Effects of Preanalytical Variables on the Detection of
Proteins by Immunohistochemistry in Formalin-Fixed,
Paraffin-Embedded Tissue
Kelly B. Engel, PhD; Helen M. Moore, PhD
Nding
Context.—While formalin fixation and paraffin embed-
has become a universal mechanism of tissue
preservation and a gold standard for immunohistochemis-
try, fixation and processing variables that may confound
assay effectiveness have received little attention from the
scientific community.
Objective.—To identify discrete steps in specimen fixation
and processing that may impact immunostaining, assess the
magnitude of reported effects in the literature, and highlight
preanalytical variables that require further investigation.
Data Sources.—Thirty-nine primary research articles
that investigated immunohistochemical effects of 1 or
more preanalytical variables were identified by our litera-
ture survey. Thresholds identified in the literature were then
compared with published immunohistochemistry guidelines
for formalin-fixed, paraffin-embedded specimens.
Conclusions.—Of the 62 preanalytical variables identi-
fied, 27 were examined in published research. Meta-analysis
RATIONALE
Routinely, formalin-fixed, paraffin-embedded (FFPE)
tissue specimens are analyzed by immunohistochemistry
within clinical and research laboratories around the world.
However, this ‘‘routine’’ process that was developed
decades ago is one that is both complex and variable.
Marked differences in specimen-processing regimens are
prevalent both within and among institutions owing to a
lack of standardization and formal standard operating
procedures. Thus, the influence of fixation and processing
variables on immunohistochemistry assay effectiveness, as
well as analytic and clinical outcomes, remains largely
unknown. In conjunction with the Biospecimen Research
Network (BRN), a program within the National Cancer
Institute’s Office of Biorepositories and Biospecimen Re-
search, we assessed the current state of the science to both
Accepted for publication December 8, 2010.
From Preferred Staffing Group for Office of Biorepositories and
Biospecimen Research, National Cancer Institute, Washington, DC (Dr
Engel); and the Office of Biorepositories and Biospecimen Research,
National Cancer Institute, Bethesda, Maryland (Dr Moore).
The authors have no relevant financial interest in the products or
companies described in this article.
Reprints: Helen M. Moore, PhD, Office of Biorepositories and
Biospecimen Research, National Cancer Institute, Bethesda, MD
20892 (e-mail: moorehe@mail.nih.gov).
Arch Pathol Lab Med—Vol 135, May 2011
revealed 15 preanalytical variables that were capable of
impacting immunohistochemistry (including fixation delay;
fixative type; time in fixative; reagents and conditions of
dehydration, clearing, and paraffin impregnation; and
conditions of slide drying and storage) and 12 variables
with no reported influence (including the type of processor
used; the number and position of specimens during
dehydration, clearing, and paraffin impregnation; and the
duration of paraffin block storage). Variables with antigen-
dependent or inconsistent effects were highlighted. Com-
parison of literature-supported thresholds with published
recommendations revealed (1) strong agreement among
preanalytical variables for optimal immunostaining, (2)
discrepancies among thresholds for adequate immunostain-
ing, and (3) the continued need for rigorous research and
comprehensive guidelines on specimen fixation, processing,
and storage.
(Arch Pathol Lab Med. 2011;135:537–543)
define and steer current and future BRN research initiatives.
We identified potential sources of preanalytical variability,
from the experience of the authors and BRN contributors,
and existing literature (Table 1). Surveys of published
primary research targeting discrete steps of specimen
fixation, processing, and storage were conducted to
evaluate their possible effects on quantitative and qualita-
tive immunohistochemical analysis. Preanalytical variables
with reported immunohistochemical effects are summa-
rized in Table 2. Variables that were not reported to elicit
changes in immunostaining (see Table 3), and those that
produced antigen-dependent or conflicting results or were
underrepresented in the literature, were also highlighted.
Evidence-based thresholds for optimal immunostaining
were compared with those cited by published immunohis-
tochemistry guidelines and recommendations (Table 4).
Although additional sources of preanalytical variability,
such as disparities among tissue type or antigen retrieval
techniques, may be capable of influencing immunohisto-
chemistry to unknown extents, the scope of the present
review was limited to FFPE processing variables.
PREFIXATION
A delay in specimen fixation at room temperature or 4uC
does not significantly alter the extent or intensity of
immunostaining when less than 12 hours, according to the
published literature.1–4 Although nonsignificant reductions
Preanalytical Variables and Immunohistochemistry—Engel & Moore 537
 Table 1. Potential Sources of Preanalytic Variation During Specimen Fixation and Processing
Prefixation
Duration and delay of temperature
Specimen size
Specimen manipulation (pathology ink)
Fixative
Formula
Concentration
pH
Age of reagent
Preparation source
Fixation
Tissue to fixative volume ratio
Method (immersion, injection, and sonication or microwave
acceleration)
Conditions of primary and secondary fixation
Movement
Light exposure
Primary container
No. and position of cofixed specimens
Postfixation
Washing conditions and duration
Storage reagent and duration
Processing
Type of processor, frequency of servicing and reagent
replacement
Tissue to reagent volume ratio
No. and position of coprocessed specimens
in progesterone receptor (12%) and estrogen receptor (ER;
3%) Q scores—an additive quantification method incorpo-
rating the intensity and proportion of immunopositive
staining—were observed after a room temperature prefixa-
tion delay of 1 hour and 2 hours, respectively, immuno-
staining for either receptor was not significantly altered even
after a delay of 8 hours at room temperature or 24 hours at
4uC.3 Effects noted after delays greater than 12 hours appear
to be antigen specific; for example, the incidence of
immunopositive cells was artificially increased,5 decreased,6
or unaffected,5,7 depending upon the targeted antigen.
Current guidelines for ER and progesterone receptor
immunohistochemistry from the American Society of
Clinical Oncology/College of American Pathologists rec-
ommend limiting prefixation delays to 1 hour or less,8 while
an acceptable prefixation delay was not discussed in the
remaining guidelines assessed.9,10
To the best of our knowledge, data on the medium used
for prefixation storage, use of pathology ink, or method
used to set the dye, and their influence on immunohisto-
chemistry, have not been reported.
FORMALIN FIXATION
Formula
The concentration, pH, and presence or absence of
buffer in the formalin solution used for specimen
preservation affects the quality of immunostaining in
FFPE specimens. Among the published reports reviewed,
most antigens yielded consistent immunostaining and
superior preservation when specimens were preserved in
10% neutral-buffered formalin (NBF) at a solution pH of 5
to 7 compared with unbuffered formalin1 or NBF at a
lower or higher pH.2,6,11 However, the optimal fixative for
immunohistochemistry may also depend upon the anti-
gen of interest. The intensity of epidermal growth factor
receptor immunostaining was superior in specimens
538 Arch Pathol Lab Med—Vol 135, May 2011
Dehydration and clearing
Reagent
Temperature
No. of changes
Duration (total and change-specific)
Paraffin impregnation
Type and melting point of wax
No. of changes
Duration (total and change-specific)
Method (immersion and sonication or microwave acceleration)
Paraffin sectioning
Type of blade and frequency of replacement
Frequency of servicing and wax replacement
Temperature of block during sectioning
Slide pretreatment
Water bath conditions, if used
Chemical adhesives, if used
Temperature and duration of slide drying
Storage
Temperature and duration of paraffin block storage
Temperature, duration, and manipulation of slide-mounted tissue sections
preserved with 4% unbuffered formalin compared with
10% unbuffered formalin or 10% NBF, although the extent
of immunostaining did not differ among differentially
fixed specimens.12 The type of buffer used for NBF may
also affect the quality of immunostaining; T-lymphocyte
surface membrane cluster of differentiation (CD) antigens
CD3, CD4, and CD8 displayed superior intensity and
reduced background staining when specimens were
preserved in 10% formalin buffered in Tris solution
compared with phosphate-buffered saline (PBS).1 While
published recommendations and guidelines for immuno-
histochemistry advise fixation in 10% NBF,8,10 phosphate
buffered saline is cited as the preferred buffer.10
We did not find evidence of published reports
investigating the potential influences of fixative age, or
commercial versus laboratory fixative preparation, on
immunohistochemistry.
Fixation
Although published recommendations agree regarding
the 1:10 volume ratio of specimen to fixative,8,10 equivalent
immunostaining results were reported for 5 antigens
assessed in specimens preserved with volume ratios
ranging from 1:1 to 1:20.2
The duration of fixation, that is, the length of time a
specimen is immersed in a fixative, has been a topic of
great interest in the scientific community owing in part to
conflicting results and recommended thresholds. While
effects associated with underfixation or overfixation hold
clinical relevance and include reductions in both the
intensity and extent of immunostaining,1,2,6,13–17 as well as
graded staining from periphery to core in underfixed
specimens,18 definitive thresholds for stable immunostain-
ing remain undefined. Published immunohistochemistry
guidelines assessed in the present review agree that
24 hours in fixative is optimal,8–10 although minimum
Preanalytical Variables and Immunohistochemistry—Engel & Moore
 Table 2. Variables in Specimen Fixation and Processing With Reported Effects on Immunohistochemistry
Antigen Conflicting
Preanalytic Variable Analytic Effect Dependent Reports Source, y
Fixation delay (.12 h) Alterations in the extent and intensity Yes No Start et al,5 1992
of immunostaining von Wasielewski et al,6 2002
Hammond et al,8 2010
Fixative Alterations in the extent and intensity Yes No Pollard et al,1 1987
Concentration of immunostaining, as well as Williams et al,2 1997
pH nonspecific background staining von Wasielewski et al,6 2002
Buffer von Wasielewski et al,11 1998
Atkins et al,12 2004
Time in fixative Alterations in the extent, distribution, Yes Yes Pollard et al,1 1987
and intensity of immunostaining Williams et al,2 1997
von Wasielewski et al,6 2002
von Wasielewski et al,11 1998
Arber,13 2002
Goldstein et al,14 2003
Middleton et al,15 2009
Shi et al,17 2007
De Marzo et al,18 2002
Ibarra et al,19 2010
Dehydration Alterations in the extent and intensity No Yes Pollard et al,1 1987
Reagent of immunostaining Williams et al,2 1997
Duration Cerio and MacDonald,29 1986
Temperature
Clearing Alterations in the extent and intensity No Yes Williams et al,2 1997
Reagent of immunostaining, as well as Cerio and MacDonald,29 1986
Temperature nonspecific background staining
Paraffin embedding Alterations in the extent and intensity No Yes Pollard et al,1 1987
Temperature of immunostaining Williams et al,2 1997
Duration Cerio and MacDonald,29 1986
Section/slide adhesion Alterations in the intensity of No Yes Pollard et al,1 1987
Temperature and immunostaining and nonspecific Williams et al,2 1997
duration background staining Jones et al,32 2001
Storage of slide-mounted Alterations in the extent and intensity Yes Yes Williams et al,2 1997
sections of immunostaining, as well as case van den Broek and van de
Temperature status Vijver,31 2000
Duration Bromley et al,33 1994
Shin et al,35 1997
Bertheau et al,37 1998
DiVito et al,38 2004
Jacobs et al,39 1996
Wester et al,40 2000
Fergenbaum et al,41 2004
Grabau et al,42 1998

and maximum fixation durations vary, with a minimum
of 6 hours8,9 versus longer than 8 hours10 and a maximum
of 48 hours9,10 versus 72 hours.8 A survey of the literature
introduces further variability, with several studies citing
antigen-specific stability with regard to time in fixative.2,13
Even for a common antigen evaluated in a particular
tissue, ER in breast specimens for example, data conflict,
with a minimum of 1 hour,19 6 hours,14,17 or 24 hours6,11 in
fixative required for uniform and reproducible immuno-
staining. Although maximum times in fixative also vary
among reports, the imminent risk for overfixation is
comparatively lower, with reductions in ER immuno-
staining intensity in breast tissue reported after 4 days,11
30 days,17 or 57 days.13 For other antigens, underfixation is
also a greater concern than overfixation.1,15,18 Formalin
fixation for 24 hours yielded reliable immunohistochem-
istry results for several antigens in biopsy or surgically
procured FFPE specimens.1,2,6,11,13,16–18,20,21 In fact, only 1
study reported a modest reduction in immunostaining
intensity after 22 hours, compared with specimens fixed
Arch Pathol Lab Med—Vol 135, May 2011
for 6 hours,22 after examination of CD5 immunostaining
on tissue specimens procured during autopsy.
Given the temporal constraints of the biochemical
fixation process, which is dependent on the equilibrium
between formaldehyde and methylene glycol,8,10 and the
desire for quick clinical turnaround of pathology labora-
tory results, several different methods to reduce fixation
time have been explored. One such strategy is accelerating
the penetration rate of the fixative by increasing the
temperature of fixation2 or prefacing specimen immersion
with injection,18 both of which result in modestly reduced
immersion times and immunostaining that is equivalent
to traditionally fixed specimens. Other novel fixation
methods, such as microwave heating 23–26 and ultrasound
acceleration,22,27 have recently been developed and claim
to shorten required fixation from hours to minutes.
However, recommendations by the Clinical and Labora-
tory Standards Institute (CLSI) for immunohistochemistry
acknowledge that while microwave irradiation can short-
en fixation times, specimens should be (1) 5 mm thick or
Preanalytical Variables and Immunohistochemistry—Engel & Moore 539
 Table 3. Reported Variables in Specimen Fixation and Processing That Do Not Influence Immunohistochemistry
Preanalytic Variable Range Examined Source, y
Fixation delay
Temperature Room temperature, 4uC Pollard et al,1 1987
Williams et al,2 1997
Khoury et al,3 2009
Di Tommaso et al,4 1999
Fixation
Tissue to fixative ratio 1:1 to 1:20 Williams et al,2 1997
Method of fixation Immersion, injection coupled with immersion, microwave De Marzo et al,18 2002
acceleration Chu et al,22 2005
Azumi et al,23 1990
Hopwood et al,24 1984
Login and Dvorak,25 1988
Morales et al,26 2004
Chu et al,27 2006
Postfixation wash
Duration 6–24 h Pollard et al,1 1987
Tissue processing
Type of automated processor Carousel, enclosed vacuum Pollard et al,1 1987
Williams et al,2 1997
No. and position of specimens Not specified Williams et al,2 1997
Duration of clearing Unspecified reduced and extended duration compared to Williams et al,2 1997
4-h control
Type of paraffin Polymer, nonpolymer, microcrystalline Williams et al,2 1997
Section/slide adhesion
Chemical adhesive Gelatin, APES, APES with glutaraldehyde, poly-L-lysine van den Broek and van de Vijver,31 2000
Storage
Duration of block storage 2–25 y Scharl et al,16 1990
Bromley et al,33 1994
Manne et al,34 1997
Shin et al,35 1997
Conditions of slide storage Paraffin coating, nitrogen desiccator, deparaffinization and Bromley et al,33 1994
immersion in PBS at 4uC DiVito et al,38 2004
Jacobs et al,39 1996
Wester et al,40 2000
Abbreviations: APES, aminopropyl-ethoxysilane; PBS, phosphate-buffered saline.

less, (2) immersed in formalin for no less than 4 hours
before microwave irradiation, and (3) microwaved for
5 minutes or less in 100 ml of formalin.10 Guidelines on
ultrasound-accelerated formalin fixation are unavailable
for immunohistochemistry, and the technique is classified
as ‘‘experimental’’ by the CLSI recommendations for
immunohistochemistry.10
While secondary fixation is common practice in many
laboratories, details concerning the reagents, duration,
and conditions of secondary fixation often associated with
automated tissue processing are frequently unreported in
published literature. To the best of our knowledge,
variables associated with secondary fixation have not
been evaluated for their influence on immunohistochem-

Table 4. Formal and Literature-Based Recommendations for Specimen Fixation and Processing Variables for Optimal
Staining for Most Antigens Via Immunohistochemistry
Preanalytic Variable Published Guidelines and Recommendations Literature-Based Recommendations
Fixation delay ,1 h8 ,12 h1–4
Fixation
Fixative formulaa 10% NBF, pH not specified8,10 10% NBF, pH 5–71,2,6,12
Time in fixativea 24 h8–10 24 h1,2,6,11,13,16–18,20–21
Tissue to fixative ratio 1:108,10 1:1 to 1:202
Dehydration (total duration) 1.25–15 h10 10 h1,2
Paraffin impregnation
Type of paraffin Low-melting-point paraffin (55uC–58uC)10 Low-melting-point paraffin (45uC)1
Total duration 0.5–4.5 h10 1–2 h1,28 or 8 h2
Slide drying 24 h at room temperature or 1 h at 50uC–60uC10 24 h at room temperature1 or overnight at 37uC2
Storage duration
Paraffin blocka Indefinitely10 #25 y16,33–35
Slide-mounted sectionsa 7 d9 or ,6 wk8 ,6 d38
Abbreviation: NBF, neutral-buffered formalin.
a
Antigen-specific effects have been reported.

540 Arch Pathol Lab Med—Vol 135, May 2011 Preanalytical Variables and Immunohistochemistry—Engel & Moore
istry, although CLSI recommends that secondary fixation
associated with automated tissue processing be included
in the reported fixation duration.10
Fixation parameters that have not yet been evaluated for
confounding effects on immunohistochemistry include
the size and manipulation of the specimen before
immersion (such as sectioning or biopsy procurement ex
vivo), the primary method of containment during fixation
(eg, a biopsy bag or cassette), and other fixation
conditions, such as movement of the specimen within
the fixative solution, exposure to light, and the number
and relative position of cofixed specimens.
TISSUE PROCESSING
Washing and Short-Term Alcohol Storage
While removal of excess fixative from specimens before
dehydration and subsequent processing is not uncommon,
little evidence is available concerning downstream analytic
effects of such treatment. A single article1 examined the
duration of such a wash and concluded that washing fixed
specimens in running tap water for 6 hours before
dehydration did not alter immunostaining of surface
membrane antigens, although a modest reduction in
staining intensity was observed after a 24-hour wash.
Although a widely accepted practice and a suggested
alternative to prolonged storage in formalin,10 to our
knowledge, the potential impacts of alcohol storage on
specimen immunoreactivity have not been evaluated in a
published report. It is our belief that potential effects
associated with short- or long-term storage of specimens
in another class of fixative warrant further attention.
Type of Processor
The type of automated processor (carousel versus
enclosed vacuum),1,2 and the number and position of
specimens within the carrier2 during dehydration, clear-
ing, and paraffin impregnation are reported to not alter
the intensity of immunostaining.
Dehydration and Clearing
Although evidence is limited, the type of reagents used
for dehydration and clearing may influence the extent and
intensity of immunostaining. A single study1 cites that both
the extent and intensity of immunostaining were superior in
specimens that underwent dehydration in isopropanol
compared with ethanol, methanol, acetone, or 8 other
reagents. Reported effects attributable to the type of clearing
agent used range in severity from no observable difference2
to altered staining intensity28 to alterations in both the
intensity and prevalence of immunostaining.1 While both
studies investigating this variable cited favorable immuno-
staining after clearing with chloroform,1,28 the success of
other clearing agents was less clear. For example, xylene
was reported to be inferior to chloroform and Inhibisol
(Bestobell Chemical Products Ltd, Mitcham, Surrey, UK)28;
equivalent to chloroform,1 Clearene (Surgipath, St Neots,
UK), and an unspecified xylene substitute2; and superior to
Histo-Clear (National Diagnostics, Atlanta, Georgia), tolu-
ene, and carbon tetrachloride.
The temperature of dehydration and clearing influences
the quality of immunostaining, although favorable effects
have been reported for temperatures both lower and higher
than ambient. Compared with controls that were dehy-
drated at room temperature, specimens dehydrated in
Arch Pathol Lab Med—Vol 135, May 2011
isopropanol at 4uC displayed an increase in the extent and
intensity of immunostaining, although temperature-medi-
ated effects were also dependent upon the reagent used.1
Dehydration and clearing of specimens at 4uC attenuated
background staining,29 while a temperature of 45uC in-
creased the intensity of immunostaining2 as compared with
controls processed at room temperature. Although the total
duration of dehydration improved the quality of immuno-
staining, with 10 hours resulting in more intense immuno-
staining and reduced background, compared with shorter
or longer durations,1,2 the duration of clearing did not
appear to influence immunohistochemistry, although data
were not shown.2
Details on specimen processing among published recom-
mendations and guidelines are scarce, suggesting only that
specimens undergo 1.25 to 15 hours of dehydration,
15 minutes to 3 hours per alcohol stage with 5 recom-
mended stages, with duration dependent upon the type and
size of the specimen and the efficiency of the processor.10
Paraffin Impregnation
While the type of paraffin wax (polymer, nonpolymer,
and microcrystalline) used for paraffin impregnation does
not affect immunostaining,2 the melting point of the
polymer paraffin may elicit alterations, as both the extent
and intensity of immunostaining were enhanced among
specimens embedded in paraffin with a low (45uC) as
opposed to a high (65uC) melting point.1 The duration of
paraffin impregnation can also impact immunostaining,
although reports conflict as to whether shorter (1–2 hours)1,29
or longer durations (8 hours)2 produce favorable results.
Conversely, recent guidelines and recommendations
cite that paraffin with a melting point of 55uC to 58uC and
durations of 0.5 to 4.5 hours, 15 minutes to 3 hours per
stage with 2 to 3 stages recommended, is preferred for
immunohistochemistry.10
Novel Techniques
While recent reports suggest that the time required for
sufficient tissue processing may be reduced when
performed in conjunction with microwave irradiation26,30
or ultrasound sonication,27 formal published recommen-
dations are not yet available.
Unaddressed Variables
Several factors associated with specimen processing
through dehydration, clearing, and paraffin have yet to be
examined in detail concerning their impact on immuno-
histochemistry. These variables include the age of the
reagents, which is also related to whether serial stage
reagents are freshly replaced or transitioned owing to
presumed evaporation or dilution; the number of stages
used and the frequency of cleaning and servicing of the
automated processor; and the temperature at which
specimen positioning within a block occurs.
PARAFFIN SECTIONING
Variables related to sectioning of FFPE specimens, such as
the type of blade and frequency of replacement, the
frequency of microtome cleaning and servicing, the temper-
ature of the block during sectioning, slide pretreatment, and
water bath conditions, if applicable, have received little
attention in the literature; however, techniques used to
promote paraffin section-slide adhesion have been investi-
gated. While chemicals used to promote section adhesion to
Preanalytical Variables and Immunohistochemistry—Engel & Moore 541
glass slides do not alter immunostaining or antigen stability,31
the temperature and duration of slide drying can elicit
significant effects. Published guidelines for immunohisto-
chemistry recommend drying slides at room temperature for
24 hours or 50uC to 60uC for a minimum of 1 hour.10 While
stable immunostaining was reported among slides dried at
room temperature for 24 hours1 or at 37uC overnight,2 drying
at 60uC for as little as 4 hours reduced immunostaining
intensity in 23% of target antigens compared with 37uC
overnight controls, and effects escalated with temperature
and duration, as 69% of target antigens displayed reduced
staining intensity or increased nonspecific staining after an 8-
hour incubation at 70uC.2 A different study32 reported
anecdotal reductions in staining intensity among sections
dried at 80uC, but not 58uC or 68uC, for 16 hours.
STORAGE
Paraffin Blocks
When compared with freshly embedded controls, para-
ffin blocks archived at room temperature for as little as
2 years or as long as 25 years exhibited stable immuno-
staining for most antigens evaluated.16,33–35 In fact, of 6
antigens investigated for storage stability, proliferating
cell nuclear antigen alone displayed storage duration–
dependent reductions in both immunostaining extent and
intensity, effects that were attenuated by antigen retriev-
al.36 Published immunohistochemistry guidelines concur
with current literature, noting that paraffin blocks may be
stored indefinitely in a ‘‘cool place.’’ 10
Paraffin Sections
Storage of slide-mounted tissue sections can alter
immunoreactivity, although both the time line and
magnitude of effect are antigen dependent. Immunostain-
ing effects that have been attributed to slide storage
include an artificial reduction2,31,33,37–40 or enhancement37 of
staining intensity as well as reductions in the number of
immunopositive cells.2,31,37–42 Reports conflict as to whether
these effects translate to alterations in case status.35,41
Although published guidelines recommend storage of
slide-mounted tissue sections be limited to 7 days or
fewer,10 time lines associated with stable immunostaining
appear to be antigen specific,2,31,37–41 with immunostaining
altered after 6 days38 or stable for up to 300 days of room-
temperature storage.31 However, thresholds for stable
immunoreactivity of paraffin sections vary widely among
published reports, even for a common antigen evaluated in
a particular tissue. For example, ER immunostaining of
slide-mounted sections was adversely affected after, but
not before, 6 days,38 8 weeks,33 12 weeks,37,39 or 6 months31
of room-temperature storage, although 1 report2 cites
stable immunostaining for up to 6 months of storage.
Storage-induced alterations to ER immunostaining include
reductions in staining intensity,31,33,37–40 the number of
immunopositive cells,37,38,40–42 and possibly case status.38,41
While several techniques have been evaluated for their
potential to preserve antigenicity of slide-mounted sec-
tions, a universal preservation protocol has yet to be
identified. Although preservation of antigenicity was supe-
rior among slides stored at 220uC compared with storage
at 4uC or 20uC,40,42 slide/tissue section separation frequently
occurred during immunohistochemistry after storage at
220uC.40 The efficacy of antigen preservation at 4uC varies
among reports, with slides stored at 4uC displaying either
542 Arch Pathol Lab Med—Vol 135, May 2011
superior31,39 or equivalent immunostaining,2,33 compared
with 20uC controls. Such conflicts regarding the success of
cold slide storage may be influenced by the antigen
targeted or the antibody used, given the differential
stability of 32 antigens evaluated with slides stored at
4uC, 21uC, and 37uC.31 While coating slides in paraffin
before room-temperature storage failed to attenuate stor-
age-induced alterations in immunoreactivity,39,40 paraffin
coating coupled with storage in a nitrogen desiccator
improved the preservation of antigenicity when compared
with untreated slides or either technique alone.38 Although
storage of deparaffinized slides in 10% sucrose in phosphate-
buffered saline at 4uC prevented the spatially graded
reduction in immunostaining intensity observed among
untreated slides stored at room temperature for 4 weeks,
the duration of stability was not extended beyond 4 weeks.33
CONCLUSIONS
Implementation of immunohistochemistry for the analy-
sis of FFPE specimens is both historic and extensive among
research and clinical laboratories. However, given the lack
of alignment among specimen fixation and processing
protocols at different institutions, a central question arises:
Could fixation and processing-related artifacts skew immu-
nohistochemistry data and resultant conclusions? The
results of our literature survey suggest that the answer is
yes. Documented effects of preanalytical variables range in
magnitude from the presence of nonspecific background
staining to alterations in immunostaining prevalence,
intensity, and possibly case status. Given the diversity of
effects associated with variations in preanalytical handling,
it is logical to consult available guidelines and recommen-
dations; however, the guidelines we used for comparison in
the present review were not comprehensive, addressing
only 26% of the preanalytical variables we have identified
(Table 1). Further, for several parameters the recommended
ranges were inclusive of values with literature-reported
effects, such as durations of dehydration and paraffin
impregnation. Notably, conflicting data were reported for
several parameters in the literature, even for a common
antigen. Variation among reported effects may be attribut-
able to (1) the tissue type used, as differences in
procurement strategy, specimen density, and cell type
may translate to differences in susceptibility; (2) the
utilization of different antigen retrieval methods, which
may introduce additional or mitigate existing effects
associated with other preanalytical variables; (3) antibody
specificity; (4) differences among the method of quantifica-
tion; (5) the type and strength of the applied statistical test;
and (6) incomplete methodology. Of the 61 preanalytical
variables identified as discrete steps in specimen fixation
and processing (Table 1), only 44% had published results,
with the remaining 56% overlooked or unreported by the
scientific community as a whole. Results of our review
highlight the importance of standardized specimen fixation
and processing, and the continued need for comprehensive
guidelines and rigorous biospecimen research.
The Biospecimen Research Network, a program within
the National Cancer Institute’s Office of Biorepositories
and Biospecimen Research, was developed to address
challenges such as these. Extramural research supported
by the BRN will elucidate effects of preanalytical variables,
as well as provide a solid foundation for evidence-based
best practices for specimen procurement, handling, and
storage. Detailed summaries of existing research on
Preanalytical Variables and Immunohistochemistry—Engel & Moore
preanalytical variables, including articles cited in this
review, can be found within the Biospecimen Research
Database (http://biospecimens.cancer.gov/brd/; accessed
December 2, 2010), a BRN initiative and a free searchable
online resource for the location of published peer-reviewed
research and review articles.
We thank Carolyn Compton, MD, PhD, and Jim Vaught, PhD,
for their support of this project and their insightful review of the
manuscript, and Stephen Hewitt, MD, PhD, and Jim Robb, MD,
for helpful discussions concerning potential sources of pre-
analytical variability.
References
1. Pollard K, Lunny D, Holgate CS, Jackson P, Bird CC. Fixation, processing,
and immunochemical reagent effects on preservation of T-lymphocyte surface
membrane antigens in paraffin-embedded tissue. J Histochem Cytochem. 1987;
35(11):1329–1338.
2. Williams JH, Mepham BL, Wright DH. Tissue preparation for immunocy-
tochemistry. J Clin Pathol. 1997;50(5):422–428.
3. Khoury T, Sait S, Hwang H, et al. Delay to formalin fixation effect on breast
biomarkers. Mod Pathol. 2009;22(11):1457–1467.
4. Di Tommaso L, Kapucuoglu N, Losi L, et al. Impact of delayed fixation on
evaluation of cell proliferation in intracranial malignant tumors. Appl Immuno-
histochem Mol Morphol. 1999;7(3):209–213.
5. Start RD, Cross SS, Clelland C, Silcocks PB, Rogers K, Smith JH. Delay in
fixation does not affect the immunoreactivity of proliferating cell nuclear antigen
(PCNA). J Pathol. 1992;168(2):197–199.
6. von Wasielewski R, Mengel M, Wiese B, Rüdiger T, Müller-Hermelink HK,
Kreipe H. Tissue array technology for testing interlaboratory and interobserver
reproducibility of immunohistochemical estrogen receptor analysis in a large
multicenter trial. Am J Clin Pathol. 2002;118(5):675–682.
7. Hendricks JB, Wilkinson EJ. Quality control considerations for Ki-67
detection and quantitation in paraffin-embedded tissue. J Cell Biochem Suppl.
1994;19:105–110.
8. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical
Oncology/College of American Pathologists guideline recommendations for
immunohistochemical testing of estrogen and progesterone receptors in breast
cancer (unabridged version). Arch Pathol Lab Med. 2010;134(7):e48–e72.
9. Wolff AC, Hammond ME, Schwartz JN, 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(1):18–43.
10. Lightfoote MM, Ball DJ, Hannon WH, Ridderhof JC, Vogt RF Jr. Quality
Assurance for Design Control and Implementation of Immunohistochemistry
Assays; Approved Guideline. 2nd ed. Wayne, PA: Clinical and Laboratory
Standards Institute (CLSI); 2010.
11. von Wasielewski R, Mengel M, Nolte M, Werner M. Influence of fixation,
antibody clones, and signal amplification on steroid receptor analysis. Breast J.
1998;4(1):33–40.
12. Atkins D, Reiffen KA, Tegtmeier CL, Winther H, Bonato MS, Störkel S.
Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues:
variation in staining intensity due to choice of fixative and storage time of tissue
sections. J Histochem Cytochem. 2004;52(7):893–901.
13. Arber DA. Effect of prolonged formalin fixation on the immunohisto-
chemical reactivity of breast markers. Appl Immunohistochem Mol Morphol.
2002;10(2):183–186.
14. Goldstein NS, Ferkowicz M, Odish E, Mani A, Hastah F. Minimum
formalin fixation time for consistent estrogen receptor immunohistochemical
staining of invasive breast carcinoma. Am J Clin Pathol. 2003;120(1):86–92.
15. Middleton LP, Price KM, Puig P, et al. Implementation of American Society
of Clinical Oncology/College of American Pathologists HER2 guideline
recommendations in a tertiary care facility increases HER2 immunohistochem-
istry and fluorescence in situ hybridization concordance and decreases the
number of inconclusive cases. Arch Pathol Lab Med. 2009;133(5):775–780.
16. Scharl A, Vierbuchen M, Conradt B, Moll W, Würz H, Bolte A.
Immunohistochemical detection of progesterone receptor in formalin-fixed and
paraffin-embedded breast cancer tissue using a monoclonal antibody. Arch
Gynecol Obstet. 1990;247(2):63–71.
17. Shi SR, Liu C, Taylor CR. Standardization of immunohistochemistry for
formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval
technique: from experiments to hypothesis. J Histochem Cytochem. 2007;55(2):
105–109.
Arch Pathol Lab Med—Vol 135, May 2011
18. De Marzo AM, Fedor HH, Gage WR, Rubin MA. Inadequate formalin
fixation decreases reliability of p27 immunohistochemical staining: probing
optimal fixation time using high-density tissue microarrays. Hum Pathol. 2002;
33(7):756–760.
19. Ibarra JA, Rogers LW, Kyshtoobayeva A, Bloom K. Fixation time does not
affect the expression of estrogen receptor. Am J Clin Pathol. 2010;133(5):747–755.
20. Jensen V, Ladekarl M. Immunohistochemical quantitation of oestrogen
receptors and proliferative activity in oestrogen receptor positive breast cancer.
J Clin Pathol. 1995;48(5):429–432.
21. Wilkens L, Werner M, Nolte M, et al. Influence of formalin fixation on the
detection of cytomegalovirus by polymerase chain reaction in immunocompro-
mised patients and correlation to in situ hybridization, immunohistochemistry,
and serological data. Diagn Mol Pathol. 1994;3(3):156–162.
22. Chu WS, Furusato B, Wong K, et al. Ultrasound-accelerated formalin
fixation of tissue improves morphology, antigen and mRNA preservation. Mod
Pathol. 2005;18(6):850–863.
23. Azumi N, Joyce J, Battifora H. Does rapid microwave fixation improve
immunohistochemistry? Mod Pathol. 1990;3(3):368–372.
24. Hopwood D, Coghill G, Ramsay J, Milne G, Kerr M. Microwave fixation:
its potential for routine techniques, histochemistry, immunocytochemistry and
electron microscopy. Histochem J. 1984;16(11):1171–1191.
25. Login GR, Dvorak AM. Microwave fixation provides excellent preserva-
tion of tissue, cells and antigens for light and electron microscopy. Histochem J.
1988;20(6–7):373–387.
26. Morales AR, Nassiri M, Kanhoush R, Vincek V, Nadji M. Experience with
an automated microwave-assisted rapid tissue processing method: validation of
histologic quality and impact on the timeliness of diagnostic surgical pathology.
Am J Clin Pathol. 2004;121(4):528–536.
27. Chu WS, Liang Q, Tang Y, et al. Ultrasound-accelerated tissue fixation/
processing achieves superior morphology and macromolecule integrity with
storage stability. J Histochem Cytochem. 2006;54(5):503–513.
28. Matthews JB. Influence of clearing agent on immunohistochemical
staining of paraffin-embedded tissue. J Clin Pathol. 1981;34(1):103–105.
29. Cerio R, MacDonald DM. Effect of routine paraffin wax processing on cell
membrane immunoreactivity in cutaneous tissue. J Clin Lab Immunol. 1986;
20(2):97–100.
30. Emerson LL, Tripp SR, Baird BC, Rohr LR. A comparison of immunohis-
tochemical stain quality in conventional and rapid microwave processed tissues.
Am J Clin Pathol. 2006;125(2):176–183.
31. van den Broek LJ, van de Vijver MJ. Assessment of problems in diagnostic
and research immunohistochemistry associated with epitope instability in stored
paraffin sections. Appl Immunohistochem Mol Morphol. 2000;8(4):316–321.
32. Jones WT, Stockard CR, Grizzle WE. Effects of time and temperature
during attachment of sections to microscope slides on immunohistochemical
detection of antigens. Biotech Histochem. 2001;76(2):55–58.
33. Bromley C, Palechek P, Benda J. Preservation of estrogen receptor in
paraffin sections. J Histotechnol. 1994;17(2):115–118.
34. Manne U, Myers RB, Srivastava S, Grizzle WE. Re: loss of tumor marker-
immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer
Inst. 1997;89(8):585–586.
35. Shin HJ, Kalapurakal SK, Lee JJ, Ro JY, Hong WK, Lee JS. Comparison of
p53 immunoreactivity in fresh-cut versus stored slides with and without
microwave heating. Mod Pathol. 1997;10(3):224–230.
36. Malmström PU, Wester K, Vasko J, Busch C. Expression of proliferative
cell nuclear antigen (PCNA) in urinary bladder carcinoma: evaluation of antigen
retrieval methods. APMIS. 1992;100(11):988–992.
37. Bertheau P, Cazals-Hatem D, Meignin V, et al. Variability of immunohis-
tochemical reactivity on stored paraffin slides. J Clin Pathol. 1998;51(5):370–374.
38. DiVito KA, Charette LA, Rimm DL, Camp RL. Long-term preservation of
antigenicity on tissue microarrays. Lab Invest. 2004;84(8):1071–1078.
39. Jacobs TW, Prioleau JE, Stillman IE, Schnitt SJ. Loss of tumor marker-
immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer
Inst. 1996;88(15):1054–1059.
40. Wester K, Wahlund E, Sundström C, et al. Paraffin section storage and
immunohistochemistry: effects of time, temperature, fixation, and retrieval
protocol with emphasis on p53 protein and MIB1 antigen. Appl Immunohis-
tochem Mol Morphol. 2000;8(1):61–70.
41. Fergenbaum JH, Garcia-Closas M, Hewitt SM, Lissowska J, Sakoda LC,
Sherman ME. Loss of antigenicity in stored sections of breast cancer tissue
microarrays. Cancer Epidemiol Biomarkers Prev. 2004;13(4):667–672.
42. Grabau D, Nielsen O, Hansen S, et al. Influence of storage temperature
and high-temperature antigen retrieval buffers on results of immunohistochem-
ical staining in sections stored for long periods. Appl Immunohistochem. 1998;
6(4):209–213.
Preanalytical Variables and Immunohistochemistry—Engel & Moore 543