Special stains and immunostains
Histology labs eye new kids on the block
William Check, PhD
To demonstrate the value of immunostains, Allen M. Gown, MD, medical director
and chief pathologist at PhenoPath Laboratories, Seattle, goes back
to his days as a resident in the 1970s.
“When we worked up a case and H&E didn’t give the final answer,
we first elected special stains,” he says. That included things
like reticulum stain. Now when a diagnostic dilemma arises, in most
laboratories the first step is to think of immunostains. “Antibodies
to type IV collagen can be used routinely to address many of the
same issues that were addressed by reticulum stains,” Dr.
Gown says. Looking for organisms might be one place where traditional
stains prevail. “Even then sometimes immunostains do a good
or even better job,” he says, citing Steiner stain for Helicobacter pylori.
Dr. Gown prefers to distinguish immunostains from special stains. “Maybe because
of the way immunostains were introduced into the laboratory, in
some people’s minds they are a kind of special stain,”
he says. In his view, special stains are empirical—they just
happen to work—whereas immunostains are defined by an antibody
of known specificity.
Because they are based on antibodies, immunostains have proved more of a challenge
to automate than special stains. Immunostains require special considerations
in tissue preservation and preparation to optimize their use. “They
are less forgiving than special stains,” Dr. Gown says. He
cites antigen retrieval as something generally required for immunostains
but not for special stains.
Forefront immunostains include antibodies to relatively esoteric tumor-specific markers,
such as the FLI-1 gene product found in Ewing’s sarcoma and
the WT-1 gene product that characterizes desmoplastic small cell
tumor. Both of these gene products represent C’ terminal elements
of chimeric fusion proteins created by tumor-specific chromosomal translocations.
For such tumor-type specific markers, says Paul Swanson, MD, director of anatomic pathology
at the University of Washington Medical Center, Seattle, “when
the translocation-defined portion of the protein is expressed in
the appropriate histologic context, it can be a reliable marker
for that disease.” In these cases, immunohistochemistry detects
what otherwise would be identified by cytogenetic analysis or rtPCR
for the chimeric fusion transcript. “We cannot claim either
sensitivity or specificity benefits with immunostains relative to
cytogenetics,” Dr. Swanson says. “But sometimes because
of tissue handling, conventional cytogenetics or molecular techniques
cannot be performed. In particular, rtPCR is extremely dependent
on fresh or properly prepared tissue,” Dr. Swanson says. If
tissue is formalin-fixed and routinely processed, it can be hard to retrieve intact mRNA.
In these situations, IHC offers an alternative. Immunostains that recognize lineage-restricted
markers composed of a family of transcription regulators have also
appeared on the forefront of diagnostic pathology. Most widely used
among these is thyroid transcription factor 1, or TTF 1, says Dr.
Swanson. “It is associated with elements of foregut development
in fetal tissues,” he says. It is also expressed in differentiated
thyroidal and pulmonary epithelium, and as such is a very sensitive
and reasonably specific marker for thyroid and pulmonary glandular
and endocrine neoplasms. It also has limited value in extrapulmonary neuroendocrine carcinoma.
A related marker, CDX-2, is preferentially expressed in midgut and hindgut epithelium.
“It is a reasonably specific and sensitive marker for gastrointestinal
carcinomas, especially carcinoma of the colon and rectum,” Dr. Swanson says.
TTF-1 and CDX-2 are particularly relevant to the diagnosis of metastatic carcinoma,
especially in those cases where primary site of involvement is uncertain
or unknown (occult clinical disease). “A lot of IHC today
revolves around that broader question,” Dr. Swanson says.
Other recent markers purport to distinguish between noninvasive proliferative
lesions and overtly invasive malignant disease. High-molecular-weight
keratins have been joined by the p53 analogue p63 as markers of
basal cells in prostate. P63 also complements antibodies to smooth
muscle determinants as a marker of myoepithelial cells in breast
and other organs. Immunoreactivity for these markers in the appropriate
context is evidence of an intact or attenuated basal cell or myoepithelial
layer (non-neoplastic or in situ disease), whereas the absence of
these markers is presumptive evidence of extension of carcinoma
beyond normal structures.
In contrast, α-methyl acyl coA racemase (p504s) is selectively expressed
in malignant epithelium of prostate cancer, ostensibly providing
a positive marker for carcinoma instead of the traditional inference
of malignancy from the loss of normal elements. Dr Swanson calls
it “somewhat controversial.”
Still other markers of increasing relevance to diagnostic pathology are those,
such as Her2/neu, topoisomerase–2α, and thymidylate
synthetase, that predict response to specific classes of chemotherapeutic
agents, a role until recently played only by estrogen and progesterone receptor proteins.
“For me, the important question posed by these markers is not whether a selective
panel of chemotherapeutic agents or other treatment options is available
to the patient,” Dr. Swanson says, “but whether such
treatment is of proven or expected clinical value.” In a similar
vein, one might question if there is a defined benefit to the physician
and the patient in making an immunohistochemical determination of the origin of metastatic disease.
“In most cases there is,” Dr. Swanson says, “but not always.
What we detect by immunohistochemistry may help dictate management,
but will it reliably predict outcome?”
William Check is a medical writer in Wilmette, Ill.