Application
Special Stains in Dermatopathology
Jameel Ahmad Brown, MD
Bruce R. Smoller, MD
Fellow: Division of Dermatopathology
University of Arkansas
for Medical Sciences
4301 W. Markham St.
Little Rock, AR, USA
Chair: Department of Pathology
University of Arkansas
for Medical Sciences
4301 W. Markham St.
Little Rock, AR, USA
Histochemical Stains
Dual staining with hematoxylin, which stains nuclei blue, and eosin,
a pink cytoplasmic stain, is the mainstay in routine examination of
cutaneous tissue under light microscopy. The ability to delineate
differences in tissue type and cellular components is greatly enhanced
by exploiting the inherent uptake properties of various tissues with these
stains. When particular characteristics are not easily identified with
hematoxylin and eosin, histochemical staining becomes of great value.
Its merit is recognized diagnostically and economically, as histochemical
staining is much less expensive than immunohistochemical staining and
is readily available in virtually all dermatopathology practice settings.
Commonly Used Special Stains
Periodic Acid-Schiff (PAS)
Periodic Acid-Schiff (PAS) is one of the most frequently employed
special stains in the dermatopathology laboratory. A positive stain is
perceived as purplish-red or magenta. PAS is used to demonstrate the
presence of neutral mucopolysaccharides, especially glycogen. The
process by which tissues are stained with PAS involves the oxidation
of hydroxyl groups in 1,2 glycols to aldehyde and subsequent staining
of the aldehydes with fuschin-sulfuric acid. PAS also has utility when
predigestion with diastase (PASD) has been performed. The diastase
removes glycogen from tissue sections but leaves other neutral
mucopolysaccharides behind. PAS stain allows for the recognition and
highlighting of basement membrane material and thickness, such as in
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lupus erythematosus. In addition, PASD accentuates fungal cell walls,
helps identify the presence of glycogen in tumor cells versus lipid,
and illuminates many PAS positive inclusion bodies, secretions, and
granules. Non-cutaneous pathology finds PAS equally useful (Fig. 1).
Alcian Blue
Alcian blue facilitates visualization of acid mucopolysaccharides
or acidic mucins. The staining procedure is performed at pH of
2.5 and 0.5 respectively. Positive staining is perceived as blue.
The mucopolysaccharide differential staining pattern is created by
and based on the principle that sulfated mucopolysaccharides, like
chondroitin sulfate and heparan sulfate, will stain at both pH values
while non-sulfated ones stain at a pH of 2.5 only. The most common
non-sulfated mucopolysaccharide of the skin is hyaluronic acid,
found normally in scant amounts in the papillary dermis, surrounding
cutaneous appendages, and encircling the vascular plexuses. Alcian
blue is also an excellent special stain when attempting to detect
acidic mucin in neoplasms, inflammatory conditions, and dermatoses
including granuloma annulare, lupus erythematosus, dermatomyositis,
scleromyxedema, pre-tibial myxedema, scleredema, and follicular
mucinosis. Additionally, sialomucin produced in extramammary Paget’s
disease demonstrates positive alcian blue staining. Of note, colloidal
iron demonstrates a staining profile which is essentially indistinguishable
from alcian blue, and the use of one over the other will be laboratorydependent; based on the familiarity of the operator with the nuances of
each stain and the quality of results obtained.
Figure 1. PAS-D showing fungal organisms
in a case of onychomycosis.
Figure 2. Fite stain demonstrating acid fast
bacilli in a case of lepromatous leprosy.
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Elastic Tissue Stain (Verhoff von Gieson)
The elastic tissue stain (ETS), a silver stain, is used to demonstrate
and evaluate the quantity and quality of tissue elastic fibers. Positive
staining is perceived as black. ETS serves as a dependable adjunct
when evaluating tissue sections for entities under the umbrella of
elastolytic granulomata; these diseases include actinic granuloma,
atypical facial necrobiosis lipoidica, and granuloma multiforme. Similarly,
when assessing a patient for anetoderma (focal dermal elastolysis)
and cutis laxa, the elastic tissue stain aids in identifying complete
elastolysis, fragmented elastic fibers in the papillary and mid-reticular
dermis, and the morphology of existing elastic fibers. Anomalous
distribution and organization of elastic-rich tissue, as in collagenoma
and other variants of connective tissue nevi, can also be detected. Most
recently, the elastic tissue stain has received a new place in the spotlight
of the dermatopathology literature, primarily because of its utility in
differentiating the invasive component of melanoma from a benign
underlying or adjacent melanocytic nevus when the two are associated.
Brown and Brenn Stain
The Brown and Brenn stain is a tissue Gram stain that is a practical
method of identifying and differentially staining bacteria and resolving
them into two primary groups – Gram-negative and Gram-positive. The
baceteria’s staining characteristics are based on the physical properties
of their cell walls. Gram-positive bacteria have thick cell walls composed
of high proportions of peptidoglycan which retain the purple color during
the staining process. Peptidoglycan comprises only a small fraction of
the thin cell walls of Gram-negative bacteria which also have a lipid
outer membrane; both characteristics contribute to their pink color as
the purple dye (crystal violet) is washed away during the decolorization
step. In addition to the color contrast created by the stain, morphology
can also be elucidated and one can appreciate bacilli, cocci, or
coccobacilli. The tissue Gram stain is not ideal for all bacterial species,
as some have ambiguous staining patterns or are simply not highlighted.
The Brown and Brenn stain is very difficult to interpret in skin sections,
thus its diagnostic value is limited in dermatopathology practice.
Additionally, cultures of suspected cutaneous infections are superior in
sensitivity and reproducibility to the Brown and Brenn stain.
Acid Fast Stain (AFB)
The classic acid-fast bacilli stain used most commonly in
dermatopathology settings is the Ziehl-Neelsen stain. Acid-fast
organisms are those whose cell walls contain high lipid in the form of
mycolic acids and long-chain fatty acids. These characteristics permit
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stains are economical tools and have
“Special
rapid turn around times, giving them significant
advantages to many more sophisticated
ancillary diagnostic techniques.
”
strong binding and retention of carbol fuchsin dye after decolorization
with acid-alcohol. An AFB positive organism stains brightly red after
completion of the procedure. Frequently, a counterstain of methylene
blue is used to serve as a contrasting background. AFB stain is
employed most often when there is a high clinical suspicion of a
mycobacterial infection and when granulomatous inflammation is seen
in the absence of foreign body material on histopathologic examination.
The stain is an excellent adjunct to light microscopy when seeking the
presence of mycobacterium tuberculosis; however, not all mycobacterial
species stain positively. Modified versions of the classic stain include
modified bleach Ziehl-Neelsen, Kinyoun, Ellis and Zabrowarny,
auramine-rhodamine (most sensitive of all), and Fite. The Fite stain
highlights mycobacteria in general but is specifically used to identify
mycobacterium leprosum. The stronger acid used in the classic method
is deemed too harsh for M. leprae and the lipid in the cell’s membrane is
washed away, making visualization of the organism difficult. Fite uses a
weaker acid in the decolorization phase of the procedure, preserving the
more delicate cell walls of the organism. The Fite and the Ziehl-Neelsen
methods share their positive acid-fast profiles; red is read as a positive
stain (Fig. 2).
Giemsa
Giemsa is a metachromatic stain in that many tissues/organisms
stain differently than the dye color itself. The dye mixture – methylene
blue, azure, and eosin compounds – is blue, but positive staining is
designated by a purplish hue. Giemsa is frequently used to identify mast
cells; their granules stain positively. Of note, the immunohistochemical
stains “mast cell tryptase” and “CD 117” are also used to detect mast
cells. Urticaria and urticaria pigmentosa are among the disease entities
marked by a significant increase in mast cell volume, thus Giemsa finds
its utility here. Further contributing to the value of Giemsa is the positive
staining of several infectious organisms; spirochetes, protozoans, and
cutaneous Leishmania in particular.
Figure 3. Mast cell tryptase highlighting
secretory granules of mast cells.
Figure 4. Fontana-Masson stain highlighting
melanin
in
epidermal
keratinocytes,
melanocytes, and dermal melanophages.
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Warthin-Starry
Warthin-Starry stain is a silver nitrate stain. Tissue sections are incubated
in a 1% silver nitrate solution followed by a developer such as hydroquinol.
Warthin-Starry is used chiefly in the identification of spirochetes in
diseases such as syphilis, Lyme disease, and acrodermatitis chronica
atrophicans. A positive stain will reveal black spirochetes. Like the
Verhoff von Gieson stain, the Warthin-Starry stain has the disadvantage
of non-specific elastic tissue fiber staining, making it difficult to interpret
in skin sections. Further complicating interpretation is positive staining
of melanocytes. The use of this stain is often supplanted by antibody
stains and/or serologies.
Masson’s Trichrome
Masson’s trichrome is a special stain which is typically used to
characterize and discriminate between various connective and soft tissue
components. Smooth muscle and keratin stain pink-red, collagen stains
blue-green, and elastic fibers appear black. Either phosphotungstic
or phosphomolybdic acid is used along with anionic dyes to create
a balanced staining solution. Masson’s trichrome stain is frequently
employed when the histopathologic differential includes leiomyomatous
and neural tumors. The finer characteristics of collagen in the dermis
can be better appreciated, as evidenced by the highlighted and welldelineated pattern demonstrated in collagenomas. Perivascular fibrosis,
scar formation, and sclerotic lesions are also better appreciated with the
use of Masson’s trichrome.
Congo Red
Detection of amyloid in tissue sections is greatly enhanced and
confirmed by positive staining with congo red. The stain itself is red-pink.
Examination of tissue sections suspected of involvement by amyloidosis
must be performed under both light microscopy and polarized light.
When polarized, amyloid has a characteristic apple green birefringence.
The color and tinctorial subtleties observed with congo red staining are
attributed to amyloid’s physical structural arrangement and antiparallel
beta-pleated sheets. The elastolytic material in colloid milium and the
degenerating keratinocytes in lichen amyloidosis also stain with congo
red. Other stains such as crystal violet, thioflavin T, and sirius red are
also capable of staining amyloid but they are far less convenient and
are less widely available.
Fontana-Masson
Silver is often a component of special stains in dermatopathology. Not
only is it used in the Verhoff von Gieson elastic tissue stain and the
Warthin-Starry spirochete stain described above, silver is a constituent
of the Fontana-Masson melanin stain as well. In the latter case, the
observer relies on the reduction of silver to form a black precipitate. In
the context of traumatized melanocytic lesions or melanocytic entities
superimposed upon hemorrhagic processes, the Fontana-Masson
can aid in the distinction between melanin and hemosiderin pigments.
Similarly, secondary dermal pigment deposits produced by specific
drug exposures can be distinguished from those with melanocytic
origins. Occasionally, Fontana-Masson is used in the evaluation of
vitiligo and post-inflammatory hyperpigmentation. Some observers
report that the Fontana-Masson stain is difficult to interpret when only
rare granular staining is present (Fig. 4).
Chloroacetate Esterase
The lineage-specific cytoplasmic granules of myeloid cells are readily
identified with the use of chloroacetate esterase. A positive stain is
observed as an intense bright red hue. The stain finds its relevance
in the investigation of malignant hematopoietic infiltrates in which the
lineage of the suspicious cells is unclear. It can help distinguish acute
lymphocytic leukemia from acute myeloid leukemia, although up to
approximately one-quarter of myeloid cases stain negatively, with false
negative findings attributed to excessive immaturity of the granulocytes
and significant monocytic expression. Lastly, mast cells also stain
positively with chloroacetate esterase.
In summary, special stains are an effective adjunct to routine staining
with hematoxylin and eosin in the practice of dermatopathology. While
some are easier to interpret than others, most find diagnostic utility in
everyday practice. Special stains are economical tools and have rapid
turn around times, giving them significant advantages to many more
sophisticated ancillary diagnostic techniques.
Selected Bibliography
1. Aroni K, Tsagroni E, Kavantzas N, Patsouris E, Ioannidis E. A study of the pathogenesis
of rosacea: how angiogenesis and mast cells may participate in a complex multifactorial
process. Archives of Dermatological Research 2008; 300:125-131.
2. Bancroft JD, Gamble M. Theory and practice of histologic techniques. 6th Edition.
Philadelphia: Churchill Livingstone Elsevier, 2008.
3. Bergey DH, Holt JH. Bergey’s Manual of Determinative Bacteriology. 9th Edition.
Philadelphia: Lippincott Williams & Wilkins, 1994.
4. Kamino H, Tam S, Tapia B, Toussaint S. The use of elastin immunostain improves the
evaluation of melanomas associated with nevi. Journal of Cutaneous Pathology 2009;
36(8):845-52.
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