Update and Troubleshooting Immunohistochemistry for Pathologists
Recommendations for Improved Standardization of Immunohistochemistry
Neal S. Goldstein MD, Stephen M. Hewitt MD PhD, Clive R. Taylor MD DPhil, Hadi Yaziji MD, David G
Hicks MD and Members of Ad-Hoc Committee On Immunohistochemistry Standardization
Background and Introduction
Immunohistochemistry (IHC) is widely used in surgical pathology and serves as a
diagnostic, prognostic, and predictive tool. It was recognized over a decade ago that IHC assay
standardization was vital for reproducible and reliable results. Agencies, including the Biologic Stain
Commission, CLSI (previously NACCLS), FDA, and the manufacturing sector have established guidelines,
standards, and recommendations for the reagents and manufacturer package inserts, which has provided a
relatively stable environmental platform of consistent, high-quality assay components on which
contemporary IHC is performed
The quality and consistency of these products has allowed
the development and use of so-called "black box" IHC stainers in which an IHC assay with preset
parameters, set by the manufacturer, produces relatively consistent high-quality stains .
Authors over the years have consistently noted the irregularities of IHC assays and the
need for increased standardization
Despite the improvements of reagents and automation,
contemporary IHC suffers from variable consistency, poor reproducibility, quality assurance disparities,
and the lack of standardization resulting in poor concordance, validation, and verification
Prior consensus conferences identified the likely causative factors (Table 1)
. The majority of the responsibility for suboptimal and variable IHC assay results lies
within the domain of the individual pathology laboratory and pathologist. Recent studies strongly
suggest that these problems are widespread and are not insignificant
A two-day ad-hoc consensus conference was convened in August 2006, charged with the goal of
making practical and feasible recommendations directed at standardizing these laboratory and pathologist
factors. The recommendations listed below are the product of this meeting that included input from the
directors of immunohistochemistry laboratories from a broad range of clinical practices and representing
the governmental, private, and academic sectors. These guidelines do not diverge from general practice
recommendations and are supported by scientific data. Unfortunately, laboratories often do not
appreciate the negative impact on their specimens and the validity of IHC performed on them created by
diverging away from these recommendations.
Recommendation 1: Tissue should be fixed in 10% neutral-pH, phosphate-buffered formalin for a
minimum of eight hours. If formalin or formalin-alcohol mixture is a component solution on the tissue
processor instrument, tissue should be fixed in formalin for six to twelve hours prior to being loaded
onto the tissue processor. Non-formalin-based fixatives and or alternative fixation methodologies are
strongly discouraged in regards to IHC, in large part because performance data are limited and
extrapolation from formalin fixed data is unreliable.
Comments: Formalin is aqueous, completely dissolved formaldehyde. The diffusion coefficient
of formaldehyde is 0.79, meaning it permeates tissue at approximately 1 mm/ hour
However, permeation is not fixation. Formaldehyde becomes available after disassociating from methylene
glycol in solution at a rate that is measured in hours, referred to as a "clock" reaction
Although complete tissue fixation requires 24 hours, a minimum of 6 pre-processor
hours of formalin fixation is required for consistent IHC assay results
Non-formalin fixatives and or alternative fixation methodologies are strongly discouraged
for several reasons. First, tissue fixed in formalin for less than eight hours is countered by
relatively greater alcohol dehydration fixation. Although tissues fixed in this manner can produce
hematoxylin and eosin stained slides that are similar in appearance to formalin-fixed tissues, the two
specimens have discrete differences in both histomorphology and staining characteristics by IHC. Many
alcohol-fixed tissue antigens do not require antigen retrieval (AR) to be sufficiently immunoreactive and
in general, the less formalin fixation, the less AR required . Tissues fixed in formalin
for over eight hours requires standard amounts of AR. Five to eight hours of formalin fixation is the
transition-zone between no-AR and complete-AR, and varies depending on the antigen, antibody, and tissue.
The majority of suboptimal and inconsistent IHC stains result from the mixing of specimens with different
AR requirements. Some antigens, especially small secreted peptides are difficult to stain and localize
in alcohol or alcohol-phase fixed tissue, as a result of a lack of cross linking and diffusion
throughout the tissue.
Second, a collective body of knowledge of expected or characteristic immunoreactivity of
neoplasms and lesions has accrued over time consisting of the accumulated weight of numerous published
studies and decades of pathologists' personal experience. It functions as the vital foundation on which
pathologists make interpretative decisions and plays a significant role in the validation of an antibody.
This knowledge base is almost entirely based on immunohistochemical assays performed on antigen
retrieved sections of formalin fixed paraffin embedded tissue. Tissues fixed in non-formalin fixatives
and or alternative fixation methodologies can produce high-quality appearing IHC slides
However, a similar appearance is not similar antigen expression. Neoplastic and lesional tissues fixed
in an alternative non-formalin method may not produce the same patterns of antigen immunoreactivity
patterns as formalin fixed tissues . Some microwave processors require using the
manufacturer's proprietary (and undisclosed) reagents . Although similar results have been
obtained in tissue microarrays, there have been no direct comparisons of antibody panel results of
formalin and non-formalin fixed standard tissue blocks, needle core or endoscopic biopsy tissue specimens
. Microwave fixation induces inhomogeneous tissue fixation reactions that varies with the
size specimen and composition of tissue types
It is a flawed assumption to think that
the patterns and shades of intensities in neoplasms are the same in formalin and non-formalin fixed
tissues. Demonstration of differences in the clinical significance between alternative fixed and
formalin fixed tissues may not be readily apparent, however side by side comparisons of statistically
significant numbers of specimens, usually requiring at least 30 specimens per variable are required to
demonstrate equivalency. Given the increasing role IHC-assays play as predictive markers of oncologic
chemotherapeutic agent response, faster turnaround times and laboratory cost-containment should not be
prioritized as goals ahead of the laboratory procedures required for reliable, accurate, and reproducible
Third, most antibodies used by laboratories in IHC assays are classified as analyte
specific reagents (ASR) and regulated as a class 1 in-vitro diagnostic device by the FDA
The Clinical Laboratory Improvement Amendments Act stipulates that the laboratory that
uses the ASR in its in-house IHC assay takes responsibility to understand, verify, and validate its performance characteristics
in lesions fixed and processed in their laboratory. It seems obvious to state that the Act means
laboratories are required to validate and verify that lesions fixed in their laboratory express a
characteristic immunophenotype using IHC assays developed in their laboratory. Validation means that the
assay performs as designed to detect the specified antigen. Verification means the assay detects the
antigen as the assay was design based on the specimen being analyzed. For many markers, verification
requires a small number of samples processed in a consistent and identical manner to the test sample to
demonstrate true positive and negative staining patterns. However, as the complexity of the IHC-assay
increases, especially to prognostic and predictive markers, verification of the IHC-assay requires a
significantly larger number of samples.
Fourth, class 2 or 3 "kits or systems" for in vitro diagnostic (IVD) use require the
laboratory that uses the kit or system to perform the assay in the precise and exact manner designated by
the manufacturer. Examples of such kits include the HER2 HercepTest™ (Dako Co., Carpenteria, CA,) and
Pathway™ (Ventana Co, Tucson, Az). Class 2 or 3 kits or systems IVDs stipulate the permitted fixatives
on which the assay can be applied, which is almost always formalin. Results of these kits or system IVDs
are invalid and deemed inaccurate and potentially unreliable when they are applied to tissues fixed in
method or solution not stipulated as allowable by the manufacturer.
Recommendation 2: The time and type of pre-processor fixative and the time, type, and
component solutions of the tissue processor should be recorded for every IHC tissue specimen.
Delineation of neutral buffered formalin alone is inadequate, rather, specification of the type of buffer
and its molar concentration is required. The same is true for reagents on the tissue processing
instrument, with special attention to xylene alternatives and type of embedding paraffin. This
information should accompany all tissue that is sent to an outside laboratory where the IHC staining is
performed. When IHC studies are performed in a reference laboratory, the originating laboratory is
responsible for recording this information for each case. The reference laboratory that performs the IHC
staining should record this information for each case. This information should be permanently maintained
as part of the daily work run logs of the IHC laboratory. This information should be checked against the
quality of the IHC stains and be incorporated into the IHC stain quality review log performed the
laboratory director or their designate.
Comment: These recommendations are in step with the NCCLS (CLSI) guidelines for Her2
IHC-assays that the type of fixative and length of fixation be included in the pathology report
Insufficient, inconsistent, and different types of fixation and tissue processing are
the key factors behind suboptimal and inconsistent IHC stains on automated IHC staining instruments. In
general, underfixation is a substantially larger problem than overfixation
recommendation plays a key role in standardizing fixation parameters for IHC assays. Ideally, the time
tissue is placed into fixative would be known, in reality, this information would be difficult to obtain
for most laboratories. An acceptable surrogate of fixation-start time is the time that the specimen was
accessioned into the laboratory computer system or when logged into the department, providing the
specimens are immersed in formalin at or before this time point. The start time of the tissue processor
instrument on which the tissue block is loaded is an acceptable end-fixation time point. If tissues are
fixed in formalin or formalin-alcohol solutions for an additional time period on the tissue processor,
this additional length of time should be added to the total fixation time of the specimen. It is widely
assumed that tissue processing is a black-box process and that differences or variations in this process
have little impact on the specimen. This is not true. Current evidence suggests that any changes in the processing system have the capacity to alter the chemical
makeup of the tissue specimen. Alternative processing systems, especially those which include alterative
reagents and microwaves have not been adequately validated. IHC can be performed on tissue from these
systems, however there are no well validated studies to demonstrate equivalency.
B. Antigen Retrieval
Recommendation 3: Antigen retrieval is presumed to "restore" the antigenicity following
The parameters of an AR protocol must be balanced to match the
unique length and type of tissue fixation of the individual laboratory and the characteristics of the
For tissues fixed in formalin for at least 6 hours before being
loaded onto a tissue processor, one antigen retrieval (AR) protocol is usually adequate. Different
types of AR, (such as low pH buffers, high pH buffers, various types of heating devices, enzyme
digestion, etc.) should be available for each AR protocol, depending on the optimization parameters of
individual antibodies (see Optimization and Validation section below). While enzyme digestion is not
generally considered as a component of AR, it functions as an alternative method for practical purposes.
It may be the preferred method of tissue pretreatment for some antibodies.
Comment: For most contemporary oncologic antigens, a minimum threshold level amount of energy
must be applied via AR procedure to all types of tissue specimens, regardless of the length of time above
6 hours the tissue specimen has been fixed
The method of applying the energy
(pressure cooker, microwave, waterbath) is substantially less important than the amount of energy applied
to the slides by the AR system . AR energy above this threshold generally does not improve
antigen detection . Poor or inconsistently optimized AR relative to tissue fixation is
responsible for most suboptimal contemporary automated IHC assay results. This creates a problem of
inadequate (either over or under) AR. Over-AR relative to the amount of formalin fixation produces
high-background staining, section fall-off, tissue section holes and rents, indistinct nuclear detail,
muddy chromatin, pseudo-nucleolar staining, and overly-strong edge staining of small biopsy specimens.
This type of imbalance most commonly occurs when an AR protocol intended for tissues fixed in formalin
for at least 8 hours is applied to tissues fixed in formalin less than 6 hours.
Under-AR relative to the amount of formalin fixation produces false negative
immunoreactivity. This type of imbalance most commonly occurs when an AR protocol intended for tissues
fixed in formalin for 4-8 hours is applied to tissues fixed for more than 16 hours (overnight fixation)
It is very difficult to optimize AR on poorly fixed and processed tissues. Adequate fixation is key
to achieving reproducible and optimal AR. It is not uncommon for laboratories to encounter a mixture of
under and over fixed tissues, resulting in a large spectrum of problems, that combine together to produce
result in inconsistent and unreliable IHC-assays. AR is a part of clinical IHC, and the best solution is
to standardize fixation in an effort to obtain consistent IHC.
Proper analysis of IHC assays is critical for correct tumor identification and
prognostic/predictive assessment. Pathologists who have documented expertise in this field should
interpret IHC assays. Expertise is attained by documenting experience with literature use and
proficiency testing. IHC is an adjunct to pathologic interpretation, and all IHC assays should always be
interpreted within the context of morphology.
A. Developing New ASR Antibodies into IHC Assays
Developing new analyte specific reagent (ASR) antibodies into clinical IHC assays is a
two-step process of optimization and validation. Optimization is the process in which the laboratory
serially tests and modifies component procedures with the endpoint of producing a consistent high-quality
assay. Antibody optimization must be completed before moving on to the validation step. Antibody
validation is the process whereby the parameters of the IHC assay, including its accuracy, reliability,
and reproducibility are established.
1. Antibody Optimization
Recommendation 4: Reagent package inserts should be read completely and in detail prior to
beginning the optimization process. The laboratory director should sign the package insert and it should
be maintained in an easily accessible laboratory manual as a reference source during the active life of
the antibody or reagent. The manufacturer recommendations listed in the package insert should be
Comments: Work by the Biologic Stain Commission, FDA regulations, and antigen retrieval
pretreatment procedures have led to a generally high level of consistency in commercial antibodies and
reagent products from manufacturers
Satisfactory antibody staining is usually
achieved by strictly following the manufacturer's package insert instructions.
Recommendation 5: The test tissue used to optimize the IHC assay should contain the target
antigen. Tissue specimens used for antibody optimization should be selected based on the intended use of
the IHC clinical assay and the level of target antigen expression in the respective tissue specimen.
Comments: The expression levels of some antigens can vary between neoplasms from different
patients, between benign and malignant tissues, and between different types of neoplasms .
Three examples of this issue are provided:
Example 1: The level of CD117 (C-Kit) expressed in gastrointestinal stromal tumors (GISTs) from
different patients can range from none to high. Mast cells within GISTs and the adjacent bowel wall
strongly express CD117, often at a substantially higher level than the adjacent neoplastic cells. The
sensitivity of the CD117 IHC assay should be set such that weak-CD117 expressing GISTs are
immunoreactive. If a strongly CD117 expressing GIST or mast cells are used as the test tissue against
which the CD117 antibody is optimized, weakly CD117 expressing GISTs may fall below the threshold of
immunoreactivity and be interpreted as CD117 negative.
Example 2: Some mantle cell lymphomas have low-levels of nuclear BCL1 (Cyclin D1) antigen, whereas
other mantle cell lymphomas and benign tissues such as breast acini have high levels of BCL1. If one of
the latter tissues is used to optimize the BCL1 antibody, mantle cell lymphomas with low-level BCL1
antigen expression can fall below this immunoreactivity cut-point and appear BCL1-negative.
Example 3: CD10 expression in follicular lymphomas and reactive germinal centers is usually
substantially lower than in some carcinomas. If CD10 IHC assay is optimized against a follicular
lymphoma or tissue abundant in reactive germinal centers, the same assay could be overly intense and
possibly uninterpretable when being used as a marker of renal cell carcinoma or to identify bile
canaliculi in hepatocellular adenocarcinoma.
The simplest method of establishing multiple IHC assays of a single antibody across a
range of clinically different types of tissue after the IHC assay has been optimized in one type of
tissue specimen (see Recommendation 7) is to vary the concentration of the antibody. When pre-diluted
antibodies are used, the duration of antibody incubation or intensity of antigen retrieval can be
manipulated to optimize the IHC assay. Regardless of whether concentrated or prediluted antibody is used
in the IHC assay, several different IHC assays should be established when one antibody is being used in
multiple clinical settings which cover a broad range of antigen expression levels.
Recommendation 6: The specific antibody clone selected for the IHC assay should be selected
based on intended clinical use of the IHC assay and the established record of immunoreactivity in
Comment: Antibodies bind to a small region of the target antigen. Different antibody clones
bind to regions on a target antigen that may be distinct and separate areas, overlap to some extent, or
be nearly identical. For many antigens, there are no detectable differences in immunoreactivity between
antibody clones when validated against a limited number of specimens. However, for some antigens, the
antigenic binding site of the specific antibody clone is of crucial importance. Examples of this issue
include: WT1 in desmoplastic small round cell tumors versus ovarian serous carcinoma and mesothelioma
the pattern and extent of membranous and cytoplasmic E-cadherin immunoreactivity in
ductal versus lobular breast carcinomas
and the marked difference in immunoreactivity
between the M2-7C10 and Melan-A/A103 clones of MART1 in the PEComa neoplasm group (perivascular
epithelioid cell tumors, angiomyolipoma, lymphangiomyomatosis, and clear cell lung neoplasms) and
endocrine cell neoplasms
Recommendation 7: New antibody optimization should include serially testing and modifying if
necessary, antigen retrieval , antibody concentration, and chromogen detection system. Serial tissue
sections from one tissue block should be used to facilitate the comparison of stain appearances. A
minimum of two antigen retrieval buffer solutions, five different antibody concentrations, including at
least one above, at, and below the manufacturer's recommended concentration, and at least two types or
incubation times of chromogen detection systems. Each component procedure should be modified
accordingly. The combination of component procedures modifications that results in the most consistent
and best immunoreactivity is the optimal IHC assay for an individual antibody.
Comments: Laboratories differ greatly in their fixation and tissue processing factors. There
is also a broad spectrum of different types and forms of antigen retrieval and IHC staining procedures.
An antigen often has several antibody clones, produced by different manufacturers, and packaged in
several different forms. The combination of component procedure parameters that results in the best IHC
stain is unique to each individual laboratory. No single IHC assay produces, consistent, high-quality
IHC assays across all antigens, every antibody, and in all types of tissues. Recognition of the inherent
variability of IHC makes optimization of new antibodies mandatory, regardless of the extent, type, and
complexity of the automated IHC platform used by a laboratory. The optimization process often requires
several rounds of testing and modifying the component procedures. Occasionally, an antibody cannot be
optimized, regardless of the modifications made to the component procedure parameters. Changing antibody
clones will frequently remedy this problem. The avidity of most antibodies is influenced by the pH of
the AR buffer solution and diluent and have optimal signal intensity within a relatively narrow pH range.
Antigen Retrieval: testing of a low-pH (example: citrate at pH=6) buffer solution and high-pH
(example: EDTA or EDTA -TRIS at pH=8 or 9) buffer solution is recommended.
Antibody: Serial dilutions of the antibody including at least one antibody concentration above
and below the manufacturer's recommended concentration. These antibody concentrations should be applied
to at least two sets of tissue sections, each of which were antigen retrieved in a different buffer
solution. The optimal combination of antibody concentration and antigen retrieval buffer should be
selected. If necessary, additional rounds of serial dilutions of more finely graded antibody
concentrations should be performed.
Chromogen Detection System: All antibodies (prediluted ready-to-use or concentrated) should be
evaluated in at least two types of chromogen detection systems. Some antibodies, including some
hybridized rabbit monoclonal antibodies produce a strong antigenic signal using a modified avidin-biotin
or two-step polymer detection system whereas one-step polymer detection systems result in a thin weak
signal (unpublished data, personal experience).
Recommendation 8: Detailed documentation of the antibody optimization parameters and factors,
type of tissue used to optimize the antibody, and the tissue block case number should be maintained in
the laboratory manual. Records of the set of conditions utilized, when and how an assay was modified
should be maintained as part of the laboratory manual pertaining to the individual antibody. It is not
unusual, over a period of years for a new clone to be introduced, requiring a new set of assay
conditions. For an individual case, laboratories should be able to document what antibody clone and
assay conditions were in place when the IHC assay stain was performed.
B. Antibody Validation
Recommendation 9: For IHC assays that are interpreted as a categorical, positive/negative
manner, a minimum of 25 separate tissue specimens (samples) tested by an alternative validated method in
the same laboratory or by a validated method performed in another laboratory should be evaluated.
At least 10 samples should have high levels of the target antigen, 10 samples should have
intermediate to low levels of target antigen, and 5 samples should have no immunohistochemical evidence
of the target antigen.
Additional validation and more restrictive standardized IHC assay parameters are required
for therapeutic predictive IHC-assays, such as Her2, estrogen receptor, and progesterone receptor
Predictive IHC-assays are strongly influenced by factors such as length of formalin
Genetically-engineered, rabbit monoclonal antibodies, such as clone SP1 ER
antibody (Lab Vision Co, Fremont, CA) have greater avidity compared to mouse monoclonal antibodies
. Validation must be to tissues with known levels of ER protein expression or valid
clinical/ therapeutic endpoint to avoid miscalibration
Parallel staining comparison to
immunoreactivity of a different but similarly, un-calibrated IHC assay is insufficient in this regard.
As complexity of interpretation increases, the number and spectrum of specimens required for validation
must increase accordingly.
Comments: Each tissue specimen is considered to be a single tissue sample for validation
purposes, regardless of the number of tissue blocks from the case. In this context, multiple tissue
blocks from a large neoplasm are considered as be a single tissue specimen. Three levels of target
antigen are selected to confirm that the signal threshold was set appropriately and define the IHC assay
parameters. The purpose for the validation procedure is to evaluate the IHC assays performance across a
range of clinical samples in terms of relative specificity and sensitivity of the IHC reaction.
For this reason it is important that the samples used for the validation procedure be handled in
terms of the pre-analytical variables described above, as close as possible to the clinical samples from
the laboratory on which the new test might potentially be offered. Tissue microarrays if available
are acceptable for the purpose of validating a new IHC assays, and depending on how they have been
constructed, have the potential for the survey of a number of different normal tissues and multiple tumor
types with the new IHC procedure.
Recommendation 10: A new IHC assay should show
a high level of concordance with the validated assays to which it is being compared. Data should be
evaluated and discrepancies investigated to determine their origin. A systematic approach of corrective
actions should be taken.
Recommendation 11: A log of the validation specimens samples and the IHC assay results should
be kept with the antibody package insert material in the antibody section of the laboratory manual.
B. New Lots of Active IHC Assays
Recommendation 12: All validated IHC assays need to be completely revalidated if significant
changes are made to the assay procedure. When new antibody lots of validated active IHC assays are
brought into the laboratory, three validation test samples should be tested. At least one sample should
have a high level of the target antigen, one sample should have intermediate to low level of target
antigen, and one sample should have no immunohistochemical evidence of the target antigen. Validation of
new antibody lots should be sufficient to determine the performance parameters of the IHC assay.
A. Interpreting IHC Assays
Recommendation 13: IHC assay immunoreactivity should be assessed with a quantitative scoring
All scoring systems should have two separate components: the extent or
proportion of the target antigen that was immunoreactive and the intensity of the immunoreactivity.
Immunoreactivity of an IHC assay should be assessed first. Following this assessment, a positive-result
cut-point threshold should be applied to the result that is clinically appropriate (i.e., contextual) and
takes into account the expected level of immunoreactivity, specimen size, and amount of target antigen in
Comment: There is no universal IHC scoring system. An example of a results classification
system with broad clinical utility and used by several members of the committee is classify 0-20%
immunoreactivity as "focally reactive", 21% - 80% as "variably reactive", and >80% immunoreactivity as
"uniformly reactive". Another system being used in clinical predictive IHC-assays is the IHC score
(range, 0 – 400) which the product of staining intensity (range, 1 - 4) and percentage of immunoreactive
There is also no universal cut-point threshold for a positive result
that can be applied across all clinical situations and types of specimens. Substantial variability in
interpretation is introduced by the clinical situation, type of specimen, amount of available target
tissue in the specimen, and adequacy of specimen fixation and processing .
Interpreting IHC assays in small specimens differs from their interpretation in large
specimens . In small specimens, the extent and intensity of immunoreactivity should be
assessed in the regional area with the highest proportion of target immunoreactivity. The positive
result cut-point should increase (right shift) as the amount of the target antigen available for
interpretation decreases. For low-sensitivity antibodies or focally expressed antigens, any level of
immunoreactivity can be significant. Numerous factors can complicate the determination of whether an IHC
assay result is classified as positive or negative. Separating the assessment and scoring of
immunoreactivity assessment from the determination of whether an IHC assay result is positive or
negative is recommended to improve reproducibility and accuracy of IHC assay interpretation.
B. Reporting IHC Assays
Recommendation 14: The following information should be included in the IHC assay report. Each
antibody should be reported separately.
1. Antibody clone. Given that the performance of different antibody clones against the same antigen
can vary significantly affecting the end result of immunoreactivity, it is critical to report the clone
name and type of antibody (polyclonal or monoclonal). For polyclonal antibodies, reference to the
vendor's catalogue number should be provided.
2. The subcellular localization (nucleus, cytoplasm) which is immunoreactive, pattern of antigen
expression (granular, dot, linear, homogeneous), intensity of immunoreactivity within the cellular
compartment, and the proportion of cells demonstrating this pattern of immunoreactivity.
3. The scoring system that was applied and the immunoreactivity cut-point threshold of a positive
4. Whether there is an internal positive control cell or structure present on the test (patient)
slide and descriptive documentation regarding the intensity and proportion of internal control cells or
structures that were immunoreactive.
5. Specimen number and block used for IHC slides.
6. Type of fixative and length of fixation.
7. Tissue specimen anatomic location and type of specimen.
Comments: Ideally, all IHC assays would be interpreted using a single, universal standardized
scoring system . The different cellular and structural antigenic targets, broad spectrum of
clinical situations in which IHC is used, and the lack of consistent, reliable, external and internal
control standards, makes this an unrealistic goal
Many oncologic-related nuclear
regulator antigens are normally present in the nucleus and cytoplasm. It is important that pathologists
understand the transport pathways of these proteins and restrict interpretation to the relevant
subcellular structures as reported in the literature by that specific antibody clone
In the absence of a universal IHC assay scoring system, responsibility rests with the pathologist to
provide this information in the report. Documentation also provides a method of verifying the
interpreting pathologist evaluated immunoreactivity in the appropriate antigenic target and applied
appropriate cut-points thresholds.
Table 1 IHC Assay Total Test Concept
|Pre-analytic ||Test selection|
| ||Specimen type, acquisition, transport time|
| ||Fixation: type and time|
| ||Tissue processing, type and temperature|
|Analytic ||AR procedure|
| ||Protocol, control selection|
| ||Regent validation|
| ||Technician training/ certification|
| ||Laboratory certification|
|Post-analytic ||Control evaluation|
| ||Results interpretation|
| ||Results reporting|
| ||Pathologist, experience and CME|
Table 2: List of factors that could be adjusted during the antibody optimization
|No pretreatment ||Some antibodies still perform best without any type of pretreatment|
|Enzyme digestion ||Few antibodies perform best only when enzyme digestion was used without the need for HIER|
|Retrieval buffer ||The combination of the type of buffer (i.e., citration, EDTA, Tris), and pH level can result in dramatically different signal intensity and signal-to-noise ratio|
|Heating device ||i.e., pressure cooker, electronic water bath, microwave, steamer, hot plate|
|Primary antibody incubation time ||This varies depending on the affinity of the antibody to its antigen target, the primary antibody concentration, incubation temperature and antigen levels in target tissue|
|Detection system ||Polymer detection systems may allow to further dilute the antibody titer, given their generally higher sensitivity than avidin-biotin systems. Tyramine amplification systems are the most sensitive, but also most cumbersome.|
|Chromogen ||Prolonging the application of chromogen often lead to more intense signal, but could also compromise the signal-to-noise ratio|
|Name ||Highest Degree(s) ||Institution ||E-mail Address|
|Neal S. Goldstein ||MD ||William Beaumont Hospital ||NGoldstein@beaumont.edu|
|Hadi Yaziji ||MD ||Ancillary Pathways ||firstname.lastname@example.org|
|Clive R. Taylor ||MD D Phil ||Keck School of Medicine, University of Southern California ||Clive.email@example.com|
|Ren L. Ridolfi ||MD ||Southern California Kaiser Permanente ||Ren.L.Ridolfi@kp.org|
|David G. Hicks ||MD ||Roswell Park Cancer Institute ||firstname.lastname@example.org|
|Stephen M Hewitt ||MD, PhD ||National Cancer Institute ||email@example.com|
|David J Dabbs ||MD ||Magee-Women's Hospital ||firstname.lastname@example.org|
|Alvin W Martin ||MD ||University of Louisville School of Medicine ||Awmart01@gwise.louisville.edu|
|Sunil Badve ||MBBS, MD (Path) ||Indiana University ||email@example.com|
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