Documentation of
Immunocytochemistry Controls in
the Cytopathologic Literature:
A Meta-Analysis of 100 Journal Articles
Carol Colasacco, M.L.I.S., S.C.T.(A.S.C.P.), C.T.(I.A.C.),1* Sharon Mount,
and Gladwyn Leiman, M.B.B.C.H., F.I.A.C., F.R.C.Path.1,2
Although a detailed description of the procedure and tissue used
as controls is considered a necessary component in surgical pa-
thology articles in which immunohistochemistry is utilized, such
documentation seems less stringent in the cytopathologic litera-
ture. A comprehensive literature search was done for articles
published in English within the last 15 years on nine of the most
widely used antibodies in cytopathology. Individual case reports
were excluded. Of the 100 articles reviewed, 13 articles were
review articles or commentaries and hence not included in the
analysis. Only 11 (13%) of the remaining 87 articles described
positive and negative controls run on identically prepared sam-
ples. Forty-seven articles (54%) either did not mention controls
or did not run controls as separate specimens. Sixteen articles
(18%) included a vague statement about controls. Twelve (14%)
commented only on the negative control, included only histology
tissue controls, or included cell block controls, but the study
also included other types of preparations, such as cytospins.
One article (1%) did not include controls because of insufficient
material. The College of American Pathologists recognizes the
impracticality of maintaining separate positive control samples
for every possible combination of fixation, processing, and speci-
men type. However, more stringent documentation of procedure
and use of controls in the cytopathologic literature will ensure
that immunocytochemistry results in diagnostic cytopathology as
well as in research are valid and reproducible. Diagn. Cytopa-
thol. 2011;39:245–250. ' 2010 Wiley-Liss, Inc.
Key Words: immunocytochemistry; immunoperoxidase; con-
trols; cytologic techniques; cytology
1
Department of Pathology and Laboratory Medicine, Fletcher Allen
Health Care, Burlington, Vermont
2
Pathology Department, University of Vermont College of Medicine,
Burlington, Vermont
*Correspondence to: Carol Colasacco, M.L.I.S., S.C.T.(A.S.C.P.),
C.T.(I.A.C.), Department of Pathology and Laboratory Medicine,
Fletcher Allen Health Care, Burlington, VT.
E-mail: Carol.Colasacco@vtmednet.org
Received 22 September 2009; Accepted 29 January 2010
DOI 10.1002/dc.21370
Published online 24 May 2010 in Wiley Online Library
(wileyonlinelibrary.com).
' 2010 WILEY-LISS, INC.
M.D.,
1,2
The use of enzyme conjugated antibodies to make possi-
ble the visualization of specific tissue antigens has
become a practical tool in both diagnostic surgical pathol-
ogy and cytopathology and is now a widely employed
technique throughout anatomic pathology laboratories.
The antibodies utilized include those directed at cytoplas-
mic and membrane-bound antigens as well as nuclear and
matrix antigenic sites. Immunocytochemical staining
allows a more specific classification of undifferentiated
neoplasms and, in some cases, a relatively specific identi-
fication of a primary tumor site for metastatic lesions. In
addition, immunocytochemistry has been employed for
the assessment of various biologic parameters that allow
for both the prediction of tumor behavior as well as
response to therapy. The popularity and usefulness of the
technique is reflected by the plethora of articles that have
appeared in the literature over the past 15 years.
In the arena of surgical pathology, the results of an im-
munohistochemical reaction are considered valid only if
proper positive and negative controls are used and eval-
uated in each procedure. The necessity of running control
tissues simultaneously with the diagnostic specimen poses
little logistical difficulty as, for the most part, histopatho-
logic tissue is processed in a similar manner. For exam-
ple, a tissue section is typically fixed in formalin and em-
bedded in paraffin prior to sectioning and staining. Hence,
the fixation and processing can be replicated in tissue sec-
tions used for controls. A detailed description of the pro-
cedure and tissue used as controls is considered a neces-
sary component in the surgical pathology literature
articles in which immunohistochemistry is used.
Cytopathologic specimens pose difficulties in obtaining
appropriate material for controls as the fixation and proc-
essing varies widely among specimens. Cytopathologic
specimens from fluids are processed quite differently from
aspirate smears, for example, which can be air-dried,
Diagnostic Cytopathology, Vol 39, No 4 245
Diagnostic Cytopathology DOI 10.1002/dc
COLASACCO ET AL.

fixed in ethyl alcohol, or processed by liquid-based meth- Table I. Search Terms Utilized
odology. Cell blocks have also been popular in diagnostic MeSH headings Keywords (‘‘text words’’)
cytopathology, adding yet another methodology to the AE3 protein, human AE1 HMB45
preparation of specimens. Anion exchange AE3 LCA
In many diagnostic cytopathology departments, includ- protein 1, erythrocyte Cell block Liquid-based
Antigens, CD45 Chromogranin S100
ing our own, histologic tissue is chosen as a positive con- Chromogranins CK7 Smear
trol as is done in surgical pathology. Histologic tissue is Cytology CK20 SurePath
also used for the negative control in cases where patient Histocytochemistry Cytocentrifuge Synaptophysin
Keratins, type I Fine needle ThinPrep
cytologic material is limited. For cytopathologic speci- Keratins, type II Histocytochemistry TTF-1
mens, with the exception of cell blocks which may be S100 protein Leukocyte common
processed in a manner analogous to histologic sections, Synaptophysin Antigen
Thyroid nuclear factor 1
this creates what is referred to in clinical chemistry as a
‘‘matrix mis-match’’1; in that there is a disparity between
the control material and the actual patient sample. In
addition, the development of commercially available anti- When a search query is entered into PubMed, a word-
bodies used in diagnostic pathology is based on their weighted algorithm is used to compare the terms in each
expression on histologic tissue sections, which may not citation, including those in the title and abstract as well
be transferrable to cytopathologic samples. This heightens as the MeSH headings assigned during the indexing func-
the importance of using appropriate controls when anti- tion. As a result, if a search term does not map to a spe-
bodies are used on cytopathologic specimens. cific MeSH heading, it will search for individual key-
Concern about this issue spurred us to investigate the words that appear in journal article titles and abstracts. In
use of controls by other cytopathology laboratories. The some cases, the search term utilized may map to a differ-
result is this meta-analysis of articles published in English ent term if it has been identified as a synonym by the
spanning a 15-year period as it pertains to immunocyto- National Library of Medicine. In our search, immunocyto-
chemistry with emphasis on the reporting of controls. chemistry mapped to immunohistochemistry, so entering
either term in the search query retrieved over 450,000
articles, many of which dealt specifically with histopathol-
Methods
ogy, not cytopathology studies. Therefore, it was impera-
To complete a comprehensive literature search for articles tive that a more thoughtful approach to developing a
focused on immunocytochemistry, it was necessary to search strategy be created to retrieve the most relevant
employ the Medical Subject Headings (MeSH) developed by articles possible. We chose to utilize MeSH headings,
the National Library of Medicine for use in MEDLINE1; when possible, in combination with focused keywords in
the bibliographic database that contains citations and author an effort to increase the relevance of the articles retrieved
abstracts from over 5,000 scientific journals. This database while reducing the total number retrieved to a manageable
is utilized by PubMed (http://www.ncbi.nlm.nih.gov/sites/ number.
entrez?db ¼ pubmed) and other databases developed by The articles were reviewed by two of the authors and
private vendors. A detailed search strategy was designed by in cases of disagreement consensus was reached among
utilizing the MeSH dictionary, available on the PubMed all three authors. The definitions used to classify the
website, to identify appropriate Medical Subject Headings articles were as follows:
(MeSH). Using these relevant MeSH headings helped
locate articles for which immunocytochemistry was a main Absent: Controls were not mentioned.
topic and in which at least one of the following common Vague: Statement such as ‘‘appropriate positive and
stains was utilized: AE1/AE3, LCA, HMB45, CK7, CK20, negative controls were included.’’
S100, synaptophysin, chromagranin, or TTF-1 (Table I). Identical: Controls identical to study samples were
Appropriate keywords (‘‘Text Words’’) were also identified described.
and used as additional search tools. The ‘‘related articles’’ Other: Controls were dissimilar or partially similar (i.e.,
link was followed for particularly descriptive articles. tissue control with smears or tissue control with cell block
Search limits were placed for articles published in English and ThinPrep samples run), or samples were too scant to
and articles published within the prior 15 years, except for include controls.
several searches conducted to identify ‘‘classic’’ articles Not applicable: Review articles and editorials, not new
covering the history of immunocytochemistry. These diagnostic research studies.
searches were constructed utilizing the MeSH terms ‘‘histo-
cytochemistry’’ and ‘‘cytodiagnosis,’’ linking the terms Results
with the Boolean operator ‘‘AND.’’ Individual case reports A total of 100 articles were reviewed from 13 different
were excluded. scientific journals1–100 (Fig. 1). Of these, 13 articles were

246 Diagnostic Cytopathology, Vol 39, No 4

 Diagnostic Cytopathology DOI 10.1002/dc
IMMUNOCYTOCHEMISTRY CONTROLS

others have clearly described the use of ideal immunocy-

tochemistry controls. Nadji and Ganjei74 included a
detailed description of ideal controls when performing
immunocytochemical staining in their review of immuno-
cytochemistry in diagnostic cytology in 1990, yet many
recently published articles that describe studies utilizing
immunocytochemistry include only a vague statement
about the controls that were used or no information at all.
The College of American Pathologists addresses the
impracticality of maintaining separate positive control
samples for every possible combination of fixation, proc-
essing, and specimen type in its comment for questions
Fig. 1. Description of immunocytochemistry controls in articles ANP 22550 in the Anatomic Pathology checklist (avail-
reviewed. able via the CAP website at http://www.cap.org/apps/cap.
portal.). In this comment, parallel testing is suggested as
Table II. Immunocytochemistry Controls by Journal one way to demonstrate equivalent immunoreactivity
between specimens of different types or specimens proc-
Journal Absent Vague Identical Other N/A
essed in different ways. This may be a straightforward,
Acta Cytologica 17 5 3 1 2 appropriate method to ensure that controls utilized for
Diagnostic Cytopathology 11 6 4 4 7
Cancer Cytopathology 9 3 2 5 1 immunocytochemistry are reacting reliably and in a man-
Cytopathology 1 2 0 0 1 ner that would be reproducible.
Modern Pathology 0 0 2 0 0 Many fine articles exist in the cytopathologic literature
AJCP 3 0 0 2 2
Other 6 0 0 1 0 that include clear descriptions regarding the process used
Total 47 16 11 13 13 when developing control slides for the published study. A
recent article discussing the use of immunohistochemistry
review articles or commentaries so were not applicable. stains on cytoscrapes (CS) by Skov et al.91 clearly
Only 11 of the remaining 87 articles (13%) described pos- describes the controls used, including the purposes of the
itive and negative controls run on identically prepared reference specimens ‘‘(1) to examine whether ancillary
samples. Forty-seven articles (54%) did not mention con- methods can be applied to cytologic material converted to
trols. Sixteen articles (18%) included a vague statement tissue fragment (CS) and (2) to examine the concordance
about controls (i.e., ‘‘appropriate negative and positive of ancillary methods made on CS and surgical biopsies.’’
controls were included.’’) Twelve articles (14%) had Liu and Farhood,61 among others, also clearly described
descriptions of controls that were partially similar (i.e., what controls were used for both positive and negative
tissue controls run when cell blocks and ThinPrep slides controls in their 2004 article. It was surprising to dis-
were stained) or dissimilar controls (i.e., tissue controls cover, however, that in our review, over half of published
when only smears were stained), and one article (1%) articles do not even mention controls.
stated that no controls were done because of insufficient It is our assertion that, while immunocytochemistry
material (Table II). controls may be different than the patient sample tested
out of necessity, the lack of detail regarding specific
Discussion immunocytochemistry controls used in published studies
A number of articles have been published in the scientific in the cytopathologic literature may impede subsequent
literature recognizing the challenges of applying immuno- research utilizing similar protocols. Inclusion of more
histochemistry stains to diagnostic cytopathology. Fetsch detail in regard to the use of immunocytochemistry con-
and coworkers33 compared staining differences between trols would better enable diagnostic cytopathology labora-
ThinPrep slides, cell blocks, and tissue controls. Mittel- tories to properly utilize the immunocytochemical stains
dorf et al.71 reported that TTF-1 staining intensity was currently available and ensure the validity and reproduci-
slightly lower in cytopathology preparations than surgical bility of results. The impetus to change may well reside
pathology samples, Gong et al.40 noted that staining within the ambit of journal editorial board staff during
results on cytopathology and tissue samples were compa- peer review of immunochemistry-based cytopathologic
rable with nonnuclear markers and Fadda et al.31 articles.
described the presence of increased background staining
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