gold standard’ method for quantitative assays in the clinical laboratory. ELISA reagents are purchased in prepared
CAP (College of American Pathologists) in the US, has a simi-
form, with all of the necessary reagents, defined protocols,
lar QA process, but requires only the return of stain results and
and reference or calibration standards, for use with specified
interpretation, not the stained slides.
instrumentation. Ready-to-use reagents, coupled with proven detection systems, fixed and validated protocols, recommend-
Some broad conclusions are possible:
ed controls and automation, represent an analogous pathway
resolution of the problem of pre-analytical sample prep-
that could, if widely adopted with appropriate controls, lead to
improved levels of reliability and performance for IHC.
aration is not imminent; the practical aspects of developing
tissue handling and fixation procedures that fit the daily
routine of every hospital are challenging. Importantly the
logistical obstacles to implementation of standardized sam-
ple preparation procedures worldwide are formidable;
Introduction to Immunohistochemistry | Chapter 1
References 1. Coons AH, Creech HJ, Jones RN. Immunological properties of an antibody containing a fluorescent group. Exp Biol Med 1941; 47:200-2. 2. Nakane PK, Pierce GB. Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol 1967; 33:307-18. 3.
Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970; 18:315-33.
4. Kawarai Y, Nakane PK. Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method. J Histochem Cytochem 1970; 18:161-6.
15. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American society of clinical oncology/college of american pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131:18-43. 16. Yaziji H, Taylor CR, Goldstein NS, Dabbs DJ, Hammond EH, Hewlett B, et al. Consensus recommendations on estrogen receptor test ing in breast cancer by immunohistochemistry. Appl Immunohis tochem Mol Morphol 2008; 16:513-20. 17. Hammond ME, Hayes DF, Wolff AC, Mangu PB, Temin S. Ameri can society of clinical oncology/college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Oncol Pract 2010; 6:195-7.
5. Taylor CR. The nature of reed-sternberg cells and other malignant "reticulum" cells. Lancet 1974;2:802-7.
18. Taylor CR. Quantifiable internal reference standards for immuno histochemistry: The measurement of quantity by weight. Appl Immunohistochem Mol Morphol 2006; 14:253-9.
6. Taylor CR, Burns J. The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embed ded tissues using peroxidase-labelled antibody. J Clin Pathol 1974; 27:14-20.
19. Taylor CR, Levenson RM. Quantification of immunohistochemistry — issues concerning methods, utility and semiquantitative as sessment ii. Histopathol 2006; 49:411-24.
Taylor CR, Mason DY. The immunohistological detection of intracellular immunoglobulin in formalin-paraffin sections from multiple myeloma and related conditions using the immunoperoxidase technique. Clin Exp Immunol 1974;18:417-29.
8. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495-7. 9. Mason DY, Sammons R. Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J Clin Pathol 1978;31:454-60. 10. McMichael AJ, Pilch JR, Galfre G, Mason DY, Fabre JW, Milstein C. A human thymocyte antigen defined by a hybrid myeloma monoclonal antibody. Eur J Immunol. 1979;9:205-10. 11. Brigati DJ, Budgeon LR, Unger ER, Koebler D, Cuomo C, Kennedy T, et al. Immunocytochemistry is automated: Development of a robotic workstation based upon the capillary action principle. J Histotech 1988; 11:165-83. 12. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraf fin-embedded tissues: An enhancement method for immunohisto chemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991; 39:741-8. 13. Taylor CR. An exaltation of experts: Concerted efforts in the stan dardization of immunohistochemistry. Appl Immunohistochem 1993; 1:232-43.
20. Shi SR, Liu C, Pootrakul L, Tang L, Young A, Chen R, et al. Evalua tion of the value of frozen tissue section used as "gold standard" for immunohistochemistry. Am J Clin Pathol 2008; 129:358-66. 21. Shi SR, Liu C, Taylor CR. Standardization of immunohistochemis try for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: From experiments to hypothe sis. J Histochem Cytochem 2007; 55:105-9. 22. Shi SR, Liu C, Balgley BM, Lee C, Taylor CR. Protein extraction from formalin-fixed, paraffin-embedded tissue sections: Quality evaluation by mass spectrometry. J Histochem Cytochem 2006; 54:739-43. 23. Taylor CR. Quality assurance and standardization in immunohis tochemistry. A proposal for the annual meeting of the biological stain commission, june, 1991. Biotech Histochem 1992;67:110-7. 24. Taylor CR. Report from the biological stain commission: Fda issues final rule for classification/reclassification of immunochem istry (ihc) reagents and kits. Biotech Histochem 1998; 73:175-7. 25. Hammond ME, Barker P, Taube S, Gutman S. Standard reference material for her2 testing: Report of a national institute of standards and technology-sponsored consensus workshop. Appl Immuno histochem Mol Morphol 2003;11:103-6. 26. Engel KB, Moore HM. Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 2011; 135:537-43.
14. Goldstein NS, Hewitt SM, Taylor CR, Yaziji H, Hicks DG. Recom mendations for improved standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol 2007; 15:124-33.